HIGHLY SENSITIVE DETECTION OF L-GLUTAMATE BY ONLINE AMPEROMETRIC MICRO-FLOW ANALYSIS BASED ON ENZYMATIC SUBSTRATE RECYCLING

A highly selective and sensitive on-line monitoring system is proposed for amperometric assay of trace amounts of L-glutamate. The system in cludes a microdialysis probe, immobilized enzyme reactor, and poly(1,2 -diaminobenzene)coated platinum electrode. The enzyme reactor prepared by the co-immobilization of L-glutamate oxidase and glutamate dehydro genase are here employed to enhance the sensitivity of L-glutamate as an on-line amplifier based on the substrate recycling. The L-glutamate in the dialysate from the probe are recycled enzymatically during pas sage through the reactor in the presence of sufficient amounts of NADH and oxygen to produce a large amount of hydrogen peroxide, which is d etected if selectively at a downstream poly(1,2-diaminobenzene)-coated platinum electrode without interference from oxidizable species such as L-ascorbate in the sample and NADH added to the carrier buffer. The cycle is also initiated with 2-oxoglutarate, and so saccharopine dehy drogenase reactor is positioned in series before the amplifier reactor to remove 2-oxoglutarate in the dialysate. By the present method, L-g lutamate is selectively assayed with a 160-fold increase in sensitivit y compared with the unamplified responses. The detection limit is 0.5 x 10(-7) M of L-glutamate. (C) 1998 Elsevier Science B.V.