Dye-ligand functionalized collagen fibers as an affinity chromatography stationary phase for efficient protein separation

Dye molecules serve as promising affinity ligands with significant potential in bioseparation applications. In this study, a series of dye-affinity collagen fibers (CFs-Dye) stationary phases was fabricated by immobilizing reactive dyes (reactive red 118, reactive blue 171, reactive blue 19, reactive blue 114, reactive blue 21, and reactive blue 116) onto CFs for the selective separation and purification of lysozyme (LyZ). The adsorption capacity was closely dependent on the molecular structure of dye ligands. Among the synthesized stationary phases, the anthraquinone dye-affinity CFs demonstrated superior adsorption performance. Owing to the fibrous structure of CFs and the specific affinity of anthraquinone structure, reactive blue 114 immobilized CFs (CFs-RB114) exhibited outstanding static (1594.23 mg/g) and dynamic (1504.28 mg/g) adsorption capacities, as well as superior selectivity for LyZ (SF LyZ/OVA : 87.59 and SF LyZ/BSA : 96.56). The inhibition experiments with NaCl, isopropanol, and urea demonstrated that the adsorption mechanism involves synergistic electrostatic attraction, hydrophobic interaction, and hydrogen bonding between CFs-RB114 and LyZ. As an affinity chromatography stationary phase, CFs-RB114 achieved complete separation of OVA and LyZ by stepwise elution. Additionally, CFs-RB114 could successfully extract LyZ directly from chicken egg white. This work presents a novel high-performance affinity adsorbent with exceptional efficacy for LyZ extraction and purification, demonstrating promising potential for biomolecule practical applications.