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translate(-50%, -50%);\n transform: translate(-50%, -50%);\n -webkit-transform-origin: center center;\n transform-origin: center center;\n}\n\n.fa-layers-counter {\n background-color: #ff253a;\n background-color: var(--fa-counter-background-color, #ff253a);\n border-radius: 1em;\n border-radius: var(--fa-counter-border-radius, 1em);\n -webkit-box-sizing: border-box;\n box-sizing: border-box;\n color: #fff;\n color: var(--fa-inverse, #fff);\n line-height: 1;\n line-height: var(--fa-counter-line-height, 1);\n max-width: 5em;\n max-width: var(--fa-counter-max-width, 5em);\n min-width: 1.5em;\n min-width: var(--fa-counter-min-width, 1.5em);\n overflow: hidden;\n padding: 0.25em 0.5em;\n padding: var(--fa-counter-padding, 0.25em 0.5em);\n right: 0;\n right: var(--fa-right, 0);\n text-overflow: ellipsis;\n top: 0;\n top: var(--fa-top, 0);\n -webkit-transform: scale(0.25);\n transform: scale(0.25);\n -webkit-transform: scale(var(--fa-counter-scale, 0.25));\n transform: 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animation-delay: var(--fa-animation-delay, 0);\n -webkit-animation-direction: normal;\n animation-direction: normal;\n -webkit-animation-direction: var(--fa-animation-direction, normal);\n animation-direction: var(--fa-animation-direction, normal);\n -webkit-animation-duration: 1s;\n animation-duration: 1s;\n -webkit-animation-duration: var(--fa-animation-duration, 1s);\n animation-duration: var(--fa-animation-duration, 1s);\n -webkit-animation-iteration-count: infinite;\n animation-iteration-count: infinite;\n -webkit-animation-iteration-count: var(--fa-animation-iteration-count, infinite);\n animation-iteration-count: var(--fa-animation-iteration-count, infinite);\n -webkit-animation-timing-function: cubic-bezier(0.4, 0, 0.6, 1);\n animation-timing-function: cubic-bezier(0.4, 0, 0.6, 1);\n -webkit-animation-timing-function: var(--fa-animation-timing, cubic-bezier(0.4, 0, 0.6, 1));\n animation-timing-function: var(--fa-animation-timing, cubic-bezier(0.4, 0, 0.6, 1));\n}\n\n.fa-flip {\n -webkit-animation-name: fa-flip;\n animation-name: fa-flip;\n -webkit-animation-delay: 0;\n animation-delay: 0;\n -webkit-animation-delay: var(--fa-animation-delay, 0);\n animation-delay: var(--fa-animation-delay, 0);\n -webkit-animation-direction: normal;\n animation-direction: normal;\n -webkit-animation-direction: var(--fa-animation-direction, normal);\n animation-direction: var(--fa-animation-direction, normal);\n -webkit-animation-duration: 1s;\n animation-duration: 1s;\n -webkit-animation-duration: var(--fa-animation-duration, 1s);\n animation-duration: var(--fa-animation-duration, 1s);\n -webkit-animation-iteration-count: infinite;\n animation-iteration-count: infinite;\n -webkit-animation-iteration-count: var(--fa-animation-iteration-count, infinite);\n animation-iteration-count: var(--fa-animation-iteration-count, infinite);\n -webkit-animation-timing-function: ease-in-out;\n animation-timing-function: ease-in-out;\n -webkit-animation-timing-function: var(--fa-animation-timing, ease-in-out);\n animation-timing-function: var(--fa-animation-timing, ease-in-out);\n}\n\n.fa-spin {\n -webkit-animation-name: fa-spin;\n animation-name: fa-spin;\n -webkit-animation-delay: 0;\n animation-delay: 0;\n -webkit-animation-delay: var(--fa-animation-delay, 0);\n animation-delay: var(--fa-animation-delay, 0);\n -webkit-animation-direction: normal;\n animation-direction: normal;\n -webkit-animation-direction: var(--fa-animation-direction, normal);\n animation-direction: var(--fa-animation-direction, normal);\n -webkit-animation-duration: 2s;\n animation-duration: 2s;\n -webkit-animation-duration: var(--fa-animation-duration, 2s);\n animation-duration: var(--fa-animation-duration, 2s);\n -webkit-animation-iteration-count: infinite;\n animation-iteration-count: infinite;\n -webkit-animation-iteration-count: var(--fa-animation-iteration-count, infinite);\n animation-iteration-count: var(--fa-animation-iteration-count, infinite);\n -webkit-animation-timing-function: linear;\n animation-timing-function: linear;\n -webkit-animation-timing-function: var(--fa-animation-timing, linear);\n animation-timing-function: var(--fa-animation-timing, linear);\n}\n\n.fa-spin-reverse {\n --fa-animation-direction: reverse;\n}\n\n.fa-pulse,\n.fa-spin-pulse {\n -webkit-animation-name: fa-spin;\n animation-name: fa-spin;\n -webkit-animation-direction: normal;\n animation-direction: normal;\n -webkit-animation-direction: var(--fa-animation-direction, normal);\n animation-direction: var(--fa-animation-direction, normal);\n -webkit-animation-duration: 1s;\n animation-duration: 1s;\n -webkit-animation-duration: var(--fa-animation-duration, 1s);\n animation-duration: var(--fa-animation-duration, 1s);\n -webkit-animation-iteration-count: infinite;\n animation-iteration-count: infinite;\n -webkit-animation-iteration-count: var(--fa-animation-iteration-count, infinite);\n animation-iteration-count: var(--fa-animation-iteration-count, infinite);\n -webkit-animation-timing-function: steps(8);\n animation-timing-function: steps(8);\n -webkit-animation-timing-function: var(--fa-animation-timing, steps(8));\n animation-timing-function: var(--fa-animation-timing, steps(8));\n}\n\n@media (prefers-reduced-motion: reduce) {\n .fa-beat,\n.fa-fade,\n.fa-flash,\n.fa-flip,\n.fa-pulse,\n.fa-spin,\n.fa-spin-pulse {\n -webkit-animation-delay: -1ms;\n animation-delay: -1ms;\n -webkit-animation-duration: 1ms;\n animation-duration: 1ms;\n -webkit-animation-iteration-count: 1;\n animation-iteration-count: 1;\n -webkit-transition-delay: 0s;\n transition-delay: 0s;\n -webkit-transition-duration: 0s;\n transition-duration: 0s;\n }\n}\n@-webkit-keyframes fa-beat {\n 0%, 90% {\n -webkit-transform: scale(1);\n transform: scale(1);\n }\n 45% {\n -webkit-transform: scale(1.25);\n transform: scale(1.25);\n -webkit-transform: scale(var(--fa-beat-scale, 1.25));\n transform: scale(var(--fa-beat-scale, 1.25));\n }\n}\n@keyframes fa-beat {\n 0%, 90% {\n -webkit-transform: scale(1);\n transform: scale(1);\n }\n 45% {\n -webkit-transform: scale(1.25);\n transform: scale(1.25);\n -webkit-transform: scale(var(--fa-beat-scale, 1.25));\n transform: scale(var(--fa-beat-scale, 1.25));\n }\n}\n@-webkit-keyframes fa-fade {\n 50% {\n opacity: 0.4;\n opacity: var(--fa-fade-opacity, 0.4);\n }\n}\n@keyframes fa-fade {\n 50% {\n opacity: 0.4;\n opacity: var(--fa-fade-opacity, 0.4);\n }\n}\n@-webkit-keyframes fa-flash {\n 0%, 100% {\n opacity: 0.4;\n opacity: var(--fa-flash-opacity, 0.4);\n -webkit-transform: scale(1);\n transform: scale(1);\n }\n 50% {\n opacity: 1;\n -webkit-transform: scale(1.125);\n transform: scale(1.125);\n -webkit-transform: scale(var(--fa-flash-scale, 1.125));\n transform: scale(var(--fa-flash-scale, 1.125));\n }\n}\n@keyframes fa-flash {\n 0%, 100% {\n opacity: 0.4;\n opacity: var(--fa-flash-opacity, 0.4);\n -webkit-transform: scale(1);\n transform: scale(1);\n }\n 50% {\n opacity: 1;\n -webkit-transform: scale(1.125);\n transform: scale(1.125);\n -webkit-transform: scale(var(--fa-flash-scale, 1.125));\n transform: scale(var(--fa-flash-scale, 1.125));\n }\n}\n@-webkit-keyframes fa-flip {\n 50% {\n -webkit-transform: rotate3d(0, 1, 0, -180deg);\n transform: rotate3d(0, 1, 0, -180deg);\n -webkit-transform: rotate3d(var(--fa-flip-x, 0), var(--fa-flip-y, 1), var(--fa-flip-z, 0), var(--fa-flip-angle, -180deg));\n transform: rotate3d(var(--fa-flip-x, 0), var(--fa-flip-y, 1), var(--fa-flip-z, 0), var(--fa-flip-angle, -180deg));\n }\n}\n@keyframes fa-flip {\n 50% {\n -webkit-transform: rotate3d(0, 1, 0, -180deg);\n transform: rotate3d(0, 1, 0, -180deg);\n -webkit-transform: rotate3d(var(--fa-flip-x, 0), var(--fa-flip-y, 1), var(--fa-flip-z, 0), var(--fa-flip-angle, -180deg));\n transform: rotate3d(var(--fa-flip-x, 0), var(--fa-flip-y, 1), var(--fa-flip-z, 0), var(--fa-flip-angle, -180deg));\n }\n}\n@-webkit-keyframes fa-spin {\n 0% {\n -webkit-transform: rotate(0deg);\n transform: 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On top of the design aspect, the field of computational biology and protein modelling is an area of interest for many engineering students, which was emphasized as well."}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"WiSTEM"}),Object(Le.jsx)("div",{className:pe.a.description,children:"This year, we decided to work with other clubs at Waterloo to engage different groups in science and synthetic biology. We worked with the University of Waterloo Women in STEM club to create a mini conference with speakers from various different scientific fields. Our team presented two presentations over the course of the conference. We spoke on the importance of GMOs and science communication as well as what synthetic biology is with reference to our 2020 project REMINE. 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Additionally, an overview of the software (Rosetta, GROMACS, etc.) and hardware (microcontrollers, photodiodes, lateral flow assays, etc.) was showcased in greater depth in a follow-up breakout session."}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"MUBO"}),Object(Le.jsx)("div",{className:pe.a.description,children:"In January of this year the Waterloo iGEM leads spoke with a Laurier student interested in creating an accessible space where university students could obtain hands-on lab experience outside of a classroom or formal research lab. Through some discussion we were able to provide some valuable information on how to form/lead a group, accessing funding and lab space, and other important details of creating a successful club. 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Although in-person events could not be completed, the team takes pride in the impactful work through online platforms in order to foster opportunities in synthetic biology. The collaborations are documented and described in the following categories: iGEM collaborations, internal university collaborations and collaborations with the greater community."})]}),Object(Le.jsx)("div",{className:pe.a.sections_div,children:tt.map((function(e,n){return Object(Le.jsx)("div",{active:t===e,onClick:function(){return i(e)},className:pe.a.section_block,children:Object(Le.jsxs)("div",{className:pe.a.sections,children:[Object(Le.jsx)("div",{className:pe.a.section_img,children:Object(Le.jsx)("img",{id:"collaborations".concat(n),src:it[n],alt:"Icon"})}),Object(Le.jsx)("div",{className:pe.a.section_text,children:e})]})},e)}))}),Object(Le.jsxs)("div",{children:[t===tt[0]&&Object(Le.jsx)(Xe,{}),t===tt[1]&&Object(Le.jsx)(et,{}),t===tt[2]&&Object(Le.jsx)($e,{})]})]})},at=i(43),rt=i.n(at),st=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Careers in Synthetic Biology"}),Object(Le.jsx)("div",{className:pe.a.description,children:"As part of Waterloo iGEM's outreach strategy this year, we wanted to create an educational event focusing on professional development and diversity in synthetic biology, as well as a discussion on how participation in iGEM can aid in preparation for a future career in the synthetic biology industry. The Human Practices team recognized that post-secondary students participate in extracurricular activities to develop skills and gain opportunities to advance their careers. We wanted to leverage this shared interest and iGEM\u2019s strong connection to the synthetic biology industry in our outreach efforts this year through the execution of a speaker panel and networking event."})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("img",{src:"",alt:"Career Speakers",className:rt.a.speakers_img})}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:"Our goals for this event were three-fold:"}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"To showcase various career paths available to life science students."}),Object(Le.jsx)("li",{children:"To demonstrate how iGEM can be a beneficial way to build a relevant network and experience."}),Object(Le.jsx)("li",{children:"To share stories from underrepresented groups in STEM."}),Object(Le.jsx)("ul",{children:Object(Le.jsx)("li",{children:"More specifically, we wanted to bring more awareness into specific challenges individuals from underrepresented demographics may face, and encourage students of similar backgrounds to pursue careers in synthetic biology. As our project focuses significantly on how to improve ADHD testing and diagnosis, especially for children in under-diagnosed groups, it seemed pertinent that we include an outreach initiative exploring diversity in synthetic biology as well."})})]}),Object(Le.jsx)("div",{className:pe.a.description,children:"To execute this event, we reached out to several individuals of underrepresented demographics in the synthetic biology industry to invite them for a discussion surrounding their professional experiences, as well as about the role of diversity in STEM. We were able to secure 3 speakers, 2 of whom were previously involved in iGEM during their undergraduate studies. Throughout the moderated discussion, we made an effort to illustrate the diversity of job opportunities and industries that synthetic biology is incorporated into. Afterwards, audience members were invited to participate in a Q&A period. This event was marketed towards university students across Canada interested in careers in biology, as well as those with an interest in the intersection of social impact and STEM. To assist with our marketing efforts, we collaborated with UWashington iGEM to help us promote our event to their audience base."})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("img",{src:"",alt:"Careers In Syn Bio Zoom",className:rt.a.zoom_img})})]})}}]),i}(n.Component),ot=i(33),ct=i.n(ot),lt=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Instagram Outreach"}),Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:"May:"}),Object(Le.jsx)("ul",{className:pe.a.description,children:Object(Le.jsx)("li",{children:"UW iGEM posted a \u201cScience and Research Talk Series\u201d where students could tune in and listen to current topics. If they had any questions or were just simply interested in learning about synthetic biology."})})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("img",{src:"",alt:"WiSTEM Research Talk Series"})}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:"June:"}),Object(Le.jsx)("ul",{className:pe.a.description,children:Object(Le.jsx)("li",{children:"June was pride month and we wanted to showcase LGBTQ+ in Science. UW iGEM wanted to place the spotlight on some amazing scientists in the LGBTQ+ community. These accomplishments have greatly impacted the world of science and we are forever grateful for their legacies!"})})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",alt:"LGBTQ+ in Science",className:ct.a.left_img}),Object(Le.jsx)("img",{src:"",alt:"Ben Barres",className:ct.a.right_img})]}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:"August:"}),Object(Le.jsx)("ul",{className:pe.a.description,children:Object(Le.jsx)("li",{children:"July/August was when the 2020 Tokyo Summer Olympics took place. This instagram post highlighted that synthetic Biology can be found everywhere! This included the ever-changing world of sports and some of the advancements in sports from synthetic biology."})})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",alt:"LGBTQ+ in Science",className:ct.a.left_img}),Object(Le.jsx)("img",{src:"",alt:"Ben Barres",className:ct.a.right_img})]}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.description,children:"For iGEM social media posts, references at the end of each post allows the audiences to further look into the information we found from credible sources, increasing accessibility to more information."}),Object(Le.jsx)("div",{className:pe.a.description,children:"When it comes to posting online, especially on social media platforms, it is important we take into account those who are visually impaired. 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With this in mind, our team set out to create an open-access and informative resource that teachers could use in their classrooms to help students get a better grasp of challenging concepts related to the field of synthetic biology. After further researching the learning barriers students with ADHD face, our team chose to create an informative coloring book to help high school students reinforce classroom lessons and introduce synbio. We were inspired by the Color Me PhD (Rorrer, 2018) project, created by Dr. Julie Rorrer, which combined the visual-first layout of coloring sheets with highly informative scientific explanations to educate and engage students. Coloring as a form of art therapy has shown to have great benefits for people of all ages with ADHD, as it can help improve attention span and decrease impulsive behaviour, while also having many social and emotional benefits for students (Loe & Feldman, 2007)."})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("img",{src:"",alt:"Coloring Book"})}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.description,children:"As we set out to create our colouring book, we further researched design considerations to ensure our coloring book served its intended purpose (McKnight, 2011). The first consideration we made was to ensure that the layout of both our colouring pages and information pages were neat and uncluttered (McKnight, 2011). This limits confusion as well as the search time required to find pertinent information, aiding students with shorter attention spans (McKnight, 2011). The second consideration we made was ensuring that our colouring book was free of any distractions or bright colours that may disrupt a student\u2019s attention while participating in the activity (McKnight, 2011). The third design consideration we made was to organize our colouring pages in an ordered and easy to understand layout in hopes of once again limiting confusion and reducing the time it takes to find information (McKnight, 2011). Finally we used large print text and easy to read fonts as well as bolding/italicizing to highlight important information (McKnight, 2011)."}),Object(Le.jsxs)("div",{className:pe.a.description,children:["With this project, we hope to create an outreach tool that would make synbio information and resources easily accessible to students and educators. Designed to help students grasp fundamental biology concepts and better understand our iGEM project, this coloring book will help to educate, engage, and inform. If you would like to use this resource,\xa0",Object(Le.jsx)("a",{href:"https://static.igem.org/mediawiki/2021/b/bf/T--Waterloo--coloringPages.zip",download:!0,children:"click here"}),"\xa0to download the file."]})]})]})}}]),i}(n.Component),ht=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("div",{className:pe.a.text_heading,children:"References"})}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"Loe, I. M., & Feldman, H. M. (2007, June 14). Academic and Educational Outcomes of Children With ADHD. Retrieved September 17, 2021 from https://academic.oup.com/jpepsy/article/32/6/643/1021192?login=true"}),Object(Le.jsx)("li",{children:"McComas, W. F., Reiss, M. J., Dempster, E., Lee, Y. C., Olander, C., Cl\xe9ment, P., . . . Waarlo, A. J. (2018, September 01). Considering Grand Challenges in Biology Education: Rationales and Proposals for Future Investigations to Guide Instruction and Enhance Student Understanding in the Life Sciences. Retrieved September 17, 2021, from https://online.ucpress.edu/abt/article/80/7/483/19020/Considering-Grand-Challenges-in-Biology-Education"}),Object(Le.jsx)("li",{children:"McKnight, M. (2011). Designing for Children with ADHD: The Search for Guidelines for Non-Experts. User Experience Magazine, 10(1). Retrieved September 17, 2021 from https://uxpamagazine.org/designing_children_adhd/"}),Object(Le.jsx)("li",{children:"Rorrer, J. (2018). Free PDF Vol.1. Retrieved September 17, 2021 from https://www.colormephd.org/coloring-pages/free-pdf-vol-1"})]})]})}}]),i}(n.Component),ut=i(52),mt=i.n(ut),pt=["Careers in Synthetic Biology","Instagram Outreach ","Biology Concepts Colouring Book","References"],ft=["","","",""],bt=function(){var e=be(Object(n.useState)(pt[0]),2),t=e[0],i=e[1];return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.heading_div,children:[Object(Le.jsxs)("div",{className:pe.a.title,children:[Object(Le.jsx)("div",{className:pe.a.page_heading,children:"NeuroDetech"}),Object(Le.jsx)("div",{className:pe.a.page_heading_colored,children:"Communication."})]}),Object(Le.jsx)("div",{className:pe.a.illustration,children:Object(Le.jsx)("img",{src:"",alt:"Communication Icon",className:mt.a.icon_img})})]}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Overview"}),Object(Le.jsx)("div",{className:pe.a.description,children:"Fundamental to any advancement of synthetic biology as an emerging field is public engagement through education. Ideal circumstances would have permitted collaboration with targeted demographics through workshops, conferences, and studies, as has been done in previous years. Unfortunately, these events were not possible to facilitate in the 2021 competition season, given jurisdictional health restrictions due to COVID-19. Despite these limitations, the need for community involvement cannot be compromised. As such, education and outreach was approached differently with the aid of digital communication platforms, with an emphasis on the following three categories: professional opportunities in synthetic biology, social media, and educational resource development."})]}),Object(Le.jsx)("div",{className:pe.a.sections_div,children:pt.map((function(e,n){return Object(Le.jsx)("div",{active:t===e,onClick:function(){return i(e)},className:pe.a.section_block,children:Object(Le.jsxs)("div",{className:pe.a.sections,children:[Object(Le.jsx)("div",{className:pe.a.section_img,children:Object(Le.jsx)("img",{id:"communication".concat(n),src:ft[n],alt:"Icon"})}),Object(Le.jsx)("div",{className:pe.a.section_text,children:e})]})},e)}))}),Object(Le.jsxs)("div",{children:[t===pt[0]&&Object(Le.jsx)(st,{}),t===pt[1]&&Object(Le.jsx)(lt,{}),t===pt[2]&&Object(Le.jsx)(dt,{}),t===pt[3]&&Object(Le.jsx)(ht,{})]})]})},jt=i(53),gt=i.n(jt),xt=function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.heading_div,children:[Object(Le.jsxs)("div",{className:pe.a.title,children:[Object(Le.jsx)("div",{className:pe.a.page_heading,children:"NeuroDetech"}),Object(Le.jsx)("div",{className:pe.a.page_heading_colored,children:"Contribution."})]}),Object(Le.jsx)("div",{className:pe.a.illustration,children:Object(Le.jsx)("img",{src:"",alt:"Contribution Icon",className:gt.a.icon_img})})]}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsxs)("div",{className:pe.a.description,children:["To meet bronze medal #4, we added documentation to the bottom of parts pages,\xa0",Object(Le.jsx)("a",{className:pe.a.text_link,href:"http://parts.igem.org/Part:BBa_K2926001",target:"_blank",rel:"noreferrer",children:"BBa_K2926001"}),"\xa0 and \xa0",Object(Le.jsx)("a",{className:pe.a.text_link,href:"http://parts.igem.org/Part:BBa_K1615022",target:"_blank",rel:"noreferrer",children:"BBa_K1615022"})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:["Part\xa0",Object(Le.jsx)("a",{className:pe.a.text_link,href:"http://parts.igem.org/Part:BBa_K2926001",target:"_blank",rel:"noreferrer",children:"BBa_K2926001"}),"\xa0is Cas13a from ",Object(Le.jsx)("i",{children:"Leptotrichia buccalis"}),". In contributing to this part, we added our findings from the literature regarding the usage of Cas13a, with a focus on its nonspecific RNase activity that is crucial for sensitive RNA detection methods such as SATORI. On that note, we also added documentation regarding the use of Cas13a in diagnostic and quantification contexts (such as detection of SARS-CoV-2 RNA and our project, NeuroDetech), which is another important application of CRISPR-Cas systems."]}),Object(Le.jsxs)("div",{className:pe.a.description,children:["Part\xa0",Object(Le.jsx)("a",{className:pe.a.text_link,href:"http://parts.igem.org/Part:BBa_K1615022",target:"_blank",rel:"noreferrer",children:"BBa_K1615022"}),"\xa0is monoamine oxidase A, which was one of the initial ADHD biomarkers that we considered (and later decided to rule out). With that said, the literature research used to inform this decision included information on the structure, function, and stability of monoamine oxidase A. Thus, in contributing to this part, we aimed to utilize this literature research to characterize the structure, function, and stability of this part more thoroughly."]})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",alt:"maoA"}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"3D structure of monoamine oxidase A, as visualized in UCSF Chimera"})]})]})},vt=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Education Within Greater Waterloo Community"}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"High School Outreach"}),Object(Le.jsxs)("div",{className:pe.a.description,children:["Many individuals don\u2019t learn about synthetic biology until they reach university, as it is not a field of science commonly talked about in high school environments. However, due to the increase in online classes, we were inspired to host online talks to high school students, teaching them about synthetic biology and exposing them to the world of iGEM. While synthetic biology may not be explicitly talked about in most high school biology courses, insulin is a topic that is taught in both grade 11 and 12 biology courses. Given that insulin production using ",Object(Le.jsx)("i",{children:"Escherichia coli"})," cell factories has had major impacts on making medicine for diabetes more accessible, we developed a presentation explaining synthetic biology by using this example. In this way, we could bridge the gap between a concept that high school students already knew about and an area of science that they likely did not know about. We included concepts relating to all three of our subteams so that the students could see just how interdisciplinary the field of synthetic biology can be. We had a great time talking to and answering questions from the students of Sir John A. MacDonald Secondary School\u2019s Grade 11 Biology class as well as Cameron Heights Collegiate Institute\u2019s Science Club and we hope to continue presentations of this kind in the future."]})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("img",{src:"",alt:"High School Outreach"})})]})}}]),i}(n.Component),yt=i(54),Ot=i.n(yt),wt=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Education Within iGEM Community"}),Object(Le.jsx)("div",{className:pe.a.description,children:"This year, we wanted part of our educational efforts to be directed towards the iGEM community. We focused on contributing to efforts that will help other teams in the future, as well as providing individuals with information on how they can turn synthetic biology into a long-term career path, even after iGEM ends."}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"iGEM Guide for Beginners"}),Object(Le.jsxs)("div",{className:pe.a.description,children:["This year, the newly formed iGEM Bolivia team spearheaded the creation of an iGEM for beginners manual. This educational and collaborative resource includes input from iGEM teams all around the world. Together, we hope that this guide provides help for teams that are just starting out in iGEM. We discussed things like how we go from an idea to a full project, tips for successfully contacting stakeholders, how to use math and modelling to make an iGEM project stronger and how to integrate stakeholder feedback into project development. We are very fortunate to have collaborated with iGEM Bolivia and many other iGEM teams on this and we hope that this guide will continue to be useful for new iGEM teams in the years to come. For more information, check out iGEM Bolivia\u2019s wiki page, or download the pdf of the guide\xa0",Object(Le.jsx)("a",{href:"https://static.igem.org/mediawiki/2021/2/25/T--Bolivia--collaborations_handbo1.pdf",download:!0,children:"here"}),"."]}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"Careers in Synthetic Biology"}),Object(Le.jsx)("div",{className:pe.a.description,children:"Waterloo iGEM has been around since 2005 and has had some team members go on to pursue careers in the field of synthetic biology. This inspired some of the Human Practices team members to plan an event surrounding what careers in synthetic biology and biotechnology are like. The goal of this initiative was to educate members of the iGEM community on how they could continue to contribute to the synthetic biology community after iGEM. We had a great turn out, with students from a variety of universities attending and contributing many questions to our panel of speakers, two of which were Waterloo iGEM alumni. Our diverse speaker panel contributed to the event attendees learning about how a career in synthetic biology can be achieved through industry, academia and start-up opportunities. In the end, we hope that we were able to provide students with insight into how they can turn synthetic biology into a long-term career path!"})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("img",{src:"",alt:"Careers In Syn Bio IG Promo",className:Ot.a.promo_img})})]})}}]),i}(n.Component),Nt=i(55),_t=i.n(Nt),kt=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Education Within University of Waterloo Community"}),Object(Le.jsx)("div",{className:pe.a.description,children:"The iGEM community at Waterloo is strong, but synthetic biology is still an area that not many individuals know about. For this reason, we collaborated with multiple clubs within the University of Waterloo community, to bring further awareness to the field of synthetic biology."}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"WiSTEM"}),Object(Le.jsx)("div",{className:pe.a.description,children:"Early on in the year, we collaborated with Waterloo\u2019s Women in STEM club to host a conference to bring awareness to different scientific fields. Our iGEM team focused on preparing 2 different presentations to educate the attendees on the field of synthetic biology. One of our presentations focused on describing our 2020 project, REMINE, and explaining how the work that each subteam did came together to form a finalized product. In particular, we emphasized how interdisciplinary synthetic biology can be. Our second presentation was on GMOs and the impact of science communication. Using the example of Golden Rice, we went through how the product was developed and why it has ultimately not been widely distributed. WIth this presentation we wanted to bring the focus towards the importance of doing science with a purpose and not just for the sake of simply doing science. For this, we emphasized the role that stakeholder feedback plays in project development."})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("img",{src:"",alt:"Golden Rice",className:_t.a.rice_img})}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.subheading,children:"BioTEC Conference"}),Object(Le.jsx)("div",{className:pe.a.description,children:"In previous years, we have hosted in-person workshops and seminars, but these types of educational initiatives were not a possibility due to pandemic restrictions. Instead, we collaborated with the annual Biotechnology and Bioengineering Conference (BioTEC) at Waterloo to co-host the Create Workshop, a two hour long technical seminar. Participants were introduced to the concepts that lay the foundation for the type of work done in the field of synthetic biology, through real life examples and previous iGEM projects. Furthermore, we hosted an introductory session on using Rosetta and GROMACS software as tools for rational protein design. Additionally, we spoke on hardware components (microcontrollers, photodiodes, lateral flow assays) and how these technologies are used to turn synthetic biology ideas into products that can be taken to market."})]})]})}}]),i}(n.Component),At=i(56),Ct=i.n(At),Tt=["Education Within iGEM Community","Education Within University of Waterloo Community","Education Within Greater Waterloo Community"],St=["","",""],Pt=function(){var e=be(Object(n.useState)(Tt[0]),2),t=e[0],i=e[1];return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.heading_div,children:[Object(Le.jsxs)("div",{className:pe.a.title,children:[Object(Le.jsx)("div",{className:pe.a.page_heading,children:"NeuroDetech"}),Object(Le.jsx)("div",{className:pe.a.page_heading_colored,children:"Education."})]}),Object(Le.jsx)("div",{className:pe.a.illustration,children:Object(Le.jsx)("img",{src:"",alt:"Education Icon",className:Ct.a.icon_img})})]}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Overview"}),Object(Le.jsx)("div",{className:pe.a.description,children:"Due to the effects of the global COVID-19 pandemic, many of the ways in which iGEM teams educate the community at large have been hindered. However, we decided to take the continuing pandemic restrictions in stride and to shift our focus to the use of virtual tools to successfully hit our Education goals. As has been outlined in our Communication and Collaborations pages, we took part in a variety of initiatives that aimed to further education within the iGEM community, within our university community and within our greater community as well."})]}),Object(Le.jsx)("div",{className:pe.a.sections_div,children:Tt.map((function(e,n){return Object(Le.jsx)("div",{active:t===e,onClick:function(){return i(e)},className:pe.a.section_block,children:Object(Le.jsxs)("div",{className:pe.a.sections,children:[Object(Le.jsx)("div",{id:n,className:pe.a.section_img,children:Object(Le.jsx)("img",{id:"education".concat(n),src:St[n],alt:"Icon"})}),Object(Le.jsx)("div",{className:pe.a.section_text,children:e})]})},e)}))}),Object(Le.jsxs)("div",{children:[t===Tt[0]&&Object(Le.jsx)(wt,{}),t===Tt[1]&&Object(Le.jsx)(kt,{}),t===Tt[2]&&Object(Le.jsx)(vt,{})]})]})},Dt=i(57),Et=i.n(Dt),Mt=i(11),Rt=i.n(Mt),Lt=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsx)("div",{className:pe.a.text_div,style:{marginBottom:40},children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Microfluidic Chip Design"})}),Object(Le.jsxs)(Ie,{open:!0,title:"Design",children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Selection of Flow Assay Type"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"When deciding on the mechanism to detect analyte in the test chamber, we considered both a competitive method and a sandwich method. In a competitive format, the analyte of interest in the free sample competes with the immobilized analyte in the test chamber to bind a binding molecule (i.e. an aptamer or fusion protein). In a sandwich assay, the analyte binds to both the binding molecule and an immobilized molecule in the test chamber. Thus, the analyte is \u201csandwiched\u201d between two binding molecules. Originally, we considered both of these detection methods with phenylethylamine (PEA) as the target analyte in mind, as PEA was our first biomolecule of focus (as discussed in the Design section of the Biomarker Detection engineering cycle). Given the small size of PEA, we determined that a sandwich detection method would not be possible, as this would require two molecules to bind to PEA. Given the small size of PEA, it would not be feasible for two molecules to simultaneously bind this analyte, thus we ruled out the sandwich format and decided to utilize the competitive format."}),Object(Le.jsx)("p",{children:"In designing NeuroDetech, we wanted to design a quick and easy to use system that would detect multiple analytes found in urine. We decided early on that a lateral flow assay detection method would be the best way to do this, as it is well-researched, quick and easy to use, and cost effective. The typical pregnancy test served as our preliminary model, and with this in mind, we began designing our system. We consider all the components involved in a lateral flow assay including a sample pad, test pad, control pad, etc. A key distinction we wanted to make between our system and that of a pregnancy test was the final output. While a pregnancy test provides a distinct yes or no answer, we wanted something that would produce a quantitative output. SInce there are multiple factors that could affect any given analyte, we knew that it would not be feasible to create a binary system. In order to get the desired output, we returned to the literature to research different types of assays we could use. We determined that a microfluidic assay was best because it has high precision for biomarker quantification and there\u2019s a significant improvement in sensitivity when compared to other methods. To read more about the different types of assays for quantification please go to the Design section of the Optical Detector and Application Development engineering cycle."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Overview of MCFA Chip Design"})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:20,width:"100%",height:"100%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Simplified diagram of the microfluidic assay chip."})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"A microfluidic assay is essentially a fancy version of a lateral flow assay (i.e. a pregnancy test) that is designed to work precisely with small volumes. The general design of the microfluidic assay used in NeuroDetech includes the following components, as can be seen in the figure above:"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"A sample application site, where the urine sample is dispensed."}),Object(Le.jsxs)("li",{children:["A binding molecule chamber, where the binding molecule is an aptamer or fusion protein that can:",Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:5,marginBottom:5},children:[Object(Le.jsx)("li",{children:"bind the biomolecule analyte of interest, and"}),Object(Le.jsx)("li",{children:"react with another molecule (i.e. a chemiluminescent substrate) to produce a quantifiable signal."})]})]}),Object(Le.jsx)("li",{children:"A chemiluminescent substrate chamber, which houses a substrate for the binding molecule; conversion or breakdown of the substrate by the binding molecule produces chemiluminescence or fluorescence that can be quantified."}),Object(Le.jsx)("li",{children:"A fluid delay system, which is implemented after the chemiluminescent substrate chamber. This ensures that the binding molecule and chemiluminescent substrate cannot intermix until the binding molecule has reached the test chamber. Otherwise, the test chamber would always produce a signal, rendering the assay useless."}),Object(Le.jsx)("li",{children:"The test chamber, where the biomarker analyte of interest is conjugated. The binding molecule may bind here, producing a quantifiable signal. Interpreting the signal/number of binding molecules that bind gives insight into the concentration of the target analyte in the urine sample. Generally, the higher the concentration of the target analyte in the sample, the lower the signal produced at the test chamber."}),Object(Le.jsx)("li",{children:"The control chamber, where streptavidin is conjugated. Binding molecules that are not bound in the test chamber will be bound in the control chamber. In effect, a signal is always produced at the control chamber. This positive control helps to prevent false negatives."}),Object(Le.jsx)("li",{children:"A capillary pump, which pulls the sample through the microfluidic assay."})]})}),Object(Le.jsx)("p",{children:"The output signal of the microfluidic assay can then be interpreted by an optical detector and application, which returns the concentration of the target analyte."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Microfluidic Chip Mechanical Design"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"The main inspiration for the design of the NeuroDetech lab chip is a similar point-of-care diagnostic device for infectious diseases created by Ghosh et al. (2020). The testing device designed by Ghosh et al. (2020) prioritized functions such as accessibility and speed which are applicable to NeuroDetech. Hence many concepts from this paper, like the device dimensions and parts sequence, are incorporated into our work. Overall, the objective of our work is to create a user-friendly tool for quantitative biomolecular detection. (Ghosh et al., 2020)"}),Object(Le.jsx)("p",{children:"While designing the device, multiple factors were taken into account, including flow and reaction rate. The concentration of PEA in the sample is low, which can be difficult to detect. In the design of the chip, both capillary and surface tension forces contribute to the generation of transient flow within the microstructures due to the pressure gradient. The high surface-to-volume ratio increases antigen binding, ensuring more precise and accurate results. In summary, this microfluidic device utilizes many principles of fundamental fluid dynamics. The model of the NeuroDetech lab chip is shown below."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:20,width:"80%",height:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Assembly of the microfluidic chip. Note that the top plane surfaces are transparent for easy visualization of the chip inner components."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:20,width:"50%",height:"50%",marginLeft:"25%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Full assembly of the microfluidic chip. Fluid flows from the top left through the capillary pump through to the spiral control chamber, then to the spiral test chamber. At the same time, fluid flows from the top right through the drying chamber and into the spiral test chamber."})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Sample Loading Chamber and Drying Chamber Design"})}),Object(Le.jsx)("p",{children:"The geometry of the NeuroDetech lab chip is critical to the chip\u2019s function. The test begins when the sample is deposited into the loading chamber through a sample loading port located at the top of the chip. A hydrophobic spot in the middle of the loading chamber splits the sample into two equal parts (Ghosh et al., 2020). Separate pathways move the samples toward a series of small tubes then into the drying chambers."}),Object(Le.jsx)("p",{children:"Path one leads to the binding protein drying chamber, and path two leads to the chemiluminescent substrate drying chamber. There are 10 tubes leading to the binding protein drying chamber and 7 tubes leading to the chemiluminescent substrate drying chamber. The tubes help maintain pressure to transfer fluid into the drying chambers and prevent unwanted backflow into the loading chamber."}),Object(Le.jsx)("p",{children:"Both drying chambers have a grid of rectangular pillars to increase mixing, providing binding proteins with sufficient time and space to bind to target biomarkers (Ghosh et al., 2020). The chemiluminescent substrate drying chamber has a row of tubes separating sections of the grid to maintain fluid pressure; this also ensures that fluid does not enter the test chamber before the binding protein fluid. Ramps descending into and ascending out of the drying chambers help retain fluid in the drying chamber for a sufficient period of time for uniform mixing and drying (Ghosh et al., 2020)."}),Object(Le.jsx)("p",{children:"The drying chambers connect to a series of delay valves with initially small diameters that progressively enlarge by widening the tube diameter; the fluid meniscus is thus increased in size, consequently decreasing capillary pressure. In turn, this helps ensure that no air bubbles form as the solution flows to the test chamber. The chemiluminescent substrate drying chamber pathway has a long serpentine delay tube in addition to the traditional delay valves, resulting in all the fluid from path two entering the reaction chamber only after all the fluid from path one has entered (Ghosh et al., 2020)."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:20,marginLeft:"5%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Annotated loading and drying chamber."})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Capillary Pump Design"})}),Object(Le.jsx)("p",{children:"Based on our previous research and referenced studies, few considerations were determined to be particularly important for fluid flow in the capillary pump. Through background research, we identified factors that affect fluid flow in a capillary pump. These include wettability, frequency of microstructures in the flow chamber, and the geometry of the microstructures used to increase flow resistance. These factors are the foundation for determining flow rates due to capillary flow (Olanrewaju et al., 2018)."}),Object(Le.jsx)("p",{children:"In general, more densely packed orientations of microstructures lead to higher flow resistance, in turn increasing flow pressure. Some of the key microstructure geometries commonly found in capillary pumps include tree lines, symmetric lines, balled lines, and posts. These structures must be small enough to generate enough capillary pressure for flow through the chamber (Olanrewaju et al., 2018)."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:10}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Various Capillary Pump Designs (Olanrewaju et al., 2018)"})]}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"We chose to compare three different microstructures and their effect on fluid flow: (1) symmetric oblong shapes (400 by 200\u03bcm); (2) treelines in the shape of a hexagon (thickness 1mm); and (3) balled lines (radius 500 \u03bcm, lines 1mm)."})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsxs)("div",{style:{display:"flex",flexDirection:"row",justifyContent:"space-around",flexWrap:"wrap"},children:[Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:20,width:"28%",height:"28%"}}),Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:20,width:"26%",height:"26%"}}),Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:20,width:"27%",height:"27%"}})]}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"From left to right: Oblong microstructures. Treeline microstructures. Balled lines microstructures."})]}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"The section housing these different microstructure designs are connected in series with a serpentine channel section and a spherical microstructure capillary chamber. The purposes of these additional chambers are to (1) prevent air bubbles from moving from the main capillary channel to the reaction chambers downstream and perhaps (2) to create a more uniform flow of fluid as it moves from a high capillary pressure area to the reaction chambers downstream (Ghosh et al., 2020) (Olanrewaju et al., 2018). The detailed motivations for each of these shapes are discussed in their respective sections below."})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:20,width:"70%",height:"70%",marginLeft:"15%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Sample of the lower common components of the capillary pump with oblong microstructures as an example."})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:Rt.a.sub_sub_heading,children:"Capillary Pump Design: Iteration 1"})}),Object(Le.jsx)("p",{children:"Our initial iteration utilized a high-pressure capillary pump consisting of oblong shapes that are 400 by 200 \u03bcm. These serve as posts in the chamber. According to Ghosh et al. (2020) [a], the motivation for a high-pressure capillary pump is to prevent reagents from accumulating in and around the edges of the posts and walls. This also facilitates rapid fluid flow from the openings to the reaction chambers."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:20,width:"100%",height:"100%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Full image of iteration 1."})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"Some benefits of this design include high-pressure creation and strong mixing of the reagent in the capillary pump chamber. Some drawbacks of this design include its relatively large size and the more condensed microstructures. This could pose challenges during manufacturing. Smaller microstructures require more precise tools and may have an impact on manufacturing methods and cost."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:Rt.a.sub_sub_heading,children:"Capillary Pump Design: Iteration 2"})}),Object(Le.jsx)("p",{children:"Our second design is quite different from our first design in terms of its shape and purpose. Treeline designs are typically used to pump fluid through chambers for assay purposes, but can result in different capillary pressures in different areas of the pump. For example, the pressure in a long branch of a chamber will have accumulated much higher pressure than in the meeting point of a few branches, where the shape is less uniform and the area is bigger (Olanrewaju et al., 2018)."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:20,width:"100%",height:"100%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Full image of iteration 2."})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"A problem that may arise with this design is lower capillary pressure due to wider branches (compared to the oblong microstructures in the previous design). However, this design is easier to manufacture in terms of precision and material costs. If we require more delays on our chip, this design could potentially provide bubbleless, even fluid flow."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:Rt.a.sub_sub_heading,children:"Capillary Pump Design: Iteration 3"})}),Object(Le.jsx)("p",{children:"The last iteration of our capillary pump design utilizes a combination of circular posts and linear walls, forming a dumbbell shape. According to literature, balled lines have lower flow rates than treelines (Zimmermann et al., 2007). It is noted that it may be filled in an orientation perpendicular to the inlet shown in Figure 8 below, with fluid flow facing the round post, parallel to the lines. This is because the presence of a wall between round posts will greatly limit the flow of a liquid."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:20,width:"100%",height:"100%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Full image of iteration 3."})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"Similar to the tree line design, a problem with this design is its lower capillary pressure compared to the symmetric oblongs. This, combined with the slightly shorter chamber length of designs 2 and 3, may cause the pump to not form any positive pressure at all, resulting in a failing capillary pump. A benefit of this design is the increased distribution of fluid flow across the whole capillary chamber. There is a lower risk of fluid only flowing along a linear path at any point in the chamber since the shape of the microstructures is so irregular. Compared to symmetric lines (iteration 2), there is a lower risk of undisturbed flow happening in the chamber (Zimmermann et al., 2007)."}),Object(Le.jsx)("p",{children:"Due to the high capillary pressure generation and suitability for reagent mixing, the oblong capillary pump structure is chosen for the final chip assembly design."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Selection of Chip Material"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"We looked into multiple materials in order to determine whether they fulfilled certain characteristics, such as surface hydrophilicity and ability to be biotinylated, while still being cost-effective and practical to fabricate. In order to evaluate possible candidates, we researched various materials used in microfluidic chips and outlined the characteristics of each candidate: glass, PDMS (polydimethylsiloxane), COC (cyclic olefin copolymer), and thiol-ene polymers."}),Object(Le.jsx)("p",{children:"The first material that we looked into was glass. On one hand, glass is chemically inert, biocompatible, and impermeable to gas. Additionally, it is easy to biotinylate and has excellent optical transparency (Ghosh et al., 2020). On the other hand, glass is relatively expensive, and its hardness makes it difficult to construct and etch (Ghosh et al., 2020)."}),Object(Le.jsx)("p",{children:"The second material that we looked into was PDMS, also known as polydimethylsiloxane. PDMS is easy to fabricate and inexpensive. It is currently the most commonly used material in microfluidic chips. Additionally, it is gas-permeable, biocompatible, and non-toxic. It can be biotinylated, and complex microfluidic designs can be made by stacking layers (Ghosh et al., 2020). PDMS is naturally hydrophobic, but it can be treated to control the hydrophobicity (Ghosh et al., 2020)."}),Object(Le.jsx)("p",{children:"Additionally, we also looked into cyclic olefin copolymer, also known as COC. On one hand, this amorphous polymer has good biocompatibility and high transparency. Additionally, it can be biotinylated (Ali Aghvami et al., 2017). Nevertheless, one disadvantage of COC is that hydrophobicity treatments are transient on this material relative to PDMS (Oh et al., 2013). Additionally, the literature on COC surface treatments is much less robust when compared to the literature regarding PDMS (Oh et al., 2013)."}),Object(Le.jsx)("p",{children:"Finally, we looked into thiol-ene polymers. These polymers are easy to fabricate and manipulate, and can be easily biotinylated (Sticker et al., 2020). These polymers, however, are prohibitively expensive, and there are fewer suppliers available for purchase (Sticker et al., 2020)."}),Object(Le.jsx)("p",{children:"We then evaluated the properties of each material, and finally decided to use PDMS as the chip material. PDMS is cost-effective, easy to fabricate and handle, and can be easily biotinylated (Ghosh et al., 2020). While PDMS\u2019s surface is naturally hydrophobic (Tan et al., 2010), it can easily be pre-treated in order to regulate and decrease its hydrophobicity (Tan et al, 2010; Trantidou et al., 2017). These pre-treatments are further explained in the Build section below. Additionally, the literature on the fabrication and pre-treatment of PDMS for microfluidics is extremely robust relative to the other materials."})]})]}),Object(Le.jsxs)(Ie,{open:!0,title:"Build",children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Computer-aided Chip Assembly Design"})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"A complete assembly of the microfluidic chip is built and shown below. The specific dimensions are also labelled and presented. The team used OnShape to create the CAD model throughout the year."})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:20}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Dimensioned drawing of the microfluidic chip."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:20,marginLeft:"5%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Assembly of the microfluidic chip. Note that the top plane surfaces are transparent for easy visualization of the chip inner components."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Pretreatment of PDMS"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"As discussed in the Design section above, PDMS, or polydimethylsiloxane, was chosen as the polymer material to line the inside of the microfluidic chip. PDMS is widely used for the fabrication and prototyping of microfluidic chips as well as for other purposes such as contact lenses. It is usually supplied with two components, a base elastomer and a curing agent. PDMS as a material is hydrophobically classified as having a water contact angle greater than 100 degrees. This has negative consequences for droplet-based microfluidic chips, such as the one utilized in our project. There have been numerous studies investigating methods to reduce the hydrophobicity of the material ranging from physical methods to chemical methods; however, many have proven to be either unsuccessful or temporary."}),Object(Le.jsx)("p",{children:"In our project hydrophobicity poses a problem as the PDMS can absorb hydrophobic molecules present in urine and thus expand and deform in the microfluidic assay. Previous attempts, such as UV irradiation and plasma oxidization, lead to the PDMS generally returning to a hydrophobic state after only a few minutes. Other treatment options last from a matter of weeks to months, but none are permanent. The treatment explored was using the deposition of polyvinyl alcohol (PVA) through plasma treatment. PVA is a hydrophobic polymer that can be irreversibly adsorbed onto a hydrophobic polymer such as PDMS. This absorption is done through a matter of means such as via heat immobilization following plasma treatment, plasma oxidation, and the covalent attachment of a 3-aminopropyl linker via the synthesis of a PVA PDMS copolymer micro-suspension. The pretreatment method that we chose to use for our microfluidic assay was to coat the PDMS microfluidic chip channels with PVA immediately after plasma treatment. Plasma treatment refers to the use of oxygen to introduce polar functional groups and change the surface properties. In the case of PDMS, it increases the -OH groups on the surface, which form strong covalent bonds. In turn, these improve bonding characteristics between the PVA and the PDMS. In previous studies, glass has also been used to bond with the PDMS. The first step involves dissolving the PVA in water, and then adding the solution to Milli-Q water (1% by weight) and stirring at room temperature for 40 minutes. The temperature is then gradually increased to 100\xb0C and stirred again. This decreases the concentration of PVA to around 87-90%, which is found to be more effective than PVA at over 99%. The temperature is then reduced to 65\xb0C, and the solution is stirred overnight. The PDMS is then degreased, blow-dried, and placed inside a plasma cleaner. From there the plasma treatment begins with an oxygen flow rate of 20 sccm and a pressure of 0.67 mbar. The specific configuration of the plasma cleaner is 100W for 1 min. The PVA solution is then poured onto the plasma-treated surface using a plastic syringe and left for 10 minutes to coat. The excess PVA solution is removed using pressurized nitrogen and heating to 110\xb0C for 15 minutes removing any residual moisture. For the channels of the chip, the PVA is passed using a syringe and pump. The residual PVA is removed the same way using pressurized nitrogen, and the device is heated to 110\xb0C for 15 minutes. Contact angle taken before treatment and after treatment is then compared, which should ideally show a decrease in contact angle and thus a greater surface wettability and hydrophilicity."}),Object(Le.jsx)("p",{children:"Through previous attempts, it was seen that this method of treating the hydrophobicity resulted in long-term and sustained hydrophilicity as compared to other methods. Average contact angles were 24.9 degrees for PVA 87%, while using higher concentrations of PVA led to a higher contact angle. Further storage under a temperature of -80\xb0C can maintain hydrophobicity for plasma exposed PDMS for almost 1000 days. At 100W this also had a positive correlation with the length of hydrophilic properties which sustain for about 30 days at a contact angle of 21 degrees. Other methods looked into involved the physical patterning of the PDMS surface; however, patterning within channels was found to be extremely difficult. Due to COVID-19, lab access was restricted and none of the procedures were able to be performed, as the pretreatment protocol required specialized equipment only present in Waterloo labs. Therefore it is our hope that this procedure for treating PDMS can be used for future projects."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Methods for the Conjugation of Biomolecules to PDMS"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"Key components of the microfluidic assay are the test and control chambers. Recall that the test chamber must contain molecules that can detect the analyte of interest and produce a signal; in a similar sense, the control chamber must contain a molecule that always produces a signal upon contact with the sample. In order to achieve this, any molecules involved in detection and signal production must be bound to the chambers in a stable fashion. Specifically, these specific molecules must be securely conjugated to the chamber material (PVA-treated PDMS) such that the internal chambers are able to successfully retain all the required attached molecules during fabrication, processing, storage and usage of the microfluidic assay."}),Object(Le.jsx)("p",{children:"We used different methods to ensure optimal binding for each unique molecule conjugating to the test and control chambers. Specifically, methods for phenylethylamine (PEA) and mRNA were needed, as these were the ADHD-associated analytes of interest that we planned to quantify using the microfluidic chip. In addition, a method for protein conjugation was needed in order to conjugate streptavidin to the control chamber, as a biotin-streptavidin interaction to a biotinylated protein would be used to elicit a control signal. In all cases, covalent conjugation was used. This was to ensure that binding would occur between the chamber and the biotinylated binding molecule we engineered."