Novel flavonoid-based fluorescent probes for site-specific differentiation of human and bovine serum albumin: Visualizing quantitative research in urinary HSA

Human serum albumin (HSA) is the most abundant protein in serum and is essential for maintaining human health. Abnormal levels have been associated with a variety of diseases, making it an important disease-diagnostic biomarker and therapeutic monitoring indicator. Due to the high structural similarity between human serum albumin (HSA) and bovine serum albumin (BSA), the detection and differentiation of these two proteins have garnered significant attention. In this study, we reported three rationally engineered flavonoid fluorescent probes capable of selectively detecting and distinguishing HSA from BSA. Among these compounds, HTN2 exhibited a markedly enhanced fluorescence response to HSA with a detection limit of 4.8 nM, while HTN3 demonstrated a significantly amplified fluorescence response to BSA with a detection limit of 1.4 nM. Notably, the interaction between HTN2 and HSA was entropy-driven, whereas HTN3 binding to BSA was enthalpy-driven. Molecular docking experiments investigated the intrinsic reasons for the differing selectivity of the nitrogen-containing six-membered heterocycles toward HSA versus BSA. Furthermore, the HTN2 probes were successfully applied to detect HSA in real urine samples, offering a reliable approach for HSA analysis in biological fluids. The results showed that introducing the nitrogen heterocycle improved selectivity for HSA and BSA. HTN2 entered the hydrophobic pocket of HSA effectively; however, when bound to BSA, the nitrogen heterocycle was partially exposed and formed interactions poorly. This provides an effective method for designing specific probes