色谱

色谱 ›› 2020, Vol. 38 ›› Issue (9): 1085-1094.DOI: 10.3724/SP.J.1123.2020.02027

• 研究论文 • 上一篇    下一篇

基于聚(2-甲基-2-噁唑啉)和聚丙烯酸的混合刷在毛细管电泳在线富集溶菌酶中的应用

张淼1, 王雨晨1, MUHAMMADAtif1, 陈丽娟2, 王延梅1,*()   

  1. 1 中国科学技术大学高分子科学与工程系, 安徽 合肥 230026
    2 皖西学院材料与化工学院, 安徽 六安 237012
  • 收稿日期:2020-02-26 出版日期:2020-09-08 发布日期:2020-12-11
  • 通讯作者: 王延梅
  • 作者简介:王延梅.Tel:(0551)63607652, E-mail:wangyanm@ustc.edu.cn
  • 基金资助:
    国家自然科学基金项目(21674102)

Application of mixed polymer brushes based on poly(2-methyl-2-oxazoline) and poly(acrylic acid) to on-line preconcentration of lysozyme by capillary electrophoresis

ZHANG Miao1, WANG Yuchen1, MUHAMMAD Atif1, CHEN Lijuan2, WANG Yanmei1,*()   

  1. 1 Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei 230026, China
    2 College of Materials and Chemical Engineering, West Anhui University, Lu'an 237012, China
  • Received:2020-02-26 Online:2020-09-08 Published:2020-12-11
  • Contact: WANG Yanmei
  • Supported by:
    National Natural Science Foundation of China(21674102)

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摘要：

制备了一种对溶菌酶具有可控吸附性能的混合刷涂层毛细管，用于毛细管电泳在线富集溶菌酶以提高其检测灵敏度。首先，分别通过阳离子开环聚合和可逆加成-断裂链转移（RAFT）聚合合成聚（2-甲基-2-噁唑啉）（PMOXA）和聚丙烯酸（PAA），然后将甲基丙烯酸缩水甘油酯（GMA）分别与PMOXA和PAA通过自由基共聚和RAFT聚合合成出聚（2-甲基-2-噁唑啉）-r-甲基丙烯酸缩水甘油酯（PMOXA-r-GMA）和聚丙烯酸-b-聚甲基丙烯酸缩水甘油酯（PAA-b-PGMA）。将PMOXA-r-GMA和PAA-b-PGMA的混合溶液以一定比例加入到毛细管内，通过加热即可制备出基于PMOXA和PAA的混合刷涂层毛细管。X射线光电子能谱（XPS）对毛细管原材料的表面组成研究结果表明，当混合溶液质量浓度为20 g/L、PMOXA-r-GMA和PAA-b-PGMA质量比为1：1时，所得涂层中羧基的含量随着PAA链长的增加而增加；异硫氰酸荧光素标记溶菌酶（FITC-溶菌酶）吸附实验结果显示，通过改变环境的pH和离子强度（I）可以调控涂层毛细管对溶菌酶的吸附和释放，在pH 7（I=10-5mol/L）条件下，毛细管可以吸附大量的溶菌酶，当条件变为pH 3（I=10-1mol/L）时，吸附的溶菌酶可以被释放出来。将这种具有溶菌酶可控吸附性能的涂层毛细管用于毛细管电泳在线富集溶菌酶，当PAA链长是PMOXA链长的2.2倍时，溶菌酶的灵敏度增强因子为17.69，检出限为8.7×10-5g/L；同一天内对溶菌酶连续测定5次以及连续测定5天，峰面积的日内、日间相对标准偏差（RSD）分别为2.9%和4.1%，迁移时间的日内、日间RSD分别为0.9%和2.1%。涂层的制备只需一步，简单易行，而且涂层具有很好的稳定性。本研究为毛细管电泳分析痕量蛋白质提供了一种简单有效的方法。

关键词: 毛细管电泳, 在线富集, 混合刷, 聚(2-甲基-2-噁唑啉), 聚丙烯酸

Abstract:

A capillary coated with mixed polymer brushes that shows switchability toward lysozyme adsorption was developed. This capillary was applied for the on-line preconcentration of lysozyme by capillary electrophoresis (CE) in order to enhance the detection sensitivity. First, poly(2-methyl-2-oxazoline) (PMOXA) and poly(acrylic acid) (PAA) were synthesized by cationic ring-opening polymerization and reversible addition-fragmentation chain transfer (RAFT) polymerization, respectively. Then, glycidyl methacrylate (GMA) and PMOXA were used to prepare poly(2-methyl-2-oxazoline)-random-glycidyl methacrylate (PMOXA-r-GMA) via radical copolymerization, and poly(acrylic acid)-block-poly(glycidyl methacrylate) (PAA-b-PGMA) was obtained by the RAFT polymerization of GMA and PAA. A mixed solution of PMOXA-r-GMA and PAA-b-PGMA at a certain mass ratio was then injected into the capillary. Subsequent annealing provided capillary materials coated with mixed polymer brushes based on PMOXA and PAA. X-ray photoelectron spectroscopy (XPS) analysis was performed to determine the surface composition of the capillary raw materials. When the mass concentration of the mixed solution was 20 g/L and the mass ratio of PMOXA-r-GMA and PAA-b-PGMA was 1:1, the carboxyl content in the coating increased with increasing chain length of PAA. Fluorescein isothiocyanate-labeled lysozyme (FITC-lysozyme) adsorption assay demonstrated that the coated capillary had switchable properties for lysozyme adsorption upon pH and ionic strength (I) trigger. At pH 7 (I=10-5mol/L), the capillary could adsorb a large amount of lysozyme, which could be released at pH 3 (I=10-1mol/L). Subsequently, this coated capillary was applied to the on-line preconcentration of lysozyme by CE. The sensitivity enhancement factor was 17.69 when the chain length of PAA was 2.2 times that of PMOXA. The limit of detection could reach 8.7×10-5g/L. On-line preconcentration of lysozyme was performed for five successive times on the same day and on five consecutive days. The intraday and interday relative standard deviations (RSDs) for the peak areas were 2.9% and 4.1%, respectively. The intraday and interday RSDs for the migration times were 0.9% and 2.1%, respectively. The developed method for the preparation of the coated capillary with good stability only needs one step in this work, and this research will supply a simple and effective way to analyze trace protein by CE.

Key words: capillary electrophoresis (CE), on-line preconcentration, mixed polymer brushes, poly(2-methyl-2-oxazoline) (PMOXA), poly(acrylic acid) (PAA)