Abstract
Abstract
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.