聚偏氟乙烯吸附功能膜的研制及其应用研究
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摘要
聚偏氟乙烯(Polyvinylidene fluoride,PVDF)膜材料具有优良的热稳定性、化学稳定性以及良好的机械性能,近年来,已被广泛应用于化工、环保、食品、医药和生化等诸多领域。同时,其化学键能高,疏水性强,对蛋白质存在强烈的吸附作用,且具有较好的生物相容性,因此被认为是生物医学分析领域极具发展前景的生物医用材料。
本论文以PVDF作为成膜材料,从蒸汽相诱导法和浸没沉淀法入手,研制高蛋白吸附量的PVDF吸附功能膜,考察各种因素对膜结构和性能的影响；通过对PVDF表面的改性,调控膜表面的亲疏水性,以拓展其在电泳转印、快速诊断、斑点印迹等领域的应用；并将氨基酸接枝到PVDF中空纤维膜的表面制成亲和吸附膜,用于脱除内毒素,获得较好的实验结果。同时运用分子动力学模拟的方法,探讨了牛血清蛋白在不同晶型的PVDF表面的吸附作用及机理。本论文的主要研究内容如下：
(1)蒸汽相诱导法制备PVDF微孔膜
通过蒸汽相诱导法制备了网络状结构对称且无皮层的PVDF膜,考察了蒸汽相的组成、温度、湿度、添加剂的类型对膜结构和性能的影响。研究发现,蒸汽相环境的变化对膜结构有着重要的影响,在蒸发气氛中添加了溶剂DMAC,可以减慢传质速率,使分相时间延长,所成膜的下表面皮层消失,呈现多孔形貌；温度较低,湿度较高时,质量传递速率下降,膜孔的联通性更好,孔隙率上升。以LiCl为添加剂的膜ML表面为开放式的网络结构,同时具有较小的孔径(1.24μm)和较大的孔隙率(86.1%),所以具有较大的蛋白吸附量160μg/cm2。
(2)浸没沉淀法制备PVDF微孔膜
在浸没沉淀法制备PVDF微孔膜的过程中,选择TEP为溶剂,从热力学和动力学两方面考察了铸膜液和凝固浴的组成、温度、添加剂的类型等对膜结构、晶型和性能的影响。结果表明,TEP凝胶浴的浓度能直接改变成膜机理,随着TEP浓度的上升,液液分相过程由瞬时分相过渡到延迟分相,皮层逐渐消失,可获得网络状无皮层对称结构的膜,该膜比表面积大,蛋白固载能力强；TEP凝胶浴的浓度的上升和温度的下降都可以使结晶速度变慢,使PVDF倾向于形成p型的晶体,而极性的β型PVDF具有更多活性吸附位点,有利于蛋白的吸附；以乙醇作为添加剂的膜M34结构均一,孔径分布均匀,在1μm左右。同时膜M34的强度、宏观形貌及蛋白吸附量(194μg/cm2)均在一个较理想的水平
(3)PVDF膜吸附功能膜用于免疫检测领域
将制备的PVDF膜、PVDF改性膜及PVDF/NC共混膜分别应用于电泳印迹、胶体金快速免疫层析(GICA)、免疫斑点印迹等三个领域。通过研究发现：不对称结构的PVDF膜由于其表面具有致密的皮层,蛋白吸附能力非常弱,无法转印蛋白。而结构对称无皮层的膜M34由于蛋白固载量大(194μg/cm2),与商品膜进行对比,转印效果好,是免疫印迹和生化分析领域的优良用膜；将膜M34经表面亲水化改性后,层析性能明显上升,应用在DOA类和hCG类快速诊断试剂时C/T线出现时间较快(T线50秒内,C线200秒内),并且显色状态较强(T线强度G6～G9,C线强度G7-G9)；将共混膜应用于免疫斑点印迹,通过调节共混聚合物的种类以及共混比例,制备出网络状、结构均匀的PVDF/NC共混膜M90,其显色效果明显优于商品的NC膜。
(4)以L-丝氨酸(Ser)为配基制备PVDF中空纤维亲和膜
通过对PVDF中空纤维膜基质进行改性,将丝氨酸配基键合到膜上,制备成PVDF-Ser亲和膜用于人、动物血液中内毒素的脱除。结果表明,PVDF-Ser对人血浆中内毒素的脱除效率高,清除率为48.3%,同时具有较为理想的血液相容性,总蛋白的损失率为14.9%,白蛋白恢复率为92.6%；放大的PVDF-Ser亲和膜组件用于实验猪的全血灌流实验,结果显示该亲和膜可以有效的脱除动物体内的内毒素,能够很好的降低肿瘤坏死因子(TNF-a),和白细胞介素(IL-6)的浓度,抑制炎症反应的发生。
(5)BSA与α和β晶型的PVDF表面相互作用的分子动力学模拟
采用MD模拟的方法讨论了蛋白质BSA与a和p晶型的PVDF之间的相互作用。由MD模拟得到BSA分别与α和β型PVDF之间的静电相互作用、范德华相互作用和总的相互作用能的大小。结果表明,β型PVDF与BSA的静电相互作用和范德华相互作用均比α型强,总的相互作用能比α型高了约200KJ/mol.BSA在β型表面参与吸附的基团比α型表面多了四个,分别为ASP85,SER465, GLU490,TYR492.正是由于β型表面呈极性,参与吸附的氨基酸基团密度高于α型,同时β型与BSA的相互作用能也更大,所以β型PVDF对蛋白质的固载能力更强。
Poly(vinylidene fluoride) membranes have been developed for a variety of fields, such as chemical and environmental engineering, food, medicine and biochemistry, due to their thermostability, chemical resistance and good mechanical property. Compared with the other materials, PVDF has the further advantage of being capable for use in protein blotting because of its high protein-binding capacity and biocompatibility. It is considered to be a very promising biomaterial in the field of biomedical analysis.
In this study, functional PVDF membranes for adsorption were prepared by vapor induced phase separation and immersion precipitation. The effects of various membrane formation parameters on their morphologies and property were investigated. Then, the applications were expanded after surface modified of these membranes. The adsorption dynamics simulation of Bovine Serum Albumin (BSA) onα-type andβ-type PVDF crystal surface were further studied.
