Simultaneous Visualization of Glutathione and Hydrogen Peroxide with a Dual-Responsive Fluorescent Probe to Elucidate Redox Homeostasis in Alzheimer’s Disease Models

Alzheimer's disease (AD) is a progressive neurodegenerative disorder in which oxidative stress plays a critical role in pathogenesis. As key reactive species governing oxidative stress, the dynamic interplay between glutathione (GSH) and hydrogen peroxide (H₂O₂) is tightly coupled to redox homeostasis. To enable simultaneous tracking of these two crucial species, we developed a dual-responsive fluorescent probe, TCS, for the discriminative visualization of GSH and H₂O₂. TCS features a methylsulfonyl group that is selectively reduced by GSH, triggering green fluorescence at 500 nm, and a boronate ester that is specifically cleaved by H₂O₂, generating red emission at 680 nm. These two optical channels operate independently with minimal spectral overlap, enabling simultaneous and discriminative detection of GSH and H₂O₂ without cross-interference. Probe TCS exhibits high sensitivity, excellent selectivity, and good photostability, and is well-suited for real-time imaging of both endogenous and exogenous GSH and H₂O₂ in complex biosystems. In okadaic acid-induced AD cell and zebrafish models, TCS revealed a time-dependent decrease in GSH levels accompanied by a concomitant increase in H₂O₂, consistent with progressive oxidative stress. Furthermore, comparative imaging of hippocampal tissues from AD model mice and wild-type mice showed markedly altered levels of both species, underscoring the utility of TCS for visualizing redox imbalance under AD-like pathological conditions. Collectively, this study establishes TCS as a powerful molecular tool for spatiotemporally resolved monitoring of GSH and H₂O₂ dynamics in living systems and provides a robust analytical platform for elucidating redox mechanisms in AD.