Analytical Sciences

Abstract − Analytical Sciences, 17(6), 703 (2001).

Micro-flow in vivo Analysis of L-Glutamate with an On-line Enzyme Amplifier Based on Substrate Recycling
Toshio YAO,*  Youko NANJYO*, and Hirohito NISHINO**
*Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuencho, Sakai, Osaka 599-8531, Japan
**Eicom Ltd., 24-2 Shimotoba-Enmendacho, Fushimi, Kyoto 612-8474, Japan
A micro-flow enzyme system with a microdialysis probe is proposed for the amperometric detection of trace amounts of neurotransmitter L-glutamate released from rat brain cells. The L-glutamate oxidase (EC dehydrogenase (EC coimmobilized reactor was used to enhance the sensitivity of L-glutamate as an on-line amplifier based on substrate recycling. A poly(1,2-diaminobenzene) film-coated platinum electrode was also used to selectively detect only the hydrogen peroxide generated into a upstream enzyme reactor, without interference from oxidizable species, such as L-ascorbate, the adsorption of low molecular-weight proteins in a dialysate, and NADPH added to the carrier solution to initiate substrate recycling. By the present in vivo system, L-glutamate was selectively assayed with about a 600-fold increase in sensitivity compared with the unamplified responses. The detection limit was 0.08 umol dm-3. This method was applied to an in vivo assay of L-glutamate in the extracellular space of rat brain; also, monitoring of the L-glutamate level changed after a continuous stimulation of KCl to demonstrate the reliability of the system.