A chemiluminometric flow-through sensor for simultaneous determination of L-glutamate (Glu) and L-lysine (Lys) in a single sample has been developed. Immobilized uricase, immobilized peroxidase, support material, coimmobilized glutamate oxidase/peroxidase, support material, and coimmobilized lysine oxidase/peroxidase were packed sequentially in a transparent PTFE tube, and the tube was placed in front of a photomultiplier tube as a flow cell. A three-peak recording was obtained by one injection of the sample solution. The peak height of the first peak was due to the concentrations of urate and other reductants in the sample; the immobilized uricase was used to decompose urate, and the hydrogen peroxide produced was decomposed with a luminol-hydrogen peroxide reaction by immobilized peroxidase. The peak heights of the second and third peaks were free from the interferences from the reductants and were dependent only on the concentrations of Glu and Lys, respectively. Calibration graphs for Glu and Lys were linear at 40-1,000 and 50-1,200 nM, respectively. The sampling rate was 11/h without carryover. The sensor was stable for two weeks. The sensor system was applied to the simultaneous determination of Glu and Lys in serum.

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The concentration of uric acid in biological fluids is an important indicator in the diagnosis and monitoring of gout and hyperuricemia. For a rapid and reliable assay of uric acid in human fluids and serum by FIA, immobilized uricase (EC 1.7.3.3, urate: oxygen oxidoreductase, UC), which is highly specific for uric acid, has been used as packed-bed reactors [1][2][3][4][5][6] or membrane. 7 These methods are based on the conversion of uric acid and oxygen to allantoin and hydrogen peroxide with UC [uric acid + O2 + 2H2O → allantoin + CO2 + H2O2], and the following chemiluminometric (CL) detections [1][2][3] of the hydrogen peroxide or amperometric detections 4-7 of the oxygen decrease.A closed-flow system with amperometric detection has been developed for the determination of uric acid in serum. 4 The method is simple, but is not highly sensitive; a relatively large amount of serum sample (>80 µl) without dilution was directly injected to the system. In the CL methods, the addition of a catalyst, such as ferricyanide ion for luminol systems 1,2 or organic solvents for peroxyoxalate system, 3 precludes the direct coupling of light-emitting reactions to the UC reactor; they are detrimental to immobilized UC.We have developed a CL hydrogen peroxide sensor on the basis of a luminol chemiluminescence reaction with immobilized peroxidase (POD) as a catalyst [2H2O2 + luminol + 2OH -→ 3-aminophthalate + N2 + 3H2O + light], which was packed into a flow cell. 8 The maximum efficiency for the luminol reaction was obtained at around pH 10. Since, fortunately, the pH optimum of the UC reaction is pH 9 -10, 9,10 it could be directly coupled to the luminol reaction.This paper describes a closed-loop single line FIA system for the CL determination of uric acid using a coimmobilized UC and POD contained in a flow cell. UC and POD were coimmobilized covalently to polymer beads and packed in a transparent PTFE tubing. The PTFE tubing was used as a CL flow cell. Enzymatic reactions were performed in a 100 µM luminol in carbonate buffer (pH 9.0) in the flow cell. It is preferable to minimize the consumption of reagent solutions so as to reduce the extent of damage to the environment. The single manifold and the use of a highly diluted sample solution permitted the circulation of the used luminol solution. The method was applied to the determination of uric acid in plasma. Experimental MaterialsUricase (from Arthrobacter globiformis, 30 U mg -1 ) and POD (EC 1.11.1.7, from Arthromyces ramosus, 250 U mg -1 ) were obtained from Asahi Kasei (Tokyo, Japan) and Suntory (Tokyo, Japan), respectively. Hydrophilic vinyl polymer beads (TSKgel Toyopearl HW-65F, particle size 50 -60 µm) was purchased from Tosoh (Tokyo, Japan). All other reagents were of analytical-reagent grade.A stock solution (1 mM) of luminol (3-aminophthalhydrazide) was prepared by dissolving in 0.1 M carbonate buffer (pH 9.0) and stored for more than three days in a refrigerator to attain stability before use. 1,11 The solution was diluted ten-fold with 0.1 M carbonate bu...