Team:JNFLS/Demonstrate

Demonstrate

We developed a biosensor based on the aptamer target on the HCV core protein, using competition rolling amplification. This device could be used for the detection of trace HCV virus in unpaid donation blood test and blood transfusion in clinic, which could shorten the window period of HCV detection.

1. The HCV C-O120 recombinant plasmid worked well

In order to express HCV C protein efficiently, the complete HCV C gene was truncated to 360bp and optimized using software. Fig.1 and Fig.2 illustrated that HCV C-O120 protein was expressed and purified very well, which allowed the following experiments could be done.

Fig.1 SDS-PAGE result showing the expression of HCV C-O173 and HCV C-O120, which were marked by GFP

Fig.2 The electrophoresis result showed purification of proteins.

2. The competition-based rolling amplification system worked well

Fig.3 illustrated that the components including circle probe, cDNA and HCV C aptamer designed in this device worked together well. By the aptamer competition cDNA with circle probe, the rolling amplification was inhibited (lane 2, 4, 6, 8, 10 and 12).

Fig.3 Rolling amplification products using designed circle probe, cDNA and HCV C aptamer (without HCV C protein)

When the purified HCV C protein was added into the system above, It still worked very well, showed as Fig.4. We can see that, at 94℃ annealing, cDNA#2 and 9ul purified protein combination achieved the best result. And at this condition, the probe’s concentration is about 0.1uM, and the purified protein concentration is 2×10-3mg/ml. The device with very low concentration working well illustrated that it could work well under the real-world conditions.

Fig.4 Competition-based rolling amplification products using circle probe, cDNA and HCV C aptamer with purified HCV C protein.

The model of our biosensor

Although we believe that our biosensor could work in the realistic world, considering the safety, we have not tested the device using HCV infected serum due to the infection danger. We will request the qualified Biological Company to finish this test. Here we have designed and made a biosensor model to illustrate our device. It will shorten the window period of HCV detection because its sensitivity is very high according to the experiment result (2×10-3mg/ml).

Fig.5 Tht structure of our biosensor model.

Fig.6 Diagram usage to our biosensor model.

At the end, we believe that our biosensor could work well in the realistic world.