A novel electrochemical micromethod for the investigation of the interactions between DNA and non-electroactive species is described. The method was developed using the system of double-stranded DNA (dsDNA) modified gold electrodes (dsDNA/Au), a synthesized water-soluble C60 derivative as a model, and [Co(phen)3]3+/2+ (phen=1, 10-phenanthroline) as an electroactive indicator. Electrochemical studies with dsDNA-modified gold electrodes suggest that the C60 derivative can interact strongly with dsDNA, with binding sites of the major groove of the double helix and phosphate backbone of dsDNA, a binding constant of (1.6

The interactions of benzyl viologen (BV) with single-and double-stranded calf-thymus DNA immobilized onto gold electrodes have been studied by electrochemical methods. Benzyl viologen interacts electrostatically with both doublestranded (ds) and single-stranded (ss) DNA, and the strength of the interactions is dependent on ionic strength (μ). The dicationic form (BV2+) binds to dsDNA 9 times more strongly than the singly reduced form, BV·+, in a pH 7.4 Tris-HCl buffer solution at μ=8.4mM. BV2+ binds to ssDNA 5 times more strongly than the BV·+ form. From measurements atμ=8.4mM, a binding constant (K2+) of 2.0 ((0.2)×104M-1 and a binding site size (s) of 1 base pair were obtained, respectively, for dsDNA. For ssDNA, at the same ionic strength, the values obtained for K and s were 3.6 ((0.4) ×104M-1 and 2 nucleotides, respectively. The amount of BV bound, whether to dsDNA or ssDNA, decreased with increasing ionic strength. Whereas the binding rate of BV to both dsDNA and ssDNA immobilized onto gold electrodes is relatively low, once immobilized, it dissociates rapidly away from the electrode surface. The electron-transfer rate constant for BV is moderately fast at both dsDNA- and ssDNA-modified gold electrodes. The application of benzyl viologen as an electroactive indicator capable of differentiating between surface-immobilized single- and double-stranded DNA in denaturation/regeneration cycles has been explored.

A novel microscale and surface-based method for the study of the interactions of DNA with other redox-active molecules using DNA-modified electrodes is described. The method is simple, convenient, reliable, reagentsaving, and applicable for DNA studies, especially those involving microsamples. Information such as binding site size (s, in base pairs), binding constant (K), ratio (KOx/KRed) of the binding constants for the oxidized and reduced forms of a bound species, binding free energy (△Gb), and interaction mode, including changes in the mode of interaction, and "limiting" ratio KOx°/KRed° at zero ionic strength can be obtained using only 3-15μg of DNA samples. The method was developed using [Co(Phen)3]3+/2+ (Phen) 1,10-phenanthroline)/doublestranded DNA (dsDNA)-modified gold electrodes and [Co(bpy)3]3+/2+ (2,2'-bipyridyl)/dsDNA-modified gold electrodes as model systems. For the [Co(Phen)3]3+/2+/dsDNA-modified gold electrode system, a K2+ of (2.5(0.3)×105M-1 and an s of 5bp were obtained in 5mM pH 7.1 Tris-HCl buffer solution containing 50mM NaCl. For [Co(bpy)3]3+/2+/dsDNA-modified gold electrodes, K3+ and s values of (1.3(0.3)×105M-1 and 3bp, respectively, were obtained. While the s values are consistent with those reported in the literature obtained by solution methods, the K values are almost an order of magnitude larger. A transition in the nature of the interaction between dsDNA and [Co(Phen)3]3+/2+, from electrostatic to intercalative with increasing ionic strength, was found in our studies. Negative values of △E°' for [Co(bpy)3]3+/2+ bound to dsDNA suggest that its interaction with dsDNA is predominantly electrostatic over the ionic strength range of 5-105mM. The "limiting" ratio K3+°/K2+° of 22 obtained for [Co(Phen)3]3+/2+ bound to dsDNA at zero ionic strength suggests that electrostatic interactions are predominant over intercalative ones under these limiting conditions. The ratio for [Co(bpy)3]3+/2+ of 16 also indicates that the 3+ form binds to dsDNA more strongly than the 2+ form at zero ionic strength. For [Co(Phen)3]3+/2+/single-stranded DNA (ssDNA)-modified gold electrodes, the nonuniformity of the surface structure of ssDNA-modified gold electrodes greatly complicates the analysis. A system consisting of a dsDNA-modified gold electrode and [Co(tppz)2]3+/2+ (tppz) tetra-2-pyridyl-1,4-pyrazine) was studied by this method, with a K2+ value of (5(1)×105M-1 and an s value of 7bp being obtained.

Gold electrodes were modified with DNA by adsorption. The DNA-modified electrodes were electrochemically characterized with Co(bpy)3+, a electroactive DNA-binding complex, as an indicator. It is interesting that the pair of redox peaks of Co(bpy)3+ split into two pairs at dsDNA-modified gold electrodes. One pair of peaks shifts negative, and the peak current increases notably; another pair of peaks shifts positive. These suggest that dsDNA has been immobilized onto gold electrode surfaces and the layer of dsDNA on the surfaces can bind Co(bpy)3+ in two different ways. Gold electrodes can be modified also with ssDNA by adsorption but only one pair of peaks of Co(bpy)3+ appears at ssDNA-modified gold electrodes. The amount of Co(bpy)3+ enriched by the layers of dsDNA or ssDNA adsorbed at gold electrodes was estimated from the peak charge of Co(bpy)3+ reduction at the electrodes obtained by CV. The stability of the DNA-modified electrodes was investigated. The DNA modification layer on gold surfaces is unstable to alkali and to heat, but stable to acid solutions and very stable in long stock in a dry. state. A comparison of modifications of gold, platinum and glassy carbon with DNA was carried out.

The modification of glassy carbon and gold electrodes with DNA by adsorption or covalent immobilization in a mono-or submonolayer has been investigated using the couple Co(bpy)3+/2+ as an indicator. It has been found that when the solution containing double stranded or single stranded DNA is evaporated to dryness, dehydrated DNA molecules can be irreversibly adsorbed on the surfaces of glassy carbon electrodes, in an amount close to that of the saturated adsorptive monolayer. The DNA-adsorbed layer on glassy carbon electrodes is unstable to bases, but stable to 1 M HCl solution. The adsorption of DNA on the electrodes can be evaluated from the increase in the peak current, the decrease in the value of △Ep, and the negative shift in the value of E for the Co(bpy)3+/2+ couple. DNA is very strongly adsorbed on the oxidized surfaces of glassy carbon electrodes, and the adsorptive layer is very stable towards heating. The covalent immobilization of DNA directly onto the electrode surfaces is impossible due to considerable steric hindrance; but if the active groups (sites) on the electrode surfaces are elongated with other suitable molecules, the covalent immobilization of DNA becomes possible on the electrode surfaces. The quantity of covalently immobilized DNA at the electrodes reported in the paper is about 31% of the saturated monolayer.