Mainly research of the thesis is focuses on the following aspects.
(1) Preparation of symmetric network PVDF membranes for protein adsorption via vapor induced phase separation
PVDF membranes for protein adsorption were prepared by vapor induced phase separation from PVDF/N,N-dimethyl acetamide (DMAC)/ water system. The effects of evaporation atmosphere, temperature and humidity during the preparation of the membranes on their morphologies were investigated by SEM. With low temperature and high humidity, polymer crystallization mechanism dominated the membrane formation process, and the casting solution formed membranes with symmetric morphologies in the vapor phase containing 0.79% DMAC. The effect of additives on the membrane structure and performance was also investigated. The results of adsorption experiment showed that the binding capacity of Bovine Serum Albumin (BSA) increased with the appearance of circular network morphology and the decrease of mean pore size of the membrane. With the addition of LiCl to the casting solution, the obtained membrane can adsorb BSA up to 160μg/cm2.
(2) Preparation of symmetric network PVDF membranes for protein adsorption via immersion precipitation
Symmetric network PVDF membranes without dense skin layer were prepared by immersion precipitation in a water/TEP/PVDF system to improve protein adsorption. In order to investigate the effects of different membrane morphologies and PVDF crystal types on protein-binding capacity, different dope, baths and preparation condition were used to modify the membrane structure and performance. By comparing the effect of TEP content in the bath on membrane morphology, it was found that the TEP in the bath hindered the phase-separation kinetics. As a result, the casting dope slowly entered the phase-separation boundaries, leading to the so-called delayed-type demixing. The porosity of PVDF membranes increased along with the increase of TEP content in the bath. For a slow crystallization process(higher TEP bath content and lower temperature of bath), crystallization was expected to take place at a lower rate such that PVDF chains had enough time to arrange themselves intoβ-type. Theβ-type PVDF crystal was polar because of it all-trans arrangement. It tended to adsorb BSA through hydrogen bonding or electrostatic force between the weak-negative F and the weak-positive H atoms. And the protein remained bound with more points of attachment. Thus, the interaction of electrostatic force between a-type and BSA is smaller than that withβ-type. M34 improved the adsorption of protein up to 194μg/cm2.
