Thermo- and pH dual-responsive protein imprinted polymers for recognition of bovine serum albumin

doi:10.16085/j.issn.1000-6613.2019-0355

Abstract

Abstract:

Using N-isopropylacrylamide (NIPAM) and chitosan (CS) as functional co-monomers and bovine serum albumin (BSA) as template protein, a temperature/pH dual-responsive protein imprinting polymers was prepared on the surface of modified SiO₂. The results of TEM, FTIR and TG results showed that the imprinted layer had been successfully grafted. The temperature/pH dual-responsive, adsorption capacity, adsorption kinetics, specificity, competitive adsorption and repeatability of the polymer were systematically studied. The results showed that the swelling rate and adsorption capacity of imprinted polymer (MIP) were greatly affected by temperature and pH. MIP shrank at high temperature and alkaline condition, and swelled at low temperature and acid condition. At pH 4.6 and 35℃, the optimized adsorption capacity of MIP for 0.6mg/mL BSA was 83.74mg/g, meanwhile the best imprinting time was 4h and the imprinting factor was 2.02. In addition, MIP had good specificity and competitive adsorption. After five repetitions, the adsorption capacity still maintained 88%, indicating good repeatability. This new temperature/pH double-sensitive protein molecularly imprinted synthesis method is simple and has a good application prospect in protein separation and recognition.

Key words: thermo- and pH dual-responsive, chitosan, NIPAM, molecular imprinted, polymer

摘要：

用N-异丙基丙烯酰胺(NIPAM)和壳聚糖为功能单体，牛血清白蛋白（BSA）为模板蛋白，在改性SiO₂表面制备温度/pH双敏蛋白质印迹聚合物。TEM、FTIR和TG等结果证明印迹层已成功接枝在载体表面。系统研究了聚合物的温度/pH双敏性、吸附容量、吸附动力学、特异性、竞争吸附性及重复性。结果表明，印迹聚合物（MIP）的溶胀率和吸附容量受温度和pH影响较大，高温碱性收缩，低温酸性溶胀。在pH 4.6和35℃下，对0.6mg/mL BSA吸附4h时获得较大的吸附容量（83.74mg/g），印迹因子为2.02。同时MIP也有较好的特异性和竞争吸附性。重复5次后，吸附容量仍能维持在88%，说明重复性良好。这种新型的温度/pH双敏蛋白质分子印迹合成方法简单，在蛋白质的分离和识别方面有较好的应用前景。

关键词: 温度/pH双敏性, 壳聚糖, N-异丙基丙烯酰胺, 分子印迹, 聚合物

CLC Number:

O658

Xiangzhi DONG,Yong MA,Chunping HOU,Baoliang ZHANG,Hepeng ZHANG,Qiuyu ZHANG. Thermo- and pH dual-responsive protein imprinted polymers for recognition of bovine serum albumin[J]. Chemical Industry and Engineering Progress, 2019, 38(11): 5040-5047.

董祥芝,马勇,侯春平,张宝亮,张和鹏,张秋禹. 用于蛋白质BSA识别的温度/pH双敏印迹聚合物[J]. 化工进展, 2019, 38(11): 5040-5047.

