Team:Queens Canada/Assay-Chemical-Composition
Chemical Composition
Overview
One of the most important design aspects of our biosensor is the chemical composition of the Lateral Flow Immunoassay (LFIA). It will dictate the effectiveness of the test and support the feasibility of our engineered biology. This section will break down and explain the various chemical components for the different parts of the test, specifically the lysis reagent, various buffers, and test strip antibodies. Chemical interaction analysis was performed to ensure the safety of our users and our future work will be detailed.
Chemical Components
Lysis Reagent
As previously described, cell lysis is required to ensure that the Borrelia Burgdorferi flows correctly through the Lateral Flow Immunoassay (LFIA). To keep the test at a low cost, easy to use, and portable, we decided to lyse the cell chemically through the use of a surfactant/detergent. These molecules are able to efficiently disrupt the bacteria’s lipid-bilayer, through inhibiting the hydrophilic-hydrophobic interactions in addition to inhibiting the following interactions; lipid-lipid, lipid-protein, and protein-protein within the bilayer [1]. There are three main types of lysis detergents; ionic, non-ionic, and zwitterionic. For our purpose, we desired a non-ionic detergent as they cause the least amount of damage to proteins (i.e., do not denature them) [1]. They are quite mild agents and are highly efficient, able to complete the lysis in just a few minutes or less [1].
For our purposes, we have chosen the non-ionic detergent Triton X-114. Triton X-114 is a popular non-ionic surfactant used to isolate specific proteins. It has been proven to be applicable for our usage by Jiang et al who studied the antibody binding domains for both OspA and OspB in Borrelia Burgdorferi [2]. To isolate OspA from the bacteria, they utilized the surfactant Triton X-114 as part of the extraction process [2]. Since Triton X-114 is proven, we believe it is the ideal lysis reagent.
One factor to consider when utilizing surfactants is to ensure the right quantity is used. If a concentration is greater than the detergent’s critical micelle concentration (CMC), the surfactants will form micelles which would negate their usage as shown through the following figure:
Triton X-114 has a CMC of 120 ppm at 25 therefore ensuring that this threshold is not exceeded is crucial to ensure the proper functionality of the assay [3]. Utilizing such small quantities in each lysis buffer helps mitigate any risk to the user should they come into contact with the buffer. The use of a detergent has the side benefit of increasing the solubility of OspA in the buffer as it has been determined that the full length of the outer surface protein is poorly soluble [2].
Buffers
Running Buffers
For our running buffer, we have decided to go with a phosphate buffered saline base solution (1xPBS) which is a widely used reagent for LFIAs [4]. It has been proven to have no ill effects when used with bacteria and has an almost neutral pH of 7.5-8, which is beneficial for user safety [5]. It is well suited to our synthetic proteins, which require a pH in the 7.5-8.2 range and require a pH above their isoelectric point of 6.2.
Sample Pad Buffer
Similar to the running buffer, the sample pad buffer will be comprised of primarily 1xPBS, containing a sodium chloride concentration of 0.1 M with 0.2% Tween-20 to help reduce background noise and 0.1% sodium azide which is a preservative to help with the test’s shelf life [4]. The sample buffer will be dried onto the material and left a few hours before usage or packaging [4]
Conjugate Pad Buffer
The conjugate pad has more of a unique composition because its job is to immobilize the conjugated antibodies and release them upon contact with the running buffer. To do this, the buffer will be comprised of our designed antibodies, conjugated with alkaline phosphatase, in a solution of sodium borate (20mM), 2% Bovine serum albumin (BSA), 3% sucrose, 0.6% sodium chloride, 0.2% Tween-20, and 0.1% sodium azide [4]. In a similar fashion to the sample pad buffer, the conjugate pad buffer will be applied to the pad and dried before usage or packaging. The conjugate pad can immobilize and release the antibodies upon rehydration due to the presence of sucrose [4].
Test Strip Antibodies
Because a competitive assay is being used, the proteins fused to the test strip will be a synthetic version of our target proteins, namely ospA. Typically, in a direct assay, an antibody binding one epitope of the target protein would be fused to the test strip and a second antibody binding a secondary location on the target protein would be present in solution with the sample along with a marker attached to it, typically a gold nanoparticle. Because our protein only has one known scFv binding site, we have the target protein fused to the test strip and thus, when the target protein is present in the sample, it will occupy all of the available scFv’s (which are tagged) and no interactions will happen along the test strip. In the event there is no target protein present in the sample, the tagged scFv’s will bind the synthetic target protein fused along the test line, yielding a visible result.
Future Work
Due to time constraints with our project, the proposed chemical composition for our LFIA is unable to be tested in practice. This is unfortunate as building an LFIA requires a series of trial-and-error experiments to determine what works best with your specific biosystem. Our proposed chemical system is to serve as a baseline for our LFIA and our team proposes future trials be undertaken to further optimize the capabilities of our test. This would include adjusting both the quantities of the current chemicals in addition to substituting chemicals to determine the best possible combination that gives the most reliable test results.
