Dual-Mode Biosensing Strategy Leveraging Single-Atom Fe Nanozyme for Cellular Glutathione Detection

Intracellular glutathione (GSH) detection is essential to elucidating the mechanism of redox dyshomeostasis in diverse physiological and pathological processes. However, conventional GSH detection strategies suffer from complicated operation, reliance on expensive instruments, inadequate sensitivity, and labor requirements. Herein, we propose a novel GSH sensing strategy that utilizes a single-atom-dispersed Fe nanozyme (Fe SAzyme) with remarkable dual enzyme–mimetic activities. The strategy enables dual-mode detection using both ultraviolet–visible (UV–vis) and smartphone-based RGB analysis. The RGB mode is designed for high-concentration GSH (150 to 1200 μM) detection and operates via simultaneous reaction of GSH with Fe SAzyme, TMB, and H 2 O 2 . It achieves a limit of detection (LOD) of 110 μM. Meanwhile, the UV–vis mode operates by introducing GSH into a preincubated Fe SAzyme/TMB mixture, providing a wide detection range of 10–150 μM with an LOD as low as 1.37 μM. Furthermore, the strategy effectively distinguishes between normal and cancer cells via GSH detection in cell lysates, demonstrating excellent agreement with GSH commercial kits. Leveraging the strategy, we observed DNAzyme-mediated GSH depletion both in vitro and in vivo, thereby demonstrating the feasibility of inhibiting GSH regeneration and disrupting the intracellular redox balance via silencing of GLUT-1. This study confirms the capacity of the Fe SAzyme-based dual-mode biosensing strategy for intracellular GSH detection, demonstrating its significant potential for point-of-care biomedical applications.