A novel electrochemical aptasensor for thrombin detection based on the hybridization chain reaction with hemin/G-quadruplex DNAzyme-signal amplification

In this work, a novel signal amplification electrochemical aptasensor for the sensitive and selective detection of thrombin was successfully fabricated. The amplification method was based on the hybridization chain reaction (HCR) and a pseudobienzyme electrocatalytic system. HCR-based double-stranded DNA (dsDNA) polymers not only constructed an effective carrier for anchoring larger amounts of electron mediator methylene blue (MB) into the DNA duplexes to produce a strong differential pulse voltammetry (DPV) signal, but also resulted in the formation of hemin/G-quadruplex DNAzymes nanowires by intercalating hemin into two induced single-stranded DNA (ssDNA). With the addition of NADH into the electrolytic cell, the hemin/G-quadruplex acting as an NADH oxidase and HRP-mimicking DNAzyme for the pseudobienzyme amplifying system could in situ biocatalyze the formation of H2O2 with local concentrations and low transfer loss resulting in dramatic signal enhancements. The binding event can be detected by a decrease in the integrated charge of MB which electrostatically absorbed onto dsDNA polymers. In the presence of thrombin, the dsDNA polymers associated with MB and hemin/G-quadruplex structures were removed from the electrode surface, leading to a significant decrease of redox current. DPV signals of MB provided quantitative measures of the concentrations of thrombin, with a linear calibration range of 0.01-50 nM and a detection limit of 2 pM. Moreover, the resulting aptasensor also exhibited good specificity, acceptable reproducibility and stability, indicating that the present strategy was promising for broad potential application in clinic assay and various protein analyses.