Rational construction of a novel ratiometric far-red fluorescent probe with excellent water solubility for sensing mitochondrial peroxynitrite

Peroxynitrite (ONOO−) is closely associated with various pathophysiological processes and mainly produced within the mitochondria. Therefore, developing an efficient method for mitochondrial ONOO− detection is crucial for better understanding of its functions. In view of the advantages of ratiometric fluorescent probes and far-red/NIR fluorescent probes, a kind of novel mitochondrion-targeting ratiometric far-red fluorescent probe (ANI-DP) was reasonably constructed by employing isophorone derivative as fluorescence group, diphenyl phosphinate as response group, and pyridinium cation as mitochondria-targetable group. Probe ANI-DP displayed a distinctive ratiometric fluorescence response toward ONOO− with a red-shifted emision in far-red to NIR region owing to the cleavage of a phosphinate group and change of intramolecular charge transfer (ICT) efficiency induced by ONOO−. In 100 % aqueous solution, probe ANI-DP was capable of quantitatively detecting ONOO− levels ranging from 0 to 10 μM with a low detection limit of 13.3 nM, and showed high specificity for ONOO− over other biological relevant substances. Besides, it featured large Stokes shift (202 nm), high photostability, low cytotoxicity as well as good mitochondria targetable ability. Encouraged by the outstanding properties described above, it was successfully applicated for ratiometric fluorescence imaging of ONOO− in mitochondria of living cells as well as visualization of ONOO− in zebrafish, which indicated that it holds great potential for revealing the functions of ONOO− in living biosystems.