A new biosensor design for determination of fish meat freshness

Access to quality food is one of the biggest requirements of modern people, but the concept of quality includes many criteria. Freshness is the most important quality criterion in terms of the consumability of these foods, especially since foods such as meat, dairy products and seafood spoil quickly due to their rich nutrient content. Determination of freshness is possible by detecting some indicators that occur as a result of reactions and changes that occur in parallel with the elapsed time, various properties of the fish and the conditions to which it is exposed. Among these substances, the amounts of xanthine, hypoxanthine, uric acid, which are formed by ATP breakdown reactions in the process following the death of the fish, are considered among the most critical parameters. Since the determination of these indicator substances by conventional analysis methods (spectrophotometric, chromatographic methods, etc.) is generally time-consuming, requires special equipment, skilled labor and intensive use of chemicals, biosensor technology is an alternative that can eliminate these disadvantages, and a biosensor was designed for the simultaneous determination of xanthine, hypoxanthine and uric acid, which are among the freshness criteria of fish. A biosensor is briefly defined as an analytical device that reacts with the target analyte and converts the response into measurable physical signals. In the biosensor design in this study, an electrochemical sensor design based on the electrowinning of polyacrylonitrile (PAN) polymer on pencil lead (PGE) electrodes was made based on the use of low-cost and abundant materials. With this design, it is aimed to obtain nanomodified electrode surfaces with high electrocatalytic properties, improved analyte interaction by increasing adsorption capacity and large reaction surface. The optimum application parameters and solution formulation in the electrospinning process used in the production of sensor electrodes were determined experimentally. Modified PGE electrode surfaces coated with nanofibers with an average diameter of 137 nm were obtained by applying 8% PAN solution with a distance of 13 cm, a flow rate of 5 µl/min and a voltage of 18 kV. The ability of the sensor to simultaneously determine hypoxanthine, xanthine and uric acid was demonstrated by controlled degradation studies with xanthine oxidase enzyme. With the nanomodified surfaces obtained with this design, limits of detection (LOD) of 0.74 µM for hypoxanthine, 1.28 µM for xanthine and 1.32 µM for uric acid were achieved. Thus, a novel electrochemical (voltammetric) sensor design based on the production of PGE electrodes by modification of electrospun PAN nanofibers for the simultaneous determination of hypoxanthine, xanthine and uric acid was realized for the first time, which has never been done before in the literature. The measurement performance of these sensor electrodes after 30 days of storage at 4 °C and 25 °C was also evaluated. The ratio of deviation in the measurements is reduced at 4 °C, concluding that the electrodes should be kept at 4 °C for a better preservation. The developed sensor electrodes were used to determine the freshness of standard sized farmed sea bream fish sold in Izmir and the results were supported by microbiological parameters and the pH measurements. By the fith day of storage at refrigerator that the fish became unacceptable, the critical hypoxanthine, xanthine and uric acid concentrations found to be 1,53 µM 9,41 µM and 1,41 µM respectively. Keywords: Fish freshness, electrochemical biosensor, electrospinning, PAN nanofibers, hypoxanthine, xanthine, uric acid