节点文献
蛋白质分子印迹核—壳微球的研究
Study on the Protein Imprinted Molecularly Core-Shell Microspheres
【作者】 闫长领;
【导师】 卢雁;
【作者基本信息】 兰州大学 , 物理化学, 2007, 博士
【摘要】 分子识别是生命过程的一种重要活动,分子印迹技术就是模拟天然分子的识别功能制备具有特异选择性聚合物的人工方法。经过人们的不懈努力,这方面的工作已取得了重大进步,特别是在小分子印迹方面,实验所需的各种方法和手段日臻完善,印迹聚合物的制备技术渐趋成熟,相关的许多应用研究正在逐步展开。但是对于生物大分子比如蛋白质分子,目前所采用的方法不理想,实验结果重现性差,水环境下的识别机理还不清楚,生物大分子在聚合物中扩散慢。因此,有必要对生物大分子印迹技术进行进一步的研究,这对大分子印迹材料在催化领域、物质分离、生物制药、分析化学和生物传感等应用方面具有重要意义。本论文首先对分子印迹技术的基本原理、应用和研究现状进行了论述。分子印迹技术来源于免疫学。1993年,Mosbach等在《Nature》上发表了有关茶碱分子印迹物的研究报道之后,分子印迹技术才得到了蓬勃发展。该技术的基本原理为:在制备分子印迹聚合物时,功能单体与模板分子通过一定的作用力结合在一起,引发聚合后模板分子被包埋在聚合物内部。当模板分子从聚合内部洗脱之后,聚合物中留下一些位点,这些位点能够记忆模板分子的功能和形状。分子印迹分为两种基本类型:预组织法(共价法)和自组织法(非共价法)。分子印迹聚合物在色谱分离、仿生催化、药物分析及分析传感等方面展现出良好的应用。分子印迹技术研究正从以小分子为模板向生物大分子为模板发展。目前用于印迹的蛋白质种类多种多样,较为常用的有牛血清蛋白(BSA)、牛血红蛋白、核糖核酸酶和溶菌酶等。对蛋白质印迹目前常采用的单体有丙烯酸、甲基丙烯酸、丙烯酰胺、N,N′-乙烯基二丙烯酰胺,N,N′-亚甲基二丙烯酰胺等。蛋白质分子印迹聚合物的物理形式可大致分为三维块体和二维薄膜两种。本论文的主要研究内容是制备聚苯乙烯微球,在水溶液环境条件下制备核-壳结构的蛋白分子印迹微球。通过再结合实验考察所制备的印迹微球的特异结合能力和分子识别性能等,并对蛋白质分子的印迹和识别原理进行了分析。首先,从各种制备微球的方法中,探索出了用悬浮聚合法制备聚苯乙烯微米级球体技术,考察了聚合温度、搅拌速率、引发剂用量、单体用量、表面活性剂浓度等条件对微球形貌的影响,并利用扫描电镜对微球的表观形貌、粒径及粒径大小分布进行了表征。实验结果表明,在适当条件下,悬浮聚合法可以制备出粒径在50-60微米的球体。该方法制备的聚苯乙烯微球大小较均匀、表面清洁,所需反应时间短、操作方便、产品易于分离。其次,在水溶液中于聚苯乙烯微球表面通过化学氧化法嫁接一层聚间氨基苯硼酸薄膜,形成核-壳结构微球。在此实验部分主要研究了乙酸溶液和磷酸缓冲溶液对嫁接的聚间氨基苯硼酸薄膜稳定性的影响。并用透射电镜、拉曼光谱、X-射线光电子能谱和氮气吸附,脱附实验对核-壳结构微球进行了表征。实验证明,聚间氨基苯硼酸通过苯环上的电子成对作用较牢固地结合在聚苯乙烯微球表面;核-壳微球在乙酸溶液和磷酸缓冲溶液中有很高的稳定性。再次,在制备核-壳微球的基础上,当蛋白分子参与嫁接过程时实现了蛋白质分子印迹。为了验证方法的可行性,我们首先以溶茵酶作为模板分子进行印迹。先将一定浓度的间氨基苯硼酸溶液和溶茵酶溶液相混合,使蛋白分子与功能单体相互作用,然后加入一定量的聚苯乙烯微球和一定浓度的过硫酸铵溶液,在22-24℃下进行聚合反应。反应完成后将印迹的微球从溶液中分离出来,并用去离子水洗涤。干燥一定时间后,用乙酸溶液洗涤除去模板分子,再于室温下进行干燥。用干燥的微球进行间歇式吸附实验,考察印迹微球达到吸收平衡的时间。结果表明该印迹微球可以在60min内达到吸收平衡,证明该微球对生物大分子具有较好的质量传输性能;印迹微球对模板分子的特异吸附性能实验表明,当实验达到吸收平衡时印迹微球比非印迹微球有较大的吸附能力;分子识别性能实验显示,印迹微球对模板分子有较强的结合能力。另外还考察了这种印迹微球的稳定性,实验表明其稳定性在两个月内没有明显变化。最后,利用这种原理分别制备了其它蛋白质(牛血红蛋白、牛血清蛋白、胰蛋白酶和木瓜蛋白酶等)的印迹微球,用多种方法对样品进行了表征,并考察了印迹微球的特异吸附能力和识别性能及质量传输性能和稳定性等。总之,实验表明我们制备的蛋白质印迹核-壳结构微球有较快的质量传输性能,对模板分子有高的吸附能力和良好的选择性识别性能。该分子印迹技术方法简便、反应条件温和,可以在水溶液环境中实现蛋白分子的印迹和识别,并具有一定的普适性。
【Abstract】 Molecular recognition is an important activity in life process. Molecular imprinting technique is an artifical method of synthesizing polymer with special selectivity by simulating recognition function of natural molecular. In this work, great advance has been made by effort. In specially, for small molecules, the various techniques have been perfect and applied to many aspects. For bio-macromolecules such as protein, however, the existed methods are not ideal. Experimental results have not good reproducibility. Recognition mechanism has not been completely understood under water environment and bio-macromolecules imprinted polymers have poor mass transport. Thus it is necessary to further study bio-macromolecules technique. This has a great significance for the application of bio-macromolecules imprinted materials in the domains of catalysis, separation, bio-pharmacy, analytical chemistry, sensor, and so on.In this paper, general principle, application and actuality of molecular imprinting technique were reviewed. Molecular imprinting technique originated from immunology. After the theophylline imprinted polymer was reported in Nature (1993), molecular imprinting technique has made breakthrough. The general principle of technique is polymerization process. Functional monomers form a complex with the template molecule. After the functional monomers are polymerized with the cross-linker, a polymeric network is formed around the template. The polymer liberates complementary binding sites by removing the template, and these sites can memorize the template. Molecular imprinting technique can be divided into tow distinct approaches: covalent (preorganization) and noncovalent (self-assembly). Molecularly imprinted polymers exhibit good behavior in the applications of chromatography separation, bio-mimetic catalysis, drug analysis and analytical sensor. The studies of molecular imprinting are transforming from small molecules to bio-macromolecules. At present, various proteins were used for molecular imprinting such as bovine serum albumin, bovine hemoglobin, ribonuclease and lysozyme etc. For protein molecular imprinting, the monomers commonly used are acrylic acid, methacrylic acid, acrylamide, N, N’-ethylenebis (acrylamide), N, N’-methylenebisacrylamide. The physical forms of protein molecular imprinting can