Observation of the Generation of ONOO– in Mitochondria under Various Stimuli with a Sensitive Fluorescence Probe

Peroxynitrite (ONOO^-), an important reactive oxygen species (ROS), may be generated in the mitochondria under various physiological and pathological conditions. In this work, we designed and synthesized a mitochondria-targetable fluorescence probe RPTPP by using rhodamine as the fluorophore, phenylhydrazine as the recognition moiety, and triphenyl-phosphonium cation as the mitochondria-targeting moiety. Upon reaction of the probe with ONOO^-, the oxidation of phenylhydrazine by ONOO^- and the subsequent hydrolysis opens the nonfluorescent spirocyclic structure and, thus, triggers a fluorescence turn-on response, which provides a sensitive and selective method for the detection of ONOO^-. The mitochondria-targeting property of RPTPP was confirmed by the colocalization experiments as well as the mitochondria uncoupling treatments. Moreover, the applications of the probe for imaging intracellular ONOO^- were performed in living cells, which reveals that the ONOO^- level in RAW264.7 cells undergoes an about 9-fold increase with the stimulation of LPS and IFN-γ for 15 h.