氧化石墨烯功能化的毛细管电色谱酶微反应器的制备及性能研究

发布时间：2018-04-04 07:28

本文选题：氧化石墨烯　切入点：毛细管电色谱　出处：《中央民族大学》2017年硕士论文

【摘要】：毛细管电泳酶微反应器是生物分子固定方法、微酶反应以及高效分离技术相结合的一种微生化反应装置。作为一种低耗高效且易于自动化的酶催化反应工具,它在蛋白质酶解、生物转化及酶抑制剂药物筛选等方面具有独特的优势。毛细管电泳酶微反应器主要由固定化酶微反应器部分和毛细管电泳分离部分构成。其中固定化酶的活性与固酶的载体基质密切相关,而其毛细管电泳分离性能与分离模式有关。本论文选择纳米材料氧化石墨烯作为固载基质,制备了一种新型的氧化石墨烯功能化毛细管电色谱酶微反应器。氧化石墨烯特有的物化和生化性能,将其引入毛细管电泳酶微反应器中,既可使毛细管电泳从常规的毛细管区带电泳分离模式转变为分离效率更高的毛细管电色谱分离模式,同时还能增强固定化酶的活性,实现毛细管电泳酶微反应器整体性能的提升。该研究既为毛细管电泳酶微反应器新方法的发展拓展了思路,也为其在酶抑制剂药物筛选等相关领域的应用提供了新的工具。论文研究内容主要包括:1.对近年来以石墨烯及其衍生物氧化石墨烯等为载体的固定化酶技术进行了评述。重点介绍了基于物理吸附、化学键合和包埋原理的石墨烯相关材料为载体基质的固定化酶的制作方法及其在生物催化等领域的最新应用。2.基于物理吸附法和化学共价法,发展了氧化石墨烯功能化毛细管电色谱的制备方法。对所制备的毛细管电色谱进行了扫描电镜、热失重、拉曼光谱等表征。对该色谱柱的稳定性和分离度进行了考察。实验结果显示氧化石墨烯修饰的毛细管电色谱可使N-苯甲酰-L-精氨酸乙酯HCl盐(BAEE)和N-苯甲酰-L-精氨酸(BA)混合物的分离度从裸管(毛细管区带电泳分离模式)的3.7提高到4.7。这表明氧化石墨烯修饰的毛细管电色谱能显著提高毛细管电泳分离性能,为复杂化合物的分离奠定了基础。3.在上述基础上,采用物理吸附和化学共价两种酶固载化技术,成功制得氧化石墨烯功能化毛细管电色谱酶微反应器。以胰蛋白酶为固定模型酶,进行了酶活及酶促动力学等研究。实验结果显示,物理吸附法制备的固定化酶的水解活性优于化学共价法。酶促动力学结果显示,基于物理吸附法制备的氧化石墨烯功能化毛细管电色谱酶微反应器的米氏常数Km为1.10mM,最大反应速率Vmax为0.32mM-1。其Vmax是自由酶溶液的3000多倍,而Km约为自由酶溶液的1/20。与未修饰氧化石墨烯的毛细管固定化酶的活性(Km 109.77 mM,Vmax 0.00046 mM·s-1)相比,氧化石墨烯为载体的固定化酶的亲和力与催化速率都有显著提高。这表明氧化石墨烯作为固酶基质能显著增强和提高酶的催化活性。4.利用所制备的微反应器从10种中药材中快速筛选有胰蛋白酶抑制剂活性成分的药材。结果显示三七和大黄中存在胰蛋白酶抑制剂活性成分,对胰蛋白酶的抑制率分别为26%和26.12%。这表明所制备的微反应器是一种潜在药物活性成分的快速筛选工具。
[Abstract]:Capillary electrophoresis enzyme micro reactor is bio molecule fixation method, a micro micro enzyme reaction and efficient separation technology combined with the chemical reaction device. As a tool of enzyme catalytic reaction of high efficiency and low energy consumption and easy automation, it in protein digestion, has unique advantages in terms of biotransformation and enzyme inhibitor drug screening. Capillary electrophoretic enzyme micro reactor by immobilized enzyme microreactor and capillary electrophoresis separation part. The immobilized enzyme activity is closely related with the carrier of immobilized enzyme, and the capillary electrophoresis separation performance and separation patterns. This paper choose graphene oxide nanomaterials as immobilization matrix, preparation a new type of functionalized graphene oxide capillary electrochromatography enzyme micro reactor. The physicochemical characteristic of graphene oxide and biochemical properties, the introduction of capillary electrophoresis enzyme micro reactor That can make the capillary electrophoresis from the conventional mode of capillary zone electrophoresis capillary into higher separation efficiency of electric separation mode, but also can enhance the activity of immobilized enzyme for capillary electrophoretic enzyme micro reactor overall performance promotion. Expand the ideas for the development of the study for capillary electrophoresis enzyme micro reactor the new method, but also provides a new tool for its application in enzyme inhibitor drug screening and other related fields. The content of this paper includes: 1. the recent fixed graphene graphene oxide and its derivatives as the carrier of enzyme technology are reviewed. The key is introduced based on physical adsorption, chemical bonding the principle and encapsulation of graphene related materials for making method of enzyme immobilization matrix and its new application in biological catalysis.2. physical adsorption and chemical covalent method based on, The development of methods for the preparation of functionalized graphene oxide capillary electrochromatography. The capillary electrochromatography prepared were analyzed by scanning electron microscope, thermogravimetric analysis, Raman spectra. The chromatographic column stability and separation were investigated. Experimental results show that the capillary electrochromatography of graphene oxide modified the N- benzene -L- benzoyl arginine ethyl HCl salt (BAEE) and N- -L- benzoyl arginine (BA) mixture separation from the bare tube (separation mode of capillary zone electrophoresis) 3.7 to 4.7. which showed the capillary electrochromatography of graphene oxide modified can greatly improve the separation performance of capillary electrophoresis separation. For complex compounds.3. provides a basis on the basis of the above, the physical adsorption and covalent immobilization of two kinds of enzyme technology, successfully prepared graphene oxide functionalized capillary electrochromatography enzyme micro reactor. With trypsin was fixed enzyme model , the enzyme activity and enzyme kinetics were studied. Experimental results show that the hydrolysis activity than covalent immobilized enzyme prepared by physical adsorption. The enzymatic kinetic results show that the physical adsorption preparation of functionalized graphene oxide capillary electrochromatography based on micro reaction enzyme Michaelis constant Km is 1.10mM, the biggest the reaction rate is 0.32mM-1. Vmax the Vmax is 3000 times more than the free enzyme solution, while Km is about free enzyme solution and 1/20. modified graphene oxide capillary immobilized enzyme activity (Km 109.77 mM, Vmax 0.00046 mM s-1) compared to graphene oxide for affinity and catalytic rate of immobilized enzyme carrier have improved significantly. This showed that the graphene oxide as a solid matrix enzyme can significantly enhance and improve the medicinal materials from 10 species in the rapid screening of trypsin inhibitor activity by micro reactor prepared by the catalytic activity of.4. The results showed that there was trypsin inhibitor in 37 and rhubarb, and the inhibition rate on trypsin was 26% and 26.12%. respectively. This indicates that the prepared microreactor is a potential screening tool for potential active components.

【学位授予单位】：中央民族大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ033

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