模板细乳液聚合法制备磁性复合微球及其在蛋白分离纯化中的应用
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摘要
近年来,有机-无机杂化复合微球,尤其是磁性复合微球正受到人们的广泛关注。由于磁性高分子微球同时具有无机磁性材料的磁响应性和有机高分子的表面功能性,在外加磁场下能方便、快速、高效的从介质中分离目标生物分子。因此,在生物医学和生物化学等领域显示广泛的应用前景。从诊断学的角度考虑,理想的磁性微球应具有均一的粒子尺寸、高的磁含量(快速的磁响应性)、功能化的表面。然而,现有制备磁性复合微球的方法要么破坏了磁性微球的单分散性,要么制备单分散的高磁含量的磁性微球的过程太复杂。并且,所制备的磁性分离载体存在吸附容量较低,偶联效率不高等问题。针对以上问题,本论文系统的围绕着磁性复合微球的制备、表面功能化修饰及其在蛋白分离纯化中的应用研究展开,主要取得了以下几个方面的结果:
1)通过磁性模板细乳液聚合法制备粒径单分散的、磁含量可控的磁性复合微球Fe_3O_4@Poly(St/DVB),整个制备过程操作简单、容易放大,具有工业化应用前景。首先,通过超声乳化构造粒径均匀的磁性模板(磁性纳米粒子簇),然后将单体St和交联剂DVB加入到反应体系使之溶胀聚合,通过动态光散射(DLS)的结果表明单体聚合的主要场所在磁性模板细乳液中,聚合机理是细乳液聚合主导的模板细乳液聚合。所制备的磁性复合微球的尺寸有一个合理的均匀分布。DLS的表征结果表明:改变乳化剂的量,可以得到粒径在70-130 nm范围内的磁性复合微球。通过调节磁性模板细乳液与单体的比例,可以有效调节磁性复合微球磁含量于40-70 wt%之间。通过研究St/DVB的比率、乳化剂的量、助稳定剂、超声功率、引发剂类型、磁流体固含量、磁流体与单体的投料比等实验参数对磁性复合微球性能的影响,得到制备磁性复合微球的最佳配方。制备得到的磁性复合微球具有超顺磁性,可以作为优良的磁性载体而用于生物医学领域。此外,所提出的模板细乳液聚合法不仅能用于制备高磁含量、单分散的磁性复合微球,而且可以作为其它的高包封率的无机-有机杂化微球的制备方法。
2)以磁性Fe_3O_4@Poly(St/DVB)复合微球为种子,通过种子乳液聚合制备得到表面富含环氧功能基团的核壳式磁性Fe_3O_4/Poly(St/DVB)@Poly(GMA/DVB)复合微球。TEM电镜的结果表明得到的核壳式磁性复合微球的粒径分布很均一。通过加入不同量的壳层单体,核壳式磁性复合微球的磁含量可控于23-43 wt%之间。硫代硫酸钠法滴定核壳式磁性复合微球表面的环氧基为0.126-0.190 mmol/g。通过亚氨基二乙酸(IDA)与磁性复合微球表面的环氧基团开环反应,在磁性复合微球表面引入IDA金属离子螯合基团,制备得到能螯合金属离子的磁性亲和分离载体。原子吸收光谱(AAS)和通过紫外光谱(UV)的分析结果表明磁性复合载体螯合Cu~(2+)的能力分别为0.068-0.072 mmol/g和0.091-0.110 mmol/g。
3)将核壳式磁性分离载体Fe_3O_4/Poly(St-DVB)@Poly(GMA-DVB)-IDA-Ni~(2+)应用于S-腺苷甲硫氨酸合成酶(SAMS)的分离提纯。在外加磁场下作用下,磁性载体可以方便、迅速的纯化SAMS。通过详细研究反应介质初始SAMS蛋白浓度、咪唑洗脱液浓度、反应介质离子强度(NaCl浓度)、pH值、螯合金属离子种类等纯化条件对蛋白分离纯化的影响,从而得到磁性微球纯化SAMS蛋白的最佳分离条件。最后,在最佳分离条件下纯化不同初始浓度的SAMS蛋白,结果表明当初始蛋白浓度为3.0 mg/mL时,蛋白纯化量最大为50.0 mg/g,SDS-PAGE电泳结果显示所纯化的蛋白纯度很高,没有杂蛋白。
In recent years, the organic/inorganic composite polymer microspheres with magnetic properties have attracted more and more attention. Because these magnetic hybrid microspheres not only exhibit high magnetic susceptibility to an external magnetic field, but also easily further functionalized and surface-modified by the attachment of various bioactive molecules, they can conveniently separate the objective biomolecules from the medium with the help of magnets. These merits make magnetic hybrid microspheres a powerful tool for application in biomedicine and biochemistry fields. However, when diagnostics are considered, an ideal magnetic microspheres used in these applications should be exhibit the following properties: (1) a narrow size distribution allowing homogeneous particle behavior, (2) a high iron oxide (superparamagnetic) content for rapid separation under a magnetic field, (3) surface functionality for the covalent binding of biomolecules. However, either most of existing methods failed to produce uniform-sized hybrid microspheres with high magnetite content to enhance their susceptibility to magnetic field or the processes used to making such materials were too complicated. In addition, the obtained magnetic carriers possess the lower adsorption capability and binding efficiency. Based on the problems mentioned above, systematical study has been carried out on the preparation, surface modification and application of magnetic microspheres in protein separation and purification in this thesis. The main results are listed as follows:
