The level of sulfur dioxide (SO2) and viscosity in mitochondria play vital roles in various of physiological and pathological processes. Abnormalities in mitochondrial SO2 and viscosity are closely associated with numerous biological diseases. It is of great significance to develop novel fluorescence probe for simultaneous detection of SO2 and viscosity within mitochondria. Herein, we have developed a water-soluble, mitochondrial-targeted and near-infrared fluorescent probe, CMBT, for the simultaneous detection of SO2 and viscosity. The probe CMBT incorporates benzothiazolium salt as a mitochondrial targeting moiety and 7-diethylaminocoumarin as a rotor for viscosity detection, respectively. Based on the prompt reaction between nucleophilic HSO3−/SO32− and the backbone of benzothiazolium salt derivative, probe CMBT displayed high sensitivity and selectivity toward SO2 with a limit of detection as low as 0.17 µM. As viscosity increased, the twisted intramolecular charge transfer (TICT) process was restricted, resulting in fluorescence emission enhancement at 690 nm. Moreover, probe CMBT demonstrated exceptional mitochondrial targeting ability and was successfully employed to image variation of SO2 and viscosity in living cells and mice. The work highlights the great potential of the probe as a convenient tool for revealing the relationship of SO2 and viscosity in biological systems.

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