Potentiometric immunoelectrode for fast assay based on direct electron transfer catalyzed by peroxidase

A potentiometric immunoelectrode based on the direct electron transfer detection of immunointeraction is described. Enzyme peroxidase is used as a label. The ability of peroxidase to catalyze the electrode reaction of hydrogen peroxide electroreduction by a direct (mediatorless) mechanism is utilized. Peroxidase attached to the electrode surface in the presence of hydrogen peroxide results in a significant (hundreds of mV) increase of potential towards the equilibrium H2O2/H2O potential due to catalytic removal of overvoltage. Electrons are transferred directly from the electrode to the substrate molecules via the active site of the enzyme. Peroxidase labeled immunoconjugate interacting with the surface of a carbon electrode modified by antigen results in the increase of electrode potential. The rate of potential increase is proportional to the concentration of free conjugate in the solution. Free antigen in the reaction media inhibits the process of conjugate binding with immobilized antigen, resulting in a decrease in the rate of change of electrode potential. The percentage of inhibition is proportional to the concentration of free antigen (analyte). Rabbit IgG has been used as a model analyte. The method allows determination of IgG in the concentration range up to 400 mu g/ml. Sensing of the immunointeraction is in a kinetic mode. The direct electron transfer detection of immunointeraction suggests a single stage analysis scheme. An analysis time does not exceed 25 min. Different kinds of carbon materials for electrode construction have been examined.