Controlled preparation of molecularly imprinted polymer microspheres with surface uncrosslinked glycoprotein binding cavities

Lijie Huang, Congguang Zheng, Huiqi Zhang^*

Chinese Science Bulletin, 2019. 64(13): 1407-1417

DOI：https://doi.org/10.1360/N972018-01000

Abstract

Molecularly imprinted polymers (MIPs) are synthetic receptors with tailor-made recognition sites for target molecules. Their high affinity and selectivity, excellent stability, easy preparation, and low cost make them promising substitutes to biological receptors (e.g., antibody and enzyme) in many applications where molecular recognition is important. Despite significant progress made in the imprinting of small templates, the imprinting of biomacromolecules (e.g., proteins) remains a big challenge because of their large sizes, complexed structures, and conformational variability. These inherent characteristics of biomacromolecules lead to many significant problems for the resulting macromolecularly imprinted polymers (mMIPs) such as laborious removal of large templates and their slow access to the binding sites.

Recent years have witnessed much efforts being devoted to the development of mMIPs due to their great potential in proteome analysis, clinical diagnostics, and biomedicine. So far, some useful strategies have been developed for the imprinting of proteins, mainly including the bulk polymerization method, epitope imprinting strategy, and surface imprinting approach. Among them, the surface imprinting approach has been most widely used because it can readily lead to mMIPs with higher efficiency for removing large templates and more rapid template binding kinetics. Nevertheless, the presently developed mMIPs normally have crosslinked template binding sites, whose rigid structures might have negative influence on the removal of large templates and template binding kinetics. In this sense, the development of mMIPs with more flexible biomacromolecular binding cavities (e.g., uncrosslinked ones) should be useful for solving the above problems. To our knowledge, however, only rather limited numbers of publications relating to mMIPs with uncrosslinked binding sites have been disclosed, which are all based on the use of self-assembled monolayer strategy. The development of versatile new approaches for preparing mMIPs with uncrosslinked binding sites and good molecular imprinting effect is still highly desirable.

We demonstrate a facile and efficient new approach for the controlled preparation of MIP microspheres with surface uncrosslinked glycoprotein binding sites. It involves the first one-pot synthesis of uniform “living” polymer microspheres with both surface-bound epoxy groups and alkyl halide groups (i.e., atom transfer radical polymerization (ATRP)-initiating groups) via atom transfer radical precipitation polymerization, their surface modification with 3-aminophenylboronic acid (APBA) for introducing surface phenylboronic acid moieties and surface immobilization of a glycoprotein (ovalbumin (OVA)), subsequent use of the “living” polymer microspheres with surface-immobilized OVA as the ATRP initiator for the controlled grafting of poly(N-isopropylacrylamide) (PNIPAAm) brushes, and final removal of OVA. A series of MIP microspheres with surface uncrosslinked OVA binding sites were readily obtained following the above procedure by just changing the polymerization time for grafting PNIPAAm brushes, and their morphologies, chemical structures, surface hydrophilicity, water dispersion stability, and template binding properties were characterized in detail. The experimental results demonstrated that the above approach could effectively provide MIPs with excellent recognition ability toward OVA in the aqueous medium. The surface hydrophilicity and water dispersion stability of MIP microspheres were largely improved due to their surface-grafting of hydrophilic polymer brushes. Moreover, the chain length of PNIPAAm brushes showed significant influence on the template binding properties of MIP microspheres, and the best template binding capacity and specific binding were achieved for MIPs only when the thickness of PNIPAAm layers was close to the total length of the diameter of OVA plus the length of the surface-attached phenylboronic acid unit. Furthermore, the optimal MIP also exhibited good selectivity toward OVA over other proteins. The strategy presented here paves a new way for controlled and efficient preparation of glycolprotein-imprinted polymer microspheres with good molecular recognition capability.