(1) Hybrid Fe_3O_4@Poly(St/DVB) microspheres with controllable magnetite content and narrow size distribution, composed of Fe_3O_4 nanoparticles encapsulated in a crosslinked polystyrene matrix, were synthesized by magneto-template (Fe_3O_4 minidroplets) miniemulsion polymerization, the synthetic procedure for fabrication of high-quality magnetic hybrid microspheres is simple and easily scaled up, which have a potential application in industry. In this process, firstly, the magneto-template (magnetic nanoparticles cluster) was fabricated by the ultrasonic emulsification. Secondly, monomer St and crosslinked agent DVB was added and polymerized. The DLS results confirmed that the formation process of the magnetic microspheres was dominated by miniemulsion polymerization that was initiated and polymerized in monomer-swollen magneto-templates (MSM). The formed magnetic microspheres had a reasonably narrow size distribution. The average particle size of the magnetic hybrid microspheres was in the range of 70-130 nm depending on the amount of the surfactant. The magnetite content of the magnetic hybrid microspheres can be effectively modulated in the range 40-70 wt % by feeding different amounts of ferrofluid. Based on the related parameters, for instance, a given St/DVB ratio, the amount of surfactant, hydrophobic agent, ultrasonic power, type of initiator, solid content of ferrofluid, the feed ratio of magneto-miniemulsion and monomer, an optimum recipe to prepare the magnetic hybrid microspheres were obtained. The magnetic hybrid microspheres are superparamagnetic, which allow them to serve as excellent candidates for biomedical applications. This simple procedure for preparation of uniform magnetic hybrid microspheres opens a new facile route for encapsulation of inorganic nanomaterials in polymer microspheres.
(2) Fe_3O_4/Poly(St/DVB)@Poly(GMA/DVB) composite microspheres with core-shell structure were rationally fabricated by the seeded emulsion polymerization. TEM images show that the size of composite microspheres is uniform. The magnetite content of the magnetic composite microspheres can be effectively modulated in the range 23-43 wt % by polymerizing the different amounts of shell-monomer. The content of available epoxy groups was determined by Na_2S_2O_8 titration method and was found to be 0.126-0.190 mmol/g microspheres. By covalent coupling of iminodiacetic acid (IDA) chelator to the surface of the magnetic microspheres, a new immobilized metal affinity magnetic carrier was achieved. The amount of chelated Cu~(2+) in magnetic carriers to was 0.068-0.072 mmol/g and 0.091-0.110 mmol/g as determined by UV spectroscopy and atomic absorption spectrophotometry (AAS), respectively.