}),Object(Le.jsx)("p",{children:"Three main conjugation methods were identified in literature pertaining to microfluidic chip assays (Kim & Herr, 2013):"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ol",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"Physisorption: Molecules are adsorbed to a surface via intermolecular forces achieved through simple immersion of the surface in a solution."}),Object(Le.jsx)("li",{children:"Bioaffinity Interaction: Molecules are attached to a surface through specific binding phenomena existing in nature (such as the biotin-streptavidin bond)."}),Object(Le.jsx)("li",{children:"Covalent Conjugation: Molecules are attached to a surface through a covalent bond and the use of many active reagents."})]})}),Object(Le.jsx)("p",{children:"We wanted our chosen method of conjugation to be independent of environmental factors, such as pH and temperature. This ruled out the physisorption method, as the resulting bond strength is heavily dependent on such factors (Kim et al., 2013). Furthermore, we wanted our conjugation method to be secure so our attached molecules would not detach from agitation. Thus, covalent bonding was chosen as the method for conjugation considering its greater strength compared to bioaffinity interactions. Through this method, we could ensure the bond between the sample pad and the binding molecule was secure. The strength of the covalent bond guarantees system durability in the face of external factors."}),Object(Le.jsx)("p",{children:"The covalent method acts through amino groups on glutaraldehyde (GA), which has two reactive ends. These ends allow GA to act as a cross linker between the PDMS chamber and the molecule we want to covalently bind. Another compound (3-Aminopropyl)triethoxysilane (APTES) is able to attach to both the PDMS chamber and GA, creating a strong interaction and bond between the cross linker and the chamber (Kim et al., 2013). GA is a common crosslinker used in the field of biochemistry given its ability to effectively stabilize biomaterials (Pal et al., 2013). Therefore, carrying out crosslinking with GA gives our system an extra degree of stability."}),Object(Le.jsx)("p",{children:"To implement covalent conjugation, a treatment containing APTES is first used to coat the surface of the chambers. APTES binds to the hydroxyl groups on the surface of the PDSM. Then a GA treatment is used, binding GA to APTES. Finally, the system is treated with our chosen molecule, which will bind to GA covalently at its free amine residues (Pal et al., 2013). Thus, this method is appropriate for conjugation of PEA and proteins (at their lysine residues or N-termini)."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Protocol for Covalent Conjugation of Biomolecules Containing Free Amine Groups:"})}),Object(Le.jsx)("p",{children:"The procedure as outlined below was used for the covalent conjugation of both PEA in the test chamber and streptavidin in the control chamber (Thomsen et al., 2007):"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ol",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"Pretreat the PDMS sample pad with pyrogenic silicic acid (silicon oxide powder in a prepared oxyhydrogen flame) to add -OH groups to the surface of the PDMS."}),Object(Le.jsx)("li",{children:"Conduct in-flow treatment (0.15 mL/min) of a 10% w/v solution of APTES at 80\xb0C for 3 hours."}),Object(Le.jsx)("li",{children:"Derivatize the silanized microstructure in a 2.5% w/v solution of glutaraldehyde (GA) for 2 hours at room temperature."}),Object(Le.jsx)("li",{children:"Recirculate 95 \u03bcg/mL of the molecule to be immobilized over the microstructure for 10-15 hours to achieve covalent immobilization. The specific amount of immobilized molecule can be determined through the difference in its concentration of the solution before and after immobilization."}),Object(Le.jsx)("li",{children:"Circulate bovine serum albumin (BSA) blocking buffer (20 mg/mL) through the microstructure for 30 minutes to ensure that the unbinded areas of PDMS are blocked from picking up extraneous molecules in the urine that may interfere with the function of the microarray."})]})}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Covalent Conjugation of RNA:"})}),Object(Le.jsxs)("p",{children:["In the case of conjugation of RNA, a slight modification is necessary, as RNA does not contain free amine groups. Prior to adding the glutaraldehyde, an aqueous solution of pyridinium chlorochromate (PCC/H",Object(Le.jsx)("sub",{children:"2"}),"O) is circulated around the pad. Following this step, SOCl is also circulated around the pad, and finally, an mRNA solution is circulated in order to achieve conjugation (during which mRNA is conjugated at its 3\u2032 OH group)."]})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Lyophilization Protocols"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"Lyophilization, or freeze-drying, is the process of removing ice or frozen solvents via sublimation as a means of preserving heat-sensitive materials such as proteins (Barley, n.d.). Lyophilization is necessary to preserve the contents of the microfluidic assay, as the assay contains proteins, small biomolecules, and potentially RNA, all of which are biomolecules that must be kept viable for storage until the time of use."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Lyophilization of Small Biomolecules"})}),Object(Le.jsx)("p",{children:"The following procedure for lyophilization of small biomolecules, such as PEA or chemiluminescence substrates, was adapted from Ghosh and Ahn (2019):"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ol",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsxs)("li",{children:["Pre-freeze the ",Object(Le.jsx)("strong",{children:"substrate solution"})," of ",Object(Le.jsx)("strong",{children:"Amplex Red"})," as well as the ",Object(Le.jsx)("strong",{children:"enhancer solution"})," for Amplex Red in ",Object(Le.jsx)("strong",{children:"liquid nitrogen"})," for 5 minutes."]}),Object(Le.jsxs)("li",{children:["Freeze-dry the above solutions in a freeze-drying system (ex: ",Object(Le.jsx)("strong",{children:"Labconco Lyophilizer HRFD-SCIL"}),") set at -54\xb0C with 0.010 mbar pressure for 24 hours."]})]})}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Protein Lyophilization"})}),Object(Le.jsx)("p",{children:"The following procedure for protein lyophilization was adapted from a previous study which tested four formulations for lyophilization of 15 different proteins over a range of molecular weights and structural properties (Roughton et al., 2013). This study used sucrose, glycine, urea, and guanidine hydrochloride as additives in the lyophilization formulation. The proteins were dialyzed to remove any extraneous additives."}),Object(Le.jsx)("p",{children:"The solution preparation procedure below was adapted from Roughton et al. (2013):"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ol",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"Dissolve protein to 2.0 mg/mL in a potassium phosphate buffer (20 mM, pH 7.4)."}),Object(Le.jsx)("li",{children:"Dialyze protein solution against a phosphate buffer (20 mM, pH 7.4) for 24h at 4\xb0C. Switch out the dialysis buffer twice within the 24h period - once after 1h elapsed time, and once more after 2h total elapsed time."}),Object(Le.jsx)("li",{children:"Filter protein solution through a 0.2 \xb5m syringe filter."}),Object(Le.jsx)("li",{children:"Prepare sucrose (2.0 mg/mL), glycine (2.0 mg/mL), urea (2.0 M), and guanidine-HCl (2.0 M) solutions in phosphate buffer. Filter these solutions through a 0.2 \xb5m syringe filter, and store at 4\xb0C."})]})}),Object(Le.jsx)("p",{children:"The lyophilization procedure was adapted from Roughton et al. (2013):"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ol",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"Mix protein stock solution with an additive solution to a final protein concentration of 1.0 mg/mL."}),Object(Le.jsx)("ul",{className:pe.a.description,style:{marginTop:5,marginBottom:5},children:Object(Le.jsx)("li",{children:"The protein to additive ratio should be 1:1 by weight for sucrose and glycine, with final urea and guanidine-HCl concentrations of 1.0 M. To encourage cake formation, 5% trehalose should be added as a key additive. This is based on the personal experience of one of our lab members, and differs from the procedure of Roughton et al. (2013)."})}),Object(Le.jsx)("li",{children:"Add 400 \xb5L of the lyophilization formulation to a lyophilization vial."}),Object(Le.jsx)("li",{children:"Lyophilize samples in a VirTis adVantage Plus freeze dryer with the following temperature cycle:"}),Object(Le.jsxs)("ol",{className:pe.a.description,style:{marginTop:5,marginBottom:5},children:[Object(Le.jsx)("li",{children:"Pre-cool shelves at -2\xb0C for 15 min."}),Object(Le.jsx)("li",{children:"Freeze samples at -40\xb0C for 50 min."}),Object(Le.jsx)("li",{children:"Dry samples under vacuum (100 mTorr) at -35\xb0C for 10 h, -20\xb0C for 8 h, -5\xb0C for 6 h, 10\xb0C for 6 h, 25\xb0C for 6 h, and 4\xb0C for 0.5 h. This removes water by sublimation."})]})]})})]})]}),Object(Le.jsxs)(Ie,{open:!0,title:"Test",children:[Object(Le.jsx)("div",{className:pe.a.description,style:{marginTop:30},children:Object(Le.jsx)("p",{children:"Due to unforeseen circumstances (one of which was a global pandemic, and the other of which was unforeseen maintenance and construction of our lab space by the University), we unfortunately did not have lab access this year. As a result, we were unable to experimentally test the microfluidic assay with all of its components. With that said, the following methodology outlines how we have used computational modelling to gain insight into the physical dynamics within the microfluidic chip, as well as our plan of action to test our microfluidic assay chip if we had lab access."})}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Fluid Dynamics Simulation with COMSOL"})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"We completed microfluidics simulations in COMSOL on parts of our chip to model the flow of the fluid through the microchannels. This was done by using the Laminar Flow physics interface in a Time Dependent Study on COMSOL. The following equations were solved by COMSOL to model the laminar flow."})}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:20}})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"Here are the velocity and pressure plots for a serpentine microchannel and for the spiral test chambers."})}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)("div",{style:{display:"flex",flexDirection:"row",justifyContent:"space-around",flexWrap:"wrap"},children:[Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:20,width:"43%"}}),Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:20,width:"43%"}})]})}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)("div",{style:{display:"flex",flexDirection:"row",justifyContent:"space-around",flexWrap:"wrap"},children:[Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:20,width:"43%"}}),Object(Le.jsx)("img",{src:"",className:Rt.a.design_img,style:{borderRadius:20,width:"43%"}})]})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"The above are preliminary examples of how the chip design can be validated in COMSOL. In the future, we would like to perform simulations on the entire assembled chip, so that we can provide an accurate estimate of the length of time the fluid will take to flow through the chip, or create the chip and conduct experimental analysis."})}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,style:{paddingBottom:0},children:"Assessment of Streptavidin Binding to Evaluate the Success of PDMS Derivatization, Conjugation, and Lyophilization"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Preliminary Assessment: Assessment of PDMS Alone"})}),Object(Le.jsx)("p",{children:"The success of our microfluidic assay is contingent on the ability of molecules to remain covalently conjugated to PDMS when desired. Thus, it would need to be verified that PDMS would be successfully derivatizated by PVA treatment, that the conjugation method would be successful, and that the lyophilization and resuspension steps would not impede the functionality of the lyophilized molecules. A way to test the success of all of these steps would involve an experiment testing the ability of streptavidin in the control chamber to bind biotin. Without successful derivatization of the PDMS surface, conjugation of streptavidin to the control chamber would not be successful. As well, assessment of binding after lyophilization by running a biotin solution would give insight into whether lyophilization is somehow damaging the effectiveness of the lyophilized molecules (in this case, streptavidin)."}),Object(Le.jsx)("p",{children:"To test these, the derivatization, conjugation, and lyophilization methods from the Build section should be adapted for a small, test-scale PDMS film. Specifically, the following procedure, adapted from Thomsen et al. (2007), should be performed:"}),Object(Le.jsx)("p",{children:"Materials:"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"PDMS film"}),Object(Le.jsx)("li",{children:"Pyrogenic silicic acid"}),Object(Le.jsx)("li",{children:"10% w/v (3-Aminopropyl)triethoxysilane (APTES)"}),Object(Le.jsx)("li",{children:"2.5% w/v Glutaraldehyde"}),Object(Le.jsx)("li",{children:"5 mg/mL streptavidin"})]})}),Object(Le.jsx)("p",{children:"Procedure:"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ol",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"Soak a PDMS film with pyrogenic silicic acid (Thomsen et al., 2007)."}),Object(Le.jsx)("li",{children:"Drain the pyrogenic silicic acid and rinse the PDMS film."}),Object(Le.jsx)("li",{children:"Incubate the PDMS film with a 10% w/v solution of APTES at 80\xb0C for 3 hours."}),Object(Le.jsx)("li",{children:"Drain the APTES solution and rinse the PDMS film."}),Object(Le.jsx)("li",{children:"Incubate the PDMS film with a 2.5% w/v solution of glutaraldehyde (GA) on a rocking shaker for 2 hours at room temperature."}),Object(Le.jsx)("li",{children:"Drain the glutaraldehyde solution and rinse the PDMS film."}),Object(Le.jsx)("li",{children:"Incubate the PDMS film with the 5 mg/mL solution of streptavidin on a rocking shaker for 10-15 hours at 4\u02daC."}),Object(Le.jsx)("li",{children:"Aliquot 1 mL of the residual solution for determination of streptavidin concentration. This can be done by absorbance at 280 nm, or using a Bradford assay."}),Object(Le.jsx)("li",{children:"Drain the rest of the residual solution and rinse the PDMS film."}),Object(Le.jsx)("li",{children:"Incubate the PDMS film with a solution of bovine serum albumin (BSA) on a rocking shaker for 30 minutes at room temperature."}),Object(Le.jsx)("li",{children:"Drain the residual solution and rinse the PDMS film."})]})}),Object(Le.jsx)("p",{children:"Knowing the initial streptavidin concentration and the determined concentration of the residual streptavidin solution, the amount of streptavidin conjugated to the PDMS film can be determined."}),Object(Le.jsx)("p",{children:"The streptavidin-conjugated PDMS film can then be lyophilized by first freezing the film in liquid nitrogen for 5 minutes, then utilizing a freeze-drying system such as the Labconco Lyophilizer HRFD-SCIL set at -54\xb0C with 0.010 mbar pressure for 24 hours."}),Object(Le.jsx)("p",{children:"In the meantime, fluorophore-conjugated biotin molecules can be prepared using principles of organic chemistry related to carboxylic acid derivatives. First, HCl should be added to a biotin solution until the pH is around 1. The molar amount of biotin should be the same as the molar amount of streptavidin conjugated to the PDMS film. The purpose of lowering pH to around 1 is as follows: biotin has a carboxylic acid/carboxylate group; adding HCl to a pH of around 1 protonates any carboxylates, thereby preparing biotin for the subsequent reaction. From here, the acidified biotin should be reacted with an equimolar amount of thionyl chloride. This produces a \u201cbiotin chloride\u201d that can then be reacted with an equimolar amount of a nucleophilic fluorophore, which effectively substitutes with the chlorine atom to produce biotin conjugated to a fluorophore."}),Object(Le.jsx)("p",{children:"From here, the freeze-dried PDMS film can be resuspended in the fluorescent biotin solution and incubated on a rocking shaker for 20 minutes (which is the same as the assay time for NeuroDetech\u2019s microfluidic assay chip). The residual solution can be drained, and its fluorescence measured using a spectrophotometer."}),Object(Le.jsx)("p",{children:"Since the fluorescent biotin and streptavidin were in equimolar amounts, and since the binding affinity of streptavidin to biotin is so strong, it is expected that there should be little to no fluorescence in the residual solution, since most or all of the fluorescent biotin should have been bound to the streptavidin on the PDMS film."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Assessment in Microfluidic Assay Chip Conditions"})}),Object(Le.jsx)("p",{children:"After construction of the chip, the general method for the preliminary assessment (described above) would need to be repeated to ensure that the success of derivatization, conjugation, and lyophilization for the PDMS film could also be replicated in the actual microfluidic assay chip."}),Object(Le.jsx)("p",{children:"To do this, a \u201csacrificial\u201d microfluidic assay chip should be prepared, such that the sample can be recovered after running the sample. Once again, fluorescent biotin should be prepared, as described in the preliminary assessment method (above)."}),Object(Le.jsx)("p",{children:"From here, the fluorescent biotin sample should be flowed through the sacrificial microfluidic assay, as if the fluorescent biotin sample was an actual urine sample. Again, the molar amount of fluorescent biotin should be the same as the molar amount of streptavidin conjugated to the control chamber. The expected result is that the fluorescent biotin sample should bind to the conjugated streptavidin at the control chamber, thereby producing fluorescence at the control chamber. As well, the sample fluid should be recovered, and its fluorescence measured; the fluorescence should be negligible, as most (if not all) of the biotin should have been bound at the control chamber. If these expected results are observed, then it would be demonstrated that the methods for PDMS derivatization and conjugation, as well as the viability of lyophilization, would be successful in the microfluidic assay chip."})]})]}),Object(Le.jsxs)(Ie,{open:!0,title:"Learn",children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Chip Design Improvements and Next Steps"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"The above drying chamber design is quite similar in structure to the source paper. While the test sample used in the source is blood, NeuroDetech uses urine as the test sample; as urine is less viscous than blood, the flow rate will be faster in the former. For this reason, the probability that air bubbles will generate increases, potentially lowering device sensitivity. Future iterations will work to decrease the flow rate within the device. This can be accomplished by increasing the quantity or intensity of delay valves and altering microstructure sizes and shapes. One way to decrease flow rate is to add a delay in both paths one and two; hence slowing the flow rate in both paths would improve substrate recovery and thus increase test sensitivity."}),Object(Le.jsx)("p",{children:"An addition of a hydrophobic vent between path two and the test chamber would decrease the probability of trapped air bubbles, thus increasing efficiency and accuracy. Another possible improvement is to shrink the scale of the microstructures, in order to increase the number of microstructures for each capillary pump and increase laminar flow. At the microscale, fluid flow will have less turbulence and bubble formation can be reduced. Lastly, it would be optimal to create all of the capillary pump exteriors with identical geometries for a future design. This design oversight should be improved for future COMSOL simulations to mitigate any errors that might have arisen."}),Object(Le.jsx)("p",{children:"Experimental results of the newly developed POC test will allow designers to adapt the reaction time, flow rate, and other factors to suit their purposes by changing channel lengths and widths."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Concerns Regarding Streptavidin Solubility"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"Although the use of streptavidin and biotin as a general-purpose method to bind binding molecules at the control chamber is, in theory, perfectly functional, a question arose during the Design phase of this engineering cycle: would a high concentration of streptavidin conjugated to the control chamber result in solubility issues? Streptavidin is a rather large protein, and is tetrameric. Would there be an alternative version of streptavidin that could be used to decrease the risk of solubility issues arising from the impracticality of shoving many tetrameric streptavidin molecules into a small space?"}),Object(Le.jsx)("p",{children:"As it turns out, an engineered monomeric form of streptavidin exists; though its binding affinity is not as high as tetrameric streptavidin, its monomeric form mitigates the aforementioned solubility issues (DeMonte et al., 2013). As a result, we decided to utilize monomeric streptavidin in the control chamber of our microfluidic assay. To address the issue of reduced affinity, we decided to utilize computational rational protein design to improve the affinity of monomeric streptavidin; this process is discussed in the Protein Optimization engineering cycle."})]})]})]})}}]),i}(n.Component),It=i(29),Bt=i.n(It),Wt=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsx)("div",{className:pe.a.text_div,style:{marginBottom:40},children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Biomarker Detection: Fusion Protein Design"})}),Object(Le.jsxs)(Ie,{open:!0,title:"Design",children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"The Big Picture"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"When initially considering how to approach developing an ADHD diagnosis tool that was noninvasive, we decided that detection of a urinary ADHD-associated biomarker would be viable for our purposes. From here, we needed to explore the literature to answer the following questions:"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"What urinary ADHD-associated biomarker should we choose to detect?"}),Object(Le.jsx)("li",{children:"What binding molecule should we use to bind/detect our chosen ADHD-associated biomarker? What considerations are there for the design of our chosen binding molecule?"}),Object(Le.jsx)("li",{children:"How would we design our binding molecule to produce a signal in response to detection of the ADHD-associated biomarker?"})]})}),Object(Le.jsx)("p",{children:"In this Design section, our considerations and answers to these \u201cbig picture questions\u201d will be discussed"})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Selection of a Target ADHD-Associated Biomarker"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"Selection of an optimal ADHD biomarker hinged on the following key characteristics:"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"The biomarker should be collected via a non-invasive sample (e.g. urine, sweat, saliva)."}),Object(Le.jsx)("li",{children:"It should be present in an appreciable quantity in the sample."}),Object(Le.jsx)("li",{children:"It should have well-defined numerical values of statistical data present in literature."}),Object(Le.jsx)("li",{children:"It should have a discernible diagnostic window relative to healthy individuals."})]})}),Object(Le.jsx)("p",{children:"While several sources report a link between ADHD and impaired regulation of brain catecholamines, quantifying these catecholamines in a non-invasive manner would be challenging for a number of reasons (Pliszka et al. 1996; Biederman 2005; Oades et al. 2005). Due to their rapid metabolism and trace levels in biological samples, analytes such as norepinephrine or dopamine would not be strong candidates to measure (Feres et al., 2014). Therefore, it was more feasible to investigate precursors or derivatives of norepinephrine or dopamine as markers of their imbalance rather than measuring the catecholamine concentrations themselves."}),Object(Le.jsx)("p",{children:"Various biomarkers that showed evidence of imbalances of the noradrenergic or dopaminergic systems were mostly found in blood, requiring a more invasive collection method and thus less ideal for an affordable point of care diagnostic tool. Additional biomarker candidates were excluded from consideration due to insufficient evidence supporting a specific linkage to ADHD as opposed to other behavioural disorders. These eliminated analytes included neuropeptide Y, homovanillic acid (HVA), monoamine oxidase, 3-methoxy-4-hydroxyphenylglycol (MHPG) and 5-hydroxyindoleacetic acid (5-HIAA) (Faraone et al., 2014; Gerra et al., 2007; Jayamohananan et al., 2019)."}),Object(Le.jsx)("p",{children:"Scassellati et al., (2012) published an extensive review of promising biomarkers of ADHD to be used in diagnostics. One such marker was phenylethylamine (PEA), a stimulant for the release of dopamine. Meta-analysis of PEA-ADHD data reports significantly lower urinary PEA levels in ADHD patients versus controls based on three unique studies (Baker et al., 1991; Kusaga et al., 2002; Zametkin et al., 1984). One such study also showed evidence of lowered PEA levels being associated with symptoms of inattentiveness (Baker et al., 1991). Kusaga et al., (2002) investigated PEA alongside MHPG, HVA and 5-HIAA, comparing against individuals with an autistic disorder as a disease control. PEA was the only tested analyte in the study that showed significant differences relative to negative and disease controls."}),Object(Le.jsx)("p",{children:"Given the consistent literature support of PEA as a viable marker linked to ADHD, readily available data and quantifiable numbers, and its presence in a relatively non-invasive biological sample, it was determined to be the strongest candidate to use as a biomarker in our detection device."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Human Physiology of PEA"})}),Object(Le.jsx)("p",{children:"PEA is a small molecule most known for its role as a neurotransmitter implicated in psychological disorders such as ADHD, depression, and schizophrenia (Irsfeld, Spadafore & Pruess, 2014). PEA is most commonly found as a trace amine in the brain of mammals, justified by its high solubility within plasma and permeability across the blood-brain barrier. In this context, it mainly functions as an inhibitor of dopamine and serotonin transporters by binding to the TAAR1 protein. As a result, PEA supplements are commonly administered as antidepressants (Sabelli, Fink, Fawcett & Tom, 1996)."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Selection of a PEA-Binding Molecule"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Consideration of Aptamers for PEA-Binding"})}),Object(Le.jsx)("p",{children:"Aptamers are single-stranded oligonucleotides or oligopeptides that bind to target molecules, and are generally between 20 and 60 units in length (Keefe et al., 2010). For aptamers consisting of nucleotides, both DNA and RNA sequences can be used. RNA aptamers offer greater structural diversity and thus more flexibility in terms of potential targets, but simultaneously suffer from lower stability, especially under high temperatures and alkaline conditions (Thiel & Giangrande, 2009). Aptamers can be found naturally in the form of riboswitches, but are more commonly engineered for a specific target. The relatively simple design and engineering process geared towards a wide range of analytes, including proteins, antibiotics, organophosphates and various low molecular weight compounds (Ilgu & Nilsen-Hamilton, 2016), has made aptamers a popular choice for molecular biologists."}),Object(Le.jsx)("p",{children:"In terms of the procedure for aptamer engineering, SELEX (Systematic Evolution of Ligands by EXponential enrichment) is an iterative method for selecting the aptamers with optimal binding to the analyte. The process starts with a randomized library of nucleotide sequences, otherwise known as the initial oligonucleotide pool, which is flanked by primer regions (Zhuo et al., 2017). This then allows for amplification via PCR (for DNA) or reverse transcription-PCR (for RNA) to build up a synthetic library, which is incubated with the target molecule. Next, a selection round is performed by separating the target-bound sequences from unbound sequences (Zhuo et al., 2017), followed by amplification of the former via PCR (for DNA) or reverse transcription-PCR (for RNA). This new enriched library pool is then used for the next screening round, and the process is repeated while steadily increasing the stringency of the selection process until only a select few high-affinity aptamers are left. This generally takes around 5-15 rounds (Zhuo et al., 2017). The high-affinity aptamers are then analyzed in detail for optimal design."}),Object(Le.jsx)("p",{children:"The key design aspect of aptamers centers on the stem-loop/hairpin folding structure (Sabri et al., 2020), which is the functional part of the aptamer that specifically binds to the target molecules. As such, computational methods have been developed to aid with the modelling of aptamer structures from primary sequences specifically by first predicting the secondary stem-loop structure (Hu et al., 2021), which can be carried out by various classes of computational software based on their underlying logic. Commonly used tools include RNAfold and Mfold (currently amalgamated into UNAfold), which are based on minimizing the free energy; CentroidFold, which is based on multiple sequence alignment; Vfold, which makes use of RNA motif-based loop entropies, and RNAstructure, which has several complicated underlying structure prediction methods, including maximum expected accuracy, stochastic sampling, exhaustive traceback and pseudoknot prediction. Next, the equivalent tertiary RNA structure is synthesized using generally fragment-based computational methods, such as RNAComposer, 3dRNA, Vfold3D or simRNA, which generally involve dividing the input secondary structure into fragments, including the helices and loops, then assembling the corresponding 3D templates, and finally applying energy minimization to the overall structure. The developed computational tools mainly focus on RNA structure modelling, and so if necessary, the output can be converted it back into the corresponding DNA structure by directly replacing uracil residues with thymine in the RNA structure .pdb file in PyMOL and fixing the distance of each H atom with the \u201cfix hydrogen\u201d mode. For evaluating the strength of binding to the target, various docking tools may be used, including Autodock, ZDOCK, HADDOCK, PatchDock or MolSoft, followed by molecular dynamics simulations and binding affinity estimations in AMBER or VMD."}),Object(Le.jsx)("p",{children:"A variant on aptamers is the molecular beacon, which is a cDNA hairpin structure with a fluorophore at one end and a quencher at the other end, with the quencher stopping the fluorophore from creating a signal in its natural state (Tyagi & Kramer, 2012). However, in the presence of the analyte, the stem and loop will open to bind to the analyte, and the quencher is no longer able to quench the fluorophore, so the fluorophore creates a fluorescent signal. Molecular beacons generally consist of 15-30 base pairs in the loop region for binding to the target analyte, which can be nucleic acid sequences, toxins, proteins, and so on, as well as 4-6 base pairs at the double stranded stem (Tyagi & Kramer, 2012). Their high specificity and selectivity in binding has enabled a wide range of functionality in messenger RNA detection, intercellular imaging, protein and small molecule analysis, biosensors, biochip development, single nucleotide polymorphism and gene expression studies. Due to the similarity in structure, the process of engineering molecular beacons is practically identical to that of aptamers."}),Object(Le.jsxs)("p",{children:["Despite how well-developed and widely-applied the SELEX method is, it depends rather heavily on lab work. The outlined ",Object(Le.jsx)("em",{children:"in silico"})," selection of aptamers cannot act as a full replacement for the ",Object(Le.jsx)("em",{children:"in vitro"})," process, given that computational methods only provide theoretical structures and binding affinities that may not approximate the true values even remotely. Any mathematical model has its exceptions and inaccuracies, and thus in general, ",Object(Le.jsx)("em",{children:"in silico"})," work is done to narrow down the possible sequences for the SELEX method from a large starting pool. For instance, Chushak and Stone proposed a method that takes around 2.5 x 10",Object(Le.jsx)("sup",{children:"8"})," sequences and selects 10",Object(Le.jsx)("sup",{children:"3"})," to 10",Object(Le.jsx)("sup",{children:"4"})," of them with potentially high binding affinity to the target molecule, and these selected sequences are then passed along for further screening in the lab (Chushak & Stone, 2009)."]}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Decision to Pursue a Protein-Based Detection Method"})}),Object(Le.jsx)("p",{children:"Unfortunately, given our complete lack of lab access due to the COVID-19 situation, in addition to our aversion for proposing an aptamer part based on purely computational structure modelling and binding affinity evaluations, we ultimately decided against aptamer design in favour of engineering existing proteins. Even so, we have documented our aptamer research in summary here in the hopes that it may be of use to any future projects considering aptamers as a potential solution."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Overview of PEA-Binding Fusion Protein Design"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"As mentioned, we decided to pursue a protein-based detection method. Specifically, in order to facilitate both detection of PEA as well as signal production, we decided to design a fusion protein to be the PEA-binding molecule. One domain of the fusion protein would consist of a PEA-binding protein domain, while the other domain of the fusion protein would consist of a chemiluminescent enzyme, which could react with a chemiluminescent substrate to produce a signal. PEA would be conjugated to the test chamber of the microfluidic assay (using the small molecule conjugation method discussed in the Design section of the Chip Design engineering cycle). This effectively allows for quantification of the amount of urinary PEA by quantification of the number of binding molecules that bind to the PEA in the test chamber. As well, in order to facilitate a signal at the control chamber, the fusion protein would also be biotinylated; by conjugating streptavidin to the control chamber, fusion proteins could be \u201ccaught\u201d at the control chamber, thereby producing a signal."}),Object(Le.jsx)("p",{children:"Overall, the flow of events of the microfluidic assay chip (with respect to PEA detection) would be as follows:"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ol",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"After applying the urine sample to the microfluidic assay chip, the fusion protein binds to any PEA in the sample."}),Object(Le.jsx)("li",{children:"Then, the sample travels to the test chamber, where any fusion proteins with open binding sites will bind to the PEA conjugated to the test chamber."}),Object(Le.jsx)("li",{children:"Any other fusion proteins will travel to the control chamber; since they are biotinylated, they will be bound by the streptavidin conjugated to the control chamber."}),Object(Le.jsx)("li",{children:"Then, the chemiluminescent substrate is released to the test and control chambers, allowing a signal to be produced in each chamber."})]})}),Object(Le.jsx)("p",{children:"In the test chamber, the higher the signal, the lower the amount of PEA in the urine sample, which would correspond to a higher risk of ADHD."}),Object(Le.jsx)("p",{children:"With the above in mind, the design of the microfluidic assay for the detection of biomarkers (such as PEA) would be as follows:"})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",alt:"MCFA overview PEA",className:Bt.a.design_img,style:{borderRadius:20,marginLeft:"5%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Diagram of the microfluidic assay designed to detect urinary biomarkers (i.e. PEA)."})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Selection of a PEA-Binding Protein Domain"})}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:Bt.a.sub_sub_heading,children:"Criteria for a Protein Domain Suitable for Use in a Fusion Protein"})}),Object(Le.jsx)("p",{children:"To ensure that the diagnostic test is as accurate as possible, the protein must have a high binding affinity for PEA in urine, and preferably only PEA, considering the array of constituents typically present in urine (Boulton & Milward, 1971). Selecting a monomeric protein that is not naturally localized within the cell membrane is also preferable to ensure proper folding of the selected domain as part of a fusion protein, as well as proper binding of PEA when incorporated into the fusion protein (Urbina et al., 2019). It was also necessary to ensure that the protein folding of the selected domain would not be altered if folding were to occur as part of the formation of a recombinant protein. Candidates that demonstrated an inability to bind without the presence of multiple subunits were excluded, as they were unlikely to be stable and functional in the aqueous environment of our assay. Integral membrane proteins were similarly excluded as their conformational state is dependent on the preservation of a membrane-embedded hydrophobic region, incompatible with our assay\u2019s aqueous format (Marinko et al., 2019)."}),Object(Le.jsx)("p",{children:"The sequence of the protein must be known to enable the use of engineering tools for development, testing, and validation, namely 3D modelling, docking, and dynamics simulations. The protein must also be stable in the environment where binding is expected to take place; specifically, the protein must demonstrate stability under conditions similar to human urine. In addition, not only is the stability of the protein important, but it also must be uninhibited by the conditions of urine. That is, in order to be considered viable for use in processing urine samples, it had to be demonstrated that the pH, temperature, and salinity of standard human urine would not interfere with normal functionality of the binding domain. Finally, the catalytic activity of the protein must be removed without compromising the protein\u2019s ability to bind to its substrate."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:Bt.a.sub_sub_heading,children:"PEA-Binding Protein Candidates"})}),Object(Le.jsx)("p",{children:"Proteins that were considered for their reported interaction with PEA included VMAT2, FeaR, TAAR1, TAAR4, TynA, and 1UTM (Eiden & Weihe, 2011; Lindemann et al., 2005; Murakawa et al., 2015; Zeng & Spiro, 2013; Zucchi et al., 2006). Using the criteria outlined above, the following proteins were ruled out:"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"VMAT2 is non-specific to PEA, and binds a multitude of urinary biogenic amines (Eiden & Weihe, 2011)."}),Object(Le.jsx)("li",{children:"FeaR is a transcription factor regulating PEA levels but does not bind PEA itself, making it unsuitable for PEA quantification ( Lindemann et al., 2005)."}),Object(Le.jsx)("li",{children:"TAAR1 and TAAR4 are transmembrane G protein coupled receptors (GPCRs) that are very unlikely to be stable in an aqueous diagnostic context and are thus inadmissible (Murakawa et al., 2015; Zeng & Spiro, 2013)."}),Object(Le.jsx)("li",{children:"TynA is a dimer, and its stability and ability to bind PEA in a fusion protein was uncertain (Zucchi et al., 2006)."})]})}),Object(Le.jsxs)("p",{children:["From this assessment of the protein candidates, the most optimal PEA-binding protein for use in our microfluidic assay was selected as 1UTM, a monomeric trypsin derived from ",Object(Le.jsx)("em",{children:"Salmo salar"}),", with a dissociation constant (Kd) of 0.000972 (RCSB PDB, n.d.)."]})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",alt:"3D structure of 1UTM",className:Bt.a.design_img,style:{borderRadius:20,marginLeft:"5%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"The 3D structure of 1UTM, as visualized using UCSF Chimera, is as follows, where 1UTM is orange and PEA is green."})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Selection of a Chemiluminescent Enzyme Domain"})}),Object(Le.jsx)("p",{children:"The method of detection selected for quantification of the marker analyte PEA in urine was based on the chemiluminescence produced by the interaction of horseradish peroxidase (HRP) and Amplex Red. HRP is a commonly used enzyme to catalyze chemiluminescent reactions, with multiple candidate substrates such as Luminol, Acridan or Amplex Red (Ghosh et al., 2020; Heo et al., 2021; Osman et al., 2000). Compared to other prevalent enzymes utilized in chemiluminescence detection, such as alkaline phosphatase, horseradish peroxidase was a smaller protein candidate (44 kDa), reported to be stable in neutral pH, and when directly evaluated against alkaline phosphatase, exhibited greater detection sensitivity (Beyzavi et al., 19987; Chattopadhyay et al., 2000). There are multiple examples of HRP specifically being used for measuring analyte concentrations in urine samples, suggesting this to be a reliable method of detection for our purposes (Chang et al., 2017; Eamudomkarn et al., 2018)."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Selection of a Chemiluminescent Substrate for HRP"})}),Object(Le.jsx)("p",{children:"Several chemiluminescent substrates for HRP were considered, including Acridan, a compound that emits light when oxidized in the presence of HRP (Roda, 2011). Unfortunately, there was insufficient data on the chemiluminescent properties of pure Acridan, particularly an expected value for a ratio of concentration to chemiluminescence. Without this knowledge, and without lab access to determine the relationship between concentration and chemiluminescence, we would not be able to confidently interpret the resulting chemiluminescent signal."}),Object(Le.jsx)("p",{children:"Instead, a compound known as Amplex Red was explored as an option. Amplex Red has definitive values for excitation and emission maxima provided by ThermoFisher Scientific, and has been found to oxidize in the presence of HRP, forming a red-fluorescent product known as resorufin (ThermoFisher Scientific, n.d.)."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Protein Biotinylation"})}),Object(Le.jsx)("p",{children:"The biotin-streptavidin complex system has one of the highest specificities and affinities for non-covalent binding observed in nature (Wilchek et al., 2006), and is exploited in our design for the detection of proteins. In particular, this requires biotinylation, which is the process of attaching biotin (a small molecule) to some target protein or other macromolecule, so that the biotinylated analyte may be recognized and bound to streptavidin (a protein) (Kay et al., 2009). Recall that in our microfluidic assay, our fusion protein is biotinylated to allow streptavidin (conjugated to the control chamber) to bind, thereby eliciting a signal at the control chamber."}),Object(Le.jsx)("p",{children:"Typically in designing fusion proteins, biotinylation of the fusion protein is preferred to fusion with streptavidin, since biotin is smaller in size than globular proteins, which allows for the minimization of steric interference and the conjugation of multiple biotin molecules to a single protein for signal amplification. When designing a fusion protein, reducing the size of the fusion is advantageous to prevent instability and aggregation, making biotinylation of a fusion protein preferable over adding another protein domain. In addition, biotin has a valeric acid side chain, which can be easily derivatized and conjugated to reactive structures without affecting its avidin-binding function (Kay et al., 2009). This feature allows for the incorporation of many biotinylation reagents that target specific functional groups or residues, including primary amines, sulfhydryls, carboxyls and carbohydrates, without inactivating the target macromolecule."}),Object(Le.jsx)("p",{children:"Biotinylation is most commonly performed through chemical means, although it can also occur through enzymatic approaches. Enzymatic methods generally require the co-expression of bacterial biotin ligase and an exogenously expressed protein of interest that is modified to carry a biotin acceptor peptide, which provides a more uniform biotinylation than chemical methods and can be cell compartment specific (Cull & Schatz, 2000). Chemical methods, on the other hand, provide much greater flexibility in the type of biotinylation needed. This is especially preferred to enzymatic methods when specific functional groups such as carbonyls, carboxyls, primary amines or sulfhydryls are targeted (Chapman-Smith & Cronan Jr, 1999)."}),Object(Le.jsx)("p",{children:"For our purposes, it suffices to biotinylate the protein at any residue, and so the enzymatic method was used by fusing the protein at its C-terminus to the AviTag or the Acceptor Peptide (Cull & Schatz, 2000), a 15-amino acid peptide (GLNDIFEAQKIEWHE). The protein is then incubated with BirA in the presence of biotin and ATP, which allows for biotinylation to occur at the C-terminus."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Fusion Protein Linker Design"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsxs)("p",{children:["Linkers are amino acid sequences that fuse two or more protein domains together - in this case, 1UTM and HRP. The design of linkers is vital to fusion protein functionality, since inappropriate linkers may lead to misfolding of the fusion proteins, low yield in protein production, or impaired bioactivity (Chen et al., 2013). Linkers are generally classified into 3 categories by structure: flexible linkers, rigid linkers, and in vivo cleavable linkers. Flexible linkers are usually used when the joined domains require a certain degree of movement or interaction. They are generally composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids, where the small size of these residues provides flexibility, and allows for mobility of the connected functional domains (Argos, 1990). The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduces the unfavorable interaction between the linker and the protein moieties. The most commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues, known as a GS linker. One of the most widely used flexible linkers has sequence (Gly-Gly-Gly-Gly-Ser)",Object(Le.jsx)("sub",{children:"n"}),". Varying the copy number n allows for different lengths of the GS linker to be constructed, so that appropriate separation of the functional domains or necessary inter-domain interactions can be achieved. The lack of rigidity of flexible linkers, however, can sometimes result in poor expression yields or loss of biological activity, due to an inefficient separation of the protein domains or insufficient reduction of their interference with each other. In these situations, rigid linkers have been successfully applied to keep a fixed distance between the domains and to maintain their independent functions. Alpha helix-forming linkers with the sequence of (EAAAK)",Object(Le.jsx)("sub",{children:"n"})," have been applied to the construction of many recombinant fusion proteins (Amet et al., 2008), where n generally takes on values between 2 and 5. The alpha helical conformation is exhibited by many natural linkers, with it having a rigid and stable structure, due to intra-segment hydrogen bonds and a closely packed backbone (Aurora et al., 1997). Therefore, the stiff alpha-helical linkers may act as rigid spacers between protein domains. Another type of rigid linker has a Pro-rich sequence, (XP)",Object(Le.jsx)("sub",{children:"n"}),", with X designating any amino acid, preferably Ala, Lys, or Glu. The incorporation of Pro in non-helical linkers can increase the stiffness, and allows for effective separation of the protein domains."]}),Object(Le.jsxs)("p",{children:["Cleavable linkers are less relevant to our discussion, since we require the fusion protein to function as a unit. For the sake of completion, cleavable linkers are linkers that allow for the release of free functional domains in vivo. They are oligopeptides without the versatility of crosslinking agents, and are designed to be cleaved under specific conditions such as the presence of reducing reagents or proteases in the ",Object(Le.jsx)("em",{children:"in vivo"})," environment. Cleavable linkers may reduce steric hindrance, improve bioactivity, or achieve independent actions/metabolism of individual domains of recombinant fusion proteins after linker cleavage."]}),Object(Le.jsx)("p",{children:"Keeping the above considerations in mind, we decided that a polar, flexible, and medium-sized linker would be best for our purposes, as this would allow for proper separation of protein domains in aqueous solution, without the risk of linker-domain interactions (namely, hydrophobic interactions) that might compromise the conformation of the domains. The flexibility and length of the linker would allow each domain to function effectively, without risk of steric hindrance between domains (especially around the active sites of each domain). The amino acid sequence of the chosen linker is as follows:"}),Object(Le.jsx)("p",{children:"N\u2019-SGSTSTSTSTSGS-C\u2019"}),Object(Le.jsx)("p",{children:"With all of the above design considerations in mind, we utilized multiple-domain homology modelling in UCSF Chimera to obtain a 3D model of our fusion protein, as pictured below, with 1UTM on the left and HRP on the right."})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("img",{src:"",alt:"3D structure of 1UTM HRP",className:Bt.a.design_img,style:{borderRadius:20}})})]}),Object(Le.jsxs)(Ie,{open:!0,title:"Build",children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Vector Selection"})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsxs)("p",{children:["We decided to use the pET28a plasmid vector, which is a general-purpose expression vector with a strong promoter (T7), making it appropriate for expression of our fusion protein. It contains a ",Object(Le.jsx)("em",{children:"lac"})," operon, allowing for induction of expression using \u200b\u200bisopropyl beta-D-1-thiogalactopyranoside (IPTG), an allolactose analog."]})}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Chassis Selection"})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsxs)("p",{children:["For the expression chassis, we decided to use ",Object(Le.jsx)("em",{children:"E. coli BL21(DE3)"}),", which is a general-purpose strain for bacterial protein expression. BL21 lacks ",Object(Le.jsx)("em",{children:"lon"})," and ",Object(Le.jsx)("em",{children:"ompT"})," proteases involved in the cleavage of extraneous proteins and T7 RNA polymerase, respectively. The lack of these proteases ensure that protein expression is possible. As well, disulfide bond formation is possible, and is largely catalyzed by natively expressed thioredoxin reductase (trxB), glutathione reductase (gor) and oxidases DsbA/DsbC."]})}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Protein Purification"})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"To enable purification of the desired fusion protein by affinity chromatography, a 6-His tag with a TEV linker was appended to the N-terminus of the fusion protein. The His-tag has a very high affinity for nickel ions, and is only outcompeted by imidazole. To purify the protein, the expression cell culture would be lysed, centrifuged to remove cell debris, then the supernatant poured into an affinity chromatography column with Ni-NTA resin. The TEV linker of the fusion protein, containing a TEV recognition site, could be cleaved by adding TEV protease. This would allow for elution of the fusion protein while simultaneously removing the 6-His tag and TEV linker. The Ni-NTA resin could then be eluted by the addition of imidazole, which would cause the bound 6-His tags and TEV linkers to elute."})})]}),Object(Le.jsxs)(Ie,{open:!0,title:"Test",children:[Object(Le.jsx)("div",{className:pe.a.description,style:{marginTop:30},children:Object(Le.jsx)("p",{children:"Due to unforeseen circumstances (one of which was a global pandemic, and the other of which was unforeseen maintenance and construction of our lab space by the University), we unfortunately did not have lab access this year. As a result, we were unable to experimentally characterize the fusion protein. With that said, the following methodology outlines our plan of action to test our fusion protein construct if we had lab access."})}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Experimental Demonstration of Fusion Protein Stability"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"The beauty of working with a fusion protein with a chemiluminescent enzyme domain is that the activity (and by extension, stability) of the fusion protein can be assayed very easily. In the case of the 1UTM-HRP fusion, where Amplex Red is the chemiluminescent substrate of HRP, Amplex Red could be added to a sample of the purified fusion protein in a cuvette. The fusion protein solution would be expected to display chemiluminescence of wavelength 563-587 nm upon addition of Amplex Red; the chemiluminescence can be measured using a spectrophotometer. A negative control would involve adding Amplex Red to the protein\u2019s buffer solution (but without the protein!) to ensure that chemiluminescence displayed by the protein solution is not due to some side reaction with the buffer solution components. A positive control would involve adding Amplex Red to a known pure solution of HRP (not in a fusion protein); this would ensure that Amplex Red is not degraded."}),Object(Le.jsx)("p",{children:"To follow up on the previous stability experiment, simulation of urine conditions (including pH, salt, and major metabolites) would allow us to demonstrate the stability and functionality of the protein in urine. Roxe (1990) determined that urine typically had the following conditions:"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"A pH range of 5-9, but usually slightly acidic. pH 6 is a reasonable generalization."}),Object(Le.jsx)("li",{children:"A protein concentration of much less than 30 mg/dL; equal or above indicates disease. To simulate protein in urine, bovine serum albumin (BSA) would be an appropriate, inert choice."}),Object(Le.jsx)("li",{children:"A glucose concentration of much less than 100 mg/dL; equal or above indicates disease."}),Object(Le.jsx)("li",{children:"Approximately 4 mM urea."}),Object(Le.jsx)("li",{children:"Approximately 20 mM salt (NaCl)."})]})}),Object(Le.jsx)("p",{children:"These components could be dissolved in DI water to simulate a urine sample. Then, the experimental method described in the previous paragraph could be done to demonstrate stability and functionality of the fusion protein in urine-like conditions."}),Object(Le.jsx)("p",{children:"From here, it would be useful to obtain an experimental standard curve for the reaction between HRP and Amplex Red in urine-like conditions. To do this, a series of dilutions of the fusion protein would be made, then a known excess concentration of Amplex Red would be added to each fusion protein dilution. In measuring the chemiluminescence produced by each dilution, a standard curve of chemiluminescence vs. fusion protein concentration can be plotted. A multiplate and multiplate analyzer can be used for this. The same positive and negative controls from the stability experiment (above) should be implemented here as well. This would simulate the conditions of chemiluminescence in the microfluidic assay chip, since in the MCFA chip, the number of fusion protein molecules that bind at the test chamber would be variable, while the amount of Amplex Red that passes through the test chamber would be a known excess amount."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,style:{paddingBottom:0},children:"Experimental Determination of PEA-Binding Affinity of the Fusion Protein"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"Binding affinity between a protein and ligand can be expressed in terms of the protein\u2019s dissociation constant, Kd. Mathematically, Kd is equivalent to the ratio of unbound protein to the ratio of bound protein. It follows that the lower the Kd, the more that protein is bound at any given time, and thus the higher the protein\u2019s affinity for the ligand."}),Object(Le.jsx)("p",{children:"To determine the Kd of the PEA-binding fusion protein, we would need to determine the ratio of unbound:bound protein after incubation with PEA. To do so, an absorbance-based method can be used, as follows:"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ol",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"Prepare a solution of the PEA-binding fusion protein (the concentration can vary, so long as it is known)."}),Object(Le.jsx)("li",{children:"Prepare an equimolar solution of PEA, prepared in the same buffer solution as the protein solution."}),Object(Le.jsx)("li",{children:"Using a spectrophotometer, measure the absorbance of a 1/2 diluted PEA solution at 257 nm."}),Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:5,marginBottom:5},children:[Object(Le.jsx)("li",{children:"PEA consists of a benzene ring and an amine group, the former of which exhibits absorbance around 257 nm due to the conjugated pi system (Tong et al., 2020)."}),Object(Le.jsx)("li",{children:"If the absorbance is over 2.0, dilute the solution and try again until the absorbance is less than 2.0. The dilution factor can be used to calculate the concentration of PEA in the undiluted solution."})]}),Object(Le.jsx)("li",{children:"Incubate the protein solution with the PEA solution in a 1:1 ratio."}),Object(Le.jsx)("ul",{className:pe.a.description,style:{marginTop:5,marginBottom:5},children:Object(Le.jsx)("li",{children:"To prevent protein loss to pipette tips during transfer, prime any tipes used with a solution of bovine serum albumin (BSA), which will prevent protein \u201csticking\u201d to the tip."})}),Object(Le.jsx)("li",{children:"Centrifuge the incubated sample to pellet all of the fusion protein, including the fusion protein molecules bound to PEA. Recover the supernatant only."}),Object(Le.jsx)("li",{children:"Using a spectrophotometer, measure the absorbance of the PEA supernatant solution at 257 nm."}),Object(Le.jsx)("ul",{className:pe.a.description,style:{marginTop:5,marginBottom:5},children:Object(Le.jsx)("li",{children:"Again, if the absorbance is over 2.0, dilute the solution and try again until the absorbance is less than 2.0. The dilution factor can be used to calculate the concentration of PEA in the undiluted solution."})})]})}),Object(Le.jsx)("p",{children:"In order to interpret the absorbance values, a standard curve must be generated. This should be done by preparing a set of dilutions of a PEA solution of known concentration, then measuring the absorbance of each dilution. The resulting standard curve of absorbance vs. concentration can then be used to determine the concentration of an unknown PEA solution, given its absorbance."}),Object(Le.jsxs)("p",{children:["Any amount of PEA that was \u201cmissing\u201d from the supernatant (relative to the original known amount of PEA) will have been bound by the fusion protein (in a 1:1 molar ratio). Therefore, ",Object(Le.jsx)("br",{}),"Kd = concentration of unbound protein / concentration of bound protein ",Object(Le.jsx)("br",{}),"By extension, ",Object(Le.jsx)("br",{}),"Kd = PEA",Object(Le.jsx)("sub",{children:"sup"})," / (original concentration of PEA - PEA",Object(Le.jsx)("sub",{children:"sup"}),") where PEA",Object(Le.jsx)("sub",{children:"sup"})," = supernatant concentration of PEA."]}),Object(Le.jsx)("p",{children:"It would be expected that the fusion protein exhibit an experimental Kd similar to the Kd of standalone 1UTM: approximately 0.000972 (RCSB PDB, n.d.). A dissociation constant much higher than the expected value (and by extension, a lower binding affinity) might indicate that the PEA-binding domain of the fusion protein is not individually stable or active."