An efficient TP-FRET-based lysosome-targetable fluorescent probe for imaging peroxynitrite with two well-resolved emission channels in living cells, tissues and zebrafish

Peroxynitrite (ONOO−), an important role of reactive oxygen species (ROS) in vivo, and studies showed abnormal of ROS can induce lysosomal membrane permeabilization (LMP) and lead to the death of cells. Thus, it is of great significance for designing an effective method for investigating relationship between physiology and pathology between ONOO− and lysosome. Herein, for the first time, we adopted a Förster resonance energy transfer (FRET) strategy to construct a lysosome-targetable small molecular ratiometric two-photon (TP) fluorescent probe (NpRh-ONOO) for detecting ONOO− in living cells, tissues and zebrafish. Specifically, a TP fluorophore and a rhodamine B fluorophore are directly connected by a flexible piperidine linker to form the TP-FRET-scaffold, a hydrazide as ONOO− reactive set, and a dimethylamino as lysosome targeting-group, which shows a target-modulated ratiometric TP fluorescence response, two well-resolved emission peaks separated by 73 nm, rapid response (<10 s), high selectivity and sensitivity with the detection limit is as low as 3.3 nM for ONOO−. These prominent features of probe were then applied for ratiometric bioimaging both exogenous and endogenous peroxynitrite in living cells, tissues and zebrafish, demonstrating it can be used as a powerful tool for biological research of lysosomal peroxynitrite in biological systems.