Electrochemical DNA hybridization signal amplification system using methylene blue and ascorbic acid

This study presents a method for real-time monitoring of heterogeneous DNA hybridization using chronoamperometry (CA). The electrochemical assay is based on methylene blue (MB)-labeled ssDNA target (tMB) hybridization with a complementary ssDNA capture probe (p) chemisorbed to a gold electrode through the Au-S linkage. Real-time monitoring of nucleic acid binding is enabled by introducing ascorbic acid (AA) during CA measurements at oxidative potential. After hybridization, the newly formed MB-labeled double stranded DNA (ptMB) oxidized AA and, as a result, shuttled the electrons directly to the electrode. AA-ptMB electrocatalytic cycle allows amplifying the signal from hybridized MB-labeled DNA through oxidation of AA. The process consists three particular processes: hybridization of tMB to the surface probe, reaction between AA and ptMB, and (c) the electron transfer from ptMB to the electrode. We have demonstrated that system is limited by the AA-ptMB redox reaction (bimolecular constant, k = 8 +/- 0.1 M-1 s(-1)). AA-ptMB cycle is sequence-specific, thus fully developed current saturation curves can be used to calculate hybridization parameters and evaluate binding kinetics under various conditions. In addition, evaluating the initial hybridization rate allows quantifying the concentration of labeled ssDNA (tMB) in several minutes. This study provides the first report of a simple electrocatalytic cycle between MB-labeled DNA and a reducer AA for monitoring DNA hybridization in real time.