Abstract:

Natural products have consistently served as an invaluable source for novel drug development， and the efficient and targeted screening of bioactive constituents from these complex mixtures is crucial for advancing drug discovery. Bioaffinity fishing techniques exploit the highly specific interactions between ligands and their biological targets—such as antigen-antibody， enzyme-substrate， and hormone-receptor interactions—to simultaneously separate and identify active compounds from complex natural product extracts. When coupled with liquid chromatography-mass spectrometry， bioaffinity fishing enables the integrated separation， screening， and analysis of bioactive molecules in a single step. This comprehensive review focuses specifically on bioaffinity fishing materials， which are fundamental to the effectiveness and reproducibility of the fishing process. The review systematically explores recent advances over the past three years， categorizing the discussion along two primary dimensions： carrier materials and immobilization methods. In terms of carrier materials， the article provides a comprehensive review of both carrier-free and carrier-based systems. Carrier-free approaches， including affinity ultrafiltration and size exclusion chromatography， utilize free targets in solution， while carrier-based systems immobilize the target onto a solid support. The review details a wide spectrum of innovative supports such as magnetic beads， which facilitate easy separation； metal-organic frameworks， known for their high surface area and tunable porosity； silica-based materials （e.g.， mesoporous silica and silica chromatographic stationary phases）， prized for their stability and versatility； gel-based materials like agarose and synthetic polymers， which offer superior biocompatibility； microchannel-based systems， including capillary columns and microfluidic chips that enable high-throughput screening with minimal sample consumption； fluorescent materials that integrate fishing with real-time activity monitoring and imaging； and other emerging materials such as graphene， carbon nanotubes， and paper-based substrates. Each material system is evaluated regarding its preparation， advantages， limitations， and representative applications in fishing active ingredients from natural products. Regarding immobilization strategies， the review compares three principal methodologies： physical adsorption， which is simple but often prone to leakage and low efficiency； physical encapsulation， which protects the target yet may incur mass transfer limitations and potential activity loss； and chemical cross-linking， which enhances stability but risks altering the target’s conformation and activity. A significant portion of the discussion is devoted to advanced directional immobilization techniques designed to preserve biological activity and improve efficiency. These include various bioorthogonal reactions such as Halo-tag/alkyl chloride， His-tag/Ni-NTA， SpyTag/SpyCatcher， SNAP-tag/benzylguanine， and the use of unnatural amino acid incorporation combined with click chemistry （e.g.， azide-alkyne cycloaddition， thiol-ene addition）. These strategies allow for precise orientation and covalent attachment of proteins， leading to improved stability， reduced nonspecific binding， enhanced chromatographic performance， and higher binding affinity. Notably， these methods enable the one-step immobilization of even low-abundance or endogenous targets without the need for prior purification， greatly expanding the technique’s applicability. Finally， the review outlines current challenges and prospective directions for the field. Key challenges include the lack of clear selection criteria for carrier materials， potential detrimental effects of immobilization on target structure and function， and the need to balance generality with cost-effectiveness. Future efforts should focus on developing more efficient and targeted immobilization protocols， creating multifunctional composite materials that combine separation， detection， and screening capabilities， fostering interdisciplinary collaborations integrating nanotechnology and bioinformatics， and adhering to green chemistry principles to ensure sustainability. As technological progress continues， bioaffinity fishing is poised to play an increasingly vital role in elucidating the material basis of natural medicine efficacy and accelerating the discovery of new therapeutic agents.

Key words: natural products, bioaffinity fishing technology, immobilization, carriers