A microtubule-localizing activity-based sensing fluorescent probe for imaging hydrogen peroxide in living cells

Hydrogen peroxide (H₂O₂) is a major reactive oxygen species (ROS) in living systems with broad roles spanning both oxidative stress and redox signaling. Indeed, owing to its potent redox activity, regulating local sites of H₂O₂ generation and trafficking is critical to determining downstream physiological and/or pathological consequences. We now report the design, synthesis, and biological evaluation of Microtubule Peroxy Yellow 1 (MT-PY1), an activity-based sensing fluorescent probe bearing a microtubule-targeting moiety for detection of H₂O₂ in living cells. MT-PY1 utilizes a boronate trigger to show a selective and robust turn-on response to H₂O₂ in aqueous solution and in living cells. Live-cell microscopy experiments establish that the probe co-localizes with microtubules and retains its localization after responding to changes in levels of H₂O₂, including detection of endogenous H₂O₂ fluxes produced upon growth factor stimulation. This work adds to the arsenal of activity-based sensing probes for biological analytes that enable selective molecular imaging with subcellular resolution.