Hirohito Nishino

High-performance liquid chromatography followed by peroxyoxalate chemiluminescence detection of acetylcholine and choline utilizing immobilized enzymes

A sensitive and selective method for the simultaneous determination of acetylcholine (ACh) and choline (Ch) is reported. ACh and Ch were separated on a reversed-phase column, passed through an immobilized enzymes (acetylcholine esterase and choline oxidase) column, and converted to hydrogen peroxide. The generated hydrogen peroxide was detected by the peroxyoxalate chemiluminescence reaction. The linear determination ranges were from 10 pmol to 10 nmol. The detection limit for both cholines was 1 pmol.

Highly sensitive detection of l-glutamate by on-line 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 includes 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 dehydrogenase 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 passage through the reactor in the presence of sufficient amounts of NADH and oxygen to produce a large amount of hydrogen peroxide, which is detected 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 dehydrogenase reactor is positioned in series before the amplifier reactor to remove 2-oxoglutarate in the dialysate. By the present method, l-glutamate is selectively assayed with a 160-fold increase in sensitivity compared with the unamplified responses. The detection limit is 0.5x10(-7) M of l-glutamate.