用于蛋白质BSA识别的温度/pH双敏印迹聚合物

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

摘要/Abstract

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

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

中图分类号:

O658

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.

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.

图/表 13

图1 GPTMS-SiO2、 MIP和NIP 的TEM图像

图2 SiO2、GPTMS-SiO2、MIP和NIP的红外谱图a—SiO2；b—GPTMS-SiO2；c—MIP；d—NIP

图3 SiO2、 GPTMS-SiO2、MIP和NIP的热失重图a—SiO2； b—GPTMS-SiO2； c—MIP； d—NIP

图4 不同温度和pH下MIP的溶胀率

图5 温度和pH对吸附容量的影响

图6 温度/pH双敏聚合物的识别机理

图7 MIP和NIP的吸附等温线

表1 吸附等温方程求得参数值

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

图8 MIP和NIP的吸附动力学曲线

图9 MIP和NIP的特异性吸附容量

表2 MIP的特异性吸附性能

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

图10 MIP和NIP的竞争性吸附容量

图11 MIP和NIP的重复使用性

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