Biomedical sciences, and in particular biomarker research, demand efficient glycoproteins enrichment platforms. In this work, we present a facile and time-saving method to synthesize phenylboronic acid and copolymer multifunctionalized magnetic nanoparticles (NPs) using a distillation-precipitation polymerization (DPP) technique. The polymer shell is obtained through copolymerization of two monomers-affinity ligand 3-acrylaminophenylboronic acid (AAPBA) and a hydrophilic functional monomer. The resulting hydrophilic Fe3O4@P(AAPBA-co-monomer) NPs exhibit an enhanced binding capacity toward glycoproteins by an additional functional monomer complementary to the surface presentation of the target protein. The effects of monomer ratio of AAPBA to hydrophilic comonomers on the binding of glycoproteins are systematically investigated. The morphology, structure, and composition of all the synthesized microspheres are characterized by transmission electron microscopy (TEM), X-ray powder diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), and vibrating sample magnetometer (VSM). The hydrophilic Fe3O4@P(AAPBA-co-monomer) microspheres show an excellent performance in the separation of glycoproteins with high binding capacity; And strong magnetic response allows them to be easily separated from solution in the presence of an external magnetic field. Moreover, both synthetic Fe3O4@P(AAPBA) and copolymeric NPs show good adsorption to glycoproteins in physiological conditions (pH 7.4). The Fe3O4@P(AAPBA-co-monomer) NPs are successfully utilized to selectively capture and identify the low-abundance glycopeptides from the tryptic digest of horseradish peroxidase (HRP). In addition, the selective isolation and enrichment of glycoproteins from the egg white samples at physiological condition is obtained by Fe3O4@P(AAPBA-co-monomer) NPs as adsorbents.

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Affinity maturation and rational design have a raised importance in the application of nanobody (VHH), and its unique structure guaranteed these processes quickly done in vitro. An anti-CD47 nanobody, Nb02, was screened via a synthetic phage display library with 278 nM of KD value. In this study, a new strategy based on homology modeling and Rational Mutation Hotspots Design Protocol (RMHDP) was presented for building a fast and efficient platform for nanobody affinity maturation. A three-dimensional analytical structural model of Nb02 was constructed and then docked with the antigen, the CD47 extracellular domain (CD47ext). Mutants with high binding affinity are predicted by the scoring of nanobody-antigen complexes based on molecular dynamics trajectories and simulation. Ultimately, an improved mutant with an 87.4-fold affinity (3.2 nM) and 7.36 °C higher thermal stability was obtained. These findings might contribute to computational affinity maturation of nanobodies via homology modeling using the recent advancements in computational power. The add-in of aromatic residues which formed aromatic-aromatic interaction plays a pivotal role in affinity and thermostability improvement. In a word, the methods used in this study might provide a reference for rapid and efficient in vitro affinity maturation of nanobodies.