本文選題：氧化石墨烯　切入點：毛細管電色譜　出處：《中央民族大學》2017年碩士論文

【摘要】：毛細管電泳酶微反應器是生物分子固定方法、微酶反應以及高效分離技術(shù)相結(jié)合的一種微生化反應裝置。作為一種低耗高效且易于自動化的酶催化反應工具,它在蛋白質(zhì)酶解、生物轉(zhuǎn)化及酶抑制劑藥物篩選等方面具有獨特的優(yōu)勢。毛細管電泳酶微反應器主要由固定化酶微反應器部分和毛細管電泳分離部分構(gòu)成。其中固定化酶的活性與固酶的載體基質(zhì)密切相關,而其毛細管電泳分離性能與分離模式有關。本論文選擇納米材料氧化石墨烯作為固載基質(zhì),制備了一種新型的氧化石墨烯功能化毛細管電色譜酶微反應器。氧化石墨烯特有的物化和生化性能,將其引入毛細管電泳酶微反應器中,既可使毛細管電泳從常規(guī)的毛細管區(qū)帶電泳分離模式轉(zhuǎn)變?yōu)榉蛛x效率更高的毛細管電色譜分離模式,同時還能增強固定化酶的活性,實現(xiàn)毛細管電泳酶微反應器整體性能的提升。該研究既為毛細管電泳酶微反應器新方法的發(fā)展拓展了思路,也為其在酶抑制劑藥物篩選等相關領域的應用提供了新的工具。論文研究內(nèi)容主要包括:1.對近年來以石墨烯及其衍生物氧化石墨烯等為載體的固定化酶技術(shù)進行了評述。重點介紹了基于物理吸附、化學鍵合和包埋原理的石墨烯相關材料為載體基質(zhì)的固定化酶的制作方法及其在生物催化等領域的最新應用。2.基于物理吸附法和化學共價法,發(fā)展了氧化石墨烯功能化毛細管電色譜的制備方法。對所制備的毛細管電色譜進行了掃描電鏡、熱失重、拉曼光譜等表征。對該色譜柱的穩(wěn)定性和分離度進行了考察。實驗結(jié)果顯示氧化石墨烯修飾的毛細管電色譜可使N-苯甲酰-L-精氨酸乙酯HCl鹽(BAEE)和N-苯甲酰-L-精氨酸(BA)混合物的分離度從裸管(毛細管區(qū)帶電泳分離模式)的3.7提高到4.7。這表明氧化石墨烯修飾的毛細管電色譜能顯著提高毛細管電泳分離性能,為復雜化合物的分離奠定了基礎。3.在上述基礎上,采用物理吸附和化學共價兩種酶固載化技術(shù),成功制得氧化石墨烯功能化毛細管電色譜酶微反應器。以胰蛋白酶為固定模型酶,進行了酶活及酶促動力學等研究。實驗結(jié)果顯示,物理吸附法制備的固定化酶的水解活性優(yōu)于化學共價法。酶促動力學結(jié)果顯示,基于物理吸附法制備的氧化石墨烯功能化毛細管電色譜酶微反應器的米氏常數(shù)Km為1.10mM,最大反應速率Vmax為0.32mM-1。其Vmax是自由酶溶液的3000多倍,而Km約為自由酶溶液的1/20。與未修飾氧化石墨烯的毛細管固定化酶的活性(Km 109.77 mM,Vmax 0.00046 mM·s-1)相比,氧化石墨烯為載體的固定化酶的親和力與催化速率都有顯著提高。這表明氧化石墨烯作為固酶基質(zhì)能顯著增強和提高酶的催化活性。4.利用所制備的微反應器從10種中藥材中快速篩選有胰蛋白酶抑制劑活性成分的藥材。結(jié)果顯示三七和大黃中存在胰蛋白酶抑制劑活性成分,對胰蛋白酶的抑制率分別為26%和26.12%。這表明所制備的微反應器是一種潛在藥物活性成分的快速篩選工具。
[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.

【學位授予單位】：中央民族大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：TQ033

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本文編號：1708961