})]})]}),Object(Le.jsx)(Ie,{open:!0,title:"Learn",children:Object(Le.jsx)("div",{className:pe.a.description,style:{marginTop:30},children:Object(Le.jsx)("p",{children:"Without lab access (as explained in the Test section), we were not able to perform the experiments outlined in the Test section. However, if we were to have performed those experiments, the standard curve experiment relating chemiluminescence to fusion protein concentration would have been valuable for quantification purposes. Specifically, it would have further informed the development of the optical detector and app (described below in the Optical Detector and Application Development engineering cycle), as the standard curve would allow for more accurate interpretation of the signal at the test chamber under simulated urine conditions, which would be most representative of true urine samples analyzed by NeuroDetech."})})})]})}}]),i}(n.Component),Ht=i(19),zt=i.n(Ht);function Ft(e,t,i){return t in e?Object.defineProperty(e,t,{value:i,enumerable:!0,configurable:!0,writable:!0}):e[t]=i,e}function Gt(e,t){var i=Object.keys(e);if(Object.getOwnPropertySymbols){var n=Object.getOwnPropertySymbols(e);t&&(n=n.filter((function(t){return Object.getOwnPropertyDescriptor(e,t).enumerable}))),i.push.apply(i,n)}return i}function Vt(e){for(var t=1;t<arguments.length;t++){var i=null!=arguments[t]?arguments[t]:{};t%2?Gt(Object(i),!0).forEach((function(t){Ft(e,t,i[t])})):Object.getOwnPropertyDescriptors?Object.defineProperties(e,Object.getOwnPropertyDescriptors(i)):Gt(Object(i)).forEach((function(t){Object.defineProperty(e,t,Object.getOwnPropertyDescriptor(i,t))}))}return e}function Ut(e,t){if(null==e)return{};var i,n,a=function(e,t){if(null==e)return{};var i,n,a={},r=Object.keys(e);for(n=0;n<r.length;n++)i=r[n],t.indexOf(i)>=0||(a[i]=e[i]);return a}(e,t);if(Object.getOwnPropertySymbols){var r=Object.getOwnPropertySymbols(e);for(n=0;n<r.length;n++)i=r[n],t.indexOf(i)>=0||Object.prototype.propertyIsEnumerable.call(e,i)&&(a[i]=e[i])}return a}var qt=i(9),Kt=i.n(qt),Yt=n.createContext({prefixes:{}});Yt.Consumer,Yt.Provider;function Jt(e,t){var i=Object(n.useContext)(Yt).prefixes;return e||i[t]||t}var $t=["bsPrefix","className","striped","bordered","borderless","hover","size","variant","responsive"],Zt=n.forwardRef((function(e,t){var i=e.bsPrefix,n=e.className,a=e.striped,r=e.bordered,s=e.borderless,o=e.hover,c=e.size,l=e.variant,d=e.responsive,h=Ut(e,$t),u=Jt(i,"table"),m=Kt()(n,u,l&&"".concat(u,"-").concat(l),c&&"".concat(u,"-").concat(c),a&&"".concat(u,"-striped"),r&&"".concat(u,"-bordered"),s&&"".concat(u,"-borderless"),o&&"".concat(u,"-hover")),p=Object(Le.jsx)("table",Vt(Vt({},h),{},{className:m,ref:t}));if(d){var f="".concat(u,"-responsive");return"string"===typeof d&&(f="".concat(f,"-").concat(d)),Object(Le.jsx)("div",{className:f,children:p})}return p})),Qt=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Protein Optimization"})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"To increase efficiency and accuracy of the detection of target biomarker concentration, we decided to improve the binding affinity and stability of the parts of the fusion proteins - trypsin for binding PEA and monomeric streptavidin for biotin. Protein modelling and improved redesign results are detailed in this section."})}),Object(Le.jsxs)(Ie,{open:!0,title:"Design",children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Protein Design Methods"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"There are multiple ways to design and modify proteins that aim to increase binding affinity and stability of the protein. Three main methods were explored by our team, directed evolution, rational protein design and de novo design. Directed evolution follows the path of natural selection, but is guided towards a specific goal that one is trying to achieve with the protein (Huang et.al, 2016). This method commonly requires in-lab testing either in-vivo or in-vitro methods. Rational protein design is the process of assessing the structure of the protein and suggesting changes in the amino acid sequence that could possibly change the folding and increase overall stability. The suggested mutations can be verified using multiple different methods, for example, peptide synthesis - where we link amino acids together, or computational modelling and prediction of folding. (Fields, 2002). De novo protein design consists of designing a protein from scratch by making a new amino acid sequence. This can also be verified through different methods similar to rational protein design."}),Object(Le.jsx)("p",{children:"For the 2021 iGEM project, the most suitable method for protein design was rational protein design incorporated with computational modelling to be able to analyze a broader scope of mutations. This is due to limited access to the lab, thus methods like peptide synthesis to test out the directed evolution method or the de novo method are not feasible."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Rational Design: Identification of Key Binding Residues"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsxs)("p",{children:["The binding pocket residues of 1UTM were identified by visualizing the unmodified protein in UCSF Chimera. Although the Protein Data Bank of the Research Collaboratory for Structural Bioinformatics (RCSB PDB) lists 5 residues as those involved in binding (Berman et al, 2000), numbering issues with 1UTM pdb file from the PDB had been noted. The PDB version of 1UTM numbers the residues starting from 16. Thus, the file had to be renumbered using Chimera\u2019s functions. Later on in the process, running the Rosetta protocols for single mutations caused a mismatch in the numbering between the original renumbered version of the proteins and the version outputted by Rosetta. The two versions were misaligned by one number; to elaborate, the amino acid located at position 171 in the original renumbered file was instead seen at 170 in the file output by Rosetta, continuing throughout the protein. It is believed that the difference in numbering was due to the presence of the Ca",Object(Le.jsx)("sup",{children:"2+"})," ligand in another part of the protein, which was still present in the original renumbered file but was removed during the Rosetta protocols."]}),Object(Le.jsxs)("p",{children:["Thus, to avoid errors in identifying the binding residues, a Chimera function was used to identify all residues within a distance of 5 \xc5 from the PEA ligand on the file with no Ca",Object(Le.jsx)("sup",{children:"2+"})," ligand present. This distance was deemed appropriate in the work of Gao and Skolnick (2012), which stated that the median minimum distance between the heavy atoms of the proteins interfaces and the ligands was 4.2 \xc5. It should be noted that while residue 40 (His) was within the 5 \xc5 distance it was not taken as a binding pocket residue since it was located on a different part of the protein entirely. It appears that the part of the protein to which the residue was attached merely folds in the direction of PEA."]})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,style:{marginBottom:20},children:Object(Le.jsx)("img",{src:"",className:zt.a.design_img})}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Rational Design: Observation and Verification with Chimera"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"UCSF Chimera, a widely used molecular chemical visualization and analysis software, was selected for analyzing the biophysical folding structures of trypsin to phenylethylamine (PEA) for its simplicity and versatility. Much like its well-known counterpart, PyMol, UCSF Chimera can be used to analyze genomic sequences and other complexed molecules in the structure. In contrast, UCSF Chimera is also capable of performing molecular dynamics simulations, sequence alignment, genome editing for designing new proteins, and ligand docking through its AutodockVina integration to name a few."}),Object(Le.jsxs)("p",{children:["For this year\u2019s project, Chimera was used extensively for both rational protein design, ligand docking, and energy conformer analysis. To start, rational protein design in Chimera consisted of visualizing the native Trypsin protein structure and selecting amino acids within a 5.0 \xc5 radius of the known binding pocket for PEA. The motivation behind this was to enhance intermolecular interactions, notably H-bonding, in order to stabilize the protein and increase binding affinity. Amino acids within this radius were selected and replaced by longer-chain similarly charged amino acids. This was followed by a structure minimization function to ensure that the swapped amino acid did not introduce any undesirable steric clashes. The mechanism behind this follows that of the MMTK program commonly seen in Python for molecular dynamics simulations, ensuring that our protein is oriented in its optimal rotamer angles. As a precursor to ",Object(Le.jsx)("em",{children:"minimization"}),", another protocol known as ",Object(Le.jsx)("em",{children:"Dock Prep"})," is also called to add missing hydrogens and charges in the structure. The resulting proteins after minimization were further evaluated using the ",Object(Le.jsx)("em",{children:"score_jd2"})," protocol for conformer energy analysis in Rosetta Commons."]}),Object(Le.jsx)("p",{children:"Due to the low throughput of proposed mutated proteins in Chimera, we turned to Rosetta Common\u2019s automated protein mutation approach. Further, as a visual verification of the protein complexes generated by Rosetta, Chimera was used."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Computation-Guided Design Approach"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"The method that we chose to go with to modify our protein is rational design with the assistance of a protein design software called Rosetta. Rosetta has functionalities that give us the ability to auto generate all possible mutations for a given protein and then assign it a Rosetta energy score which is a weighted average of multiple different factors such as repulsive and attractive forces, and hydrogen bond energy among many more. The unit is in Rosetta Energy Units (REU). This functionality only requires an input of the PDB file of the protein, and it is optional to specify which mutations to make. This gives us a comprehensive overview of all the possible mutations that can be made on 1UTM. With this information we can do further analysis and propose double and multi mutations to arrive at a protein that is stable and is specific to the ligand. (Rosetta Commons, n.d.)"}),Object(Le.jsx)("p",{children:"We chose to put an emphasis on the computational method of Rosetta, instead of using a pure rational protein design method because there is a lack of literature on the 1UTM protein. In order to be able to propose adequate mutations for the 1UTM protein, we must be able to understand its structure and how it behaves when folding. Due to the lack of literature on 1UTM and restricted lab access, there is no clear way of making good predictions on which amino acids to change in the sequence."}),Object(Le.jsx)("p",{children:"First, we incorporated rational protein design into the choice of single mutations. As such, we considered the biochemical properties of the residues and PEA in order to determine possible advantageous mutations for binding affinity, even if they did not specifically improve the stability of the protein."}),Object(Le.jsx)("p",{children:"We then assessed the Rosetta scores for the biochemically-chosen single mutations to ensure they were reasonably stable. A mix of these and the mutations with the best Rosetta scores were then passed to Rosetta to produce double mutations."}),Object(Le.jsx)("p",{children:"Finally, the top double mutation pairs were combined to make the multi-mutated protein candidates. These top candidates were later assessed by other more computationally intensive software such as GROMACS and AutoDock Vina to calculate interaction energy and binding energy."})]})]}),Object(Le.jsxs)(Ie,{open:!0,title:"Build",children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Rosetta: Protein Mutations"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"Rosetta is a software tool that was primarily designed to predict structures of proteins. It now has many other applications such as determining macromolecular interactions, molecular docking and protein design (Kaufmann et. al., 2010)."}),Object(Le.jsx)("p",{children:"Specifically for this project we utilize a protocol, which is a combination of algorithms, called Point Mutant Scan (pmut_scan_parallel) which allows us to input the original protein data bank (PDB) file that contains the sequence of amino acids of the entire protein, and the protocol will output PDBs with all possible combinations of single mutations."}),Object(Le.jsx)("p",{children:"The algorithm that Rosetta uses is, once given the original PDB file, it generates a list of every possible mutation of all residues in the protein. The algorithm does this by swapping out the existing amino acid at that residue and replacing it with all other available amino acids. It then makes the mutation and predicts the structure of the newly mutated protein (Rosetta Commons, n.d.). The way that Rosetta is able to predict the structure of the protein is by taking small parts of the protein that is known to create the structure that is most similar to how the protein behaves in nature using the Monte Carlo approach (Rohl et. al., 2004). The Monte Carlo method in brief is the process of sampling a multitude of random combinations, and taking the average of those combinations to form a prediction of the outcome (Zhang et. al., 1992)."}),Object(Le.jsx)("p",{children:"The outcome of running this protocol is a list of new PDB files, which are then passed onto a scoring protocol. The way that Rosetta determines a score for each mutated protein is by using a weighted average of different energies that it calculates, such as repulsive, attractive and hydrogen bond energies. The lower the Rosetta score, the more stable the protein is, thus the goal is to design the protein to have a lower score compared to the original protein."}),Object(Le.jsx)("span",{className:zt.a.list_heading,children:"Workflow for Mutating Proteins"}),Object(Le.jsxs)("ol",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"Obtain the 1UTM PDB file from the protein data bank."}),Object(Le.jsx)("li",{children:"Run a python script to \u201cclean\u201d the PDB file. This removes all the headers and descriptions from the PDB file and leaves it with only the amino acids and the ligand."}),Object(Le.jsx)("li",{children:"Use Rosetta\u2019s Point Mutant Scan protocol to generate all the possible single mutations for 1UTM."}),Object(Le.jsx)("li",{children:"Score each new mutated protein using Rosetta\u2019s scoring protocol to quantify the mutation and perform further analysis and verification."})]}),Object(Le.jsx)("p",{style:{marginTop:20},children:"Since the point mutant scan protocol and the scoring protocol employ the Monte Carlo method, a high performance computer was used to be able to support the computational power that is needed to generate all the random combinations. We used the Graham cluster from Compute Canada to utilize the Rosetta Software."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Improvement of Trypsin"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Results: Trypsin Single Mutation"})}),Object(Le.jsx)("p",{children:"There were a total of 4143 single mutations made from using Rosetta\u2019s Point Mutant Scan protocol. Listed below are the top 50 lowest scoring single mutations. The original score for the un-mutated 1UTM with PEA was -322.402 REU."}),Object(Le.jsxs)("p",{children:["Top 10 Rosetta Scores for 1UTM single mutations and the original protein score are listed in the table below. Please check the full top 50 scores on the spreadsheet ",Object(Le.jsx)("a",{href:"https://docs.google.com/spreadsheets/u/1/d/1r_Wf-dGtYojBKM7i1tRiwdZFUIioxGey/edit",target:"_blank",children:"here"}),". For this and all following score tables, the mutations are written in the format \u201cOriginal Amino Acid|Residue Number|New Amino Acid\u201d."]})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)(Zt,{bordered:!0,className:pe.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:"Score (REU)"}),Object(Le.jsx)("th",{children:"Mutation"})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-322.402"}),Object(Le.jsx)("td",{children:"Original"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-344.859"}),Object(Le.jsx)("td",{children:"A205T"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-344.706"}),Object(Le.jsx)("td",{children:"I81N"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-344.222"}),Object(Le.jsx)("td",{children:"I81H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-343.811"}),Object(Le.jsx)("td",{children:"I81W"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-343.794"}),Object(Le.jsx)("td",{children:"I81R"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-343.771"}),Object(Le.jsx)("td",{children:"I81K"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-343.657"}),Object(Le.jsx)("td",{children:"I81Y"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-343.647"}),Object(Le.jsx)("td",{children:"A205C"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-343.253"}),Object(Le.jsx)("td",{children:"I81F"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-343.191"}),Object(Le.jsx)("td",{children:"I81Q"})]})]})]})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsxs)("p",{children:["However, our main goal for our newly designed protein is to increase its ability to bind to the analyte which is PEA. Therefore we must look at the mutations made near the binding pocket residues. Those residues are 169, 170, 171, 172, 175, 189, 190, 191,192,194, 195, 202, 203, and 204. These mutations should have a stronger impact on 1UTM\u2019s binding affinity with PEA, thus are analyzed in more detail. The mutations that are considered for each residue are listed in the spreadsheet ",Object(Le.jsx)("a",{href:"https://docs.google.com/spreadsheets/u/1/d/1r_Wf-dGtYojBKM7i1tRiwdZFUIioxGey/edit",target:"_blank",children:"here"}),"."]}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Single Mutation Result Interpretation"})}),Object(Le.jsx)("p",{children:"Double mutations for the binding residues were proposed using a combination of rational design and simply evaluating the Rosetta scores of the single mutations for stability. The characteristics of PEA were considered with those of an individual residue in order to make suggestions."}),Object(Le.jsx)("p",{children:"The criteria for rational design-proposed mutations depended on the properties of the original amino acid as well as those of the ligand. PEA is positively charged (EMBL-EBI, 2021), thus we proposed changing positively-charged, neutral, or non-polar residues such as serine, cysteine, glutamine, (Merck KGaA, 2021) , and glycine (PubChem Identifier: CID 750, 2021), to negatively-charged amino acids such as aspartic acid and glutamic acid to better accommodate the ligand (Foulquier, 2020). Phenylalanine and Tyrosine, which both possess aromatic side chains, were suggested as changes for any amino acid in the binding pocket, in particular Valine, which possesses an aliphatic side chain (PubChem Identifier: CID 6287, 2021), (Merck KGaA, 2021). These changes were proposed to hopefully result in pi-pi stacking between 1UTM and PEA\u2019s aromatic ring. (Hardinger, 2017a). Trp, which also has an aromatic side chain, was not tested in these circumstances due to its bulkier structure, which could result in increased steric interference (Hardinger, 2017b). As such, when it appeared on the unmodified version of 1UTM, a change from Tryptophan to Phenylalanine or Tyrosine was suggested."}),Object(Le.jsx)("p",{children:"A comparison of the original 1UTM protein and the mutation of residue 170 from serine to Aspartic acid was visualized in Chimera, as seen in the images below. The original protein is brown, while the mutated protein is blue. Residue 170 is seen enclosed in the red circle on both images."})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("img",{src:"",className:zt.a.design_img})}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("img",{src:"",className:zt.a.design_img})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"Next, we checked the Rosetta scores for these biochemically chosen single mutations to ensure they were reasonably stable. If a mutation received a score of -300 or lower it was considered for double mutations, otherwise, it was discarded. A maximum of four mutations per residue was passed to Rosetta to synthesize double mutations. When four candidates were passed on, the top two Rosetta scores and two best-scoring rational design candidates were selected, if all four were distinct from each other. If there were less than two suitable rational design candidates, the top three Rosetta-scoring mutations were submitted for double mutations. These groups of top three scorers were accompanied by a rational design candidate if a suitable one was present."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Results: Double Mutations with Rosetta"})}),Object(Le.jsx)("p",{children:"In order to generate the double mutations, we used the same Rosetta protocol, however this time we defined a list of all the double mutations that we want to make. The outcome of this is a PDB file for each double mutation that was passed into the protocol."}),Object(Le.jsx)("p",{children:"A heat map was then generated to allow us to visually see which double mutation combination gave us the lowest scoring protein."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:zt.a.design_img}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Heat Map of 1UTM Double Mutations"})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"From the heat map above, we picked out the strongest (lowest scoring) double mutations and combined them with other double mutations that also performed well but not the strongest. This led to a total of 56 triple and quadruple mutations that were made on 1UTM, using the same Point Mutant Scan protocol used for the single and double mutations. We again defined the triple and quadruple mutations that were selected and the output is a PDB file for each mutation made."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Results: Trypsin Multiple Mutations"})}),Object(Le.jsx)("p",{children:"The top 15 1UTM multi-mutations will be further considered and analyzed using other tools like Auto-dock Vina and Gromacs. However, the best scoring designed protein based on Rosetta scores is a tie between four triple mutations, which are"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ol",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"Y204F C195D S190T"}),Object(Le.jsx)("li",{children:"Y204F C195E S190T"}),Object(Le.jsx)("li",{children:"Y204F C195F S190T"}),Object(Le.jsx)("li",{children:"Y204F C195Y S190T"})]})}),Object(Le.jsx)("p",{children:"They all have a score of -368.553 REU."}),Object(Le.jsxs)("p",{children:["Top 15 1UTM Multi-Mutations are listed in the table below. Full tables of modified 1UTM scores are also uploaded ",Object(Le.jsx)("a",{href:"https://docs.google.com/spreadsheets/u/1/d/1r_Wf-dGtYojBKM7i1tRiwdZFUIioxGey/edit",target:"_blank",children:"here"}),"."]})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)(Zt,{bordered:!0,className:pe.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:"Score (REU)"}),Object(Le.jsx)("th",{children:"Mutation 1"}),Object(Le.jsx)("th",{children:"Mutation 2"}),Object(Le.jsx)("th",{children:"Mutation 3"}),Object(Le.jsx)("th",{children:"Mutation 4"})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-368.553"}),Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"-"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-368.553"}),Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"-"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-368.553"}),Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"-"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-368.553"}),Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195Y"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"-"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.779"}),Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"C171D"}),Object(Le.jsx)("td",{children:"Y204H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.779"}),Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"C171E"}),Object(Le.jsx)("td",{children:"Y204H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.779"}),Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"C171F"}),Object(Le.jsx)("td",{children:"Y204H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.779"}),Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195Y"}),Object(Le.jsx)("td",{children:"C171Y"}),Object(Le.jsx)("td",{children:"Y204H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.616"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"C171E"}),Object(Le.jsx)("td",{children:"Y204H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.616"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"C171F"}),Object(Le.jsx)("td",{children:"Y204H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.616"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195Y"}),Object(Le.jsx)("td",{children:"C171Y"}),Object(Le.jsx)("td",{children:"Y204H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.616"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"C171D"}),Object(Le.jsx)("td",{children:"Y204H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.421"}),Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.421"}),Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.421"}),Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-"})]})]})]})}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Improvement of Monomeric Streptavidin"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"Monomeric streptavidin, conjugated to the control chamber, binds to biotin on the binding molecule when it reaches the control chamber. For streptavidin, the same workflow as 1UTM was used to design this protein. First we auto-generated a list of all possible single mutations using Rosetta."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Results: Monomeric Streptavidin Single Mutation"})}),Object(Le.jsxs)("p",{children:["The top 10 lowest scoring proteins are given in the following table. The original score for un-mutated streptavidin with biotin was -104.826 REU. Full top 50 mutations are recorded ",Object(Le.jsx)("a",{href:"https://docs.google.com/spreadsheets/u/1/d/1SgsB72_eUh2hm3cXvlslBUDv0N5JtfM7/edit",target:"_blank",children:"here"}),"."]})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)(Zt,{bordered:!0,className:pe.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:"Score (REU)"}),Object(Le.jsx)("th",{children:"Mutation"})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-104.826"}),Object(Le.jsx)("td",{children:"Original"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-141.239"}),Object(Le.jsx)("td",{children:"Y52F"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-139.972"}),Object(Le.jsx)("td",{children:"L57M"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-139.911"}),Object(Le.jsx)("td",{children:"C41A"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-139.911"}),Object(Le.jsx)("td",{children:"C41D"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-139.911"}),Object(Le.jsx)("td",{children:"C41E"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-139.911"}),Object(Le.jsx)("td",{children:"C41F"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-139.911"}),Object(Le.jsx)("td",{children:"C41G"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-139.911"}),Object(Le.jsx)("td",{children:"C41H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-139.911"}),Object(Le.jsx)("td",{children:"C41I"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-139.911"}),Object(Le.jsx)("td",{children:"C41K"})]})]})]})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsxs)("p",{children:["We also took a closer look at the binding pocket residues for streptavidin which are residues 12, 16, 32, 34, 36, 72, 74 and 92. The mutations that scored higher than the original protein for each binding pocket residue are further analyzed and recorded ",Object(Le.jsx)("a",{href:"https://docs.google.com/spreadsheets/u/1/d/1SgsB72_eUh2hm3cXvlslBUDv0N5JtfM7/edit",target:"_blank",children:Object(Le.jsx)("span",{className:zt.a.link,children:"here"})}),"."]}),Object(Le.jsx)("p",{children:"Then double mutations were chosen based on the Rosetta scores similar to the process used for 1UTM. Those double mutations were passed into Rosetta and a heat map was generated for Streptavidin as well."})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("img",{src:"",className:zt.a.design_img})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"From the heat map, the strongest double mutations were chosen and then combined with other less strong mutations as well as a few of the top 50 single mutations to generate triple and quadruple mutations. This list was also passed into Rosetta to generate the PDB files and scores. The scores for the 12 suggested triple, quadruple, quintuple and sextuple mutations for streptavidin are listed below."})}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)(Zt,{bordered:!0,className:pe.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:"Score (REU)"}),Object(Le.jsx)("th",{children:"Mutation"}),Object(Le.jsx)("th",{}),Object(Le.jsx)("th",{}),Object(Le.jsx)("th",{}),Object(Le.jsx)("th",{})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-167.751"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"Y52F"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-166.862"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"L57M"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-157.12"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"T49K"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-154.14"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"N64S"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-153.232"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"R60T"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-133.52"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"A36C"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-132.144"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"N34A"}),Object(Le.jsx)("td",{children:"A36C"}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-132.051"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"N34A"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-131.989"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"A36C"}),Object(Le.jsx)("td",{children:"Y32A"}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-130.956"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"Y32C"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-129.267"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"N34A"}),Object(Le.jsx)("td",{children:"A36C"}),Object(Le.jsx)("td",{children:"Y32A"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-128.766"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"N34A"}),Object(Le.jsx)("td",{children:"Y32A"}),Object(Le.jsx)("td",{})]})]})]})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"Based on Rosetta scores, the best designed protein consisted of the 3 mutations, T74C, N12A, Y52F."})})]}),Object(Le.jsxs)(Ie,{open:!0,title:"Test",children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Docking Analysis with AutoDock Vina and Chimera"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"To test that the modified proteins from Rosetta protocols do have a higher binding energy with the ligand, docking analysis and binding energies are calculated for the top mutated proteins produced in the Build section. Autodock Vina is a program for performing molecular docking simulations using input receptor-ligand pairs. Performing ligand docking by AutoDock Vina results in the generation of multiple PDBQT files containing the coordinates in space for the docked protein complex. These PDBQT files can further be loaded into the previously mentioned UCSF Chimera for verification of binding in the desired pocket. Compared to its original counterpart, AutoDock Vina is 59% more accurate than AutoDock 4 in yielding binding mode predictions with an RMSD less than 2. Similarly, AutoDock Vina is nearly 450, and 65 times faster than AutoDock 4, running on an 8-, and 1- core CPU, respectively. Conveniently, the functionalities of AutoDock Vina are integrated into the Chimera GUI and can also be run using Chimera Scripts, so long as you have downloaded the AutoDock Vina executable on your local device. Previously, docking simulations were run manually using this functionality in Chimera by selecting a volumetric box for the binding pocket, setting the appropriate binding mode parameters, and running the docking one protein at a time. To circumvent the issue of long process times using this manual method, an automated method using Chimera\u2019s support for Python scripts was implemented."}),Object(Le.jsx)("p",{children:"The Python script serves several purposes including, opening the mutated protein output from Rosetta, deleting the ligand from the complex, adding the native ligand file to the environment, and performing ligand docking, saving only the docking coordinate of the highest scoring conformer. These scores were then copied and written to a master energy score text file automatically and saved. Both singly- and doubly- mutated proteins generated by Rosetta were used for docking. The doubly mutated proteins combined mutations from both the binding pocket and a non-binding region in efforts to decrease the energy score of the overall structure for enhanced stability. Implementation of this process revealed that singly mutated trypsin-PEA complexed proteins outperformed doubly-mutated trypsin-PEA protein complexes on average. To illustrate this, the following distributions were observed for singly- vs doubly- mutated trypsin proteins. As you can see, the maximum energy score for doubly mutated proteins was -4.8 kcal/mole, while an energy score of -4.9 kcal/mole was frequently seen in singly-mutated proteins. From these preliminary energy score results we can deduce that double mutations on the complex did not have any effect on decreasing its energy score, and thus stability was not affected. To further confirm this a secondary energy analysis should be performed using the score_jd2 protocol in Rosetta or structure scoring in GROMACS."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("div",{className:pe.a.figure_text,children:"1UTM-PEA Docking Scores"}),Object(Le.jsxs)(Zt,{bordered:!0,className:pe.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:"Mutation 1"}),Object(Le.jsx)("th",{children:"Mutation 2"}),Object(Le.jsx)("th",{children:"Mutation 3"}),Object(Le.jsx)("th",{children:"Mutation 4"}),Object(Le.jsx)("th",{children:"Interaction Energy (kcal/mole)"})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Native"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-4.6"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"C171E"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"C171F"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"S190"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-4.7"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-4.7"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195Y"}),Object(Le.jsx)("td",{children:"C171Y"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-4.9"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"C171D"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195Y"}),Object(Le.jsx)("td",{children:"C171Y"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195Y"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"C171D"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"C171E"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"C171F"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-4.8"})]})]})]})]}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"A similar energy conformer analysis using the above-mentioned automated approach using a Python script was implemented for mutation candidates generated by Rosetta on the monomeric streptavidin-biotin complex. The lowest scoring multi-mutant streptavidin proteins yielded an energy score of -7.4 kcal/mole."})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Streptavidin-Biotin Docking Scores"}),Object(Le.jsxs)(Zt,{bordered:!0,className:pe.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:"Mutation 1"}),Object(Le.jsx)("th",{children:"Mutation 2"}),Object(Le.jsx)("th",{children:"Mutation 3"}),Object(Le.jsx)("th",{children:"Mutation 4"}),Object(Le.jsx)("th",{children:"Mutation 5"}),Object(Le.jsx)("th",{children:"Interaction Energy (kcal/mole)"})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"L57M"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-5.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"T49K"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-5.9"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"Y52F"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-7.4"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"A36C"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-7.1"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"R60T"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-5.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"N64S"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-5.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Native"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-5.6"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"A-N12A"}),Object(Le.jsx)("td",{children:"N34A"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-7.1"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"A-N12A"}),Object(Le.jsx)("td",{children:"A-N34A"}),Object(Le.jsx)("td",{children:"A36C"}),Object(Le.jsx)("td",{children:"Y32A"}),Object(Le.jsx)("td",{children:"-5.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"A-N12A"}),Object(Le.jsx)("td",{children:"A-Y32C"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-7.2"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"A-N12A"}),Object(Le.jsx)("td",{children:"A-N34A"}),Object(Le.jsx)("td",{children:"A-Y32A"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-5.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"A-N12A"}),Object(Le.jsx)("td",{children:"A-A36C"}),Object(Le.jsx)("td",{children:"A-Y32A"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-5.7"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"A-N12A"}),Object(Le.jsx)("td",{children:"A-N34A"}),Object(Le.jsx)("td",{children:"A-A36C"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-7.4"})]})]})]})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Molecular Dynamics Analysis with GROMACS"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"We used molecular dynamics simulations to test the relative binding affinities of the top 15 1UTM multi-mutation proteins with PEA. We did this by using GROMACS to find the total interaction energy between each protein and PEA and comparing the relative magnitudes of these interaction energies to compare binding strength. The GROMACS simulations were performed on the Graham computing cluster hosted by ComputeCanada."}),Object(Le.jsxs)("p",{children:["To complete the GROMACS simulations, first, we prepared the protein and ligand topologies using the CHARMM36 force field and TIP3P water model (CHARMM force field files, MacKerell Lab). Then, we completed a solvation process to place the system in solution, and then added Na",Object(Le.jsx)("sup",{children:"+"})," ions to the solution to balance out the net charge of the system. Next, we carried out an energy minimization step, to relax the system to the closest local energy minimum and avoid extreme behaviour that could occur due to collisions between particles. The next step was using standard NVT and NPT equilibration procedures in GROMACS to equilibrate the system to the desired temperature and pressure. Finally, we ran the production MD simulation. When this step was completed, we performed analysis to collect the total interaction energy for each pair of protein and PEA. We used the magnitudes of these interaction energies as a way to compare the relative strength of the interaction between PEA and each protein (Lemkul, 2018)."]}),Object(Le.jsx)("p",{children:"Here are the total interaction energies we collected for the original 1UTM and the top 15 multiple mutation proteins:"})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)(Zt,{bordered:!0,className:pe.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:"Mutation 1"}),Object(Le.jsx)("th",{children:"Mutation 2"}),Object(Le.jsx)("th",{children:"Mutation 3"}),Object(Le.jsx)("th",{children:"Mutation 4"}),Object(Le.jsx)("th",{children:"Total Interaction Energy (kJ/mol)"})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-108.7289"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-125.7208"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-94.3676"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-96.4218"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195Y"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-89.3934"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"C171D"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-106.7369"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"C171E"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-93.5768"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"C171F"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-111.2318"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195Y"}),Object(Le.jsx)("td",{children:"C171Y"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-118.3681"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"C171E"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-120.5737"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"C171F"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-105.4843"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195Y"}),Object(Le.jsx)("td",{children:"C171Y"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-112.5343"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"C171D"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-115.1761"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-119.752"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-94.7425"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-126.5064"})]})]})]})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"From the table, we can see that eight out of fifteen of our proposed mutations had higher total interaction energies than the original 1UTM, indicating that these proteins have a higher affinity to PEA than the original. The candidate S190A C195F Y204H had the total interaction energy with the greatest magnitude (126.5064 kJ/mol), which corresponds to it having the highest relative binding affinity to PEA. It was closely followed by the candidate Y204F C195D S190T with a total interaction energy of magnitude 125.7208 kJ/mol. This candidate also tied for the best score based on the Rosetta analysis and the second best based on AutoDock analysis, so based on all three analyses, it is likely a good option for our NeuroDetech chip."}),Object(Le.jsx)("p",{children:"The final MD simulation in GROMACS was run for 10 hours per protein candidate. In the future, we would like to run this simulation for multiple days to produce more accurate results. Additionally, we would like to test the candidate proteins in the lab to confirm these molecular dynamics results before building our chip."})]})]}),Object(Le.jsxs)(Ie,{open:!0,title:"Learn",children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Improved Proteins"})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsxs)("p",{children:["Based on Rosetta scores and AutoDock scores, the improved protein for streptavidin consisted of the 3 mutations, T74C, N12A, Y52F. This improved protein constitutes the new BioBrick Part ",Object(Le.jsx)("a",{href:"http://parts.igem.org/Part:BBa_K3843005",children:"BBa_K3843005"}),". The 3D structure of the improved streptavidin, visualized using UCSF Chimera, is as follows, where monomeric streptavidin is in blue, and biotin is in green."]})}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("img",{src:"",className:zt.a.design_img})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsxs)("p",{children:["The improved protein for 1UTM based on Rosetta scores, AutoDock scores, and GROMACS analysis also contains 3 mutations, which are Y204F, C195D, S190T. This improved protein constitutes the new BioBrick Part ",Object(Le.jsx)("a",{href:"http://parts.igem.org/Part:BBa_K3843001",children:"BBa_K3843001"}),". The 3D structure of the improved 1UTM, visualized using UCSF Chimera, is as follows, where 1UTM is in orange, and PEA is in green."]})}),Object(Le.jsx)("div",{className:pe.a.figure_div,style:{marginBottom:20},children:Object(Le.jsx)("img",{src:"",className:zt.a.design_img})}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Discussion"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"One advantage of the rational design approach is that it can be specifically focused on optimizing the binding affinity of the protein. While Rosetta scores allow one to filter out candidates that are unacceptable based on overall molecular stability, the scores do not serve as proof that the binding itself has been improved."}),Object(Le.jsx)("p",{children:"However, the trial and error nature of the process means that rational design is more time-consuming than using Rosetta to generate all possible mutations for individual residues. Moreover, the basic process of rational protein design does not account for everything. Repulsive forces, for instance, may not be observed until the mutated protein is either docked or tested for stability in Rosetta."}),Object(Le.jsx)("p",{children:"The energy scores from AutoDock Vina revealed that the most stable mutant protein for Streptavidin was observed when Threonine was mutated to Cysteine on residue 74, coupled with Asparagine to Alanine on residue 12, and Tyrosine to Phenylalanine on residue 52. Similarly, mutations from Threonine to Cysteine on residue 74, coupled with Asparagine to Alanine on residue 12, Asparagine to Alanine on residue 34, and Alanine to Cysteine on residue 36 yielded the same energy score of -7.4 kcal/mole. Meanwhile, the lowest scoring mutations for trypsin occurred when the Cysteine on residue 171 was switched to either Phenylalanine, Glutamine, Glutamic Acid, Arginine, Serine, Aspartic Acid, Tryptophan, Alanine, Valine, Threonine, Asparagine, Leucine, Methionine, or Isoleucine. This may suggest that residue 171 in its native structure as a Cysteine is very unstable, as all these mutations on residue 171 led to an energy score of -4.9 kcal/mole, 0.3 kcal/mole lower than the wild type."}),Object(Le.jsx)("p",{children:"AutoDock Vina provides a quick and facile method for automating the docking process for a specified protein-ligand pair. Compared to the manual method, using an automated method reduces time by over 50-fold. This method is able to both write new summary files and increase accessibility for future analysis through multiple levels of organization. With this in mind, this method is prone to error in cases where the docking occurs in an undesired binding pocket. However, through careful design and selection of a protein, to target high selectivity and specificity, this can be overlooked. For this reason, AutoDock Vina presents itself as a suitable software for protein-ligand docking simulations."}),Object(Le.jsx)("p",{children:"Compared to using the AutoDock vina executable in a command line, the integration within Chimera is able to perform all the same functionalities. This includes defining volumetric boxes for docking searches, selection of a number of binding modes to generate, hydration, and structure editing prior to docking. The flexibility in selection of operating parameters provided by both the native and integrated AutoDock Vina within Chimera was advantageous for our application of ligand docking both for Trypsin and Streptavidin, enabling us to allocate our time to complete higher priority tasks."})]})]})]})}}]),i}(n.Component),Xt=i(58),ei=i.n(Xt),ti=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsx)("div",{className:pe.a.text_div,style:{marginBottom:40},children:Object(Le.jsx)("span",{className:pe.a.text_heading,style:{paddingBottom:0},children:"Gene Marker Detection: Design of mRNA-Binding CRISPR-Cas System"})}),Object(Le.jsxs)(Ie,{open:!0,title:"Design",children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Rationale for Switching to Detection of Genetic Markers"})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsxs)("p",{children:["Attention deficit hyperactivity disorder (ADHD) is a common yet complex neurological disorder, and while PEA was (serendipitously) a suitable biomarker for a diagnostic application, continuing on the path of biomarkers was not feasible. First of all, there is much conflicting and limited recent evidence of urinary biomarker variances within ADHD patients, and furthermore, genetic markers are generally better candidates for diagnostics given the genetic nature of ADHD (Faraone et al., 2014). Firstly, biomarkers such as 5-HIAA, MHPG, NPY, DBH, and HVA are indeed relevant to ADHD (Faraone et al., 2014), but conflicting evidence and limited case studies makes it difficult to use for diagnostics. As an example, a paper from ",Object(Le.jsx)("em",{children:"Biology Psychiatry"})," observed a decrease in urinary MHPG in patients with ADHD, but a more recent paper from ",Object(Le.jsx)("em",{children:"Molecular Chemistry"})," ",Object(Le.jsx)("em",{children:"Neuropathology"})," observed no such variances (Shekim et al., 1983 & Baker et al., 1993). Due to much conflicting evidence, diagnostics using biomarkers became a recurring issue. Secondly, genetic markers are strong candidates for detecting ADHD (Faraone et al., 2014). Genes of the dopaminergic and serotonergic systems (particularly ",Object(Le.jsx)("em",{children:"DAT1, DRD4, DBH, SNAP25, CHRNA4"}),", and ",Object(Le.jsx)("em",{children:"TPH-2"}),") make for good candidates, and there is recent evidence linking their genetic behaviour to ADHD (Zhang et al., 2012). Alongside this, CRISPR-Cas13 methods of quantifying RNA, particularly the SATORI method, makes diagnostics possible without the need for prior amplification (Shinoda et al., 2021). With the literature pointing us away from biomarkers and towards genetic markers, we decided to adapt our microfluidic assay for the quantification of genetic markers - specifically, the detection of ADHD-associated mutations present in mRNA."]})}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Review of RNA Stability in a Diagnostic Context"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"When initially considering an mRNA detection system, we were quite worried about the stability of mRNA - would it be even possible to quantify mRNA in urine? To address this concern, we looked into the literature to determine the following:"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"What RNases are present in urine that would need to be inactivated for our system to work?"}),Object(Le.jsx)("li",{children:"Do any successful urinary mRNA quantification methods exist in literature?"})]})}),Object(Le.jsx)("p",{children:"In this section, we will review our findings from the literature, and also discuss the experience of one of our lab members with mRNA quantification using a lateral flow assay."}),Object(Le.jsx)("p",{children:"Ribonucleases (RNases) belong to a large class of enzymes, ubiquitous across both bacterial and eukaryotic organisms (Eun, 1996). Found in nearly all mammalian tissues and fluids, they are critical to a number of processes such as digestion, RNA metabolism, RNA processing and serve an integral role in protection against infection (Eun, 1996; Gotte & Menegazzi, 2019). Most human secretary RNases serve vital to anti-inflammatory, antibacterial and antiviral actions, catalysing the degradation of foreign intracellular RNA, DNA:RNA hybrids and dsRNA as indicators of infection (Gotte & Menegazzi, 2019). For this reason, an initial concern of using urinary RNA as a source to detect ADHD-associated mutations was the risk of RNase activity compromising RNA integrity (Becknell et al., 2019)."}),Object(Le.jsx)("p",{children:"The functionality of our device in the detection of ADHD-associated mutations is contingent on RNA being stable in a biological sample from the point of collection to the point at which the sample reaches the test chamber. While literature on use of urine as a source of RNA suggests that proper sample storage conditions circumvent these concerns, we did not have the opportunity to test this given our lack of lab access. One of our lab members, however, has had extensive experience working with RNA in the context of COVID-19 diagnostics from saliva."}),Object(Le.jsx)("p",{children:"The company at which she worked, aimed to detect SARS-CoV2 RNA from saliva samples in an analogous lateral-flow assay microfluidic chip, while also incorporating detection of human RNA as a control for RNA stability. The results that our lab member collected highly supported the use of biological samples as a source of RNA, despite the risk of RNase activity potentially compromising the RNA quality. The use of RNase inhibitors during sample collection was sufficient in protecting the RNA, providing a reliable signal for the human RNA control at the test point. The significance of proper sample storage was further tested in negative controls lacking the RNase inhibitor. Absence of the inhibitor yielded consistently negative results in detecting the human RNA control gene, demonstrating that the RNase inhibitor was necessary in stabilizing RNA in saliva. Similar efforts of SARS-CoV2 RNA detection in saliva using the same methods of detection have been reported with success since then (Aoki et al., 2021; Lalli et al., 2021)."}),Object(Le.jsxs)("p",{children:["In addition to the personal experience of our lab member, numerous literature sources suggest that despite the general instability of RNA, absolute and relative methods of mRNA quantification in urine are viable, given careful handling and storage of RNA samples as well as proper inactivation of RNases. This literature is summarized below: ",Object(Le.jsx)("br",{}),"Seo et al. (2017) successfully performed RT-qPCR to quantify the mRNA of three genes associated with kidney transplant rejection. To ensure stability of the RNA, they added their urine samples to RNAlater, an RNA inhibitor cocktail, and stored these samples at -80\u02daC (Seo et al., 2017). Sample-to-sample variation in measured mRNA concentrations could be circumvented by normalizing the absolute quantity of mRNA to the quantity of a constitutively expressed housekeeping gene (Seo et al., 2017). Note that this is a common practice for mRNA quantification in general, not just in urine samples."]}),Object(Le.jsx)("p",{children:"Bradley et al. (2019) were also successful in extracting and quantifying mRNA by RT-qPCR across multiple RNA extraction kits. Under clinical settings, urine samples cannot always be processed immediately or stored under -80\xb0C conditions, making RNA degradation a concern for clinical processing. To test the efficacy of RNA extraction under clinical settings, the RNA samples were processed using centrifugation, filtration, and aliquoting methods under clinical settings (Bradley et al., 2019). With the use of RNAlater, the subsequent RNA extractions were successful in isolating RNA for quantification by RT-qPCR (Bradley et al., 2019)."}),Object(Le.jsx)("p",{children:"Overall, from the personal experience of our lab member, replicated results in the field, and literature supporting the viability of RNA quantification in urine, we found it safe to state that detection of RNA in urine would be achievable for our purposes in a microfluidic assay."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Overview of CRISPR-Cas Design"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"Initially, to allow for generation of a quantifiable signal, we considered constructing a Cas-HRP fusion protein, similarly to the PEA-binding fusion protein. However, we realized that a simpler method that is commonly used in literature is to utilize the inherent RNase activity of CRISPR-Cas systems to cleave a fluorophore and quencher, thereby producing fluorescence. In effect, the design of the microfluidic assay for gene marker detection remains very similar to that for PEA detection; the only changes include the following:"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"Instead of the chemiluminescent substrate, a fluorophore and quencher would be used, linked by ssRNA."}),Object(Le.jsx)("li",{children:"Instead of a fusion protein, a simple CRISPR-Cas complex would be used. The Cas protein should be biotinylated to allow for a signal to be produced at the control chamber."}),Object(Le.jsx)("li",{children:"At the test chamber, the target mRNA should be conjugated (instead of PEA)."}),Object(Le.jsx)("li",{children:"At the control chamber, in addition to conjugated streptavidin, a known sample of the target mRNA (not conjugated) should also be lyophilized in order to activate the RNase activity of any CRISPR-Cas that binds to the control chamber."})]})}),Object(Le.jsx)("p",{children:"This design is able to detect specific ADHD-associated mutations present in the target mRNA. If the target mRNA (containing the mutation) in the urine is detected and bound by the CRISPR-Cas complex, then less CRISPR-Cas complexes with \u201copen binding sites\u201d are available to bind to the mRNA conjugated to the test chamber, resulting in a lower signal. Overall, if the signal is a certain threshold level lower than the signal for an individual without ADHD, then the person is deemed to have the mutation."}),Object(Le.jsx)("p",{children:"With the above in mind, the design of the microfluidic assay for the detection of gene markers would be as follows:"})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,style:{marginBottom:30},children:[Object(Le.jsx)("img",{src:"",alt:"MCFA overview CRISPR",className:ei.a.design_img,style:{borderRadius:20}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Diagram of the microfluidic assay designed to detect gene markers (ADHD-associated mRNA mutations)."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"The Big Picture"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"With the above design considerations in mind, the outstanding questions to be answered for the design of an mRNA-quantifying CRISPR-Cas system include the following:"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"What specific genes (and by extension, their ADHD-associated mutations) should be detected?"}),Object(Le.jsx)("li",{children:"What Cas protein should be used? Would we need to adapt the Cas protein to our system in any way?"}),Object(Le.jsx)("li",{children:"How should we design the CRISPR guide RNA sequences?"})]})}),Object(Le.jsx)("p",{children:"In the following sections, our considerations and answers to these \u201cbig picture questions\u201d will be discussed."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Selection of ADHD-associated genes to be quantified by mRNA transcript detection"})}),Object(Le.jsx)("p",{children:"ADHD is often comorbid with other psychological disorders, such as schizophrenia, depression, anxiety, and bipolar disorder, to name a few. In a similar sense, biomarkers and gene markers associated with ADHD can often be associated with other psychological disorders as well. In order to reduce the likelihood that our microfluidic assay system produces false positive results associated with other psychological disorders, we aimed to detect multiple ADHD-associated gene markers. In selecting the most viable gene candidates, we conducted literature research into the following ADHD-associated genes. We favoured genes whose ADHD-associated mutations would also be present in their corresponding mRNA transcripts; that is, mutations in the promoter region or introns were ruled out. We also favoured genes whose ADHD-associated mutations were well-characterized and had known sequences. As well, variable number of tandem repeat mutations (VNTRs) were ruled out, as these would not be easily detected by hybridization."}),Object(Le.jsx)("p",{children:"In searching the literature for suitable gene markers, we considered the following genes:"}),Object(Le.jsxs)("p",{children:[Object(Le.jsx)("em",{children:"DBH"})," codes for dopamine beta-hydroxylase, which catalyzes the conversion of dopamine into norepinephrine. However, the major ADHD-associated mutations for DBH occur within introns, making it impossible to detect these mutations in the corresponding mRNA transcript (Roman et al., 2002)."]