Another suggestion for future testing is to determine the effectiveness of chemical lysing through the use of Triton X-114. Further tests should be done to optimize the concentration to achieve the desired extent of lysing without harmful ramifications. If the chemical lysing is unsuccessfully optimized, alternative methods could be explored, these include the use of electronics such as a handheld corona device, as designed by Escobedo et al [6].
Chemical Interactions Analysis
Our proposed chemicals for our buffers and lysis reagent were all identified and analyzed in Table 1. They were assessed in terms of the hazardous properties and summary limits of each of the substances. The hazardous properties that were analyzed include flammability/explosivity, reactivity, toxicity to humans and the environment, and NFPA rating.
| Material | Hazardous Property | Summary and Limits |
|---|---|---|
| Phosphate Buffered Saline (1xPBS) [7] | Flammable/Explosive | Not flammable |
| Reactivity | - Stable at normal conditions - Incompatible with strong oxidizing agents |
|
| Toxicity to Humans | - None | |
| Environmental Toxicity | Some components pose issues in large quantities to fresh water algae and fish but quantities in PBS are so small that there are no concerns for toxicity | |
| NFPA Rating | - Health: 1 - Flammability: 0 - Instability: 0 - Physical Hazard: N/A |
|
| Triton X-114 [8] | Flammable/Explosive | Store away from sources of heat and ignition |
| Reactivity | - Stable under normal conditions - Hygroscopic - Air and light sensitive |
|
| Toxicity to Humans | - Severe eye irritant - Acutely toxic |
|
| Environmental Toxicity | Toxic to aquatic organisms and environment, specifically freshwater fish | |
| NFPA Rating | - Health: 2 - Flammability: 1 - Instability: 1 - Physical Hazard: N/A |
|
| Tween 20 [9] | Flammable/Explosive | Slightly flammable |
| Reactivity | - Stable under normal conditions - Incompatible with strong oxidizing agents |
|
| Toxicity to Humans | - None | |
| Environmental Toxicity | None | |
| NFPA Rating | - Health: 0 - Flammability: 1 - Instability: 0 - Physical Hazard: N/A |
|
| Sodium Azide [10] | Flammable/Explosive | Slighlty flammable |
| Reactivity | - Reactive - Incompatible with acids, oxidizing agents, peroxides, acid chlrides and metals |
|
| Toxicity to Humans | - Health hazard can cause extreme toxicity or death - Fatal if swallowed - Fatal if in contact with skin or inhaled |
|
| Environmental Toxicity | Highly toxid to aquatic organisms and could have harmful long-term effects in the aquatic environment | |
| NFPA Rating | - Health: 4 - Flammability: 1 - Instability: 4 - Physical Hazard: N/A |
|
| Sodium Borate [11] | Flammable/Explosive | Not flammable |
| Reactivity | - Stable under normal conditions - Incompatible with alkaloid salts, metallic salts and strong acids |
|
| Toxicity to Humans | - No, just an irritant | |
| Environmental Toxicity | Not available | |
| NFPA Rating | - Health: 2 - Flammability: 0 - Instability: 0 - Physical Hazard: N/A |
|
| Bovine Serum Albumin (BSA) [12] | Flammable/Explosive | None |
| Reactivity | - Stable under normal conditions - Incompatible with strong oxidizing agents |
|
| Toxicity to Humans | - Can cause respiratory irritation | |
| Environmental Toxicity | None | |
| NFPA Rating | Not available | |
| Sucrose [13] | Flammable/Explosive | None |
| Reactivity | - Stable - Incompatible with strong oxidizers |
|
| Toxicity to Humans | - None | |
| Environmental Toxicity | None | |
| NFPA Rating | - Health: 0 - Flammability: 0 - Instability: 0 - Physical Hazard: N/A |
|
| Sodium Chloride [14] | Flammable/Explosive | None |
| Reactivity | - Hygroscopic - Incompatible with metals, strong oxidizers, strong acids, bromine trifluoride, nitro compounds, dichloromaleic anhydride and urea |
|
| Toxicity to Humans | - None, just a mild irritant | |
| Environmental Toxicity | None | |
| NFPA Rating | - Health: 1 - Flammability: 0 - Instability: 1 - Physical Hazard: N/A |
Through examining the various reagents found in Table 1, there are a few that could pose some health concerns. Sodium borate, BSA, and sodium chloride are all irritants but don’t cause any harmful or deadly consequences. The two most dangerous chemicals are Triton X-114 and sodium azide. Both possess significant health concerns to those who work with them. This indicates that proper PPE must be worn by the LFIA manufacturers. These chemicals, while might possess harmful properties, they do not pose a significant risk to users as they are used in minimal quantities in a dilute aqueous solution and the user should have little to no direct contact with the chemical dried into the strip.
