Team:HKIS/Engineering
Engineering Success
Optimizing the Lateral Flow Assay
Design
Research/ Imagine
Build/Test
Learn
We have successfully designed and built a highly sensitive, portable Vibrio detection kit. This kit is capable of detecting real Vibrio bacterium in any solution. We utilized unique design principles and methodologies not present in existing Vibrio detection devices, such as our kit's pam independency and on-site incubation portability. Throughout our experiments, we have faced many challenges and issues, below are a few vital experimental iterations we had to go through in order to develop our working system.
Check out these links! These are early experimental or extra lab work that is not documented in other part of the wiki!
Click the link for the journey of designing the crRNA and RPA primers! PDF link to the Design Proof of Concept.
Click the link for the optimization of buffer to achieve 10X efficiency! Also for some pilot test results PDF link to the Early optimization and pilot test!.
Click the link for more novel implementation of asymmetric RPA primer which increased the signal 2-5 fold, also check out the session in the wiki! Photo for the Asymmetric fluorescence for TDH (crRNA 1 and 2) and vcgC gene (crRNA 3 and 4).
Iteration 1
Research/Imagine
When first exploring our idea for a portable detection system, we researched many different ways to display it. First and foremost, we wanted our detection system to be affordable and easy to use. We looked into multiple ways to display a result. We researched a fluorescence based visualization extensively. However, it would be impractical in on-field conditions due to the equipment required. We decided on a lateral flow assay. Not only do lateral flow assays display a clear one/two band result, but they also show varying band intensity levels for a rough estimate of bacteria concentration in the oyster. Additionally, this technology is expected to be somewhat intuitive for users, as it is also the display method used for pregnancy tests.
Design/Build/Test
Our first test of the lateral flow assay was to determine the minimum concentration of Cas12a enzyme required to show a result. We created several tubes of reagents with different concentrations of Cas12a, and once they were prepared, we submerged our lateral flow assay dipsticks in them for our first test. However, we had a problem. While the assays showed a result, there was no change in the test (positive) band. Even the control sample showed a positive result. In the image below, you can see that from the left (high concentration) to the right (control), there is always a positive testing band, even though in control (farthest right), there shouldn’t be. The fact that even negative control LFAs resulted in positive results was a serious issue: we did not want our system to show false-positive results. We decided to investigate this issue to come up with a possible solution.
Iteration 2
Research/Imagine
Our central hypothesis for this result’s cause was that either the biotin was degraded/overflowed or the gold nanoparticles were oversaturated. We first decided to test for the degradation/overflow of biotin to rule it out as a cause.
Design
First, we designed an experiment with varying concentrations of biotin to test for biotin degradation. This protocol would hopefully show whether the biotin overflowed or if it had degraded. We tested at four different concentrations to get enough variety for accurate results.
Build/Test
Our test ended up showing similar results to the first one: there was minimal variation in concentrations, to the point where the 2 µL biotin tube had almost the same results as the 0.25 µL biotin tube. In this image, it is difficult to discern which assay was used with which concentration at first glance. We had to solve this issue since it would make using our kit very difficult, as it would be nigh-impossible to discern positive and negative results.
Learn/Improve
We realized that while the biotin might have degraded, it was more likely that the gold nanoparticles in the lateral flow assay were oversaturated. The oversaturation explained the strong results, even with lower concentrations of biotin or Cas12a. Now that we realized the issue was due to oversaturation, we had to find a way to reduce gold nanoparticle quantities.
Iteration 3
Design
To combat the oversaturation of gold nanoparticles, we came up with the solution of physically cutting the lateral flow assay testing section. We thought that by reducing the amount of gold, we would reduce the strength of the result.
Build/Test
We took specific measurements and cut off the end of the test (where the gold particles were situated). We left one dipstick whole as a control, then cut off 8mm, 10mm, 12mm, and 14mm. We created five tubes of the same reagent mix for the experiment to evaluate the change in the results best. Our results were generally as expected (as seen in the image below). As the amount cut off increased, the brightness of the result decreased. However, it is clear from the image that this method was very crude, even though you can see a clarity gradient in the testing band. This was as expected since the gold nanoparticles play a significant role in the function of the lateral flow assay.
Learn/Improve
While we had a functional result, this method was still very unrefined. There had to be better solutions than just cutting each dipstick before use. We decided to continue researching to see if we could develop a more sophisticated method of improving the lateral flow assay.
Iteration 4
Imagine/Research
We researched several different lateral flow assays and possible reasons for oversaturation and bright results. Finally, we came upon the idea to adjust the buffer viscosity of the lateral flow assay. We realized that by making the buffer more viscous, we could adjust how the reagents flowed up through the assay and possibly make the result more accurate.
