Biosensor for direct determination of organophosphate nerve agents using recombinant Escherichia coli with surface-expressed organophosphorus hydrolase. 2. Fiber-optic microbial biosensor.

A fiber-optic microbial biosensor suitable for direct measurement of organophosphate nerve agents was developed. The unique features of this novel microbial biosensor were the recombinant Escherichia coli cells expressing the enzyme organophosphorus hydrolase on the cell surface and the optical detection of the products of enzyme-catalyzed organophosphate hydrolysis. The use of cells with the metabolic enzyme expressed on the cell surface as a biological sensing element provides advantages of no resistance 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. The use of an optical transducer allows the detection of different organophosphates in a mixture, presently not feasible with acetylcholinesterase-based biosensors. E. coli cells expressing organophosphorus hydrolase (OPH) on the cell surface were immobilized in low melting temperature agarose on a nylon membrane and attached to the common end of a bifurcated fiber-optic bundle. OPH-expressing E. coli cells catalyzed the hydrolysis of organophosphorus pesticides to form stoichiometric amounts of chromophoric products that absorb light at specific wavelengths. The backscattered radiation of the specific wavelength incident light was measured using a photomultiplier detector and correlated to the organophosphate concentration. The best sensitivity and response time were obtained using a sensor constructed with 1.5 mg of cells operating in pH 9, 50 mM HEPES buffer with 100 mM NaCl and 0.05 mM CoCl2 at 30 degrees C. At optimized conditions, the biosensor measured paraoxon, parathion, and coumaphos pesticides with high selectivity against triazine and carbamate pesticides in approximately 10 min. The lower detection limits were 3 microM for paraoxon and parathion and 5 microM for coumaphos. When stored in the buffer at 22 degrees C, the biosensor was stable for over a 1-month period and showed no decline in the response for over 75 repeated usages. The new fiber-optic microbial biosensor is an ideal tool for on-line monitoring of the detoxification process for organophosphate pesticides-contaminated wastewaters but may not be suitable for environmental monitoring.