There are no chemicals that pose risk through their flammable properties as well are either slightly at risk or not flammable. Those that are flammable, it is more applicable for when in their solid phase as opposed to when in solution as is our case. In addition, the LFIA should not be used around flame due to the risk of flame with the assay materials. In terms of manufactures, those assembling and making the LFIA should take precautionary safety measures in terms of PPE and fire safety. They should ensure to keep all sources of ignition away from their assembling area.
Overall, the most dangerous chemical that will be utilized is sodium azide since it is reactive and poses serious and dangerous health risks. Despite its dangerous properties, this chemical does not pose a risk to our users since it is used in very minimal quantities (0.1%) that when in solution/dried into the sample pad, it is dilute enough to still perform its task (it is a preservative) but not pose any health issues should someone accidentally brush the surface of the test. To ensure the safety of our users, the test should be run before distribution to confirm user safety and make any necessary adjustments if required.
References
1. M. Johnson, "Detergent: Triton X-100, Tween-20, and MOre," Labome, 14 May 2021. [Online]. Available: https://www.labome.com/method/Detergents-Triton-X-100-Tween-20-and-More.html. [Accessed 24 July 2021].
2. W. Jiang, P. D. Gorevic, R. J. Dattwyler, J. J. Dunn and B. Luft, "Purification of Borrelia Burgdorgeri Outer Surface Protein A (OspA) and Analysis of Antibody Binding Domains," Clinical and Diagnostic Labratorry Immunology , vol. 1, pp. 406-412, 1994.
3. DOW, "Triton X-114 Surfactant," [Online]. Available: https://www.dow.com/content/dam/dcc/documents/en-us/productdatasheet/119/119-01884-01-triton-x114-surfactant-tds.pdf?iframe=true. [Accessed 21 July 2021].
4. D. ÇAM and H. A. ÖKTEM, "Optimiizations needed for lateral flow assay for rapid detection of pathogenic E. coli.," Turkish Journal of Biology, vol. 41, pp. 954-968, 2017.
5. C. Parolo , A. Sena-Torralba, J. Francisco Bergua, E. Calucho, C. Fuentes-CHust, L. Hu, L. Rivas, R. Álvarez-Diduk, E. P. Nguyen, S. Ciniti, D. Quesada-González and A. Merkoçi, "Tutorial: Design and fabrication of nanoparticle-based lateral flowimmunoassays," Nature Protocols, vol. 15, pp. 3788-3816, 2020.
6. C. Escobedo, S. Bürgel, S. Kemmerling, N. Sauter, T. Braun and A. Hierlemann, "On-chip lysis of mammalian cells through a handheld corona device," Lab on a Chip, no. 15, p. 2990, 2015.
7. ThermoFisher Scientific, "Phosphate Buffered Salien Solution Safety Data Sheet," ThermoFisher Scientific , 18 January 2018. [Online]. Available: http://biocompare.com. [Accessed 29 July 2021].
8. Thermofisher Scientific, "Triiton X-114 Safety Data Sheet," Thermofisher Scientific, 23 January 2018. [Online]. Available: https://www.fishersci.com/store/msds?partNumber=AC422365000&productDescription=TRITON+X-114+500ML&vendorId=VN00032119&countryCode=US&language=en. [Accessed 29 July 2021].
9. TherrmoFisher Scientific, "Tween 20 Safety Data Sheet," TherrmoFisher Scientific, 21 July 2021. [Online]. Available: https://www.fishersci.com/msdsproxy%3FproductName%3DBP337500%26productDescription%3DTWEEN%2B20%2B500ML%26catNo%3DBP337-500%2B%26vendorId%3DVN00033897%26storeId%3D10652. [Accessed 30 July 2021].
10. ThermoFisher Scientific, "Sodium Azide Safety Data Sheet," ThermoFisher Scientific, 25 April 2019. [Online]. Available: https://www.fishersci.com/store/msds?partNumber=S227I25&productDescription=SOD+AZIDE+GRAN+PURIF+25G+IND&vendorId=VN00033897&countryCode=US&language=en. [Accessed 30 July 2021].
11. ThermoFisher Scientific, "Sodium Tetraborate Decahydrate MSDS," Thermofisher Scientific, 15 February 2008. [Online]. Available: https://fscimage.fishersci.com/msds/21010.htm. [Accessed 30 July 2021].
12. ThermoFisher Scientific, "Bovine Serum Albumin Safety Data Sheet," ThermoFisher Scientific, 24 Deecember 2020. [Online]. Available: https://www.fishersci.se/chemicalProductData_uk/wercs?itemCode=12877172&lang=EN. [Accessed 30 July 2021].
13. ThermoFisher Scientific, "Sucrose Material Safety Datat Sheet," ThermoFisher Scientific, 3 October 2005. [Online]. Available: https://fscimage.fishersci.com/msds/01576.htm. [Accessed 30 July 2021].
14. ThermoFisher Scientific, "Sodium Chloride Material Safety Data Sheet," ThermoFisher Scientific, 15 February 2008. [Online]. Available: https://fscimage.fishersci.com/msds/21105.htm. [Accessed 30 July 2021].