(3) The application of functional adsorption PVDF membranes
PVDF merbranes prepared in this work were used for Western-blot, Colloidal gold enhanced immunochromatography assay and Dot-blot. It was found that the asymmetric PVDF membrane can not be used in Western-blot due to its asymmetric structure with a skin layer and weak protein binding capacity. Proteins on the sodium dodecyl sulfate polyacrylamide gel electrophoresis gels were successfully electro-blotted onto M34. Compared with commercial PVDF membranes, it was feasible for the PVDF membranes M34 prepared in this work be used for Western-blot; Hydrophilic surface modification of membrane M34 was performed by 1%STP treatment. Then used in colloidal gold enhanced immunochromatography assay, which had a good chromatographic property (T line< 50s, C line< 200s) and strong color development(T line:G6-G9, C line:G7-G9); The PVDF/NC blend membrane M90 was successfully used in Dot-blotting as the solid membrane. The brown dot can be seen on the membrane when the positive sample was used. Compared with commercial NC membranes, the effect of color development was stronger.
(4) PVDF-Ser affinity membrane
We firstly used L-serine (Ser) as ligands separately, PVDF hollow-fiber membrane as the bare membrane,1,6-hexanediamine as the spacer arm to make new types of affinity membrane for endotoxin removal from human plasma. For human plasma sample, the clearance efficiency of lab-made PVDF-Ser affinity membrane module could reach 48.3% at lml/min velocity. The endotoxin adsorption capability was 0.027EU/cm2 with little effect on other biochemical indicators, the loss of total protein (including the various types of enzyme) was 14.9% and the recovery rate of albumin is 92.4%; Then, amplified lab-made PVDF-Ser affinity membrane modules were used in animal experiments. The results showed that PVDF-Ser affinity membrane has good blood compatibility and the amplified module could effectively remove endotoxin from domestic pig, it could reduce concentrations of tumor necrosis factor(TNF-a) and interleukin(IL-6), thereby the inflammatory reaction was inhabited.
(5) The adsorption dynamics simulation of BSA onα-type andβ-type PVDF crystal surface
The adsorption dynamics of BSA on a-type andβ-type PVDF crystal surface were studied in detail. It was showed that electrostatic and VDW interaction between β-type PVDF crystal and BSA was 200KJ/mol stronger than a-type PVDF crystal. There are four types of residues (ASP 85, SER 465, GLU 490, TYR 492) which participated in the adsorption of BSA onto theβ-type PVDF crystal, but they were not participated onto the a-type. The combine of more residues participated in adsorption and larger interaction energy betweenβ-type PVDF crystal and BSA made the protein binding capacity ofβ-type PVDF crystal was larger than a-type.
本论文以PVDF作为成膜材料,从蒸汽相诱导法和浸没沉淀法入手,研制高蛋白吸附量的PVDF吸附功能膜,考察各种因素对膜结构和性能的影响；通过对PVDF表面的改性,调控膜表面的亲疏水性,以拓展其在电泳转印、快速诊断、斑点印迹等领域的应用；并将氨基酸接枝到PVDF中空纤维膜的表面制成亲和吸附膜,用于脱除内毒素,获得较好的实验结果。同时运用分子动力学模拟的方法,探讨了牛血清蛋白在不同晶型的PVDF表面的吸附作用及机理。本论文的主要研究内容如下：
(1)蒸汽相诱导法制备PVDF微孔膜
通过蒸汽相诱导法制备了网络状结构对称且无皮层的PVDF膜,考察了蒸汽相的组成、温度、湿度、添加剂的类型对膜结构和性能的影响。研究发现,蒸汽相环境的变化对膜结构有着重要的影响,在蒸发气氛中添加了溶剂DMAC,可以减慢传质速率,使分相时间延长,所成膜的下表面皮层消失,呈现多孔形貌；温度较低,湿度较高时,质量传递速率下降,膜孔的联通性更好,孔隙率上升。以LiCl为添加剂的膜ML表面为开放式的网络结构,同时具有较小的孔径(1.24μm)和较大的孔隙率(86.1%),所以具有较大的蛋白吸附量160μg/cm2。
