Gold nanoparticles based sandwich electrochemical immunosensor.

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.

Direct electrochemistry of horseradish peroxidase-gold nanoparticles conjugate.

We have studied the direct electrochemistry of horseradish peroxidase (HRP) coupled to gold nanoparticles (AuNP) using electrochemical techniques, which provide some insight in the application of biosensors as tools for diagnostics because HRP is widely used in clinical diagnostics kits. AuNP capped with (i) glutathione and (ii) lipoic acid was covalently linked to HRP. The immobilized HRP/AuNP conjugate showed characteristic redox peaks at a gold electrode. It displayed good electrocatalytic response to the reduction of H(2)O(2), with good sensitivity and without any electron mediator. The covalent linking of HRP and AuNP did not affect the activity of the enzyme significantly. The response of the electrode towards the different concentrations of H(2)O(2) showed the characteristics of Michaelis Menten enzyme kinetics with an optimum pH between 7.0 to 8.0. The preparation of the sensor involves single layer of enzyme, which can be carried out efficiently and is also highly reproducible when compared to other systems involving the layer-by-layer assembly, adsorption or encapsulation of the enzyme. The immobilized AuNP-HRP can be used for immunosensor applications.