A cyclization-driven ratiometric fluorescent probe for hypochlorite detection: targeted sensing and visualization in plant samples and living cells

Most ClO^- sensing probes are limited by having only a single active site, which often results in structural breakdown after ClO^- interaction. To overcome this challenge, a novel ratiometric fluorescent probe, PBT-2, featuring a phenothiazine-modified thiophene-benzothiazole hydrazone scaffold, was synthesized and characterized for efficient ClO^- sensing. The probe operates via an internal charge transfer (ICT) "turn-off" mechanism, exhibiting rapid fluorescence quenching from light yellow to colorless within 15 seconds under optimized conditions. It offers high selectivity and an ultra-low detection limit of 2.2 nM. Mechanistic insights revealed that the sensing involves ClO^--triggered oxidation followed by intramolecular cyclization, supported by ¹H-NMR titration, DFT calculations, and ESI-MS analysis. The probe demonstrated its utility in visualizing ClO^- in plant roots, aiding in the study of hypochlorite's physiological roles, and was further validated in living cells, where it enabled real-time fluorescence imaging of intracellular ClO^- with excellent biocompatibility. In addition, PBT-2 was successfully applied to detect ClO^- in commercial bleach and environmental water samples, showcasing outstanding photophysical and sensing performance across diverse matrices.