THE DEVELOPMENT OF A NEW BIOSENSOR BASED ON RECOMBINANT ESCHERICHIA-COLI FOR THE DIRECT-DETECTION OF ORGANOPHOSPHORUS NEUROTOXINS

A new biosensor for the direct detection of organophosphorus (OP) neur otoxins has been developed utilizing cryoimmobilized, recombinant E. c oli cells capable of hydrolyzing a wide spectrum of OP pesticides and chemical warfare agents. The biological transducer was provided by the enzymatic hydrolysis of OP neurotoxins by organophosphate hydrolase w hich generates two protons through a reaction in which P-O, P-F, PS or P-CN bonds are cleaved, and the proton release corresponded with the quantity of organophosphate hydrolyzed. This stoichiometric relationsh ip permitted the creation of a potentiometric biosensor for detection of OP neurotoxins and a pH-based assay was developed as a direct funct ion of the concentration of OP neurotoxins and the immobilized biomass . In these studies utilizing paraoxon as the substrate, neurotoxin con centration was determined with two different types of measuring units containing immobilized cells: (1) a stirred batch reactor; and (2) a f low-through column minireactor. A pH glass electrode was used as the p hysical transducer. The linear detection range for paraoxon spanned a concentration range of 0.25-250 ppm (0.001-1.0 mM). The response times were 10 min for the batch reactors and 20 min for the flow-through sy stems. It was possible to use the same biocatalyst repetitively for 25 analyses with a 10 min intermediate washing of the biocatalyst requir ed for reestablishing the starting conditions. The cryoimmobilized E. coli cells exhibited stable hydrolytic activity for over 2 months unde r storage in 50 mM potassium-phosphate buffer at +4 degrees C and prov ide the potential for the development of a stable biotransducer for de tecting various OP neurotoxins. (C) 1996 Elsevier Science Limited.