Team:Queens Canada/Assay-Design
Assay Design
Description
The biotechnology utilized by the team was a lateral flow immunoassay (LFIA) strip housed within a custom device. The LFIA biotechnology was chosen due to its lightweight, the feasibility of mass production, simplicity, and how it best compliments our designed detection biology. Since our system utilizes the enzyme alkaline phosphatase, an enzyme that can synthesize large quantities of a clear substrate to create a coloured product visible to the human eye, the test is indicative of the presence of OspA rather than the quantity of OspA. This makes an LFIA the perfect choice as it is a positive/negative test, indicating only the presence of a certain analyte. This test also does not require any electronics parts or expensive materials. A common example of LFIA currently available in the market would be the SNAP test or a pregnancy test. An LFIA works by applying an aqueous sample to the sample pad of the test. The analytes then flow using capillary action into the conjugate pad, releasing antibodies conjugated with a coloured conjugate. The flow continues into the membrane where the sample will interact with the test and control lines to provide the test results. The liquid is then caught by the wicking pad which helps drive the sample flow.
For LFIA’s there are two distinctive designs: a direct and a competitive. A pregnancy test would be an example of a direct test where the presence of the analyte is shown by the appearance of a coloured test line in conjunction with a control line. The second design, which will be employed by the SUBLyme test, is a competitive design typically used for smaller-scale molecules which are unable to bind to multiple antibodies at the same time [1]. This is due to the antibodies only have a single antigenic determinate which is the case for our target molecule, OspA [1]. The competitive test’s results are the direct reverse of those for a direct test. In a competitive assay, the presence of the target analyte is indicated by the lack of or movement of the test line. This is because the target analyte is competing with antibodies with a coloured conjugate to bind to the immobilized antibodies on the test line. The target analyte will have a stronger binding affinity to the antibody so when it is present within the tested sample, it can beat out the coloured conjugate, resulting in the colour flowing down the pad and no line. However, if the target analyte is not within the sample, the coloured conjugate is then able to bind to the immobilized antigen and produce the coloured test line that indicates a negative test.
Components
The assay is broken down into the following components:
Sample Pad
The sample pad is the first component of the test to interact with the test solution. It catches the applied solution and can evenly distribute it across the pad and guide the solution to the next section, the conjugate pad [1].
The sample pad will be comprised of cellulose fiber to reduce the risk of any hindering interactions between the solution and the pad material, resulting in the target analyte not binding to the material [2, 1]. It will contain a phosphate buffer which will assist in adjusting the sample flow speed and the pores will be able to filter any redundant materials, increasing test sensitivity [2]. The chemical composition of the sample pad is detailed in Assay Chemical Composition.
Conjugate Pad
Its purpose is to store the conjugated antibodies (the molecules that when bound, produce the coloured signal) until the test is used. The conjugate pad possesses its specific buffer, which is responsible for these antibodies' immobilization, which is usually dried into the material so that when rehydrated, the antibodies are released into the moving solution [2]. This is detailed in Assay Chemical Composition.
To ensure proper immobilization and ensure no hindrance to the target molecules, the conjugate pad will be composed of glass fiber [2]. Similar to the sample pad, glass fiber exhibits minimal and non-specific binding to ensure that the target analyte remains mobile within the test [2].
Membrane
The membrane is arguably the most important component of the LFIA. Nitrocellulose was chosen as the material for this component as it allows for easing binding when immobilizing antibodies and proteins and is ideal to support capillary flow [1, 2]. The time it takes the sample solution to travel and fill the whole membrane section is called capillary flow and is often used to characterize various nitrocellulose membranes due to inconsistencies in pore size that arise due to manufacturing [2]. It is important to specify and characterize capillary flow time as it influences the tests' completion time, specificity, and sensitivity [2]. This parameter is modelled and characterized in fluid dynamics, using flow tests to determine capillary flow time as well as assay dimensions. More details can be found on the Fluid Dynamicspage.
