Micro-flow in vivo analysis of L-glutamate with an on-line enzyme amplifier based on substrate recycling.
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 1.4.3.11)/glutamate dehydrogenase (EC 1.4.1.4) 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 mumol 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.
Yao
T
T
Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuencho, Sakai, Osaka 599-8531, Japan.
Nanjyo
Y
Y
Nishino
H
H
D000818:Animals; D019149:Bioreactors; D015374:Biosensing Techniques / Q000379:methods*; D001923:Brain Chemistry; D004800:Enzymes, Immobilized; D005969:Glutamate Dehydrogenase; D018698:Glutamic Acid / Q000032:analysis*; D008991:Monitoring, Physiologic / Q000379:methods; D009862:Online Systems; D010088:Oxidoreductases; D051381:Rats
D004800:Enzymes, Immobilized; D018698:Glutamic Acid; D010088:Oxidoreductases; D005969:Glutamate Dehydrogenase