Two-photon ESIPT-based fluorescent probe using 4-hydroxyisoindoline-1,3-dione for the detection of peroxynitrite

Excited-state intramolecular proton transfer (ESIPT)-based fluorophores with two-photon excitation fluorescence (TPEF) are rare. Our aim with this research was to develop ESIPT-based fluorophores exhibiting TPEF. Herein, we used 4-hydroxyisoindoline-1,3-dione as a scaffold to develop a two-photon fluorescent probe BHID-Bpin, for the detection of peroxynitrite (ONOO^-). BHID-Bpin exhibits excellent selectivity, sensitivity, and fast response towards ONOO^- in PBS buffer solution (10 mM, pH = 7.40). Additionally, BHID-Bpin displays high photo-stability under two-photon irradiation at 750 nm. Furthermore, the probe can image endogenous ONOO^- in HeLa cells and exogenous ONOO^- in rat hippocampal slices at a depth of 110 μm.