PDMA-b-PEO-b-PDMA的合成及其用于毛细管电泳分离蛋白质的研究
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摘要
毛细管电泳(capillary electrophoresis, CE)由于其分离速度快,分离效率高,及样品消耗量少等特点,而成为多肽、蛋白质、DNA和氨基酸等生物分子分离的一个有力工具,并在生物工程、医学药物等生命科学领域以及食品检测等化学领域显示了极其重要的应用前景。但是毛细管表面的硅羟基(Si-OH)电离为Si-O-,在毛细管电泳分离蛋白质过程中,管壁与蛋白质尤其是碱性蛋白质会产生强烈的吸附,从而造成蛋白质分离效率下降、峰对称性差、迁移时间重现性差、蛋白质回收率低等问题。
目前,为了抑制蛋白质在毛细管壁的吸附,人们常采用的方法主要有极端pH值法、添加剂法、外加电场和聚合物涂层等。极端pH法和添加剂法不利于分离选择性,甚至会引起蛋白质变性;外加电场则受目前实验仪器条件的限制,且实验结果受焦耳热影响;相较而言聚合物涂层法不仅高效而且稳定,可以有效是解决这个问题。毛细管涂层的形成大致可分为两类:共价键合和物理吸附。共价键合涂层法制备复杂、耗时耗力。其中共价键合涂层稳定性好但制备过程复杂且制备过程难以控制;物理吸附涂层的制作简单且稳定性较好,因而备受青睐。目前已用于物理吸附改性毛细管壁的聚合物主要有中性聚合物和阳离子聚合物。其中有聚N, N-二甲基丙烯酰胺(PDMA),聚乙烯醇(PVA),聚乙烯亚胺(PEI)聚羟乙基丙烯酰胺(PHEA),聚环氧乙烷(PEO)等。
本论文通过原子转移自由基聚合(atom transfer radical polymerization, ATRP)方法合成了三嵌段共聚物聚((N, N-二甲基丙烯酰胺)-b-乙二醇-b-(N, N-二甲基丙烯酰胺))(PDMA-b-PEO-b-PDMA),并将其作为物理涂层分离蛋白质,取得了较好效果,主要研究工作如下:
一、通过ATRP法合成了一组不同分子量的PDMA-b-PEO-b-PDMA三嵌段共聚物,并用核磁(1H NMR)对其结构,凝胶渗透色谱(GPC)对其分子量及分子量分布进行表征。
二、PDMA-b-PEO-b-PDMA中PEO链段和PDMA链段均对毛细管具有涂覆作用,特别是相对疏水的PDMA链段,它可以使聚合物涂层的稳定性大大增加。将PDMA-b-PEO-b-PDMA三嵌段共聚物用作物理涂层材料,研究了PDMA分子量、缓冲溶液pH值及共聚物结构对碱性蛋白质分离效率的影响,同时考察了该三嵌段共聚物对酸、碱、中性混合蛋白质,及生物大分子的分离效果。
三、测量PDMA-b-PEO-b-PDMA三嵌段共聚物涂层管、PEO涂层管和空管在pH 3.17-8.00范围内的电渗流(EOF),以考察pH值分别对它们EOF的影响。以EOF的变化为蛋白质吸附的一个表征手段,多次测量EOF,比较PDMA-b-PEO-b-PDMA涂层管与空管,发现PDMA-b-PEO-b-PDMA涂层能有效的抑制蛋白质吸附,并且控制EOF的大小和稳定性,是性能优良的涂层材料。
Capillary electrophoresis (CE), which offers high separation efficiency, less sample and reagent consumption and short analysis time, has emerged as a powerful analytical method for separation of biomacromolecules, such as peptides, proteins, DNA and nucleic acids. However, the separation of proteins is complicated for their tendency to adsorb onto the negatively charged surface of fused silica capillaries, and this is particularly true for the separation of basic proteins. The adsorption will seriously damage separation efficiency. Firstly, adsorption causes tailing of the sample peak, which leads to loss of resolution and sensitivity. Secondly it will change theζ-potential of the capillary which affects the electroosmotic flow (EOF) rate, giving improper migration times and poor repeatability. Also theζ-potential caused by local adsorption of proteins causes additional peak broadening.
