A Sequentially Activated Probe for Imaging of Superoxide Anion and Peroxynitrite in PC12 Cells under Oxidative Stress

Superoxide anion (O₂^·-) and peroxynitrite (ONOO^-), two important oxidants under oxidative stress, coexist in complex cell and organism systems, playing crucial roles in various physiological and pathological processes, particularly in neurodegenerative diseases. Despite the absence of robust molecular tools capable of simultaneously visualizing O₂^·- and ONOO^- in biosystems, the relationship between these two species remains understudied. Herein, we present sequentially activated fluorescent probe, DHX-SP, which exhibits exceptional selectivity and sensitivity toward O₂^·- and ONOO^-. This probe enables precise imaging of these species in living PC12 cells under oxidative stress conditions using distinct fluorescence signal combinations. Furthermore, the probe DHX-SP has the ability to visualize changes in O₂^·- and ONOO^- levels during ferroptosis of PC12 cells and in the Parkinson's disease model. These findings establish a connection between the crosstalk of the phosphorus group of O₂^·- and ONOO^- in PC12 cells under oxidative stress.