The development and experimental verification of a theoretical model for immobilized enzyme inhibition biosensors is reported. The model predicts that the percentage of inhibited enzyme (I %), after exposure to an inhibitor, is linearly related to both the inhibitor concentration ([I] B ) and the square root of incubation time (t 1=2 ):
The model is based on diffusion limited inhibitor transport and takes into account the heterogeneous nature of enzyme inhibition that results from immobilization of an enzyme at the surface of a transducer. Experimental verification of the model was achieved using an electrochemical (amperometric) acetylcholine sensor. The immobilized enzyme used in this study was acetylcholinesterase, while the organophosphorus pesticide, paraoxon, was employed as the inhibitor. A second enzyme, choline oxidase, was co-immobilized to facilitate electrochemical detection of the substrate. To satisfy the conditions required by the proposed model a specially designed biosensing device was fabricated employing a microdisk array as the transducer. Acetylcholinesterase (0.00025 units) and choline (1.0 units) were coimmobilized onto the array using a water-based polyurethane=polyethylene oxide colloidal dispersion matrix. A dialysis membrane was used as an active barrier to prevent fouling of the array and to restrict enzyme leaching. The device was used for both batch and flow injection analysis and experimental results verifying the proposed model are presented using both modes.