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.