Flow-through chemiluminescence sensor using immobilized oxidases for the selective determination of L-glutamate in a flow-injection system

A selective and sensitive chemiluminometric flow sensor for the determinati on 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 oxidas es, UC/POD/GOD/POD in a flow cell. The immobilized UC was employed to decom pose urate, which is one of the major interfering components in serum for a luminol-H2O2 chemiluminescence reaction. The H2O2 produced from the UC rea ction readily reacted with reducing components, such as ascorbate and gluta thione, and then the excess H2O2 was decomposed by the immobilized POD. L-G lutamate in the sample plug was enzymatically converted to H2O2 with immobi lized GOD. Subsequently, the peroxide reacts with luminol on the immobilize d POD to produce chemiluminescenece, proportional to glutamate concentratio n. The enzymes were immobilized on tresylated poly(vinyl alcohol beads). Th e immobilized enzymes were packed into a TPFE tube (1.0 mm i.d. x 60 cm), i n turn, and used as a flow cell. The sampling rate was 30 h(-1). The calibr ation graph for L-glutamate is linear for 20 nM - 5 muM; the detection limi t (signal-to-noise = 3) is 10 nM.