Triple-Channel fluorescent probe for simultaneous detection of Cys, SO₂, and H₂S to discriminate Cancer cells via Transsulfuration pathway metabolic profiling.

Early and accurate cancer diagnosis is essential for improving patient prognosis and guiding effective treatment. Metabolic reprogramming of the transsulfuration pathway is a hallmark of malignancy, characterized by elevated intracellular levels of cysteine (Cys), sulfur dioxide (SO₂), and hydrogen sulfide (H₂S). Here, we report the design and synthesis of TAC, a coumarin-based triple-channel fluorescent probe incorporating three distinct reactive sites for the simultaneous detection of Cys, SO₂, and H₂S. TAC exhibits high sensitivity, excellent selectivity, robust photostability, and negligible spectral crosstalk among the three emission channels. In live-cell imaging, TAC enabled real-time visualization of both endogenous and exogenous analytes with high spatial-temporal resolution. Importantly, TAC accurately discriminated between normal and cancer cells by capturing their distinct sulfur metabolic fingerprints, with cancer cells exhibiting significantly higher fluorescence in all channels. This work introduces the first fluorescent probe capable of triple-analyte imaging within the transsulfuration pathway, providing a powerful platform for non-invasive metabolic profiling and early cancer screening.