离子型聚合物的制备及在蛋白质分离中的应用
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摘要
毛细管电泳(capillary electrophoresis, CE)是指荷电粒子或离子依靠直流电场驱动力作用,在毛细管中以不同速度定向运动的行为。由于其具有分离效率高、样品消耗量少、分析速度快、操作简便等特点被广泛的应用于各种分离分析领域,其中如应用于氨基酸、核酸、多肽、蛋白质等生物大分子的分离。我们通常使用的熔融硅毛细管是由具有良好的导热和紫外透过性的熔融硅制备。在缓冲溶液的作用下,未经修饰的熔融硅毛细管内壁上的Si-OH会发生解离,形成带有负电荷的内表面(Si-O-),当这类毛细管用于分离蛋白质类生物大分子时,会因为毛细管内壁和蛋白质间的静电或疏水等相互作用而产生十分严重的吸附现象。从而导致蛋白质的分离效率下降,迁移时间重现性差、峰拖尾、变形,甚至无法实现有效分离等问题。
为解决或缓解这种现象,研究人员已进行了很多尝试,如采用极端pH值,两性离子添加剂,高离子强度等方法,但目前最常见有效的办法是通过共价键合或物理吸附两种途径在毛细管内壁上形成聚合物涂层。聚合物涂层可以起到掩蔽毛细管内壁上的Si-OH,抑制Si-OH的解离,从而阻抗蛋白质在内壁上吸附等作用。从涂层的稳定性方面来说,通过共价键合到毛细管内壁上的涂层要好于物理吸附涂层,但是共价键合涂层管制备过程繁琐且难控制,涂层再生性差。相比之下,物理吸附涂层管则制备简单易于控制,再生性强,因而近年来物理吸附涂层越来越受到研究人员的青睐。
围绕物理吸附涂层的制备,取得的主要研究成果如下：
1.利用硝酸铈铵(CAN)在硝酸溶液中引发的普通自由基聚合反应合成了不同接枝率的接枝共聚物羟乙基纤维素接枝聚甲基丙烯酸N，N-二甲氨基乙酯(HEC-g-PDMAEMA),然后使其通过物理吸附在毛细管内壁上形成HEC-g-PDMAEMA涂层。研究结果表明,相对于未经修饰的毛细管,HEC-g-PDMAEMA涂层管能有效地抑制电渗流,且涂层毛细管的电渗流不仅可以通过缓冲溶液的pH值来调节,还可以通过选择不同接枝率的接枝共聚物HEC-g-PDMAEMA来调节,这使得酸性蛋白质和碱性蛋白质可以在同一毛细管中实现分离。我们还详细研究了不同的接枝率和缓冲溶液pH值的改变对蛋白质分离结果的影响。最后,我们使用HEC-g-PDMAEMA涂层管对现实生活中的样品鸡蛋清蛋白和奶粉蛋白进行了定性定量分析。
2.通过ATRP方法制备了三嵌段共聚物(聚甲基丙烯酸N,N-二甲氨基乙酯-b-聚氧乙烯-b-聚甲基丙烯酸N,N-二甲氨基乙酯,PDMAEMA-b-PEO-b-PDMAEMA),并用碘甲烷使三嵌段共聚物PDMAEMA链段上的叔胺完全季铵化。季铵化的三嵌段共聚物两端是带正电荷的PDMAEMA链段,中间链段为具有优异阻抗蛋白吸附性能的PEO。季铵化的三嵌段共聚物(QDED)与毛细管内壁通过静电和氢键相互作用结合在一起,形成物理吸附涂层。比较不同pH值的缓冲溶液和不同PDMAEMA链段长度的QDED涂层管的电渗流变化,选择合适的分离条件进行蛋白质的分离。研究结果表明：相同pH值下PDMAEMA链段越长,电渗流(绝对值)就越大；不同PDMAEMA嵌段长度的三嵌段共聚物QDED在pH值3.0到7.0范围内可实现蛋白质混合物的快速高效分离。和未经修饰的毛细管相比较,QDED涂层管能够一次性分离蛋白质的混合物(酸性,中性和碱性)。并使用QDED涂层管对鸡蛋清中的相应蛋白做了定量分析。
3.通过可逆加成断裂链转移(RAFT)聚合合成了单体比例不同的无规共聚物poly(DMA-co-SBMA),这种共聚物是由抗污性能优异的聚(甲基丙烯酸甲酯磺基甜菜碱)(PSBMA)和具有自涂覆性能的聚合物Ⅳ,N-二甲基丙烯酰胺(PDMA)组成的。用接触角仪和X射线光电子能谱仪分别研究了聚合物在玻璃片上形成涂层的亲水性和组成。研究了不同离子强度和不同pH值对蛋白质分离效率的影响,并比较了无规共聚物涂层管、未经修饰的毛细管以及PSBMA涂层管蛋白质分离效率的差异。结果表明,对于无规共聚物涂层玻璃片,随着SBMA含量的增加,涂层的亲水性和涂覆能力都在逐渐增加。在80mM的缓冲溶液中,poly(DMA-co-SBMA)涂层管拥有最高的分离效率,最高可达到1509000N/m；在pH值3.0～5.0范围内,10min即可实现四种碱性蛋白质的分离；蛋白质迁移时间的RSD在0.23%～2.92%之间。
Capillary electrophoresis is refer to the charged particle or ion depends on the high voltage electric field drive, directional movement behavior at different speeds in the capillary. Due to its high separation efficiency, less sample consumption, rapid separation time, easy operation and other characteristics, it is applied in various kinds of separation and analysis fields, such as applied to bio-macromolecules separation such as amino acid, nucleic acid, peptide and protein, etc. Fused-silica is a kind of material which is used to manufacture the capillary owing to its good thermal conductivity and ultraviolet transparency. However, under the effect of buffer solution, the capillary inner wall would be negatively charged due to the dissociation of silanol groups. When the capillary was used to separate protein, the interaction between capillary inner wall and proteins would result in protein adsorption. It would lead to the decrease of the separation efficiency and reproducibility of migration time, peak trailing, deformation, and even unable to achieve effective separation and so on.
