Illumination of Hydroxyl Radical Generated in Cells during Ferroptosis, <i>Arabidopsis thaliana</i>, and Mice Using a New Turn-On Near-Infrared Fluorescence Probe

Hydroxyl radical (·OH), taken as the most active and aggressive reactive oxygen species (ROS), plays an important role in cell redox regulation and ferroptosis processes. It is a great challenge to develop methods for highly selective and sensitive detection and imaging of ·OH. A new near-infrared (NIR) fluorescence probe <b>Probe-HMP</b> was designed and synthesized by introducing 3-methylpyrazolone as the specific recognition moiety to the hemicyanine backbone of the NIR fluorophore <b>AXPI-NH</b>_<b>2</b>, which formed with the hydrazine group. <b>Probe-HMP</b> exhibited excellent detection performance <i>in vitro</i>, such as instantaneous response, low detection limit of 24 nM, and excellent selectivity without the interference from other ROS. Based on the actions of ·OH promoter phenylmercuric acetate (PMA) and ·OH scavenger 4-hydroxy-TEMPO (Tempol), <b>Probe-HMP</b> was successfully applied to obtain images of endogenous ·OH in HepG2 cells, <i>Arabidopsis thaliana</i>, and mice. The results show that <b>Probe-HMP</b> can stably and efficiently image endogenous ·OH, the fluorescence intensity of the experimental group incubated with PMA was higher than that of the control group incubated with <b>Probe-HMP</b> only, and a significant decrease could be observed in the inhibitor group incubated with Tempol. More importantly, <b>Probe-HMP</b> can achieve the detection of endogenous ·OH in HepG2 cells during ferroptosis by using erastin and deferoxamine mesylate (DFO) to induce or inhibit ferroptosis, revealing that the fluorescence intensity change of <b>Probe-HMP</b> was caused by ·OH generated and ferroptosis is accompanied by significant ·OH generation. The excellent performance of <b>Probe-HMP</b> makes it a promising candidate for exploring the physiological and pathological processes associated with ferroptosis.