Mitochondria-targeted bifunctional probes for monitoring SO₂ and viscosity in diverse environments

Sulfur dioxide (SO₂) and mitochondrial viscosity are critical indicators of both environmental and physiological health. However, simultaneous detection of these parameters remains challenging due to limitations in probe sensitivity and spectral overlap. In this study, we report the design and synthesis of two mitochondrial-targeted fluorescent probes, HVTI and HVTB, capable of simultaneously detecting SO₂ derivatives and viscosity both in vitro and in vivo. These bifunctional probes utilize the Michael addition reaction and the twisted intramolecular charge transfer (TICT) mechanism to achieve high selectivity and sensitivity. Upon reacting with SO₂ derivatives, the probes display a pronounced fluorescence shift from red to blue-specifically from 614 nm to 456 nm for HVTI and from 642 nm to 463 nm for HVTB. In addition, increased viscosity results in a significant enhancement of red fluorescence intensity. eliminate spectral crosstalk and enable colorimetric detection discernible to the naked eye. Using advanced spectroscopic methods, the probes demonstrated robust performance in real water samples, achieving recovery rates of 94.29-104.30 % and detection limits as low as 0.25 µM. Moreover, imaging studies in RAW 264.7 cells and zebrafish validated their ability to visualize SO₂ and viscosity changes in vivo, with excellent biocompatibility and low cytotoxicity. The probes exhibit exceptional functionality across diverse environments, offering unprecedented precision in monitoring cellular microenvironments and aquatic ecosystems. This study represents a significant advancement in fluorescence-based detection, positioning HVTI and HVTB as versatile tools for both environmental analysis and biomedical research.