A Biomimetic Protein Immobilization Method for Studying Drug–Protein Interactions Based on Affinity Capillary Electrochromatography

In complex systems, a universal and effective method for accurately determining the dissociation constant (K_d) of drug-protein interactions (DPI) has not been identified yet. In this study, a novel biomimetic affinity capillary electrochromatography (ACEC) platform was developed. Polydopamine (PDA) was employed not only as a coating material but also as an immobilization agent for human serum albumin (HSA), resulting in the fabrication of the PDA/PDA/HSA@capillary. A series of characterization experiments on the PDA/PDA/HSA@capillary showed that PDA was successfully coated on the inner wall of the capillary, and HSA was also successfully immobilized on the capillary column. The optimal concentration of PDA for preparing the PDA/PDA/HSA@capillary was determined to be 0.8 mg mL^-1, and the optimal concentration for immobilizing HSA was 0.25 mM, respectively. Moreover, the PDA/PDA/HSA@capillary showed good separation effects in the complex systems of different drugs. Furthermore, the electrophoretic performance of individual and mixed samples was compared across different capillary columns. Through interaction studies between proteins and compounds, the K_d value of rutin was 1.09 × 10³ mol L^-1 that of quercitrin was 2.78 × 10³ mol L^-1, and that of quercetin was 7.44 × 10⁴ mol L^{- 1}, aligning with results from other established methods. The method was applied to the Sophora japonica extract, and the K_d was consistent with the rutin in the mixed system. Reproducibility studies demonstrated that high separation efficiency was maintained even after 50 consecutive runs. By integrating biomimetic material science with chromatographic innovation, this work overcomes critical bottlenecks in traditional affinity capillary electrophoresis (ACE), providing a universal tool for high-throughput drug screening and structure-guided therapeutic design.