Analytical Sciences

Flow-through Chemiluminescence Sensor Using Immobilized Oxidases for the Selective Determination of L-Glutamate in a Flow-Injection System

Nobutoshi KIBA,  Seiji ITO, Masaki TACHIBANA, Kazue TANI, and Hitoshi KOIZUMI

Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Yamanashi University, Kofu 400-8511, Japan

A selective and sensitive chemiluminometric flow sensor for the determination of L-glutamate in serum, based on immobilized oxidases such as glutamate oxidase (GOD), uricase (UC) and peroxidase (POD), is described herein. The principle for the selective chemiluminometric detection for L-glutamate is based on coupled reactions of four sequentially aligned immobilized oxidases, UC/POD/GOD/POD in a flow cell. The immobilized UC was employed to decompose urate, which is one of the major interfering components in serum for a luminol-H₂O₂ chemiluminescence reaction. The H₂O₂ produced from the UC reaction readily reacted with reducing components, such as ascorbate and glutathione, and then the excess H₂O₂ was decomposed by the immobilized POD. L-Glutamate in the sample plug was enzymatically converted to H₂O₂ with immobilized GOD. Subsequently, the peroxide reacts with luminol on the immobilized POD to produce chemiluminescenece, proportional to glutamate concentration. The enzymes were immobilized on tresylated poly(vinyl alcohol beads). The immobilized enzymes were packed into a TPFE tube (1.0 mm i.d. x 60 cm), in turn, and used as a flow cell. The sampling rate was 30 h^-1. The calibration graph for L-glutamate is linear for 20 nM - 5 uM; the detection limit (signal-to-noise = 3) is 10 nM.

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