Improved binding induced self-assembled DNA to achieve ultrasensitive electrochemical proximity assay

We presented here binding induced self-assembly of DNA to regulate the distance between the two aptamers that capture the thrombin to achieve ultrasensitive electrochemical proximity assay (ECPA). Three complementary DNA sequences (S1, S2 and Substrate) were applied to construct the aptasensor. One end of two sequences (S1, S2 served as probes) was thrombin's aptamer, while remaining end of S2 was modified with methylene blue (MB) for signal readout. The substrate sequence containing poly adenine (polyA) can combine with the gold-plated substrate for adjusting the distance between S1 and S2. By using this approach, self-assembly of DNA was achieved by only three complementary sequences, which had the advantage of simpler operation and less time consuming. It was worth noting that through regulating the length of the polyA, the optimal binding distance between the aptamers for capturing target obtained, resulting in increased specificity. Under optimum conditions, the electrochemical proximity assay sensor with excellent reproducibility, high specificity, reliable stability and promising sensitivity revealed an ultralow detection limit of 53.70 aM (S/N = 3) and a wide linear range from 1 fM to 100 nM for thrombin detection. This strategy was extremely flexible, which offered potential applications for detecting various kinds of protein targets via direct electrochemical readout.