Mediator-free triple-enzyme cascade electrocatalytic aptasensor with exonuclease-assisted target recycling and hybridization chain reaction amplification

• We designed a simple and ultrasensitive mediator-free triple-enzyme cascade electrocatalytic aptasensor for thrombin detection.
• The exonuclease-assisted target recycling and hybridization chain reaction (HCR) amplification strategy were also used for signal amplification.
• As a result, the proposed method can detect thrombin specifically with a detection limit as low as 20 fM.

The amplified sensitive detection of protein is essential to biomedical research as well as clinical diagnosis. Here, we developed an ultrasensitive mediator-free triple-enzyme cascade electrocatalytic aptasensor for thrombin detection on the basis of exonuclease-assisted target recycling and hybridization chain reaction (HCR) amplification strategy. The double strands constructed by the hybridization of thrombin binding aptamer (S1) with its complementary strand (S2) were firstly assembled on the electrode. Upon addition of target to the system, the S1 recognized thrombin and left off electrode to make space for assembly of hybrid-primer probe (H0). Then, the H0 triggered the HCR to form the multi-functional hemin/G-quadruplex DNAzyme nanowires. In the mediator-free triple-enzyme cascade electrocatalytic amplification system, the hemin/G-quadruplex DNAzyme nanowires here simultaneously played three roles: the redox probe, NADH oxidase and HRP-mimicking DNAzyme, respectively, which effectively avoided the fussy redox probe and enzyme labeling process, serving a useful alternative or supplement to conventional assays that typically suffer from complexity and poor sensitivity. Additionally, in order to improve the assembly amount of hemin/G-quadruplex DNAzyme nanowire, the exonuclease-assisted target recycling amplification was used for the continuous removal of S1. As a result, the proposed method can detect thrombin specifically with a detection limit as low as 20 fM.