Unlabeled hairpin-DNA probe for the detection of single-nucleotide mismatches by electrochemical impedance spectroscopy

An unlabeled hairpin-DNA probe was used for the detection of eight single-nucleotide mismatches by electrochemical impedance spectroscopy (EIS). Upon hybridization of the target strand with the hairpin DNA probe, the stem-loop structure is opened and forms a duplex DNA. Accordingly, the film thickness is increased, which causes differences in the electrical properties of the film before and after hybridization. Randles equivalent circuits were employed to evaluate the EIS result. The differences in the charge-transfer resistance DeltaR(CT) between hairpin DNA (before hybridization) and duplex DNA (after hybridization) shows the consequence of a large structural rearrangement from hairpin to duplex. If a single-nucleotide mismatch is present in the center of the duplex, the difference in charge-transfer resistance DeltaR(CT) between B-DNA in the absence and presence of Zn(2+) allows the unequivocal detection of all eight single-nucleotide mismatches. The detection limit was measured, and DeltaR(CT) allows the discrimination of a single-nucleotide mismatch with the concentration of the target strand as low as 10 pM.