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 t...