The membrane also will contain the test lines containing the immobilized ‘catching’ antigens. The protein of interest found on Borrelia Burgdorferi is immobilized on the test line. When the protein of interest is present in the sample added to the pad, the bioreceptor will bind all available target proteins. This will prevent the bioreceptor from binding the target protein fused along the test line, resulting in the lack of a line appearing on the test. In the situation where the target protein is not present in the sample, the bioreceptors will be unoccupied and will bind the target protein fused to the test line as it flows over. These bioreceptors consist of our engineered single-chain variable fragment (scFv) and alkaline phosphatase.
Wicking Pad
The last component, the wicking pad (also known as the absorbent pad), is used to catch the fluid as it exits the membrane and wicks the fluid through the assay. The wicking pad dictates what volume of the sample should be applied; often larger pads allow for larger volumes to be applied which in turn can increase the sensitivity of the test [1]. In addition, it can prevent backwashing back into the membrane which would disrupt the test results [2]. Similar to the sample pad, the wicking pad will be comprised of cellulose fibers [2].
Design Considerations
Sensitivity
One concern that arose during the preliminary stages of the prototype design is the sensitivity of the immunoassay. This concern arose due to the location of the targeted bacteria, Borrelia Burgdorferi. This bacteria is located within the midgut of a tick, whose length range from 3mm unfed to 12.7mm fed [1]. This means that only a small sample size is available and subsequently a very limited portion of the target analyte, ospA can be retrieved. OspA is an outer surface protein located on Borrelia Burgdorferi and is the target analyte as it is an indicator of Lyme disease.
It was determined by Piesman et al through the use of quantitative PCR (qPCR), that on average there are approximately 998 Borrelia Burgdorferi spirochetes in a tick’s midgut before feeding [2]. Upon feeding/attachment for 48 hours, the quantity increases to 5,884 spirochetes [2]. Statistically, there will be a multitude of ospA molecules per spirochetes, indicating that there should be well over 998 analyte molecules to be tested for within a given sample. The proposed assay design utilizes the competitive design as previously described, which has been proven with a precision of 10% or better by Woodley et al. It is estimated that 131 molecules is the assay’s lower limit of quantification [3]. Comparing the lower limit of quantification to that of the proposed quantity of ospA within a given analyte proves that the proposed assay design should be more than capable of sensing the presence of the analytes within the small quantity of sample.
Bacteria Size Restrictions
The pore size of the nitrocellulose membrane was considered when selecting the components of the LFIA. Nitrocellulose can come in a range of pores sizes, ranging from 0.2-15. Initially, we hoped that the entire Borrelia Burgdorferi spirochete would be able to flow unrestricted through the membrane and the various pads, but unfortunately, while the width of the spirochete is only 0.3, its length ranges from 10-20 [6]. This means that potentially 50% of the spirochetes, assuming a uniform distribution of lengths among the spirochetes tests, would be unable to fit through the pores. This could cause a potential blockage within the assay, which would hinder the flow and therefore null the effect of the results.
To remedy this, our team has decided to lyse the bacteria into smaller components to ensure flow within the assay. The idea is to break the outer membrane into pieces, releasing the contents into our lysis buffer along with our target analyte, the outer surface protein OspA. This protein is located on the outer portion of the cell membrane and will have a wider range of mobility and potentially increase test sensitivity due to the lysing. This is because more OspA will be available to bind, as now multiple OspAs are available per lysed bacterium. Previously, only one OspA per bacterium was able to bind to a receptor antibody. While there are many techniques available to lyse cell membranes, our team decided to lyse the cell chemically to ensure the portability and low cost for our detection device. Specifically, we chose the detergent Triton X-114 to accomplish this task due to its favourable characteristics of being non-ionic, having a quick lysis time, requiring low concentrations, and its ability to not denature our target protein. Additionally, Triton X-114 is favoured amongst Lyme disease scientists to isolate OspA. A more thorough chemical profiling and supporting information about the choice of Triton X-114 can be seen on our Assay Chemical Composition.
Usage
Some key design criteria that were outlined as essential at the beginning of the design process included portability, ability to be reused, accessibility, ease of use, and safety. All these elements were incorporated into the assay’s casing final design iteration.
