表面具有非交联结构糖蛋白识别位点的分子印迹聚合物微球的可控制备
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摘要
将原子转移自由基沉淀聚合技术、表面锚定糖蛋白策略及表面引发的可控自由基聚合方法相结合,发展了一种简便高效地制备表面具有(由亲水性聚合物刷形成的)非交联结构糖蛋白(卵清蛋白(OVA))识别位点的分子印迹聚合物(MIP)微球的新方法.对所得具有不同非交联印迹壳层厚度的MIP微球的形貌、化学结构、表面亲水性及模板吸附性能进行了系统研究.结果表明,该方法可高效制备在水溶液中对OVA具有优异识别性能的MIPs.随着MIP微球表面亲水性聚合物刷的引入,其表面亲水性与水相分散稳定性明显提高;同时亲水性聚合物刷的长度亦对MIPs的模板吸附性能有显著影响:只有当亲水性聚合物刷长度与OVA粒径加上微球表面修饰的苯硼酸基的总长度相近时, MIP微球对OVA的吸附容量与专一性吸附方能达到最优;此外,该MIP还具有良好的OVA选择性.
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 m MIPs 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.
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 m MIPs 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.
引文
1 Zhang H,Ye L,Mosbach K.Non-covalent molecular imprinting with emphasis on its application in separation and drug development.J Mol Recognit,2006,19:248-259
2 Hoshino Y,Shea K J.The evolution of plastic antibodies.J Mater Chem,2011,21:3517-3521
3 Wulff G,Liu J.Design of biomimetic catalysts by molecular imprinting in synthetic polymers:The role of transition state stabilization.Acc Chem Res,2012,45:239-247
4 Chen L,Wang X,Lu W,et al.Molecular imprinting:Perspectives and applications.Chem Soc Rev,2016,45:2137-2211
5 Wackerlig J,Schirhagl R.Applications of molecularly imprinted polymer nanoparticles and their advances toward industrial use:A review.Anal Chem,2016,88:250-261
6 Pan J,Chen W,Ma Y,et al.Molecularly imprinted polymers as receptor mimics for selective cell recognition.Chem Soc Rev,2018,47:5574-5587
7 Li S,Cao S,Whitcombe M J,et al.Size matters:Challenges in imprinting macromolecules.Prog Polym Sci,2014,39:145-163
8 Zhang H.Recent advances in macromolecularly imprinted polymers by controlled radical polymerization techniques.Mol Imprint,2015,3:35-46
9 Li D,Chen Y,Liu Z.Boronate affinity materials for separation and molecular recognition:Structure,properties and applications.Chem Soc Rev,2015,44:8097-8123
10 Wang Y,Zhou Y,Sokolov J,et al.A potentiometric protein sensor built with surface molecular imprinting method.Biosens Bioelectron,2008,24:162-166
11 Zhang X,Du X,Huang X,et al.Creating protein-imprinted self-assembled monolayers with multiple binding sites and biocompatible imprinted cavities.J Am Chem Soc,2013,135:9248-9251
12 Stephenson-Brown A,Acton A L,Preece J A,et al.Selective glycoprotein detection through covalent templating and allosteric click-imprinting.Chem Sci,2015,6:5114-5119
13 Zhang H.Controlled/“living”radical precipitation polymerization:A versatile polymerization technique for advanced functional polymers.Eur Polym J,2013,49:579-600
14 Xing R,Wang S,Bie Z,et al.Preparation of molecularly imprinted polymers specific to glycoproteins,glycans and monosaccharides via boronate affinity controllable-oriented surface imprinting.Nat Protoc,2017,12:964-987
15 Zoppe J O,Ataman N C,Mocny P,et al.Surface-initiated controlled radical polymerization:State-of-the-art,opportunities,and challenges in surface and interface engineering with polymer brushes.Chem Rev,2017,117:1105-1318
16 Zhang H,Jiang J,Zhang H,et al.Efficient synthesis of molecularly imprinted polymers with enzyme inhibition potency by the controlled surface imprinting approach.ACS Macro Lett,2013,2:566-570
17 Ma Y,Pan G,Zhang Y,et al.Narrowly dispersed hydrophilic molecularly imprinted polymer nanoparticles for efficient molecular recognition in real aqueous samples including river water,milk,and bovine serum.Angew Chem Int Ed,2013,52:1511-1514
