Paper-Based Enzyme Biosensor for One-Step Detection of Hypoxanthine in Fresh and Degraded Fish

Food freshness monitoring, which can reflect the quality of the product at the time of use, remains a great challenge for consumers and the food industry. Herein, we report the development of a cost-effective enzyme-based paper biosensor, which can monitor fish freshness and predict spoilage. The biosensor measures the release of hypoxanthine (HX), an indicator of meat and fish degradation, using the enzymatic conversion of HX by xanthine oxidase (XOD). We demonstrate that the entrapment of XOD and an organic dye, nitro blue tetrazolium chloride (NBT), within a sol–gel biohybrid enables their stabilization on paper and promotes the enzymatic reaction with further retention of the reaction products within the cellulosic network . Linearity in the micromolar concentration range with a detection limit of 3.7 μM for HX is obtained. The biosensor has high selectivity toward HX and is manufactured in few steps from inexpensive widely available materials. The applicability of the biosensor is demonstrated by following fish degradation over time and measuring HX concentrations ranging from 117 (±9) to 198 (±5) μM within 24 h of degradation, at levels that are comparable with those measured by a commercial enzymatic kit for HX detection. As compared to the commercial kit, our biosensors are more cost-effective, do not require addition of exogenous reagents and are portable, having all of the reagents needed for analysis embedded within the sensing platform. This proof-of-concept work demonstrates that the paper-based HX biosensor has potential as a robust reagentless device for real-time monitoring of food freshness and for other applications in which HX plays an important role.