Imaging viscosity and peroxynitrite by a mitochondria-targeting two-photon ratiometric fluorescent probe

The irregular viscosity and peroxynitrite (ONOO–) concentration in mitochondria would result in mitochondria disorders. However, fluorescent probes that can detect both of viscosity and peroxynitrite have not been explored. For this reason, a two-photon fluorescent molecule TP-1Bz was designed and synthesized in this work for the dual-ratiometric-imaging of viscosity and exogenous and endogenous ONOO– in mitochondria. Results indicated that TP-1Bz has two emissions at 407 nm and 650 nm respectively, their ratio (I650/I407) showed linear relationship to the viscosity of the media in the logarithmic plot, and its two-photon action cross-section (Φσ) at 880 nm was increased from 1.2 GM in aqueous solution to be 86.3 GM (72-fold) in 99% glycerol. With a good targeting capability to mitochondria, TP-1Bz allowed us to measure the average viscosity in the mitochidria of HepG2 cells (73.3 cp) and the mitochondria viscosity change upon the addition of ionpores. TP-1Bz also showed the high sensitivity to ONOO– (LOD close to 12.1 nM) in water/glycerol (2/3) solvent as well as the high selectivity over other ROS. With the advantages of its lower cytotoxicity, pH- and photo- stabilities, TP-1Bz was successfully employed for imaging ONOO– in living cells and even in tissue samples of rat liver through the monitoring the emission intensities of the blue and red channels. As the first example of two-photon ratiometric fluorescent probe for dual-imaging viscosity and peroxynitrite in mitochondria, TP-1Bz has great potential to be developed for clinical applications.