(3) When magnetic carriers were used to purify S-adenosylmethionine synthetase (SAMS), the purified SAMS protein can be separated conveniently and rapidly with an external magnet. Based on the detailed investigation on the purification condition such as the initial SAMS concentration, the concentration of imidazole elution, the ionic strength (NaCl concentration), pH, the type of metal ion, an optimum purification condition for SAMS protein was obtained. By the optimum purification condition, various initial concentrations of SAMS protein was purified by the magnetic carriers. The experimental results indicated that the maximum protein purification capacity was 50.0 mg/g marries as for the 3.0 mg/L initial SAMS concentration. SDS-PAGE results shown that purified SAMS protein had high purity and low nonspecific adsorption.
1)通过磁性模板细乳液聚合法制备粒径单分散的、磁含量可控的磁性复合微球Fe_3O_4@Poly(St/DVB),整个制备过程操作简单、容易放大,具有工业化应用前景。首先,通过超声乳化构造粒径均匀的磁性模板(磁性纳米粒子簇),然后将单体St和交联剂DVB加入到反应体系使之溶胀聚合,通过动态光散射(DLS)的结果表明单体聚合的主要场所在磁性模板细乳液中,聚合机理是细乳液聚合主导的模板细乳液聚合。所制备的磁性复合微球的尺寸有一个合理的均匀分布。DLS的表征结果表明:改变乳化剂的量,可以得到粒径在70-130 nm范围内的磁性复合微球。通过调节磁性模板细乳液与单体的比例,可以有效调节磁性复合微球磁含量于40-70 wt%之间。通过研究St/DVB的比率、乳化剂的量、助稳定剂、超声功率、引发剂类型、磁流体固含量、磁流体与单体的投料比等实验参数对磁性复合微球性能的影响,得到制备磁性复合微球的最佳配方。制备得到的磁性复合微球具有超顺磁性,可以作为优良的磁性载体而用于生物医学领域。此外,所提出的模板细乳液聚合法不仅能用于制备高磁含量、单分散的磁性复合微球,而且可以作为其它的高包封率的无机-有机杂化微球的制备方法。
2)以磁性Fe_3O_4@Poly(St/DVB)复合微球为种子,通过种子乳液聚合制备得到表面富含环氧功能基团的核壳式磁性Fe_3O_4/Poly(St/DVB)@Poly(GMA/DVB)复合微球。TEM电镜的结果表明得到的核壳式磁性复合微球的粒径分布很均一。通过加入不同量的壳层单体,核壳式磁性复合微球的磁含量可控于23-43 wt%之间。硫代硫酸钠法滴定核壳式磁性复合微球表面的环氧基为0.126-0.190 mmol/g。通过亚氨基二乙酸(IDA)与磁性复合微球表面的环氧基团开环反应,在磁性复合微球表面引入IDA金属离子螯合基团,制备得到能螯合金属离子的磁性亲和分离载体。原子吸收光谱(AAS)和通过紫外光谱(UV)的分析结果表明磁性复合载体螯合Cu~(2+)的能力分别为0.068-0.072 mmol/g和0.091-0.110 mmol/g。
3)将核壳式磁性分离载体Fe_3O_4/Poly(St-DVB)@Poly(GMA-DVB)-IDA-Ni~(2+)应用于S-腺苷甲硫氨酸合成酶(SAMS)的分离提纯。在外加磁场下作用下,磁性载体可以方便、迅速的纯化SAMS。通过详细研究反应介质初始SAMS蛋白浓度、咪唑洗脱液浓度、反应介质离子强度(NaCl浓度)、pH值、螯合金属离子种类等纯化条件对蛋白分离纯化的影响,从而得到磁性微球纯化SAMS蛋白的最佳分离条件。最后,在最佳分离条件下纯化不同初始浓度的SAMS蛋白,结果表明当初始蛋白浓度为3.0 mg/mL时,蛋白纯化量最大为50.0 mg/g,SDS-PAGE电泳结果显示所纯化的蛋白纯度很高,没有杂蛋白。
In recent years, the organic/inorganic composite polymer microspheres with magnetic properties have attracted more and more attention. Because these magnetic hybrid microspheres not only exhibit high magnetic susceptibility to an external magnetic field, but also easily further functionalized and surface-modified by the attachment of various bioactive molecules, they can conveniently separate the objective biomolecules from the medium with the help of magnets. These merits make magnetic hybrid microspheres a powerful tool for application in biomedicine and biochemistry fields. However, when diagnostics are considered, an ideal magnetic microspheres used in these applications should be exhibit the following properties: (1) a narrow size distribution allowing homogeneous particle behavior, (2) a high iron oxide (superparamagnetic) content for rapid separation under a magnetic field, (3) surface functionality for the covalent binding of biomolecules. However, either most of existing methods failed to produce uniform-sized hybrid microspheres with high magnetite content to enhance their susceptibility to magnetic field or the processes used to making such materials were too complicated. In addition, the obtained magnetic carriers possess the lower adsorption capability and binding efficiency. Based on the problems mentioned above, systematical study has been carried out on the preparation, surface modification and application of magnetic microspheres in protein separation and purification in this thesis. The main results are listed as follows:
