Development of a new biosensor system for the determination of the hypoxanthine ratio, an indicator of fish freshenss

Abstract

A biosensor system has been develoed to measure the hypoxanthine concentration ration [Hx]/[Hx + INO + IMP], an indicator for fish freshness. The biosensory system consisted of a detection chamber equipped with an amperometric electrode (platinum anode vs silver/silver chloride cathode polarized at + 0.7 volt). After being cross-linked with bovine serum albumin and then deposited on a preactivated nylon membrane, immobilized xanthine oxidase was attached to the sensing area of the electrode and used for determination of hypoxanthine (Hx). The amperometric probe detected both hydrogen peroxide and uric acid released from the biooxidation of hypoxanthine. Nucleotidase immobilized on the all of a polystyrene tube precoated with polyethylenimine was used for conversion of inosine-5′-monophosphate (IMP) to inosine (INO). After injection of soluble nucleoside phosphorylase (NP) and phosphate to the detection chamber, the resulting INO was introduced for determination of [Hx + INO + IMP]. For repeated analyses, guanosine was added during the course of hypoxanthine measurement to prevent contaminating reactions from residual NP as well as to reduce the level phosphate. In the presence of phosphate, the residual NP catalysed guanosine to guanine and ribose-1-phosphate, which are known as the strong NP inhibitors. When applied to cod and trout, the hypoxanthine ratio (H value) obtained by the biosensor system agreed well with that of the conventional enzymatic assay.