Application of Redox Enzymes for Probing the Antigen−Antibody Association at Monolayer Interfaces: Development of Amperometric Immunosensor Electrodes

Abstract

Insulation of the electrical contact between a redox protein and an electrode surface upon association of an antibody to an antigen monolayer assembled on the electrode is used to develop immunosensor devices. In one configuration, a mixed monolayer consisting of the N epsilon-(2,4-dinitrophenyl)lysine antigen and ferrocene units acting as electron transfer mediators is applied to sense the dinitrophenyl antibody (DNP-Ab) in the presence of glucose oxidase (GOx) and glucose. In the absence of DNP-Ab, the mixed monolayer electrode stimulates the mediated electrocatalyzed oxidation of glucose that results in an amplified amperometric response. Association of the DNP-Ab to the modified electrode blocks the electrocatalytic transformation. The extent of the electrode insulation by the DNP-Ab is controlled by the Ab concentration in the sample. In the second configuration, a N epsilon-(2,4-dinitrophenyl)lysine antigen monolayer assembled on a Au electrode is applied to sense the DNP-Ab in the presence of a redox-modified GOx, exhibiting electrical communication with the electrode surface. Two kinds of redox-modified "electrically wired" GOx are applied: GOx modified by N-(ferrocenylmethyl)caproic acid, Fc-GOx, and a novel electrobiocatalyst generated by reconstitution of apo-GOx with a ferrocene-modified FAD semisynthetic cofactor. Electrocatalytic oxidation of glucose by the electrically wired biocatalysts proceeds in the presence of the antigen monolayer electrode, giving rise to an amplified amperometric signal. The electrocatalytic transformation is blocked upon association of the DNP-Ab to the monolayer electrode. The extent of electrode insulation toward the bioelectrocatalytic oxidation of glucose is controlled by the DNP-Ab concentrations in the samples. The application of biocatalysts for amperometric sensing of antigen-antibody interactions at the electrode surface makes the electrode insensitive to microscopic pinhole defects in the monolayer assembly. The antigen monolayer electrode is applied to sense the DNP-Ab in the concentration range 1-50 micrograms mL-1.