Novel protein detection method based on proximity-dependent polymerase reaction and aptamers

In recent years, specific detection of proteins is one of the hot issues about aptamers in proteomics. Here we reported a simple, sensitive and specific proximity-dependent protein assay with dual DNA aptamers. Thrombin was used as the model protein, and two aptamer probes with complementary sequence at 3′-end were designed for the two distinct epitopes of the protein. Association of the two aptamers with thrombin resulted in stable hybrids due to the proximity of 3′-end, then polymerase reaction was induced. The amount of obtained dsDNA was indicated using the fluorescence dye Sybr Green I. The results showed that the initial velocity of polymerase reaction had a positive correlation with concentration of thrombin. The advantages of this dual-aptamer-based approach included simple and flexible design of aptamer probes, high selectivity and high sensitivity. The detection limit was 6.9 pmol/L. Supported by the National Key Basic Research Program of China (Grant No. 2002CB513110), Key Technologies Research and Development Program of China (Grant No. 2003BA310A16 and 2005EP090026), Key Project of International Technologies Collaboration Program of China (Grant No. 2003DF000039), and the National Natural Science Foundation of China (Grant No. 90606003, 20475015 and 20620120107)