Label-Free Detection of DNA Hybridization in Presence of Intercalators Using Electrochemical Impedance Spectroscopy



Actinomycin D or proflavine which are known to intercalate within the helix of double-stranded DNA (dsDNA) are used as label-free control to unequivocally prove complementary DNA hybridization by means of electrochemical impedance spectroscopy (EIS). Based on a carefully designed interface comprising a thiol-tethered (20mer) oligonucleotide capture probe which forms a self-assembled monolayer on a gold electrode together with a short chain hydroxyl-terminated alkylthiol, formation of dsDNA can be monitored by an increase of the charge-transfer resistance of a free-diffusing negatively charged redox species ([Fe(CN)6]3−/4−). The increase of the charge transfer resistance due to complementary hybridization was about 10 times from the unmodified Au surface to the dsDNA modified electrode. Specific interaction of intercalators with dsDNA leads to a decrease in charge transfer resistance due to the conformational changes in the dsDNA monolayer and partial charge compensation caused by the positively charged intercalators. No shift in the charge transfer resistance was observed in case of incubation of a ssDNA surface with intercalators or when hybridization was invoked using a noncomplementary DNA sequence. Thus, hybridization can be unambiguously detected using EIS by first recording the increase in charge-transfer resistance due to hybridization with the matching target strand followed by recording a decrease in charge transfer resistance caused by intercalation. Nonspecific adsorption can hence be doubtlessly excluded as a reason for the observed changes in the impedance spectrum.