Single-enzyme level inorganic–organic engineering enables long-term stabilization of paper-based enzyme biosensors for hypoxanthine detection in food samples

A single-enzyme level inorganic–organic engineering strategy was developed. • XOD@Si with enhanced activity and stability was achieved using silica precursors. • XOD@Si was integrated with pullulan on nitrocellulose membrane. • The XOD@Si-Pul biosensor achieve high stability under room temperature. • The XOD@Si-Pul biosensor enables on-site detectin of Hx in various food samples. Hypoxanthine (Hx) is an important biomarker for food freshness and clinical diagnosis, necessitating rapid and reliable on-site detection methods. Paper-based enzyme sensors offer a promising solution, yet their practical application is severely constrained by poor storage stability at room temperature. Herein, we report a bioinspired paper-based enzyme sensor for Hx detection based on enzyme stabilization at the single-enzyme level. Each xanthine oxidase (XOD) molecule was encapsulated within a conformal silica shell with three different silica precursors, achieving single-enzyme-level silicification and yielding XOD@Si with enhanced catalytic activity and structural stability. Further integration of XOD@Si with pullulan network on nitrocellulose membrane resulted in excellent color uniformity and signal intensity. The sensor exhibited a linear response of 19.5–625 μM and a limit of detection of 6.7 μM. With good selectivity and reliable quantitative capability, the sensor performed well in liquid meat soup and enabled “attach-and-read” assessment of meat freshness. Notably, the biomimetic dual-protection strategy conferred excellent storage stability, maintaining stable signal output for several weeks at room temperature, thereby overcoming a major limitation of conventional paper-based enzyme sensors. This work provides a versatile and scalable strategy for stabilizing enzymes on paper substrates, contributing to the development of stable, low-cost, and field-deployable biosensing platforms.