Determination of surface-immobilized double-stranded DNA using a metallointercalator and catalytic voltammetry

DNA films immobilized on an indium tin oxide (ITO) electrode surface were detected and determined employing a high-affinity intercalator, Ru(bpy)(2)(dppz)(2+) (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine), as a redox indicator, and oxalate as a sacrificial electron donor in solution to chemically amplify the voltammetric signal of the indicator. Nucleic acids were immobilized on ITO by layer-by-layer electrostatic adsorption, using avidin as the first layer and nucleic acid as the second layer. In quartz crystal microbalance (QCM) measurements on an avidin-coated gold surface, the amount of adsorption from a 200 mu g L-1 nucleic acid solution was found to be 3.2 ng mm(-2) for both double-stranded (ds-) and single-stranded (ss-) calf-thymus DNA as well as polycytidylic acid (Poly-C). After binding with Ru(bpy)(2)(dppz)(2+) (Ru-dppz), voltammetry of the ds-DNA film on ITO was carried out in an indicator-free phosphate buffer. An anodic peak at about 1.15V was observed, and it was assigned to Ru-dppz oxidation. When measured in an oxalate buffer, however, a catalytic current was observed due to the oxidation of oxalate by electrochemically generated Ru(bpy)(2)(dppz)(3+), resulting in a 120-fold increase in the signal. Since oxalate itself produces a very low oxidation current on ITO, catalytic voltammetry produces about a 14-fold improvement in the signal-to-blank ratio over the non-amplified determination. As a result, ds-DNA adsorbed from 20ng mL(-1) solution could be detected, which was estimated by QCM to be 160 pg mm(-2) on the surface. The catalytic current of ds-DNA was substantially higher than that of ss-DNA and poly-C, indicative of selective binding of the redox indicator to ds-DNA. The results serve as a basis for the catalytic voltammetric detection of DNA hybridization in future work.