In this report we have used gold nanoparticles (AuNPs) to covalently attach an antibody (Ab(1)) using a spacer arm. The AuNPs/Ab(1) modified gold electrode was used for a sandwich electrochemical immunoassay. The detection was done using cyclic voltammetry and impedance measurements using Horse Radish Peroxidase (HRP) as enzyme label on secondary antibody (Ab(2)) and 3,3', 5,5'-tertramethyl benzidine (TMB) as an electroactive dye. The cyclic voltammetric experiments showed three clear peaks at potentials 154 mV, -33 mV and -156 mV. There was an increase in the both anodic and cathodic current values for the peak at potential -33 mV, when H2O2 was added and the other peaks observed at potential 154 mV and -156 mV resulted due to the oxidation and reduction of TMB. The detection limit of this electrode was 2 ng/mL or 10 pg/5 microL of the analyte. The electrochemical impedance spectroscopy studies demonstrate that the formation of antigen-antibody complexes increases the series resistance and thus confirms the assembly on the electrode. This study showed that AuNPs was efficient in preserving the activity and orientation of the antibody and it can form a major platform in many clinical immunoassays.
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