Team:Lubbock TTU/Implementation

Implementation

During our project development, we received feedback from multiple experts and integrated their feedback into the design of our project. The main focus of our project is to identify our user interface and device design to help detect the chytridiomycosis pandemic. We considered different aspects of the project such as the safety aspects, project envisionment, and end-user target audience when developing our ChiSPY project. We reviewed articles to gain an understanding of the current population status of amphibians and why mitigating this disease would be beneficial to the world. Articles and meetings with experts helped us center our focus on a biosensor project that is further explained in the Human Practicespage.

Who Will Be Our End-User?

When considering our project and how it could be applied in the real world, we decided the best place for our biosensor to be used would be in the field such as a lake or a pond. This decision was made based on experist feedback and peer review articles. Our project’s end-users will be conservationists and field ecologists need a diagnostic device to identify the presence of Bd . The ChiSPY biosensor device will use a modified swabbing protocol to test the presence of Bd

Our Vision of User Application:

Our ChiSPY biosensor will utilize a modified swabbing protocol on the amphibian’s skin to detect the presence of Bd . The standard swabbing protocol calls for detecting zoospores, so one swab per amphibian is sufficient. This allows users to utilize their resources and not waste any precious materials with the one swab method. However, since we are detecting chitin left behind by the shells of the zoosporangia, we needed a swabbing protocol capable of adopting a corrective control [2]. Thus, we created our own swabbing protocol. One where you could obtain an overall measurement for environmental chitin and another measurement for the sporangia chitin. Once the swabbing technique was complete, the user would apply it to the ChiSPY biosensor to test for the presence of the fungus. The swabbing protocol, and a more in-depth description of it can be found in the Human Practicespage.

During one of our discussions with Dr. Rodriguez, we learned that current detection methods for chytridiomycosis are $4,000+. We wanted something that would ensure that money wasn’t going to waste.

Real-Life Implementation

There has been a lack of detection systems that can be applied towards in-situ experiments. Current detection systems such as MiniPCR and qPCR are good DNA sequencing devices; MiniPCR is a thermocycler blueGel device that helps detect single deoxyribonucleic acids to sequence. qPCR is a technology used for measuring DNA using PCR. It monitors the amplification of a target DNA molecule [5]. However, there are some limitations to them. Conservationists would benefit from a more feasible, accessible, and affordable detection system to help gather information for their research. If our ChiSPY biosensor project is successful, it would raise research value in the eyes of conservationists and ecologists as a device that can better help them detect this pathogen.

The ChiSPY project implementation is focused on two values: Research and Finance

In terms of the research aspect, it gives end users more accessible data for their research interpretation and review. It allows them to obtain information about the presence of infected and uninfected amphibians which can help them treat and isolate infected amphibians. Having this outcome allows better communication in terms of project results and finding other conservationists that are reviewing over the same fungus.

The financial value of the ChiSPY biosensor helps alleviate some money complications that researchers may have. While speaking with Dr. David Rodrgiuz, a field ecologist and population genetics, we discussed the current detection methods such as Minion, Mic PCR, and qPCR [1]. He mentioned the limitations of them such as cost. When discussing the current prices, he mentioned MiniPCR to cost nearly $16,000 for one trial run [1] . Although these DNA sequencers are mobile and can be done in the field, the quality of data is much lower. There is more room for errors in terms of using the detection system as of right now. In comparison, our project can provide more reliable results while being cost-efficient in terms of detecting Bd . The cost-efficient feature of our biosensor will ease end-users about not spending thousands of dollars to use a device to detect the fungus. The ChiSPY biosensor also provides resource availability to the low-economic area where conservationists are in need of a cheaper detection system

Bd is already known to be present. However, the method to quantify a Bd outbreak is qPCR, and not all regions of the world have access to this machinery. Additionally, one of the main issues regarding determining where field ecologists should focus their efforts in mitigating the Bd pandemic is that the intensity of infection differs across Bd strains. This is due to the copy number variation found in the ITS-1 region of the genome. However, since ChiSPY detects something free from DNA, it does not run into this problem.

Safety Aspects Of The ChiSPY Project

In terms of the safety aspects of our biosensor, we brainstormed on how we could ensure safety within our biosensor in an in-field experiment. Since our biosensor is cell based, we decided to use E. coli as our host organism. Most E. coli strains are harmless and E. coli strains used in the lab have their infections DNA sequences taken out. We wanted to assure users that our biosensor does not content any sonstruts that would warrant a safety risk within the field. Another factor of E. coli is that it has a high efficiency of introduction of DNA molecules into the cell [4] . Another safety aspect of our biosensor that we considered is selectivity. It’s an important feature of the ChiSPY biosensor that allows it to detect a specific analyte in a sample containing the other contaminants and admixtures [3] . This ensures us and end users that that biosensor is specific to chitin and can’t detect other potential external inputs.

Implementation Challenges ChiSPY Has Considered

We have considered and would need to further investigate many challenges that still need to be solved before our project can be truly implemented in the real world

- Tuning: This challenge made us aware of the precision and accuracy of our hardware. It also made us aware of misreadings when it comes to the detection process within our biosensor.

- Faulty triggers: Identifying potential triggers that can set off the hardware detection system in terms of producing a green fluorescence. Review over other analytes that have a similar structure to chitin or other triggers would help prevent this from occurring.

- Price of the project: Calculating a real-lifetime price can be difficult. We have to consider factors such as market analysis and the level of increased hardware pieces when reviewing over our biosensor.

- Environmental: Since we are using a live-cell biosensor, there’s a chance that our cell construct could be altered from the outside environment such as temperature, climate, and more. We considered what potential external outputs could alter and/or affect our biosensor.

Sources

[1] Rodriguez, David. (2021). Meeting discussed on September 29, 2021 over qPCR, MiniPCR, Mic PCR detection devices.

[2] JE;, Berger L;Hyatt AD;Speare R;Longcore. “Life Cycle Stages of the Amphibian Chytrid Batrachochytrium Dendrobatidis.” Diseases of Aquatic Organisms, U.S. National Library of Medicine, Dec. 2005, pubmed.ncbi.nlm.nih.gov/16465834/.

[3] Bhalla, N., Jolly, P., Formisano, N., & Estrela, P. (2016). Introduction to biosensors. Essays in biochemistry, 60(1), 1–8. https://doi.org/10.1042/EBC20150001

[4] Cronan, John. (2014). Escherichia coli as an Experimental Organism. https://doi.org/10.1002/9780470015902.a0002026.pub2

[5]Staff, Ask A Scientist, et al. “What Is QPCR?” Ask a Scientist, 7 Feb. 2020, https://www.thermofisher.com/blog/ask-a-scientist/what-is-qpcr/.