Amperometric aptasensor for thrombin detection using enzyme-mediated direct electrochemistry and DNA-based signal amplification strategy

In this work, a new electrochemical aptasensor based on direct electron transfer and electrocatalysis of horseradish peroxidase (HRP) using exonuclease-catalyzed target recycling and hybridization chain reaction (HCR) for signal amplification was developed for highly sensitive detection of thrombin. The electrochemical signal was originated from HRP without the addition or labeling of redox probes. To construct the aptasensor, the capture probe was immobilized on gold nanoparticles (AuNPs) modified electrode for the following hybridization with the complementary thrombin binding aptamer. In the presence of thrombin, the formation of aptamer-thrombin complex would result in the dissociation of aptamer from the double-strand DNA (dsDNA). Subsequently, with the employment of exonuclease, aptamer was selectively digested and thrombin could be released for analyte recycling. The capture probe and two hairpin helper DNAs lead to the formation of extended dsDNA polymers through HCR on the electrode surface. Then the biotin-labeled dsDNA polymers could introduce numerous avidin-labeled HRP, resulting in significantly amplified electrochemical signal through the direct electrochemistry and electrocatalysis of HRP. The proposed strategy combined the amplification of analyte recycling and HCR, as well as the inherent electroactivity and catalytic activity of HRP, which exhibited high sensitivity for thrombin determination with an ultra-low detection limit of 1.2×10-13 M. Moreover, the detection scheme could be easily extended to the detection of other biomolecules.