A ratiometric NIR two-photon probe for biothiols detection in cellular autophagy and liver injury models

Biothiols, as crucial players in biological processes, are critical biomarkers in various physiological and pathological states. Real-time monitoring of the dynamic changes of biothiols in vivo is of great significance for elucidating their roles in disease onset and progression. In this study, a novel near-infrared two-photon ratiometric fluorescent probe, NADCO-A, was developed based on the NADCO platform by introducing an electron-deficient acrylate group as a specific response unit for biothiols. The probe operates via an ingenious "ICT weakening-restoration" mechanism, achieving a significant emission red shift of 136 nm (from 570 nm to 706 nm), accompanied by pronounced ratiometric fluorescence signal changes. NADCO-A was successfully applied to real-time ratiometric imaging of biothiols in living cells, revealing a dynamic trend of initial decrease followed by an increase in biothiol content during cellular autophagy. Furthermore, the probe enabled dynamic monitoring of biothiol level fluctuations in drug-induced liver injury (DILI) models (at both cellular and murine levels), revealing the depletion of biothiols during liver injury. It also demonstrated promising application potential in evaluating the efficacy of hepatoprotective drugs, notably identifying silybin (Sil) and scoparone (Sco) as having favorable therapeutic effects on liver injury. These results demonstrate that NADCO-A is a reliable and versatile chemical tool with significant value for exploring biothiol-related physiological functions and disease mechanisms.