Label-free detection of protein–DNA interactions using electrochemical impedance spectroscopy

Abstract

In this work, the applicability of an impedimetric DNA sensor has been investigated for the detection of protein–DNA interactions. The sensor is based on short thiol-modified single-stranded DNA, which is chemisorbed to gold chip electrodes. In the presence of the redox system ferri-/ferrocyanide impedance measurements show an increase in charge transfer resistance after immobilization and hybridization of ssDNA to the sensor surface. The use of a longer capture oligonucleotide (a 25-mer instead of an 18-mer) results in a decreasing probe concentration on the surface. Furthermore it causes an increase of the charge transfer resistance for both ssDNA and dsDNA. The hybridization event, however, can be detected with a similar sensitivity compared to an 18-mer (with the same surface concentration) and allows a good discrimination between ssDNA and dsDNA. This electrode system is used to follow an enzyme reaction on the surface electrochemically. The cleavage of a double-stranded DNA by restriction endonuclease BamHI could be verified by cyclic voltammetry and impedance spectroscopy. The investigations are performed in dithiothreitol (DTT) free buffer solution since the incubation with DTT results in an alteration of the surface impedance. The sequence specific DNA-binding of the transcription factor NF-κB p50 is found to cause a decrease in charge transfer resistance. The signal change is concentration dependent and occurs due to a neutralization effect of the negatively charged DNA backbone.