Enhanced electrochemical activity of redox-labels in multi-layered protein films on indium tin oxide nanoparticle-based electrode

Facile electrical communication between redox-active labeling molecules and electrode is essential in the electrochemical detection of bio-affinity reactions. In this report, nanometer-sized indium tin oxide (ITO) particles were employed in the fabrication of porous thick film electrodes to enhance the otherwise impeded electrochemical activity of redox labels in multi-layered protein films, and to enable quantitative detection of avidin/biotin binding interaction. To carry out the affinity reaction, avidin immobilized on an ITO electrode was reacted with mouse IgG labeled with both biotin and ruthenium Tris-(2,2′-bipyridine) (Ru–bipy). The binding reaction between avidin and biotin was detected by the catalytic voltammetry of Ru–bipy in an oxalate-containing electrolyte. On sputtered ITO thin film electrode, although a single layer of Ru–bipy labeled avidin exhibited substantial anodic current, attaching the label to the outer IgG layer of the avidin/biotin–IgG binding pair resulted in almost complete loss of the signal. However, electrochemical current was recovered on ITO film electrodes prepared from nanometer-sized particles. The surface of the nanoparticle structured electrode was found by scanning electron microscopy to be very porous, and had twice as much surface binding capacity for avidin as the sputtered electrode. The results were rationalized by the assumption of different packing density of avidin inner layer on the two surfaces, and consequently different electron transfer distance between the electrode and Ru–bipy on the IgG outer layer. A linear relationship between electrochemical current and IgG concentration was obtained in the range of 40–4000 nmol L−1 on the nanoparticle-based electrode. The approach can be employed in the electrochemical detection of immunoassays using non-enzymatic redox labels.