(2)浸没沉淀法制备PVDF微孔膜
在浸没沉淀法制备PVDF微孔膜的过程中,选择TEP为溶剂,从热力学和动力学两方面考察了铸膜液和凝固浴的组成、温度、添加剂的类型等对膜结构、晶型和性能的影响。结果表明,TEP凝胶浴的浓度能直接改变成膜机理,随着TEP浓度的上升,液液分相过程由瞬时分相过渡到延迟分相,皮层逐渐消失,可获得网络状无皮层对称结构的膜,该膜比表面积大,蛋白固载能力强；TEP凝胶浴的浓度的上升和温度的下降都可以使结晶速度变慢,使PVDF倾向于形成p型的晶体,而极性的β型PVDF具有更多活性吸附位点,有利于蛋白的吸附；以乙醇作为添加剂的膜M34结构均一,孔径分布均匀,在1μm左右。同时膜M34的强度、宏观形貌及蛋白吸附量(194μg/cm2)均在一个较理想的水平
(3)PVDF膜吸附功能膜用于免疫检测领域
将制备的PVDF膜、PVDF改性膜及PVDF/NC共混膜分别应用于电泳印迹、胶体金快速免疫层析(GICA)、免疫斑点印迹等三个领域。通过研究发现：不对称结构的PVDF膜由于其表面具有致密的皮层,蛋白吸附能力非常弱,无法转印蛋白。而结构对称无皮层的膜M34由于蛋白固载量大(194μg/cm2),与商品膜进行对比,转印效果好,是免疫印迹和生化分析领域的优良用膜；将膜M34经表面亲水化改性后,层析性能明显上升,应用在DOA类和hCG类快速诊断试剂时C/T线出现时间较快(T线50秒内,C线200秒内),并且显色状态较强(T线强度G6～G9,C线强度G7-G9)；将共混膜应用于免疫斑点印迹,通过调节共混聚合物的种类以及共混比例,制备出网络状、结构均匀的PVDF/NC共混膜M90,其显色效果明显优于商品的NC膜。
(4)以L-丝氨酸(Ser)为配基制备PVDF中空纤维亲和膜
通过对PVDF中空纤维膜基质进行改性,将丝氨酸配基键合到膜上,制备成PVDF-Ser亲和膜用于人、动物血液中内毒素的脱除。结果表明,PVDF-Ser对人血浆中内毒素的脱除效率高,清除率为48.3%,同时具有较为理想的血液相容性,总蛋白的损失率为14.9%,白蛋白恢复率为92.6%；放大的PVDF-Ser亲和膜组件用于实验猪的全血灌流实验,结果显示该亲和膜可以有效的脱除动物体内的内毒素,能够很好的降低肿瘤坏死因子(TNF-a),和白细胞介素(IL-6)的浓度,抑制炎症反应的发生。
(5)BSA与α和β晶型的PVDF表面相互作用的分子动力学模拟
采用MD模拟的方法讨论了蛋白质BSA与a和p晶型的PVDF之间的相互作用。由MD模拟得到BSA分别与α和β型PVDF之间的静电相互作用、范德华相互作用和总的相互作用能的大小。结果表明,β型PVDF与BSA的静电相互作用和范德华相互作用均比α型强,总的相互作用能比α型高了约200KJ/mol.BSA在β型表面参与吸附的基团比α型表面多了四个,分别为ASP85,SER465, GLU490,TYR492.正是由于β型表面呈极性,参与吸附的氨基酸基团密度高于α型,同时β型与BSA的相互作用能也更大,所以β型PVDF对蛋白质的固载能力更强。
Poly(vinylidene fluoride) membranes have been developed for a variety of fields, such as chemical and environmental engineering, food, medicine and biochemistry, due to their thermostability, chemical resistance and good mechanical property. Compared with the other materials, PVDF has the further advantage of being capable for use in protein blotting because of its high protein-binding capacity and biocompatibility. It is considered to be a very promising biomaterial in the field of biomedical analysis.
In this study, functional PVDF membranes for adsorption were prepared by vapor induced phase separation and immersion precipitation. The effects of various membrane formation parameters on their morphologies and property were investigated. Then, the applications were expanded after surface modified of these membranes. The adsorption dynamics simulation of Bovine Serum Albumin (BSA) onα-type andβ-type PVDF crystal surface were further studied.
Mainly research of the thesis is focuses on the following aspects.
(1) Preparation of symmetric network PVDF membranes for protein adsorption via vapor induced phase separation
PVDF membranes for protein adsorption were prepared by vapor induced phase separation from PVDF/N,N-dimethyl acetamide (DMAC)/ water system. The effects of evaporation atmosphere, temperature and humidity during the preparation of the membranes on their morphologies were investigated by SEM. With low temperature and high humidity, polymer crystallization mechanism dominated the membrane formation process, and the casting solution formed membranes with symmetric morphologies in the vapor phase containing 0.79% DMAC. The effect of additives on the membrane structure and performance was also investigated. The results of adsorption experiment showed that the binding capacity of Bovine Serum Albumin (BSA) increased with the appearance of circular network morphology and the decrease of mean pore size of the membrane. With the addition of LiCl to the casting solution, the obtained membrane can adsorb BSA up to 160μg/cm2.