}),Object(Le.jsxs)("p",{children:[Object(Le.jsx)("em",{children:"MAOA"})," codes for monoamine oxidase A, a mitochondrial enzyme which degrades monoamines such as serotonin, norepinephrine and dopamine (known ADHD-associated biomarkers). However, the major ADHD-associated mutations for MAOA occurred within the promoter region or introns, making it impossible to detect these mutations in the corresponding mRNA transcript. As well, differential expression of MAOA between males and females made it unfavourable for universal use (Rommelse et al., 2008)."]}),Object(Le.jsxs)("p",{children:[Object(Le.jsx)("em",{children:"GRIN2A"})," codes for a subunit of the NMDA receptor complex, which is implicated in memory and learning. However, it is not certain whether there truly is an association between ADHD and GRIN2A; thus it was ruled out (Adams et al., 2004)."]}),Object(Le.jsxs)("p",{children:[Object(Le.jsx)("em",{children:"DRD4"})," codes for a major dopamine receptor. A predictable 7-repeat VNTR is associated with ADHD (this is different from VNTRs where the number of repeats is not predictable). Notably, 7-repeat VNTR alleles containing missense mutations led to an observed three-fold increase in likelihood of hyperactivity (Tovo-Rodrigues et al., 2013). This makes sense, as a lack of DRD4 functionality in the DRD4 dopamine receptor would lead to an inability to bind dopamine; with dopamine being an ADHD biomarker, the association between ADHD and the DRD4 missense mutations makes sense (Tovo-Rodrigues et al., 2013). Thus, targeting these missense mutations within the 7-repeat region could be a viable strategy. Specifically, the following missense mutations are viable targets:"]}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"A single base deletion of cytosine in VNTR motif 3"}),Object(Le.jsx)("li",{children:"A single base deletion of cytosine in VNTR motif 1"})]})}),Object(Le.jsxs)("p",{children:[Object(Le.jsx)("em",{children:"HTR1B"})," codes for 5-hydroxytryptamine receptor 1B, and polymorphisms in HTR1B have been implicated in ADHD; however, it is only implicated in some ADHD subtypes and thus is not a good universal indicator of ADHD (Ickowicz et al., 2006)."]}),Object(Le.jsxs)("p",{children:[Object(Le.jsx)("em",{children:"TPH-2"})," codes for tryptophan hydroxylase, which is involved in the synthesis of serotonin, an ADHD-associated biomarker. Multiple SNPs in TPH-2 are statistically significant with regard to ADHD association, including the ones listed below. However, these SNPs are intronic, making them unsuitable for detection in mRNA."]}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"rs1843809-T allele"}),Object(Le.jsx)("li",{children:"rs1386497-A allele"}),Object(Le.jsx)("li",{children:"rs1386493-C allele"})]})}),Object(Le.jsxs)("p",{children:[Object(Le.jsx)("em",{children:"CHRNA4"})," codes for a nicotinic acetylcholine receptor, where the following SNP mutations were found to be ADHD-associated (Lee et al., 2007). However, these SNPs are intronic, making them unsuitable for detection in mRNA."]}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"rs2273505-G allele"}),Object(Le.jsx)("li",{children:"rs3787141-T allele"})]})}),Object(Le.jsxs)("p",{children:[Object(Le.jsx)("em",{children:"SNAP25"})," codes for a presynaptic membrane protein involved in the regulation of neurotransmitter release. The SNP T1065T>G is known to be ADHD-associated (Faraone & Mick, 2010). This SNP occurs in the 3\u2032 untranslated region and is thus present and detectable in mRNA."]}),Object(Le.jsx)("p",{children:"Of the above mutations, the ADHD-associated genes that we chose to detect using our microfluidic assay included the following:"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:Object(Le.jsx)("em",{children:"DRD4"})}),Object(Le.jsx)("li",{children:Object(Le.jsx)("em",{children:"SNAP25"})})]})}),Object(Le.jsx)("p",{children:"To detect the ADHD-associated genes selected above, we decided to utilize a CRISPR-Cas-based system. CRISPR-Cas would allow for sensitive detection of mRNA, with enough specificity to distinguish single nucleotide polymorphisms (SNPs) (Shinoda et al., 2021). As a result, mutations associated with ADHD could be quantified, even SNP mutations. The following describes the design considerations of employing a CRISPR-based system in a microfluidic assay."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Selection of Cas protein"})}),Object(Le.jsx)("p",{children:"CRISPR Cas systems involve a Cas protein and a CRISPR guide RNA (gRNA); the Cas protein exhibits endonuclease activity when bound to its guide RNA. In selecting the appropriate Cas protein for detection of urinary mRNA, it is important to consider the distinctions between the Cas proteins - specifically, their targets:"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"CRISPR-Cas9 cleaves double-stranded DNA (dsDNA), creating blunt ends. It cleaves a single, specific target (Koonin & Makarova, 2019)."}),Object(Le.jsx)("li",{children:"CRISPR-Cas12 cleaves double-stranded DNA (dsDNA), creating sticky (staggered) ends. It cleaves and remains bound to its target. When bound, it also exhibits nonspecific nuclease activity, indiscriminately cleaving DNA other than the target (Koonin & Makarova, 2019)."}),Object(Le.jsx)("li",{children:"CRISPR-Cas13 cleaves single-stranded RNA (ssRNA). Similarly to Cas12, it cleaves its target and remains bound, and will also exhibit nonspecific nuclease activity, indiscriminately cleaving ssRNA other than the target (Koonin & Makarova, 2019)."})]})}),Object(Le.jsx)("p",{children:"Due to the ability of CRISPR-Cas13 to target ssRNA, such as mRNA transcripts, we chose to utilize a Cas13-based system."}),Object(Le.jsxs)("p",{children:["There are several relatively well-characterized Cas13 proteins that originate from different species of the ",Object(Le.jsx)("em",{children:"Leptotrichia"})," genus, such as Cas13a from ",Object(Le.jsx)("em",{children:"L. wadei, L. buccalis"}),", and ",Object(Le.jsx)("em",{children:"L. shahii"}),". Of these Cas13a variants, ",Object(Le.jsx)("em",{children:"L. shahii"})," was the earliest discovered; however, diagnostics tools have favoured ",Object(Le.jsx)("em",{children:"L. wadei"})," and ",Object(Le.jsx)("em",{children:"L. buccalis"})," for their increased analyte sensitivity. In fact, Fozouni et al. (2020) utilized Cas13a from ",Object(Le.jsx)("em",{children:"L. buccalis"})," to detect SAR-CoV-2 RNA, favouring the ",Object(Le.jsx)("em",{children:"L. buccalis"})," variant as it had the highest sensitivity compared to the other Cas13a variants (Fozouni et al., 2020). Inspired by this, we decided to adapt Cas13a from ",Object(Le.jsx)("em",{children:"L. buccalis"})," (henceforth referred to as LbuCas13a) for the detection of ADHD-associated mRNA transcripts in our microfluidic assay."]}),Object(Le.jsxs)("p",{children:["Overall, Cas13a from ",Object(Le.jsx)("em",{children:"L. buccalis"})," was chosen for the detection of ADHD-associated mRNA."]}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Adaptation of LbuCas13a for use in a microfluidic assay"})}),Object(Le.jsx)("p",{children:"Recall that the microfluidic assay consists of a test chamber and control chamber. The analyte of interest (in this case, the target ADHD-associated mutant mRNA transcript) is covalently conjugated to the test chamber. This would allow LbuCas13a to bind to these conjugated target mRNA transcripts, thereby activating LbuCas13a\u2019s nonspecific RNAse activity."}),Object(Le.jsx)("p",{children:"To utilize this nonspecific RNAse activity to produce fluorescence, we decided to use a fluorophore/quencher system, connected via an RNA linker. When the fluorophore is in close proximity to the quencher, the fluorophore is \u201cquenched\u201d by the quencher and does not produce fluorescence. However, when the fluorophore is separated from the quencher, the fluorophore produces fluorescence. The nonspecific RNAse activity of LbuCas13 (when bound to the target mRNA transcript) cleaves any surrounding RNA, including the RNA linker between the fluorophore and quencher. Ultimately, this allows for the production of fluorescence upon detection of the target mRNA, even for small quantities of mRNA."}),Object(Le.jsx)("p",{children:"The control chamber contains covalently conjugated streptavidin. In order to allow Cas13 to bind at the control chamber, LbuCas13a must be biotinylated, which would facilitate a strong biotin-streptavidin interaction at the control chamber. Biotinylation of LbuCas13a would be facilitated in the same manner as biotinylation of 1UTM, the PEA-binding protein."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Design of CRISPR guide RNA sequences"})}),Object(Le.jsxs)("p",{children:["CRISPR guide RNA consists of a stem sequence that forms a hairpin loop and is recognized by the Cas protein. In the case of LbuCas13a, Fozouni et al. (2020) suggests that the following 30-nucleotide sequence is appropriate as a stem sequence for the CRISPR guide RNA (Fozouni et al., 2020): ",Object(Le.jsx)("br",{}),"5\u2032-GACCACCCCAAAAAUGAAGGGGACUAAAAC-3\u2032"]}),Object(Le.jsx)("p",{children:"This stem sequence would be added to the 5\u2032 end of a recognition sequence. This recognition sequence would be complementary to the target mRNA of interest, allowing for target mRNA binding by the CRISPR-Cas13 system."}),Object(Le.jsx)("p",{children:"Recall that the ADHD-associated genes whose mRNA transcripts would be detected include the following:"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:Object(Le.jsx)("em",{children:"DRD4"})}),Object(Le.jsx)("li",{children:Object(Le.jsx)("em",{children:"SNAP25"})})]})}),Object(Le.jsx)("p",{children:"The sequences of the ADHD-associated mutations for each gene were obtained from the papers describing the association of each gene with ADHD (the papers referenced can be seen with the guide RNA sequences below). These sequences were translated to RNA sequences by replacing all thymine residues with uracil, and converted to the complementary sequence for use as a CRISPR recognition sequence. Then, the stem sequence was appended to the 5\u2032 end of each recognition sequence. Listed below are the guide RNA sequences for each ADHD-associated gene:"}),Object(Le.jsxs)("p",{children:[Object(Le.jsx)("em",{children:"DRD4"})," missense deletion in VNTR motif 3 (recognition sequence obtained from Tovo-Rodrigues et al., 2013): ",Object(Le.jsx)("br",{}),"5\u2032-GACCACCCCAAAAAUGAAGGGGACUAAAACGGCGCGGGCCGGAGGGGGGCCUGGGGACGCCGGG-3\u2032 ",Object(Le.jsx)("br",{}),"This sequence was used to create a new BioBrick Part: ",Object(Le.jsx)("a",{href:"http://parts.igem.org/Part:BBa_K3843005",target:"_blank",children:"BBa_K3843006"})]}),Object(Le.jsxs)("p",{children:[Object(Le.jsx)("em",{children:"DRD4"})," missense deletion in VNTR motif 1 (recognition sequence obtained from Tovo-Rodrigues et al., 2013): ",Object(Le.jsx)("br",{}),"5\u2032-GACCACCCCAAAAAUGAAGGGGACUAAAACGCGCGGGGCGGAGGGGGUCCUGGGGACGCCGGGG-3\u2032 ",Object(Le.jsx)("br",{}),"This sequence was used to create a new BioBrick Part: ",Object(Le.jsx)("a",{href:"http://parts.igem.org/Part:BBa_K3843005",target:"_blank",children:"BBa_K3843008"})]}),Object(Le.jsxs)("p",{children:[Object(Le.jsx)("em",{children:"SNAP25"})," SNP T1065T>G (recognition sequence obtained from Barr et al., 2000): ",Object(Le.jsx)("br",{}),"5\u2032-GACCACCCCAAAAAUGAAGGGGACUAAAACCACCGAGAUUGAGGAACUCCAGAACUCAAAGUAA-3\u2032 ",Object(Le.jsx)("br",{}),"This sequence was used to create a new BioBrick Part: ",Object(Le.jsx)("a",{href:"http://parts.igem.org/Part:BBa_K3843005",target:"_blank",children:"BBa_K3843007"})]})]})]}),Object(Le.jsxs)(Ie,{open:!0,title:"Build",children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Vector and Chassis Selection"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsxs)("p",{children:["The Zhang Lab, based in Massachusetts, USA, is highly experienced with the use of CRISPR-Cas13a; they have developed an excellent ",Object(Le.jsx)("a",{href:"https://zlab.bio/cas13",target:"_blank",children:"resource page"})," for CRISPR-Cas13 work."]}),Object(Le.jsxs)("p",{children:["In this CRISPR-Cas13 guide, the Zhang Lab provides a pET-based plasmid vector for the expression of Cas13a from ",Object(Le.jsx)("em",{children:"Leptotrichia wadei"}),". In our case, for the expression of Cas13a from ",Object(Le.jsx)("em",{children:"Leptotrichia buccalis"}),", we would swap the insert with the sequence for LbuCas13a. The vector contains a 6-His tag at the N-terminus, suitable for affinity chromatography using an Ni-NTA resin column (Abudayyeh & Gootenberg, n.d.)."]}),Object(Le.jsxs)("p",{children:["The Zhang Lab suggests the use of ",Object(Le.jsx)("em",{children:"E. coli Rosetta 2(DE3)pLysS Singles"})," for Cas13a expression. With that said, this strain is a BL21 derivative for expression of human proteins (though bacterial expression is reportedly fine using the plasmid above). For bacterial expression, simply using ",Object(Le.jsx)("em",{children:"E. coli BL21"})," would likely be sufficient (Abudayyeh & Gootenberg, n.d.)."]}),Object(Le.jsx)("p",{children:"The CRISPR guide RNA sequences would be ordered from a synthesis company, such as IDT, as the Zhang Lab reported better performance using synthesized guide RNAs (Abudayyeh & Gootenberg, n.d.)."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Cas13a Purification"})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"Since the Zhang Lab\u2019s plasmid allows for expression of a 6-His tag along with Cas13a, the expressed protein can be purified by affinity chromatography, similarly to the purification of the PEA-binding fusion protein. An Ni-NTA resin would be used to capture the His-tagged Cas13a. Then, elution of Cas13a would occur through the addition of imidazole."})})]}),Object(Le.jsxs)(Ie,{open:!0,title:"Test",children:[Object(Le.jsx)("div",{className:pe.a.description,style:{marginTop:30},children:Object(Le.jsx)("p",{children:"Due to unforeseen circumstances (one of which was a global pandemic, and the other of which was unforeseen maintenance and construction of our lab space by the University), we unfortunately did not have lab access this year. As a result, we were unable to experimentally test the functionality of the CRISPR-Cas13a constructs. With that said, the following methodology outlines our plan of action to test our CRISPR-Cas13a constructs if we had lab access."})}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Experimental Demonstration of CRISPR-Cas13a Success"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Demonstration of CRISPR-Cas13a Stability in Urine-Like Conditions"})}),Object(Le.jsx)("p",{children:"In a solution containing ssRNA-linked fluorophores/quenchers, the nonspecific RNase activity of CRISPR-Cas13 after target ssRNA detection allows for the rapid production of a fluorescent signal. The stability and functionality of CRISPR-Cas13 in urine-like conditions can be tested in a similar way as described for the PEA-binding fusion protein. Once again, the expected conditions of urine include pH ~6, a low protein concentration (that can be simulated using BSA), a low glucose concentration, around 4 mM urea, and around 20 mM NaCl (Roxe, 1990). After preparing a solution in DI water with these conditions and adding CRISPR-Cas13a, the fluorophores/quenchers, and the target mRNA sequence, the simulated urine solution would be expected to display fluorescence of wavelength 535 nm that could be measured using a spectrophotometer (Shinoda et al., 2021). A negative control would involve only the simulated urine."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Experimental Calibration Curve"})}),Object(Le.jsx)("p",{children:"Just like with the PEA-binding fusion protein, it would be useful to obtain an experimental standard curve for the fluorescence produced upon separation of the fluorophores and quenchers by CRISPR-Cas13a cleavage of the linker ssRNA. To do this, a series of dilutions of CRISPR-Cas13a would be made, then a known excess concentration of the ssRNA-linked fluorophores and quenchers would be added to each CRISPR-Cas13a dilution. In measuring the fluorescence produced by each dilution, a standard curve of fluorescence vs. CRISPR-Cas13a concentration can be plotted. A multiplate and multiplate analyzer can be used for this. This would simulate the conditions of fluorescence in the microfluidic assay chip, since in the MCFA chip, the number of CRISPR-Cas13a molecules that bind at the test chamber would be variable, while the amount of ssRNA-linked fluorophores and quenchers that pass through the test chamber would be a known excess amount."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Experimental Calibration Curve"})}),Object(Le.jsx)("p",{children:"Proper identification of ADHD-associated gene markers by NeuroDetech relies on the ability of CRISPR-Cas13a to distinguish between two mRNA sequences differing only by a single nucleotide polymorphism (SNP). Shinoda et al. (2021) has demonstrated this to be possible and reliable; however, as a component of the proof of concept, we would need to test this for our specific mRNA sequences. To do so, mRNA sequences for the \u2018normal\u2019 allele as well as the ADHD-associated mutant allele would need to be obtained, likely by synthesis by a company like IDT. From here, the stability experiment described above should be repeated for the \u2018normal\u2019 and ADHD-associated sequences. It would be expected that the \u2018normal\u2019 mRNA sequences would not be recognized by CRISPR-Cas13a; thus, its nonspecific RNase activity would not be activated, and no fluorescence would be produced. For the ADHD-associated sequences, fluorescence should be expected, since these mutant sequences are the desired targets of the designed CRISPR-Cas13a guide RNAs."})]})]}),Object(Le.jsx)(Ie,{open:!0,title:"Learn",children:Object(Le.jsx)("div",{className:pe.a.description,style:{marginTop:30},children:Object(Le.jsx)("p",{children:"Without lab access (as explained in the Test section), we were not able to perform the experiments outlined in the Test section. However, similarly to the PEA-binding protein engineering cycle, if we were to have performed those experiments, the standard curve experiment relating fluorescence to CRISPR-Cas13a concentration would have been valuable for quantification purposes. Specifically, it would have further informed the development of the optical detector and app (described below in the Optical Detector and Application Development engineering cycle), as the standard curve would allow for more accurate interpretation of the signal at the test chamber under simulated urine conditions, which would be most representative of true urine samples analyzed by NeuroDetech."})})})]})}}]),i}(n.Component),ii=i(3),ni=i.n(ii),ai=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,style:{paddingBottom:0},children:"Optical Detector and Application Development"})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"In this section, the development of the optical detector and desktop application used to quantify the biomarker and visualize the results to users is described in detail. Alternative biomarker quantification assays are discussed and the reason for selecting a microfluidic assay for this project is explained. The hardware and software development process with final prototypes is also presented."})}),Object(Le.jsxs)(Ie,{open:!0,title:"Design",children:[Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Biomarkers and Lateral Flow Assays"}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"A biomarker is an objective indicator of a patient\u2019s medical state. Some examples include pulse, blood pressure, and concentrations of molecules associated with various health conditions. Biomarkers are widely used in basic and clinical research to help predict relevant clinical outcomes (Strimbu & Tavel, 2010). Similarly, our project applies the concept of biomarkers to validate clinical diagnoses. To do this, we need to detect the presence of and quantify specific biomarkers. One of the most prevalent ways to do this is with a lateral flow assay."}),Object(Le.jsx)("p",{children:"Lateral flow assays (LFAs) are tools widely used to detect and quantify analytes in samples. The tester applies a liquid sample to the sample pad located at the end of the LFA strip. It flows to the detection zone, where biological components bind to the target analyte in the sample. After, it flows to the test and control lines. Visualization of the detection result is typically done by conjugating the binding molecules to coloured nanoparticles. The intensity of the test line corresponds to the analyte\u2019s concentration and can be read by eye or reader equipment. Typically paper-based, the device displays results within 5 to 30 minutes. Portability, simplicity, and low manufacturing costs are some of the many reasons why LFAs are popular in several fields where rapid testing is needed. A pregnancy test is a well-known example of an LFA (Koczula & Gallotta, 2016)."})]}),Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Biomarker Quantification Methods"}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"Our project required a quantitative result. Our original approach was to design an assay in the form of a traditional LFA and develop an image processing software to quantify the test result based on the test line intensity. After some research, we discovered several other ways to quantify LFA results, so we analyzed the different methods and tried to pick the best one for our purposes. Some of the qualities we examined included:"}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"Accessibility, simplicity, and portability"}),Object(Le.jsx)("li",{children:"Readout time: the amount of time needed to display accurate test results"}),Object(Le.jsx)("li",{children:"Limit of detection: the lowest analyte concentration that can be distinguished from a sample with no analyte (Zhan et al., 2020)"}),Object(Le.jsx)("li",{children:"Sensitivity"}),Object(Le.jsx)("li",{children:"Whether or not the size of the target analyte matters"}),Object(Le.jsx)("li",{children:"Feasibility of building a prototype"})]})}),Object(Le.jsx)("p",{children:"We researched a total of five methods and compared their advantages and disadvantages. Note that all quantification methods vary from paper to paper, so some points may be specific to one paper. The following gives a general look at each method\u2019s properties and its potential for improvement."})]}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Method 1: Traditional LFA and colorimetric analysis"})}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Description"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"Image processing software quantifies the analyte based on the colour intensity of the LFA test line in a photo."}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Advantages"})}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"Analyte size does not matter"}),Object(Le.jsx)("li",{children:"User-friendly and portable since readings can be done with a smartphone (Foysal et al., 2019)"}),Object(Le.jsx)("li",{children:"Quick interpretation time (Foysal et al., 2019)"}),Object(Le.jsx)("li",{children:"Low cost (Foysal et al., 2019)"})]})}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Disadvantages"})}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"Ambient lighting affects accuracy (Foysal et al., 2019)"}),Object(Le.jsx)("li",{children:"Can only quantify nanoparticles on the surface of the test line (Qu et al., 2020)"}),Object(Le.jsx)("li",{children:"Common LFA labels such as gold nanoparticle labels are not very sensitive (Ghosh & Ahn, 2019)"})]})}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Readout time"})}),Object(Le.jsx)("p",{children:"Since interpretation time is short, the total readout time depends on the LFA readout time."}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Method 2: LFA ruler"})}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Description"})}),Object(Le.jsx)("p",{children:"Contains a built-in quantification method so that the amount of analyte can be read from the device. For example, the analyte concentration could correspond to the distance that ink advances in a microfluidic channel (Li et al., 2019)."}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Advantages"})}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"Analyte size does not matter"}),Object(Le.jsx)("li",{children:"No external readers are needed since quantitative detection is done by eye (Wu et al., 2016)"}),Object(Le.jsx)("li",{children:"Low cost (Wu et al., 2016)"}),Object(Le.jsx)("li",{children:"Portable (Wu et al., 2016)"})]})}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Disadvantages"})}),Object(Le.jsx)("p",{children:Object(Le.jsx)("ul",{className:pe.a.description,style:{marginTop:0},children:Object(Le.jsx)("li",{children:"Results are read by eye, which limits the precision"})})}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Readout time"})}),Object(Le.jsx)("p",{children:"Including the LFA readout time and the quantification time, the entire process takes approximately 20 to 30 minutes (Lin et al., 2018; Li et al., 2019)."}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Method 3: Magnetic LFA"})}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Description"})}),Object(Le.jsx)("p",{children:"Magnetic nanoparticles act as the label, replacing gold nanoparticles in traditional LFAs. Particles on the test line are quantified by an external reader (Moyano et al., 2020)."}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Advantages"})}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"Analyte size does not matter (Yan et al., 2019)"}),Object(Le.jsx)("li",{children:"Quantifies nanoparticles throughout the entire thickness of the LFA membrane (Moyano et al., 2020)"}),Object(Le.jsx)("li",{children:"Magnetic signals remain stable for longer than optical signals (Moyano et al., 2020)"}),Object(Le.jsx)("li",{children:"May have improved sensitivity and limit of detection compared to a traditional LFA and visual detection (Jacinto et al., 2018)"})]})}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Disadvantages"})}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"Might be difficult to implement in protein modelling software"}),Object(Le.jsx)("li",{children:"Limited research"})]})}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Readout time"})}),Object(Le.jsx)("p",{children:"Due to design similarities, the readout time is likely similar to a traditional LFA, plus a few minutes for quantification."}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Notes"})}),Object(Le.jsx)("p",{children:"External reader needed, which may or may not be portable (Moyano et al., 2020)"}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Method 4: Traditional LFA and thermal sensor"})}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Description"})}),Object(Le.jsx)("p",{children:"A laser shines on the test line and a thermal sensor measures the heat generated by the nanoparticles (Qu et al., 2020)."}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Advantages"})}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"Analyte size does not matter"}),Object(Le.jsx)("li",{children:"Results are more stable since this method quantifies nanoparticles throughout the entire thickness of the LFA membrane (Qu et al., 2020)"}),Object(Le.jsx)("li",{children:"Short detection time of approximately 120 seconds (Qu et al., 2020)"}),Object(Le.jsx)("li",{children:"Improved limit of detection compared to visual detection (Zhan et al., 2020)"}),Object(Le.jsx)("li",{children:"Portable external reader, which consists of the laser and thermal sensor (Qu et al., 2020)"})]})}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Disadvantages"})}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"Measures nanoparticles indirectly, resulting in lower sensitivity compared to visual detection (Qu et al., 2020)"}),Object(Le.jsx)("li",{children:"Ambient temperature affects accuracy (Qu et al., 2020)"})]})}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Readout time"})}),Object(Le.jsx)("p",{children:"The total time is equal to the readout time of a traditional LFA plus 120 seconds for detection."}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Method 5: Chemiluminescent Microfluidic Capillary Flow Assay"})}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Description"})}),Object(Le.jsx)("p",{children:"Microchannels mix the sample, detecting agent, and chemiluminescent reagents. An optical detection system quantifies the analyte based on the light intensity generated by the reaction (Ghosh et al., 2020)."}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Advantages"})}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"Higher sensitivity and improved limit of detection compared to traditional LFA and visual detection (Ghosh & Ahn, 2019; Bhattacharyya & Klapperich, 2007)"}),Object(Le.jsx)("li",{children:"Low cost (Ghosh et al., 2020)"}),Object(Le.jsx)("li",{children:"Portable, due to small device and reader size (Ghosh et al., 2020)"}),Object(Le.jsx)("li",{children:"Longer shelf life due to lyophilization (freeze-drying) of reagents (Ramachandran et al., 2014)"}),Object(Le.jsx)("li",{children:"Reduced sample volume required (Ghosh et al., 2020)"}),Object(Le.jsx)("li",{children:"Real-time detection, which can decrease response time (Do & Ahn, 2008)"})]})}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Disadvantages"})}),Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:"Complicated design compared to traditional LFA"}),Object(Le.jsx)("li",{children:"Less user-friendly compared to other methods"})]})}),Object(Le.jsx)("div",{style:{margin:"20px 0 20px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Readout time"})}),Object(Le.jsx)("p",{children:"The total time takes about 20 to 25 minutes. While the chemiluminescent reaction takes about 1 minute to begin and the signal remains for 20 to 25 minutes, our sources recorded results towards the end of that period (Ghosh et al., 2020; Hemmig et al., 2020). It is worth noting that with real-time detection, it is possible to get results within a shorter period."})]}),"=",Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Deciding on a Quantification Method"}),"= ",Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"Since Method 1 was our original plan, it became the standard to which we compared all the other methods. Methods 3 and 4 are similar to Method 1. All three methods use the format of a traditional LFA, with little to no modifications made to the strip. The only difference between Methods 1 and 3 is the reader. All methods require some external reader, but Method 1 is the most accessible and user-friendly since it can be done with a phone."}),Object(Le.jsx)("p",{children:"The main benefit that Methods 3 and 4 had over Method 1 was the ability to quantify nanoparticles throughout the entire thickness of the LFA strip. This likely results in an improved limit of detection. We planned on designing a competitive LFA, meaning a fainter signal would correspond to a higher concentration. In our case, an improvement in the limit of detection would mean that our design would be able to test samples with greater analyte concentrations. Our plan at the time involved testing if a patient had lower levels of a specific biomarker, which would correspond to a more intense signal. Thus we decided that an improved limit of detection was not a priority. Since Method 4 quantifies the nanoparticles indirectly, it is not as sensitive as visual detection. We agreed that sensitivity is a priority, so we eliminated Method 4."}),Object(Le.jsx)("p",{children:"Method 3, on the other hand, could be more sensitive than visual detection. Unfortunately, Method 3 is a relatively new quantification method, so it would be difficult to research due to limited available information. Meanwhile, Method 1 would be much easier to research as it is already widely used in several fields. In addition, Method 3 would be difficult, if not impossible, to model in the protein modelling software we use. Since we did not have lab access, we knew we would need to rely on modelling software. In addition, the existing research specifies that it requires an external reader. It is unlikely that this method would be more portable and user-friendly than Method 1, which involves taking a photo with a phone. For these reasons, we decided to eliminate Method 3. The possibility of higher sensitivity did not justify the added research needed to understand the complexities of the method."}),Object(Le.jsx)("p",{children:"We also decided to eliminate Method 2. The quantification time is longer than the quantification times in the other methods, making its total readout time on the longer side. Methods 1 and 5 use equipment to quantify the test results, likely yielding more consistent and precise readings from test to test. Meanwhile, Method 2 relies on measurements by eye, which limits precision and may increase error."}),Object(Le.jsx)("p",{children:"Finally, we needed to decide between Method 1 and Method 5. The advantages of Method 5 were clear since it was developed as a solution to address the limitations of Method 1, specifically their lack of sensitivity and quantitative accuracy. The assay in Method 5 is still a one-step process, with the only added complication being having to measure out a specific sample volume. We thought that Method 5 might require analytes of smaller size due to the size of its microchannels. However, articles showed that microfluidics-based approaches work with analytes comparable in size to our target analyte, so we decided that that would not be an issue. In the end, we chose Method 5 because we thought the significant improvement in sensitivity justified the additional research needed to understand this method and its complicated design."})]}),"=",Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Introduction to Microfluidic Assay"}),"= ",Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"Our primary reference for a chemiluminescent microfluidic capillary flow assay (referred to in the source as a microchannel capillary flow assay, or MCFA) describes a design in which a microfluidic chip emits light through chemiluminescence on the addition of a sample that is negative for a malaria biomarker. Custom hardware measures the produced light and sends the resulting data to a smartphone through its USB port for analysis and display to the user (Ghosh et al., 2020). This model served as a basis for our project design."}),Object(Le.jsx)("p",{children:"The microfluidic chip consists of a sample loading chamber, two parallel chambers, one of which holds lyophilized (freeze-dried) fusion protein and the other a chemiluminescent substrate, a reaction chamber from which the light is emitted, and a capillary pump that passively moves the sample through the system. Our design is a competitive lateral flow assay. The reaction chamber initially contains only immobile analyte molecules. When the sample is added to the loading chamber, it is split and passes separately through the two parallel chambers, in one chamber mixing with fusion protein and in the other mixing with a chemiluminescent substrate. Next, the sample-fusion protein mix reaches the reaction chamber. The substrate-sample mix moves through a longer delaying path and only reaches the reaction chamber after binding molecules with biomarkers (path 1 sample fluid) fill the chamber. Finally, the substrate binds to the fusion proteins present in the reaction chamber, which causes chemiluminescence. This design results in a dimmer output indicating a higher concentration of analyte in the sample, and a brighter output indicating a lower concentration of the analyte."}),Object(Le.jsxs)("p",{children:["Our fusion protein consists of a domain that binds to the target analyte in sample, and a horseradish peroxidase (HRP) domain, which acts as the enzyme that binds the chemiluminescent substrate. The substrate we chose is Amplex Red. For more info about the design of the fusion protein, please refer to the ",Object(Le.jsx)("strong",{children:"Biomarker Detection: PEA-Binding Fusion Protein Design"})," section on the Engineering page."]})]}),"=",Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Hardware Design"}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Simulation"})}),Object(Le.jsx)("p",{children:"We started off by creating a circuit simulation in TinkerCAD. We adapted some microcontroller code and a simple photodiode circuit taken from various sources. The purpose of this was to get a general idea of how the circuit and microcontroller will work and which parts to order."})]})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:ni.a.design_img,style:{borderRadius:10,width:"80%",height:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Labelled TinkerCAD circuit simulation."})]}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"In this early simulated prototype, the integers printed to the serial monitor increased as the photodiode was exposed to less light and decreased as the light level increased. When the photodiode detected light, it became a conductor and the current flowed to the ground, so the microcontroller read a low voltage level. At a low light level, the photodiode became a resistor and prevented current from flowing to the ground. Instead, the current flowed to the analog pin and the microcontroller read a high voltage (Kranthi, 2013)."})}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("iframe",{src:"https://static.igem.org/mediawiki/2021/8/84/T--Waterloo--TinkerCADVidFinal.mp4",style:{width:900,height:500}})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Circuit Part Selection"})}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Light Sensor"})}),Object(Le.jsxs)("p",{children:["Initially, we considered the use of a photomultiplier tube (PMT) for the detection of resorufin. Resorufin is the fluorescent compound produced in the reaction chambers and has emission maxima of 563 nm. Additionally, the area of the MCFA reaction chamber was 9 mm",Object(Le.jsx)("sup",{children:"2"}),". These two design specifications in peak spectral sensitivity and radiant sensitive area were the main priorities for selecting an optical sensor."]}),Object(Le.jsx)("p",{children:"The high internal amplification characteristic of PMTs, as well as its ability to detect low light levels, prompted the consideration for Hamatsu\u2019s R12900U-01 Micro-PMT. However, with a lower peak spectral sensitivity of 420 nm, the sensitivity and the cost were not aligned with our design goals\u2014a similar trend we observed across all PMT devices (Hamamatsu, 2021). Having to look towards cheaper alternatives in photodiodes instead, we decided upon the PDB-C156 from Advanced Photonix for our medium-fidelity prototype. The PDB-C156 is a through-hole PIN silicon photodiode with a peak spectral sensitivity of 660 nm and response time of 15 ns (Advanced Photonix, n.d.)."}),Object(Le.jsx)("p",{children:"Having no access to the lab this year and no physical fabrication of the MCFA lab chip to test with, the peak spectral sensitivity of the photodiode was not our main priority for the medium-fidelity prototype. Instead, our goal was to test the functionality of the rest of the circuit on the breadboard, and the communication between the microcontroller and the software."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Transimpedance Amplifier"})}),Object(Le.jsx)("p",{children:"Operating the photodiode in reverse and using a general-purpose OPA350PA as a transimpedance amplifier, we wanted to amplify the 90 \u03bcA of short-circuit current of the PDB-C156 photodiode to an output voltage swing of 0V to 5V. To achieve the desired gain, we used a 47k\u03a9 and a 10k\u03a9 resistor in series."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsxs)("div",{className:ni.a.formula_div,children:[Object(Le.jsxs)("div",{className:ni.a.formula_center,children:["R",Object(Le.jsx)("sub",{children:"f"})," = \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsxs)("div",{className:ni.a.top,children:["V",Object(Le.jsx)("sub",{children:"max"})," - V",Object(Le.jsx)("sub",{children:"min"})]}),Object(Le.jsxs)("div",{className:ni.a.bottom,style:{marginLeft:"50%"},children:["I",Object(Le.jsx)("sub",{children:"sc"})]})]}),Object(Le.jsx)("div",{className:ni.a.formula_center,children:"\xa0 = \xa0"}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,children:"5V - 0V"}),Object(Le.jsx)("div",{className:ni.a.bottom,style:{marginLeft:"10%"},children:"90 \u03bcA"})]}),Object(Le.jsx)("div",{className:ni.a.formula_center,children:"\xa0 = \xa055555.5 \u03a9"})]}),Object(Le.jsxs)("div",{className:ni.a.formula_description,children:[Object(Le.jsxs)("div",{children:["R",Object(Le.jsx)("sub",{children:"f"})," = feedback resistor"]}),Object(Le.jsxs)("div",{children:["V",Object(Le.jsx)("sub",{children:"max"})," = maximum output voltage"]}),Object(Le.jsxs)("div",{children:["V",Object(Le.jsx)("sub",{children:"min"})," = minimum output voltage"]}),Object(Le.jsxs)("div",{children:["I",Object(Le.jsx)("sub",{children:"sc"})," = short circuit current of photodiode"]})]})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Analog-to-Digital Converter"})}),Object(Le.jsx)("p",{children:"To digitize the output voltage signal, we selected the MCP3302-CI/P Analog-to-Digital converter (ADC) for its high resolution and ability to interface over serial peripheral interface (SPI) protocol. With 13-bits of resolution, we favoured the Successive Approximation Register (SAR) architecture of the MCP3303-CI/P over delta-sigma, pipeline, flash, and dual slope architectures due to the perfect balance in bit resolution and sampling rate for photosensing applications. Additionally, the 4 single-ended inputs of the ADC left room for two additional photodiodes on top of the two photodiodes we were prototyping with (Microchip Technology, 2011). We decided to communicate over SPI due to the fast data transmission rate and simplicity. Another reason was that our system consisted of only one master and one slave device in the microcontroller and MCP3302 ADC respectively, which required only one set of communication lines."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Microcontroller"})}),Object(Le.jsx)("p",{children:"For the microcontroller, we decided upon the Arduino Micro as our development board for its compact size, low cost, support for SPI devices, sufficient memory, and clock speed. In addition, existing code for interfacing over SPI with a family of MCP ADCs was available for use and modification."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:ni.a.sub_sub_heading,children:"Developing the High-Fidelity Prototype"})}),Object(Le.jsxs)("p",{children:["To move forward with a higher fidelity prototype, we decided on using Vishay Intertechnology\u2019s BPW21R as our primary optical sensor over the surface-mount SFH 2430-Z from OSRAM Opto Semiconductors. Though a PIN photodiode is favoured over PN photodiodes in this application due to greater responsivity and response speed, the BPW21R most closely met our design goals with a radiant sensitive area of 7.5 mm",Object(Le.jsx)("sup",{children:"2"})," and a peak spectral sensitivity of 565 nm (Vishay, 2011). In comparison, the greater sensitivity, the size of the radiant sensitive area, and the faster response speed were held as advantages over the SFH 2430-Z."]}),Object(Le.jsx)("p",{children:"Recalculating the gain required for the BPW21R:"})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)("div",{className:ni.a.formula_div,children:[Object(Le.jsxs)("div",{className:ni.a.formula_center,children:["R",Object(Le.jsx)("sub",{children:"f"})," = \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsxs)("div",{className:ni.a.top,children:["V",Object(Le.jsx)("sub",{children:"max"})," - V",Object(Le.jsx)("sub",{children:"min"})]}),Object(Le.jsxs)("div",{className:ni.a.bottom,style:{marginLeft:"50%"},children:["I",Object(Le.jsx)("sub",{children:"sc"})]})]}),Object(Le.jsx)("div",{className:ni.a.formula_center,children:"\xa0 = \xa0"}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,children:"5V - 0V"}),Object(Le.jsx)("div",{className:ni.a.bottom,style:{marginLeft:"15%"},children:"9 \u03bcA"})]}),Object(Le.jsx)("div",{className:ni.a.formula_center,children:"\xa0 = \xa0555555.5 \u03a9"})]})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"Calculating the bandwidth and feedback capacitor value to implement a low-pass filter for attenuation of signals with undesired frequencies in the trans-impedance configuration:"})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsxs)("div",{className:ni.a.formula_div,style:{marginLeft:"15%"},children:[Object(Le.jsxs)("div",{className:ni.a.formula_center,children:["f",Object(Le.jsx)("sub",{children:"b"})," = \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,children:"0.35"}),Object(Le.jsxs)("div",{className:ni.a.bottom,style:{marginLeft:"50%"},children:["t",Object(Le.jsx)("sub",{children:"r"})]})]}),Object(Le.jsx)("div",{className:ni.a.formula_center,children:"\xa0 = \xa0"}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,children:"0.35"}),Object(Le.jsx)("div",{className:ni.a.bottom,children:"3.1 \u03bcs"})]}),Object(Le.jsx)("div",{className:ni.a.formula_center,children:"\xa0 = \xa0112903.226 Hz"})]}),Object(Le.jsxs)("div",{className:ni.a.formula_description,style:{marginBottom:20},children:[Object(Le.jsxs)("div",{children:["f",Object(Le.jsx)("sub",{children:"b"})," = bandwidth"]}),Object(Le.jsxs)("div",{children:["t",Object(Le.jsx)("sub",{children:"r"})," = rise time of photodiode"]})]}),Object(Le.jsxs)("div",{className:ni.a.formula_div,children:[Object(Le.jsxs)("div",{className:ni.a.formula_center,children:["C",Object(Le.jsx)("sub",{children:"f"})," \u2264 \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,style:{borderBottom:"none",marginLeft:"40%"},children:"1"}),Object(Le.jsxs)("div",{className:ni.a.new_bottom,children:["2\u03c0R",Object(Le.jsx)("sub",{children:"f"}),"f",Object(Le.jsx)("sub",{children:"b"})]})]}),Object(Le.jsx)("div",{className:ni.a.formula_center,children:"\xa0 = \xa0"}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,style:{borderBottom:"none",marginLeft:"40%"},children:"1"}),Object(Le.jsx)("div",{className:ni.a.new_bottom,children:"2\u03c0(555.5 k\u03a9)(112.903 kHz)"})]}),Object(Le.jsx)("div",{className:ni.a.formula_center,children:"\xa0 = \xa02.5374 pF"})]}),Object(Le.jsx)("div",{className:ni.a.formula_description,children:Object(Le.jsxs)("div",{children:["C",Object(Le.jsx)("sub",{children:"f"})," = feedback capacitor"]})})]}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"In addition, we also switched the transimpedance amplifier to the LMP7717MAE/NOPB to better fit our design specifications. Specifically, we sought a surface-mount amplifier with minimal input offset voltage, input bias current, and a gain-bandwidth product (GBW) of 55 MHz or greater, which the LMP7717MAE/NOPB satisfied. Here are the calculations used to determine these values:"})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("div",{className:ni.a.formula_div,style:{marginLeft:"10%"},children:Object(Le.jsxs)("div",{children:["C",Object(Le.jsx)("sub",{children:"i"})," = C",Object(Le.jsx)("sub",{children:"s"})," + C",Object(Le.jsx)("sub",{children:"d"})," + C",Object(Le.jsx)("sub",{children:"cm"})," = 1200pF + 15pF = 1215pF"]})}),Object(Le.jsxs)("div",{className:ni.a.formula_description,style:{marginBottom:20},children:[Object(Le.jsxs)("div",{children:["C",Object(Le.jsx)("sub",{children:"i"})," = total input capacitance"]}),Object(Le.jsxs)("div",{children:["C",Object(Le.jsx)("sub",{children:"s"})," = input source (diode) capacitance"]}),Object(Le.jsxs)("div",{children:["C",Object(Le.jsx)("sub",{children:"d"})," = differential input capacitance of the amplifier"]}),Object(Le.jsxs)("div",{children:["C",Object(Le.jsx)("sub",{children:"cm"})," = common mode input capacitance"]})]}),Object(Le.jsxs)("div",{className:ni.a.formula_div,children:[Object(Le.jsxs)("div",{className:ni.a.formula_center,children:["GBW ",">"," \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsxs)("div",{className:ni.a.top,children:["C",Object(Le.jsx)("sub",{children:"i"})," + C",Object(Le.jsx)("sub",{children:"f"})]}),Object(Le.jsxs)("div",{className:ni.a.bottom,children:["2\u03c0R",Object(Le.jsx)("sub",{children:"f"}),"C",Object(Le.jsx)("sub",{children:"f"}),Object(Le.jsx)("sup",{children:"2"})]})]}),Object(Le.jsx)("div",{className:ni.a.formula_center,children:"\xa0 = \xa0"}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,style:{borderBottom:"none",marginLeft:"10%"},children:"1215 pF + 2.5374 pF"}),Object(Le.jsxs)("div",{className:ni.a.new_bottom,children:["2\u03c0(555.5 k\u03a9)(2.5374 pF)",Object(Le.jsx)("sup",{children:"2"})]})]}),Object(Le.jsx)("div",{className:ni.a.formula_center,children:"\xa0 = \xa054.175 MHz"})]})]}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"Finally, we placed standard 0.1 \u03bcF capacitors near the supply pins of the system to serve as a bypass filter and reduce noise. With the new photodiode, transimpedance amplifier, the MCP3302-CI/SL (surface-mount equivalent ADC), and the Arduino Micro, we aimed to design a printed circuit board (PCB) to mount above the header pins of the Arduino Micro as a single unit for the high-fidelity prototype. Using Altium Designer as the PCB design software, we included an additional photodiode and transimpedance amplifier to the schematic, to connect to a separate analog input channel of the four that the MCP3302-CI/SL had available for quantification of a second analyte."})}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Software Design"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Desktop vs. Mobile"})}),Object(Le.jsx)("p",{children:"The first thing we had to decide was the platform our software would be on. A mobile application would be excellent for portability, but as previously mentioned, the Human Practices team decided that our project fits best in a clinic lab, so there was no main reason to make the software portable. Labs typically use desktop computers rather than mobile devices for test processing, so it would make sense that our project supports existing lab infrastructure. Doing the data processing on a desktop would make it easier for users to integrate the test results directly into the clinic\u2019s database. Since our plan involved graphing the concentration as the reaction occurred, a bigger screen would allow users to more easily examine graph details."}),Object(Le.jsx)("p",{children:"From the build perspective, developing a desktop application was simpler than developing a mobile one. We already had a general framework and class structure for a desktop application that could easily be adapted to suit our purposes."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Tech Stack and Libraries"})}),Object(Le.jsxs)("p",{children:["We used Python 3 to handle the graphics and user interaction, the ",Object(Le.jsx)("a",{href:"https://matplotlib.org/",target:"_blank",children:"matplotlib"})," and ",Object(Le.jsx)("a",{href:"https://numpy.org/",target:"_blank",children:"numpy"})," libraries to handle data processing and graphing, ",Object(Le.jsx)("a",{href:"https://pythonhosted.org/pyserial/",target:"_blank",children:"pyserial"})," to read data from the microcontroller, and ",Object(Le.jsx)("a",{href:"https://www.pyinstaller.org/",target:"_blank",children:"pyinstaller"})," to bundle the entire app into an executable file. We wrote the entire application in pure Python to prioritize compactness and ease of development. Due to this design choice, our app is no longer than 500 lines of code, and can be easily read, understood, and extended."]}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Features"})}),Object(Le.jsx)("p",{children:"Our app supports graphing multiple analyte concentrations simultaneously, and provides the option to toggle which analytes are displayed, allowing a user to choose any combination of analyte concentrations to view at a time. All analytes present are automatically detected, and a toggle option is automatically created to handle them. The displayed graph supports scrolling, and will pause updates if clicked. The app will calculate and display the maximum concentration of each analyte over the entire test, and can export the raw data, the maxima, and an image of the graphs. A legend and color-coding is used to distinguish the analytes being graphed, and will dynamically change and reassign colors based on user needs and preference, or as new analytes are detected or added."}),Object(Le.jsx)("p",{children:"Any number of analytes may be examined by our app simultaneously, as it will automatically handle new analytes without the need for manual reconfiguration."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Calculating the Number of Target Analyte Binding Molecules"})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"To determine the number of binding molecules that should be used in the microfluidic assay, the amount of PEA expected in a microfluidic assay sample should first be determined. To find this, we searched the literature for typical urinary PEA values. We found the following data from Kusaga et al. (2002):"})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Various analyte levels present in urine among ADHD and control subjects, adapted from Kusaga et al. (2002)."}),Object(Le.jsxs)(Zt,{bordered:!0,className:pe.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:Object(Le.jsx)("strong",{children:"Subject"})}),Object(Le.jsx)("th",{children:Object(Le.jsx)("strong",{children:"Number of patients"})}),Object(Le.jsx)("th",{children:Object(Le.jsx)("strong",{children:"Age (year)"})}),Object(Le.jsx)("th",{children:Object(Le.jsx)("strong",{children:"PEA"})}),Object(Le.jsx)("th",{children:Object(Le.jsx)("strong",{children:"MHPG"})}),Object(Le.jsx)("th",{children:Object(Le.jsx)("strong",{children:"HVA"})}),Object(Le.jsx)("th",{children:Object(Le.jsx)("strong",{children:"5HIAA"})})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"ADHD"}),Object(Le.jsx)("td",{children:"37"}),Object(Le.jsx)("td",{children:"8.76 \xb1 2.19"}),Object(Le.jsx)("td",{children:"21.66 \xb1 20.49"}),Object(Le.jsx)("td",{children:"2.12 \xb1 0.68"}),Object(Le.jsx)("td",{children:"7.44 \xb1 4.95"}),Object(Le.jsx)("td",{children:"4.31 \xb1 2.21"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Control (\u201cnormal\u201d patient)"}),Object(Le.jsx)("td",{children:"21"}),Object(Le.jsx)("td",{children:"7.67 \xb1 2.51"}),Object(Le.jsx)("td",{children:"46.61 \xb1 46.55"}),Object(Le.jsx)("td",{children:"2.17 \xb1 0.91"}),Object(Le.jsx)("td",{children:"5.21 \xb1 3.25"}),Object(Le.jsx)("td",{children:"4.38 \xb1 5.31"})]})]})]})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsxs)("p",{children:["The PEA units are in micrograms/gram of creatinine and the upper range of PEA level in urine for the control group is 93.16 micrograms PEA /gram of creatinine. Kusaga et al. (2002) collected 1 mL from each subject (Kusaga et al., 2002).",Object(Le.jsx)("br",{}),"In order to determine the control levels of creatinine, the normal amount of urine was first determined. According to Weatherspoon (2017), urine output is approximately 800 - 2000 mL per day and the urine creatinine amount is 500 to 2000 mg/24 hours (Weatherspoon, 2017). From this, we found that there is approximately 1 mg creatinine/mL of urine when we assume the PEA levels of urine based on the control group\u2019s upper range of PEA levels from the table above."]}),Object(Le.jsx)("p",{children:"To calculate the number of binding molecules, we will be taking 15 microliters each pathway for a total of 30 microliters."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsxs)("div",{className:ni.a.formula_div,style:{marginLeft:"20%"},children:[Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,children:"93.16ug PEA"}),Object(Le.jsx)("div",{className:ni.a.bottom,style:{marginLeft:"5%"},children:"1g creatine"})]}),Object(Le.jsxs)("div",{style:{marginTop:10},children:["\xa0 ",Object(Le.jsx)("strong",{children:"."})," \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,children:"0.001g creatine"}),Object(Le.jsx)("div",{className:ni.a.bottom,style:{marginLeft:"20%"},children:"1mL urine"})]}),Object(Le.jsx)("div",{className:ni.a.formula_center,children:"\xa0 = \xa00.09316ug PEA/1mL urine"})]}),Object(Le.jsxs)("div",{className:ni.a.formula_div,style:{marginLeft:"15%"},children:[Object(Le.jsx)("div",{className:ni.a.formula_center,children:"30uL urine"}),Object(Le.jsxs)("div",{style:{marginTop:10},children:["\xa0 ",Object(Le.jsx)("strong",{children:"."})," \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,style:{borderBottom:"none",marginLeft:"20%"},children:"1ml"}),Object(Le.jsx)("div",{className:ni.a.new_bottom,children:"1000uL"})]}),Object(Le.jsxs)("div",{style:{marginTop:10},children:["\xa0 ",Object(Le.jsx)("strong",{children:"."})," \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,children:"0.09316ug PEA"}),Object(Le.jsx)("div",{className:ni.a.bottom,style:{marginLeft:"20%"},children:"1ml urine"})]}),Object(Le.jsx)("div",{className:ni.a.formula_center,children:"\xa0 = \xa00.0027948ug PEA in our sample"})]}),Object(Le.jsxs)("div",{className:ni.a.formula_div,children:[Object(Le.jsx)("div",{className:ni.a.formula_center,children:"0.0027948ug"}),Object(Le.jsxs)("div",{style:{marginTop:10},children:["\xa0 ",Object(Le.jsx)("strong",{children:"."})," \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,style:{borderBottom:"none",marginLeft:"30%"},children:"1g"}),Object(Le.jsxs)("div",{className:ni.a.new_bottom,children:["1x10",Object(Le.jsx)("sup",{children:"6"}),"ug"]})]}),Object(Le.jsxs)("div",{style:{marginTop:10},children:["\xa0 ",Object(Le.jsx)("strong",{children:"."})," \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,children:"1mol PEA"}),Object(Le.jsx)("div",{className:ni.a.bottom,style:{marginLeft:"15%"},children:"121.18g"})]}),Object(Le.jsxs)("div",{style:{marginTop:10},children:["\xa0 ",Object(Le.jsx)("strong",{children:"."})," \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsxs)("div",{className:ni.a.top,children:["6.02x10",Object(Le.jsx)("sup",{children:"23"}),"molecules"]}),Object(Le.jsx)("div",{className:ni.a.bottom,style:{marginLeft:"20%"},children:"1mol PEA"})]}),Object(Le.jsxs)("div",{className:ni.a.formula_center,children:["\xa0 = \xa01.388x10",Object(Le.jsx)("sup",{children:"13"}),"molecules PEA found in 30ug urine"]})]})]}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsxs)("p",{children:["Thus we need 1.388x10",Object(Le.jsx)("sup",{children:"13"})," binding molecules in each assay."]})}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,style:{paddingBottom:0},children:"Correlate Voltage to Biomarker Concentration: Mathematical Conversion"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"A crucial part of this project was converting the voltage value read by the microcontroller to the target analyte concentration."}),Object(Le.jsx)("p",{children:"The microcontroller we used, an Arduino Micro, maps voltage values of 0V to 5V to integer values from 0 to 1023 (Arduino, n.d.). The integer values are what the software receives from the serial port. This is a linear relationship, so to convert the integer, i, back into the voltage, we simply multiply the integer value by 5V and divide by 1024."})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)("div",{className:ni.a.formula_div,children:[Object(Le.jsx)("div",{className:ni.a.formula_center,children:"V = \xa0"}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,style:{borderBottom:"none",marginLeft:"15%"},children:"5V"}),Object(Le.jsx)("div",{className:ni.a.new_bottom,children:"1024"})]}),Object(Le.jsx)("div",{className:ni.a.formula_center,children:"\xa0i"})]})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"In other words, the rate per unit is 5V/1024, which is approximately 0.0049 V per unit (Arduino, n.d.)."}),Object(Le.jsx)("p",{children:"The microcontroller reads values after the current from the photodiode passes through the transimpedance amplifier, so we need to use Ohm\u2019s law (V = IR where V is voltage, I is current, and R is resistance) to obtain the current directly from the photodiode. The resistance we used for the amplifier was 57 k\u03a9 (calculated by adding 47 k\u03a9 and 10 k\u03a9, which were resistors wired in series). The current is calculated using the following:"})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)("div",{className:ni.a.formula_div,children:[Object(Le.jsx)("div",{className:ni.a.formula_center,children:"I = \xa0"}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,style:{borderBottom:"none",marginLeft:"40%"},children:"V"}),Object(Le.jsx)("div",{className:ni.a.new_bottom,children:"57000\u03a9"})]})]})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"We can substitute in first equation for V to get the following:"})}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)("div",{className:ni.a.formula_div,children:[Object(Le.jsx)("div",{className:ni.a.formula_center,children:"I = \xa0"}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,style:{borderBottom:"none",marginLeft:"15%"},children:"5V"}),Object(Le.jsx)("div",{className:ni.a.new_bottom,children:"1024"})]}),Object(Le.jsxs)("div",{style:{marginTop:10},children:["\xa0 ",Object(Le.jsx)("strong",{children:"."