A Novel Pyrazoline-Based Fluorescent Turn-On Probe for Biothiol Detection and Live-Cell Imaging.

A novel fluorescent turn-on probe for biothiol detection was designed and synthesized. The probe consists of a pyrazoline fluorophore and a maleimide group, which serves as both the recognition unit and a fluorescence quencher. In the absence of biothiols, the probe’s fluorescence is quenched via photoinduced electron transfer (PET) from the fluorophore to the maleimide moiety. Upon addition of biothiols, the maleimide moiety undergoes Michael addition with the sulfhydryl group of the biothiols, resulting in a dramatic increase in fluorescence intensity at 460 nm. This probe exhibits exceptional sensitivity and selectivity for biothiols over other tested amino acids in solution, with an impressive detection limit of 8.88 nM for glutathione. Notably, the response time of this probe to glutathione is 40 s. The detection mechanism was elucidated using high-resolution mass spectrometry (HRMS), density functional theory (DFT), and time-dependent density functional theory (TD-DFT). Moreover, the probe has been successfully applied to image endogenous biothiols in living cells.