Figures/Tables 13

References 34

1	VLATAKISG, ANDERSSONL I, MULLERR, et al. Drug assay using antibody mimics made by molecular imprinting[J]. Nature,1993, 361(6413): 645-647.
2	WULFFG. Molecular imprinting in cross-linked materials with the aid of molecular templates—A way towards artificial antibodies[J]. Angewandte Chemie： International Edition, 1995, 34(17): 1812-1832.
3	LUH Z, XUS F. Hollow mesoporous structured molecularly imprinted polymers for highly sensitive and selective detection of estrogens from food samples[J]. Journal of Chromatography A, 2017, 1501: 10-17.
4	VIVEIROSR, KARIMK, PILETSKYS A, et al. Development of a molecularly imprinted polymer for a pharmaceutical impurity in supercritical CO₂: rational design using computational approach[J]. Journal of Cleaner Production, 2017, 168: 1025-1031.
5	WHITCOMBEM J, CHIANELLAI, LARCOMBEL, et al. The rational development of molecularly imprinted polymer-based sensors for protein detection[J], Chemical Society Reviews, 2011, 40(3): 1547-1571.
6	ANIRUDHANT S, ALEXANDERS. A potentiometric sensor for the trace level determination of hemoglobin in real samples using multiwalled carbon nanotube based molecular imprinted polymer[J]. European Polymer Journal, 2017, 97: 84-93.
7	HANSEND F. Recent developments in the molecular imprinting of proteins[J]. Biomaterials, 2007, 28(29): 4178-4191.
8	LVY Q, TANT W，SVECF. Molecular imprinting of proteins in polymers attached to the surface of nanomaterials for selective recognition of biomacromolecules[J]. Biotechnology Advances, 2013, 31(8): 1172-1186.
9	MAX T, HEX W, LIW Y, et al. Epitope molecularly imprinted polymer coated quartz crystal microbalance sensor for the determination of human serum albumin[J]. Sensors and Actuators B: Chemical, 2017, 246: 879-886.
10	LID Y, HEX W, CHENY, et al. Novel hybrid structure silica/CdTe/molecularly imprinted polymer: synthesis, specific recognition, and quantitative fluorescence detection of bovine hemoglobin[J]. ACS Applied Materials & Interfaces, 2013, 5(23): 12609-12616.
11	GAOR X, MUX R, HAOY, et al. Combination of surface imprinting and immobilized template techniques for preparation of core-shell molecularly imprinted polymers based on directly amino-modified Fe₃O₄ nanoparticles for specific recognition of bovine hemoglobin[J]. Journal of Materials Chemistry B, 2014, 2(12): 1733-1741.
12	郭天瑛, 夏永清, 郝广杰, 等. 蛋白质分子印迹技术的研究进展[J]. 化工进展, 2003, 22(7): 713-716.
	GUOT Y, XIAY Q, HAOG J, et al. Progress in the study on proteins imprinted technique[J]. Chemical Industry and Engineering Progress, 2003, 22(7): 713-716.
13	徐菲菲, 段玉清, 张海晖, 等. 表面分子印迹聚合物载体研究新进展[J]. 化工进展, 2011, 30(5): 1033-1038.
	XUF F, DUANY Q, ZHANGH H, et al. Advance in surface molecularly imprinted carriers[J]. Chemical Industry and Engineering Progress, 2011, 30(5): 1033-1038.
14	WANGY F, ZHOUJ J, ZHANGB L, et al. Fabrication and characterization of glutathione-imprinted polymers on fibrous SiO₂ microspheres with high specific surface[J]. Chemical Engineering Journal, 2017, 327: 932-940.
15	LIUM M, PIJ Y, WANGX J, et al. A sol-gel derived pH-responsive bovine serum albumin molecularly imprinted poly(ionic liquids) on the surface of multiwall carbon nanotubes[J]. Analytica Chimica Acta, 2016, 932: 29-40.
16	LINJ T, LIUZ K, ZHUQ L, et al. Redox-responsive nanocarriers for drug and gene co-delivery based on chitosan derivatives modified mesoporous silica nanoparticles[J]. Colloids and Surfaces B: Biointerfaces, 2017, 155: 41-50.
17	SEDGHIR, YASSARIM, HEIDARIB. Thermo-responsive molecularly imprinted polymer containing magnetic nanoparticles: synthesis, characterization and adsorption properties for curcumin[J]. Colloids and Surfaces B: Biointerfaces, 2018, 162: 154-162.
