Ultrasensitive detection of hydrogen peroxide at the single-particle nanozyme level

Hydrogen peroxide (H₂O₂) is widely used but highly toxic at elevated concentrations, necessitating ultrasensitive detection for safety and diagnostics. Current peroxidase nanozyme approaches for H₂O₂ detection and kinetic analysis are limited to ensemble measurements, where collective signals mask single-particle heterogeneity, compromise sensitivity, and preclude precise structure-activity correlations. To address this limitation, we herein employ Fe₃O₄@Fe/C as a model single-particle nanozyme and implement single-molecule fluorescence imaging to monitor, in situ, the catalytic oxidation of a non-fluorescent substrate to a highly fluorescent product by H₂O₂ at the single-particle level, thereby constructing an ultrasensitive H₂O₂ detection platform. Through correlating kinetic parameters with H₂O₂ response, we demonstrate that the single-particle assay achieves a limit of detection (LOD) of 24.17 nM with a linear range of 0.2-3 μM, representing a ~58-fold improvement in sensitivity over solution phase ensemble measurements (LOD=1.39 μM). In addition, quantitative analysis of the kinetic parameters reveals pronounced catalytic non-uniformity among individual nanozyme particles. This quantitative analysis system at the single-particle level provides a new method for ultrasensitive detection, and further offers new insights into revealing the microscopic processes of the detection reaction and guiding the study of structure-activity relationships.