Rational design of FexNy@C nanozymes enables a pH-independent dual-mode biosensing platform for H2O2 monitoring in living cells

Real-time monitoring of hydrogen peroxide (H 2 O 2 ) within the cellular microenvironment plays a pivotal role in early cancer diagnosis. Conventional H 2 O 2 detection methods are largely constrained by their narrow pH adaptability and limited operational modes, failing to meet the demands of complex physiological environments. We developed a rationally designed nanozyme-based platform capable of dual-mode H 2 O 2 detection across acidic and neutral pH conditions. The Fe x N y @C nanozyme, synthesized through electrospinning and controlled pyrolysis of MIL-53-derived precursors, exhibits tunable peroxidase-like activity that can be optimized by adjusting the mass ratio of MOF to polymers. Designed to simulate the horseradish peroxidase (HRP) structure, the Fe x N y @C system allows for quantitative H 2 O 2 detection in acidic environments through the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to oxidized TMB, resulting in a color change from colorless to blue. Simultaneously, a smartphone-assisted visual detection platform based on red-green-blue (RGB) color analysis was developed and thus serves as a practical tool for point-of-care testing (POCT). Owing to the facilitated Fe 2+ /Fe 3+ redox cycling and rapid electron transfer within its conductive framework, the Fe x N y @C nanozyme demonstrates high electrocatalytic activity for H 2 O 2 reduction in phosphate-buffered saline (PBS, pH 6.8). This enabled the development of an electrochemical sensor for real-time detection of H 2 O 2 released from cancer cells, highlighting its potential for tracking disease progression and monitoring therapeutic responses. This dual-mode sensor achieves high-performance H 2 O 2 detection by combining the strengths of electrochemical and colorimetric readouts. The electrochemical mode provides rapid response (1 s) and high sensitivity (1049.82 μA∙mM −1 ∙cm −2 ), while the colorimetric mode offers unique simplicity and visualizability, enabling rapid quantitative analysis within 10 min without the need for sophisticated instrumentation. Together, both modes demonstrate excellent selectivity, reproducibility and stability, making the platform adaptable to diverse application scenarios from laboratory precision to POCT.