18	LIX J, ZHANGB L, LIW, et al. Preparation and characterization of bovine serum albumin surface imprinted thermosensitive magnetic polymer microsphere and its application for protein recognition[J]. Biosensors & Bioelectronics, 2014, 51: 261-267.
19	LID Y, ZHANGX M, YANY J, et al. Thermo-sensitive imprinted polymer embedded carbon dots using epitope approach[J]. Biosensors & Bioelectronics, 2016, 9: 187-192.
20	杨紫淳, 高云玲, 姚克俭. 温敏型分子印迹水凝胶的研究进展[J]. 化工进展, 2014, 33 (1): 117-123.
	YANGZ C, GAOY L, YAOK J. Temperature-sensitive molecularly imprinted hydrogels[J]. Chemical Industry and Engineering Progress, 2014, 33 (1): 117-123.
21	许龙, 黄运安, 朱秋劲, 等. 基于壳聚糖的分子印迹聚合物的制备和应用[J]. 化工进展, 2016, 35 (3): 847-855.
	XUL, HUANGY A, ZHUQ J, et al. Preparation and application of molecularly imprinted polymers based on chitosan[J]. Chemical Industry and Engineering Progress, 2016, 35 (3): 847-855.
22	SHIQ H, TIANY, DONGX Y, et al. Chitosan-coated silica beads as immobilized metal affinity support for protein adsorption[J]. Biochemical Engineering Journal, 2003, 16(3): 317-322.
23	YUANQ, SHAHJ, HEINS, et al. Controlled and extended drug release behavior of chitosan-based nanoparticle carrier[J]. Acta Biomaterialia, 2010, 6(3): 1140-1148.
24	DENGZ W, ZHENZ P, HUX X, et al. Hollow chitosan-silica nanospheres as pH-sensitive targeted delivery carriers in breast cancer therapy[J]. Biomaterials, 2011, 32(21): 4976-4986.
25	LIC X, MAY, NIUH, et al. Hydrophilic hollow molecularly imprinted polymer microparticles with photo- and thermoresponsive template binding and release properties in aqueous media[J]. ACS Applied Materials & Interfaces, 2015, 7(49): 27340-27350.
26	DEMIRELG, ÖZÇETING, TURANE, et al. pH/Temperature-sensitive imprinted ionic poly(N-tert-butylacrylamide-co-acrylamide/ maleic acid) hydrogels for bovine serum albumin[J]. Macromolecular Bioscience, 2005, 5(11): 1032-1037.
27	WANGX L, YAOH F, LIX Y, et al. pH/Temperature-sensitive hydrogel-based molecularly imprinted polymers (hydroMIPs) for drug delivery by frontal polymerization[J]. RSC Advance, 2016, 6(96): 94038-94047.
28	GAOF X, ZHAOX L, HEX W, et al. A pH and temperature dual-responsive macroporous molecularly imprinted cryogel for enhanced recognition capability towards ovalbumin[J]. Analytical Methods, 2013, 5(23): 6700-6708.
29	DONGX Z, MAY, HOUC P, et al. Preparation of pH and temperature dual-sensitive molecularly imprinted polymers based on chitosan and N-isopropylacrylamide for recognition of bovine serum albumin[J]. Polymer International, 2019, 68(5): 955-963.
30	乔娟, 齐莉. 刺激-响应型蛋白质分子印迹材料的研究进展[J]. 科学通报, 2019, 64(13): 1330-1339.
	QIAOJ, QIL. Progress of stimuli-responsive molecular imprinted materials for capture/release of proteins (in Chinese)[J]. Chin. Sci. Bull., 2019, 64(13): 1330-1339.
31	STŐBERW, FINKA, BOHNE. Controlled growth of monodisperse silica spheres in the micron size range[J]. Journal of Colloid and Interface Science, 1968, 26(1): 62-69.
32	BOURGEAT-LAMIE, LANGJ. Encapsulation of inorganic particles by dispersion polymerization in polar media: 1. Silica nanoparticles encapsulated by polystyrene[J]. Journal of Colloid and Interface Science, 1998,197(2): 293-308.
33	CHENK M, WANGS Y, LIL, et al. Binding between proteins and cationic spherical polyelectrolyte brushes: effect of pH, ionic strength, and stoichiometry[J]. Biomacromolecules, 2013, 14(3): 818-827.
34	倪永年, 葛成相. 多元校正分光光度法同时测定兔血清内的氨甲苯酸和酚磺乙胺[J]. 分析科学学报, 2006, 22(6): 683-686.
	NIY N, GEC X. Simultaneous spectrophotometric determination of aminomethylbenzoic acid and etamsylate in rabbit serum using multivariate calibration methods[J]. Journal of Analytical Science, 2006, 22(6): 683-686.

