微流控芯片上单细胞分析的研究

发布时间：2018-04-17 12:51

本文选题：单细胞分析 + 微芯片电泳　；参考：《浙江大学》2006年博士论文

【摘要】：检测单个细胞内化学组分,以及测定单细胞对外界刺激的成分变化,有助于理解基本细胞功能以及细胞内外联系,有助于检测和鉴别大量细胞群体中少量的不正常细胞,因此单细胞的研究在重大疾病早期诊断、治疗、药物筛选和细胞生理、病理过程的研究方面有重要意义,已成为目前生命科学、生化分析等学科研究的热门课题之一。由于单细胞体积小,胞内物质浓度低,需要高灵敏度的检测方法,如激光诱导荧光(LIF)法。但是大多数胞内组分浓度低又无天然荧光,因此在解决微衍生的问题上还存有相当的空间。最近,用微流控芯片技术对生物细胞的研究引起了广泛注意,在近些年来得到快速发展。微流控芯片微米尺寸的通道适于单细胞引入,操纵,反应,分离及检测。本论文首次用芯片毛细管电泳—激光诱导荧光法定量测定了单个人血红细胞内的活性氧(ROS),将细胞进样、单细胞定位、溶膜和毛细管电泳分离,集成到一块玻璃微芯片完成。ROS包括超氧阴离子(O_2~(·-))、羟自由基(·OH)、过氧化氢等,它不但与衰老、动脉硬化、关节炎、细胞凋亡及癌变等密切相关,并且由于它易扩散和降解,普遍存在于各类细胞中,它还起细胞间信使分子的作用。因此,细胞内ROS的测定已受到普遍重视。我们用可透膜的双氢罗丹明123(DHR 123)作为衍生试剂,DHR 123无荧光,进入细胞后,以细胞本身作为微反应器,和细胞内ROS反应生成荧光产物罗丹明123(Rh 123),通过微芯片电泳激光诱导荧光法检测细胞内的Rh123,间接对细胞内ROS进行了定量。我们讨论了Rh123迁移时间随pH的变化,得到最佳检测条件,通过标准曲线法,定量测定了单个人血红细胞内ROS。该法由于避免了制备细胞悬液时溶剂的稀释作用,使分析灵敏度大大提高。方法检测下限为0.74amol,每分钟检测两个细胞,连续测定6个单细胞迁移时间的精密度为2.1%。为研究单细胞受外界刺激前后ROS的变化提供了方法和工具。在微流控芯片单细胞分析中,细胞能够进入微通道是微流控芯片分析细胞的
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R329

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