Enzyme-free fluorescence aptasensor for amplification detection of human thrombin via target-catalyzed hairpin assembly

Abstract

Aptamers have many advantages, such as simple synthesis, good stability, high binding affinity and wide applicability, making them suitable candidates for protein detection. Since the disease-related protein may be present in very small amounts in biological samples, the development of amplification paths for aptasensors is essential. In this paper, we develop a simple and enzyme-free amplified aptasensor for protein detection via target-catalyzed hairpin assembly. This aptasensor contains two DNA hairpins termed as H1 and H2. H1, which is modified at its 5' and 3' ends with a fluorophore and a quencher respectively, consists of the aptamer sequence of human thrombin. Meanwhile, H2 is partially complementary to H1. These two hairpins H1 and H2 interact slowly with each other. Upon the addition of target protein, it can facilitate the opening of the hairpin structure of H1 and thus accelerate the hybridization between H1 and H2, resulting in the significant fluorescence enhancement of the system. By monitoring the change in fluorescence intensity, we could detect the target protein with high sensitivity. The detection limit of this aptasensor is 20 pM, which is more than two orders of magnitude lower than that of reported unamplified aptasensors. Furthermore, this amplified aptasensor shows high selectivity toward its target protein. Thus, the proposed aptasensor could be used as a simple, sensitive and selective platform for target protein detection.