Development of Redox-Responsive ESIPT-Based Ratiometric Fluorescent Probes for Detection of Biothiols

Biothiols including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) play pivotal roles in pathological cells. The abnormal level of intracellular biothiols is commonly associated with diseases. Therefore, developing an efficient fluorescent probe to detect biothiols is beneficial for the early diagnosis of diseases. Herein, we report two redox-responsive ESIPT-based ratiometric fluorescent probes denoted by AO-3-DSF and TF-AO-3-DSF, respectively, in which ESIPT/ESICT fluorophore 3-hydroxy flavone (3-HF) is chosen as a dual-colored reporter. Thio-specific cleavable ethyl-disulfide group is introduced to the hydroxyl group of flavone probe at the 3-position to block ESIPT pathway at the initial state exclusively giving blue ESICT fluorescence. Upon exposure to biothiols, AO-3-DSF and TF-AO-3-DSF undergo the thiol exchange reaction followed by an intramolecular cyclization to generate demasked AO-3-OHF and TF-AO-3-OHF, respectively, resulting in a green ESIPT-dominative fluorescence. It is noteworthy that the introduction of trifluoromethyl group on 2’-position of 3-HF fluorophore not only effectively enhances the water solubility, but also rationally modulate TF-AO-3-DSF to maintain proton transfer band in aqueous media. Therefore, when TF-AO-3-DSF reaction with biothiols under biological environment, it can demonstrate a significant change of fluorescence. Both of AO-3-OHF and TF-AO-3-DSF show blue-to-green ratiometric detection of GSH with a 97- and 69-fold enhancement, respectively, and thus successfully distinguished between normal and pathological levels (2 uM to 10 mM) of GSH with a detection limit of 0.013 uM and 0.61 uM, respectively. Furthermore, AO-3-OHF and TF-AO-3-DSF both displayed remarkable reactivity toward biothiols over other amino acids. Confocal imaging show that TF-AO-3-DSF can get into HeLa cell and detect intracellular biothiols.