conveniently be subdivided into either 3D (bulk) or 2D (thin film).The experiment mainly includes the preparation of polystyrene microspheres, the preparation of imprinted core-shell microspheres in aqueous solutions for proteins, and the studies of the specific binding ability and the recognition of the imprinted microspheres. The mechanisms of imprinting and recognition for protein were analyzed.Firstly, suspension polymerization was selected for the preparation of polystyrene microspheres from the approaches to synthesize microspheres. The effects of temperature, stirring speed, initial amounts of initiator and monomer, and surfactant concentration on morphology of polystyrene microspheres were investigated. The morphology, size and size distribution of the microspheres were characterized using scanning electron microscope (SEM). The experimental results indicate that polystyrene microspheres with the diameter of 50-60μm can be prepared by the method under appropriate conditions. The microspheres have uniform size and clean surfaces. The advantages of the method are that the speed of reaction is rapid, manipulation is convenient and product is easily separated.Secondly, the thin films of poly-3-amonophenylboronic acid in aqueous environment were grafted on polystyrene microspheres by oxidation for forming core-shell microspheres. The effects of acetic acid solution and sodium phosphate buffers on stability of grafted thin films were valued. The samples were characterized using transmission electron microscope (TEM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and nitrogen adsorption/desorption methods. The results show that poly-3-amonophenylboronic acid can be grafted upon surfaces of the polystyrene microspheres by aromatic ring electron-pairing interaction, and the surfaces of core-shell microspheres have high stability in acetic acid solution and sodium phosphate buffers.Thirdly, based on the core-shell microspheres, protein molecular imprinting was implemented when protein was presented during grafting. For examining the feasibility of the strategy, lysozyme imprinted core-shell microspheres were first prepared. The 3-amonophenylboronic acid and lysozyme solutions of definite concentration were mixed for them interacting. Then definite polystyrene microspheres and the ammonium persulfate solution of definite concentration were added. After the polymerization was carried out at 22-24℃, the imprinted microbeads were separated from the solution and washed repeatedly with deioned water and dried for definite time. Finally, they were washed repeatedly with the acetic acid solution for removing template molecules and dried at room temperature. The batchwise adsorption experiments were carried out for detecting the time to reach equilibrium of adsorption, the abilities of specific adsorption for the template protein, and selective recognition for the template protein and the stability for the imprinted core-shell microspheres. The imprinted microspheres can reach equilibrium of adsorption in 60 min, and this proves the imprinted microspheres have good mass transport. The imprinted microspheres have higher specific adsorption than the nonimprinted microspheres when adsorption reaches equilibrium. The imprinted microspheres have higher adsorption ability for the template protein than non-template proteins. In additionally, the adsorption ability of imprinted microspheres didn’t obviously decrease for template protein over two months, and this shows that the imprinted microspheres possess high stability.Finally, the imprinted microspheres for various proteins such as bovine hemoglobin, bovine serum albumin, trypsin and papain were prepared using the strategy and batchwise adsorption experiments were carried out for detecting the abilities of specific adsorption for the template protein, and selective recognition for the template protein, mass transport and the stability for the imprinted core-shell microspheres.In collusion, the protein imprinted core-shell microspheres prepared by us possess good mass transport, high ability of adsorption and good recognition for template proteins. The molecularly imprinted technique can be used in aqueous solution for protein imprinting under the mild conditions. The technique is convenient and has a certain extent generality.