(1) Hybrid Fe_3O_4@Poly(St/DVB) microspheres with controllable magnetite content and narrow size distribution, composed of Fe_3O_4 nanoparticles encapsulated in a crosslinked polystyrene matrix, were synthesized by magneto-template (Fe_3O_4 minidroplets) miniemulsion polymerization, the synthetic procedure for fabrication of high-quality magnetic hybrid microspheres is simple and easily scaled up, which have a potential application in industry. In this process, firstly, the magneto-template (magnetic nanoparticles cluster) was fabricated by the ultrasonic emulsification. Secondly, monomer St and crosslinked agent DVB was added and polymerized. The DLS results confirmed that the formation process of the magnetic microspheres was dominated by miniemulsion polymerization that was initiated and polymerized in monomer-swollen magneto-templates (MSM). The formed magnetic microspheres had a reasonably narrow size distribution. The average particle size of the magnetic hybrid microspheres was in the range of 70-130 nm depending on the amount of the surfactant. The magnetite content of the magnetic hybrid microspheres can be effectively modulated in the range 40-70 wt % by feeding different amounts of ferrofluid. Based on the related parameters, for instance, a given St/DVB ratio, the amount of surfactant, hydrophobic agent, ultrasonic power, type of initiator, solid content of ferrofluid, the feed ratio of magneto-miniemulsion and monomer, an optimum recipe to prepare the magnetic hybrid microspheres were obtained. The magnetic hybrid microspheres are superparamagnetic, which allow them to serve as excellent candidates for biomedical applications. This simple procedure for preparation of uniform magnetic hybrid microspheres opens a new facile route for encapsulation of inorganic nanomaterials in polymer microspheres.
(2) Fe_3O_4/Poly(St/DVB)@Poly(GMA/DVB) composite microspheres with core-shell structure were rationally fabricated by the seeded emulsion polymerization. TEM images show that the size of composite microspheres is uniform. The magnetite content of the magnetic composite microspheres can be effectively modulated in the range 23-43 wt % by polymerizing the different amounts of shell-monomer. The content of available epoxy groups was determined by Na_2S_2O_8 titration method and was found to be 0.126-0.190 mmol/g microspheres. By covalent coupling of iminodiacetic acid (IDA) chelator to the surface of the magnetic microspheres, a new immobilized metal affinity magnetic carrier was achieved. The amount of chelated Cu~(2+) in magnetic carriers to was 0.068-0.072 mmol/g and 0.091-0.110 mmol/g as determined by UV spectroscopy and atomic absorption spectrophotometry (AAS), respectively.
(3) When magnetic carriers were used to purify S-adenosylmethionine synthetase (SAMS), the purified SAMS protein can be separated conveniently and rapidly with an external magnet. Based on the detailed investigation on the purification condition such as the initial SAMS concentration, the concentration of imidazole elution, the ionic strength (NaCl concentration), pH, the type of metal ion, an optimum purification condition for SAMS protein was obtained. By the optimum purification condition, various initial concentrations of SAMS protein was purified by the magnetic carriers. The experimental results indicated that the maximum protein purification capacity was 50.0 mg/g marries as for the 3.0 mg/L initial SAMS concentration. SDS-PAGE results shown that purified SAMS protein had high purity and low nonspecific adsorption.
引文
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