18 Zhao M,Chen X,Zhang H,et al.Well-defined hydrophilic molecularly imprinted polymer microspheres for efficient molecular recognition in real biological samples by facile RAFT coupling chemistry.Biomacromolecules,2014,15:1663-1675
19 Li C,Ma Y,Niu H,et al.Hydrophilic hollow molecularly imprinted polymer microparticles with photo-and thermoresponsive template binding and release properties in aqueous media.ACS Appl Mater Interfaces,2015,7:27340-27350
20 Gorman C B,Petrie R J,Genzer J.Effect of substrate geometry on polymer molecular weight and polydispersity during surface-initiated polymerization.Macromolecules,2008,41:4856-4865
21 Pan G,Zhang Y,Guo X,et al.An efficient approach to obtaining water-compatible and stimuli-responsive molecularly imprinted polymers by the facile surface-grafting of functional polymer brushes via RAFT polymerization.Biosens Bioelectron,2010,26:976-982
22 Wu T,Zhang Y,Wang X,et al.Fabrication of hybrid silica nanoparticles densely grafted with thermoresponsive poly(N-isopropylacrylamide)brushes of controlled thickness via surface-initiated atom transfer radical polymerization.Chem Mater,2008,20:101-109
23 He M J,Chen W X,Dong X X.Polymer Physics(Revised edition)(in Chinese).Shanghai:Fudan University Press,2005[何曼君,陈维孝,董西侠.高分子物理(修订版).上海:复旦大学出版社,2005]
24 Stein P E,Leslie A G W,Finch J T,et al.Crystal structure of ovalbumin as a model for the reactive centre of serpins.Nature,1990,347:99-102
25 Gao F X,Zhao X L,He X W,et al.A pH and temperature dual-responsive macroporous molecularly imprinted cryogel for enhanced recognition capability towards ovalbumin.Anal Methods,2013,5:6700-6708
26 Gao F X,Ma X T,He X W,et al.Smart surface imprinting polymer nanospheres for selective recognition and separation of glycoprotein.Colloids Surfaces A-Physicochem Eng Aspects,2013,433:191-199
27 Xie J,Zhong G,Cai C,et al.Rapid and efficient separation of glycoprotein using pH double-responsive imprinted magnetic microsphere.Talanta,2017,169:98-103
28 Sun X Y,Ma R T,Chen J,et al.Synthesis of magnetic molecularly imprinted nanoparticles with multiple recognition sites for the simultaneous and selective capture of two glycoproteins.J Mater Chem B,2018,6:688-696
2 Hoshino Y,Shea K J.The evolution of plastic antibodies.J Mater Chem,2011,21:3517-3521
3 Wulff G,Liu J.Design of biomimetic catalysts by molecular imprinting in synthetic polymers:The role of transition state stabilization.Acc Chem Res,2012,45:239-247
4 Chen L,Wang X,Lu W,et al.Molecular imprinting:Perspectives and applications.Chem Soc Rev,2016,45:2137-2211
5 Wackerlig J,Schirhagl R.Applications of molecularly imprinted polymer nanoparticles and their advances toward industrial use:A review.Anal Chem,2016,88:250-261
6 Pan J,Chen W,Ma Y,et al.Molecularly imprinted polymers as receptor mimics for selective cell recognition.Chem Soc Rev,2018,47:5574-5587
7 Li S,Cao S,Whitcombe M J,et al.Size matters:Challenges in imprinting macromolecules.Prog Polym Sci,2014,39:145-163
8 Zhang H.Recent advances in macromolecularly imprinted polymers by controlled radical polymerization techniques.Mol Imprint,2015,3:35-46
9 Li D,Chen Y,Liu Z.Boronate affinity materials for separation and molecular recognition:Structure,properties and applications.Chem Soc Rev,2015,44:8097-8123
10 Wang Y,Zhou Y,Sokolov J,et al.A potentiometric protein sensor built with surface molecular imprinting method.Biosens Bioelectron,2008,24:162-166
11 Zhang X,Du X,Huang X,et al.Creating protein-imprinted self-assembled monolayers with multiple binding sites and biocompatible imprinted cavities.J Am Chem Soc,2013,135:9248-9251
