An ESIPT-based fluorescent probe for the determination of hypochlorous acid (HClO): mechanism study and its application in cell imaging

A highly selective and sensitive probe for the detection of hypochlorous acid (HClO) in real samples was designed and synthesized by using the specific reaction between HClO and phenyl azo group. Upon reaction with HClO, the nonfluorescent probe generated a highly fluorescent 2-(2-hydroxy-4-chlorophenyl)benzimidazole (HBI-Cl) fluorophore, which underwent the excited state intramolecular proton transfer process to give strong fluorescence turn-on. The sensing mechanism, conversion of the nonfluorescent azo moiety into the fluorescent derivative of HBI upon reaction with HClO, was verified by independent synthesis of HBI-Cl (ϕfl ≈ 0.75). The theoretical computing results were in agreement with the experimental results that the azo moiety was the reactive site to realize fluorescence detection for HClO. Additionally, the probe was successfully utilized to determine HClO in tap water, exogenous HClO in HeLa cells, and endogenous HClO in MCF-7 cells with a low detection limit and cytotoxicity.