Team:CSMU Taiwan/Demonstrate
Overview
In addition to comply to all rules and policies approved by the iGEM Safety Committee (please see our Safety page), we self-evaluate the safety of our project with the Risk Mitigation Plan referring the selective agent and dual use research management regulations of TCDC.
This year, we design an aptamer-type rapid diagnostic kit called influsensor, trying to solve the disadvantages of screening on the influenza nowadays. (please see our Description page)
There are two main products in our project:
- The aptamer with high affinity and specificity designed for rapid diagnostic test
- Influensor (aptamer-type rapid diagnostic kit) that is extremely easy for doctors to use
Overall, our the aptamers selected out by the target proteins NPA BBa_K2951004 and HA3 BBa_K2951014 have been proved to possess the properties of high affinity and specificity through our experiments, indicateing that there is a potential to replacing the current antibody-type rapid influenza diagnostic strip. (please see our Entrepreneurship page)
Aptamers
This year, we acquire four successful experimental results related to produce aptamers with high affinity and specificity in our project:
Target protein with optimized sequence
The production of the target protein is the preprocess of SELEX. However, our original yield of NPA protein and NPB protein is too low to supply the need for SELEX. We optimized the sequence and compare the absorbance standard curve before and after the sequence optimization.
The figures indicate that the protein yield of both NPA protein and NPB protein significantly increased after the sequence optimization.
SELEX
In order to acquire the aptamers of target proteins, we conducted the SELEX experiment. After 6 rounds of SELEX for every protein respectively, the aptamers of each target proteins were obtained. The PDF files below are the sequenced results of the aptamers:
ELISA
After conducting the SELEX experiment. Using ELISA to confirm the titer value of the aptamer towards target protein enables to test two important characters we intend to understand about the selected aptamers: the affinity and the specificity.
On the figure of NPA(200X)-NPA, all of the aptamer of NPA protein tested in affinity test have significantly dose-dependent property. As a result, all of the aptamer have high affinity toward the NPA protein. On the figure of NPA(200X)-NPB, all of the aptamer of NPB protein tested in specificity test have significantly lower value comparing with the test of NPA protein. Then, we found that the NPA-4 aptamer and NPA-5 aptamer have higher affinity toward NPA protein and lower affinity toward NPB, which means NPA-4 aptamer and NPA-5 aptamer have higher specificity. As a result, we chose NPA-4 aptamer and NPA-5 aptamer to experiment in the next stage.
On the figure of ApHA3-4, there is a significant logarithmic relevance between HA3-4 aptamer and the target protein HA3, indicating the high affinity of HA3-4 aptamer. In addition, there is a lower relevance between the aptamer and the non-target protein, indicating the high specificity of HA3-4 aptamer. Also, there is a lower value when comparing with the target protein. As a result, we found that HA3-4 aptamer can effectively distinguish the two protein.
Rapid diagnostic test strip
After the confirmation of ELISA, we want to make the selected aptamer commercialized and use two different methods to test.
The two methods are direct-competition and sandwich competition.
(About the two different methods, please see our Experiment page for more details.)
Because we are in the test phage currently, we use the method of direct-competition first to confirm the feasibility and we focus on the experiment of the test group.
We have not conducted the experiment of the control group because of the limitation of time, but we have confirmed that the aptamer can be bind with Nanogold.
On this figure, 1ug, 2.5ug and 5ug of NPA-4 aptamer conjugated to Nanogold all showed a peak at 260nm, indicating that three of them are all successfully conjugated.
Although no color is shown under naked eyes when 2.5 ug of NPA-4 aptamer is conjugated to Nanogold particles.
After adjusting the contrast value, the color of test line with the sample of PBS shows up.
On this figure,the color turned lighter when the concentration of NPA rises, indicating the NPA-4 aptamer bind the NPA protein indeed.
The darkest was PBS, then 800 fold diluted, and the lightest was 100 fold diluted.
The result double-proved our experiment was successful. The color goes lighter when the concentration of target protein rises.
About the test strip experiment of the HA3-4 aptamer, we have not conduct the experiment because of the limitation of time.
However, the high affinity and the high speficity of HA3-4 was confirmed by ELISA.
We expect the result will be as successful as the result of NPA-4 aptamer.
(Please see our Results page)
Influsensor
Strip
Casing
Swab
The assembly of the swab
Final product of the swab
Influsensor is an aptamer-type rapid diagnostic kit with the characteristics of low cost, fast production and development, high stability, low false-negative rate, and animal free. (Please see our Design page)
Influsensor is made of aptamer-type rapid diagnostic strip, casing, and swab. We design every component of the kit with ingenuity. We also demonstrate the user manual and the video of user guide. All of our designs are aim for making doctors manipulate the kit easily. (Please see our Design page)
User manual
User guide
Because of all the advantages and features of the aptamer, we integrate all the benefits of atpamers and design a model containing all the aspects of a disease: Rapid screening, Prediction, Prevention, and Treatment. The model is called CSMU_Taiwan Influsystem.
CSMU_Taiwan Influsystem
Rapid screening- Aptamer-type rapid diagnostic strip (Influsensor)Prediction- AI system
Prevention- Air purifier
Treatment- Anti-hemagglutinin drug
(Please see our Description page and Design page)