})," \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,style:{borderBottom:"none",marginLeft:"40%"},children:"V"}),Object(Le.jsx)("div",{className:ni.a.new_bottom,children:"57000\u03a9"})]}),Object(Le.jsx)("div",{className:ni.a.formula_center,children:"\xa0i \xa0 = \xa0"}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,style:{borderBottom:"none",marginLeft:"40%"},children:"5V"}),Object(Le.jsx)("div",{className:ni.a.new_bottom,children:"58368000\u03a9"})]}),Object(Le.jsx)("div",{className:ni.a.formula_center,children:"\xa0i"})]})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsxs)("p",{children:["From here, further information is needed. We reached out to Vishay, the company that designed the silicon photodiode we used and asked how to convert the current response into a light intensity value. The senior manager of application engineering responded with an example of how to calculate the spectral responsivity, R",Object(Le.jsx)("sub",{children:"\u03bb"}),", of our photodiode, which gives us a value in amperes per watt (S. Ahmed, personal communication, September 20, 2021). The equation for this is"]})}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)("div",{className:ni.a.formula_div,children:[Object(Le.jsxs)("div",{className:ni.a.formula_center,children:["R",Object(Le.jsx)("sub",{children:"\u03bb"})," = \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsxs)("div",{className:ni.a.top,children:["I",Object(Le.jsx)("sub",{children:"ra"})]}),Object(Le.jsxs)("div",{className:ni.a.bottom,children:["P",Object(Le.jsx)("sub",{children:"T"})]})]})]})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsxs)("p",{children:["where I",Object(Le.jsx)("sub",{children:"ra"})," is the reverse light current and P",Object(Le.jsx)("sub",{children:"T"})," is the total power incident on the photodiode."]}),Object(Le.jsxs)("p",{children:["To calculate P",Object(Le.jsx)("sub",{children:"T"}),", we take the radiant sensitive area, A",Object(Le.jsx)("sub",{children:"p"}),", (given as 7.5 mm",Object(Le.jsx)("sup",{children:"2"})," by the data sheet) of the photodiode and multiply it by the chemiluminescent light intensity at the surface of the photodiode, which is expressed in watts per area unit (Vishay, 2011; S. Ahmed, personal communication, October 11, 2021). Assuming that the reaction chamber emits light equally in all directions, the formula for light intensity is"]})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)("div",{className:ni.a.formula_div,children:[Object(Le.jsx)("div",{className:ni.a.formula_center,children:"L = \xa0"}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,style:{borderBottom:"none",marginLeft:"40%"},children:"P"}),Object(Le.jsxs)("div",{className:ni.a.new_bottom,children:["4\xa0\u03c0\xa0r",Object(Le.jsx)("sup",{children:"2"})]})]})]})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"where P is the light wattage and r is the distance of the photodiode radiant area from the reaction chamber (Schwartz, 2018). If calculating the intensity of a lightbulb at a certain point, one can simply take the wattage listed on the packaging. However, our reaction does not have a \u201cwattage\u201d. Instead, we would need to figure out the amount of light energy produced by the reaction and divide that by the amount of time over which the light energy was produced. Putting everything together, we end up with the following equation for responsivity:"})}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)("div",{className:ni.a.formula_div,children:[Object(Le.jsxs)("div",{className:ni.a.formula_center,children:["R",Object(Le.jsx)("sub",{children:"\u03bb"})," = \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsxs)("div",{className:ni.a.top,style:{borderBottom:"none",marginLeft:"20%"},children:["I",Object(Le.jsx)("sub",{children:"ra"})]}),Object(Le.jsx)("div",{className:ni.a.new_bottom,children:Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("div",{className:ni.a.formula_div,children:Object(Le.jsxs)("div",{children:[Object(Le.jsxs)("div",{className:ni.a.top,style:{borderBottom:"none",marginLeft:"10%"},children:["A",Object(Le.jsx)("sub",{children:"p"}),"P"]}),Object(Le.jsxs)("div",{className:ni.a.new_bottom,children:["4\xa0\u03c0\xa0r",Object(Le.jsx)("sup",{children:"2"})]})]})})})})]})]})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsxs)("p",{children:["where I",Object(Le.jsx)("sub",{children:"ra"})," is the reverse light current, A",Object(Le.jsx)("sub",{children:"p"})," = 7.5 mm",Object(Le.jsx)("sup",{children:"2"})," is the radiant sensitive area of the photodiode, P is the amount of light energy produced by the reaction per time unit, and r is the distance of the radiant sensitive area from the reaction chamber."]}),Object(Le.jsxs)("p",{children:["We could not complete this calculation because of the missing values. The first value we did not have was r. This is because we did not design a casing for the hardware and assay so we did not know how far the photodiode would be from the reaction chamber. The second value we did not have was I",Object(Le.jsx)("sub",{children:"ra"}),". We would need to determine this experimentally by using a quantity of chemiluminescent substrate to generate a known amount of light energy per second, which would be the P value. Using a known quantity of chemiluminescence to transfer a known amount of power to the photodiode, we would then record the amount of current outputted by the photodiode. This would be the I",Object(Le.jsx)("sub",{children:"ra"})," value. In other words, we needed lab access and more data to complete the calculation. Then using the responsivity, we could determine the light intensity of a test sample reaction based on the photodiode\u2019s output current. However, this conversion would have limited accuracy. There are several environmental factors that affect hardware performance, and more specifically, photodiode performance, which are not included within the equation. In addition, there was not enough information available to determine the relationship between light intensity and substrate concentration, so this would need to be determined experimentally as well. Although we cannot complete any voltage-to-concentration conversion without this piece of information, we can perform a more accurate conversion if we implement a calibration step instead."]})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,style:{paddingBottom:0},children:"Correlate Voltage to Biomarker Concentration: Experimental Calibration"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"Due to a lack of information and numerous varying environmental factors that could affect the photodiode\u2019s performance, a calibration step done before the test would likely be more accurate than calculations. This is because the information it provides would better reflect the environment in which the test reaction is occurring."}),Object(Le.jsxs)("p",{children:["To do this, the assay would perform a separate reaction with a calibration sample containing a known quantity of target analyte. Let C",Object(Le.jsx)("sub",{children:"1"})," stand for the target analyte concentration of the calibration sample. Microfluidic assays require a specific sample volume so the calibration sample and the test sample will have the same volume. Since the protein binds to the target analyte and the chemiluminescent substrate reacts with the protein, the concentration of the chemiluminescent substrate that reacts is equal to the concentration of the target analyte. In our design, only half of the sample is mixed with the protein, so we would be finding the analyte concentration for that half of the sample."]}),Object(Le.jsx)("p",{children:"Another thing to note is that the amount of current flowing through a photodiode is proportional to the light intensity (Wang, n.d.). As previously mentioned, we do not know the relationship between chemiluminescent substrate concentration and light intensity and would have to determine this experimentally. For the sake of simplicity, we can assume that concentration and light intensity is a linear relationship where no substrate corresponds to no light. This would make sense if each molecule emits an equal and constant amount of light."}),Object(Le.jsxs)("p",{children:["If this assumption holds, then there would be a linear relationship between analyte concentration and current outputted by the photodiode. To find the slope of this relationship, we would divide the known target analyte concentration by the amount of current measured, which we will call I",Object(Le.jsx)("sub",{children:"1"}),". This gives us a rate in concentration units per ampere."]}),Object(Le.jsx)("p",{children:"Recall that our design is based on a competitive LFA. The amount of light emitted by the reaction depends on the amount of protein that binds to the analyte placed in the reaction chamber. This is the protein that does not bind to the analyte in the test sample. We can use the concentration units per ampere rate to determine the concentration of the protein in the reaction chamber. From the previous section, we determined the current by multiplying the integer value from the microcontroller by 5 V / 58368000 \u03a9. Therefore the equation for C, the concentration of the protein binded to analyte in the reaction chamber is"})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)("div",{className:ni.a.formula_div,children:[Object(Le.jsx)("div",{className:ni.a.formula_center,children:"C = \xa0"}),Object(Le.jsxs)("div",{children:[Object(Le.jsxs)("div",{className:ni.a.top,children:["C",Object(Le.jsx)("sub",{children:"1"})]}),Object(Le.jsxs)("div",{className:ni.a.bottom,style:{marginLeft:"30%"},children:["I",Object(Le.jsx)("sub",{children:"1"})]})]}),Object(Le.jsxs)("div",{style:{marginTop:10},children:["\xa0 ",Object(Le.jsx)("strong",{children:"."})," \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,style:{borderBottom:"none",marginLeft:"40%"},children:"5V"}),Object(Le.jsx)("div",{className:ni.a.new_bottom,children:"58368000\u03a9"})]}),Object(Le.jsx)("div",{className:ni.a.formula_center,children:"\xa0i"})]})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"where i is the integer given to the software by the microcontroller."}),Object(Le.jsx)("p",{children:"Since we would know the total amount of the protein in the assay, we divide this by half of the sample volume to find the total concentration of protein. Then the concentration of protein binded to the analyte in half of the test sample is"})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)("div",{className:ni.a.formula_div,children:[Object(Le.jsxs)("div",{className:ni.a.formula_center,children:["C",Object(Le.jsx)("sub",{children:"sample"})," = \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,style:{borderBottom:"none",marginLeft:"20%"},children:"A"}),Object(Le.jsx)("div",{className:ni.a.new_bottom,children:Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("div",{className:ni.a.formula_div,children:Object(Le.jsxs)("div",{children:[Object(Le.jsxs)("div",{className:ni.a.top,children:["V",Object(Le.jsx)("sub",{children:"sample"})]}),Object(Le.jsx)("div",{className:ni.a.bottom,style:{marginLeft:"40%"},children:"2"})]})})})})]}),Object(Le.jsx)("div",{style:{marginTop:14},children:"\xa0 - \xa0"}),Object(Le.jsxs)("div",{children:[Object(Le.jsxs)("div",{className:ni.a.top,children:["C",Object(Le.jsx)("sub",{children:"1"})]}),Object(Le.jsxs)("div",{className:ni.a.bottom,style:{marginLeft:"30%"},children:["I",Object(Le.jsx)("sub",{children:"1"})]})]}),Object(Le.jsxs)("div",{style:{marginTop:10},children:["\xa0 ",Object(Le.jsx)("strong",{children:"."})," \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:ni.a.top,style:{borderBottom:"none",marginLeft:"40%"},children:"5V"}),Object(Le.jsx)("div",{className:ni.a.new_bottom,children:"58368000\u03a9"})]}),Object(Le.jsx)("div",{className:ni.a.formula_center,children:"\xa0i"})]})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsxs)("p",{children:["where A is the number of proteins in the assay, V",Object(Le.jsx)("sub",{children:"sample"})," is the volume of a sample, C",Object(Le.jsx)("sub",{children:"1"})," is the target analyte concentration of the calibration sample, I",Object(Le.jsx)("sub",{children:"1"})," is the current outputted by the photodiode during the calibration step, and ",Object(Le.jsx)("sub",{children:"i"})," is the integer given to the software by the microcontroller."]}),Object(Le.jsx)("p",{children:"Assuming that analyte is equally distributed throughout the sample, this is equal to the analyte concentration of the entire sample."}),Object(Le.jsx)("p",{children:"This equation operates under the assumption that all proteins are either binded to analyte in the sample or analyte in the reaction chamber."})]})]}),Object(Le.jsxs)(Ie,{open:!0,title:"Build",children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Hardware Development"})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"After the design phase, the initial prototype is developed with chosen circuit parts. Below shows the schematic diagrams, breadboard configuration of the prototype and the final PCB Design of the optical detector."})}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("img",{src:"",className:ni.a.design_img,style:{borderRadius:10,width:"40%",marginLeft:"30%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Breadboard configuration"})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)(Zt,{bordered:!0,className:pe.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:"Cable colour"}),Object(Le.jsx)("th",{children:"Meaning "})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Red"}),Object(Le.jsx)("td",{children:"+5V "})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Black"}),Object(Le.jsx)("td",{children:"GND "})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Yellow"}),Object(Le.jsxs)("td",{children:["Signal V",Object(Le.jsx)("sub",{children:"OUT"})," "]})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Blue"}),Object(Le.jsx)("td",{children:"SCK "})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Green"}),Object(Le.jsx)("td",{children:"MISO "})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Purple"}),Object(Le.jsx)("td",{children:"MOSI "})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"White"}),Object(Le.jsx)("td",{children:"SS "})]})]})]})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:ni.a.design_img,style:{borderRadius:10,width:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Schematic capture (optical detector)"})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:ni.a.design_img,style:{borderRadius:10,width:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Schematic capture (system)"})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:ni.a.design_img,style:{borderRadius:10,width:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"PCB Layout 3D Top View"})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:ni.a.design_img,style:{borderRadius:10,width:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"PCB Layout 3D Bottom View"})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:ni.a.design_img,style:{borderRadius:10,width:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"PCB Layout 2D Top Layer View"})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:ni.a.design_img,style:{borderRadius:10,width:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"PCB Layout 2D Bottom Layer View"})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"The optical detector captures the chemiluminescence signal by the use of a PN photodiode. The photodiode reacts to the brightness by generating electric current under reverse conditions. This current is then converted to voltage and amplified by the transimpedance amplifier. The output signal from the amplifier passes through an analog-to-digital converter to undergo digitization and processing. With the signal in digital form, the microcontroller reads the voltage at the pin and sends the value to the desktop running the software. In our set-up, the microcontroller reads and sends a thousand voltage values per second. The software converts the voltage values into the analyte concentration using the appropriate conversion and displays the values in an analyte concentration vs. time graph."}),Object(Le.jsx)("p",{children:"This proof of concept demo video shows that the software application graphs change with the light intensity detected by the optical detection circuit. Please note that the current graphed concentration values only correlate with detected voltage, and that conversion to concentration has not been implemented."})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("iframe",{src:"https://static.igem.org/mediawiki/2021/d/d9/T--Waterloo--ProofOfConceptDemoVideo.mp4",style:{width:900,height:500}})})]}),Object(Le.jsxs)(Ie,{open:!0,title:"Test",children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Hardware and Serial Port Communication"})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"The proof of concept demo video shows that the connection between desktop application and the optical detection circuit is successful, and that displayed graph varies with detected light intensity."})}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Software"})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsxs)("p",{children:["When run with the ",Object(Le.jsx)("code",{children:"--no-serial"})," argument, the user may manually simulate microcontroller input by entering data in the format ",Object(Le.jsx)("code",{children:"[ANALYTE] [VOLTAGE]"})," and watching the graph respond in real time. We tested the software by inputting various data points and verified that they are plotted as expected."]})}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("img",{src:"",className:ni.a.design_img,style:{borderRadius:10}})}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("img",{src:"",className:ni.a.design_img,style:{borderRadius:10,width:"80%",height:"80%",marginLeft:"10%"}})}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("img",{src:"",className:ni.a.design_img,style:{borderRadius:10}})}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("img",{src:"",className:ni.a.design_img,style:{borderRadius:10,width:"80%",height:"80%",marginLeft:"10%"}})})]}),Object(Le.jsxs)(Ie,{open:!0,title:"Learn",children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Errors and Optimization"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Hardware"})}),Object(Le.jsx)("p",{children:"Vishay stated that the values on the photodiode data sheet are for a CIE\u200b\u200b Illuminant A, which is a typical tungsten-filament light (S. Ahmed, personal communication, October 11, 2021; International Commision on Illumination, n.d.). Although we were not able to test the full set-up which would include the microfluidic assay, hardware, and software, it is safe to say that this photodiode was not designed with the purpose of detecting chemiluminescence. In future versions of our project, it would be best to replace this photodiode with a photomultiplier tube that is designed to detect chemiluminescence. If other photodiodes do not exist for this purpose, we could do more research on circuits designed specifically to detect chemiluminescence to better adapt our current circuit for our purposes. The emission spectra for luminescent reagents provided with the reagents may be helpful information in designing a better circuit."}),Object(Le.jsx)("p",{children:"Naturally, circuit components can vary in performance due to limitations of the hardware. A possible optimization we can make is to replace existing parts with more advanced, precise, and sensitive parts. We could even commission circuit parts custom-made for our purposes if we had the budget to do so."}),Object(Le.jsx)("p",{children:"There are plenty of factors that affect the hardware performance. It is important to keep the factors consistent so tests do not vary in their result for any factor other than sample analyte concentration. This can be done by building a casing for the microfluidic assay and hardware. We would need to take these factors into account when designing the casing. One factor is the angle of the photodiode relative to the reaction chamber. The photodiode is most sensitive when the light hits perpendicular to the radiant sensitive area so we would need to be careful when constructing a casing for the setup to ensure that the reaction chamber is perpendicular to the diode. Another factor is the temperature, which affects how much current can flow through a photodiode. We could design the casing so that the diode is insulated from the rest of the circuit, keeping the temperature near the diode as constant as possible. The calibration step also helps ensure consistent performance by determining the voltage-to-concentration conversion in the test environment."}),Object(Le.jsx)("p",{children:"For possible improvements that can be made for the circuit, applying a standard +5V DC bias to the photodiode will be necessary in future designs to operate the photodiode in reverse bias under photoconductive mode. This will improve the response time, signal-to-noise ratio, and maintain linearity in the relationship between the intensity of incident light and output voltage. With the bias, the consequence of increasing the dark current must be carefully considered in the design to retain signal integrity."}),Object(Le.jsx)("p",{children:"With the external voltage applied, it is recommended to increase the output voltage to a standard swing of 0V to +10V. This can be accomplished by using a new, +10V single-supply operational amplifier with specifications that match the newly calculated gain-bandwidth product. In addition, increasing the reference voltage of the ADC to +10V is recommended to include the full output range for digitization."}),Object(Le.jsx)("p",{children:"In the current system, all components are being supplied from the +5V pin of the Arduino Micro, which acquires power from its USB port. This source can involve lots of noise that will directly affect the accuracy of the ADC as it uses this supply as its reference voltage. Therefore, considerations for an external, non-USB power supply and voltage regulation will provide a steady and clean supply for the circuit components and improve the signal-to-noise ratio. With now two lines of supplies in +5V and +10V in the system, implementing boost/buck converters and regulating the respective voltages will be necessary."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Software"})}),Object(Le.jsx)("p",{children:"While the desktop app can be extended as needed if a usage requires it, some new features which would be generally useful are supporting a togglable per-analyte function to map from voltage to concentration and more robust data analytics tools (such as the ability to easily plot moving averages)."}),Object(Le.jsx)("p",{children:"We could do more to reduce noise in the data. Most, if not all, of the noise reduction is done by the circuit parts. We could optimize this by getting parts with better noise-reducing qualities, but we could also look into noise-reduction algorithms. Implementing an existing algorithm or designing and implementing our own would provide the tester with more refined and readable data."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:0},children:"Calibration"})}),Object(Le.jsx)("p",{children:"As mentioned in the calibration section, the calculation depends on the assumption that all proteins either bind to analyte in the sample or analyte in the reaction chamber. It is possible that a protein does not bind to any analyte. To decrease the chance of this, the microfluidic chip should maximize mixing so most, if not all, analyte molecules in the sample are binded to. In addition, the reaction chamber should also contain an equal, if not greater, amount of analyte to maximize the chance that free-floating proteins bind to an analyte molecule."}),Object(Le.jsx)("p",{children:"The calibration also relies on very precise amounts of reagents. There is a limit to how accurate manufacturing equipment can be so there will inevitably be some error. The calibration also relies on a very precise sample volume. Since testers input the sample, there is a higher chance of error. Testers can minimize this by ensuring only trained individuals conduct the test and by using precise lab tools, taking care to minimize air bubbles. We could also design the microfluidic assay such that it can only contain the exact volume of sample needed. The testers would simply need to fill the chip until it overflows. The excess would not contribute to the reaction."}),Object(Le.jsx)("p",{children:"As previously mentioned, the calibration step helps ensure consistent performance by determining the conversion in the test environment. However, since it is done before the test, it would not adjust for changes in the test environment during the test. For example, the circuit could heat up during the test, causing a rise in ambient temperature, which would affect the diode\u2019s performance. We could implement a sort of \u201creal-time calibration\u201d. This would mean that the calibration reaction would occur at the same time or slightly ahead of the actual test so the setup could recalculate the conversion during the test. This way, the conversion would better reflect the test conditions at that point in time."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Next Steps"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"As mentioned in the previous section, designing, building, and testing a casing for the assay and the hardware is a next step necessary for building a fully-working setup."}),Object(Le.jsx)("p",{children:"There is also more to be done about the voltage-to-analyte concentration conversion. Firstly, we need to experimentally determine the relationship between chemiluminescent substrate and light intensity, which we currently assume to be linear. The other step is to implement the calibration step as described previously so we could test and optimize this step."}),Object(Le.jsx)("p",{children:"With lab access, proper materials, and a fully-functional setup, we could experimentally determine more details about our design, such as the limit of detection, sensitivity, and a more precise readout time. We could use this information to further optimize our design and circuit parts. This would also inform users about test limitations so they can make more informed diagnoses."})]})]})]})}}]),i}(n.Component),ri=(i(91),function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsx)("div",{className:pe.a.text_div,style:{marginBottom:40},children:Object(Le.jsx)("span",{className:pe.a.text_heading,style:{paddingBottom:0},children:"References"})}),Object(Le.jsx)(Ie,{open:!0,title:"Microfluidic Chip Design",children:Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:Object(Le.jsxs)("ul",{className:pe.a.description,style:{marginTop:0},children:[Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Aghvamia, S. A., Opathalagea, A., Zhang, Z. K., Ludwig, M., Heymann M., Norton, M., Wilkins, N., & Fraden, S. (August 2017). Rapid prototyping of cyclic olefin copolymer (COC) microfluidic devices. ",Object(Le.jsx)("em",{children:"Sensors and Actuators B: Chemical, 247"}),"(4), ",Object(Le.jsx)("a",{href:"https://doi.org/10.1016/j.snb.2017.03.023",children:"https://doi.org/10.1016/j.snb.2017.03.023"}),"."]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Barley, J. (n.d.). ",Object(Le.jsx)("em",{children:"Basic principles of freeze drying"}),". SP Scientific. Retrieved October 10, 2021, from ",Object(Le.jsx)("a",{href:"https://www.spscientific.com/freeze-drying-lyophilization-basics/",children:"https://www.spscientific.com/freeze-drying-lyophilization-basics/"})]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Beyzavi, K., Hampton, S., Kwasowski, P., Fickling, S., Marks, V., & Clift, R. (1987). Comparison of horseradish peroxidase and alkaline phosphatase-labelled antibodies in enzyme immunoassays. ",Object(Le.jsx)("em",{children:"Annals of Clinical Biochemistry"}),", ",Object(Le.jsx)("em",{children:"24"}),", 145-152. doi:10.1177/000456328702400204"]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Chang, Y., Xu, J., & Zhang, Q. (2017). Microplate magnetic chemiluminescence immunoassay for detecting urinary survivin in bladder cancer. ",Object(Le.jsx)("em",{children:"Oncology Letters"}),", ",Object(Le.jsx)("em",{children:"14"}),"(4), 4043-4052. doi:10.3892/ol.2017.6675"]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Chattopadhyay, K., & Mazumdar, S. (2000). Structural and conformational stability of horseradish peroxidase:\u2009 effect of temperature and pH. ",Object(Le.jsx)("em",{children:"Biochemistry"}),", ",Object(Le.jsx)("em",{children:"39"}),"(1), 263-270. doi:10.1021/bi990729o"]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Eamudomkarn, C., Sithithaworn, P., Kamamia, C., Yakovleva, A., Sithithaworn, J., Kaewkes, S., Techasen, A., Loilome, W., Yongvanit, P., Wangboon, C., Saichua, P., Itoh, M., & Bethony, J. M. (2018). Diagnostic performance of urinary IGG Antibody Detection: A novel approach for population screening of strongyloidiasis. ",Object(Le.jsx)("em",{children:"PloS One"}),", ",Object(Le.jsx)("em",{children:"13"}),"(7), e0192598-e0192598. doi:10.1371/journal.pone.0192598 ",Object(Le.jsx)("a",{href:"https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0192598",children:"https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0192598"}),"."]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Ghosh, S., & Ahn, C. (2019). Lyophilization of chemiluminescent substrate reagents for high-sensitive microchannel-based lateral flow assay (MLFA) in point-of-care (POC) diagnostic system. ",Object(Le.jsx)("em",{children:"Analyst"}),", ",Object(Le.jsx)("em",{children:"144"}),"(6): 2109."]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Ghosh, S., Aggarwal, K., Vinitha, T. U., Nguyen, T., Han, J., & Ahn, C. H. (2020). A new microchannel capillary flow assay (MCFA) platform with lyophilized chemiluminescence reagents for a smartphone-based POCT detecting malaria. ",Object(Le.jsx)("em",{children:"Microsystems & Nanoengineering"}),", ",Object(Le.jsx)("em",{children:"6"}),"(1), 5. doi:10.1038/s41378-019-0108-8"]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Heo, Y., Shin, K., Park, M. C., & Kang, J. Y. (2021). Photooxidation-induced fluorescence amplification system for an ultra-sensitive enzyme-linked immunosorbent assay (ELISA). 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Transposing Lateral Flow Immunoassays to Capillary-Driven Microfluidics Using Self-Coalescence Modules and Capillary-Assembled Receptor Carriers [Abstract]. Analytical Chemistry, 92(1), 940-946. ",Object(Le.jsx)("a",{href:"https://doi.org/10.1021/acs.analchem.9b03792",children:"https://doi.org/10.1021/acs.analchem.9b03792"})]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["International Commission on Illumination. (n.d.). Colorimetric Illuminants. ",Object(Le.jsx)("a",{href:"https://cie.co.at/publications/colorimetric-illuminants",children:"https://cie.co.at/publications/colorimetric-illuminants"})]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Jacinto, M. J., Trabuco, J. R. C., Vu, B. V., Garvey, G., Khodadadi, M., Azevedo, A. M., Aires-Barros, M. R., Chang, L., Kourentzi, K., Litvinov, D., & Willson, R. C. (2018). Enhancement of lateral flow assay performance by electromagnetic relocation of reporter particles. PLOS ONE, 14(3). 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Increased urine phenylethylamine after methylphenidate treatment in children with ADHD. ",Object(Le.jsx)("em",{children:"Annals of neurology, 52"}),"(3), 372\u2013374. ",Object(Le.jsx)("a",{href:"https://doi.org/10.1002/ana.10302",children:"https://doi.org/10.1002/ana.10302"})," "]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Li, Zhao., Chen, H., & Wang, P. (2019). Lateral Flow Assay Ruler for Quantitative and Rapid Point-of-care Testing. Analyst, 144(10), 3314-3322. ",Object(Le.jsx)("a",{href:"https://doi.org/10.1039/c9an00374f",children:"https://doi.org/10.1039/c9an00374f"})]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Lin, B., Guan, Z., Song, Y., Song, E., Lu, Z., Liu, D., An, Y., Zhu, Z., Zhou, L., & Yang, C. (2018). Lateral flow assay with pressure meter readout for rapid point-of-care detection of disease-associated protein [Abstract]. Lab chip, 18(6), 965-970. ",Object(Le.jsx)("a",{href:"https://doi.org/10.1039/c8lc00010g",children:"https://doi.org/10.1039/c8lc00010g"})]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Microchip Technology. (2011). MCP3302/04. ",Object(Le.jsx)("a",{href:"https://ww1.microchip.com/downloads/en/DeviceDoc/21697F.pdf",children:"https://ww1.microchip.com/downloads/en/DeviceDoc/21697F.pdf"})]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Moyano, A., Serrano-Pertierra, E., Salvador, M., Mart\xednez-Garc\xeda, J. C., Rivas, M., & Blanco-L\xf3pez, M. C. (2020). Magnetic Lateral Flow Immunoassays. Diagnostics, 10(5), 288. ",Object(Le.jsx)("a",{href:"https://doi.org/10.3390/diagnostics10050288",children:"https://doi.org/10.3390/diagnostics10050288"})]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Qu, Z., Wang K., Alfranca, G., de la Fuente, J. M., & Cui, D. (2020). A plasmonic thermal sensing based portable device for lateral flow assay detection and quantification. Nanoscale Research Letters, 15(1), 10. ",Object(Le.jsx)("a",{href:"https://doi.org/10.1186/s11671-019-3240-3",children:"https://doi.org/10.1186/s11671-019-3240-3"})]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Ramachandran, S., Fu, E., Lutz, B., & Yager, P. (2014). Long-term dry storage of an enzyme-based reagent system for ELISA in point-of-care devices [Abstract]. Analyst, 139, 1456-1462. ",Object(Le.jsx)("a",{href:"https://doi.org/10.1039/C3AN02296J",children:"https://doi.org/10.1039/C3AN02296J"})]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Strimbu, K., & Tavel, J. A. (2010). What are biomarkers? Current Opinion in HIV and AIDS, 5(6), 463-466. ",Object(Le.jsx)("a",{href:"https://doi.org/10.1097/COH.0b013e32833ed177",children:"https://doi.org/10.1097/COH.0b013e32833ed177"})]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Schwartz, E. (2018, April 30). How to Calculate Light Intensity. Sciencing. ",Object(Le.jsx)("a",{href:"https://sciencing.com/calculate-light-intensity-7240676.html",children:"https://sciencing.com/calculate-light-intensity-7240676.html"})]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Vishay. (2011, August 23). TEMD5080X01 [PDF file]. ",Object(Le.jsx)("a",{href:"https://www.vishay.com/docs/81643/temd5080x01.pdf",children:"https://www.vishay.com/docs/81643/temd5080x01.pdf"})]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Vishay. (2011, November 23). BPW21R [PDF file]. ",Object(Le.jsx)("a",{href:"https://www.vishay.com/docs/81519/bpw21r.pdf",children:"https://www.vishay.com/docs/81519/bpw21r.pdf"})]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Wang, W. (n.d.). Optical Detectors [PowerPoint slides]. University of Washington. ",Object(Le.jsx)("a",{href:"https://depts.washington.edu/mictech/optics/me557/detector.pdf",children:"https://depts.washington.edu/mictech/optics/me557/detector.pdf"})]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Weatherspoon, D. (2017). ",Object(Le.jsx)("em",{children:"Urine 24-Hour Volume Test"}),". Healthline."]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Wu, Z., Fu, Q., Yu, S., Sheng, L., Xu, M., Yao, C., Xiao, W., Li, X., & Tang, Y. (2016). Pt@AuNPs integrated quantitative capillary-based biosensors for point-of-care testing application [Abstract]. Biosensors and Bioelectronics, 85, 657-663. ",Object(Le.jsx)("a",{href:"https://doi.org/10.1016/j.bios.2016.05.074",children:"https://doi.org/10.1016/j.bios.2016.05.074"})]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Yan W., Wang, K., Xu H., Huo, X., Jin, Q., & Cui D. (2019). Machine Learning Approach to Enhance the Performance of MNP-Labeled Lateral Flow Immunoassay. Nano-Micro Letters, 11(1), 7. \u200b\u200b",Object(Le.jsx)("a",{href:"https://doi.org/10.1007/s40820-019-0239-3",children:"https://doi.org/10.1007/s40820-019-0239-3"})]})}),Object(Le.jsx)("li",{children:Object(Le.jsxs)("p",{children:["Zhan, L., Granade, T., Liu, Y., Wei, X., Youngpairoj, A., Sullivan, V., Johnson, J., & Bischof, J. (2020). Development and optimization of thermal contrast amplification lateral flow immunoassays for ultrasensitive HIV p24 protein detection. Microsystems & Nanoengineering, 6, 54. ",Object(Le.jsx)("a",{href:"https://doi.org/10.1038/s41378-020-0168-9",children:"https://doi.org/10.1038/s41378-020-0168-9"})]})})]})})})})]})}}]),i}(n.Component)),si=["Microfluidic Chip Design","Biomarker Detection: Fusion Protein Design","Protein Optimization","Gene Marker Detection: Design of mRNA-Binding CRISPR-Cas System","Optical Detector and Application Development","References"],oi=["","","","","",""],ci=function(){var e=be(Object(n.useState)(si[0]),2),t=e[0],i=e[1];return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.heading_div,children:[Object(Le.jsxs)("div",{className:pe.a.title,children:[Object(Le.jsx)("div",{className:pe.a.page_heading,children:"NeuroDetech"}),Object(Le.jsx)("div",{className:pe.a.page_heading_colored,children:"Engineering."})]}),Object(Le.jsx)("div",{className:pe.a.illustration,children:Object(Le.jsx)("img",{src:"",alt:"Engineering Icon",className:Et.a.icon_img})})]}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("span",{className:pe.a.text_heading,children:"Overview"}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"The engineering design cycle was used numerous times to facilitate the design of the key components of NeuroDetech, where an engineering design cycle often led into another distinct but related cycle. Firstly, an engineering design cycle was used in the design, CAD modelling, and fluid dynamics testing of the microfluidic assay chip (Microfluidic Chip Design). After finalizing the design of the microfluidic assay, it then became necessary to use the engineering design cycle to design the fusion protein for biomolecule detection (Biomarker Detection: Fusion Protein Design, which leads into Protein Optimization), a CRISPR-Cas system for gene marker detection (Gene Marker Detection: Design of mRNA-Binding CRISPR-Cas System), and the optical detector/app used for signal interpretation and quantification (Optical Detector and Application Development)."})})]}),Object(Le.jsx)("div",{className:pe.a.sections_div,children:si.map((function(e,n){return Object(Le.jsx)("div",{active:t===e,onClick:function(){return i(e)},className:pe.a.section_block,children:Object(Le.jsxs)("div",{className:pe.a.sections,children:[Object(Le.jsx)("div",{className:pe.a.section_img,children:Object(Le.jsx)("img",{id:"engineering".concat(n),src:oi[n],alt:"Icon"})}),Object(Le.jsx)("div",{className:pe.a.section_text,children:e})]})},e)}))}),Object(Le.jsxs)("div",{children:["Microfluidic Chip Design"===t&&Object(Le.jsx)(Lt,{}),"Biomarker Detection: Fusion Protein Design"===t&&Object(Le.jsx)(Wt,{}),"Protein Optimization"===t&&Object(Le.jsx)(Qt,{}),"Gene Marker Detection: Design of mRNA-Binding CRISPR-Cas System"===t&&Object(Le.jsx)(ti,{}),"Optical Detector and Application Development"===t&&Object(Le.jsx)(ai,{}),"References"===t&&Object(Le.jsx)(ri,{})]})]})},li=i(59),di=i.n(li),hi=i(36),ui=i.n(hi),mi=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Potential Cost to Patients"}),Object(Le.jsx)("div",{className:pe.a.description,children:"To a working individual earning minimum wage in Ontario, this is equivalent to 140 hours of time at work before tax. Unfortunately, the cost of an ADHD assessment may lead to some individuals having no option but to opt out of the assessment due to affordability issues and financial strain (Centre for ADHD Awareness Canada [CADDAC], 2013). This potential financial hurdle can result in further under-diagnoses of both children and adults with ADHD, preventing them from receiving the treatment they need (CADDAC, 2013). As such, the design process of NeuroDetech required us to consider and assess factors such as the financial impact on patients, lab manufacturing, and governmental assistance."}),Object(Le.jsx)("div",{className:pe.a.description,children:"Individuals currently undergoing an assessment for ADHD in Ontario, Canada must pay for all expenses themselves, unless they have private insurance, for any psychological tests done (Government of Ontario, n.d). All out of pocket costs paid by the patient include clinical assessment, and further possible standardized assessments such as a neurocognitive or psychodiagnostic testing (Government of Ontario, n.d). The only tests covered by OHIP for patients undergoing the ADHD assessment are the laboratory blood tests ordered for inpatients at hospitals to rule out any additional physiological conditions (Government of Ontario, n.d)."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("div",{className:pe.a.figure_text,children:" Table 1: Potential costs to individuals undergoing ADHD Assessment"}),Object(Le.jsxs)(Zt,{bordered:!0,className:ui.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:"Cost Source"}),Object(Le.jsx)("th",{children:"Costs"})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Initial Clinical Assessment"}),Object(Le.jsx)("td",{children:"$ 2,000.00"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Standardized Testing"}),Object(Le.jsx)("td",{children:"$ 2,500.00"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Average Time Cost (Considering Minimum Wage)"}),Object(Le.jsx)("td",{children:"$ 181.77"})]})]})]})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("div",{className:pe.a.description,children:"Table 1 displays a sum of the potential costs that an individual undergoing the ADHD assessment may have to pay. The sum in the table varies, as standardized testing and time of cost may be an additional cost to the initial clinical assessment depending on the severity of ADHD, according to stakeholder feedback. The standardized testing cost refers to the cost of a neurocognitive test or psychodiagnostic test. The time cost refers to the time given up from an individual\u2019s workplace. The average time of the cost was derived from expert stakeholder information."})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("div",{className:ui.a.crop,children:Object(Le.jsx)("img",{src:"",alt:"Cost Factors",className:ui.a.cost_img})}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Cost factors of ADHD diagnosis (Created in BioRender)"})]}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.description,children:"At first, NeuroDetech was trying to tackle the problem of reducing the cost of the clinical assessment by making a \u201ccheaper test\u201d. However, it is apparent from stakeholders in the industry that a single quantitative test alone is not adequate in diagnosing ADHD. Therefore, due to needing a qualitative test such as the clinical assessment, the current psychological expenses will still remain what they are for the patient. Nonetheless, NeuroDetech will be used as a quantitative test to aid in the diagnosis of ADHD to provide reassurance, as well as added verification to the patient and the assessor."}),Object(Le.jsx)("div",{className:pe.a.description,children:"After assessing how NeuroDetech will financially impact patients, two possible situations are plausible. The first is that the patient will pay the full cost of the NeuroDetech test. This is not the outcome NeuroDetech aimed at financially, but with only limited time and resources to fully assess this possibility, it is a scenario that remains possible. The second option is that all NeuroDetech costs are covered by OHIP, leaving no expense to patients."}),Object(Le.jsx)("div",{className:pe.a.description,children:"Let\u2019s consider the former scenario, in which NeuroDetech is not covered under OHIP. Assuming that basic chemicals such as buffers are readily available and don\u2019t add drastically to the cost of NeuroDetech. Using minimal protein purification equipment would approximately cost ~$1700, along with one time lab costs of ~$400-500 for PEA (Phenethylamine) and ~$1,100-1,200 for the CRISPR Cas13 fusion protein (LabOnTheCheap, 2018). Without considering protein purification costs, the cost of producing 50 microfluidic assays approximately comes out to $11.67 considering the per litre cell culture cost. Including protein purification materials which can cost around ~$550, this brings the one time costs to ~$3400 (for protein purification equipment, PEA, and CRISPR Cas13 fusion protein). Thus, the cost of producing 50 microfluidic assays is ~$22.67. Hence the price for producing one microfluidic assay, aside from the one time cost is 0.45$ per chip."}),Object(Le.jsx)("div",{className:pe.a.description,children:"If covered by OHIP, assuming that we use the government's existing protein purification equipment and other lab facilities, the cost of manufacturing mentioned above would be transferred to the government, which is still far cheaper than if OHIP were to cover any other psychological expenses for ADHD diagnosis."}),Object(Le.jsx)("div",{className:pe.a.description,children:"To further analyze the plausibility of OHIP coverage for Neurodetech, we have established a realistic case for it. In order for a test to be covered by OHIP, it must be deemed medically necessary ( Ministry of Health, n.d). Since Neurodetech would be considered a laboratory test, we looked into if the current laboratory tests that are ordered for ADHD assessment are covered by OHIP. After inquiring with Dynacare Laboratories, we determined that current ADHD laboratory tests are covered by OHIP, as well as a comparable fractionated catecholamine urine test. We have hypothesized that if the current laboratory tests are covered by OHIP, as well as similar urine sample tests, then NeuroDetech should be deemed essential as well."})]})]})}}]),i}(n.Component),pi=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Manufacturing and Legal Considerations"}),Object(Le.jsx)("div",{className:pe.a.description,children:"In order to establish a standardized and safe methodology of implementing NeuroDetech, it is essential that this diagnostic test is protected by a patent. Existing under a patent ensures that the exact same product is produced with the same accuracy by one manufacturer. Additionally, protecting NeuroDetech with a patent comes with the ability to outsource manufacturing costs to a 3rd-party pharmaceutical diagnostics company with the technology to produce it at a much lower cost, to ultimately lower the cost of the test to patients. This provides a profitable exchange to both manufacturer (who obtains a share of patent rights) and patient, at the expense of the designer. These costs, however, are absorbed by the fact that more tests are able to be sold, and the fact that it is typically cheaper for the designer to employ contract manufacturing than to build multiple units themselves."})]})})}}]),i}(n.Component),fi=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"References"}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"Centre for ADHD Awareness Canada. (2013, October, 14). Paying Attention to the Cost of ADHD\u2026 The Price Paid by Canadian Families, Governments and Society. https://caddac.ca/wp-content/uploads/2017/01/Socioeconomic-Policy-Paper-1.pdf"}),Object(Le.jsx)("li",{children:"Government of Ontario. (n.d). What OHIP Covers. https://www.ontario.ca/page/what-ohip-covers#section-9"}),Object(Le.jsx)("li",{children:"Ministry of Health. (n.d). Ontario Health Insurance Plan. https://www.health.gov.on.ca/en/public/programs/ohip/ohipfaq_mn.aspx"}),Object(Le.jsx)("li",{children:"LabOnTheCheap. (2018, July 10). Technical Tuesday: Protein purification on a budget: Lab on the cheap. Lab On The Cheap |. Retrieved October 7, 2021, from https://www.labonthecheap.com/protein-purification-on-a-budget/. "})]})]})})}}]),i}(n.Component),bi=["Potential Cost to Patients","Manufacturing and Legal Considerations","References"],ji=["","",""],gi=function(){var e=be(Object(n.useState)(bi[0]),2),t=e[0],i=e[1];return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.heading_div,children:[Object(Le.jsxs)("div",{className:pe.a.title,children:[Object(Le.jsx)("div",{className:pe.a.page_heading,children:"NeuroDetech"}),Object(Le.jsx)("div",{className:pe.a.page_heading_colored,children:"Finance."})]}),Object(Le.jsx)("div",{className:pe.a.illustration,children:Object(Le.jsx)("img",{src:"",alt:"Finance Icon",className:di.a.icon_img})})]}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Overview"}),Object(Le.jsx)("div",{className:pe.a.description,children:"For many people, having an extra $2,000 dollars in their pocket could mean paying for school tuition, 2 months worth of rent, a laptop, or a flight to Asia. To an individual with possible underlying ADHD symptoms, this could mean covering the cost of an initial clinical assessment, as a first step towards receiving an official diagnosis. We knew that implementing NeuroDetech into the diagnosis procedures for ADHD would contribute an additional cost to the patient. As such, we decided to investigate what that cost would be and whether we could minimize it through the use of health insurance."})]}),Object(Le.jsx)("div",{className:pe.a.sections_div,children:bi.map((function(e,n){return Object(Le.jsx)("div",{active:t===e,onClick:function(){return i(e)},className:pe.a.section_block,children:Object(Le.jsxs)("div",{className:pe.a.sections,children:[Object(Le.jsx)("div",{className:pe.a.section_img,children:Object(Le.jsx)("img",{id:"finance".concat(n),src:ji[n],alt:"Icon"})}),Object(Le.jsx)("div",{className:pe.a.section_text,children:e})]})},e)}))}),Object(Le.jsxs)("div",{children:[t===bi[0]&&Object(Le.jsx)(mi,{}),t===bi[1]&&Object(Le.jsx)(pi,{}),t===bi[2]&&Object(Le.jsx)(fi,{})]})]})},xi=i(14),vi=i.n(xi),yi=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Microfluidic Capillary Assay Chip Design"}),Object(Le.jsx)("div",{className:pe.a.description,children:"In order to establish a standardized and safe methodology of implementing NeuroDetech, it is essential that this diagnostic test is protected by a patent. Existing under a patent ensures that the exact same product is produced with the same accuracy by one manufacturer. Additionally, protecting NeuroDetech with a patent comes with the ability to outsource manufacturing costs to a 3rd-party pharmaceutical diagnostics company with the technology to produce it at a much lower cost, to ultimately lower the cost of the test to patients. This provides a profitable exchange to both manufacturer (who obtains a share of patent rights) and patient, at the expense of the designer. These costs, however, are absorbed by the fact that more tests are able to be sold, and the fact that it is typically cheaper for the designer to employ contract manufacturing than to build multiple units themselves."}),Object(Le.jsxs)(Ie,{open:!0,title:"Overall Chip Mechanical Design",children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:"0"},children:"Microfluidic Chip Mechanical Design"})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"The main inspiration for the design of the NeuroDetech lab chip is a similar point-of-care diagnostic device for infectious diseases created by Ghosh et al. (2020). The testing device designed by Ghosh et al. (2020) prioritized functions such as accessibility and speed which are applicable to NeuroDetech. Hence many concepts from this paper, like the device dimensions and parts sequence, are incorporated into our work. Overall, the objective of our work is to create a user-friendly tool for quantitative biomolecular detection. (Ghosh et al., 2020)"}),Object(Le.jsx)("p",{children:"While designing the device, multiple factors were taken into account, including flow and reaction rate. The concentration of PEA in the sample is low, which can be difficult to detect. In the design of the chip, both capillary and surface tension forces contribute to the generation of transient flow within the microstructures due to the pressure gradient. The high surface-to-volume ratio increases antigen binding, ensuring more precise and accurate results. In summary, this microfluidic device utilizes many principles of fundamental fluid dynamics. The model of the NeuroDetech lab chip is shown below."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:vi.a.design_img,style:{borderRadius:20,width:"80%",height:"80%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Assembly of the microfluidic chip. Note that the top plane surfaces are transparent for easy visualization of the chip inner components."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:vi.a.design_img,style:{borderRadius:20,width:"50%",height:"50%",marginLeft:"25%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Full assembly of the microfluidic chip. Fluid flows from the top left through the capillary pump through to the spiral control chamber, then to the spiral test chamber. At the same time, fluid flows from the top right through the drying chamber and into the spiral test chamber."})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subsubheading,children:"Sample Loading Chamber and Drying Chamber Design"})}),Object(Le.jsx)("p",{children:"The geometry of the NeuroDetech lab chip is critical to the chip\u2019s function. The test begins when the sample is deposited into the loading chamber through a sample loading port located at the top of the chip. A hydrophobic spot in the middle of the loading chamber splits the sample into two equal parts (Ghosh et al., 2020). Separate pathways move the samples toward a series of small tubes then into the drying chambers."}),Object(Le.jsx)("p",{children:"Path one leads to the binding protein drying chamber, and path two leads to the chemiluminescent substrate drying chamber. There are 10 tubes leading to the binding protein drying chamber and 7 tubes leading to the chemiluminescent substrate drying chamber. The tubes help maintain pressure to transfer fluid into the drying chambers and prevent unwanted backflow into the loading chamber."}),Object(Le.jsx)("p",{children:"Both drying chambers have a grid of rectangular pillars to increase mixing, providing binding proteins with sufficient time and space to bind to target biomarkers (Ghosh et al., 2020). The chemiluminescent substrate drying chamber has a row of tubes separating sections of the grid to maintain fluid pressure; this also ensures that fluid does not enter the test chamber before the binding protein fluid. Ramps descending into and ascending out of the drying chambers help retain fluid in the drying chamber for a sufficient period of time for uniform mixing and drying (Ghosh et al., 2020)."}),Object(Le.jsx)("p",{children:"The drying chambers connect to a series of delay valves with initially small diameters that progressively enlarge by widening the tube diameter; the fluid meniscus is thus increased in size, consequently decreasing capillary pressure. In turn, this helps ensure that no air bubbles form as the solution flows to the test chamber. The chemiluminescent substrate drying chamber pathway has a long serpentine delay tube in addition to the traditional delay valves, resulting in all the fluid from path two entering the reaction chamber only after all the fluid from path one has entered (Ghosh et al., 2020)."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:vi.a.design_img,style:{borderRadius:20,marginLeft:"5%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Annotated loading and drying chamber."})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subsubheading,children:"Capillary Pump Design"})}),Object(Le.jsx)("p",{children:"Based on our previous research and referenced studies, few considerations were determined to be particularly important for fluid flow in the capillary pump. Through background research, we identified factors that affect fluid flow in a capillary pump. These include wettability, frequency of microstructures in the flow chamber, and the geometry of the microstructures used to increase flow resistance. These factors are the foundation for determining flow rates due to capillary flow (Olanrewaju et al., 2018)."}),Object(Le.jsx)("p",{children:"In general, more densely packed orientations of microstructures lead to higher flow resistance, in turn increasing flow pressure. Some of the key microstructure geometries commonly found in capillary pumps include tree lines, symmetric lines, balled lines, and posts. These structures must be small enough to generate enough capillary pressure for flow through the chamber (Olanrewaju et al., 2018)."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:vi.a.design_img,style:{borderRadius:10}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Various Capillary Pump Designs (Olanrewaju et al., 2018)"})]}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"We chose to compare three different microstructures and their effect on fluid flow: (1) symmetric oblong shapes (400 by 200\u03bcm); (2) treelines in the shape of a hexagon (thickness 1mm); and (3) balled lines (radius 500 \u03bcm, lines 1mm)."})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsxs)("div",{style:{display:"flex",flexDirection:"row",justifyContent:"space-around",flexWrap:"wrap"},children:[Object(Le.jsx)("img",{src:"",className:vi.a.design_img,style:{borderRadius:20,width:"28%",height:"28%"}}),Object(Le.jsx)("img",{src:"",className:vi.a.design_img,style:{borderRadius:20,width:"26%",height:"26%"}}),Object(Le.jsx)("img",{src:"",className:vi.a.design_img,style:{borderRadius:20,width:"27%",height:"27%"}})]}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"From left to right: Oblong microstructures. Treeline microstructures. Balled lines microstructures."})]}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"The section housing these different microstructure designs are connected in series with a serpentine channel section and a spherical microstructure capillary chamber. The purposes of these additional chambers are to (1) prevent air bubbles from moving from the main capillary channel to the reaction chambers downstream and perhaps (2) to create a more uniform flow of fluid as it moves from a high capillary pressure area to the reaction chambers downstream (Ghosh et al., 2020) (Olanrewaju et al., 2018). The detailed motivations for each of these shapes are discussed in their respective sections below."})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:vi.a.design_img,style:{borderRadius:20,width:"70%",height:"70%",marginLeft:"15%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Sample of the lower common components of the capillary pump with oblong microstructures as an example."})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subsubheading,children:"Capillary Pump Design: Iteration 1"})}),Object(Le.jsx)("p",{children:"Our initial iteration utilized a high-pressure capillary pump consisting of oblong shapes that are 400 by 200 \u03bcm. These serve as posts in the chamber. According to Ghosh et al. (2020) [a], the motivation for a high-pressure capillary pump is to prevent reagents from accumulating in and around the edges of the posts and walls. This also facilitates rapid fluid flow from the openings to the reaction chambers."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:vi.a.design_img,style:{borderRadius:20,width:"100%",height:"100%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Full image of iteration 1."})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"Some benefits of this design include high-pressure creation and strong mixing of the reagent in the capillary pump chamber. Some drawbacks of this design include its relatively large size and the more condensed microstructures. This could pose challenges during manufacturing. Smaller microstructures require more precise tools and may have an impact on manufacturing methods and cost."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subsubheading,children:"Capillary Pump Design: Iteration 2"})}),Object(Le.jsx)("p",{children:"Our second design is quite different from our first design in terms of its shape and purpose. Treeline designs are typically used to pump fluid through chambers for assay purposes, but can result in different capillary pressures in different areas of the pump. For example, the pressure in a long branch of a chamber will have accumulated much higher pressure than in the meeting point of a few branches, where the shape is less uniform and the area is bigger (Olanrewaju et al., 2018)."