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. • 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.