重力自平衡自由流电泳装置的研制与应用

发布时间：2018-02-23 13:24

本文关键词： 自由流电泳半导体散热蛋白质分离细胞分离　出处：《上海交通大学》2014年硕士论文　论文类型：学位论文

【摘要】：自由流电泳（free flow electrophoresis, FFE）指具有电荷差异的待分离混合物随液相背景缓冲液（carrier buffer, CB）通过多通道泵输入由两块绝缘板构成的薄腔后，受到垂直于液体流动方向的不同大小的电场力，实现分离分析的电泳技术。FFE具有操作连续、分离模式多样、无固体支持介质、分离条件温和等特点，既可以用做制备工具，也可以作为分析检测工具。比较大型的FFE装置常用于制备研究，而微流控芯片级FFE（microfluidic FFE, μFFE）常用来对物质进行分析检测。可以说，自从德国科学家Sevsson在1949年首次提出FFE原型以来，有关FFE的研究从未停止。FFE凭着自身强大的优势，已经在小分子（无机或有机化合物）、大分子（多糖、多肽、蛋白质、核酸）、（亚）细胞等对象的分离分析上得到了很多应用。随着科技的发展、技术的进步，FFE技术在多年的发展历程中也不断成熟，各种商业化的FFE仪器也出现在市场上。最早型号的Elphor Vap21FFE装置于1994年由BenderHobein研制成功，并被用作多种生物样本的制备。随后，德国科学家Gerhard Weber对装置进行改进，研发了新一代的FFE，并专门成立了FFE Service GmbH公司在全球范围内提供专门的FFE服务。后来，美国医疗器械巨头BD公司也研制了BDTMFFE型号的FFE仪器并将其投入市场，授权北京博奥生物有限公司为中国地区的独家代理。然而，一台进口FFE仪器售价在120~140万人民币，非常昂贵，限制了FFE技术的推广应用。中国对于FFE的重视程度与国外相比，仍相距甚远。到目前为止，国内并没有具有自主知识产权的商业化FFE问世。国内做FFE研究方向的科学家也为数不多，对FFE的重视程度尚显不足。国内较为引人注意的FFE样机是20世纪80年代末研制的A3-2型FFE仪器，当时在神州四号飞船上对细胞色素C（cytochrome C, Cyt C）和牛血红蛋白（hemoglobin, Hb）进行过失重电泳分离实验，但并没有商业化。生物分离与分析实验室作为国际上FFE研究方向的佼佼者，自2009年以来一直在尝试国产FFE仪器的商业化，成功研制出基于重力自平衡原理的FFE装置，并在国际上首次提出移动反应界面自由流电泳（free flow moving reactionboundary electrophoresis, FFMRBE）概念，，将FFE技术与移动反应界面原理相结合。该FFE装置被成功应用于吩嗪-1-羧酸（phenazine-1-carboxylic acid, PCA）的分离纯化、蛋白质和细胞的分离。然而，该实验室研制的FFE也存在一些问题，比如：（1）装置操作很不方便；（2）容易漏液；（3）外形丑陋，距离商业化仪器的标准甚远。因此，本论文的研究内容就是针对旧FFE装置存在的问题进行改进，力争研制出达到商业化标准的低成本国产FFE装置，同时也对装置的性能进行了全方位的检测，并将新一代的FFE应用到蛋白质、细胞的分离。研制出的新型FFE装置包括4部分：（1）自动化的电路集成系统（包括输液系统、散热系统、温控系统、供电系统）；（2）气液缓冲器（gas cusion injector,GCI）（具备排气泡功能）；（3）分离室（分离腔体积为45mm*120mm*0.6-0.8mm，12个进口、24个出口）；（4）自平衡回收装置（self-balance collector, SBC）。实验表明，装置具备优良的性能，整体设计上基本符合商业化FFE仪器的标准。曲线实验和气泡产生实验表明装置具有很好的散热效果，在使用10mMpH8.0Tris-HCl背景缓冲液（carrier buffer，CB）（电导率为720μS·cm-1）、50mMpH8.0Tris-HCl电极缓冲液（electrode buffer，EB）（电导率为2530μS·cm-1）的条件下，最大可施加电压为400V，不散热条件下的电流较之散热条件下的电流高-5.30%~70.26%。在散热条件下，装置可以使用20mM CB、100mM EB电解质溶液，并将电压施加到300V。同时，装置可以成功分离氨基黑（amino black）、反应红（reactive red）、苏木色精（hematoxylin）3种染料的混合物，牛血清白蛋白（bovine serum albumin, BSA）、Cyt C、肌红蛋白（myoglobin, Mb）的混合物，基本实现大肠杆菌（Escherichia coli）、嗜热链球菌（Streptococcus thermophilus）的分离，部分实现嗜热链球菌和酿酒酵母（Saccharomyces cerevisiae）的分离。
[Abstract]:Free flow electrophoresis ( FFE ) means that the mixture to be separated which has a charge difference is input by a multi - channel pump into a thin cavity composed of two insulating plates through a multi - channel pump . It can be used as a preparation tool and can be used as an analytical detection tool . The FFE has the advantages of continuous operation , various separation modes , no solid support medium , mild separation condition and the like . With the development of science and technology , the technology advances , the FFE technology has been mature in many years . All kinds of commercial FFE instruments have also appeared in the market . The first model Elphor - 21FFE has been developed by Bender Hoskins in 1994 and has been used as a variety of biological samples . Later , American medical device giant BD has developed a new generation of FFE instruments and put it into the market , and authorized Beijing Boao Bio Inc . to serve as the sole agent in China . However , an imported FFE instrument is sold at 120 - 1.4 million yuan , which is very expensive and restricts the application and application of FFE technology . China ' s attention to FFE is far from that of foreign countries . So far , there is no commercial FFE with independent intellectual property rights . In China , there are few scientists with independent intellectual property rights . In China , there are few scientists in FFE research . The FFE has been developed in the late 1980 ' s . At the time , the experiments were carried out on cytochrome C ( Cyt C ) and bovine hemoglobin ( hemoglobin , Hb ) in Shenzhou No . 4 spacecraft , but it was not commercialized . Since 2009 , FFE has been successfully applied to the separation and purification of phenazine - 1 - carboxylic acid ( PCA ) and the separation of proteins and cells . However , the FFE developed by the laboratory has some problems , such as : ( 1 ) the operation of the device is very inconvenient ; ( 2 ) it is easy to leak liquid ; ( 3 ) the appearance is ugly and the standard of the distance commercial instrument is very far . Therefore , the research content of this paper is to improve the existing FFE device with low cost and to develop a low - cost domestic FFE device which meets the commercial standard , and meanwhile , the performance of the device is comprehensively tested , and the new generation FFE is applied to the separation of the protein and the cell . The newly developed FFE device includes four parts : ( 1 ) automatic circuit integration system ( including infusion system , heat dissipation system , temperature control system , power supply system ) ; ( 2 ) gas - liquid buffer ( gas cusion injector , GCI ) ( with bubble discharge function ) ; ( 3 ) separation chamber ( separation chamber volume 45 mm * 120 * 0.6 - 0.8mm , 12 inlets , 24 outlets ) ; ( 4 ) self - balance collector ( SBC ) . The experimental results show that the device has excellent performance and is basically in conformity with the standard of commercial FFE instrument . The experiment of curve and bubble generation show that the device has good heat dissipation effect . Under the condition of heat radiation , 20 mM CB , 100 mM EB electrolyte solution can be applied , and the mixture of bovine serum albumin ( BSA ) , Cyt C and Myoglobin ( Mb ) can be successfully separated .

【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：Q503

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