(2) Preparation of symmetric network PVDF membranes for protein adsorption via immersion precipitation
Symmetric network PVDF membranes without dense skin layer were prepared by immersion precipitation in a water/TEP/PVDF system to improve protein adsorption. In order to investigate the effects of different membrane morphologies and PVDF crystal types on protein-binding capacity, different dope, baths and preparation condition were used to modify the membrane structure and performance. By comparing the effect of TEP content in the bath on membrane morphology, it was found that the TEP in the bath hindered the phase-separation kinetics. As a result, the casting dope slowly entered the phase-separation boundaries, leading to the so-called delayed-type demixing. The porosity of PVDF membranes increased along with the increase of TEP content in the bath. For a slow crystallization process(higher TEP bath content and lower temperature of bath), crystallization was expected to take place at a lower rate such that PVDF chains had enough time to arrange themselves intoβ-type. Theβ-type PVDF crystal was polar because of it all-trans arrangement. It tended to adsorb BSA through hydrogen bonding or electrostatic force between the weak-negative F and the weak-positive H atoms. And the protein remained bound with more points of attachment. Thus, the interaction of electrostatic force between a-type and BSA is smaller than that withβ-type. M34 improved the adsorption of protein up to 194μg/cm2.
(3) The application of functional adsorption PVDF membranes
PVDF merbranes prepared in this work were used for Western-blot, Colloidal gold enhanced immunochromatography assay and Dot-blot. It was found that the asymmetric PVDF membrane can not be used in Western-blot due to its asymmetric structure with a skin layer and weak protein binding capacity. Proteins on the sodium dodecyl sulfate polyacrylamide gel electrophoresis gels were successfully electro-blotted onto M34. Compared with commercial PVDF membranes, it was feasible for the PVDF membranes M34 prepared in this work be used for Western-blot; Hydrophilic surface modification of membrane M34 was performed by 1%STP treatment. Then used in colloidal gold enhanced immunochromatography assay, which had a good chromatographic property (T line< 50s, C line< 200s) and strong color development(T line:G6-G9, C line:G7-G9); The PVDF/NC blend membrane M90 was successfully used in Dot-blotting as the solid membrane. The brown dot can be seen on the membrane when the positive sample was used. Compared with commercial NC membranes, the effect of color development was stronger.
(4) PVDF-Ser affinity membrane
We firstly used L-serine (Ser) as ligands separately, PVDF hollow-fiber membrane as the bare membrane,1,6-hexanediamine as the spacer arm to make new types of affinity membrane for endotoxin removal from human plasma. For human plasma sample, the clearance efficiency of lab-made PVDF-Ser affinity membrane module could reach 48.3% at lml/min velocity. The endotoxin adsorption capability was 0.027EU/cm2 with little effect on other biochemical indicators, the loss of total protein (including the various types of enzyme) was 14.9% and the recovery rate of albumin is 92.4%; Then, amplified lab-made PVDF-Ser affinity membrane modules were used in animal experiments. The results showed that PVDF-Ser affinity membrane has good blood compatibility and the amplified module could effectively remove endotoxin from domestic pig, it could reduce concentrations of tumor necrosis factor(TNF-a) and interleukin(IL-6), thereby the inflammatory reaction was inhabited.
(5) The adsorption dynamics simulation of BSA onα-type andβ-type PVDF crystal surface
The adsorption dynamics of BSA on a-type andβ-type PVDF crystal surface were studied in detail. It was showed that electrostatic and VDW interaction between β-type PVDF crystal and BSA was 200KJ/mol stronger than a-type PVDF crystal. There are four types of residues (ASP 85, SER 465, GLU 490, TYR 492) which participated in the adsorption of BSA onto theβ-type PVDF crystal, but they were not participated onto the a-type. The combine of more residues participated in adsorption and larger interaction energy betweenβ-type PVDF crystal and BSA made the protein binding capacity ofβ-type PVDF crystal was larger than a-type.
引文
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