Multiplexed electrochemical coding of DNA-protein bindings

A simple, sensitive and multiplexed electrochemical sensor for the detection of DNA-protein binding based on the exonuclease protection strategy is described. Two electroactive species, methylene blue (MB)- and ferrocene (Fc)-labeled dsDNA probes are self-assembled on a gold electrode to prepare the sensor surface. The target proteins, vascular endothelial growth factor (VEGF) and estrogen receptor (ERα), bind to the dsDNA probes and protect the probes from digesting by exonuclease III due to the steric hindrance of the bound proteins. These protein-protected, MB/Fc-labeled sequences remaining on the sensor surface display two distinct voltammetric peaks, whose peak potentials (MB: −0.27 V; Fc: +0.27 V) and intensities reflect the identities and amounts of the corresponding target proteins, for simultaneous and multiplexed detection of DNA-protein bindings. The proposed sensor is also selective to the target proteins against other interference molecules. By using labels with distinct voltammetric peaks, the developed method can be easily expanded for simultaneous detection of multiple DNA-protein bindings.