A pattern-free paper enzyme biosensor for one-step detection of fish freshness indicator hypoxanthine with a microfluidic aggregation effect

In this study, a paper-based enzyme biosensor for hypoxanthine (Hx) was developed, enabling visual and one-step fish freshness detection. Xanthine oxidase and horseradish peroxidase were immobilized on nitrocellulose membranes with 3,3′,5,5′-tetramethylbenzidine to output the colour signal. Chitosan oligosaccharide lactate-modified nitrocellulose membranes entrapped the dual-enzyme system and exhibited excellent microfluidic aggregation effect. The developed enzyme biosensor produced a linear response of 0.01–0.16 mmolL -1 with a detection limit of 8.22 μmolL -1 , and was selective for Hx with recoveries of 96.13–103.11 % for fish samples. These biosensors were attached directly to the surface of fish samples and the colour was revealed within 3 min. Colour signals can be judged by the naked-eye to distinguish between fresh and spoiled fish samples and analyzed by a smartphone for quantitative analysis. The biosensor shows great potential as a powerful pattern- and reagent-free device for on-site freshness evaluation of fish.