An ESIPT-based near-infrared ratiometric reversible fluorescent probe for dynamic in situ monitoring of glutathione in diabetic models

Glutathione (GSH) is a critical antioxidant that is closely associated with a variety of diseases, including diabetes. Accurate and dynamic monitoring of GSH levels is essential for effective clinical diagnosis. Although numerous fluorescent probes have been developed for GSH detection, traditional irreversible single-channel probes suffer from several significant limitations, such as unavoidable excitation-emission crosstalk, the absence of self-calibration capability, and the inability to differentiate between chronic depletion and transient fluctuations. To address these challenges, we developed a reversible ratiometric GSH probe named BBTD. This probe utilized 2-(2-hydroxyphenyl)benzothiazole (HBT), a fluorophore based on excited-state intramolecular proton transfer (ESIPT), and incorporated 1H-indene-1,3(2H)-dione as the Michael addition site for GSH. A thiophene unit was introduced between these components to extend the conjugated system and shift the emission wavelength into the near-infrared region. Probe BBTD exhibited reversible, ratiometric, highly sensitive (Detection limit: 0.23 μM) and large Stokes shift responses to GSH, with ESIPT-based emission reaching up to 750 nm (Stokes shift: 320 nm; HBT-COU: 621 nm), making it highly suitable for real-time in vivo monitoring of GSH fluctuations. In a diabetes model, BBTD effectively quantified GSH depletion in the liver and assessed the therapeutic efficacy of N-acetylcysteine (NAC).