聚合物	K/mL·mg^-1	Q_max/mg·g^-1	相关系数R
MIP	16.91	89.03	0.9969
NIP	5.51	53.43	0.9825

聚合物	K/mL·mg^-1	Q_max/mg·g^-1	相关系数R
MIP	16.91	89.03	0.9969
NIP	5.51	53.43	0.9825

蛋白质种类	紫外吸收波长/nm	分子量	等电点（PI）	印迹因子（IF）	选择因子（β）
BSA	278	66000	4.9	2.02	—
BHb	405	68000	6.8	1.08	1.82
HSA	277	66000	4.7～4.9	1.25	1.58
OVA	277	43000	4.7	1.18	1.67
Lyz	280.5	14300	11	1.16	1.70

蛋白质种类	紫外吸收波长/nm	分子量	等电点（PI）	印迹因子（IF）	选择因子（β）
BSA	278	66000	4.9	2.02	—
BHb	405	68000	6.8	1.08	1.82
HSA	277	66000	4.7～4.9	1.25	1.58
OVA	277	43000	4.7	1.18	1.67
Lyz	280.5	14300	11	1.16	1.70

[1]	LIN Xiaopeng, XIAO Youhua, GUAN Yichen, LU Xiaodong, ZONG Wenjie, FU Shenyuan. Recent progress of flexible electrodes for ion polymer-metal composites (IPMC) [J]. Chemical Industry and Engineering Progress, 2023, 42(9): 4770-4782.
[2]	QIAN Sitian, PENG Wenjun, ZHANG Xianming. Comparative analysis of forming cyclic oligomers via PET melt polycondensation and cyclodepolymerization [J]. Chemical Industry and Engineering Progress, 2023, 42(9): 4808-4816.
[3]	WANG Shaofan, ZHOU Ying, HAO Kang’an, HUANG Anrong, ZHANG Ruju, WU Chong, ZUO Xiaoling. Self-healing and blue-light hydrogel with pH responsiveness [J]. Chemical Industry and Engineering Progress, 2023, 42(9): 4837-4846.
[4]	ZHU Chuanqiang, RU Jinbo, SUN Tingting, XIE Xingwang, LI Changming, GAO Shiqiu. Characteristics of selective non-catalytic reduction of NO _x with solid polymer denitration agent [J]. Chemical Industry and Engineering Progress, 2023, 42(9): 4939-4946.
[5]	LI Bogeng, LUO Yingwu, LIU Pingwei. Consideration on research content and method of polymer product engineering [J]. Chemical Industry and Engineering Progress, 2023, 42(8): 3905-3909.
[6]	WANG Baoying, WANG Huangying, YAN Junying, WANG Yaoming, XU Tongwen. Research progress of polymer inclusion membrane in metal separation and recovery [J]. Chemical Industry and Engineering Progress, 2023, 42(8): 3990-4004.
[7]	CHEN Junjun, FEI Chang’en, DUAN Jintang, GU Xueping, FENG Lianfang, ZHANG Cailiang. Research progress on chemical modification of polyether ether ketone for the high bioactivity [J]. Chemical Industry and Engineering Progress, 2023, 42(8): 4015-4028.
[8]	YU Jingwen, SONG Luna, LIU Yanchao, LYU Ruidong, WU Mengmeng, FENG Yu, LI Zhong, MI Jie. An indole-bearing hypercrosslinked polymer In-HCP for iodine adsorption from water [J]. Chemical Industry and Engineering Progress, 2023, 42(7): 3674-3683.
[9]	YU Xixi, ZHANG Jinshuai, LEI Wen, LIU Chengguo. Research progress of self-healing photocuring polymeric materials based on dynamic covalent bonds [J]. Chemical Industry and Engineering Progress, 2023, 42(7): 3589-3599.
[10]	SUN Zhengnan, LI Hongjing, JING Guolin, ZHANG Funing, YAN Biao, LIU Xiaoyan. Application of EVA and its modified polymer in crude oil pour point depressant field [J]. Chemical Industry and Engineering Progress, 2023, 42(6): 2987-2998.
[11]	YU Dingyi, LI Yuanyuan, WANG Chenyu, JI Yongsheng. Preparation of lignin-based pH responsive hydrogel and its application in controlled drug release [J]. Chemical Industry and Engineering Progress, 2023, 42(6): 3138-3146.
[12]	YANG Farong, GU Lili, LIU Yang, LI Weixue, CAI Jieyun, WANG Huiping. Preparation and application of molecularly imprinted polymers of terbutylazine assisted by computer simulation [J]. Chemical Industry and Engineering Progress, 2023, 42(6): 3157-3166.
[13]	YANG Jiatian, TANG Jinming, LIANG Zirong, LI Yinhong, HU Huayu, CHEN Yuan. Preparation and application of novel starch-based super absorbent polymer dust suppressant [J]. Chemical Industry and Engineering Progress, 2023, 42(6): 3187-3196.
[14]	WANG Lin, XIN Meihua, LI Mingchun, CHEN Qi, MAO Yangfan. Preparation of quaternized/sulfonated chitosan and its anti-biofilm activity [J]. Chemical Industry and Engineering Progress, 2023, 42(5): 2577-2585.
[15]	HE Zhiyong, GUO Tianfo, WANG Jinli, LYU Feng. Progress of CO₂/epoxide copolymerization catalyst [J]. Chemical Industry and Engineering Progress, 2023, 42(4): 1847-1859.