In order to minimize protein adsorption and stabilize EOF, numerous approaches have been explored, including the use of extreme pH, high ionic strength and zwitterionic additives. However, the most common approach has been to coat the capillary wall, and the major objective of capillary coating is to modify or mask the silanol groups on the capillary surface by presenting a more inert surface. Nowadays, polymer coatings for capillary become more popular, in which the capillary can be modified with polymers by covalent bond or physical adsorption. However, the procedures employed to prepare a covalent coating are laborious and time consuming. Physically adsorbed coating becomes more attractive due to its fast coating procedure and reproducibility. Several polymers such as poly (N, N-dimethylacrylamide) (PDMA), poly (vinyl alcohol) (PVA), polyethyleneimine (PEI), poly (ethylene oxide) (PEO) and poly (N-hydroxy- ethylacrylamide) (PHEA) performed as physical adsorbed coatings can reduce wall-protein interaction and effectively control the EOF.
In this paper, a range of well-defined triblock copolymer, poly (N, N-dimethylacrylamide)-block-poly (ethylene oxide)-block-poly (N, N-dimethylacryl- amide) (PDMA-b-PEO-b-PDMA) was synthesized by atom transfer radical polymerization (ATRP), which was used as physical coating of capillaries for proteins separation. Work was on the following aspects:
1. A series of copolymers of PDMA-b-PEO-b-PDMA was synthesized with different relative molecular mass of PDMA, and characterized by 1H NMR and GPC;
2. Copolymers of PDMA-b-PEO-b-PDMA were used as physical coating of CE for proteins separation. The effect of relative molecular mass of PDMA of copolymers, pH value and structure of copolymers on proteins separation was studied;
3. The influence of pH value on the EOF of PDMA-b-PEO-b-PDMA coated capillary, PEO coated capillary and bare capillary were investigated. The variation of EOF can be used as a measure of the protein adsorption. By comparing the EOF of bare capillary with PDMA-b-PEO-b-PDMA coated capillary, the PDMA-b-PEO- b-PDMA coating showed good ability on minimizing protein adsorption and stabilizing EOF.
目前,为了抑制蛋白质在毛细管壁的吸附,人们常采用的方法主要有极端pH值法、添加剂法、外加电场和聚合物涂层等。极端pH法和添加剂法不利于分离选择性,甚至会引起蛋白质变性;外加电场则受目前实验仪器条件的限制,且实验结果受焦耳热影响;相较而言聚合物涂层法不仅高效而且稳定,可以有效是解决这个问题。毛细管涂层的形成大致可分为两类:共价键合和物理吸附。共价键合涂层法制备复杂、耗时耗力。其中共价键合涂层稳定性好但制备过程复杂且制备过程难以控制;物理吸附涂层的制作简单且稳定性较好,因而备受青睐。目前已用于物理吸附改性毛细管壁的聚合物主要有中性聚合物和阳离子聚合物。其中有聚N, N-二甲基丙烯酰胺(PDMA),聚乙烯醇(PVA),聚乙烯亚胺(PEI)聚羟乙基丙烯酰胺(PHEA),聚环氧乙烷(PEO)等。
本论文通过原子转移自由基聚合(atom transfer radical polymerization, ATRP)方法合成了三嵌段共聚物聚((N, N-二甲基丙烯酰胺)-b-乙二醇-b-(N, N-二甲基丙烯酰胺))(PDMA-b-PEO-b-PDMA),并将其作为物理涂层分离蛋白质,取得了较好效果,主要研究工作如下:
一、通过ATRP法合成了一组不同分子量的PDMA-b-PEO-b-PDMA三嵌段共聚物,并用核磁(1H NMR)对其结构,凝胶渗透色谱(GPC)对其分子量及分子量分布进行表征。
二、PDMA-b-PEO-b-PDMA中PEO链段和PDMA链段均对毛细管具有涂覆作用,特别是相对疏水的PDMA链段,它可以使聚合物涂层的稳定性大大增加。将PDMA-b-PEO-b-PDMA三嵌段共聚物用作物理涂层材料,研究了PDMA分子量、缓冲溶液pH值及共聚物结构对碱性蛋白质分离效率的影响,同时考察了该三嵌段共聚物对酸、碱、中性混合蛋白质,及生物大分子的分离效果。
三、测量PDMA-b-PEO-b-PDMA三嵌段共聚物涂层管、PEO涂层管和空管在pH 3.17-8.00范围内的电渗流(EOF),以考察pH值分别对它们EOF的影响。以EOF的变化为蛋白质吸附的一个表征手段,多次测量EOF,比较PDMA-b-PEO-b-PDMA涂层管与空管,发现PDMA-b-PEO-b-PDMA涂层能有效的抑制蛋白质吸附,并且控制EOF的大小和稳定性,是性能优良的涂层材料。
