Enzyme-Triggered In Situ Assembly of Fe3O4 Nanozyme Synthesis Enables Portable Point-of-Care Detection of Acid Phosphatase

Acid phosphatase (ACP) is a clinically important enzyme whose early-stage detection is hindered by its extremely low abundance, nonspecific tissue distribution, and rapid loss of activity under conventional analytical conditions. Herein, we present a target-driven in situ nanozyme synthesis strategy that enables rapid and ultrasensitive point-of-care testing (POCT) of ACP. In this approach, ACP catalyzes the hydrolysis of L-ascorbic acid 2-phosphate sesquimagnesium (AAPS), producing ascorbic acid (AA). The generated AA partially reduces Fe3+ ions to Fe2+, thereby initiating alkaline co-precipitation and in situ formation of Fe3O4 nanoparticles. Polyvinylpyrrolidone (PVP) stabilizes the nanoparticles and preserves catalytic accessibility, while their intrinsic magnetism allows for efficient magnetic separation to eliminate matrix interference. The resulting Fe3O4@PVP nanozymes display pronounced peroxidase-like activity, catalyzing hydrogen-peroxide-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB). Quantitative readout can be achieved using either spectrophotometric analysis or smartphone imaging. The sensing platform achieves a detection limit of 0.021 U/L within 40 min and demonstrates excellent sensitivity, selectivity, and operational robustness. Successful validation in human serum confirms its clinical feasibility, while smartphone-based imaging enables portable and low-cost quantification suitable for decentralized diagnostics. Collectively, this work establishes a generalizable paradigm for target-triggered nanozyme generation aimed at detecting low-abundance and labile biomarkers.