Highly sensitive detection and live cell imaging of human serum albumin using functionalized naphthalimide probes

In this study, we rationally designed and synthesized three novel naphthalimide-based fluorescent probes, Nap-A1, Nap-A2, and Nap-A3, by introducing cationic pyridinium units functionalized with distinct peripheral groups (ester, boronate ester, and nitro) into a classical donor-acceptor (D-A) scaffold. These probes exhibited prominent intramolecular charge transfer (ICT), as confirmed by systematic solvatochromism, viscosity-dependent fluorescence enhancement, and density functional theory (DFT) calculations. Nap-A2 demonstrated the most effective binding to human serum albumin (HSA) with a low detection limit of 0.072 mg/mL, Nap-A2 exhibits the highest binding affinity (K_b = 1.2862 × 10⁶ M^-1) among the three probes, together with a broad linear range (0-6 mg/mL) and a unique 1: 2 binding stoichiometry with HSA. Competitive binding assays and molecular docking simulations further revealed distinct interaction modes for Nap-A1, Nap-A2, and Nap-A3. All probes maintained high selectivity, excellent ionic strength/pH stability, and performed reliably in artificial urine. Nap-A2 exhibited low cytotoxicity and enabled successful real-time visualization of exogenous and endogenous HSA in live HepG-2 cells. Although boronate esters are known to react with H₂O₂, control experiments in H₂O₂-rich environments and structural analysis confirm that the fluorescence response observed in cells is primarily driven by HSA binding rather than H₂O₂-mediated transformation.