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.

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