BIOSENSOR FOR DIRECT DETERMINATION OF ORGANOPHOSPHATE NERVE AGENTS USING RECOMBINANT ESCHERICHIA-COLI WITH SURFACE-EXPRESSED ORGANOPHOSPHORUS HYDROLASE - 1 - POTENTIOMETRIC MICROBIAL ELECTRODE

A potentiometric microbial biosensor for the direct measurement of org anophosphate (OP) nerve agents was developed by modifying a pH electro de with an immobilized layer of Escherichia coli cells expressing orga nophosphorus hydrolase (OPH) on the cell surface, OPH catalyzes the hy drolysis of organophosporus pesticides to release protons, the concent ration of which is proportional to the amount of hydrolyzed substrate. The sensor signal and response time were optimized with respect to th e buffer pH, ionic concentration of buffer, temperature, and weight of cells immobilized using paraoxon as substrate. The best sensitivity a nd response time were obtained using a sensor constructed with 2.5 mg of cells and operating in pH 8.5, 1 mM HEPES buffer. Using these condi tions, the biosensor was used to measure as low as 2 mu M of paraoxon, methyl parathion, and diazinon, The biosensor had very good storage a nd multiple use stability, The use of cells with the metabolic enzyme expressed on cell surface as biological transducer provides advantages of no resistances to mass transport of the analyte and product across the cell membrane and low cast due to elimination of enzyme purificat ion, over the conventional microbial biosensors based on cells express ing enzyme intracellularly and enzyme-based sensors, respectively.