Development of a ratiometric nitric oxide probe with baseline resolved emissions by an ESIPT and rhodol ring opened-closed integrated two-photon platform

In recent years, reflecting the degree of cellular inflammation through in situ monitoring of nitric oxide using fluorescence sensing has received much attention due to many merits such as non-invasiveness and easy operation. In particular, two-photon excitation microscopy can significantly improve the imaging resolution and visualization time. In the meantime, a ratiometric-based nitric oxide fluorescent sensor can avoid the interference of many factors, including light source intensity, solvent scattering degree, solvent color, solvent viscosity, probe distribution, and instrument performance, and improve the accuracy of the result. However, the mutual interference of two emission peaks is still an issue restricting the development of this field. In this work, the Rh-NO-F dye obtained by modifying the rhodol dye with benzothiazole exhibited excited state intramolecular proton transfer (ESIPT) in the closed ring state. In the open ring state, however, the emission wavelength can be significantly red-shifted by increasing the degree of dye conjugation. By introducing o-phenylenediamine, the recognition domain of NO, we successfully designed and synthesized a ratiometric two-photon NO fluorescent probe, Rh-NO-P, which showed a 154 nm increase in the maximum emission wavelength before and after the response and almost no interference between the two emission peaks. Confocal imaging showed that the probe could achieve in situ detection of exogenous NO fluctuations in cells. The probe was also successfully applied to detect the changes in NO content during wound healing in mice.