FRET-Based Mito-Specific Fluorescent Probe for Ratiometric Detection and Imaging of Endogenous Peroxynitrite: Dyad of Cy3 and Cy5

Peroxynitrite (OONO^-) is profoundly implicated in health and disease. The physiological and pathological outcome of OONO^- is related to its local concentration, and hence, a reliable OONO^- assay is highly desired. We have developed a FRET-based small-molecule fluorescent probe (PNCy3Cy5), harnessing the differential reactivity of Cy3 and Cy5 toward OONO^- by fine-tuning. It exhibits high detection sensitivity and yields a ratiometric fluorescent signal. We have exemplified that it can be applied in semiquantitative determination of OONO^- in living cells. Notably, it specifically localizes in mitochondria, where endogenous OONO^- is predominantly generated. Thus, PNCy3Cy5 is a promising molecular tool for peroxynitrite biology.