Homogeneous Electrochemical Assay for Real-time Monitoring of Exonuclease III Activity Based on a Graphene Monolayer

A homogeneous electrochemical assay based on a graphene monolayer electrode was developed for simple, sensitive, rapid and quantitative analysis of the exonuclease III (Exo III) activity. The method utilized a methylene blue (MB) tagged DNA substrate with hairpin structure, and a graphene monolayer attached on the working electrode. Before digestion, the hairpin structure prevents the adsorption of the DNA substrate to the graphene surface. Degradation of the substrate by the 3'-5' Exo III, however, yields single-stranded DNA (ssDNA), resulting in its subsequent binding to the graphene surface through pi-pi stacking, which produces the voltammetric current from electrochemical reduction of the MB tag anchoring at the end of ssDNA. A direct quantification of the Exo III activity can be achieved by measuring the reductive peak current of the MB tag under easily attainable potential (similar to -0.1V vs Ag/AgCl) range comparably sensitive to the conventional methods such as a gel-based or fluorescence-based assays. Our approach can be applied to measure various exonucleases activity by adjusting the structure of DNA substrate suggesting a new assay method in drug screening and basic research related to the enzymes.