Design
We decided to use polyethylene glycol (PEG) as our thickening agent for the buffer. Additionally, we consulted the Milenia protocols and reduced the amount of biotin used from 10 pmol to 5 pmol. We hoped that this alteration to the buffer would result in a more sophisticated solution to our problem.
Build/Test
We first made the PEG buffer mix. For this experiment, we tested a 4% PEG concentration and a 5% PEG concentration. We made them by mixing 8000 weight PEG with TBS buffer and filling them to 10 mL. From there, we developed a controlled positive and negative result for each. A clear positive result and a clear negative result were expected for each. We also expected that the buffer viscosity would have an impact on the results.
Learn
Our results were excellent. The controlled negative showed no testing band, and the controlled positive had an unmistakably clear positive band. We found that the 4% PEG had the best result, with an extremely clear positive and negative. We decided to move forward with the 4% PEG Assay buffer instead of the provided assay buffer we had been using previously. In the image below, the LFA on the left has no visible testing band, while the LFA on the right has a very clear positive testing band. We had finally optimized our lateral flow assay.
Optimizing the Cas12a Ratio
Iteration 1
Research/Imagine
When planning our project, we researched several ways we could produce a successful detection system. We decided on a Cas12a-RPA one-pot assay. The functionality of Cas12a, regardless of the presence of a PAM site, allowed for a broader scope of detection. We chose RPA because of its relatively low reaction temperature and its rapid signal amplification speed. Furthermore, these reagents can be freeze-dried and can therefore be used in the field. Once this was established, we wanted to determine the ideal ratio of RPA solution quantity to Cas12a enzyme for optimized result brightness.
Design
We knew it was possible to directly oversaturate the solution with Cas12a to get an extremely positive result, but we wanted to develop a more sophisticated system by being more economical in our use of Cas12a. This would also add to the affordability of our detection system since the Cas12a is one of the more expensive reagents. We designed the experiment by varying our RPA volume to change the ratio of Cas12a to RPA. We decided to run this first experiment using fluorescence since it would be easier to indicate the best ratio.
Build/Test
From our first experiment, we found that after only five minutes, the brightness of the fluorescence was so high that it was difficult to discern the optimal ratio of Cas12a to RPA. While we were able to identify the best ratio after careful examination, it was quite hard to differentiate with the naked eye. In the image below, at first glance, it is very difficult to tell which is the brightest, even though some are slightly brighter than others.
Learn/Improve
We thought that this bright result might have been due to an error in the RPA. It was also possible that the issue was the Cas12a, where a small quantity with high concentration led to the error in results. We decided to troubleshoot this high fluorescence to see if it was due to an error or if it was an accurate result.
Iteration 2
Design
For our new experiment, we decided to make a negative RPA reaction to rule out the possibility of RPA error. We also readjusted the quantity of Cas12a, but we did not change the concentration. Since this experiment was to determine if the Cas12a ratio was optimal, we wanted to maintain it to test if it was a legitimate result or the product of error. We wanted to test this because we thought the amount of Cas12a might have created a higher than expected yield of the Cas-crRNA complex.
Build/Test
We essentially ran the same experiment except with an altered negative control and Cas12a quantity. We again ran it with fluorescence to have a point of reference against our previous results. However, we also ran it using the lateral flow assay. Our negative control for the fluorescence had no RPA FQ, while the LFA had no RPA Biotin. From this, we were able to see how this reaction displayed in our actual testing kit. In the image below, the LFA and the FQ setup clearly show negative and positive results similar to the previous experiment.
Learn
Our results were excellent. The lateral flow dipstick experiment was purely cosmetic and was kept to determine whether the issue lay with fluorescence or lateral flow assay. We found that the brightness was the same after five minutes, while the controls in both the fluorescence and the LFA showed negative results (with no brightness or testing band). We, therefore, realized that the brightness was due to the use of abnormally high concentrations of fluorescent-quenchers. Our positive result for the fluorescence was similarly bright after five to ten minutes, proving that our previous results accurately represented the experiment. These results also showed a clear testing band on the lateral flow assay. Overall, the accuracy and success of this experiment ensured that we had found the optimal ratio of Cas12a to RPA, which would be used in all later builds of our experiments.
Conclusion
By following the proper experimental cycles, we managed to produce an extremely specific and highly intuitive Vibrio detection kit that functions as intended. Learn more about proposed implementation of our product on the implementation page.
Sources
“An Engineer's Mindset: Creativity in Engineering.” · NES Fircroft, www.nesfircroft.com/blog/2021/09/an-engineers-mindset-creativity-in-engineering.