An amperometric biosensor for real-time analysis of molecular recognition

An amperometric, enzyme-channeling immunosensor was used to study the qualitative and quantitative aspects of molecular biorecognition in real time. The immunosensor consisted of a polyethylenimine-modified carbon electrode on which glucose oxidase was coimmobilized with a specific antibody or with calmodulin. The immunological reactions were monitored electrochemically in situ, and the binding curves were directly visualized on a computer screen. This approach was applied for estimating the kinetic constants of the reaction between IgG and its specific anti-IgG antibodies, as well as for mapping of sites and the interaction of calmodulin and its target peptides and proteins via its monoclonal antibodies. The respective association and dissociation rate constants for IgG/antiIgG were calculated as 1.3 x 10(6) M-1 s(-1) and 0.2 x 10(-3) s(-1). The site-mapping of phosphodiesterase, melittin, calcineurin, or mastoparan on the calmodulin molecule were estimated using two conformation-dependent mAbs, CAM1 and CAM4, whose epitopes were previously located. The data presented confirm that the amperometric, enzyme-channeling immunosensor may be used as a tool for valuable characterizing of biospecific interactions, such as determining the concentration of molecules, selecting different antibodies, and characterizing the binding sites of biomolecules. (C) 1998 Elsevier Science S.A.