An electrochemical in vivo flow-injection system for highly selective and sensitive detection of L-glutamate using enzyme reactor involving amplification

A bioelectrochemical in vivo flow-injection system with an on-line microdialysis sampling is proposed for the assay of the trace amounts of L-glutamate released from rat brain cells. The system includes a microdialysis probe, two immobilized enzyme reactors, a six-way auto-injector, and a poly(1,2-diaminobenzene) film-coated platinum electrode. In the first stage of the operation, the dial sate from the probe was delivered to the sample loop of the six-way autoinjector by perfusing Ringer's solution for 3 min at 2 muL min(-1). The calibration of the detection system including enzyme reactors was performed simultaneously. In the second stage, the dialysate in sample loop was automatically injected for 30s into the flow-injection line and assayed. An L-glutamate oxidase/glutamate dehydrogenase coimmobilized reactor was used as an on-line amplifier based on the substrate recycling. To improve the substrate specificity of this reactor, acidic pretreatment and 6-diazo-5-oxo-L-norleucine treatment were done. The resulting enzyme reactor worked as a specific reactor of L-glutamate involving amplification (amplification factor was about 60 at a carrier flow-rate of 100 muL min(-1)). The cycle was also initiated with 2-oxoglutarate, and so saccharopine dehydrogenase reactor was positioned in series before the amplifier reactor to remove 2-oxoglutarate. By the present method, the variations of L-glutamate level released from rat brain cells were assayed in vivo and also after continuous stimulation of KCI, to demonstrate the reliability of the system. Analytical speed was 20 dialysates h(-1) and the detection limit was less than 0.5 muM.