Effect of π‐bridge unit on human serum albumin binding properties of D‐π‐A ‐based fluorescent dyes

Abstract Currently, various molecular scaffolds are employed to develop novel organic push‐pull dyes for detecting human serum albumin (HSA). This study synthesized two D‐π‐A type dyes, LXH‐H and LXH‐Ph, differing in their π‐bridges, to investigate their binding interactions with HSA using spectroscopic, thermodynamic, and molecular docking approaches. LXH‐H exhibited a selective fluorescence response to HSA, with approximately 36‐fold fluorescence enhancement observed compared to other common proteins and duplex DNA. In contrast, negligible spectral changes occurred for LXH‐Ph upon HSA interaction, indicating the critical role of the π‐bridge in HSA recognition. Experimental and docking results demonstrated that both dyes bind to site 3 on HSA, forming complexes primarily driven by hydrogen‐bonding forces, with LXH‐H displaying higher binding affinity than LXH‐Ph. Furthermore, the potential application of LXH‐H for detecting HSA in urine‐containing samples was explored.