Cyclic voltammetry of submicrogram quantities of supercoiled, linear and denatured DNAs with DNA-modified mercury electrode.

Abstract

We have previously shown that a DNA-modified electrode can be prepared by immersing the hanging mercury drop electrode (HMDE) in a small volume (about 5-10 microliters) of a DNA solution. Within a short period of time the DNA is irreversibly adsorbed at the electrode, resisting subsequent washing. The electrode is then transferred into a voltammetric cell filled with the background electrolyte (that does not contain any nucleic acid) in which the voltammetric measurements are performed. This procedure is called adsorptive transfer stripping voltammetry (AdTSV). In this paper AdTS cyclic voltammetry (CV) peaks of DNA were measured to report on the stability of the attachment of plasmid DNA molecules to the electrode surface. It was shown that the attachment of plasmid DNA to the electrode was, like the case of calf thymus DNA, sufficiently stable. If the DNA-modified electrode was immersed in a protein solution no significant exchange between the DNA and protein was observed. Submicrogram amounts of DNA were sufficient to attain full coverage of the electrode at relatively short waiting times and the detection limit of the denatured DNA was below 2 ng. The intensity of the AdTS CV signals of supercoiled, linearized and thermally denatured linear DNAs differed from one another. It was shown that alkaline denaturation of linear and supercoiled DNA in solution can be studied using AdTS CV technique. It was further shown that at neutral pH the linearized plasmid undergoes denaturation due to a prolonged contact with the electrode charged to potentials around -1.2 V. Such a surface denaturation has been previously observed with calf thymus DNA and synthetic double-stranded polynucleotides. Our results show that in contrast to linear DNA, supercoiled DNA was not significantly denatured as a result of its prolonged contact with the electrode charged to various potentials in the range between -0.1 to -1.5 V.