Facile Synthesis of pH-Adaptive Ascorbate Peroxidase-like Nanozyme for One-Step Enzyme-Nanozyme Cascade Biosensing

Nanozymes feature structural stability, functional diversity, and tunable activity, demonstrating promise in enzyme-nanozyme cascade systems for small molecule detection. However, the cascade performance is typically hindered by pH incompatibility and the cumbersome preparation of nanozymes. Herein, copper-doped zeolitic imidazolate framework-90 (Cu/Zn-ZIF-90) was developed through a facile synthesis within 10 min of reaction, which was established as a promising ascorbate peroxidase (APX)-like nanozyme for pH-adaptive cascades. Cu/Zn-ZIF-90 oxidizes ascorbic acid using H2O2 as cosubstrate and demonstrates higher affinity for H2O2 than most APX-like or POD-like nanozymes, enabling effective intermediate conversion in cascade reactions. Notably, Cu/Zn-ZIF-90 nanozyme exhibits optimal activity under neutral pH, resolving pH mismatch when coupled with acid-denatured oxidases. As proof of concept, we integrated APX-like Cu/Zn-ZIF-90 with choline oxidase to develop a one-step cascade fluorescence biosensor for choline detection. The sensor achieved a broad linear range (1–1000 μM) and a low detection limit (0.85 μM), outperforming most existing methods while demonstrating robust applicability in the analysis of complex food matrices. This work establishes APX-mimicking nanozymes as key components to overcome cascade pH barriers, enabling one-step small-molecule biosensing.