Chemiluminometric Determination of Uric Acid in Plasma by Closed-loop FIA with a Coimmobilized Enzyme Flow Cell

Abstract

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...

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“…The sensors have been applied to the assay of a few real samples, , because the luminol−H 2 O 2 reaction is seriously subjected to interference from reducing substances, which depress results by interacting with the H 2 O 2 and the intermediates of the luminol reaction. − …”

“…8 A uricase/POx coimmobilized on the polymer beads in a flow cell was used to develop a sensor for uric acid in a closedloop FI system. 9 Lower detection limits for the sensors were 10 -8 M levels.…”