Oxidation of guanine residues in nucleic acids using a metal mediator, tris(bipyridyl) ruthenium(II) (Ru(bpy)(3)(2+)), provides a sensitive and simple method for detection of unmodified nucleic acids. This method can be implemented in a hybridization-based assay, in which nucleic acid probes are immobilized at the electrode surface to capture the nucleic acid target of interest. In this paper, the effect of the tin-doped indium. oxide (ITO) surface preparation on the immobilization of nucleic acid probes and electron-transfer kinetics of Ru(bpy)(3)(2+) was examined. ITO preparation methods have a profound effect on the amount of material immobilized on the electrode surface via either phosphonate or silane linkage. Common cleaning procedures that utilize phosphate-containing detergents cannot be employed in this system because of the strong adsorption of phosphate to the ITO surface, which was shown to reduce the amount of nucleic acid bound to the ITO surface. It was also found that a negative charge at the electrode surface significantly enhances the electron-transfer kinetics between Ru(bPY)(3)(2+) and the ITO electrode. Phosphate ions adsorbed at the ITO surface were found to be the most effective way to enhance the electron-transfer kinetics of Ru(bpy)(3)(2+). As expected for a charge-related phenomenon, phosphate ions adsorbed at the ITO surface had the opposite effect on the negatively charged redox probe, Fe(CN)(6)(4-). The presence of adventitious carbon on the electrode surface was shown to adversely impact solid-phase immobilization by decreasing the stability of both phosphonate and silane immobilized materials exposed to hybridization conditions. UV treatment of ITO surfaces prior to solid-phase immobilization results in the removal of adventitious carbon yielding a more stable solid phase.