Biosensor for Direct Determination of Organophosphate Nerve Agents Using RecombinantEscherichia coliwith Surface-Expressed Organophosphorus Hydrolase. 1. Potentiometric Microbial Electrode

A potentiometric microbial biosensor for the direct measurement of organophosphate (OP) nerve agents was developed by modifying a pH electrode with an immobilized layer of Escherichia coli cells expressing organophosphorus hydrolase (OPH) on the cell surface. OPH catalyzes the hydrolysis of organophosporus pesticides to release protons, the concentration of which is proportional to the amount of hydrolyzed substrate. The sensor signal and response time were optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and weight of cells immobilized using paraoxon as substrate. The best sensitivity and 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 conditions, the biosensor was used to measure as low as 2 microM of paraoxon, methyl parathion, and diazinon. The biosensor had very good storage and multiple use stability. The use of cells with the metabolic enzyme expressed on cell surface as a biological transducer provides advantages of no resistances to mass transport of the analyte and product across the cell membrane and low cost due to elimination of enzyme purification, over the conventional microbial biosensors based on cells expressing enzyme intracellularly and enzyme-based sensors, respectively.