Capillary electrophoresis (CE), which offers high separation efficiency, less sample and reagent consumption and short analysis time, has emerged as a powerful analytical method for separation of biomacromolecules, such as peptides, proteins, DNA and nucleic acids. However, the separation of proteins is complicated for their tendency to adsorb onto the negatively charged surface of fused silica capillaries, and this is particularly true for the separation of basic proteins. The adsorption will seriously damage separation efficiency. Firstly, adsorption causes tailing of the sample peak, which leads to loss of resolution and sensitivity. Secondly it will change theζ-potential of the capillary which affects the electroosmotic flow (EOF) rate, giving improper migration times and poor repeatability. Also theζ-potential caused by local adsorption of proteins causes additional peak broadening.
In order to minimize protein adsorption and stabilize EOF, numerous approaches have been explored, including the use of extreme pH, high ionic strength and zwitterionic additives. However, the most common approach has been to coat the capillary wall, and the major objective of capillary coating is to modify or mask the silanol groups on the capillary surface by presenting a more inert surface. Nowadays, polymer coatings for capillary become more popular, in which the capillary can be modified with polymers by covalent bond or physical adsorption. However, the procedures employed to prepare a covalent coating are laborious and time consuming. Physically adsorbed coating becomes more attractive due to its fast coating procedure and reproducibility. Several polymers such as poly (N, N-dimethylacrylamide) (PDMA), poly (vinyl alcohol) (PVA), polyethyleneimine (PEI), poly (ethylene oxide) (PEO) and poly (N-hydroxy- ethylacrylamide) (PHEA) performed as physical adsorbed coatings can reduce wall-protein interaction and effectively control the EOF.
In this paper, a range of well-defined triblock copolymer, poly (N, N-dimethylacrylamide)-block-poly (ethylene oxide)-block-poly (N, N-dimethylacryl- amide) (PDMA-b-PEO-b-PDMA) was synthesized by atom transfer radical polymerization (ATRP), which was used as physical coating of capillaries for proteins separation. Work was on the following aspects:
1. A series of copolymers of PDMA-b-PEO-b-PDMA was synthesized with different relative molecular mass of PDMA, and characterized by 1H NMR and GPC;
2. Copolymers of PDMA-b-PEO-b-PDMA were used as physical coating of CE for proteins separation. The effect of relative molecular mass of PDMA of copolymers, pH value and structure of copolymers on proteins separation was studied;
3. The influence of pH value on the EOF of PDMA-b-PEO-b-PDMA coated capillary, PEO coated capillary and bare capillary were investigated. The variation of EOF can be used as a measure of the protein adsorption. By comparing the EOF of bare capillary with PDMA-b-PEO-b-PDMA coated capillary, the PDMA-b-PEO- b-PDMA coating showed good ability on minimizing protein adsorption and stabilizing EOF.
引文
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