Fluorescent Probe for Simultaneous Monitoring of ONOO ^– and NAD(P)H Reveals Dynamic Cellular Redox Fluctuations

The redox homeostasis is essential for sustaining normal cellular activities, and its disruption underlies the onset and progression of diverse pathologies. Among the diverse redox-active species, peroxynitrite (ONOO^-, a highly reactive oxidant) and NAD(P)H (a central reducing cofactor) could serve as representative biomarkers of oxidative and reductive states, respectively, and simultaneous detection of ONOO^- and NAD(P)H is therefore of great significance for deep redox biology interpretation. Here, we developed a highly sensitive and specific fluorescent probe, <b>BQ</b>, for simultaneously monitoring mitochondrial ONOO^- and NAD(P)H. <b>BQ</b> enabled real-time visualization of redox dynamics in living cells and in vivo, allowing assessment of oxidative stress, cancer-normal cell discrimination, and evaluation of antioxidant drugs. With the aid of <b>BQ</b>, we uncovered a pronounced redox imbalance in an epileptic zebrafish model, which was effectively ameliorated by conventional antiepileptic agents and further alleviated by a clinical stroke drug. We expect that <b>BQ</b> could be a versatile tool to established robust imaging strategy for redox homeostasis research.