Electrochemical Detection of Single-Nucleotide Mismatches Using an Electrode Microarray

Gold electrode arrays with electrode diameters of 10 μm were used for the detection of eight single-nucleotide mismatches in unlabeled and prehybridized DNA by electrochemical impedance spectroscopy (EIS). Because of the differences in the electrical properties of films of duplex DNA (normal duplex DNA in B-form) in the presence and absence of Zn2+ at pH ≥ 8.6, Randles equivalent circuits were employed to evaluate the EIS results. The difference in the charge-transfer resistance (ΔR CT) between B-DNA (absence of Zn2+ at pH ≥ 8.6) and M-DNA (presence of Zn2+ at pH ≥ 8.6) allows unequivocal detection of all eight single-nucleotide mismatches within a 20-mer DNA sequence. After dehybridization/rehybridization with target DNA, ΔR CT allows the discrimination of single-nucleotide mismatches with concentrations of the target strand as low as 10 fM. Although the presence of protein impurities (bovine serum albumin, 10 μg/mL) interferes with the detection of the target strand (1 pM detection limit), the presence of nontarget DNA (calf thymus DNA, 10-8 M) does not interfere, and the detection limit for recognition of the target strand remains at 10 fM.