A Noncovalent Fluorescent Probe for Dynamic Monitoring of Hemoglobin Accumulation in Living Cells

Dynamic assessment of intracellular hemoglobin (Hb) levels enables a deeper understanding of Hb-induced cytotoxicity, supports the evaluation of disease progression, and informs the development of targeted therapeutic strategies. However, traditional Hb detection methods typically require cell lysis or tissue fixation, which precludes the observation of dynamic processes in living cells. Herein, we reported a noncovalent fluorescent probe, DMBM, which enabled the dynamic monitoring of bovine hemoglobin (BHb) accumulation in living cells. DMBM exhibited strong intrinsic fluorescence and demonstrated a significant selective quenching response toward BHb (LOD = 0.23 μM). This specific fluorescence response originated from noncovalent interactions between DMBM and BHb, as evidenced by the significant fluorescence recovery observed upon BHb denaturation induced by high concentrations of urea. Furthermore, DMBM possessed a strong binding affinity for BHb (K_a = (1.7 ± 0.06) × 10⁵ M^-1), and this interaction remained unaffected by common drugs. Additionally, DMBM exhibited negligible cytotoxicity at concentrations below 60 μM, and its intrinsic fluorescence signal was inversely proportional to the intracellular accumulation levels. DMBM serves as a reliable fluorescent tool for the visualization and dynamic monitoring of Hb accumulation in living cells, offering a promising strategy for diagnosing Hb-related physiological and pathological processes.