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:vi.a.design_img,style:{borderRadius:20,width:"100%",height:"100%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Full image of iteration 2."})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"A problem that may arise with this design is lower capillary pressure due to wider branches (compared to the oblong microstructures in the previous design). However, this design is easier to manufacture in terms of precision and material costs. If we require more delays on our chip, this design could potentially provide bubbleless, even fluid flow."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subsubheading,children:"Capillary Pump Design: Iteration 3"})}),Object(Le.jsx)("p",{children:"The last iteration of our capillary pump design utilizes a combination of circular posts and linear walls, forming a dumbbell shape. According to literature, balled lines have lower flow rates than treelines (Zimmermann et al., 2007). It is noted that it may be filled in an orientation perpendicular to the inlet shown in Figure 8 below, with fluid flow facing the round post, parallel to the lines. This is because the presence of a wall between round posts will greatly limit the flow of a liquid."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:vi.a.design_img,style:{borderRadius:20,width:"100%",height:"100%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Full image of iteration 3."})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"Similar to the tree line design, a problem with this design is its lower capillary pressure compared to the symmetric oblongs. This, combined with the slightly shorter chamber length of designs 2 and 3, may cause the pump to not form any positive pressure at all, resulting in a failing capillary pump. A benefit of this design is the increased distribution of fluid flow across the whole capillary chamber. There is a lower risk of fluid only flowing along a linear path at any point in the chamber since the shape of the microstructures is so irregular. Compared to symmetric lines (iteration 2), there is a lower risk of undisturbed flow happening in the chamber (Zimmermann et al., 2007)."}),Object(Le.jsx)("p",{children:"Due to the high capillary pressure generation and suitability for reagent mixing, the oblong capillary pump structure is chosen for the final chip assembly design."})]})]}),Object(Le.jsx)(Ie,{open:!0,title:"Selection of Chip Material",children:Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"We looked into multiple materials in order to determine whether they fulfilled certain characteristics, such as surface hydrophilicity and ability to be biotinylated, while still being cost-effective and practical to fabricate. In order to evaluate possible candidates, we researched various materials used in microfluidic chips and outlined the characteristics of each candidate: glass, PDMS (polydimethylsiloxane), COC (cyclic olefin copolymer), and thiol-ene polymers."}),Object(Le.jsx)("p",{children:"The first material that we looked into was glass. On one hand, glass is chemically inert, biocompatible, and impermeable to gas. Additionally, it is easy to biotinylate and has excellent optical transparency (Ghosh et al., 2020). On the other hand, glass is relatively expensive, and its hardness makes it difficult to construct and etch (Ghosh et al., 2020)."}),Object(Le.jsx)("p",{children:"The second material that we looked into was PDMS, also known as polydimethylsiloxane. PDMS is easy to fabricate and inexpensive. It is currently the most commonly used material in microfluidic chips. Additionally, it is gas-permeable, biocompatible, and non-toxic. It can be biotinylated, and complex microfluidic designs can be made by stacking layers (Ghosh et al., 2020). PDMS is naturally hydrophobic, but it can be treated to control the hydrophobicity (Ghosh et al., 2020)."}),Object(Le.jsx)("p",{children:"Additionally, we also looked into cyclic olefin copolymer, also known as COC. On one hand, this amorphous polymer has good biocompatibility and high transparency. Additionally, it can be biotinylated (Ali Aghvami et al., 2017). Nevertheless, one disadvantage of COC is that hydrophobicity treatments are transient on this material relative to PDMS (Oh et al., 2013). Additionally, the literature on COC surface treatments is much less robust when compared to the literature regarding PDMS (Oh et al., 2013)."}),Object(Le.jsx)("p",{children:"Finally, we looked into thiol-ene polymers. These polymers are easy to fabricate and manipulate, and can be easily biotinylated (Sticker et al., 2020). These polymers, however, are prohibitively expensive, and there are fewer suppliers available for purchase (Sticker et al., 2020)."}),Object(Le.jsx)("p",{children:"We then evaluated the properties of each material, and finally decided to use PDMS as the chip material. PDMS is cost-effective, easy to fabricate and handle, and can be easily biotinylated (Ghosh et al., 2020). While PDMS\u2019s surface is naturally hydrophobic (Tan et al., 2010), it can easily be pre-treated in order to regulate and decrease its hydrophobicity (Tan et al, 2010; Trantidou et al., 2017). These pre-treatments are further explained in the Build section below. Additionally, the literature on the fabrication and pre-treatment of PDMS for microfluidics is extremely robust relative to the other materials."})]})}),Object(Le.jsxs)(Ie,{open:!0,title:"Computer-aided Chip Assembly Design",children:[Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"A complete assembly of the microfluidic chip is built and shown below. The specific dimensions are also labelled and presented. The team used OnShape to create the CAD model throughout the year."})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:vi.a.design_img,style:{borderRadius:20}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Dimensioned drawing of the microfluidic chip."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:vi.a.design_img,style:{borderRadius:20}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Assembly of the microfluidic chip. Note that the top plane surfaces are transparent for easy visualization of the chip inner components."})]})]})]})})}}]),i}(n.Component),Oi=i(60),wi=i.n(Oi),Ni=i(6),_i=i.n(Ni),ki=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Optical Detector Development"})}),Object(Le.jsxs)(Ie,{open:!0,title:"Hardware Design",children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:"0"},children:"Simulation"})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"We started off by creating a circuit simulation in TinkerCAD. We adapted some microcontroller code and a simple photodiode circuit taken from various sources. The purpose of this was to get a general idea of how the circuit and microcontroller will work and which parts to order."})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:_i.a.design_img,style:{borderRadius:10,width:"80%",height:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Labelled TinkerCAD circuit simulation."})]}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"In this early simulated prototype, the integers printed to the serial monitor increased as the photodiode was exposed to less light and decreased as the light level increased. When the photodiode detected light, it became a conductor and the current flowed to the ground, so the microcontroller read a low voltage level. At a low light level, the photodiode became a resistor and prevented current from flowing to the ground. Instead, the current flowed to the analog pin and the microcontroller read a high voltage (Kranthi, 2013)."})}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("iframe",{src:"https://static.igem.org/mediawiki/2021/8/84/T--Waterloo--TinkerCADVidFinal.mp4",style:{width:900,height:500}})}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:"0"},children:"Circuit Part Selection"})}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subsubheading,children:"Light Sensor"})}),Object(Le.jsxs)("p",{children:["Initially, we considered the use of a photomultiplier tube (PMT) for the detection of resorufin. Resorufin is the fluorescent compound produced in the reaction chambers and has emission maxima of 563 nm. Additionally, the area of the MCFA reaction chamber was 9 mm",Object(Le.jsx)("sup",{children:"2"}),". These two design specifications in peak spectral sensitivity and radiant sensitive area were the main priorities for selecting an optical sensor."]}),Object(Le.jsx)("p",{children:"The high internal amplification characteristic of PMTs, as well as its ability to detect low light levels, prompted the consideration for Hamatsu\u2019s R12900U-01 Micro-PMT. However, with a lower peak spectral sensitivity of 420 nm, the sensitivity and the cost were not aligned with our design goals\u2014a similar trend we observed across all PMT devices (Hamamatsu, 2021). Having to look towards cheaper alternatives in photodiodes instead, we decided upon the PDB-C156 from Advanced Photonix for our medium-fidelity prototype. The PDB-C156 is a through-hole PIN silicon photodiode with a peak spectral sensitivity of 660 nm and response time of 15 ns (Advanced Photonix, n.d.)."}),Object(Le.jsx)("p",{children:"Having no access to the lab this year and no physical fabrication of the MCFA lab chip to test with, the peak spectral sensitivity of the photodiode was not our main priority for the medium-fidelity prototype. Instead, our goal was to test the functionality of the rest of the circuit on the breadboard, and the communication between the microcontroller and the software."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subsubheading,children:"Transimpedance Amplifier"})}),Object(Le.jsx)("p",{children:"Operating the photodiode in reverse and using a general-purpose OPA350PA as a transimpedance amplifier, we wanted to amplify the 90 \u03bcA of short-circuit current of the PDB-C156 photodiode to an output voltage swing of 0V to 5V. To achieve the desired gain, we used a 47k\u03a9 and a 10k\u03a9 resistor in series."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsxs)("div",{className:_i.a.formula_div,children:[Object(Le.jsxs)("div",{className:_i.a.formula_center,children:["R",Object(Le.jsx)("sub",{children:"f"})," = \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsxs)("div",{className:_i.a.top,children:["V",Object(Le.jsx)("sub",{children:"max"})," - V",Object(Le.jsx)("sub",{children:"min"})]}),Object(Le.jsxs)("div",{className:_i.a.bottom,style:{marginLeft:"50%"},children:["I",Object(Le.jsx)("sub",{children:"sc"})]})]}),Object(Le.jsx)("div",{className:_i.a.formula_center,children:"\xa0 = \xa0"}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:_i.a.top,children:"5V - 0V"}),Object(Le.jsx)("div",{className:_i.a.bottom,style:{marginLeft:"10%"},children:"90 \u03bcA"})]}),Object(Le.jsx)("div",{className:_i.a.formula_center,children:"\xa0 = \xa055555.5 \u03a9"})]}),Object(Le.jsxs)("div",{className:_i.a.formula_description,children:[Object(Le.jsxs)("div",{children:["R",Object(Le.jsx)("sub",{children:"f"})," = feedback resistor"]}),Object(Le.jsxs)("div",{children:["V",Object(Le.jsx)("sub",{children:"max"})," = maximum output voltage"]}),Object(Le.jsxs)("div",{children:["V",Object(Le.jsx)("sub",{children:"min"})," = minimum output voltage"]}),Object(Le.jsxs)("div",{children:["I",Object(Le.jsx)("sub",{children:"sc"})," = short circuit current of photodiode"]})]})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subsubheading,children:"Analog-to-Digital Converter"})}),Object(Le.jsx)("p",{children:"To digitize the output voltage signal, we selected the MCP3302-CI/P Analog-to-Digital converter (ADC) for its high resolution and ability to interface over serial peripheral interface (SPI) protocol. With 13-bits of resolution, we favoured the Successive Approximation Register (SAR) architecture of the MCP3303-CI/P over delta-sigma, pipeline, flash, and dual slope architectures due to the perfect balance in bit resolution and sampling rate for photosensing applications. Additionally, the 4 single-ended inputs of the ADC left room for two additional photodiodes on top of the two photodiodes we were prototyping with (Microchip Technology, 2011). We decided to communicate over SPI due to the fast data transmission rate and simplicity. Another reason was that our system consisted of only one master and one slave device in the microcontroller and MCP3302 ADC respectively, which required only one set of communication lines."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subsubheading,children:"Microcontroller"})}),Object(Le.jsx)("p",{children:"For the microcontroller, we decided upon the Arduino Micro as our development board for its compact size, low cost, support for SPI devices, sufficient memory, and clock speed. In addition, existing code for interfacing over SPI with a family of MCP ADCs was available for use and modification."}),Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subsubheading,children:"Developing the High-Fidelity Prototype"})}),Object(Le.jsxs)("p",{children:["To move forward with a higher fidelity prototype, we decided on using Vishay Intertechnology\u2019s BPW21R as our primary optical sensor over the surface-mount SFH 2430-Z from OSRAM Opto Semiconductors. Though a PIN photodiode is favoured over PN photodiodes in this application due to greater responsivity and response speed, the BPW21R most closely met our design goals with a radiant sensitive area of 7.5 mm",Object(Le.jsx)("sup",{children:"2"})," and a peak spectral sensitivity of 565 nm (Vishay, 2011). In comparison, the greater sensitivity, the size of the radiant sensitive area, and the faster response speed were held as advantages over the SFH 2430-Z."]}),Object(Le.jsx)("p",{children:"Recalculating the gain required for the BPW21R:"})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)("div",{className:_i.a.formula_div,children:[Object(Le.jsxs)("div",{className:_i.a.formula_center,children:["R",Object(Le.jsx)("sub",{children:"f"})," = \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsxs)("div",{className:_i.a.top,children:["V",Object(Le.jsx)("sub",{children:"max"})," - V",Object(Le.jsx)("sub",{children:"min"})]}),Object(Le.jsxs)("div",{className:_i.a.bottom,style:{marginLeft:"50%"},children:["I",Object(Le.jsx)("sub",{children:"sc"})]})]}),Object(Le.jsx)("div",{className:_i.a.formula_center,children:"\xa0 = \xa0"}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:_i.a.top,children:"5V - 0V"}),Object(Le.jsx)("div",{className:_i.a.bottom,style:{marginLeft:"15%"},children:"9 \u03bcA"})]}),Object(Le.jsx)("div",{className:_i.a.formula_center,children:"\xa0 = \xa0555555.5 \u03a9"})]})}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"Calculating the bandwidth and feedback capacitor value to implement a low-pass filter for attenuation of signals with undesired frequencies in the trans-impedance configuration:"})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsxs)("div",{className:_i.a.formula_div,style:{marginLeft:"15%"},children:[Object(Le.jsxs)("div",{className:_i.a.formula_center,children:["f",Object(Le.jsx)("sub",{children:"b"})," = \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:_i.a.top,children:"0.35"}),Object(Le.jsxs)("div",{className:_i.a.bottom,style:{marginLeft:"50%"},children:["t",Object(Le.jsx)("sub",{children:"r"})]})]}),Object(Le.jsx)("div",{className:_i.a.formula_center,children:"\xa0 = \xa0"}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:_i.a.top,children:"0.35"}),Object(Le.jsx)("div",{className:_i.a.bottom,children:"3.1 \u03bcs"})]}),Object(Le.jsx)("div",{className:_i.a.formula_center,children:"\xa0 = \xa0112903.226 Hz"})]}),Object(Le.jsxs)("div",{className:_i.a.formula_description,style:{marginBottom:20},children:[Object(Le.jsxs)("div",{children:["f",Object(Le.jsx)("sub",{children:"b"})," = bandwidth"]}),Object(Le.jsxs)("div",{children:["t",Object(Le.jsx)("sub",{children:"r"})," = rise time of photodiode"]})]}),Object(Le.jsxs)("div",{className:_i.a.formula_div,children:[Object(Le.jsxs)("div",{className:_i.a.formula_center,children:["C",Object(Le.jsx)("sub",{children:"f"})," \u2264 \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:_i.a.top,style:{borderBottom:"none",marginLeft:"40%"},children:"1"}),Object(Le.jsxs)("div",{className:_i.a.new_bottom,children:["2\u03c0R",Object(Le.jsx)("sub",{children:"f"}),"f",Object(Le.jsx)("sub",{children:"b"})]})]}),Object(Le.jsx)("div",{className:_i.a.formula_center,children:"\xa0 = \xa0"}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:_i.a.top,style:{borderBottom:"none",marginLeft:"40%"},children:"1"}),Object(Le.jsx)("div",{className:_i.a.new_bottom,children:"2\u03c0(555.5 k\u03a9)(112.903 kHz)"})]}),Object(Le.jsx)("div",{className:_i.a.formula_center,children:"\xa0 = \xa02.5374 pF"})]}),Object(Le.jsx)("div",{className:_i.a.formula_description,children:Object(Le.jsxs)("div",{children:["C",Object(Le.jsx)("sub",{children:"f"})," = feedback capacitor"]})})]}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"In addition, we also switched the transimpedance amplifier to the LMP7717MAE/NOPB to better fit our design specifications. Specifically, we sought a surface-mount amplifier with minimal input offset voltage, input bias current, and a gain-bandwidth product (GBW) of 55 MHz or greater, which the LMP7717MAE/NOPB satisfied. Here are the calculations used to determine these values:"})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("div",{className:_i.a.formula_div,style:{marginLeft:"10%"},children:Object(Le.jsxs)("div",{children:["C",Object(Le.jsx)("sub",{children:"i"})," = C",Object(Le.jsx)("sub",{children:"s"})," + C",Object(Le.jsx)("sub",{children:"d"})," + C",Object(Le.jsx)("sub",{children:"cm"})," = 1200pF + 15pF = 1215pF"]})}),Object(Le.jsxs)("div",{className:_i.a.formula_description,style:{marginBottom:20},children:[Object(Le.jsxs)("div",{children:["C",Object(Le.jsx)("sub",{children:"i"})," = total input capacitance"]}),Object(Le.jsxs)("div",{children:["C",Object(Le.jsx)("sub",{children:"s"})," = input source (diode) capacitance"]}),Object(Le.jsxs)("div",{children:["C",Object(Le.jsx)("sub",{children:"d"})," = differential input capacitance of the amplifier"]}),Object(Le.jsxs)("div",{children:["C",Object(Le.jsx)("sub",{children:"cm"})," = common mode input capacitance"]})]}),Object(Le.jsxs)("div",{className:_i.a.formula_div,children:[Object(Le.jsxs)("div",{className:_i.a.formula_center,children:["GBW ",">"," \xa0"]}),Object(Le.jsxs)("div",{children:[Object(Le.jsxs)("div",{className:_i.a.top,children:["C",Object(Le.jsx)("sub",{children:"i"})," + C",Object(Le.jsx)("sub",{children:"f"})]}),Object(Le.jsxs)("div",{className:_i.a.bottom,children:["2\u03c0R",Object(Le.jsx)("sub",{children:"f"}),"C",Object(Le.jsx)("sub",{children:"f"}),Object(Le.jsx)("sup",{children:"2"})]})]}),Object(Le.jsx)("div",{className:_i.a.formula_center,children:"\xa0 = \xa0"}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("div",{className:_i.a.top,style:{borderBottom:"none",marginLeft:"10%"},children:"1215 pF + 2.5374 pF"}),Object(Le.jsxs)("div",{className:_i.a.new_bottom,children:["2\u03c0(555.5 k\u03a9)(2.5374 pF)",Object(Le.jsx)("sup",{children:"2"})]})]}),Object(Le.jsx)("div",{className:_i.a.formula_center,children:"\xa0 = \xa054.175 MHz"})]})]}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"Finally, we placed standard 0.1 \u03bcF capacitors near the supply pins of the system to serve as a bypass filter and reduce noise. With the new photodiode, transimpedance amplifier, the MCP3302-CI/SL (surface-mount equivalent ADC), and the Arduino Micro, we aimed to design a printed circuit board (PCB) to mount above the header pins of the Arduino Micro as a single unit for the high-fidelity prototype. Using Altium Designer as the PCB design software, we included an additional photodiode and transimpedance amplifier to the schematic, to connect to a separate analog input channel of the four that the MCP3302-CI/SL had available for quantification of a second analyte."})})]}),Object(Le.jsxs)(Ie,{open:!0,title:"Prototype Demo",children:[Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"After the design phase, the initial prototype is developed with chosen circuit parts. Below shows the schematic diagrams, breadboard configuration of the prototype and the final PCB Design of the optical detector."})}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("img",{src:"",className:_i.a.design_img,style:{borderRadius:10,width:"40%",marginLeft:"30%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Breadboard configuration"})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)(Zt,{bordered:!0,className:pe.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:"Cable colour"}),Object(Le.jsx)("th",{children:"Meaning "})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Red"}),Object(Le.jsx)("td",{children:"+5V "})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Black"}),Object(Le.jsx)("td",{children:"GND "})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Yellow"}),Object(Le.jsxs)("td",{children:["Signal V",Object(Le.jsx)("sub",{children:"OUT"})," "]})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Blue"}),Object(Le.jsx)("td",{children:"SCK "})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Green"}),Object(Le.jsx)("td",{children:"MISO "})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Purple"}),Object(Le.jsx)("td",{children:"MOSI "})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"White"}),Object(Le.jsx)("td",{children:"SS "})]})]})]})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:_i.a.design_img,style:{borderRadius:10,width:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Schematic capture (optical detector)"})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:_i.a.design_img,style:{borderRadius:10,width:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Schematic capture (system)"})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:_i.a.design_img,style:{borderRadius:10,width:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"PCB Layout 3D Top View"})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:_i.a.design_img,style:{borderRadius:10,width:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"PCB Layout 3D Bottom View"})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:_i.a.design_img,style:{borderRadius:10,width:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"PCB Layout 2D Top Layer View"})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:_i.a.design_img,style:{borderRadius:10,width:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"PCB Layout 2D Bottom Layer View"})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"The optical detector captures the chemiluminescence signal by the use of a PN photodiode. The photodiode reacts to the brightness by generating electric current under reverse conditions. This current is then converted to voltage and amplified by the transimpedance amplifier. The output signal from the amplifier passes through an analog-to-digital converter to undergo digitization and processing. With the signal in digital form, the microcontroller reads the voltage at the pin and sends the value to the desktop running the software. In our set-up, the microcontroller reads and sends a thousand voltage values per second. The software converts the voltage values into the analyte concentration using the appropriate conversion and displays the values in an analyte concentration vs. time graph."}),Object(Le.jsx)("p",{children:"This proof of concept demo video shows that the software application graphs change with the light intensity detected by the optical detection circuit. Please note that the current graphed concentration values only correlate with detected voltage, and that conversion to concentration has not been implemented."})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("iframe",{src:"https://static.igem.org/mediawiki/2021/d/d9/T--Waterloo--ProofOfConceptDemoVideo.mp4",style:{width:900,height:500}})})]})]})}}]),i}(n.Component),Ai=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"References"}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsxs)("li",{children:["Advanced Photonix. (n.d.). ",Object(Le.jsx)("i",{children:"PDB-C156"})," [PDF file]. https://datasheet.octopart.com/PDB-C156-Advanced-Photonix-datasheet-140068.pdf"]}),Object(Le.jsx)("li",{children:"Arduino. (n.d.). analogRead(). https://www.arduino.cc/reference/en/language/functions/analog-io/analogread/"}),Object(Le.jsxs)("li",{children:["Bhattacharyya, A., & Klapperich, C. M. (2007). Design and testing of a disposable microfluidic chemiluminescent immunoassay for disease biomarkers in human serum samples. ",Object(Le.jsx)("i",{children:"Biomedical Microdevices"}),", 9, 245. https://doi.org/10.1007/s10544-006-9026-2"]}),Object(Le.jsxs)("li",{children:["Digi-Key Electronics. (n.d.). ",Object(Le.jsx)("i",{children:"SFH 2430-Z \u200b\u200bOSRAM Opto Semiconductors Inc"}),". https://www.digikey.ca/en/products/detail/osram-opto-semiconductors-inc/SFH-2430-Z/1668853"]}),Object(Le.jsxs)("li",{children:["Foysal, K. H., Seo, S. E., Kim, M. J., Kwon, O. S., & Chong, J. W. (2019). Analyte Quantity Detection from Lateral Flow Assay Using a Smartphone. ",Object(Le.jsx)("i",{children:"Sensors, 19"}),"(21), 4812. https://doi.org/10.3390/s19214812"]}),Object(Le.jsxs)("li",{children:["Ghosh, S., Aggarwal, K. Upaassana, V. T., Nguyen, T., Han, J., & Ahn, C. H. (2020). A new microchannel capillary flow assay (MCFA) platform with lyophilized chemiluminescence reagents for a smartphone-based POCT detecting malaria. ",Object(Le.jsx)("i",{children:"Microsystems & Nanoengineering, 6"}),", 5. https://doi.org/10.1038/s41378-019-0108-8"]}),Object(Le.jsxs)("li",{children:["Ghosh, S., & Ahn, C. H. (2019). Lyophilization of Chemiluminescent Substrate Reagents for High Sensitive Microchannel-based Lateral Flow Assay (MLFA) in Point-of-Care (POC) Diagnostic System. ",Object(Le.jsx)("i",{children:"The Analyst, 144"}),"(6), 2109-2119. https://doi.org/10.1039/C8AN01899E"]}),Object(Le.jsxs)("li",{children:["Hamamatsu. (2021, January). ",Object(Le.jsx)("i",{children:"R12900U_H15710_TPMH1378E"})," [PDF file]. https://www.hamamatsu.com/resources/pdf/etd/R12900U_H15710_TPMH1378E.pdf"]}),Object(Le.jsxs)("li",{children:["Hemmig, E., Temiz, Y., G\xf6k\xe7e, O., Lovchik, R. D., & Delamarche, E. (2020). Transposing Lateral Flow Immunoassays to Capillary-Driven Microfluidics Using Self-Coalescence Modules and Capillary-Assembled Receptor Carriers [Abstract]. ",Object(Le.jsx)("i",{children:"Analytical Chemistry, 92"}),"(1), 940-946. https://doi.org/10.1021/acs.analchem.9b03792"]}),Object(Le.jsxs)("li",{children:["International Commission on Illumination. (n.d.). ",Object(Le.jsx)("i",{children:"Colorimetric Illuminants"}),". https://cie.co.at/publications/colorimetric-illuminants"]}),Object(Le.jsxs)("li",{children:["Jacinto, M. J., Trabuco, J. R. C., Vu, B. V., Garvey, G., Khodadadi, M., Azevedo, A. M., Aires-Barros, M. R., Chang, L., Kourentzi, K., Litvinov, D., & Willson, R. C. (2018). Enhancement of lateral flow assay performance by electromagnetic relocation of reporter particles. ",Object(Le.jsx)("i",{children:"PLOS ONE, 14"}),"(3). https://doi.org/10.1371/journal.pone.0186782"]}),Object(Le.jsx)("li",{children:"Koczula, K. M., & Gallotta, A. (2016). Lateral flow assays. Essays in Biochemistry, 60(1), 111-120. https://doi.org/10.1042/EBC20150012"}),Object(Le.jsxs)("li",{children:["Kranthi. (2013, May 31). How to Use IR LED and Photodiode with Arduino. ",Object(Le.jsx)("i",{children:"Robotix"}),". http://startrobotics.blogspot.com/2013/05/how-to-use-ir-led-and-photodiode-with-arduino.html."]}),Object(Le.jsxs)("li",{children:["Li, Zhao., Chen, H., & Wang, P. (2019). Lateral Flow Assay Ruler for Quantitative and Rapid Point-of-care Testing. ",Object(Le.jsx)("i",{children:"Analyst, 144"}),"(10), 3314-3322. https://doi.org/10.1039/c9an00374f"]}),Object(Le.jsxs)("li",{children:["Lin, B., Guan, Z., Song, Y., Song, E., Lu, Z., Liu, D., An, Y., Zhu, Z., Zhou, L., & Yang, C. (2018). Lateral flow assay with pressure meter readout for rapid point-of-care detection of disease-associated protein [Abstract]. ",Object(Le.jsx)("i",{children:"Lab chip, 18"}),"(6), 965-970. https://doi.org/10.1039/c8lc00010g"]}),Object(Le.jsxs)("li",{children:["Microchip Technology. (2011). ",Object(Le.jsx)("i",{children:"MCP3302/04"}),". https://ww1.microchip.com/downloads/en/DeviceDoc/21697F.pdf"]}),Object(Le.jsxs)("li",{children:["Moyano, A., Serrano-Pertierra, E., Salvador, M., Mart\xednez-Garc\xeda, J. C., Rivas, M., & Blanco-L\xf3pez, M. C. (2020). Magnetic Lateral Flow Immunoassays. ",Object(Le.jsx)("i",{children:"Diagnostics, 10"}),"(5), 288. https://doi.org/10.3390/diagnostics10050288"]}),Object(Le.jsxs)("li",{children:["Olanrewaju, A., Beaugrandm M., Yafia, M., Juncker, D. Capillary microfluidics in microchannels: from microfluidic networks to capillaries circuits. ",Object(Le.jsx)("i",{children:"Lab on a Chip "}),"16, 2323-2347 (2018). https://doi.org/10.1039/C8LC00458G"]}),Object(Le.jsxs)("li",{children:["Qu, Z., Wang K., Alfranca, G., de la Fuente, J. M., & Cui, D. (2020). A plasmonic thermal sensing based portable device for lateral flow assay detection and quantification. ",Object(Le.jsx)("i",{children:"Nanoscale Research Letters, 15"}),"(1), 10. https://doi.org/10.1186/s11671-019-3240-3"]}),Object(Le.jsxs)("li",{children:["Ramachandran, S., Fu, E., Lutz, B., & Yager, P. (2014). Long-term dry storage of an enzyme-based reagent system for ELISA in point-of-care devices [Abstract]. ",Object(Le.jsx)("i",{children:"Analyst, 139"}),", 1456-1462. https://doi.org/10.1039/C3AN02296J"]}),Object(Le.jsxs)("li",{children:["Strimbu, K., & Tavel, J. A. (2010). What are biomarkers? ",Object(Le.jsx)("i",{children:"Current Opinion in HIV and AIDS, 5"}),"(6), 463-466. https://doi.org/10.1097/COH.0b013e32833ed177"]}),Object(Le.jsxs)("li",{children:["Schwartz, E. (2018, April 30). How to Calculate Light Intensity. ",Object(Le.jsx)("i",{children:"Sciencing"}),". https://sciencing.com/calculate-light-intensity-7240676.html"]}),Object(Le.jsx)("li",{children:"Vishay. (2011, August 23). TEMD5080X01 [PDF file]. https://www.vishay.com/docs/81643/temd5080x01.pdf"}),Object(Le.jsxs)("li",{children:["Vishay. (2011, November 23). ",Object(Le.jsx)("i",{children:"BPW21R"})," [PDF file]. https://www.vishay.com/docs/81519/bpw21r.pdf"]}),Object(Le.jsxs)("li",{children:["Wang, W. (n.d.). ",Object(Le.jsx)("i",{children:"Optical Detectors"})," [PowerPoint slides]. 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Development and optimization of thermal contrast amplification lateral flow immunoassays for ultrasensitive HIV p24 protein detection. ",Object(Le.jsx)("i",{children:"Microsystems & Nanoengineering, 6"}),", 54. https://doi.org/10.1038/s41378-020-0168-9"]}),Object(Le.jsxs)("li",{children:["Zimmermann, M., Schmid, H., Hunziker, P., Delamarche, E. Capillary pumps for autonomous capillary systems. 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",Object(Le.jsx)("b",{children:"an inability to focus"}),", or ",Object(Le.jsx)("b",{children:"restlessness"})," and ",Object(Le.jsx)("b",{children:"impulsive decision-making"})]})}),Object(Le.jsxs)("div",{className:Di.a.center_content_div,children:[Object(Le.jsx)("div",{className:Di.a.categories_intro_text,children:"Impacts aspects of an individual's life such 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professionals."]}),Object(Le.jsx)("div",{className:"mt-5"}),Object(Le.jsxs)("div",{className:Di.a.intro_text,style:{maxWidth:"50%",paddingTop:"3%"},children:["Which can be influenced by the patient's ",Object(Le.jsx)("b",{children:"gender"}),", ",Object(Le.jsx)("b",{children:"race"})," and ",Object(Le.jsx)("b",{children:"culture"}),"."]})]}),Object(Le.jsxs)("div",{className:Di.a.heading_div,style:{marginTop:"-5%"},children:[Object(Le.jsxs)("div",{className:Di.a.pie_chart_text,children:["Gender biases are especially prevalent, with males being ",Object(Le.jsx)("b",{children:"3 times"})," more likely to be diagnosed than females."]}),Object(Le.jsx)("img",{src:"",alt:"Pie Chart",className:Di.a.pie_chart_img})]}),Object(Le.jsx)("div",{className:Di.a.goal_background_div,children:Object(Le.jsx)("div",{className:Di.a.goal_background,children:Object(Le.jsxs)("div",{className:Di.a.goal_text,children:["The goal of ",Object(Le.jsx)("b",{children:"NeuroDetech"})," is to create a quantitative tool to aid the diagnosis of ADHD and reduce the margin of bias."]})})}),Object(Le.jsxs)("div",{className:Di.a.heading_div,children:[Object(Le.jsx)("div",{className:Di.a.question_text,children:"How do we reduce bias in ADHD diagnoses?"}),Object(Le.jsx)("img",{src:"",alt:"Lightbulb",className:Di.a.lightbulb_img})]}),Object(Le.jsxs)("div",{className:Di.a.heading_div,children:[Object(Le.jsx)("img",{src:"",alt:"Biomarkers",className:Di.a.biomarkers_img}),Object(Le.jsxs)("div",{className:Di.a.pie_chart_text,children:["ADHD can be correlated to a statistically significant ",Object(Le.jsx)("b",{children:"lack or surplus"})," of specific biomarkers."]})]}),Object(Le.jsx)("div",{className:Di.a.center_content_div,children:Object(Le.jsx)("div",{className:Di.a.intro_text,style:{maxWidth:"75%"},children:"By quantifying the concentration of these biomarkers in urine, a non-invasive assessment of the risk of having ADHD can be provided."})}),Object(Le.jsx)("div",{className:Di.a.center_content_div,style:{marginBottom:"3%"},children:Object(Le.jsx)("div",{className:Di.a.discover_text,children:"Click below to learn more:"})}),Object(Le.jsxs)("div",{className:Di.a.icons_div,children:[Object(Le.jsxs)(ce,{to:"/Team:Waterloo/Engineering",className:Di.a.icons,style:{marginLeft:"-30%",marginRight:"30%"},children:[Object(Le.jsx)("div",{className:Di.a.icons_img,style:{backgroundImage:"var(--main-page-engineering-icon)"}}),Object(Le.jsx)("div",{className:Di.a.icons_text,children:"Engineering"})]}),Object(Le.jsxs)(ce,{to:"/Team:Waterloo/Human_Practices",className:Di.a.icons,style:{marginLeft:"30%",marginRight:"-30%"},children:[Object(Le.jsx)("div",{className:Di.a.icons_img,style:{backgroundImage:"var(--main-page-human-practices-icon)"}}),Object(Le.jsx)("div",{className:Di.a.icons_text,children:"Human 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These goals aim to end poverty, protect the planet, and ensure that people enjoy peace and prosperity (Department of Social & Economic Affairs, n.d.). NeuroDetech focuses more on this ensuring that people enjoy peace and prosperity by addressing four values identified to be the most relevant to our project: Good Health and Well-Being, Quality Education, Reduced Inequalities, and Partnerships for the Goals."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",alt:"Relevant Values",className:Gi.a.values_img}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"(Logos courtesy of the United Nations Department of Social & Economic Affairs 17 Goals, n.d.)"})]}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.subheading,children:"Good Health and Well-Being"}),Object(Le.jsx)("div",{className:pe.a.description,children:"To this day, ADHD has been a condition that has been diagnosed qualitatively, and therefore, it can sometimes be misdiagnosed (Ford-Jones, P. C., 2015). We want to help improve the well-being of ADHD patients by developing a procedure that reduces the stress and struggles associated with the arduous process."}),Object(Le.jsx)("div",{className:pe.a.description,children:"When designing NeuroDetech, it was important for us to ensure that our microfluidic device would not only be reliable, but also easy to use for patients and medical professionals. Point-of-care devices have become extremely relevant in today's society because of their user-friendly features (St John, A., & Price, C. P., 2014). Additionally, testing urine instead of blood made the process a lot simpler for patients. Keeping these things in mind while designing our project allowed us to make NeuroDetech an all-around effective device."}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"Quality Education"}),Object(Le.jsx)("div",{className:pe.a.description,children:"Despite the amount of scientific evidence collected about ADHD, the spread of misinformation regarding its presentation in individuals and how it is diagnosed is still prevalent [1]. We wanted to ensure that NeuroDetech would be a way to educate more people about what ADHD is and how medical research (such as the research done by the Waterloo iGEM team!) continually aims to develop a novel and reliable method that will make the diagnosis process more comfortable for patients."}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"Reduced Inequalities"}),Object(Le.jsx)("div",{className:pe.a.description,children:"People with ADHD, especially kids, often exhibit behaviour that leads to higher risks of being bullying victims. Impulsive remarks, clumsiness, poor understanding of social cues, and being overly affectionate are just a few behaviours that ADHD patients get made fun of for (Keder et al., 2013).. When designing NeuroDetech, developing a device that would help reduce these inequalities was vital. We want to help ADHD patients become diagnosed effectively so interventions can be implemented as soon as possible to reduce the negative impacts of ADHD patients feeling excluded from society."}),Object(Le.jsx)("div",{className:pe.a.description,children:"ADHD often presents differently in males in comparison to females, with literature displaying a higher prevalence in males. Generally, these are qualitative differences that are seen to be more prominent in men than women with ADHD (Rucklidge, 2010). Current diagnostic methods identify symptoms displayed by men more often than women and this can lead to biases in ADHD diagnosis. By providing a quantitative method that aids in the diagnosis of ADHD with NeuroDetech, we aim to eliminate some of these possible biases in ADHD diagnosis."}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"Partnerships for the Goals"}),Object(Le.jsx)("div",{className:pe.a.description,children:"Our project may be designed by a small group of inspired students from Waterloo, but our goal is to showcase the possibilities for synthetic biology solutions in medical diagnosis. ADHD affects millions of people globally and several of these patients have had to go through a series of assessments that may have been subject to clinician bias (Fadus et al., 2019)."})]})]})}}]),i}(n.Component),Ui=i(39),qi=i.n(Ui),Ki=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Understanding the Current ADHD Diagnosis Process in Canada"}),Object(Le.jsx)("div",{className:pe.a.description,children:"The initial goal for our project was to create a device that would aid in the diagnosis of ADHD. During project development, we realized we needed to have an understanding of the current diagnosis procedures for ADHD in Canada, so that we could successfully develop a product that would have the potential for real-life implementation."}),Object(Le.jsx)("div",{className:pe.a.description,children:"Our Human Practices subteam began by conducting research into ADHD diagnosis procedures and established a diagnosis pipeline, which allowed us to visualize the process in a sequential and organized way. From the research conducted, the team established a diagnosis pipeline using research collected from various online sources, such as research articles, ADHD centre websites, and clinical centre resources. From the literature, we established that the diagnosis 'pipeline' consisted of a combination of seven steps as seen in the figure below. After the original pipeline was created, the next step was contacting various stakeholders (psychologists, psychiatrists, general practitioners, etc.) about their experience with the diagnosis process to confirm its accuracy. Stakeholders include but are not limited to ADHD assessors such as psychologists, psychiatrists and general practitioners. Stakeholder testimonials allowed us to determine that the diagnosis process varied vastly between different medical professions and even among those working in the same discipline."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",alt:"Current Pipeline",className:qi.a.pipeline_img}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"The current ADHD diagnosis procedure in Canada."})]}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.description,children:"For instance, between the medical professionals that were consulted, the assessment process ranged from a total of 4 to 20 hours, with some medical professionals having assessments that spanned over multiple days. Although it appeared to be common practice to assess for and rule out other disorders that may be contributing to ADHD-like symptoms, the methods were not consistent among the medical professionals. While psychologist assessors used standardized checklists to assess patients for learning disabilities and cognitive skills, our discussions with Vera Roncon, a clinical psychologist from Mt Pleasant Therapy Centre, informed us that some assessors also requested bloodwork to rule out factors such as thyroid dysfunction (2021). In addition, by discussing with Dr. Megan Smith, a clinical child and school psychologist, it was determined that other less common tests also include sleep studies (2021)."}),Object(Le.jsx)("div",{className:pe.a.description,children:"Through the research conducted, it became evident to the team that the current processes and tests in place were mainly qualitative, thus creating the need for NeuroDetech- a quantitative test to aid in ADHD diagnosis."}),Object(Le.jsx)("div",{className:pe.a.description,children:"Based on our initial research about ADHD diagnosis, there are many issues and biases that surround the diagnostic process. An example of this stems from the fact that males tend to show prototypical symptoms of ADHD while females do not (Ford-Jones, P. C., 2015). Many females can go undiagnosed for ADHD because their symptoms typically manifest as inattentiveness, rather than the noticeable hyperactivity often seen in males. As such, the gender bias mainly causes a disparity of ADHD testing referrals (Ramtekkar, 2010)."}),Object(Le.jsx)("div",{className:pe.a.description,children:"Another example of a diagnosis issue is age - adults are less likely to be diagnosed with ADHD. One reason for this problem is that ADHD symptoms for adults generally overlap with the symptoms for other mental illnesses, such as depression, bipolar disorder, anxiety disorder, and many others (MediLexicon, 2021). In addition, many studies show a decrease in ADHD symptoms as an individual ages. This result goes on to impact the diagnosis procedures, as can be noted by the lack of ADHD symptom criteria specific to adults used for diagnosis (Ramtekkar, 2010)."}),Object(Le.jsx)("div",{className:pe.a.description,children:"Race inequality is also evident in ADHD diagnostic practices. Studies suggest that African American and Hispanic children are underdiagnosed with ADHD (Morgan, 2013). African American children show more symptoms of the disorder than white children, yet they are being diagnosed at only 66% the rate of White children. Although the root cause of this disparity is still being researched, it is clear that a bias exists in diagnostic procedures and that the process needs to be improved."}),Object(Le.jsx)("div",{className:pe.a.description,children:"Given some of the weaknesses with current diagnostic practices, our goal was to implement NeuroDetech within the existing diagnostic pipeline as a supporting tool to aid the process and mitigate the effects of these biases. The quantitative nature of NeuroDetech allows an unbiased metric to be used for diagnosis, allowing individuals affected by ADHD in under-diagnosed populations to get a less biased diagnosis."}),Object(Le.jsx)("div",{className:pe.a.description,children:"With all this determined, the next step of our project was to determine the best implementation of NeuroDetech within the established diagnosis pipeline. Based on our research, the preliminary ideas for implementation were 1) as a pre-screening test before official ADHD assessment, or 2) conducted along with the lab tests that screen for other disorders. Diagnosis visuals were created to showcase the two options of implementation location as seen in the figures below."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",alt:"Prescreen Pipeline",className:qi.a.pipeline_img}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"The proposed ADHD diagnosis procedure with NeuroDetech as a pre-screen test."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",alt:"Lab Testing Pipeline",className:qi.a.pipeline_img}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"The proposed ADHD diagnosis procedure with the NeuroDetech test implemented with the other lab tests."})]})]})}}]),i}(n.Component),Yi=i(65),Ji=i.n(Yi),$i=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Determining Implementation"}),Object(Le.jsx)("div",{className:pe.a.description,children:"To aid in the diagnostic process of ADHD, NeuroDetech aimed to measure related biomarkers. In-vitro studies involved the use of live cell culture and the experimentation being executed outside an organism. This approach brought advantages such as lower costs and easier to control variables, thus resulting in highly reproducible outcomes (National Research Council, 1999), which was an asset to NeuroDetech\u2019s goal of testing for biomarkers of ADHD. Although using an in-vitro model improved the accessibility of this testing device to its intended audience, the results were not directly translatable to subjects (National Research Council, 1999)."}),Object(Le.jsx)("div",{className:pe.a.description,children:"Conversely, an in-vivo approach would result in higher accuracy and would be more reliable and relatable to the test subjects, in this case humans (National Research Council, 1999; Cook et al., 2015). This approach raised concerns in research ethics through its reliance on human subjects. Additionally, its time-consuming nature would likely not allow results to be seen in an 8-month time frame (Cook et al., 2015). Finally, the materials required to complete in-vivo studies are more expensive, thus decreasing product accessibility and affordability."}),Object(Le.jsx)("div",{className:pe.a.description,children:"Under the aforementioned conditions, it would be difficult to implement an in-vivo approach; associated ethical concerns are too ambiguous and complex to analyze through the lens of integrated human practices. As a result, an in-vitro solution should be considered, as it would be easier to implement within the time frame of approximately 8 months, and with heightened lab restrictions due to the COVID-19 pandemic. The in-vitro approach was executed to aid in developing our diagnostic test using creativity and literature reviews of existing protocols."}),Object(Le.jsxs)("div",{className:pe.a.description,children:["With the final stance being in-vitro testing, determining the implementation of the tool was one of the main discussions our team had. This discussion revolved around implementing our tool either as ",Object(Le.jsx)("b",{children:"a)"})," a prescreening device or ",Object(Le.jsx)("b",{children:"b)"})," an additional step in a series of standard tests required by a primary healthcare provider. These \u2018lab testing days\u2019 are requested to benchmark an individual\u2019s physiological conditions prior to diagnosis with blood work, urine analysis, etc."]})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",alt:"In Vitro In Vivo testing",className:Ji.a.vitro_vivo_img}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"(Digital art image courtesy of NfERnOv2, 2011)"})]}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.description,children:"Moreover, implementing our tool during the lab day testing allowed patients to not undergo multiple testings over a span of days. This has a huge impact on those who are not comfortable with labs (may be similar to that of white coat syndrome) (Lerwick, 2016). On the other hand, with our tool being an additional test, it required training and a specific setting to be used. However, this was seen as beneficial as there would trained professionals such as lab technicians who administer the test. Furthermore, unlike the pre-screening implementation, the lab day testing allowed our tool to be an added confirmation that could aid in the diagnosis of ADHD rather than a sole answer for consumers. This allowed professionals to view our tool from a quantitative stand-point in addition to their qualitative results from formal assessments."}),Object(Le.jsx)("div",{className:pe.a.description,children:'With the current pandemic, many hospitals have adapted the \u201cpre-screening and covid testing\u201d method for world-wide communities and can be somewhat compared to our test. The screening done for COVID-19 includes many questions such as whether one has symptoms, been in close contact with a potential exposure, etc (Government of Canada, 2021). These questions allow members of the community to decide if a COVID test is warranted. However, unlike COVID-19 pre screening methods, there is no simple yes or no answer to whether one has ADHD or not (Davidovitch et al., 2017). Thus, it further indicates that using NeuroDetech as a sole prescreening tool would not be effective. Clinical psychologist, Megan Smith, states \u201cI definitely do NOT think it should be used as a pre-screening tool. Nothing that is going to catch the vast majority of cases should be\u201d. Thus, implementing our tool during lab day testing would be more effective as \u201cusing it as part of an assessment allows it to be weighed with other info more generally" (M. Smith, personal communication, June 7, 2021).'}),Object(Le.jsx)("div",{className:pe.a.description,children:"The main intention behind the pre-screening test was to use it for populations without healthcare coverage. Since many people forgo ADHD testing due the financial burden, the pre-screening option would help people determine if they need to complete the entire ADHD assessment process or stop after the pre-screen returns a negative. The second option was a lab-day test. In the original pipeline it was seen that during the ADHD diagnosis there can be a lab-day where patients are to complete blood work. Our idea was to combine NeuroDetech within the rest of the lab testing as it would be logistically more efficient. In the end, the option chosen was to include the NeuroDetech test along with the other lab tests."})]})]})}}]),i}(n.Component),Zi=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"References"}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"Department of Economic and Social Affairs. (n.d.). The 17 Goals. United Nations. Retrieved October 20, 2021, from https://sdgs.un.org/goals."}),Object(Le.jsx)("li",{children:"Ford-Jones, P. C. (2015, May). Misdiagnosis of attention deficit hyperactivity disorder: 'normal behaviour' and relative maturity. Paediatrics & child health. Retrieved September 17, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4443828/."}),Object(Le.jsx)("li",{children:"St John, A., & Price, C. P. (2014). Existing and Emerging Technologies for Point-of-Care Testing. The Clinical biochemist. Reviews, 35(3), 155\u2013167. Retrieved October 20, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4204237/."}),Object(Le.jsx)("li",{children:"Keder, R. , Sege, R. , Raffalli, P. & Augustyn, M. (2013). Bullying and ADHD. Journal of Developmental & Behavioral Pediatrics, 34 (8), 623-625. Retrieved October 20, 2021, from: https://oce.ovid.com/article/00004703-201310000-00012/HTML. doi: 10.1097/DBP.0000000000000011."}),Object(Le.jsx)("li",{children:"Fadus, M.C., Ginsburg, K.R., Sobowale, K. et al. Unconscious Bias and the Diagnosis of Disruptive Behavior Disorders and ADHD in African American and Hispanic Youth. Acad Psychiatry 44, 95\u2013102 (2020). Retrieved October 20, 2021, from: https://link.springer.com/article/10.1007/s40596-019-01127-6#citeas. https://doi.org/10.1007/s40596-019-01127-6."}),Object(Le.jsx)("li",{children:"Rucklidge, J. Gender differences in attention-deficit/hyperactivity disorder. Psychiatr Clin North Am. 2010 Jun;33(2):357-73. doi: 10.1016/j.psc.2010.01.006. PMID: 20385342. Retrieved October 20, 2021, from: https://pubmed.ncbi.nlm.nih.gov/20385342/."}),Object(Le.jsx)("li",{children:"Ramtekkar, U. P., Reiersen, A. M., Todorov, A. A., & Todd, R. D. (2010, March). Sex and age differences in attention-deficit/hyperactivity disorder symptoms and diagnoses: Implications for DSM-V and ICD-11. Journal of the American Academy of Child and Adolescent Psychiatry. Retrieved September 17, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3101894/."}),Object(Le.jsx)("li",{children:"MediLexicon International. (n.d.). ADHD misdiagnosis: Why might it happen? Medical News Today. Retrieved September 17, 2021, from https://www.medicalnewstoday.com/articles/325595#autism-spectrum-disorder."}),Object(Le.jsx)("li",{children:"Morgan, P. L., Staff, J., Hillemeier, M. M., Farkas, G., & Maczuga, S. (2013, July). Racial and ethnic disparities in ADHD diagnosis from kindergarten to eighth grade. Pediatrics. Retrieved September 17, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3691530/."}),Object(Le.jsx)("li",{children:"Davidovitch, M., Koren, G., Fund, N., Shrem, M., & Porath, A. (2017, December 29). Challenges in defining the rates of ADHD diagnosis and treatment: Trends over the last decade. BMC Pediatrics. Retrieved September 17, 2021, from https://bmcpediatr.biomedcentral.com/articles/10.1186/s12887-017-0971-0."}),Object(Le.jsx)("li",{children:"Government of Canada. (2021, August 9). COVID-19 screening questions used by federal corrections. Canada.ca. Retrieved September 17, 2021, from https://www.canada.ca/en/correctional-service/campaigns/covid-19/visits/screening-questions.html."}),Object(Le.jsx)("li",{children:"Lerwick, J. L. (2016). Minimizing pediatric healthcare-induced anxiety and trauma. World Journal of Clinical Pediatrics, 5(2), 143\u2013150. https://doi.org/10.5409/wjcp.v5.i2.143"}),Object(Le.jsx)("li",{children:"National Research Council (US) Committee on Methods of Producing Monoclonal Antibodies. Monoclonal Antibody Production. Washington (DC): National Academies Press (US); 1999. 4, Summary of Advantages and Disadvantages of In Vitro and In Vivo Methods. Available from: https://www.ncbi.nlm.nih.gov/books/NBK100200/"}),Object(Le.jsx)("li",{children:"Cook, K., Synder, J., & Calvert, J. (2015). Canadian Research Ethics Board Members\u2019 attitudes toward benefits from clinical trials. BMC Medical Ethics, 16:84. https://doi.org/10.1186/s12910-015-0079-8"}),Object(Le.jsx)("li",{children:"NfERnOv2 (2011). In vitro vs in vivo. [Digital Artwork]. DeviantArt. https://www.deviantart.com/nfernov2/art/In-vitro-versus-in-vivo-271424868"})]})]})})}}]),i}(n.Component),Qi=["Project Values","Understanding Current Diagnosis Process","Determining Implementation","References"],Xi=["","","",""],en=function(e){var t=be(Object(n.useState)(Qi[0]),2),i=t[0],a=t[1];return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.heading_div,children:[Object(Le.jsxs)("div",{className:pe.a.title,children:[Object(Le.jsx)("div",{className:pe.a.page_heading,children:"NeuroDetech"}),Object(Le.jsx)("div",{className:pe.a.page_heading_colored,children:"Human Practices."})]}),Object(Le.jsx)("div",{className:pe.a.illustration,children:Object(Le.jsx)("img",{src:"",alt:"Human Practices Icon",className:zi.a.icon_img})})]}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Overview"}),Object(Le.jsx)("div",{className:pe.a.description,children:"The development of NeuroDetech is a synthetic biology and scientific endeavour, which is deeply interwoven with societal, economic, and regulatory implications. To truly assess optimal user experiences, biomarker detection, and hardware configuration, what is now coined \u201cintegrated human practices\u201d is essential. From initial project selection to various implementation options, current frameworks and legislative precedents were characterized and evaluated. Stakeholder consultations from medical professionals and diagnostic equipment manufacturers shaped project direction along each and every stage. The following page dives deep into the impact of human practices on this year's project, which allows Neurodetech to extend beyond a synthetic biology proof of concept."})]}),Object(Le.jsx)("div",{className:pe.a.sections_div,children:Qi.map((function(e,t){return Object(Le.jsx)("div",{active:i===e,onClick:function(){return a(e)},className:pe.a.section_block,children:Object(Le.jsxs)("div",{className:pe.a.sections,children:[Object(Le.jsx)("div",{className:pe.a.section_img,children:Object(Le.jsx)("img",{id:"hp".concat(t),src:Xi[t],alt:"Icon"})}),Object(Le.jsx)("div",{className:pe.a.section_text,children:e})]})},e)}))}),Object(Le.jsxs)("div",{children:[i===Qi[0]&&Object(Le.jsx)(Vi,{}),i===Qi[1]&&Object(Le.jsx)(Ki,{}),i===Qi[2]&&Object(Le.jsx)($i,{}),i===Qi[3]&&Object(Le.jsx)(Zi,{})]})]})},tn=i(66),nn=i.n(tn),an=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Current Challenges and Future Improvements"}),Object(Le.jsxs)("div",{className:pe.a.description,children:["While we have worked hard over the course of the past year to explore every aspect of NeuroDetech\u2019s implementation, there are always areas that can be improved. Understanding the costs associated with NeuroDetech is an area that we explored this year (check out our\xa0",Object(Le.jsx)(ce,{to:"/Team:Waterloo/Finance",className:pe.a.text_link,children:"Finance"}),"\xa0page!), but in the future we would like to deepen the extent of this analysis. Efforts to reach out to stakeholders at life sciences technology companies such as Bio-Rad Laboratories and Thermo Fisher Scientific were unfortunately unsuccessful. This prevented us from having direct conversations about the manufacturing costs of a product like NeuroDetech, which could have provided us with real-life estimates. As such, we believe that this is an area that merits further investigation."]}),Object(Le.jsx)("div",{className:pe.a.description,children:"Secondly, receiving first-hand feedback from individuals with ADHD is something we would like to explore in the future. Due to complexities surrounding patient privacy, we were unable to get ethics approval to conduct interviews with individuals with ADHD to get their first-hand feedback on NeuroDetech. For us, it is important to know how a product like this would impact individuals undergoing ADHD diagnosis procedures. Would the product make them feel more secure about their diagnosis? Would they be indifferent? These are questions that we consider to be worth asking, because a product like ours is just as much about the individuals receiving the diagnosis as it is about the individuals determining the diagnosis."}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"Lab Access: What We Would Have Done..."}),Object(Le.jsx)("div",{className:pe.a.description,children:"Due to unforeseen circumstances (one of which was a global pandemic, and the other of which was unforeseen maintenance and construction of our lab space by the University), we unfortunately did not have lab access this year. As a result, though we were able to model and test some of the components of NeuroDetech using computational methods and prototyping, we were not able to physically test and demonstrate a proof of concept for NeuroDetech."}),Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:"Given lab access, we would have liked to have done the following:"}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"We would have tested our microfluidic assay chip in terms of proper functionality and sample fluid manipulation."}),Object(Le.jsx)("li",{children:"We would have characterized and tested the functionality of our phenylethylamine (PEA) binding fusion protein. This would have included cloning, expression, purification, and activity assays of our PEA-binding fusion protein, to confirm that the fusion protein is stable in urine conditions, that the fusion protein can bind PEA, and that the fusion protein can produce a predictable, quantifiable signal when reacted with Amplex Red (the chemiluminescent substrate)."}),Object(Le.jsx)("li",{children:"We would have tested that our affinity-improved versions of the streptavidin and trypsin proteins did, in fact, display improved affinity in wet lab experiments."}),Object(Le.jsx)("li",{children:"We would have demonstrated the success of the CRISPR-Cas13a system as a quantification method for ADHD-associated urinary mRNA, using a simulated urine sample spiked with the target RNA sequence of interest."})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:["Methodologies related to how we would have performed the above tests given lab access are outlined in our\xa0",Object(Le.jsx)(ce,{to:"/Team:Waterloo/Engineering",className:pe.a.text_link,children:"Engineering"}),"\xa0page."]