Fluorescent Probe HKSOX-1 for imaging and detection of endogenous superoxide in live cells and in vivo

Superoxide anion radical (O₂^-) is undoubtedly the most important primary reactive oxygen species (ROS) found in cells, whose formation and fate are intertwined with diverse physiological and pathological processes. Here we report a highly sensitive and selective O₂^- detecting strategy involving O₂^- cleavage of an aryl trifluoromethanesulfonate group to yield a free phenol. We have synthesized three new O₂^- fluorescent probes (HKSOX-1, HKSOX-1r for cellular retention, and HKSOX-1m for mitochondria-targeting) which exhibit excellent selectivity and sensitivity toward O₂^- over a broad range of pH, strong oxidants, and abundant reductants found in cells. In confocal imaging, flow cytometry, and 96-well microplate assay, HKSOX-1r has been robustly applied to detect O₂^- in multiple cellular models, such as inflammation and mitochondrial stress. Additionally, our probes can be efficiently applied to visualize O₂^- in intact live zebrafish embryos. These probes open up exciting opportunities for unmasking the roles of O₂^- in health and disease.