Light- and H2O2-Switchable Dual-Enzyme Carbon Dot Nanozymes with Fe–Pyrrolic N Dual Sites for Orthogonally Activated Multimodal Detection of Glutathione and Hydrogen Peroxide

Redox biomarkers play pivotal roles in maintaining cellular homeostasis and are closely associated with the onset and progression of numerous diseases. However, constructing nanozyme-based sensing systems capable of selectively quantifying different redox biomarkers through independent catalytic pathways remains challenging. Herein, we report Fe-doped carbon dot (Fe-CD) nanozymes featuring Fe-pyrrolic N dual active sites that enable orthogonally activated dual-enzyme catalytic behavior for the selective detection of glutathione (GSH) and hydrogen peroxide (H2O2). Mechanistic studies reveal that modulation of the ethylenediamine precursor regulates the abundance of pyrrolic N species and the Fe2+/Fe3+ ratio, thereby governing the switching between light-activated oxidase-like (OXD-like) activity and H2O2-activated peroxidase-like (POD-like) activity. The optimized Fe-CD2.0 nanozymes exhibit balanced dual catalytic performance, allowing the construction of two independently activated sensing pathways based on the inhibition and promotion of TMB oxidation for GSH and H2O2 detection, respectively. Furthermore, the characteristic absorbance, fluorescence-quenching capability, and photothermal conversion property of oxidized TMB enable the development of a multimodal sensing platform integrating fluorescence, colorimetric, and photothermal readouts. The platform achieves sensitive determination of GSH and H2O2 with limits of detection (LODs) of less than 3.16 μM and satisfactory recoveries ranging from 93.54% to 109.11% in multiple cell lines. The excellent agreement among multiple detection modes further enhances analytical confidence and reliability. This work establishes an Fe-pyrrolic N dual-site engineering strategy for constructing stimulus-switchable carbon dot nanozymes and provides a versatile framework for orthogonally activated multimodal sensing of redox biomarkers.