Differential recognition and quantification of HSA and BSA based on two red-NIR fluorescent probes

It is always a challenge to achieve the selective recognition between human serum albumin (HSA) and bovine serum albumin (BSA) because of their highly homologous primary structure. In this work, we reported two red-NIR fluorescence probes, BI-FPI and NTPS-FPI, for HSA and BSA differential recognition. BI-FPI showed remarkable emission enhancement toward HSA over BSA, while NTPS-FPI exhibited the opposite selectivity toward BSA. The obvious different interaction for probes with HSA and BSA were demonstrated by site specific competitive binding experiments and molecular docking studies. BI-FPI located within site I of HSA, which mainly depended on the hydrophobic residues and pi-pi interaction. While NTPS-FPI docked into the interface between subdomains II and IIIA of BSA, and the strong hydrogen bond interaction was the main contributor for the binding. Furthermore, the good calibration graphs of BI-FPI to HSA enabled us to develop the attractive multiple functional probe for qualitatively and quantitatively detecting HSA, and the detection limit (0.01 mu M, 0.66 mg/L) met the requirements of traditional HSA assay in serum or in urine (30 mg/L).