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 use d as a label. The ability of peroxidase to catalyze the electrode reac tion 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 potentia l due to catalytic removal of overvoltage. Electrons are transferred d irectly from the electrode to the substrate molecules via the active s ite 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 pro portional to the concentration of free conjugate in the solution. Free antigen in the reaction media inhibits the process of conjugate bindi ng with immobilized antigen, resulting in a decrease in the rate of ch ange of electrode potential. The percentage of inhibition is proportio nal to the concentration of free antigen (analyte). Rabbit IgG has bee n used as a model analyte. The method allows determination of IgG in t he concentration range up to 400 mu g/ml. Sensing of the immunointerac tion is in a kinetic mode. The direct electron transfer detection of i mmunointeraction suggests a single stage analysis scheme. An analysis time does not exceed 25 min. Different kinds of carbon materials for e lectrode construction have been examined.