Theoretical characterization of two-photon fluorescent probes for nitric oxide detection: sensing mechanism, photophysical properties and protonation effects

-diamino aromatics have been designed and synthesized for NO detection utilizing the principle of photoinduced electron transfer (PET). However, the underlying PET mechanism has rarely been validated, and a systematic computational study on the photophysical properties is urgently desired. In this study, we used a theoretical protocol to comparatively investigate the sensing mechanism, photophysical properties and protonation effects of two emblematic probes NINO and PYSNO in aqueous solution, which combines a polarizable continuum model (PCM), time-dependent density functional theory (TD-DFT) and thermal vibration correlation function formalism (TVCF). Our findings reveal that the weak emission of NINO is due to activated PET with negative driving energy and blocked fluorescence with significant charge separation. In contrast, the poor luminescence of PYSNO is caused by the facilitated non-radiative dissipation, even though the fluorescence emission remains unobstructed. Although NINO has been successfully used in two-photon microscopy for detecting NO, we suggest that PYSNO possesses a superior two-photon absorption (TPA) cross section in the near-infrared region. The protonation effects suggest that both probes can function effectively in practical acidic lysosomal environments. Our study opens a new avenue for understanding the mechanism and predicting the properties of two-photon fluorescent probes for NO detection, thus aiding the rational design of efficient fluorescent sensors.