}),Object(Le.jsxs)("div",{className:pe.a.description,children:["Finally, as a proof of concept, we would have liked to put it all together by testing the ability of our set of microfluidic assay chips to quantify our target ADHD-associated biomarkers and gene markers, where the microfluidic assay chip contains all of the necessary components and is tested using a simulated urine sample spiked with the ADHD-associated biomarkers/gene markers. This would be the proof of concept to demonstrate the functionality of NeuroDetech. Our methodology related to how we would have demonstrated our proof of concept given lab access is outlined in our\xa0",Object(Le.jsx)(ce,{to:"/Team:Waterloo/Proof_Of_Concept",className:pe.a.text_link,children:"Proof of Concept"}),"\xa0page."]})]})})}}]),i}(n.Component),rn=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"General Safety Considerations for NeuroDetech Usage"}),Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:"To properly determine how NeuroDetech would be implemented in a real life medical laboratory setting, our team developed a list of safety considerations that must be taken into account when our tool is being used."}),Object(Le.jsxs)("ol",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"As with any other flow assay tests, the results from NeuroDetech should not be taken to be 100% accurate (TWinFM). The chance of the tool malfunctioning, or giving misleading results always exists, thus only properly trained individuals, such as lab technicians, should use NeuroDetech. This will ensure proper sample and MCFA chip handling, as well as the ability to troubleshoot, should it be necessary."}),Object(Le.jsx)("li",{children:"Furthermore, the test contains a variety of chemicals, and should not be broken/tampered with to reveal the inner contents, at any point in the life of the tool. Chemicals such as Amplex Red that can cause harm if in contact with the user. Thus the user must take precautions to not break or alter the structure of the MCFA chip at any point during its usage to expose these chemicals in an unsafe manner. (Iacobucci, 2021)"}),Object(Le.jsx)("li",{children:"Once the test has been completed, post-handling is required to interpret the results (eg: running the chip on an app to yield a graph). Since the test would contain traces of urine, it will have to be handled with care to ensure that there is no contamination from the chip to the user, or external environment, such as the computer."}),Object(Le.jsx)("li",{children:"Lastly, the chip and the test must be discarded safely (biohazardous waste bin), and must not be left laying in the open."})]})]})})}}]),i}(n.Component),sn=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"NeuroDetech Implementation in Lab Settings"}),Object(Le.jsx)("div",{className:pe.a.description,children:"From the beginning of NeuroDetech\u2019s development, we were focused on creating an efficient assay that could provide high accuracy results, while still being minimally invasive. Our team had the goal of developing a project that would serve as a resource for healthcare workers providing ADHD diagnoses. Initially, we were unsure of whether NeuroDetech would be most effective as a pre-screening tool or as a tool used in medical laboratory settings. Through our Integrated Human Practices work, stakeholder testimonials helped us determine that the strength of NeuroDetech was in its ability to provide a quantitative metric as a part of standardized lab testing, instead of as a pre-screening."}),Object(Le.jsxs)("div",{className:pe.a.description,children:["Once stakeholder feedback pointed us away from the direction of a pre-screening tool, we began looking at the different ways in which we could develop a tool that would be used in medical laboratory settings. The first major decision was choosing a method of testing. Both ",Object(Le.jsx)("i",{children:"in vivo"})," and ",Object(Le.jsx)("i",{children:"in vitro"})," methods of analysis were advertised as favourable approaches. However, it became clear that an ",Object(Le.jsx)("i",{children:"in vivo"})," method would likely entail cell culture, an expensive, time inefficient, and inaccessible approach given restricted lab access. In addition, the circulation of a test containing products from cell culture would be locked behind professional distribution and expensive mass production given the high level of maintenance required, contradicting our goals for accessibility (O\u2019Neill et al., 2021). Therefore, it was decided that an ",Object(Le.jsx)("i",{children:"in vitro"})," method of testing, namely a lateral flow assay (LFA), would be preferred."]}),Object(Le.jsx)("div",{className:pe.a.description,children:"Once we had our initial LFA idea, we began exploring the specifics of what our product would look like. Copious amounts of research done on biomarkers related to ADHD eventually led us to phenylethylamine (PEA). We also decided on using a microfluidic assay, rather than a traditional lateral flow assay, due to their increased accuracy and sensitivity (Cui & Wang, 2019). When developing the chip itself, we made decisions such as using a Polydimethylsiloxane (PMDS) microfluidic chip for its ease of fabrication and affordability. We also wanted to increase the amount of information that NeuroDetech could provide, as using more than 1 biomarker would improve the efficacy of our bioassay and our confidence in the ADHD risk assessment (Araz, Tentori & Herr, 2013). By using a CRISPR-Cas13a system, we could also provide information regarding the detection of ADHD-associated mutations in mRNA transcripts via the quantification of gene markers. Lastly, our team focused on ensuring that the test would be minimally invasive and decided to use urine analysis for metabolites. Feedback from Dr. Janet Patterson (MD) informed us that in inpatient units, psychiatrists will often ask for certain tests (such as liver function, renal function, and thyroid function tests, as well as urinalysis and an electrocardiogram) to be conducted on the patient, as that medical, as well as psychological, information can be available for the diagnosis process. This allows us to easily implement NeuroDetech as part of the medical work-up procedure that is already in place. The setup of the device also requires minimal device supply from the test center: a laptop for connecting with the optical detector specifically designed for NeuroDetech lab chip. This allows healthcare professionals to interpret data conveniently without the need to be in a fixed laboratory setting, since the optical detector device is light and portable."})]})})}}]),i}(n.Component),on=i(18),cn=i.n(on),ln=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Proper Usage of NeuroDetech"}),Object(Le.jsx)("div",{className:pe.a.description,children:"Proper usage of NeuroDetech is contingent on preprocessing of the urine sample, proper usage of the microfluidic chip and associated quantification app, and disposal of the system."}),Object(Le.jsxs)(Ie,{open:!0,title:"Preliminary Urine Processing",children:[Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.subheading,children:"RNase Inhibition"}),Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:"Preserving the intactness of mRNA in the urine is crucial if gene marker detection is to be carried out in an accurate manner. In order to achieve this, RNAlater, an RNase inhibitor cocktail from Invitrogen, can be used. As per its use instructions, 5 volumes of RNAlater should be added to preserve a sample (ThermoFisher Scientific, n.d.). Considering that the four microfluidic assay chips only require 30 \xb5L of sample each, a 600 \xb5L sample of preserved urine can be prepared as follows:"}),Object(Le.jsxs)("ol",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"Aliquot 100 \xb5L of urine sample."}),Object(Le.jsx)("li",{children:"To the urine aliquot, add 500 \xb5L of RNAlater."})]}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"Urinalysis"}),Object(Le.jsx)("div",{className:pe.a.description,children:"A study conducted by researchers at the University of Alberta revealed that human urine is composed of more than 3000 metabolites, making it a highly complex specimen to analyze (University of Alberta News Staff, 2013). Since NeuroDetech utilizes urine specimens, it is important to consider other factors that could affect PEA levels of the patient\u2019s urine. Therefore, performing a urinalysis as a screening procedure prior to using NeuroDetech can ensure a higher level of accuracy when analyzing the PEA levels in urine samples. Additionally, taking certain precautions prior to the urinalysis will decrease the likelihood of inaccurate test results. The main objective of the urinalysis is to ensure that the urine sample is as normal as possible and that there are no pre-existing conditions that could render the results of the NeuroDetech test unreliable."}),Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:Object(Le.jsx)("b",{children:"The following precautions must be taken prior to conducting a urine test that tests for catecholamines such as PEA:"})}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"Discuss prescribed medications, non-prescription medications, vitamins, and supplements with your physician (Mayo Clinic Staff, 2019). "}),Object(Le.jsx)("li",{children:"If it is safe to do so, avoid consumption of all medications for three days prior to urine collection. Exceptions include: common hypertensives including diuretics, ACE inhibitors, calcium channel blockers, alpha and beta blockers, etc. (\u201cDietary Restrictions for Urine Tests\u201d, n.d.)."}),Object(Le.jsx)("li",{children:"Avoid consumption of the following 24 hours prior to urine collection: acetaminophen, alcohol, antihistamines, caffeine, aspirin, and vitamin B (\u201cDietary Restrictions for Urine Tests\u201d, n.d.)."}),Object(Le.jsx)("li",{children:"Avoid strenuous exercise 24 hours prior to urine collection (\u201cDietary Restrictions for Urine Tests\u201d, n.d.)."})]}),Object(Le.jsx)("div",{className:cn.a.procedure_text,children:Object(Le.jsx)("b",{children:"The following 3-step laboratory procedure describes how a urinalysis is performed:"})}),Object(Le.jsx)("div",{className:cn.a.procedure_text,children:Object(Le.jsx)("i",{children:Object(Le.jsx)("b",{children:"1. Visual Exam"})})}),Object(Le.jsx)("div",{className:cn.a.procedure_text,children:"Visually observe and make note of the urine sample's appearance based on its colour and clarity (Mayo Clinic Staff, 2019)."}),Object(Le.jsxs)("div",{className:cn.a.procedure_text,children:[Object(Le.jsx)("b",{children:"Colour:"})," shades of yellow described as pale, colourless, dark, or amber",Object(Le.jsx)("br",{}),Object(Le.jsx)("b",{children:"Clarity:"})," clear, slightly cloudy, cloudy, or turbid"]}),Object(Le.jsx)("div",{className:cn.a.procedure_text,children:Object(Le.jsx)("i",{children:Object(Le.jsx)("b",{children:"2. Chemical Exam: Dipstick Test"})})}),Object(Le.jsx)("div",{className:cn.a.procedure_text,children:"A dipstick is essentially a plastic stick with reagent pads that change colour depending on the presence of certain chemicals. Dip the dipstick in the urine and interpret the results by using the urinalysis guide on the dipstick test container. The dipsticks tests for: acidity (pH), concentration (specific gravity), protein, glucose, ketones, bilirubin, evidence of infection (nitrites), and blood (Mayo Clinic Staff, 2019)."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("div",{className:pe.a.figure_text,children:Object(Le.jsx)("b",{children:"Normal Ranges of Urine Dipstick Test Results"})}),Object(Le.jsxs)(Zt,{bordered:!0,className:cn.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:Object(Le.jsx)("b",{children:"Reagent Pad Test"})}),Object(Le.jsx)("th",{children:Object(Le.jsx)("b",{children:"Normal Range"})})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Acidity (pH)"}),Object(Le.jsx)("td",{children:"5 to 9"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Concentration (specific gravity)"}),Object(Le.jsx)("td",{children:"1.003 to 1.040"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Protein"}),Object(Le.jsx)("td",{children:"< 30 mg/dL"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Glucose"}),Object(Le.jsx)("td",{children:"< 100 mg/dL"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Ketones"}),Object(Le.jsx)("td",{children:"Variable"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Bilirubin"}),Object(Le.jsx)("td",{children:"None"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Nitrites"}),Object(Le.jsx)("td",{children:"None"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Blood"}),Object(Le.jsx)("td",{children:"None"})]})]})]})]}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:cn.a.procedure_text,children:"The table above displays the normal ranges of urine dipstick results (Roxe, 1990). Any results outside of these ranges should be analyzed further."}),Object(Le.jsx)("div",{className:cn.a.procedure_text,children:Object(Le.jsx)("i",{children:Object(Le.jsx)("b",{children:"3. Microscopic Exam"})})}),Object(Le.jsx)("div",{className:cn.a.procedure_text,children:"Centrifuge the urine and examine the resuspended sediment under a microscope (Roxe, 1990). Identify if there are abnormal levels of white blood cells, red blood cells, bacteria, yeast, casts, or crystals (Mayo Clinic Staff, 2019)."}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"Removal of Naturally Occurring Biotin from Urine"}),Object(Le.jsxs)("div",{className:pe.a.description,children:["As part of the pre-treatment process, the urine sample is passed through a biotin filtration system prior to performing the microfluidic assay. The purpose of this step is to prevent ambient urinary biotin from binding to streptavidin molecules in the control chamber. Streptavidin has a high affinity for biotinylated molecules (with a dissociation constant Kd = 10",Object(Le.jsx)("sup",{children:"-14"})," M), and we wanted to use this binding capacity to bind the biotinylated fusion protein to the streptavidin in the control chamber (Chivers et al., 2011). However, if urinary biotin molecules are passed through the control chamber, they could \u201cblock\u201d the streptavidin binding sites, disrupting our fusion protein\u2019s ability to bind to streptavidin."]}),Object(Le.jsxs)("div",{className:pe.a.description,children:["To perform the biotin filtration step, the urine sample is passed through a resin column of Pierce Monomeric Avidin, obtained from ThermoFisher Scientific (ThermoFisher Scientific, n.d.). The biotin molecules in the urine will bind to the monomeric avidin beads, filtering the biotin out of the sample. The affinity between monomeric avidin and biotin is not as strong (compared to streptavidin and biotin), so biotin can be easily eluted by passing a solution known as \u201cRegeneration Buffer,\u201d provided in the Pierce Monomeric Avidin kit, through the column. The resin beads can be reused up to ten times without significant loss of biotin binding capacity. The complete protocol describing the use of the biotin filtration system is detailed in\xa0",Object(Le.jsx)("a",{href:"https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011221_Pierce_Monomeric_Avidin_UG.pdf",target:"_blank",rel:"noreferrer",children:"these instructions for the ThermoFisher Scientific Pierce Monomeric Avidin Kit"}),"."]})]})]}),Object(Le.jsx)(Ie,{open:!0,title:"Usage and Disposal of Microfluidic Assay",children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.subheading,children:"Operation of the Microfluidic Assay: User Guide"}),Object(Le.jsx)("div",{className:pe.a.description,children:"Once the urine sample has been properly preprocessed (including urinalysis, ambient biotin removal, and addition of RNase inhibitor), analysis of the urine sample for the concentration of an ADHD-associated biomarker or gene marker can be performed. The following steps outline the general protocol that would be carried out by a user of NeuroDetech:"}),Object(Le.jsxs)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:["For the usage of a single microfluidic assay chip:",Object(Le.jsx)("br",{}),Object(Le.jsx)("i",{children:"All steps should be performed with proper PPE for handling of urine, which poses a biohazard risk. Gloves, goggles, and a protective apron are a minimum requirement for PPE."})]}),Object(Le.jsxs)("ol",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"Download the NeuroDetech computer application."}),Object(Le.jsx)("li",{children:"Connect the optical detector of the microfluidic assay to the computer via USB."}),Object(Le.jsx)("li",{children:"Pipette 30 \xb5L of the RNAlater-preserved urine sample onto the sample application site of the microfluidic assay chip."}),Object(Le.jsx)("ul",{children:Object(Le.jsx)("li",{children:"Through the action of the capillary pump, the sample will immediately begin to flow through the microfluidic assay."})}),Object(Le.jsx)("li",{children:"Wait 20 minutes for the sample to flow through the assay. A graph of analyte concentration (i.e. PEA or ADHD-associated mRNA) will be visualized in real time on the computer application as the sample passes through the test and control chambers."}),Object(Le.jsx)("ul",{children:Object(Le.jsx)("li",{children:"The 20-minute assay time is based on the design of Ghosh et al. (2020), from which the design of NeuroDetech\u2019s microfluidic assay is inspired. Dimensions of the NeuroDetech chip follow the dimensions as outlined in the paper."})}),Object(Le.jsx)("li",{children:"Once the assay is complete, disconnect the optical detector from the computer, then dispose of the microfluidic assay in the appropriate biohazard waste. The optical detector hardware is reusable."})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:["The above steps can be repeated for the other three microfluidic assays to determine the concentrations of the other analytes. After completing all assays, concentrations for PEA, as well as the presence of ADHD-associated mutations in ",Object(Le.jsx)("i",{children:"DRD4"})," VNTR motif 3, ",Object(Le.jsx)("i",{children:"DRD4"})," VNTR motif 1, and ",Object(Le.jsx)("i",{children:"SNAP25"})," will have been determined."]}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"Safe Disposal of the Microfluidic Assay"}),Object(Le.jsx)("div",{className:pe.a.description,children:"There exists a need to identify a safe usage and waste protocol for used microfluidic assays. Our test contains a variety of chemicals, and should not be broken/tampered with to reveal the inner contents, at any point in the life of the tool. Chemicals such as Amplex Red can cause harm if in contact with the user. Furthermore, used tests will contain urine samples, which can pose a threat to people handling the assay. These chemicals have the potential to induce effects such as skin and eye irritation (Thermo Fisher, 2019). Thus, the user must take precautions to not break or alter the structure of the microfluidic assay at any point during its usage to expose these chemicals in an unsafe manner (Iacobucci, 2021)."}),Object(Le.jsx)("div",{className:pe.a.description,children:"Once the test has been completed, post-handling is required to interpret the results (e.g., running the chip on an application to yield a graph). Since the test would contain traces of urine, it will have to be handled with care to ensure that there is no contamination from the chip to the user, or external environment, such as the computer. Safe disposal methods would include discarding a used assay in a timely manner immediately after it has done its job in an appropriate waste bin/biohazard bin."}),Object(Le.jsx)("div",{className:pe.a.description,children:"By exercising caution during its handling, usage, and waste processes, we can ensure that NeuroDetech is being utilized in a safe and efficient manner within a clinical setting."})]})})]})})}}]),i}(n.Component),dn=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"References"}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"AMPLEX\u2122 Red reagent. Thermo Fisher Scientific - US. (2019). Retrieved October 8, 2021, from https://www.thermofisher.com/order/catalog/product/A12222#/A12222."}),Object(Le.jsx)("li",{children:"Araz, M. K., Tentori, A. M., & Herr, A. E. (2013). Microfluidic Multiplexing in Bioanalyses. Journal of Laboratory Automation, 18(5), 350\u2013366. https://doi.org/10.1177/2211068213491408"}),Object(Le.jsxs)("li",{children:["Chivers, C. E., Koner, A. L., Lowe, E. D., & Howarth, M. (2011). How the biotin\u2013streptavidin interaction was made even stronger: Investigation via crystallography and a chimaeric tetramer. ",Object(Le.jsx)("i",{children:"Biochemical Journal, 435"}),"(1), 55\u201363. https://doi.org/10.1042/bj20101593."]}),Object(Le.jsx)("li",{children:"Cui, P., & Wang, S. (2019). Application of microfluidic chip technology in pharmaceutical analysis: A review. Journal of pharmaceutical analysis, 9(4), 238\u2013247. https://doi.org/10.1016/j.jpha.2018.12.001"}),Object(Le.jsxs)("li",{children:[Object(Le.jsx)("i",{children:"Dietary Restrictions for Urine Tests"}),". (n.d.). MidMichigan Health. Retrieved October 3, 2021, from https://www.midmichigan.org/conditions-treatments/tests-procedures/dietary-restrictions-for-urine-tests/."]}),Object(Le.jsx)("li",{children:"Ghosh, S., Aggarwal, K. Upaassana, V. T., Nguyen, T., Han, J., & Ahn, C. H. (2020). A new microchannel capillary flow assay (MCFA) platform with lyophilized chemiluminescence reagents for a smartphone-based POCT detecting malaria. Microsystems & Nanoengineering, 6, 5. https://doi.org/10.1038/s41378-019-0108-8"}),Object(Le.jsxs)("li",{children:["Iacobucci, G. (2021). Covid-19: US regulator raises \u201csignificant concerns\u201d over safety of rapid lateral flow tests. ",Object(Le.jsx)("i",{children:"BMJ"}),". https://doi.org/10.1136/bmj.n1514"]}),Object(Le.jsxs)("li",{children:["Mayo Clinic Staff. (2019, October 23). ",Object(Le.jsx)("i",{children:"Urinalysis"}),". Mayo Clinic. Retrieved October 3, 2021, from https://www.mayoclinic.org/tests-procedures/urinalysis/about/pac-20384907."]}),Object(Le.jsx)("li",{children:"O'Neill, E. N., Cosenza, Z. A., Baar, K., & Block, D. E. (2021). Considerations for the development of cost\u2010effective cell culture media for cultivated meat production. Comprehensive Reviews in Food Science and Food Safety, 20(1), 686-709. "}),Object(Le.jsxs)("li",{children:["Roxe, D. M. (1990). Urinalysis. In ",Object(Le.jsx)("i",{children:"Clinical methods: The history, physical, and Laboratory Examinations"}),". essay, Butterworths. Retrieved October 3, 2021, from https://www.ncbi.nlm.nih.gov/books/NBK302/."]}),Object(Le.jsxs)("li",{children:["Thermo Fisher Scientific. (n.d.). ",Object(Le.jsx)("i",{children:"Pierce\u2122 monomeric AVIDIN AGAROSE Kit, 2 ML"}),". Retrieved July 1, 2021, from https://www.thermofisher.com/order/catalog/product/20227?SID=srch-srp-20227#/20227?SID=srch-srp-20227."]}),Object(Le.jsxs)("li",{children:["ThermoFisher Scientific. (n.d.). ",Object(Le.jsx)("i",{children:"Pierce\xae Monomeric Avidin Kit"}),". Retrieved July 1, 2021, from https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011221_Pierce_Monomeric_Avidin_UG.pdf."]}),Object(Le.jsx)("li",{children:"TWinFM. \u201cLateral Flow Testing for Workplaces - Are They Safe?\u201d TWinFM, www.twinfm.com/article/lateral-flow-testing-for-workplaces-are-they-safe."}),Object(Le.jsxs)("li",{children:["University of Alberta News Staff. (2013, September 9). ",Object(Le.jsx)("i",{children:"Researchers find the key to what's in our pee"}),". Faculty of Medicine & Dentistry. Retrieved October 3, 2021, from https://www.ualberta.ca/medicine/news/2013/september/urinechemicals.html."]})]})]})})}}]),i}(n.Component),hn=["NeuroDetech Implementation in Lab Settings","Proper Usage of NeuroDetech","General Safety Considerations for NeuroDetech Usage","Current Challenges and Future Improvements","References"],un=["","","","",""],mn=function(){var e=be(Object(n.useState)(hn[0]),2),t=e[0],i=e[1];return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.heading_div,children:[Object(Le.jsxs)("div",{className:pe.a.title,children:[Object(Le.jsx)("div",{className:pe.a.page_heading,children:"NeuroDetech"}),Object(Le.jsx)("div",{className:pe.a.page_heading_colored,children:"Implementation."})]}),Object(Le.jsx)("div",{className:pe.a.illustration,children:Object(Le.jsx)("img",{src:"",alt:"Implementation Icon",className:nn.a.icon_img})})]}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Overview"}),Object(Le.jsx)("div",{className:pe.a.description,children:"Throughout the entirety of NeuroDetech\u2019s development, we kept its implementation at the forefront of our minds. Continuous stakeholder feedback and thorough research from all 3 subteams allowed us to create a product that could realistically be manufactured and employed in laboratory settings."})]}),Object(Le.jsx)("div",{className:pe.a.sections_div,children:hn.map((function(e,n){return Object(Le.jsx)("div",{active:t===e,onClick:function(){return i(e)},className:pe.a.section_block,children:Object(Le.jsxs)("div",{className:pe.a.sections,children:[Object(Le.jsx)("div",{className:pe.a.section_img,children:Object(Le.jsx)("img",{id:"implementation".concat(n),src:un[n],alt:"Icon"})}),Object(Le.jsx)("div",{className:pe.a.section_text,children:e})]})},e)}))}),Object(Le.jsxs)("div",{children:[t===hn[0]&&Object(Le.jsx)(sn,{}),t===hn[1]&&Object(Le.jsx)(ln,{}),t===hn[2]&&Object(Le.jsx)(rn,{}),t===hn[3]&&Object(Le.jsx)(an,{}),t===hn[4]&&Object(Le.jsx)(dn,{})]})]})},pn=i(67),fn=i.n(pn),bn=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"References"}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"5 tips on designing colorblind-friendly visualizations. Tableau. (n.d.). Retrieved October 21, 2021, from https://www.tableau.com/about/blog/examining-data-viz-rules-dont-use-red-green-together."}),Object(Le.jsx)("li",{children:"Blue Light has a dark side. Harvard Health. (2020, July 7). Retrieved October 21, 2021, from https://www.health.harvard.edu/staying-healthy/blue-light-has-a-dark-side?_ga=2.89087004.623627247.1634565693-1834042642.1634565693."}),Object(Le.jsx)("li",{children:"Fottrell, Q. (2018, August 4). People spend most of their waking hours staring at screens. MarketWatch. Retrieved October 21, 2021, from https://www.marketwatch.com/story/people-are-spending-most-of-their-waking-hours-staring-at-screens-2018-08-01?_ga=2.151606590.623627247.1634565693-1834042642.1634565693."})]})]})})}}]),i}(n.Component),jn=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Wiki Design"}),Object(Le.jsxs)("div",{className:pe.a.description,children:["To read about the design of our wiki and how our design increases accessibility for all users and for ADHD, please refer to our wiki design handbook\xa0",Object(Le.jsx)("a",{href:"https://static.igem.org/mediawiki/2021/6/68/T--Waterloo--InclusivityDesignHandbook.pdf",download:!0,children:"here"}),". The description below outlines the key features that were considered. Please note that the features are fully designed but not fully implemented. Development will be completed in future iterations."]}),Object(Le.jsx)(Ie,{open:!0,title:"Accessibility Toolbar",children:Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("div",{className:pe.a.description,children:"Although perfect accessibility is unrealistic, there are many opportunities for improvement in the areas of inclusion. In this year's wiki, we have developed and completed our accessibility toolbar with an additional feature - the day and night mode. The accessibility toolbar can be enabled by mouse clicks or keyboard shortcuts which are visibly displayed under each label."})})}),Object(Le.jsx)(Ie,{open:!0,title:"The Benefits of Dark Mode",children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.description,children:"We introduce the switch mode options, a simple toggle that lets users change the theme of the website into a darker colour. Exposure to light for a long period of time can suppress the secretion of melatonin\u2013in particular, the effects of blue light are stronger at night (Harvard Health Publishing, 2020). Moreover, the symptoms of digital eye strain can be caused through bright screens (Fottrell, 2018). Therefore, we want to implement the option of dark mode to help protect our users\u2019 vision when browsing our wiki."}),Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:"Our previous year version of the Accessibility Toolbar features:"}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"Read-aloud screen-reader (play, pause, restart)"}),Object(Le.jsx)("li",{children:"Adjustable font size controller"})]}),Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:"Our new version of the Accessibility Toolbar features:"}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"Adjustable font size controller"}),Object(Le.jsx)("li",{children:"Change Mode (day/night)"}),Object(Le.jsx)("li",{children:"Read-aloud screen-reader (play, pause, restart)"})]}),Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:"In future interactions of the Accessibility Toolbar, we want to include:"}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"Word-highlighter (as screen-reader plays)"}),Object(Le.jsx)("li",{children:"Active voice search and search bar by keywords"}),Object(Le.jsx)("li",{children:"Customizable colour palettes(for various types of colour-blindness)"})]})]})}),Object(Le.jsx)(Ie,{open:!0,title:"Navigability",children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.description,children:"Easy navigation around a website is one of the most important considerations when it comes to designing a website. The cornerstone of web usability is navigation, and users can better navigate the website and discover information more quickly if it is well-designed."}),Object(Le.jsx)("div",{className:pe.a.description,children:'We added the "up to the top" arrow options so that when the information gets too lengthy, users have the option to scroll up to the top of the page faster instead of manually scrolling up.'}),Object(Le.jsx)("div",{className:pe.a.description,children:"To improve the website's functionality, we added the index section for users to quickly find information."})]})}),Object(Le.jsx)(Ie,{open:!0,title:"Chatbot",children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.description,children:"We want to introduce the chat bot feature of wiki, which conducts conversations without human intervention. The chatbot allows users to access all the necessary knowledge with keywords attached, leading to the information. We believe that the chatbot would offer convenience to the users with fast and efficient communication as to where things are and address their concerns on existing problems."}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"How can we make our chatbot user-friendly?"}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"Chatbot Function"}),Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:"For our future wiki, we want to focus on the two main functions for our chatbot, which include:"}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"Searching for information: when users input a keyword, the chatbot will automatically return them a list of pages containing the keywords and allow them to click on the page from the conversation."}),Object(Le.jsx)("li",{children:"Contacting the team: the users can also send us messages/emails through the chatbot when they have any concerns or questions for our team."})]}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"Chatbot Personality"}),Object(Le.jsx)("div",{className:pe.a.description,children:"Incorporating the human-like characteristics of the bot through programming, the chatbot's personality is built on its ability to keep a consistent tone of voice throughout the conversation. This entails carefully selecting the phrases used in a chatbot. It involves directing how our chatbot maintains interactions and responds to irrelevant or perplexing questions. The Chatbot personality helps define the user experience and is key to the way users interact and learn about our information."}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"Chatbot Future Development"}),Object(Le.jsx)("div",{className:pe.a.description,children:"Besides finishing the development of the chatbot, we want the future wiki to expand more on the functionality and characteristics of the chatbot. 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Instead, the current flowed to the analog pin and the microcontroller read a high voltage (Kranthi, 2013)."})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("video",{width:"100%",controls:!0,children:Object(Le.jsx)("source",{src:"https://static.igem.org/mediawiki/2021/8/84/T--Waterloo--TinkerCADVidFinal.mp4"})}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"TinkerCAD Demo"})]})]})}}]),i}(n.Component),_r=i(73),kr=i.n(_r),Ar=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Microfluidic Chip Modelling"}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"Computer-aided Chip Assembly Design"}),Object(Le.jsx)("div",{className:pe.a.description,children:"A complete assembly of the microfluidic chip model is built and shown below. The specific dimensions are also labelled and presented. The team used OnShape to create the CAD model throughout the year."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",alt:"Dimensioned Full Chip"}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Dimensioned drawing of the microfluidic chip."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",alt:"Corner View Assembly",className:kr.a.assembly_img}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Assembly of the microfluidic chip. Note that the top plane surfaces are transparent for easy visualization of the chip inner components."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("div",{className:pe.a.description,children:"For further information on the details of the chip model design, please navigate to the Microfluidic Chip Design module under the Engineering page. With an initial model built in Computer-Aided Design software, the chip can be built later on to prototype the process in which sample flows through the chip and improve the design of the chip model."})})]})}}]),i}(n.Component),Cr=i(16),Tr=i.n(Cr),Sr=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Protein Optimization Modelling"}),Object(Le.jsx)("div",{className:pe.a.description,children:"The selected proteins to be optimized, trypsin and monomeric streptavidin, are modelled and analyzed using software such as Rosetta, Chimera and Autodock. In the end, proteins with improved stability and affinity are designed."}),Object(Le.jsx)(Ie,{open:!0,title:"Rosetta: Protein Mutations",children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.description,children:"Rosetta is a software tool that was primarily designed to predict structures of proteins. It now has many other applications such as determining macromolecular interactions, molecular docking and protein design (Kaufmann et. al., 2010)."}),Object(Le.jsx)("div",{className:pe.a.description,children:"Specifically for this project we utilize a protocol, which is a combination of algorithms, called Point Mutant Scan (pmut_scan_parallel) which allows us to input the original protein data bank (PDB) file that contains the sequence of amino acids of the entire protein, and the protocol will output PDBs with all possible combinations of single mutations."}),Object(Le.jsx)("div",{className:pe.a.description,children:"The algorithm that Rosetta uses is, once given the original PDB file, it generates a list of every possible mutation of all residues in the protein. The algorithm does this by swapping out the existing amino acid at that residue and replacing it with all other available amino acids. It then makes the mutation and predicts the structure of the newly mutated protein (Rosetta Commons, n.d.). The way that Rosetta is able to predict the structure of the protein is by taking small parts of the protein that is known to create the structure that is most similar to how the protein behaves in nature using the Monte Carlo approach (Rohl et. al., 2004). The Monte Carlo method in brief is the process of sampling a multitude of random combinations, and taking the average of those combinations to form a prediction of the outcome (Zhang et. al., 1992)."}),Object(Le.jsx)("div",{className:pe.a.description,children:"The outcome of running this protocol is a list of new PDB files, which are then passed onto a scoring protocol. The way that Rosetta determines a score for each mutated protein is by using a weighted average of different energies that it calculates, such as repulsive, attractive and hydrogen bond energies. The lower the Rosetta score, the more stable the protein is, thus the goal is to design the protein to have a lower score compared to the original protein."}),Object(Le.jsx)("div",{className:pe.a.subheading,style:{marginLeft:"5%"},children:"Workflow for Mutating Proteins"}),Object(Le.jsxs)("ol",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"Obtain the 1UTM PDB file from the protein data bank. "}),Object(Le.jsx)("li",{children:"Run a python script to \u201cclean\u201d the PDB file. This removes all the headers and descriptions from the PDB file and leaves it with only the amino acids and the ligand."}),Object(Le.jsx)("li",{children:"Use Rosetta\u2019s Point Mutant Scan protocol to generate all the possible single mutations for 1UTM."}),Object(Le.jsx)("li",{children:"Score each new mutated protein using Rosetta\u2019s scoring protocol to quantify the mutation and perform further analysis and verification."})]}),Object(Le.jsx)("div",{className:pe.a.description,children:"Since the point mutant scan protocol and the scoring protocol employ the Monte Carlo method, a high performance computer was used to be able to support the computational power that is needed to generate all the random combinations. We used the Graham cluster from Compute Canada to utilize the Rosetta Software."})]})}),Object(Le.jsxs)(Ie,{open:!0,title:"Improvement of Trypsin Protein",children:[Object(Le.jsxs)("div",{className:Tr.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.subheading,style:{marginLeft:"5%"},children:"Results: Trypsin Single Mutation"}),Object(Le.jsx)("div",{className:pe.a.description,children:"There were a total of 4143 single mutations made from using Rosetta\u2019s Point Mutant Scan protocol. Listed below are the top 50 lowest scoring single mutations. The original score for the un-mutated 1UTM with PEA was -322.402 REU."}),Object(Le.jsxs)("div",{className:pe.a.description,children:["Top 10 Rosetta Scores for 1UTM single mutations and the original protein score are listed in the table below. Please check the full top 50 scores on the spreadsheet\xa0",Object(Le.jsx)("a",{className:pe.a.text_link,href:"https://docs.google.com/spreadsheets/u/1/d/1r_Wf-dGtYojBKM7i1tRiwdZFUIioxGey/edit",target:"_blank",rel:"noreferrer",children:"here"}),". For this and all following score tables, the mutations are written in the format \u201cOriginal Amino Acid|Residue Number|New Amino Acid\u201d."]})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)(Zt,{bordered:!0,className:pe.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:"Score (REU)"}),Object(Le.jsx)("th",{children:"Mutation"})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-322.402"}),Object(Le.jsx)("td",{children:"Original"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-344.859"}),Object(Le.jsx)("td",{children:"A205T"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-344.706"}),Object(Le.jsx)("td",{children:"I81N"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-344.222"}),Object(Le.jsx)("td",{children:"I81H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-343.811"}),Object(Le.jsx)("td",{children:"I81W"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-343.794"}),Object(Le.jsx)("td",{children:"I81R"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-343.771"}),Object(Le.jsx)("td",{children:"I81K"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-343.657"}),Object(Le.jsx)("td",{children:"I81Y"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-343.647"}),Object(Le.jsx)("td",{children:"A205C"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-343.253"}),Object(Le.jsx)("td",{children:"I81F"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-343.191"}),Object(Le.jsx)("td",{children:"I81Q"})]})]})]})}),Object(Le.jsxs)("div",{className:Tr.a.text_div,children:[Object(Le.jsxs)("div",{className:pe.a.description,children:["However, our main goal for our newly designed protein is to increase its ability to bind to the analyte which is PEA. Therefore we must look at the mutations made near the binding pocket residues. Those residues are 169, 170, 171, 172, 175, 189, 190, 191,192,194, 195, 202, 203, and 204. These mutations should have a stronger impact on 1UTM\u2019s binding affinity with PEA, thus being analyzed in more detail. The mutations that are considered for each residue are listed in the spreadsheet\xa0",Object(Le.jsx)("a",{className:pe.a.text_link,href:"https://docs.google.com/spreadsheets/u/1/d/1r_Wf-dGtYojBKM7i1tRiwdZFUIioxGey/edit",target:"_blank",rel:"noreferrer",children:"here"}),"."]}),Object(Le.jsx)("div",{className:pe.a.subheading,style:{marginLeft:"5%"},children:"Single Mutation Result Interpretation"}),Object(Le.jsx)("div",{className:pe.a.description,children:"Double mutations for the binding residues were proposed using a combination of rational design and simply evaluating the Rosetta scores of the single mutations for stability. The characteristics of PEA were considered with those of an individual residue in order to make suggestions."}),Object(Le.jsx)("div",{className:pe.a.description,children:"The criteria for rational design-proposed mutations depended on the properties of the original amino acid as well as those of the ligand. PEA is positively charged (EMBL-EBI, 2021), thus we proposed changing positively-charged, neutral, or non-polar residues such as serine, cysteine, glutamine, (Merck KGaA, 2021) , and glycine (PubChem Identifier: CID 750, 2021), to negatively-charged amino acids such as aspartic acid and glutamic acid to better accommodate the ligand (Foulquier, 2020). Phenylalanine and Tyrosine, which both possess aromatic side chains, were suggested as changes for any amino acid in the binding pocket, in particular Valine, which possesses an aliphatic side chain (PubChem Identifier: CID 6287, 2021), (Merck KGaA, 2021). These changes were proposed to hopefully result in pi-pi stacking between 1UTM and PEA\u2019s aromatic ring. (Hardinger, 2017a). Trp, which also has an aromatic side chain, was not tested in these circumstances due to its bulkier structure, which could result in increased steric interference (Hardinger, 2017b). As such, when it appeared on the unmodified version of 1UTM, a change from Tryptophan to Phenylalanine or Tyrosine was suggested."}),Object(Le.jsx)("div",{className:pe.a.description,children:"A comparison of the original 1UTM protein and the mutation of residue 170 from serine to Aspartic acid was visualized in Chimera, as seen in the images below. The original protein is brown, while the mutated protein is blue. Residue 170 is seen enclosed in the red circle on both images."}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",alt:"1UTM Mutation Overlap 1b",className:Tr.a.mutation_img}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"1UTM Original 170 Residue"})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",alt:"1UTM Mutation Overlap 2",className:Tr.a.mutation_img}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"1UTM Mutated 170 Residue"})]}),Object(Le.jsx)("div",{className:pe.a.description,children:"Next, we checked the Rosetta scores for these biochemically chosen single mutations to ensure they were reasonably stable. If a mutation received a score of -300 or lower it was considered for double mutations, otherwise, it was discarded. A maximum of four mutations per residue was passed to Rosetta to synthesize double mutations. When four candidates were passed on, the top two Rosetta scores and two best-scoring rational design candidates were selected, if all four were distinct from each other. If there were less than two suitable rational design candidates, the top three Rosetta-scoring mutations were submitted for double mutations. These groups of top three scorers were accompanied by a rational design candidate if a suitable one was present."}),Object(Le.jsx)("div",{className:pe.a.subheading,style:{marginLeft:"5%"},children:"Results: Double Mutations with Rosetta"}),Object(Le.jsx)("div",{className:pe.a.description,children:"In order to generate the double mutations, we used the same Rosetta protocol, however this time we defined a list of all the double mutations that we want to make. The outcome of this is a PDB file for each double mutation that was passed into the protocol."}),Object(Le.jsx)("div",{className:pe.a.description,children:"A heat map was then generated to allow us to visually see which double mutation combination gave us the lowest scoring protein."}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",alt:"1UTM Double Mutations Heatmap",className:Tr.a.mutation_img}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Heat Map of 1UTM Double Mutations"})]}),Object(Le.jsx)("div",{className:pe.a.description,children:"From the heat map above, we picked out the strongest (lowest scoring) double mutations and combined them with other double mutations that also performed well but not the strongest. This led to a total of 56 triple and quadruple mutations that were made on 1UTM, using the same Point Mutant Scan protocol used for the single and double mutations. We again defined the triple and quadruple mutations that were selected and the output is a PDB file for each mutation made."}),Object(Le.jsx)("div",{className:pe.a.subheading,style:{marginLeft:"5%"},children:"Results: Trypsin Multiple Mutations"}),Object(Le.jsx)("div",{className:pe.a.description,children:"The top 15 1UTM multi-mutations will be further considered and analyzed using other tools like Auto-dock Vina and Gromacs. However, the best scoring designed protein based on Rosetta scores is a tie between four triple mutations, which are"}),Object(Le.jsxs)("ol",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"Y204F C195D S190T"}),Object(Le.jsx)("li",{children:"Y204F C195E S190T"}),Object(Le.jsx)("li",{children:"Y204F C195F S190T"}),Object(Le.jsx)("li",{children:"Y204F C195Y S190T"})]}),Object(Le.jsx)("div",{className:pe.a.description,children:"They all have a score of -368.553 REU."}),Object(Le.jsxs)("div",{className:pe.a.description,children:["Top 15 1UTM Multi-Mutations are listed in the table below. Full tables of modified 1UTM scores are also uploaded\xa0",Object(Le.jsx)("a",{className:pe.a.text_link,href:"https://docs.google.com/spreadsheets/u/1/d/1r_Wf-dGtYojBKM7i1tRiwdZFUIioxGey/edit",target:"_blank",rel:"noreferrer",children:"here"}),"."]})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)(Zt,{bordered:!0,className:pe.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:"Score (REU)"}),Object(Le.jsx)("th",{children:"Mutation 1"}),Object(Le.jsx)("th",{children:"Mutation 2"}),Object(Le.jsx)("th",{children:"Mutation 3"}),Object(Le.jsx)("th",{children:"Mutation 4"})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-368.553"}),Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"-"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-368.553"}),Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"-"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-368.553"}),Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"-"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-368.553"}),Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195Y"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"-"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.779"}),Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"C171D"}),Object(Le.jsx)("td",{children:"Y204H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.779"}),Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"C171E"}),Object(Le.jsx)("td",{children:"Y204H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.779"}),Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"C171F"}),Object(Le.jsx)("td",{children:"Y204H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.779"}),Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195Y"}),Object(Le.jsx)("td",{children:"C171Y"}),Object(Le.jsx)("td",{children:"Y204H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.616"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"C171E"}),Object(Le.jsx)("td",{children:"Y204H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.616"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"C171F"}),Object(Le.jsx)("td",{children:"Y204H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.616"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195Y"}),Object(Le.jsx)("td",{children:"C171Y"}),Object(Le.jsx)("td",{children:"Y204H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.616"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"C171D"}),Object(Le.jsx)("td",{children:"Y204H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.421"}),Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.421"}),Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-366.421"}),Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-"})]})]})]})})]}),Object(Le.jsxs)(Ie,{open:!0,title:"Improvement of Monomeric Streptavidin",children:[Object(Le.jsxs)("div",{className:Tr.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.description,children:"Monomeric streptavidin section of the binding molecule binds to biotin when it reaches the control chamber. For streptavidin, the same workflow as 1UTM was used to design this protein. First we auto-generated a list of all possible single mutations using Rosetta."}),Object(Le.jsx)("div",{className:pe.a.subheading,style:{marginLeft:"5%"},children:"Results: Monomeric Streptavidin Single Mutation"}),Object(Le.jsxs)("div",{className:pe.a.description,children:["The top 10 lowest scoring proteins are given in the following table. The original score for un-mutated streptavidin with biotin was -104.826 REU. Full top 50 mutations are recorded\xa0",Object(Le.jsx)("a",{className:pe.a.text_link,href:"https://docs.google.com/spreadsheets/u/1/d/1SgsB72_eUh2hm3cXvlslBUDv0N5JtfM7/edit",target:"_blank",rel:"noreferrer",children:"here"}),"."]})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)(Zt,{bordered:!0,className:pe.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:"Score (REU)"}),Object(Le.jsx)("th",{children:"Mutation"})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-104.826"}),Object(Le.jsx)("td",{children:"Original"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-141.239"}),Object(Le.jsx)("td",{children:"Y52F"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-139.972"}),Object(Le.jsx)("td",{children:"L57M"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-139.911"}),Object(Le.jsx)("td",{children:"C41A"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-139.911"}),Object(Le.jsx)("td",{children:"C41D"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-139.911"}),Object(Le.jsx)("td",{children:"C41E"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-139.911"}),Object(Le.jsx)("td",{children:"C41F"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-139.911"}),Object(Le.jsx)("td",{children:"C41G"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-139.911"}),Object(Le.jsx)("td",{children:"C41H"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-139.911"}),Object(Le.jsx)("td",{children:"C41I"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-139.911"}),Object(Le.jsx)("td",{children:"C41K"})]})]})]})}),Object(Le.jsxs)("div",{className:Tr.a.text_div,children:[Object(Le.jsxs)("div",{className:pe.a.description,children:["We also took a closer look at the binding pocket residues for streptavidin which are residues 12, 16, 32, 34, 36, 72, 74 and 92. The mutations that scored higher than the original protein for each binding pocket residue are further analyzed and recorded\xa0",Object(Le.jsx)("a",{className:pe.a.text_link,href:"https://docs.google.com/spreadsheets/u/1/d/1SgsB72_eUh2hm3cXvlslBUDv0N5JtfM7/edit",target:"_blank",rel:"noreferrer",children:"here"}),"."]}),Object(Le.jsx)("div",{className:pe.a.description,children:"Then double mutations were chosen based on the Rosetta scores similar to the process used for 1UTM. Those double mutations were passed into Rosetta and a heat map was generated for Streptavidin as well."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",alt:"Strep Heatmap",className:Tr.a.mutation_img}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Heat Map of Monomeric Streptavidin Double Mutations"})]}),Object(Le.jsx)("div",{className:Tr.a.text_div,children:Object(Le.jsx)("div",{className:pe.a.description,children:"From the heat map, the strongest double mutations were chosen and then combined with other less strong mutations as well as a few of the top 50 single mutations to generate triple and quadruple mutations. This list was also passed into Rosetta to generate the PDB files and scores. The scores for the 12 suggested triple, quadruple, quintuple and sixtuple mutations for streptavidin are listed below."})}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)(Zt,{bordered:!0,className:pe.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:"Score (REU)"}),Object(Le.jsx)("th",{children:"Mutation"}),Object(Le.jsx)("th",{}),Object(Le.jsx)("th",{}),Object(Le.jsx)("th",{}),Object(Le.jsx)("th",{})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-167.751"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"Y52F"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-166.862"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"L57M"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-157.12"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"T49K"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-154.14"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"N64S"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-153.232"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"R60T"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-133.52"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"A36C"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-132.144"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"N34A"}),Object(Le.jsx)("td",{children:"A36C"}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-132.051"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"N34A"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-131.989"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"A36C"}),Object(Le.jsx)("td",{children:"Y32A"}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-130.956"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"Y32C"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-129.267"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"N34A"}),Object(Le.jsx)("td",{children:"A36C"}),Object(Le.jsx)("td",{children:"Y32A"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"-128.766"}),Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"N34A"}),Object(Le.jsx)("td",{children:"Y32A"}),Object(Le.jsx)("td",{})]})]})]})}),Object(Le.jsx)("div",{className:Tr.a.text_div,children:Object(Le.jsx)("div",{className:pe.a.description,children:"Based on Rosetta scores, the best designed protein consisted of the mutations, T74C, N12A, Y52F."})})]}),Object(Le.jsxs)(Ie,{open:!0,title:"Docking Analysis with AutoDock Vina and Chimera",children:[Object(Le.jsxs)("div",{className:Tr.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.description,children:"To test that the modified proteins from Rosetta protocols do have a higher binding energy with the ligand, docking analysis and binding energies are calculated for the top mutated proteins produced in the Build section. Autodock Vina is a program for performing molecular docking simulations using input receptor-ligand pairs. Performing ligand docking by AutoDock Vina results in the generation of multiple PDBQT files containing the coordinates in space for the docked protein complex. These PDBQT files can further be loaded into the previously mentioned UCSF Chimera for verification of binding in the desired pocket. Compared to its original counterpart, AutoDock Vina is 59% more accurate than AutoDock 4 in yielding binding mode predictions with an RMSD less than 2. Similarly, AutoDock Vina is nearly 450, and 65 times faster than AutoDock 4, running on an 8-, and 1- core CPU, respectively. Conveniently, the functionalities of AutoDock Vina are integrated into the Chimera GUI and can also be run using Chimera Scripts, so long as you have downloaded the AutoDock Vina executable on your local device. Previously, docking simulations were run manually using this functionality in Chimera by selecting a volumetric box for the binding pocket, setting the appropriate binding mode parameters, and running the docking one protein at a time. To circumvent the issue of long process times using this manual method, an automated method using Chimera\u2019s support for Python scripts was implemented."}),Object(Le.jsx)("div",{className:pe.a.description,children:"The Python script serves several purposes including, opening the mutated protein output from Rosetta, deleting the ligand from the complex, adding the native ligand file to the environment, and performing ligand docking, saving only the docking coordinate of the highest scoring conformer. These scores were then copied and written to a master energy score text file automatically and saved. Both singly- and doubly- mutated proteins generated by Rosetta were used for docking. The doubly mutated proteins combined mutations from both the binding pocket and a non-binding region in efforts to decrease the energy score of the overall structure for enhanced stability. Implementation of this process revealed that singly mutated trypsin-PEA complexed proteins outperformed doubly-mutated trypsin-PEA protein complexes on average. To illustrate this, the following distributions were observed for singly- vs doubly- mutated trypsin proteins. As you can see, the maximum energy score for doubly mutated proteins was -4.8 kcal/mole, while an energy score of -4.9 kcal/mole was frequently seen in singly-mutated proteins. From these preliminary energy score results we can deduce that double mutations on the complex did not have any effect on decreasing its energy score, and thus stability was not affected. To further confirm this a secondary energy analysis should be performed using the score_jd2 protocol in Rosetta or structure scoring in GROMACS."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("div",{className:pe.a.figure_text,children:"1UTM-PEA Docking Scores"}),Object(Le.jsxs)(Zt,{bordered:!0,className:pe.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:"Mutation 1"}),Object(Le.jsx)("th",{children:"Mutation 2"}),Object(Le.jsx)("th",{children:"Mutation 3"}),Object(Le.jsx)("th",{children:"Mutation 4"}),Object(Le.jsx)("th",{children:"Interaction Energy (kcal/mole)"})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Native"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-4.6"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"C171E"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"C171F"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"S190"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-4.7"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-4.7"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195Y"}),Object(Le.jsx)("td",{children:"C171Y"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-4.9"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"C171D"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195Y"}),Object(Le.jsx)("td",{children:"C171Y"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195Y"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195D"}),Object(Le.jsx)("td",{children:"C171D"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{children:"C195E"}),Object(Le.jsx)("td",{children:"C171E"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"C171F"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Y204F"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"S190T"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-4.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"S190A"}),Object(Le.jsx)("td",{children:"C195F"}),Object(Le.jsx)("td",{children:"Y204H"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-4.8"})]})]})]})]}),Object(Le.jsx)("div",{className:Tr.a.text_div,children:Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"A similar energy conformer analysis using the above-mentioned automated approach using a Python script was implemented for mutation candidates generated by Rosetta on the monomeric streptavidin-biotin complex. The lowest scoring multi-mutant streptavidin proteins yielded an energy score of -7.4 kcal/mole."})})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Streptavidin-Biotin Docking Scores"}),Object(Le.jsxs)(Zt,{bordered:!0,className:pe.