Towards immunoassay in whole blood: separationless sandwich-type electrochemical immunoassay based on in-situ generation of the substrate of the labeling enzyme

An 18 minute separationless amperometric ELISA-type sandwich immunoassay, utilizing only stable reagents and having no washing steps is described. The platform for the assay was an electron conducting redox hydrogel on a vitreous carbon electrode. Avidin and choline oxidase were co-immobilized on the redox hydrogel and the biotinylated antibody to the antigen to be assayed (the biotin-labeled F(ab')(2) fragment of goat anti-rabbit IgG) was bound to the gel. When the antigen (goat anti-rabbit IgG) was present in the analyzed solution, then its binding to the immobilized antibody made the electrode receptive to the complementary peroxidase-labeled antibody (horseradish peroxidase-labeled F(ab')(2) fragment of goat anti-rabbit IgG). Its binding resulted in electrical contact ("wiring") of the horseradish peroxidase label to the redox hydrogel, and converted the non-catalytic hydrogel into an electrocatalyst for the reduction of hydrogen peroxide to water at -0.07 V (SCE) and resulted in the flow of a cathodic current. The electroreduced hydrogen peroxide was not added to the solution and was therefore not significantly accessible to hydrogen peroxide decomposing agents such as catalase. Instead, it was generated within the coating of the electrode through reacting dissolved choline with oxygen. This reaction was catalyzed by the immobilized choline oxidase. The reaction centers of choline oxidase, unlike those of horseradish peroxidase, are not connected to the electrode by the redox hydrogel.