A dual-fluorophore and dual-site multifunctional fluorescent sensor for real-time visualization of mitochondrial ONOO-/GSH cross-talk in living cells

The redox homeostasis between glutathione (GSH) and peroxynitrite (ONOO-) is essential for maintaining normal physiological functions in biological system, and the imbalance of GSH and ONOO- is closely related to the pathogenesis of diverse diseases. Herein, we presented a dual-fluorophore and dual-site probe for the simultaneous detection of mitochondrial GSH and ONOO- via the integrated mechanism of ICT and FRET. The probe exhibited favorable response performance toward GSH and ONOO- at pH 7.4 including high sensitivity, fast response and superior selectivity. Especially, there was no interfering fluorescent signals of ONOO- (or GSH) during the detection of GSH (or ONOO-) alone, while significant fluorescence variation was observed during detecting ONOO- (or GSH) in the presence of GSH (or ONOO-). Therefore, the probe could monitor the coexistence of GSH and ONOO- not just the separate existence of GSH or ONOO-. Given these advantages, Mito-CM-CD has been successfully utilized for real-time visualization of GSH and ONOO- in normal HepG2 cells and drug-induced HepG2 cells. Importantly, it has been found that depletion of GSH and indirect formation of ONOO- are associated with the APAP-induced liver injury which can be effectively relieved by supplementing GSH. We anticipated that this research will promote to understand the interrelation of GSH and ONOO- under different physiological and pathological settings. [Display omitted] •Mito-CM-CD exhibited rapid response, high selectivity and low LOD to ONOO- and GSH.•The probe displayed quenched signal to ONOO- but enhanced signal to GSH at 640 nm under single-wavelength excitation.•Mito-CM-CD could sense ONOO- (or GSH) with enhanced blue but decreased red fluorescence in the presence of GSH (or ONOO-).•Confocal microscopy experiments indicated that Mito-CM-CD can be used for visualizing GSH and ONOO- in living cells.