12 Stephenson-Brown A,Acton A L,Preece J A,et al.Selective glycoprotein detection through covalent templating and allosteric click-imprinting.Chem Sci,2015,6:5114-5119
13 Zhang H.Controlled/“living”radical precipitation polymerization:A versatile polymerization technique for advanced functional polymers.Eur Polym J,2013,49:579-600
14 Xing R,Wang S,Bie Z,et al.Preparation of molecularly imprinted polymers specific to glycoproteins,glycans and monosaccharides via boronate affinity controllable-oriented surface imprinting.Nat Protoc,2017,12:964-987
15 Zoppe J O,Ataman N C,Mocny P,et al.Surface-initiated controlled radical polymerization:State-of-the-art,opportunities,and challenges in surface and interface engineering with polymer brushes.Chem Rev,2017,117:1105-1318
16 Zhang H,Jiang J,Zhang H,et al.Efficient synthesis of molecularly imprinted polymers with enzyme inhibition potency by the controlled surface imprinting approach.ACS Macro Lett,2013,2:566-570
17 Ma Y,Pan G,Zhang Y,et al.Narrowly dispersed hydrophilic molecularly imprinted polymer nanoparticles for efficient molecular recognition in real aqueous samples including river water,milk,and bovine serum.Angew Chem Int Ed,2013,52:1511-1514
18 Zhao M,Chen X,Zhang H,et al.Well-defined hydrophilic molecularly imprinted polymer microspheres for efficient molecular recognition in real biological samples by facile RAFT coupling chemistry.Biomacromolecules,2014,15:1663-1675
19 Li C,Ma Y,Niu H,et al.Hydrophilic hollow molecularly imprinted polymer microparticles with photo-and thermoresponsive template binding and release properties in aqueous media.ACS Appl Mater Interfaces,2015,7:27340-27350
20 Gorman C B,Petrie R J,Genzer J.Effect of substrate geometry on polymer molecular weight and polydispersity during surface-initiated polymerization.Macromolecules,2008,41:4856-4865
21 Pan G,Zhang Y,Guo X,et al.An efficient approach to obtaining water-compatible and stimuli-responsive molecularly imprinted polymers by the facile surface-grafting of functional polymer brushes via RAFT polymerization.Biosens Bioelectron,2010,26:976-982
22 Wu T,Zhang Y,Wang X,et al.Fabrication of hybrid silica nanoparticles densely grafted with thermoresponsive poly(N-isopropylacrylamide)brushes of controlled thickness via surface-initiated atom transfer radical polymerization.Chem Mater,2008,20:101-109
23 He M J,Chen W X,Dong X X.Polymer Physics(Revised edition)(in Chinese).Shanghai:Fudan University Press,2005[何曼君,陈维孝,董西侠.高分子物理(修订版).上海:复旦大学出版社,2005]
24 Stein P E,Leslie A G W,Finch J T,et al.Crystal structure of ovalbumin as a model for the reactive centre of serpins.Nature,1990,347:99-102
25 Gao F X,Zhao X L,He X W,et al.A pH and temperature dual-responsive macroporous molecularly imprinted cryogel for enhanced recognition capability towards ovalbumin.Anal Methods,2013,5:6700-6708
26 Gao F X,Ma X T,He X W,et al.Smart surface imprinting polymer nanospheres for selective recognition and separation of glycoprotein.Colloids Surfaces A-Physicochem Eng Aspects,2013,433:191-199
27 Xie J,Zhong G,Cai C,et al.Rapid and efficient separation of glycoprotein using pH double-responsive imprinted magnetic microsphere.Talanta,2017,169:98-103
28 Sun X Y,Ma R T,Chen J,et al.Synthesis of magnetic molecularly imprinted nanoparticles with multiple recognition sites for the simultaneous and selective capture of two glycoproteins.J Mater Chem B,2018,6:688-696