Activation and Diffusion in the Kinetics of Adsorption and Molecular Recognition on Surfaces. Enzyme-Amplified Electrochemical Approach to Biorecognition Dynamics Illustrated by the Binding of Antibodies to Immobilized Antigens

An electrochemical method is proposed for investigating the dynamics of recognition between a biomolecule and an immobilized receptor. It involves redox labeling of the solute molecule and monitoring the binding by the electrochemical response of the electrode onto which the receptor is immobilized. With large biomolecules, as, for example, antigens and antibodies, leading to small surface concentrations, simple redox labeling may prove insufficient to obtain detectable responses. Redox enzymes are then advantageously used as labels thanks to the signal amplification offered by their catalytic properties. The applicability of the method is illustrated by the reaction of an immobilized monolayer of goat IgG antigen (or of one Fab fragment) with an antigoat antibody labeled with glucose oxidase. Particular care is taken to free the kinetic data from the effect of diffusion. The latter factor may interfere whatever the detection technique. A full account of the combination between recognition kinetics and diffusion is therefore given in terms of a kinetic zone diagram leading to diagnostic criteria and data processing procedures that allow a proper extraction of the recognition thermodynamic and kinetic constants. The theory applies as well to the dynamics of adsorption of nonbiological molecules on surfaces.