Hypochlorite sensing and real-time imaging with XY-01: A red-emitting fluorescent turn-on probe for living cells and colorectal cancer organoids

Hypochlorite (ClO⁻), a major reactive oxygen species generated in inflammation, is a potent biological oxidant involved in diverse physiological and pathological processes; therefore, sensitive detection of ClO⁻ is important for understanding disease pathophysiology and supporting early diagnosis and prevention. Here, we aimed to develop a physiologically compatible fluorescent tool for specific ClO⁻ sensing and imaging. We designed and synthesized a novel A-D-A type molecular fluorescent probe, XY-01, and characterized it by NMR, HRMS, UV-Vis and fluorescence spectroscopy. XY-01 operates through ClO⁻-triggered oxidation of a thioformyl group (C=S) to a carbonyl (C=O), which restores intramolecular charge transfer and produces a prominent fluorescence turn-on signal. In PBS (pH 7.4), XY-01 responded to ClO⁻ within 1 min with strong red emission at 666 nm and a large Stokes shift (~167 nm), showed high selectivity against common ions and reactive species, and achieved a detection limit of 3.39 µM within the biologically relevant range. Cytotoxicity assays indicated negligible toxicity, enabling real-time confocal imaging of ClO⁻ distribution in HCT-116 cells and colorectal cancer organoids. Collectively, XY-01 is a simple, sensitive, and low-toxicity probe that provides a promising platform for optical sensing and imaging of hypochlorite in living cells and organoids.