本文關(guān)鍵詞： 自由流電泳 半導(dǎo)體散熱 蛋白質(zhì)分離 細(xì)胞分離　出處：《上海交通大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：自由流電泳（free flow electrophoresis, FFE）指具有電荷差異的待分離混合物隨液相背景緩沖液（carrier buffer, CB）通過多通道泵輸入由兩塊絕緣板構(gòu)成的薄腔后，受到垂直于液體流動(dòng)方向的不同大小的電場(chǎng)力，實(shí)現(xiàn)分離分析的電泳技術(shù)。FFE具有操作連續(xù)、分離模式多樣、無固體支持介質(zhì)、分離條件溫和等特點(diǎn)，既可以用做制備工具，也可以作為分析檢測(cè)工具。比較大型的FFE裝置常用于制備研究，而微流控芯片級(jí)FFE（microfluidic FFE, μFFE）常用來對(duì)物質(zhì)進(jìn)行分析檢測(cè)�？梢哉f，自從德國(guó)科學(xué)家Sevsson在1949年首次提出FFE原型以來，有關(guān)FFE的研究從未停止。FFE憑著自身強(qiáng)大的優(yōu)勢(shì)，已經(jīng)在小分子（無機(jī)或有機(jī)化合物）、大分子（多糖、多肽、蛋白質(zhì)、核酸）、（亞）細(xì)胞等對(duì)象的分離分析上得到了很多應(yīng)用。 隨著科技的發(fā)展、技術(shù)的進(jìn)步，F(xiàn)FE技術(shù)在多年的發(fā)展歷程中也不斷成熟，各種商業(yè)化的FFE儀器也出現(xiàn)在市場(chǎng)上。最早型號(hào)的Elphor Vap21FFE裝置于1994年由BenderHobein研制成功，并被用作多種生物樣本的制備。隨后，德國(guó)科學(xué)家Gerhard Weber對(duì)裝置進(jìn)行改進(jìn)，研發(fā)了新一代的FFE，并專門成立了FFE Service GmbH公司在全球范圍內(nèi)提供專門的FFE服務(wù)。后來，美國(guó)醫(yī)療器械巨頭BD公司也研制了BDTMFFE型號(hào)的FFE儀器并將其投入市場(chǎng)，授權(quán)北京博奧生物有限公司為中國(guó)地區(qū)的獨(dú)家代理。然而，一臺(tái)進(jìn)口FFE儀器售價(jià)在120~140萬(wàn)人民幣，非常昂貴，限制了FFE技術(shù)的推廣應(yīng)用。 中國(guó)對(duì)于FFE的重視程度與國(guó)外相比，仍相距甚遠(yuǎn)。到目前為止，國(guó)內(nèi)并沒有具有自主知識(shí)產(chǎn)權(quán)的商業(yè)化FFE問世。國(guó)內(nèi)做FFE研究方向的科學(xué)家也為數(shù)不多，對(duì)FFE的重視程度尚顯不足。國(guó)內(nèi)較為引人注意的FFE樣機(jī)是20世紀(jì)80年代末研制的A3-2型FFE儀器，當(dāng)時(shí)在神州四號(hào)飛船上對(duì)細(xì)胞色素C（cytochrome C, Cyt C）和牛血紅蛋白（hemoglobin, Hb）進(jìn)行過失重電泳分離實(shí)驗(yàn)，但并沒有商業(yè)化。 生物分離與分析實(shí)驗(yàn)室作為國(guó)際上FFE研究方向的佼佼者，自2009年以來一直在嘗試國(guó)產(chǎn)FFE儀器的商業(yè)化，成功研制出基于重力自平衡原理的FFE裝置，并在國(guó)際上首次提出移動(dòng)反應(yīng)界面自由流電泳（free flow moving reactionboundary electrophoresis, FFMRBE）概念，，將FFE技術(shù)與移動(dòng)反應(yīng)界面原理相結(jié)合。該FFE裝置被成功應(yīng)用于吩嗪-1-羧酸（phenazine-1-carboxylic acid, PCA）的分離純化、蛋白質(zhì)和細(xì)胞的分離。 然而，該實(shí)驗(yàn)室研制的FFE也存在一些問題，比如：（1）裝置操作很不方便；（2）容易漏液；（3）外形丑陋，距離商業(yè)化儀器的標(biāo)準(zhǔn)甚遠(yuǎn)。因此，本論文的研究?jī)?nèi)容就是針對(duì)舊FFE裝置存在的問題進(jìn)行改進(jìn)，力爭(zhēng)研制出達(dá)到商業(yè)化標(biāo)準(zhǔn)的低成本國(guó)產(chǎn)FFE裝置，同時(shí)也對(duì)裝置的性能進(jìn)行了全方位的檢測(cè)，并將新一代的FFE應(yīng)用到蛋白質(zhì)、細(xì)胞的分離。 研制出的新型FFE裝置包括4部分：（1）自動(dòng)化的電路集成系統(tǒng)（包括輸液系統(tǒng)、散熱系統(tǒng)、溫控系統(tǒng)、供電系統(tǒng)）；（2）氣液緩沖器（gas cusion injector,GCI）（具備排氣泡功能）；（3）分離室（分離腔體積為45mm*120mm*0.6-0.8mm，12個(gè)進(jìn)口、24個(gè)出口）；（4）自平衡回收裝置（self-balance collector, SBC）。 實(shí)驗(yàn)表明，裝置具備優(yōu)良的性能，整體設(shè)計(jì)上基本符合商業(yè)化FFE儀器的標(biāo)準(zhǔn)。曲線實(shí)驗(yàn)和氣泡產(chǎn)生實(shí)驗(yàn)表明裝置具有很好的散熱效果，在使用10mMpH8.0Tris-HCl背景緩沖液（carrier buffer，CB）（電導(dǎo)率為720μS·cm-1）、50mMpH8.0Tris-HCl電極緩沖液（electrode buffer，EB）（電導(dǎo)率為2530μS·cm-1）的條件下，最大可施加電壓為400V，不散熱條件下的電流較之散熱條件下的電流高-5.30%~70.26%。在散熱條件下，裝置可以使用20mM CB、100mM EB電解質(zhì)溶液，并將電壓施加到300V。同時(shí)，裝置可以成功分離氨基黑（amino black）、反應(yīng)紅（reactive red）、蘇木色精（hematoxylin）3種染料的混合物，牛血清白蛋白（bovine serum albumin, BSA）、Cyt C、肌紅蛋白（myoglobin, Mb）的混合物，基本實(shí)現(xiàn)大腸桿菌（Escherichia coli）、嗜熱鏈球菌（Streptococcus thermophilus）的分離，部分實(shí)現(xiàn)嗜熱鏈球菌和釀酒酵母（Saccharomyces cerevisiae）的分離。
[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 .

【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：Q503

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