Numerous approaches have been applied to minimize this adsorption, such as extreme pH, zwitterionic additives, high ionic strength, but by far the most common and effective way is using covalent bonding or physical adsorption to form polymer coating on the capillary wall. Silanol groups on the capillary inner wall can be masked by the polymer coating, the electroosmotic flow is suppressed, and the interaction of protein-wall would be decreased, etc. From the aspects of stability of the coating, coating which covalently bonded onto the inner wall of capillary is more stability than the physically adsorbed coating, however, process of the preparation of covalently linked coating is cumbersome and difficult to control, and coating reproducibility is poor. Compared to the covalently bonded coating, process of the preparation of physically adsorbed coating is simple, easy to control, strong regeneration, and therefore in recent years the physically adsorbed coating is more and more get the favour of scientists.
Around the preparation of physically adsorbed polymer coatings, the main results are as follows:
1. The graft copolymers of hydroxyethylcellulose-graft-poly(2-(dimethylamino)ethyl methacrylate) (HEC-g-PDMAEMA) with different graft ratio were synthesized by using ceric ammonium nitrate initiator in aqueous nitric acid solution. Electroosmotic flow measurement results showed that the synthesized HEC-g-PDMAEMA graft copolymer coated capillary could suppress EOF effectively compared to the bare fused-silica capillary, and efficient separations of basic proteins were also achieved. The electrical charge of the coated capillary wall could be modulated by varying not only the pH of the running buffer, but also the grafting ratio of poly(2-(dimethylamino)ethyl methacrylate) grafts, which makes it is possible to analyze the basic and acidic proteins in the same capillary. The effects of poly(2-(dimethylamino)ethyl methacrylate) grafting ratio in HEC-g-PDMAEMA and buffer pH on the separation of basic proteins for capillary electrophoresis were investigated in detail. Furthermore, egg white proteins and milk powder samples were separated by the HEC-g-PDMAEMA coated capillary, respectively.
