APPLICATION OF REDOX ENZYMES FOR PROBING THE ANTIGEN-ANTIBODY ASSOCIATION AT MONOLAYER INTERFACES - DEVELOPMENT OF AMPEROMETRIC IMMUNOSENSOR ELECTRODES

Insulation of the electrical contact between a redox protein and an el ectrode surface upon association of an antibody to an antigen monolaye r 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 electro n transfer mediators is applied to sense the dinitrophenyl antibody (D NP-Ab) in the presence of glucose oxidase (GOx) and glucose, In the ab sence of DNP-Ab, the mixed monolayer electrode stimulates the mediated electrocatalyzed oxidation of glucose that results in an amplified am perometric response. Association of the DNP-Ab to the modified electro de blocks the electrocatalytic transformation, The extent of the elect rode insulation by the DNP-Ab is controlled by the Ab concentration in the sample. In the second configuration, a N epsilon-(2,4-dinitrophen yl)lysine antigen monolayer assembled on a Au electrode is applied to sense the DNP-Ab in the presence of a redox-modified GOx, exhibiting e lectrical communication with the electrode surface, Two kinds of redox -modified ''electrically wired'' GOx are applied: GOx modified by N-(f errocenylmethyl)caproic acid, Fc-GOx, and a novel electrobiocatalyst g enerated by reconstitution of apo-GOx with a ferrocene-modified FAD se misynthetic cofactor, Electrocatalytic oxidation of glucose by the ele ctrically wired biocatalysts proceeds in the presence of the antigen m onolayer electrode, giving rise to an amplified amperometric signal. T he 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 t he DNP-Ab concentrations in the samples. The application of biocatalys ts for amperometric sensing of antigen-antibody interactions at the el ectrode 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 mu g mL(- 1).