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.