2. A novel noncovalent adsorbed coating for capillary electrophoresis has been prepared and explored. This coating was based on quaternized poly(2-(dimethylamino)ethyl methacrylate)-block-poly(ethylene oxide)-block-poly(2-(dimethylamino)ethyl methacrylate)(QDED) triblock copolymer which was synthesized by atomic transfer radical polymerization(ATRP) in our laboratory. The polycationic polymer and the negatively charged fused-silica surface attracted each other through electrostatic interactions and hydrogen bonds. It was demonstrated that the coated capillaries provided an electroosmotic flow with reverse direction, and the magnitude of the electroosmotic flow can be modulated by varying the molecular mass of poly(2-(dimethylamino)ethyl methacrylate)(PDMAEMA) block and pH value of the buffer, respectively. The effects of the molecular mass of PDMAEMA block in QDED triblock copolymer and pH value of the buffer on the separation of basic proteins were investigated in detail. The triblock copolymer coatings showed higher separation efficiency, better migration time repeatability and would apply to wider range of pH than bare fused-silica capillary when used in separating proteins. Proteins from egg white were also separated respectively through this QDED triblock copolymer coated capillary. These results demonstrated that the QDED triblock copolymer coatings are suitable for analyzing biosample.
3. Firstly, the poly(DMA-co-SBMA)s with different feed ratio (SBMA/DMA) were synthesized via the reversible addition fragmentation chain transfer (RAFT) polymerization. And then, X-ray photoelectron spectroscopy (XPS) and water contact angel (CA) were used to investigate the composition and hydrophilicity of poly(DMA-co-SBMA) coating formed on the glass slide surfaces. CA measurements revealed that the poly(DMA-co-SBMA) coating became more hydrophilic with the increment of feed ratio (SBMA/DMA), and at the same time the XPS results showed that the coating ability was also increased with the increment of feed ratio. Followed, the copolymer was applied to coat the fused-silica capillary inner wall and the coated capillary was used to separate the mixture of proteins (lysozyme, cytochrome c, ribonuclease A and α-chymotrypsinogen A) in a pH range from3.0to5.0. Under the optimum conditions, an excellent separation of basic proteins with peak efficiencies ranging from551000to1509000N/m had been accomplished within10min. Furthermore, the compare of separation efficiency among the bare, PSBMA and poly(DMA-co-SBMA) coated capillary was also investigated.
