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

Abstract

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 ] 3À/4À ). 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.

Controlled Orientation of DNA in a Binary SAM as a Key for the Successful Determination of DNA Hybridization by Means of Electrochemical Impedance Spectroscopy

“…The obtained electrodes were further incubated with proflavine as specific intercalator for dsDNA, as previously reported. [28] The decrease of the concentration of the MCH molecules co-assembled at the gold electrode endows an increase of R ct when the hybridization was carried out for 1.5 h. A hybridization time of only 45 min did not reveal significant changes in the R ct (Figure 2 B). This tendency is even more pronounced at higher dilutions of MCH in the monolayer (Figures 2 C, D).…”

“…layers; [28,29] iv) the roughness of the electrode surface, which determines the packing density in the SAM; [30,31] v) the Debye length, which reflects the decay length of the electric field and is a measure of the counterion screening; and vi) the electric field in front of the electrode surface and the formation of the electrochemical double layer. [32] It has been shown that the orientation of grafted DNA strands can be efficiently controlled by the electric field.…”

Metal-containing peptide nucleic acid conjugates

“…DNA biosensors probe and/or detect the hybridization event, which is then transduced, for example, into an electrochemical [17][18][19][20][21][22] or optical [23][24][25][26] signal (Figure 3). In principle, an analytical sample, which contains, amongst others, the DNA sequence-of-interest (the target), is deposited onto the recognition layer of a transducer.…”

A novel cobalt complex for enhancing amperometric and impedimetric DNA detection

“…Indirect labels like intercalating molecules can offer a useful compromise between easier sample preparation and improved detection sensitivity, whereby direct labelling of the target nucleic acid sequence is not required. have also been used in conjunction with EIS to serve as signal validation tools to exclude potential false positives by non-specific adsorption [20,21]. These molecules cause a decrease in R ct upon binding to dsDNA, but cause little or no change with ssDNA.…”