A sensitive xanthine oxidase electrode with silk net for estimating fish freshness

Abstract

An enzyme biosensor was constructed using a plate platinum electrode and immobilked xanthine oxidase (XOD). Only a very small quantity of enzyme was chemically immobilized on a special silk net. Hydrogen peroxide released during the enzymatic reaction waa detected by the electrode at +0.65 V (VE. Ag/AgCl). The electrode wbs very sensitive to hypoxanthine and its detection limit was lxlO-' mol/L. W h e n it was applied to the determination of 6sh freshness, the results agreed well with those obtained by traditional methods-determination of total volatile basic nitrogen (TVB-N) and microbial count. A range for estimating the freshness of river 6sh waa suggested. KeywordsXanthine oxidase electrode, biosensor, amperometry, fish freshness Biosensor technology is entering a very active ~h a s e . l -~ In resent years, it was a p plied widely in the growing biotechnology and food processing areas, in the clinical laboratories, and in pollution control. It is very important to estimate the freshness of fish and meat for food industry. Adenqsine triphosphate (ATP) in the fish tissue begins to degrade after the death of a fish, ATP is decomposed quickly to adenosine diphosphate (ADP), adenosine monophosphate (AMP), and inosine monophosphate (IMP). IMP is an important factor of the pleasant flavor of fresh fish. Under the existence of enzymes, IMP is further decomposed to inosine (-), hypoxanthine (Hx), xanthine (X), and uric acid (UA). In general, enzymatic decomposition of inosine or hypoxanthine is the rate-determining step, depending on the kind of fish. Therefore, hypoxanthine is accumulated in fish tissue and its amount can be used to indicate the fish In order to determine hypoxanthine, some complicated methods, such as paper chromatography,8 anion-exchange chr~matography,~ HPLClO and precipitation" have been employed. Recently, some enzyme biosensors have been designed for this purpose. The method was relatively simple and the determination was rapid. In these designs, a basic solid electrode detected consumed oxygen or generated hydrogen peroxide amperometricdy during the following enzymatic reactions: Hypoxanthine + 1 / 2 0 2 + H20 xzD Xanthine + H202 XOD Xanthine + 1/202 + HzO Uric acid + H202