Highly Specific and Ultrasensitive Two-Photon Fluorescence Imaging of Native HOCl in Lysosomes and Tissues Based on...

Hypochlorous acid (HOCl) acts as a weak acid distributed mainly in acidic organelle lysosomes of phagocytes and plays a crucial role in the immune defense. The elaborate interrelation between the variations of HOCl levels in lysosomes and different physiological and pathological processes remains unclear. Thus, the accurate determination of lysosomal HOCl in living cells and in vivo is very important. Because of extremely low concentration and difficulty in distinguishing HOCl from OCl^- under the physiological environment, it is still a great challenge to specifically monitor the intracellular intrinsic HOCl levels without exogenous stimulation, which impedes an exact understanding of its biological roles. In this paper, based on the electrophilic addition of Cl⁺ to sulfide moiety, we have developed a two-photon fluorescent probe O-(N-butyl-1,8-naphthalimide)-4-yl-N,N-dimethylthiocarbamate (NDMTC) for the specific determination of HOCl over OCl^- and other bioactive molecules. Our results show that NDMTC possesses a detection limit of 7.6 pM, and it is the first fluorescent probe for detecting HOCl at the picomolar level. Furthermore, by introducing an alkylmorpholine group to the NDMTC framework, the lysosome-targetable derivative Lyso-NDMTC was obtained, and its ability to image HOCl in the lysosome organelles was clearly confirmed. Combined with two-photon fluorescence imaging of background suppression and deeper tissue penetration, NDMTC and Lyso-NDMTC were used to successfully visualize intracellular native HOCl and discern tumor tissue in mice. This study offers two perfect fluorescence imaging probes for further investigation of pathological roles of HOCl in various diseases.