为解决或缓解这种现象,研究人员已进行了很多尝试,如采用极端pH值,两性离子添加剂,高离子强度等方法,但目前最常见有效的办法是通过共价键合或物理吸附两种途径在毛细管内壁上形成聚合物涂层。聚合物涂层可以起到掩蔽毛细管内壁上的Si-OH,抑制Si-OH的解离,从而阻抗蛋白质在内壁上吸附等作用。从涂层的稳定性方面来说,通过共价键合到毛细管内壁上的涂层要好于物理吸附涂层,但是共价键合涂层管制备过程繁琐且难控制,涂层再生性差。相比之下,物理吸附涂层管则制备简单易于控制,再生性强,因而近年来物理吸附涂层越来越受到研究人员的青睐。
围绕物理吸附涂层的制备,取得的主要研究成果如下：
1.利用硝酸铈铵(CAN)在硝酸溶液中引发的普通自由基聚合反应合成了不同接枝率的接枝共聚物羟乙基纤维素接枝聚甲基丙烯酸N，N-二甲氨基乙酯(HEC-g-PDMAEMA),然后使其通过物理吸附在毛细管内壁上形成HEC-g-PDMAEMA涂层。研究结果表明,相对于未经修饰的毛细管,HEC-g-PDMAEMA涂层管能有效地抑制电渗流,且涂层毛细管的电渗流不仅可以通过缓冲溶液的pH值来调节,还可以通过选择不同接枝率的接枝共聚物HEC-g-PDMAEMA来调节,这使得酸性蛋白质和碱性蛋白质可以在同一毛细管中实现分离。我们还详细研究了不同的接枝率和缓冲溶液pH值的改变对蛋白质分离结果的影响。最后,我们使用HEC-g-PDMAEMA涂层管对现实生活中的样品鸡蛋清蛋白和奶粉蛋白进行了定性定量分析。
2.通过ATRP方法制备了三嵌段共聚物(聚甲基丙烯酸N,N-二甲氨基乙酯-b-聚氧乙烯-b-聚甲基丙烯酸N,N-二甲氨基乙酯,PDMAEMA-b-PEO-b-PDMAEMA),并用碘甲烷使三嵌段共聚物PDMAEMA链段上的叔胺完全季铵化。季铵化的三嵌段共聚物两端是带正电荷的PDMAEMA链段,中间链段为具有优异阻抗蛋白吸附性能的PEO。季铵化的三嵌段共聚物(QDED)与毛细管内壁通过静电和氢键相互作用结合在一起,形成物理吸附涂层。比较不同pH值的缓冲溶液和不同PDMAEMA链段长度的QDED涂层管的电渗流变化,选择合适的分离条件进行蛋白质的分离。研究结果表明：相同pH值下PDMAEMA链段越长,电渗流(绝对值)就越大；不同PDMAEMA嵌段长度的三嵌段共聚物QDED在pH值3.0到7.0范围内可实现蛋白质混合物的快速高效分离。和未经修饰的毛细管相比较,QDED涂层管能够一次性分离蛋白质的混合物(酸性,中性和碱性)。并使用QDED涂层管对鸡蛋清中的相应蛋白做了定量分析。
3.通过可逆加成断裂链转移(RAFT)聚合合成了单体比例不同的无规共聚物poly(DMA-co-SBMA),这种共聚物是由抗污性能优异的聚(甲基丙烯酸甲酯磺基甜菜碱)(PSBMA)和具有自涂覆性能的聚合物Ⅳ,N-二甲基丙烯酰胺(PDMA)组成的。用接触角仪和X射线光电子能谱仪分别研究了聚合物在玻璃片上形成涂层的亲水性和组成。研究了不同离子强度和不同pH值对蛋白质分离效率的影响,并比较了无规共聚物涂层管、未经修饰的毛细管以及PSBMA涂层管蛋白质分离效率的差异。结果表明,对于无规共聚物涂层玻璃片,随着SBMA含量的增加,涂层的亲水性和涂覆能力都在逐渐增加。在80mM的缓冲溶液中,poly(DMA-co-SBMA)涂层管拥有最高的分离效率,最高可达到1509000N/m；在pH值3.0～5.0范围内,10min即可实现四种碱性蛋白质的分离；蛋白质迁移时间的RSD在0.23%～2.92%之间。
Capillary electrophoresis is refer to the charged particle or ion depends on the high voltage electric field drive, directional movement behavior at different speeds in the capillary. Due to its high separation efficiency, less sample consumption, rapid separation time, easy operation and other characteristics, it is applied in various kinds of separation and analysis fields, such as applied to bio-macromolecules separation such as amino acid, nucleic acid, peptide and protein, etc. Fused-silica is a kind of material which is used to manufacture the capillary owing to its good thermal conductivity and ultraviolet transparency. However, under the effect of buffer solution, the capillary inner wall would be negatively charged due to the dissociation of silanol groups. When the capillary was used to separate protein, the interaction between capillary inner wall and proteins would result in protein adsorption. It would lead to the decrease of the separation efficiency and reproducibility of migration time, peak trailing, deformation, and even unable to achieve effective separation and so on.
