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

Hydroxyl radical (<middle dot>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 <middle dot>OH. A new near-infrared (NIR) fluorescence probe Probe-HMP was designed and synthesized by introducing 3-methylpyrazolone as the specific recognition moiety to the hemicyanine backbone of the NIR fluorophore AXPI-NH 2 , which formed with the hydrazine group. Probe-HMP exhibited excellent detection performance in vitro, such as instantaneous response, low detection limit of 24 nM, and excellent selectivity without the interference from other ROS. Based on the actions of <middle dot>OH promoter phenylmercuric acetate (PMA) and <middle dot>OH scavenger 4-hydroxy-TEMPO (Tempol), Probe-HMP was successfully applied to obtain images of endogenous <middle dot>OH in HepG2 cells, Arabidopsis thaliana, and mice. The results show that Probe-HMP can stably and efficiently image endogenous <middle dot>OH, the fluorescence intensity of the experimental group incubated with PMA was higher than that of the control group incubated with Probe-HMP only, and a significant decrease could be observed in the inhibitor group incubated with Tempol. More importantly, Probe-HMP can achieve the detection of endogenous <middle dot>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 Probe-HMP was caused by <middle dot>OH generated and ferroptosis is accompanied by significant <middle dot>OH generation. The excellent performance of Probe-HMP makes it a promising candidate for exploring the physiological and pathological processes associated with ferroptosis.