本文選題：單細(xì)胞分析 + 微芯片電泳　； 參考：《浙江大學(xué)》2006年博士論文

【摘要】：檢測單個細(xì)胞內(nèi)化學(xué)組分,以及測定單細(xì)胞對外界刺激的成分變化,有助于理解基本細(xì)胞功能以及細(xì)胞內(nèi)外聯(lián)系,有助于檢測和鑒別大量細(xì)胞群體中少量的不正常細(xì)胞,因此單細(xì)胞的研究在重大疾病早期診斷、治療、藥物篩選和細(xì)胞生理、病理過程的研究方面有重要意義,已成為目前生命科學(xué)、生化分析等學(xué)科研究的熱門課題之一。 由于單細(xì)胞體積小,胞內(nèi)物質(zhì)濃度低,需要高靈敏度的檢測方法,如激光誘導(dǎo)熒光(LIF)法。但是大多數(shù)胞內(nèi)組分濃度低又無天然熒光,因此在解決微衍生的問題上還存有相當(dāng)?shù)目臻g。 最近,用微流控芯片技術(shù)對生物細(xì)胞的研究引起了廣泛注意,在近些年來得到快速發(fā)展。微流控芯片微米尺寸的通道適于單細(xì)胞引入,操縱,反應(yīng),分離及檢測。 本論文首次用芯片毛細(xì)管電泳—激光誘導(dǎo)熒光法定量測定了單個人血紅細(xì)胞內(nèi)的活性氧(ROS),將細(xì)胞進樣、單細(xì)胞定位、溶膜和毛細(xì)管電泳分離,集成到一塊玻璃微芯片完成。ROS包括超氧陰離子(O_2~(·-))、羥自由基(·OH)、過氧化氫等,它不但與衰老、動脈硬化、關(guān)節(jié)炎、細(xì)胞凋亡及癌變等密切相關(guān),并且由于它易擴散和降解,普遍存在于各類細(xì)胞中,它還起細(xì)胞間信使分子的作用。因此,細(xì)胞內(nèi)ROS的測定已受到普遍重視。我們用可透膜的雙氫羅丹明123(DHR 123)作為衍生試劑,DHR 123無熒光,進入細(xì)胞后,以細(xì)胞本身作為微反應(yīng)器,和細(xì)胞內(nèi)ROS反應(yīng)生成熒光產(chǎn)物羅丹明123(Rh 123),通過微芯片電泳激光誘導(dǎo)熒光法檢測細(xì)胞內(nèi)的Rh123,間接對細(xì)胞內(nèi)ROS進行了定量。我們討論了Rh123遷移時間隨pH的變化,得到最佳檢測條件,通過標(biāo)準(zhǔn)曲線法,定量測定了單個人血紅細(xì)胞內(nèi)ROS。該法由于避免了制備細(xì)胞懸液時溶劑的稀釋作用,使分析靈敏度大大提高。方法檢測下限為0.74amol,每分鐘檢測兩個細(xì)胞,連續(xù)測定6個單細(xì)胞遷移時間的精密度為2.1%。為研究單細(xì)胞受外界刺激前后ROS的變化提供了方法和工具。 在微流控芯片單細(xì)胞分析中,細(xì)胞能夠進入微通道是微流控芯片分析細(xì)胞的
[Abstract]:The detection of chemical components within a single cell, as well as changes in the components stimulated by a single cell to the outside, contribute to the understanding of basic cellular functions and to the internal and external connection of the cell, and to the detection and identification of a small number of abnormal cells in a large number of cell populations,Therefore, the study of single cell plays an important role in the early diagnosis, treatment, drug screening and the study of cellular physiology and pathological process of major diseases, and has become one of the hot topics in the field of life science, biochemical analysis and so on.Because of the small size of single cell and the low concentration of intracellular substance, high sensitive detection methods, such as laser induced fluorescence assay (LIF), are needed.However, most of the intracellular components have low concentration and no natural fluorescence, so there is still considerable room for solving the problem of micro-derivation.Recently, microfluidic chip technology has attracted wide attention in biological cells, and has been developed rapidly in recent years.Microfluidic chip micron-sized channels are suitable for single cell introduction, manipulation, reaction, separation and detection.In this paper, the reactive oxygen species (Ros) in a single human erythrocyte were quantitatively determined by microarray capillary electrophoresis and laser-induced fluorescence method. The cells were injected, located, separated by membrane and capillary electrophoresis.Integrated into a glass microchip, Ros includes superoxide anion O _ 2, hydroxyl radical (OHN), hydrogen peroxide, etc. It is not only closely related to aging, arteriosclerosis, arthritis, apoptosis and canceration, but also because of its easy diffusion and degradation.Ubiquitous in various types of cells, it also acts as an intercellular messenger molecule.Therefore, the detection of intracellular ROS has been paid more and more attention.We used dihydrorhodamine (123(DHR 123), a permeable membrane, as a derivative reagent, DHR123, as a non-fluorescent reagent, and after entering the cell, we used the cell itself as a microreactor.Rhodamine 123(Rh 123 was produced by intracellular ROS reaction. The intracellular ROS was detected by microchip electrophoresis laser-induced fluorescence assay.We discussed the change of Rh123 migration time with pH and obtained the best detection conditions. The ros in single human erythrocyte were quantitatively determined by standard curve method.The method avoids the dilution effect of solvent in the preparation of cell suspension, so the analytical sensitivity is greatly improved.Methods the detection limit was 0.74 amol, two cells were detected every minute, and the precision of 6 single cell migration times was 2.1.It provides a method and a tool for studying the changes of ROS before and after the single cell is stimulated by external environment.In microfluidic chip single cell analysis, cells can enter the microchannel, which is used to analyze cells by microfluidic chips.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R329

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