Dual-color reversible fluorescent carbon dots designed for dynamic monitoring of cellular superoxide anion radicals

The superoxide anion radical (O₂˙^-) represents the primary reactive oxygen species generated in biological systems. Real-time monitoring of its dynamic fluctuations provides valuable insights into disease progression and enables early diagnosis of hepatic ischemia-reperfusion injury (HIRI). In this work, we developed a novel dual-color fluorescent carbon dot (CD) probe through a one-step hydrothermal synthesis for reversible O₂˙^- detection. The CDs demonstrated excellent sensitivity, dynamically detecting O₂˙^- concentrations ranging from 0 to 60 μM with a detection limit of 0.56 μM. The probe exhibited remarkable reversibility, maintaining stable performance through at least three complete oxidation-reduction cycles following glutathione (GSH) treatment. In practical applications, the CDs achieved 95.2-104% recovery rates when detecting O₂˙^- in serum samples. Cellular imaging experiments confirmed the probe's effectiveness in normal hepatocytes (LO2), showing clear reversible responses to O₂˙^- fluctuations. Application in a HIRI cell model revealed significant elevation of O₂˙^- levels and provided new evidence for its role in HIRI-related signaling pathways. This study not only presents an effective dual-color fluorescent probe for dynamic O₂˙^- monitoring but also establishes a versatile synthetic strategy that could be adapted for imaging other biologically relevant molecules in living cells.