Direct electron transfer based tri-enzyme electrode for monitoring of organophosphorus pesticides

A potentiometric biosensor has been developed based on the ability of organophosphorus pesticides to inhibit the catalytic activity of the enzyme choline esterase. The detection of choline esterase activity is based on the principle of molecular transduction. Immobilized peroxidase acting as the molecular transducer, catalyzes the electroreduction of hydrogen peroxide by direct (mediatorless) electron transfer. The sensing element consists of a carbon based electrode covered by a layer of three co-immobilized enzymes: choline esterase, choline oxidase and peroxidase. A butyryl choline sensitive tri-enzyme electrode has been developed employing highly dispersed teflonized carbon black as an electrode material. The immobilization procedure is based on physical adsorption of peroxidase and co-immobilization of choline oxidase and choline esterase using glutaraldehyde as a binding agent. The electrode retains 95% of its initial activity after 1 month of storage at 4 degrees. The parameters of the inhibitor assay procedure have been optimized. The procedure for measuring the electrode activity requires 3-5 min. Incubation of the electrode in a solution containing the organophosphorus pesticide, trichlorfon, for 10 min results in a notable decrease of electrode activity. This allows for the determination of trichlorfon in a nanomolar concentration range with a low detection limit of 5 nM (1.3 ppb).