A two-photon near-infrared fluorescent probe for imaging endogenous hypochlorite in cells, tissue and living mouse

Hypochlorite (HClO/ClO-) plays an important role in the body's immune function system and helps destroy invading bacteria and pathogens. Therefore, real-time and visual detection of hypochlorous acid in the living body is necessary. Here, we describe the first example of a two-photon excitation near-infrared light-emitting fluorescent probe (Nil-ClO) for HClO/ClO- based on Nile Red derivatives. Nil-ClO offers a large fluorescence response near 650 nm and good selectivity to HClO/ClO- in solution. The probe showed an approximately 8-fold increase in fluorescence within 1 min when it was detected at 650 nm in the presence of 30 equivalents of HClO/ClO-. The probe was further used for the fluorescence-based detection of HClO/ClO- in cells, tissues, and mice in a liver injury model. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) experiments showed that the probe had almost no cytotoxicity at the concentrations needed for imaging. The Nil-ClO can response to HClO/ClO- in both HeLa cells and RAW cells quickly. In tissue imaging experiments, Nil-ClO was used to detect HClO/ClO- with a significant near-infrared fluorescence response down to 100 pm of depth. Finally, the obvious fluorescence enhancement in liver injury mouse model indicated a burst of endogenously generated HClO/ClO- in mouse during in vivo imaging experiments.