The test was designed to be lightweight and compact, easy for portability. To ensure no components shift and to prevent any leakage, a case for the device and its extra components was designed. The case allows for the device to be easily packed or clipped onto the bag in the outdoors without worry about how the movement might affect it. The device casing was designed to allow the assay to be disassembled and allow the lateral flow strip to be replaced easily. In addition, extra cartridges of lysis buffer solution were included, to make the test reusable. To ensure ease of use, the test comes with step-by-step instructions and is quite simple to use, ensuring that users of all backgrounds can easily use the test. The test eliminates the use of any electronics and is made with cost-effective materials to make the test easily affordable for all social and economic demographics. In addition to the casing, the test was designed with the user’s safety in mind, as it eliminates or greatly mitigates the risk of the user coming into contact with the specimen and required lysis buffer. These attributes are better outlined on the Device Design and CAD Modelling Iterations pages.
Fluid Distribution
As described previously, an important characteristic of the nitrocellulose membrane, and the assay as a whole, is the capillary flow time. Therefore, to help choose our membrane, defining characteristics like test time and the capillary flow time were modelled. This was done through modelling the capillary action of the sample volume to determine the length travelled with respect to time. These tests were performed for multiple pore sizes and the processes. To confirm the results of our modelling, a series of flow rate tests, using a variety of volumes and pore sizes were completed. The details and results of the modelling and flow tests can be found on the Fluid Dynamicspage.
Assay Dimentions
One of the final design aspects taken into consideration is the dimensions of the assay. All three directional factors (thickness, width, and length) can impact how the solution flows through the assay. As discussed on the fluid dynamics page, the flow slows down as it moves through the nitrocellulose membrane section, meaning the length impacts the flow speed [7]. The length of an assay requires optimization so that enough binding time is given for the analytes (the flow slows down enough) while minimizing the materials used. The wicking pad also impacts flow as it dictates the quantity of solution that can be applied to the test [2]. In addition, the thickness of the sample pad impacts the consistency of the flow [7]. A good parameter to measure the sample pad is bed volume which correlates to the quantity of air contained within the pad [7]. It can be used to determine the amount of liquid required to wet the pad [7]. Bed volume can be defined by the following equation:
Bed Volume = Total volume of pad x Porosity (%)
An assay’s required dimensions are very subjective to the biotechnology and the chemistry that is being employed in it. Therefore, tests are required to correctly determine and optimize dimensions. Our team will be performing the first of these tests to determine the base assay dimension. These tests can be further tested when our biotechnology is at the stage that it can be implemented into the device.
References
1. K. M. Koczula and A. Gallotta, "Lateral Flow Assays," Essays in Biochemistry, vol. 60, pp. 111-120, 2016.
2. Innova Biosciences Guide, "Guide to Lateral Flow Immunoassays," [Online]. Available: https://fnkprddata.blob.core.windows.net/domestic/download/pdf/IBS_A_guide_to_lateral_flow_immunoassays.pdf. [Accessed 11 June 2021].
3. Illinois Department of Public Health, "Prevention and Control, Common Ticks," Illinois Department of Public Health, [Online]. Available: http://www.idph.state.il.us/envhealth/pccommonticks.htm. [Accessed 8 June 2021].
4. J. Piesman, B. S. Schneider and N. Zeidner, "Use of Quantitative PCR to Measure Density of Borrelia Burgdorferi in the Midgut an Salivary Glands of Feeding Tick Vectors," Journal of Clinical Microbiology , pp. 4145-4148, 2001.
5. C. F. Woolley, M. A. Hayes, P. Mahantl, S. D. Gilman and T. Taylor, "Theoretical Limitations of Quantification for Noncompetitive Sandwhich Immunoassays," Analytical and Bioanalytical Chemistry, vol. 407, no. 28, pp. 8605-8615, 2015.
6. J. A. Hyde, "Borrelia Burgdorgeri Keeps Moving and Carries on: A Review of Borrelial Dissemination and Invasion," Fronteir Immunology, vol. 8, no. 114, 2017.
7. C. Parolo, A. Sena-Torralba, J. Fancisco Bergua, E. Calucho, C. Fuentes-Chust, L. Hu, L. Rivas, R. Álvarez-Diduk, E. P. Nguyen, S. Cinti, D. Quesada-González and A. Merkoçi, "Tutorial: Design and Fabrication of Nanoparticle-based lateral-flow immunoassays," Nature Protocols, vol. 15, pp. 3788-3816, 20201.