Numerous approaches have been applied to minimize this adsorption, such as extreme pH, zwitterionic additives, high ionic strength, but by far the most common and effective way is using covalent bonding or physical adsorption to form polymer coating on the capillary wall. Silanol groups on the capillary inner wall can be masked by the polymer coating, the electroosmotic flow is suppressed, and the interaction of protein-wall would be decreased, etc. From the aspects of stability of the coating, coating which covalently bonded onto the inner wall of capillary is more stability than the physically adsorbed coating, however, process of the preparation of covalently linked coating is cumbersome and difficult to control, and coating reproducibility is poor. Compared to the covalently bonded coating, process of the preparation of physically adsorbed coating is simple, easy to control, strong regeneration, and therefore in recent years the physically adsorbed coating is more and more get the favour of scientists.
Around the preparation of physically adsorbed polymer coatings, the main results are as follows:
1. The graft copolymers of hydroxyethylcellulose-graft-poly(2-(dimethylamino)ethyl methacrylate) (HEC-g-PDMAEMA) with different graft ratio were synthesized by using ceric ammonium nitrate initiator in aqueous nitric acid solution. Electroosmotic flow measurement results showed that the synthesized HEC-g-PDMAEMA graft copolymer coated capillary could suppress EOF effectively compared to the bare fused-silica capillary, and efficient separations of basic proteins were also achieved. The electrical charge of the coated capillary wall could be modulated by varying not only the pH of the running buffer, but also the grafting ratio of poly(2-(dimethylamino)ethyl methacrylate) grafts, which makes it is possible to analyze the basic and acidic proteins in the same capillary. The effects of poly(2-(dimethylamino)ethyl methacrylate) grafting ratio in HEC-g-PDMAEMA and buffer pH on the separation of basic proteins for capillary electrophoresis were investigated in detail. Furthermore, egg white proteins and milk powder samples were separated by the HEC-g-PDMAEMA coated capillary, respectively.
2. A novel noncovalent adsorbed coating for capillary electrophoresis has been prepared and explored. This coating was based on quaternized poly(2-(dimethylamino)ethyl methacrylate)-block-poly(ethylene oxide)-block-poly(2-(dimethylamino)ethyl methacrylate)(QDED) triblock copolymer which was synthesized by atomic transfer radical polymerization(ATRP) in our laboratory. The polycationic polymer and the negatively charged fused-silica surface attracted each other through electrostatic interactions and hydrogen bonds. It was demonstrated that the coated capillaries provided an electroosmotic flow with reverse direction, and the magnitude of the electroosmotic flow can be modulated by varying the molecular mass of poly(2-(dimethylamino)ethyl methacrylate)(PDMAEMA) block and pH value of the buffer, respectively. The effects of the molecular mass of PDMAEMA block in QDED triblock copolymer and pH value of the buffer on the separation of basic proteins were investigated in detail. The triblock copolymer coatings showed higher separation efficiency, better migration time repeatability and would apply to wider range of pH than bare fused-silica capillary when used in separating proteins. Proteins from egg white were also separated respectively through this QDED triblock copolymer coated capillary. These results demonstrated that the QDED triblock copolymer coatings are suitable for analyzing biosample.
3. Firstly, the poly(DMA-co-SBMA)s with different feed ratio (SBMA/DMA) were synthesized via the reversible addition fragmentation chain transfer (RAFT) polymerization. And then, X-ray photoelectron spectroscopy (XPS) and water contact angel (CA) were used to investigate the composition and hydrophilicity of poly(DMA-co-SBMA) coating formed on the glass slide surfaces. CA measurements revealed that the poly(DMA-co-SBMA) coating became more hydrophilic with the increment of feed ratio (SBMA/DMA), and at the same time the XPS results showed that the coating ability was also increased with the increment of feed ratio. Followed, the copolymer was applied to coat the fused-silica capillary inner wall and the coated capillary was used to separate the mixture of proteins (lysozyme, cytochrome c, ribonuclease A and α-chymotrypsinogen A) in a pH range from3.0to5.0. Under the optimum conditions, an excellent separation of basic proteins with peak efficiencies ranging from551000to1509000N/m had been accomplished within10min. Furthermore, the compare of separation efficiency among the bare, PSBMA and poly(DMA-co-SBMA) coated capillary was also investigated.
引文
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