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:"Mutation 1"}),Object(Le.jsx)("th",{children:"Mutation 2"}),Object(Le.jsx)("th",{children:"Mutation 3"}),Object(Le.jsx)("th",{children:"Mutation 4"}),Object(Le.jsx)("th",{children:"Mutation 5"}),Object(Le.jsx)("th",{children:"Interaction Energy (kcal/mole)"})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"L57M"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-5.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"T49K"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-5.9"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"Y52F"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-7.4"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"A36C"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-7.1"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"R60T"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-5.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"N12A"}),Object(Le.jsx)("td",{children:"N64S"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-5.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Native"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-5.6"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"A-N12A"}),Object(Le.jsx)("td",{children:"N34A"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-7.1"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"A-N12A"}),Object(Le.jsx)("td",{children:"A-N34A"}),Object(Le.jsx)("td",{children:"A36C"}),Object(Le.jsx)("td",{children:"Y32A"}),Object(Le.jsx)("td",{children:"-5.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"A-N12A"}),Object(Le.jsx)("td",{children:"A-Y32C"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-7.2"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"A-N12A"}),Object(Le.jsx)("td",{children:"A-N34A"}),Object(Le.jsx)("td",{children:"A-Y32A"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-5.8"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"A-N12A"}),Object(Le.jsx)("td",{children:"A-A36C"}),Object(Le.jsx)("td",{children:"A-Y32A"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-5.7"})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"T74C"}),Object(Le.jsx)("td",{children:"A-N12A"}),Object(Le.jsx)("td",{children:"A-N34A"}),Object(Le.jsx)("td",{children:"A-A36C"}),Object(Le.jsx)("td",{}),Object(Le.jsx)("td",{children:"-7.4"})]})]})]})]})]})]})})}}]),i}(n.Component),Pr=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"References"}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"European Molecular Biology Laboratory (EMBL-EBI). (2021). PEA : Summary. PDBeChem: Ligand Dictionary (PDB Ligand Chemistry - chemical component dictionary). Retrieved October 13, 2021, from https://www.ebi.ac.uk/pdbe-srv/pdbechem/chemicalCompound/complete/PEA."}),Object(Le.jsx)("li",{children:"Fields G. B. (2002). Introduction to peptide synthesis. Current protocols in protein science, Chapter 18, Unit\u201318.1. https://doi.org/10.1002/0471140864.ps1801s26"}),Object(Le.jsx)("li",{children:"Foulquier, E. (2020, January 20). Amino acids. IMGT Education. Retrieved October 8, 2021, from http://www.imgt.org/IMGTeducation/Aide-memoire/_UK/aminoacids/charge/."}),Object(Le.jsx)("li",{children:"Gao, M., & Skolnick, J. (2012). The distribution of ligand-binding pockets around protein-protein interfaces suggests a general mechanism for pocket formation. Proceedings of the National Academy of Science of the United States of America , 109(10), 3784\u20133789. https://doi.org/10.1073/pnas.1117768109"}),Object(Le.jsx)("li",{children:"Hardinger, S. A. (2017). Aromatic-aromatic interaction (aromatic stacking, pi stacking): Illustrated Glossary of Organic Chemistry . Retrieved October 8, 2021, from http://www.chem.ucla.edu/~harding/IGOC/A/aromatic_aromatic_interaction.html."}),Object(Le.jsx)("li",{children:"Huang, PS., Boyken, S. & Baker, D. The coming of age of de novo protein design. Nature 537, 320\u2013327 (2016). https://doi.org/10.1038/nature19946"}),Object(Le.jsx)("li",{children:"Kaufmann, K. W., Lemmon, G. H., DeLuca, S. L., Sheehan, J. H., & Meiler, J. (2010). Practically Useful: What the Rosetta Protein Modeling Suite Can Do for You. Biochemistry (Easton), 49(14), 2987\u20132998. https://doi.org/10.1021/bi902153g"}),Object(Le.jsx)("li",{children:"Leiros, H.-K. S., Brandsdal, B. O., Andersen, O. A., Os, V., I Leiros, Helland, R., Otlewski, J., Willassen, N. P., & Smalas, A. O. (2003). Trypsin specificity as elucidated by LIE calculations, X-ray structures and association constant measurements. Doi: 10.2210/pdb1UTM/pdb"}),Object(Le.jsx)("li",{children:"Merck KGaA. (2021). Amino acids reference chart. MilliporeSigma. Retrieved October 8, 2021, from https://www.sigmaaldrich.com/CA/en/technical-documents/technical-article/protein-biology/protein-structural-analysis/amino-acid-reference-chart."}),Object(Le.jsx)("li",{children:"National Center for Biotechnology Information (2021). PubChem Compound Summary for CID 6287, Valine. Retrieved October 8, 2021 from https://pubchem.ncbi.nlm.nih.gov/compound/Valine"}),Object(Le.jsx)("li",{children:"National Center for Biotechnology Information (2021). PubChem Compound Summary for CID 750, Glycine. Retrieved October 8, 2021 from https://pubchem.ncbi.nlm.nih.gov/compound/Glycine"}),Object(Le.jsx)("li",{children:"Packer, M., Liu, D. Methods for the directed evolution of proteins. Nat Rev Genet 16, 379\u2013394 (2015). https://doi.org/10.1038/nrg3927"}),Object(Le.jsx)("li",{children:"Rohl CA, Strauss CE., Misura KM., Baker D. Protein Structure Prediction Using Rosetta. Methods in Enzymology. 2004;383:66-93. doi:10.1016/S0076-6879(04)83004-0"}),Object(Le.jsx)("li",{children:'Rosetta Commons. (n.d.) Point mutant ("pmut") scan application, pmut_scan_parallel. Retreived from https://www.rosettacommons.org/docs/latest/application_documentation/design/'}),Object(Le.jsx)("li",{children:"Rosetta Commons. (n.d.). Energy Terms in Rosetta. Retrieved from https://www.rosettacommons.org/docs/latest/rosetta_basics/scoring/score-types"}),Object(Le.jsx)("li",{children:"Zhang CT, Chou KC. Monte Carlo simulation studies on the prediction of protein folding types from amino acid composition. 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However, streptavidin is tetrameric and rather bulky, making solubility and stability a concern in the context of a microfluidic assay. An engineered monomeric form of streptavidin exists, albeit at some expense of binding affinity, where the monomeric streptavidin and biotin interaction has a Kd of 10",Object(Le.jsx)("sup",{children:"-9"})," M (DeMonte et al., 2013). As a result, we sought to improve the affinity of monomeric streptavidin using computational rational protein design."]}),Object(Le.jsxs)("div",{className:pe.a.description,children:["The existing part for monomeric streptavidin (known as mSA2) is\xa0",Object(Le.jsx)("a",{className:pe.a.text_link,href:"http://parts.igem.org/Part:BBa_K3843007",target:"_blank",rel:"noreferrer",children:"BBa_K3843007"}),". Our affinity-improved version of mSA2, dubbed mSA2+, is documented under part\xa0",Object(Le.jsx)("a",{className:pe.a.text_link,href:"http://parts.igem.org/Part:BBa_K3843005",target:"_blank",rel:"noreferrer",children:"BBa_K3843005"}),"."]}),Object(Le.jsxs)("div",{className:pe.a.description,children:["Overall, through our computational rational protein design process (described in our\xa0",Object(Le.jsx)(ce,{to:"/Team:Waterloo/Engineering",className:pe.a.text_link,children:"Engineering"}),"\xa0page), we improved mSA2\u2019s biotin binding affinity by inducing the following mutations: T74C, N12A, and Y52F. These mutations were able to decrease the individual energy of mSA2 by 62.925 REU (Rosetta Energy Units) compared to the unmutated mSA2, based on Rosetta's scoring function. As well, the energy score of the mSA2-biotin complex decreased by 1.8 kcal/mol, or 7.5 kJ/mol (on AutoDock Vina), corresponding to an increase in binding affinity."]}),Object(Le.jsx)("div",{className:pe.a.description,children:"The following image depicts the improved mSA2, with mSA2+ coloured in blue and biotin highlighted in green, as visualized in UCSF Chimera:"})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("img",{src:"",alt:"mSA2+",className:Br.a.msa2_img})})]})}}]),i}(n.Component),Hr=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"References"}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsxs)("li",{children:["\u200b\u200bChivers, C. E., Koner, A. L., Lowe, E. D., & Howarth, M. (2011). How the biotin\u2013streptavidin interaction was made even stronger: Investigation via crystallography and a chimaeric tetramer. ",Object(Le.jsx)("i",{children:"Biochemical Journal, 435"}),"(1), 55\u201363. https://doi.org/10.1042/bj20101593."]}),Object(Le.jsxs)("li",{children:["DeMonte, D., Drake, E. J., Lim, K. H., Gulick, A. M., & Park, S. (2013, June 17). Structure\u2010based engineering of streptavidin monomer with a reduced biotin dissociation rate. 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Current ADHD diagnosis procedures require a series of psychological assessments that are qualitative in nature and subject to potential clinician bias."}),Object(Le.jsx)("div",{className:pe.a.description,children:"During the process of project development, we were inspired by the stories of some of our team members who have ADHD and first-hand experience with the diagnosis procedure. Upon further research, we learned about how many biases there are in the ADHD diagnosis process. For example, females are typically underdiagnosed because the assessments upon which ADHD diagnosis relies are based on data from males (Skogli et al., 2013). This, along with other areas of biases inspired us to develop a tool that could quantitatively aid in the diagnosis of ADHD."}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("b",{children:"The result of our efforts is NeuroDetech."})}),Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:"NeuroDetech is a quantitative tool that aids in the diagnosis of ADHD and reduces the margin of bias. NeuroDetech consists of a series of microfluidic capillary flow assay (MCFA) lab chips designed using process engineering methods. Each MCFA lab chip contains a binding molecule (a fusion protein or CRISPR-Cas13a complex) that detects an ADHD-associated biomarker or gene marker in urine. Through protein engineering and computational protein redesign methods, the binding molecule can be adapted for the sensitive detection of a variety of ADHD-associated markers. Specifically, we have designed MCFA chips that detect the following:"}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"A deficiency in phenylethylamine (PEA), which is excreted in the urine and is a known indicator of ADHD. A fusion protein is used to facilitate the detection of urinary PEA."}),Object(Le.jsxs)("li",{children:["The presence of ADHD-associated mutations in the mRNA transcripts of the ",Object(Le.jsx)("i",{children:"DRD4"})," and ",Object(Le.jsx)("i",{children:"SNAP25"})," genes. To detect these mutations in mRNA, CRISPR-Cas13a is used."]})]}),Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:"The signal produced by each lab chip can be analyzed by an optical detector, which emits a signal over time that is visualized on a software app. All in all, NeuroDetech provides healthcare professionals with insight into an individual\u2019s risk for ADHD."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("div",{className:Ur.a.video_text,children:"Promotion Video"}),Object(Le.jsx)("video",{controls:!0,className:Ur.a.video,children:Object(Le.jsx)("source",{src:"https://static.igem.org/mediawiki/2021/5/51/T--Waterloo--DescriptionPromotionVideo.mp4"})})]}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"References"}),Object(Le.jsx)("ul",{className:pe.a.description,children:Object(Le.jsx)("li",{children:"Skogli, E. W., Teicher, M. H., Andersen, P. N., Hovik, K. T., & \xd8ie, M. (2013). ADHD in girls and boys\u2013gender differences in co-existing symptoms and executive function measures. BMC psychiatry, 13(1), 1-12."})})]})]})}}]),i}(n.Component),Kr=i(20),Yr=i.n(Kr),Jr=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Microfluidic Chip"}),Object(Le.jsxs)(Ie,{open:!0,title:"Chip Model",children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"The main inspiration for the design of the NeuroDetech lab chip is a similar point-of-care diagnostic device for infectious diseases created by Ghosh et al. (2020). The testing device designed by Ghosh et al. (2020) prioritized functions such as accessibility and speed which are applicable to NeuroDetech. Hence many concepts from this paper, like the device dimensions and parts sequence, are incorporated into our work. Overall, the objective of our work is to create a user-friendly tool for quantitative biomolecular detection. (Ghosh et al., 2020)"}),Object(Le.jsx)("p",{children:"While designing the device, multiple factors were taken into account, including flow and reaction rate. The concentration of PEA in the sample is low, which can be difficult to detect. In the design of the chip, both capillary and surface tension forces contribute to the generation of transient flow within the microstructures due to the pressure gradient. The high surface-to-volume ratio increases antigen binding, ensuring more precise and accurate results. In summary, this microfluidic device utilizes many principles of fundamental fluid dynamics. The model of the NeuroDetech lab chip is shown below."})]})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Yr.a.design_img,style:{borderRadius:20,width:"80%",height:"80%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Assembly of the microfluidic chip. Note that the top plane surfaces are transparent for easy visualization of the chip inner components."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Yr.a.design_img,style:{borderRadius:20,width:"50%",height:"50%",marginLeft:"25%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Full assembly of the microfluidic chip. Fluid flows from the top left through the capillary pump through to the spiral control chamber, then to the spiral test chamber. At the same time, fluid flows from the top right through the drying chamber and into the spiral test chamber."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:"0"},children:"Sample Loading Chamber and Drying Chamber Design"})}),Object(Le.jsx)("p",{children:"The geometry of the NeuroDetech lab chip is critical to the chip\u2019s function. The test begins when the sample is deposited into the loading chamber through a sample loading port located at the top of the chip. A hydrophobic spot in the middle of the loading chamber splits the sample into two equal parts (Ghosh et al., 2020). Separate pathways move the samples toward a series of small tubes then into the drying chambers."}),Object(Le.jsx)("p",{children:"Path one leads to the binding protein drying chamber, and path two leads to the chemiluminescent substrate drying chamber. There are 10 tubes leading to the binding protein drying chamber and 7 tubes leading to the chemiluminescent substrate drying chamber. The tubes help maintain pressure to transfer fluid into the drying chambers and prevent unwanted backflow into the loading chamber."}),Object(Le.jsx)("p",{children:"Both drying chambers have a grid of rectangular pillars to increase mixing, providing binding proteins with sufficient time and space to bind to target biomarkers (Ghosh et al., 2020). The chemiluminescent substrate drying chamber has a row of tubes separating sections of the grid to maintain fluid pressure; this also ensures that fluid does not enter the test chamber before the binding protein fluid. Ramps descending into and ascending out of the drying chambers help retain fluid in the drying chamber for a sufficient period of time for uniform mixing and drying (Ghosh et al., 2020)."}),Object(Le.jsx)("p",{children:"The drying chambers connect to a series of delay valves with initially small diameters that progressively enlarge by widening the tube diameter; the fluid meniscus is thus increased in size, consequently decreasing capillary pressure. In turn, this helps ensure that no air bubbles form as the solution flows to the test chamber. The chemiluminescent substrate drying chamber pathway has a long serpentine delay tube in addition to the traditional delay valves, resulting in all the fluid from path two entering the reaction chamber only after all the fluid from path one has entered (Ghosh et al., 2020)."})]})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Yr.a.design_img,style:{borderRadius:20,marginLeft:"5%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Annotated loading and drying chamber."})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("div",{style:{margin:"20px 0 40px 0"},children:Object(Le.jsx)("span",{className:pe.a.subheading,style:{marginLeft:"0"},children:"Capillary Pump Design"})}),Object(Le.jsx)("p",{children:"Based on our previous research and referenced studies, few considerations were determined to be particularly important for fluid flow in the capillary pump. Through background research, we identified factors that affect fluid flow in a capillary pump. These include wettability, frequency of microstructures in the flow chamber, and the geometry of the microstructures used to increase flow resistance. These factors are the foundation for determining flow rates due to capillary flow (Olanrewaju et al., 2018)."}),Object(Le.jsx)("p",{children:"In general, more densely packed orientations of microstructures lead to higher flow resistance, in turn increasing flow pressure. Some of the key microstructure geometries commonly found in capillary pumps include tree lines, symmetric lines, balled lines, and posts. These structures must be small enough to generate enough capillary pressure for flow through the chamber (Olanrewaju et al., 2018)."})]})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Yr.a.design_img,style:{borderRadius:10}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Various Capillary Pump Designs (Olanrewaju et al., 2018)"})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"We chose to compare three different microstructures and their effect on fluid flow: (1) symmetric oblong shapes (400 by 200\u03bcm); (2) treelines in the shape of a hexagon (thickness 1mm); and (3) balled lines (radius 500 \u03bcm, lines 1mm)."}),Object(Le.jsx)("p",{children:"For a complete explanation of the comparison of designs, please navigate to the Microfluidic Chip Design section under the Engineering page. Due to the high capillary pressure generation and suitability for reagent mixing, the oblong capillary pump structure is chosen for the final chip assembly design. "})]})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsxs)("div",{style:{display:"flex",flexDirection:"row",justifyContent:"space-around",flexWrap:"wrap"},children:[Object(Le.jsx)("img",{src:"",className:Yr.a.design_img,style:{borderRadius:20,width:"28%",height:"28%"}}),Object(Le.jsx)("img",{src:"",className:Yr.a.design_img,style:{borderRadius:20,width:"26%",height:"26%"}}),Object(Le.jsx)("img",{src:"",className:Yr.a.design_img,style:{borderRadius:20,width:"27%",height:"27%"}})]}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"From left to right: Oblong microstructures. Treeline microstructures. Balled lines microstructures."})]})]}),Object(Le.jsxs)(Ie,{open:!0,title:"Computer-aided Chip Assembly Design",children:[Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"A complete assembly of the microfluidic chip is built and shown below. The specific dimensions are also labelled and presented. The team used OnShape to create the CAD model throughout the year."})})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Yr.a.design_img,style:{borderRadius:20}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Dimensioned drawing of the microfluidic chip."})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Yr.a.design_img,style:{borderRadius:20}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Assembly of the microfluidic chip. Note that the top plane surfaces are transparent for easy visualization of the chip inner components."})]})]})]})})}}]),i}(n.Component),$r=i(26),Zr=i.n($r),Qr=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Optical Detector and Visualization Application"}),Object(Le.jsx)("div",{className:pe.a.subheading,style:{marginLeft:"0"},children:"Hardware Development"}),Object(Le.jsx)("div",{className:pe.a.description,children:Object(Le.jsx)("p",{children:"After the design phase, the initial prototype is developed with chosen circuit parts. Below shows the schematic diagrams, breadboard configuration of the prototype and the final PCB Design of the optical detector."})}),Object(Le.jsxs)("div",{children:[Object(Le.jsx)("img",{src:"",className:Zr.a.design_img,style:{borderRadius:10,width:"40%",marginLeft:"30%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Breadboard configuration"})]})]}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsxs)(Zt,{bordered:!0,className:pe.a.table,children:[Object(Le.jsx)("thead",{children:Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("th",{children:"Cable colour"}),Object(Le.jsx)("th",{children:"Meaning "})]})}),Object(Le.jsxs)("tbody",{children:[Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Red"}),Object(Le.jsx)("td",{children:"+5V "})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Black"}),Object(Le.jsx)("td",{children:"GND "})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Yellow"}),Object(Le.jsxs)("td",{children:["Signal V",Object(Le.jsx)("sub",{children:"OUT"})," "]})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Blue"}),Object(Le.jsx)("td",{children:"SCK "})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Green"}),Object(Le.jsx)("td",{children:"MISO "})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"Purple"}),Object(Le.jsx)("td",{children:"MOSI "})]}),Object(Le.jsxs)("tr",{children:[Object(Le.jsx)("td",{children:"White"}),Object(Le.jsx)("td",{children:"SS "})]})]})]})}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Zr.a.design_img,style:{borderRadius:10,width:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Schematic capture (optical detector)"})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Zr.a.design_img,style:{borderRadius:10,width:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"Schematic capture (system)"})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Zr.a.design_img,style:{borderRadius:10,width:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"PCB Layout 3D Top View"})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Zr.a.design_img,style:{borderRadius:10,width:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"PCB Layout 3D Bottom View"})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Zr.a.design_img,style:{borderRadius:10,width:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"PCB Layout 2D Top Layer View"})]}),Object(Le.jsxs)("div",{className:pe.a.figure_div,children:[Object(Le.jsx)("img",{src:"",className:Zr.a.design_img,style:{borderRadius:10,width:"80%",marginLeft:"10%"}}),Object(Le.jsx)("div",{className:pe.a.figure_text,children:"PCB Layout 2D Bottom Layer View"})]}),Object(Le.jsx)("div",{className:pe.a.text_div,children:Object(Le.jsxs)("div",{className:pe.a.description,children:[Object(Le.jsx)("p",{children:"The optical detector captures the chemiluminescence signal by the use of a PN photodiode. The photodiode reacts to the brightness by generating electric current under reverse conditions. This current is then converted to voltage and amplified by the transimpedance amplifier. The output signal from the amplifier passes through an analog-to-digital converter to undergo digitization and processing. With the signal in digital form, the microcontroller reads the voltage at the pin and sends the value to the desktop running the software. In our set-up, the microcontroller reads and sends a thousand voltage values per second. The software converts the voltage values into the analyte concentration using the appropriate conversion and displays the values in an analyte concentration vs. time graph."}),Object(Le.jsx)("p",{children:"This proof of concept demo video shows that the software application graphs change with the light intensity detected by the optical detection circuit. Please note that the current graphed concentration values only correlate with detected voltage, and that conversion to concentration has not been implemented."})]})}),Object(Le.jsx)("div",{className:pe.a.figure_div,children:Object(Le.jsx)("iframe",{src:"https://static.igem.org/mediawiki/2021/d/d9/T--Waterloo--ProofOfConceptDemoVideo.mp4",style:{width:900,height:500}})})]})}}]),i}(n.Component),Xr=i(75),es=i.n(Xr),ts=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"References"}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"Ghosh, S., Aggarwal, K., U., V.T. et al. A new microchannel capillary flow assay (MCFA) platform with lyophilized chemiluminescence reagents for a smartphone-based POCT detecting malaria. Microsyst Nanoeng 6, 5 (2020). https://doi.org/10.1038/s41378-019-0108-8"}),Object(Le.jsx)("li",{children:"Olanrewaju, A., Beaugrandm M., Yafia, M., Juncker, D. Capillary microfluidics in microchannels: from microfluidic networks to capillaries circuits. Lab on a Chip 16, 2323-2347 (2018). https://doi.org/10.1039/C8LC00458G"}),Object(Le.jsxs)("li",{children:["RCSB PDB. (n.d.). ",Object(Le.jsx)("i",{children:"1UTM"}),". Retrieved October 17, 2021, from https://www.rcsb.org/structure/1UTM"]}),Object(Le.jsxs)("li",{children:["Roxe, D. M. (1990). Urinalysis. In ",Object(Le.jsx)("i",{children:"Clinical methods: The history, physical, and Laboratory Examinations"}),". essay, Butterworths. Retrieved October 3, 2021, from https://www.ncbi.nlm.nih.gov/books/NBK302/."]}),Object(Le.jsxs)("li",{children:["Shinoda, H., Taguchi, Y., Nakagawa, R., Makino, A., Okazaki, S., Nakano, M., Muramoto, Y., Takahashi, C., Takahashi, I., Ando, J., Noda, T., Nureki, O., Nishimasu, H., & Watanabe, R. (2021, April 19). Amplification-free RNA detection with CRISPR\u2013Cas13. ",Object(Le.jsx)("i",{children:"Communications Biology, 4"}),", 476. doi: https://doi.org/10.1038/s42003-021-02001-8"]}),Object(Le.jsx)("li",{children:"Tong, A., Tang, X., Zhang, F., & Wang, B. (2020, March 16). Study on the shift of ultraviolet spectra in aqueous solution with variations of the solution concentration. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 234, 118259. https://www.sciencedirect.com/science/article/pii/S1386142520302377?via%3Dihub."}),Object(Le.jsx)("li",{children:"Zimmermann, M., Schmid, H., Hunziker, P., Delamarche, E. Capillary pumps for autonomous capillary systems. Lab on a Chip 1, 119-125 (2007). https://doi.org/10.1039/B609813D"})]})]})})}}]),i}(n.Component),is=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Wet Lab Experiments"}),Object(Le.jsx)("div",{className:pe.a.description,children:"Due to unforeseen circumstances (one of which was a global pandemic, and the other of which was unforeseen maintenance and construction of our lab space by the University), we unfortunately did not have lab access this year. As a result, we were unable to experimentally test the microfluidic assay with all of its components. With that said, the following set of experiments outlines how we have tested each component of NeuroDetech to demonstrate proof of concept of these individual components, as well as how we would have demonstrated a proof of concept of the NeuroDetech system as a functioning whole."}),Object(Le.jsx)(Ie,{open:!0,title:"Assessment of Streptavidin Binding to Evaluate the Success of PDMS Derivatization, Conjugation, and Lyophilization",children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.subheading,children:"Preliminary Assessment: Assessment of PDMS Alone"}),Object(Le.jsx)("div",{className:pe.a.description,children:"The success of our microfluidic assay is contingent on the ability of molecules to remain covalently conjugated to PDMS when desired. Thus, it would need to be verified that PDMS would be successfully derivatized by PVA treatment, that the conjugation method would be successful, and that the lyophilization and resuspension steps would not impede the functionality of the lyophilized molecules. A way to test the success of all of these steps would involve an experiment testing the ability of streptavidin in the control chamber to bind biotin. Without successful derivatization of the PDMS surface, conjugation of streptavidin to the control chamber would not be successful. As well, assessment of binding after lyophilization by running a biotin solution would give insight into whether lyophilization is somehow damaging the effectiveness of the lyophilized molecules (in this case, streptavidin)."}),Object(Le.jsx)("div",{className:pe.a.description,children:"To test these, the derivatization, conjugation, and lyophilization methods from the Build section should be adapted for a small, test-scale PDMS film. Specifically, the following procedure, adapted from Thomsen et al. (2007), should be performed:"}),Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:"Materials:"}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"PDMS film"}),Object(Le.jsx)("li",{children:"Pyrogenic silicic acid"}),Object(Le.jsx)("li",{children:"10% w/v (3-Aminopropyl)triethoxysilane (APTES)"}),Object(Le.jsx)("li",{children:"2.5% w/v Glutaraldehyde"}),Object(Le.jsx)("li",{children:"5 mg/mL streptavidin"})]}),Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:"Procedure:"}),Object(Le.jsxs)("ol",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"Soak a PDMS film with pyrogenic silicic acid."}),Object(Le.jsx)("li",{children:"Drain the pyrogenic silicic acid and rinse the PDMS film."}),Object(Le.jsx)("li",{children:"Incubate the PDMS film with a 10% w/v solution of APTES at 80\xb0C for 3 hours."}),Object(Le.jsx)("li",{children:"Drain the APTES solution and rinse the PDMS film."}),Object(Le.jsx)("li",{children:"Incubate the PDMS film with a 2.5% w/v solution of glutaraldehyde (GA) on a rocking shaker for 2 hours at room temperature."}),Object(Le.jsx)("li",{children:"Drain the glutaraldehyde solution and rinse the PDMS film."}),Object(Le.jsx)("li",{children:"Incubate the PDMS film with the 5 mg/mL solution of streptavidin on a rocking shaker for 10-15 hours at 4\u02daC."}),Object(Le.jsx)("li",{children:"Aliquot 1 mL of the residual solution for determination of streptavidin concentration. This can be done by absorbance at 280 nm, or using a Bradford assay."}),Object(Le.jsx)("li",{children:"Drain the rest of the residual solution and rinse the PDMS film."}),Object(Le.jsx)("li",{children:"Incubate the PDMS film with a solution of bovine serum albumin (BSA) on a rocking shaker for 30 minutes at room temperature."}),Object(Le.jsx)("li",{children:"Drain the residual solution and rinse the PDMS film."})]}),Object(Le.jsx)("div",{className:pe.a.description,children:"Knowing the initial streptavidin concentration and the determined concentration of the residual streptavidin solution, the amount of streptavidin conjugated to the PDMS film can be determined."}),Object(Le.jsx)("div",{className:pe.a.description,children:"The streptavidin-conjugated PDMS film can then be lyophilized by first freezing the film in liquid nitrogen for 5 minutes, then utilizing a freeze-drying system such as the Labconco Lyophilizer HRFD-SCIL set at -54\xb0C with 0.010 mbar pressure for 24 hours."}),Object(Le.jsx)("div",{className:pe.a.description,children:"In the meantime, fluorophore-conjugated biotin molecules can be prepared using principles of organic chemistry related to carboxylic acid derivatives. First, HCl should be added to a biotin solution until the pH is around 1. The molar amount of biotin should be the same as the molar amount of streptavidin conjugated to the PDMS film. The purpose of lowering pH to around 1 is as follows: biotin has a carboxylic acid/carboxylate group; adding HCl to a pH of around 1 protonates any carboxylates, thereby preparing biotin for the subsequent reaction. From here, the acidified biotin should be reacted with an equimolar amount of thionyl chloride. This produces a \u201cbiotin chloride\u201d that can then be reacted with an equimolar amount of a nucleophilic fluorophore, which effectively substitutes with the chlorine atom to produce biotin conjugated to a fluorophore."}),Object(Le.jsx)("div",{className:pe.a.description,children:"From here, the freeze-dried PDMS film can be resuspended in the fluorescent biotin solution and incubated on a rocking shaker for 20 minutes (which is the same as the assay time for NeuroDetech\u2019s microfluidic assay chip). The residual solution can be drained, and its fluorescence measured using a spectrophotometer."}),Object(Le.jsx)("div",{className:pe.a.description,children:"Since the fluorescent biotin and streptavidin were in equimolar amounts, and since the binding affinity of streptavidin to biotin is so strong, it is expected that there should be little to no fluorescence in the residual solution, since most or all of the fluorescent biotin should have been bound to the streptavidin on the PDMS film."}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"Assessment in Microfluidic Assay Chip Conditions"}),Object(Le.jsx)("div",{className:pe.a.description,children:"After construction of the chip, the general method for the preliminary assessment (described above) would need to be repeated to ensure that the success of derivatization, conjugation, and lyophilization for the PDMS film could also be replicated in the actual microfluidic assay chip."}),Object(Le.jsx)("div",{className:pe.a.description,children:"To do this, a \u201csacrificial\u201d microfluidic assay chip should be prepared, such that the sample can be recovered after running the sample. Once again, fluorescent biotin should be prepared, as described in the preliminary assessment method (above)."}),Object(Le.jsx)("div",{className:pe.a.description,children:"From here, the fluorescent biotin sample should be flowed through the sacrificial microfluidic assay, as if the fluorescent biotin sample was an actual urine sample. Again, the molar amount of fluorescent biotin should be the same as the molar amount of streptavidin conjugated to the control chamber. The expected result is that the fluorescent biotin sample should bind to the conjugated streptavidin at the control chamber, thereby producing fluorescence at the control chamber. As well, the sample fluid should be recovered, and its fluorescence measured; the fluorescence should be negligible, as most (if not all) of the biotin should have been bound at the control chamber. If these expected results are observed, then it would be demonstrated that the methods for PDMS derivatization and conjugation, as well as the viability of lyophilization, would be successful in the microfluidic assay chip."})]})}),Object(Le.jsx)(Ie,{open:!0,title:"Experimental Demonstration of Fusion Protein Success",children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.subheading,children:"Demonstration of Fusion Protein Stability"}),Object(Le.jsx)("div",{className:pe.a.description,children:"The beauty of working with a fusion protein with a chemiluminescent enzyme domain is that the activity (and by extension, stability) of the fusion protein can be assayed very easily. In the case of the 1UTM-HRP fusion, where Amplex Red is the chemiluminescent substrate of HRP, Amplex Red could be added to a sample of the purified fusion protein in a cuvette. The fusion protein solution would be expected to display chemiluminescence of wavelength 563-587 nm upon addition of Amplex Red; the chemiluminescence can be measured using a spectrophotometer. A negative control would involve adding Amplex Red to the protein\u2019s buffer solution (but without the protein!) to ensure that chemiluminescence displayed by the protein solution is not due to some side reaction with the buffer solution components. A positive control would involve adding Amplex Red to a known pure solution of HRP (not in a fusion protein); this would ensure that Amplex Red is not degraded."}),Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:"To follow up on the previous stability experiment, simulation of urine conditions (including pH, salt, and major metabolites) would allow us to demonstrate the stability and functionality of the protein in urine. Roxe (1990) determined that urine typically had the following conditions:"}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"A pH range of 5-9, but usually slightly acidic. pH 6 is a reasonable generalization."}),Object(Le.jsx)("li",{children:"A protein concentration of much less than 30 mg/dL; equal or above indicates disease. To simulate protein in urine, bovine serum albumin (BSA) would be an appropriate, inert choice."}),Object(Le.jsx)("li",{children:"A glucose concentration of much less than 100 mg/dL; equal or above indicates disease."}),Object(Le.jsx)("li",{children:"Approximately 4 mM urea."}),Object(Le.jsx)("li",{children:"Approximately 20 mM salt (NaCl)."})]}),Object(Le.jsx)("div",{className:pe.a.description,children:"From here, it would be useful to obtain an experimental standard curve for the reaction between HRP and Amplex Red in urine-like conditions. To do this, a series of dilutions of the fusion protein would be made, then a known excess concentration of Amplex Red would be added to each fusion protein dilution. In measuring the chemiluminescence produced by each dilution, a standard curve of chemiluminescence vs. fusion protein concentration can be plotted. A multiplate and multiplate analyzer can be used for this. The same positive and negative controls from the stability experiment (above) should be implemented here as well. This would simulate the conditions of chemiluminescence in the microfluidic assay chip, since in the MCFA chip, the number of fusion protein molecules that bind at the test chamber would be variable, while the amount of Amplex Red that passes through the test chamber would be a known excess amount."}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"Experimental Demonstration of PEA-Binding Success of the Fusion Protein"}),Object(Le.jsx)("div",{className:pe.a.description,children:"In order for the fusion protein to adequately bind PEA, it should exhibit a similar binding affinity for PEA as the standalone PEA-binding protein, 1UTM."}),Object(Le.jsx)("div",{className:pe.a.description,children:"Binding affinity between a protein and ligand can be expressed in terms of the protein\u2019s dissociation constant, Kd. Mathematically, Kd is equivalent to the ratio of unbound protein to the ratio of bound protein. It follows that the lower the Kd, the more that protein is bound at any given time, and thus the higher the protein\u2019s affinity for the ligand."}),Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:"To determine the Kd of the PEA-binding fusion protein, we would need to determine the ratio of unbound:bound protein after incubation with PEA. To do so, an absorbance-based method can be used, as follows:"}),Object(Le.jsxs)("ol",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"Prepare a solution of the PEA-binding fusion protein (the concentration can vary, so long as it is known)."}),Object(Le.jsx)("li",{children:"Prepare an equimolar solution of PEA, prepared in the same buffer solution as the protein solution."}),Object(Le.jsx)("li",{children:"Using a spectrophotometer, measure the absorbance of a 1/2 diluted PEA solution at 257 nm."}),Object(Le.jsxs)("ul",{children:[Object(Le.jsx)("li",{children:"PEA consists of a benzene ring and an amine group, the former of which exhibits absorbance around 257 nm due to the conjugated pi system (Tong et al., 2020)."}),Object(Le.jsx)("li",{children:"If the absorbance is over 2.0, dilute the solution and try again until the absorbance is less than 2.0. The dilution factor can be used to calculate the concentration of PEA in the undiluted solution."})]}),Object(Le.jsx)("li",{children:"Incubate the protein solution with the PEA solution in a 1:1 ratio."}),Object(Le.jsx)("ul",{children:Object(Le.jsx)("li",{children:"To prevent protein loss to pipette tips during transfer, prime any tipes used with a solution of bovine serum albumin (BSA), which will prevent protein \u201csticking\u201d to the tip."})}),Object(Le.jsx)("li",{children:"Centrifuge the incubated sample to pellet all of the fusion protein, including the fusion protein molecules bound to PEA. Recover the supernatant only."}),Object(Le.jsx)("li",{children:"Using a spectrophotometer, measure the absorbance of the PEA supernatant solution at 257 nm."}),Object(Le.jsx)("ul",{children:Object(Le.jsx)("li",{children:"Again, if the absorbance is over 2.0, dilute the solution and try again until the absorbance is less than 2.0. The dilution factor can be used to calculate the concentration of PEA in the undiluted solution."})})]}),Object(Le.jsx)("div",{className:pe.a.description,children:"In order to interpret the absorbance values, a standard curve must be generated. This should be done by preparing a set of dilutions of a PEA solution of known concentration, then measuring the absorbance of each dilution. The resulting standard curve of absorbance vs. concentration can then be used to determine the concentration of an unknown PEA solution, given its absorbance."}),Object(Le.jsxs)("div",{className:pe.a.description,children:["Any amount of PEA that was \u201cmissing\u201d from the supernatant (relative to the original known amount of PEA) will have been bound by the fusion protein (in a 1:1 molar ratio). Therefore,",Object(Le.jsx)("br",{}),Object(Le.jsx)("div",{style:{textAlign:"center"},children:"Kd = concentration of unbound protein / concentration of bound protein"}),"By extension,",Object(Le.jsx)("br",{}),Object(Le.jsxs)("div",{style:{textAlign:"center"},children:["Kd = PEA",Object(Le.jsx)("sub",{children:"sup"})," / (original concentration of PEA - PEA",Object(Le.jsx)("sub",{children:"sup"}),")"]})]}),Object(Le.jsx)("ul",{className:pe.a.description,style:{marginTop:"-1%"},children:Object(Le.jsxs)("li",{children:["where PEA",Object(Le.jsx)("sub",{children:"sup"})," = supernatant concentration of PEA."]})}),Object(Le.jsx)("div",{className:pe.a.description,children:"It would be expected that the fusion protein exhibit an experimental Kd similar to the Kd of standalone 1UTM: approximately 0.000972 (RCSB PDB, n.d.). A dissociation constant much higher than the expected value (and by extension, a lower binding affinity) might indicate that the PEA-binding domain of the fusion protein is not individually stable or active."})]})}),Object(Le.jsx)(Ie,{open:!0,title:"Experimental Demonstration of CRISPR-Cas13a Success",children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.subheading,children:"Demonstration of CRISPR-Cas13a Stability in Urine-Like Conditions"}),Object(Le.jsx)("div",{className:pe.a.description,children:"In a solution containing ssRNA-linked fluorophores/quenchers, the nonspecific RNase activity of CRISPR-Cas13 after target ssRNA detection allows for the rapid production of a fluorescent signal. The stability and functionality of CRISPR-Cas13 in urine-like conditions can be tested in a similar way as described for the PEA-binding fusion protein. Once again, the expected conditions of urine include pH ~6, a low protein concentration (that can be simulated using BSA), a low glucose concentration, around 4 mM urea, and around 20 mM NaCl (Roxe, 1990). After preparing a solution in RNase-free water with these conditions and adding CRISPR-Cas13a, the fluorophores/quenchers, and the target mRNA sequence, the simulated urine solution would be expected to display fluorescence of wavelength 535 nm that could be measured using a spectrophotometer (Shinoda et al., 2021). A negative control would involve only the simulated urine, and should not display fluorescence; otherwise, the simulated urine may have confounding factors that cause a false positive result."}),Object(Le.jsx)("div",{className:pe.a.subheading,children:"Experimental Demonstration of SNP Discernment by CRISPR-Cas13a"}),Object(Le.jsx)("div",{className:pe.a.description,children:"Proper identification of ADHD-associated gene markers by NeuroDetech relies on the ability of CRISPR-Cas13a to distinguish between two mRNA sequences differing only by a single nucleotide polymorphism (SNP). Shinoda et al. (2021) has demonstrated this to be possible and reliable; however, as a component of the proof of concept, we would need to test this for our specific mRNA sequences. To do so, mRNA sequences for the \u2018normal\u2019 allele as well as the ADHD-associated mutant allele would need to be obtained, likely by synthesis by a company like IDT. From here, the stability experiment described above should be repeated for the \u2018normal\u2019 and ADHD-associated sequences. It would be expected that the \u2018normal\u2019 mRNA sequences would not be recognized by CRISPR-Cas13a; thus, its nonspecific RNase activity would not be activated, and no fluorescence would be produced. For the ADHD-associated sequences, fluorescence should be expected, since these mutant sequences are the desired targets of the designed CRISPR-Cas13a guide RNAs."})]})}),Object(Le.jsx)(Ie,{open:!0,title:"Experimental Proof of Concept of NeuroDetech",children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.description,children:"With the success of the proof of concepts for the individual components of NeuroDetech (as described in the above sections), we would finally put together all of the pieces and demonstrate a proof of concept for NeuroDetech as a whole."}),Object(Le.jsxs)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:["To demonstrate that the PEA-detecting microfluidic assay chip can quantify PEA accurately, a set of dilutions of a known concentration of PEA dissolved in simulated urine samples should first be prepared. Again, the simulated conditions of urine include pH ~6, a low protein concentration (that can be simulated using BSA), a low glucose concentration, around 4 mM urea, and around 20 mM NaCl (Roxe, 1990). Then, each dilution should be analyzed using a microfluidic assay chip as if the sample was a true urine sample. A protocol for the proper usage of the microfluidic assay can be found under \u201cUsage and Disposal of Microfluidic Assay\u201d in the\xa0",Object(Le.jsx)(ce,{to:"/Team:Waterloo/Implementation",className:pe.a.link_text,children:"Implementation"}),"\xa0page. The steps relevant to this proof of concept are shown below:"]}),Object(Le.jsxs)("ol",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:"Download and open the NeuroDetech computer application."}),Object(Le.jsx)("li",{children:"Connect the optical detector of the microfluidic assay to the computer via USB."}),Object(Le.jsx)("li",{children:"Pipette 30 \xb5L of the urine sample onto the sample application site of the microfluidic assay chip."}),Object(Le.jsx)("ul",{children:Object(Le.jsx)("li",{children:"Through the action of the capillary pump, the sample will immediately begin to flow through the microfluidic assay."})}),Object(Le.jsx)("li",{children:"Wait 20 minutes for the sample to flow through the assay. A graph of PEA concentration will be visualized in real time on the computer application as the sample passes through the test and control chambers."}),Object(Le.jsx)("ul",{children:Object(Le.jsx)("li",{children:"The 20-minute assay time is based on the design of Ghosh et al. (2020), from which the design of NeuroDetech\u2019s microfluidic assay is inspired. Dimensions of the NeuroDetech chip follow the dimensions as outlined in the paper."})}),Object(Le.jsx)("li",{children:"Once the assay is complete, disconnect the optical detector from the computer, then dispose of the microfluidic assay in the appropriate biohazard waste. The optical detector hardware is reusable."})]}),Object(Le.jsx)("div",{className:pe.a.description,children:"The output concentration of each microfluidic assay chip should correspond well to the true, known concentrations of each PEA dilution in simulated urine."}),Object(Le.jsx)("div",{className:pe.a.description,children:"A similar process can be used to demonstrate that the microfluidic assays designed to detect ADHD-associated mRNA mutants would be successful. To do this, mRNA for the \u2018normal\u2019 allele as well as the ADHD-associated mutant allele would be prepared in a simulated urine solution. Then, 5 volumes of RNAlater, an RNase inhibitor cocktail, would be added, as per manufacturer instructions (ThermoFisher Scientific, n.d.). Then, the samples would be analyzed by the microfluidic assay chip as per the procedure described above."}),Object(Le.jsx)("div",{className:pe.a.description,children:"For each ADHD-associated gene, the microfluidic assay would be successful if for the mutant mRNA sample, it displays a fluorescence readout such that the fluorescence is lower than the baseline fluorescence by at least the limit of detection of the microfluidic assay. This would correspond to CRISPR-Cas13a binding at the test chamber in a quantity greater than the detection limit, indicating that the mutant mRNA has been detected. Conversely, for the normal mRNA sample, the fluorescence readout should be highly similar to the baseline fluorescence, indicating no detection of ADHD-associated mutant mRNA. In this way, we would demonstrate that the microfluidic assay has the ability to distinguish between normal vs. ADHD-associated mRNA, and that the assay can successfully detect the latter."})]})})]})})}}]),i}(n.Component),ns=["Optical Detector and Visualization Application","Microfluidic Chip","Wet Lab Experiments","References"],as=["","","",""],rs=function(){var e=be(Object(n.useState)(ns[0]),2),t=e[0],i=e[1];return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.heading_div,children:[Object(Le.jsxs)("div",{className:pe.a.title,children:[Object(Le.jsx)("div",{className:pe.a.page_heading,children:"NeuroDetech"}),Object(Le.jsx)("div",{className:pe.a.page_heading_colored,children:"Proof Of Concept."})]}),Object(Le.jsx)("div",{className:pe.a.illustration,children:Object(Le.jsx)("img",{src:"",alt:"PoC Icon",className:es.a.icon_img})})]}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Overview"}),Object(Le.jsx)("div",{className:pe.a.description,children:"We have demonstrated proof of concept of various components of the project, including prototyping the optical detector and visualization application and the design of the microfluidic chip. As well, although we did not have lab access this year, we outline our plan of action to demonstrate the experimental proof of concept for each component of the microfluidic assay, as well as proof of concept for the assay as a whole."})]}),Object(Le.jsx)("div",{className:pe.a.sections_div,children:ns.map((function(e,n){return Object(Le.jsx)("div",{active:t===e,onClick:function(){return i(e)},className:pe.a.section_block,children:Object(Le.jsxs)("div",{className:pe.a.sections,children:[Object(Le.jsx)("div",{className:pe.a.section_img,children:Object(Le.jsx)("img",{id:"poc".concat(n),src:as[n],alt:"Icon"})}),Object(Le.jsx)("div",{className:pe.a.section_text,children:e})]})},e)}))}),Object(Le.jsxs)("div",{children:[t===ns[0]&&Object(Le.jsx)(Qr,{}),t===ns[1]&&Object(Le.jsx)(Jr,{}),t===ns[2]&&Object(Le.jsx)(is,{}),t===ns[3]&&Object(Le.jsx)(ts,{})]})]})},ss=i(76),os=i.n(ss),cs=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Laboratory"}),Object(Le.jsx)("div",{className:pe.a.description,children:"Our lab is located on the third floor of the ESC (Earth Science and Chemistry) building at the University of Waterloo. We have our own tabletop centrifuges, thermal cycler, analytical and top-loading balances, -20\xb0C freezer, and 4\xb0C refrigerator. We share the -80\xb0C freezer and 37\xb0C incubator with the host lab, under the guidance and supervision of Dr. Roderick Slavcev at the Science Innovation Hub."})]})})}}]),i}(n.Component),ls=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Project Safety Considerations"}),Object(Le.jsx)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:"The following safety considerations were accounted for during the design of our project:"}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsxs)("li",{children:["We have chosen non-pathogenic chassis organisms which are described as Biosafety Level 1 (",Object(Le.jsx)("i",{children:"E. coli DH5alpha, E. coli BL21(DE3)"}),", and ",Object(Le.jsx)("i",{children:"E. coli Rosetta 2(DE3)pLysS Singles"}),"). "]}),Object(Le.jsx)("li",{children:"Our project utilizes CRISPR-Cas13 to target mRNA from human genes. Though the risk of accidental targeting of mRNA within the body is low (unless CRISPR-Cas13 is accidentally ingested), caution should be exerted to avoid any such situations. Any work with the CRISPR guide RNAs or Cas13 would be done in a separate area, and the guide RNAs and Cas13 would only be combined only once absolutely ready. Proper disposal of gloves and used CRISPR-Cas13-related solutions into biohazard waste would be followed. Gloves, a lab coat, and goggles would be minimum PPE."}),Object(Le.jsx)("li",{children:"Glutaraldehyde and (3-Aminopropyl)triethoxysilane, or APTES, are both used in the process of covalently conjugating biomolecules to polydimethylsiloxane (PDMS), the microfluidic assay material. Glutaraldehyde and APTES are both toxic and highly irritating to mucous membranes (such as the eyes or respiratory tract). As a result, when carrying out covalent conjugation to PDMS, we would ensure to handle these chemicals only in a fume hood, with gloves, goggles, and a lab coat being minimum PPE."}),Object(Le.jsx)("li",{children:"Phenylethylamine (PEA) is one of the ADHD-associated analytes of interest, as it is an ADHD-associated neurotransmitter. Regarding safety, PEA is highly flammable, toxic upon ingestion, and poses a risk to aquatic life. All work with PEA would be done in the fume hood, and PEA-containing waste would be collected in a designated waste container to be disposed of at the University\u2019s hazardous waste disposal centre.. To minimize exposure to pure PEA, PEA would be dissolved into aqueous solution as soon as possible."})]}),Object(Le.jsxs)("div",{className:pe.a.description,children:["For more information about safe design and usage in relation to the proposed implementation plan for this project, see the",Object(Le.jsx)(ce,{to:"/Team:Waterloo/Implementation",children:Object(Le.jsx)("span",{children:" Implementation "})}),"page."]})]})})}}]),i}(n.Component),ds=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return Object(Le.jsx)("div",{className:pe.a.container,children:Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Safety Guidelines"}),Object(Le.jsx)("div",{className:pe.a.subheading,style:{marginLeft:"5%"},children:"Canadian Guidelines"}),Object(Le.jsxs)("div",{className:pe.a.description,children:["In Canada, all research practices involving biological materials are strictly regulated by the\xa0",Object(Le.jsx)("a",{className:pe.a.text_link,href:"https://www.canada.ca/en/public-health.html",target:"_blank",rel:"noreferrer",children:"Public Health Agency of Canada"}),". In addition, there are certain regulations that must be followed in every work place, as recorded by the\xa0",Object(Le.jsx)("a",{className:pe.a.text_link,href:"https://www.canada.ca/en/employment-social-development/services/health-safety/reports/workplace-health-safety-facts.html",target:"_blank",rel:"noreferrer",children:"Workplace Health & Safety Regulations"}),"."]}),Object(Le.jsx)("div",{className:pe.a.subheading,style:{marginLeft:"5%"},children:"University Guidelines"}),Object(Le.jsxs)("div",{className:pe.a.description,style:{marginBottom:"-2%"},children:["All laboratory practices operated within the University of Waterloo iGEM Team are regulated by the\xa0",Object(Le.jsx)("a",{className:pe.a.text_link,href:"https://uwaterloo.ca/safety-office/",target:"_blank",rel:"noreferrer",children:"University of Waterloo\u2019s Safety Office"}),". As such, everyone in the lab had been appropriately trained with the safety modules required by the University of Waterloo before they were permitted to work in the lab. The training included WHMIS 2015, Laboratory Biosafety Training, and General Lab Safety. The training was based on the following safety guidelines:"]}),Object(Le.jsxs)("ul",{className:pe.a.description,children:[Object(Le.jsx)("li",{children:Object(Le.jsx)("a",{className:pe.a.text_link,href:"https://uwaterloo.ca/safety-office/laboratory-safety",target:"_blank",rel:"noreferrer",children:"University of Waterloo Laboratory Safety Guidelines"})}),Object(Le.jsx)("li",{children:Object(Le.jsx)("a",{className:pe.a.text_link,href:"\u200b\u200bhttps://uwaterloo.ca/safety-office/laboratory-safety/biosafety",target:"_blank",rel:"noreferrer",children:"University of Waterloo Biological Safety Guidelines"})}),Object(Le.jsx)("li",{children:Object(Le.jsx)("a",{className:pe.a.text_link,href:"\u200b\u200bhttps://uwaterloo.ca/safety-office/occupational-health-safety/whmis",target:"_blank",rel:"noreferrer",children:"University of Waterloo Workplace Hazardous Materials Information System"})}),Object(Le.jsx)("li",{children:Object(Le.jsx)("a",{className:pe.a.text_link,href:"\u200b\u200bhttps://uwaterloo.ca/safety-office/occupational-health-safety/workplace-violence-and-harassment",target:"_blank",rel:"noreferrer",children:"University of Waterloo Workplace Violence and Harassment"})})]}),Object(Le.jsx)("div",{className:pe.a.description,children:"Lastly, all protocols conducted by the Waterloo iGEM team at the University of Waterloo are described in Safe Operating Procedures (SOPs) approved by advisors in the Science Innovation Hub."}),Object(Le.jsx)("div",{className:pe.a.subheading,style:{marginLeft:"5%"},children:"Team Guidelines"}),Object(Le.jsx)("div",{className:pe.a.description,children:"Lab goggles, coats, and gloves are mandatory PPE in the lab. All of our chemical solutions are labelled and safely stored in appropriate containers. Flammable substances and acids are stored separately in appropriate cupboards. From our broken glass waste bin and regularly checked fire extinguisher, to our fully stocked first aid kit and highly visible safety information signs on the door, we take safety awareness and procedures seriously. We also dispose of our biohazardous waste properly -- materials such as contaminated pipette tips, microtubes, KimWipes, etc. are put in biohazardous waste containers and compiled into our large bin. Once the bin is full, we safely transport it to the hazardous waste disposal site on the university campus, where it is properly disposed of by qualified employees. We have also implemented a weekly eyewash maintenance program to ensure that stagnant, dead-leg water is removed from the tubing and that the apparatus is always clear and properly functional in the case of an emergency. Overall, our lab and project had many safety features implemented to ensure that if we had lab access, everyone involved inside and outside of the lab would have been kept safe."})]})})}}]),i}(n.Component),hs=["Project Safety Considerations","Laboratory","Safety Guidelines"],us=["","",""],ms=function(){var e=be(Object(n.useState)(hs[0]),2),t=e[0],i=e[1];return Object(Le.jsxs)("div",{className:pe.a.container,children:[Object(Le.jsxs)("div",{className:pe.a.heading_div,children:[Object(Le.jsxs)("div",{className:pe.a.title,children:[Object(Le.jsx)("div",{className:pe.a.page_heading,children:"NeuroDetech"}),Object(Le.jsx)("div",{className:pe.a.page_heading_colored,children:"Safety."})]}),Object(Le.jsx)("div",{className:pe.a.illustration,children:Object(Le.jsx)("img",{src:"",alt:"Safety Icon",className:os.a.icon_img})})]}),Object(Le.jsxs)("div",{className:pe.a.text_div,children:[Object(Le.jsx)("div",{className:pe.a.text_heading,children:"Overview"}),Object(Le.jsx)("div",{className:pe.a.description,children:"Due to unforeseen circumstances (one of which was a global pandemic, and the other of which was unforeseen maintenance and construction of our lab space by the University), we unfortunately did not have lab access this year. Although we were unable to perform any experiments, we still considered safety when writing our suggested procedures. We also state our lab\u2019s safety equipment and the university and national guidelines we must follow while working in it."})]}),Object(Le.jsx)("div",{className:pe.a.sections_div,children:hs.map((function(e,n){return Object(Le.jsx)("div",{active:t===e,onClick:function(){return i(e)},className:pe.a.section_block,children:Object(Le.jsxs)("div",{className:pe.a.sections,children:[Object(Le.jsx)("div",{className:pe.a.section_img,children:Object(Le.jsx)("img",{id:"safety".concat(n),src:us[n],alt:"Icon"})}),Object(Le.jsx)("div",{className:pe.a.section_text,children:e})]})},e)}))}),Object(Le.jsxs)("div",{children:[t===hs[0]&&Object(Le.jsx)(ls,{}),t===hs[1]&&Object(Le.jsx)(cs,{}),t===hs[2]&&Object(Le.jsx)(ds,{})]})]})},ps=i(7),fs=i.n(ps),bs=function(e){Ue(i,e);var t=Je(i);function i(){return ze(this,i),t.apply(this,arguments)}return Ge(i,[{key:"render",value:function(){return 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