毛细管电泳在线富集方法的研究与应用

发布时间：2018-08-03 17:12

【摘要】：毛细管电泳(Capillary Electrophoresis,CE),是在毛细管(分离通道)中,分析物在电场下中依据各个组成之间的淌度差异或分配行为完成分离检测。由于其具有效率高、成本低、小剂量等特点,因此,这些年在分析分离领域越来越受到科研工作者的重视与青睐,另外,不同检测器成功与毛细管电泳技术联用,给CE检测技术的应用范围带来更大的空间,经过科研工作者的不懈努力下,毛细管电泳相关技术已经在食品、药物、环境、生化等领域蓬勃发展和广泛应用。然而,由于进样量小和检测器等的限制,CE的检测灵敏度达不到生物体系中痕量组分检测的需求,为了解决这一问题,除了改进和完善各类检测器外,科研工作者们还致力于研究各种在线富集技术并与CE联用,期待能够进一步提高灵敏度,并且更好地完成对复杂样品的检测。本文将毛细管电泳分别与非接触式电导检测器(C4D)和激光诱导荧光检测器(LIF)联用,通过探索并建立不同的在线富集体系,进一步研究在线富集技术,并且应用于不同的实际样品中,主要内容如下:第一章为绪论,绪论包括CE的发展综述、分离模式和进样方法、检测技术、在线富集技术及应用,并介绍了本论文的研究目的和意义。第二章将场放大进样与毛细管电泳-非接触式电导检测联用(FESI-CE-C4D),实现了五种人工添加剂(亚硝酸盐、异抗坏血酸盐、山梨酸盐硝酸盐、硫酸盐)的在线富集和分离检测。在最佳条件下,五种物质的检测限为0.05-1mgL-1(S/N=3)。为了评估该模型的应用潜力,把该模型运用到火腿肠中人工添加剂的检测当中,样品处理操作简单,且回收率结果良好(91.9%-106.2%)。第三章将毛细管电泳(CE)-激光诱导荧光(LIF)联用,结合动态pH界面和瞬时捕获两种在线富集技术,成功地实现了谷胱甘肽的有效富集和分离检测。实验对在线富集和分离检测的各种参数进行了探究,在动态pH界面下,分析物会被压缩成一个狭窄的区域,同时被SDS胶束在样品区带和胶束相边界捕获,最后被分离。最优条件下,还原型谷胱甘肽,甘氨酸,氧化型谷胱甘肽,半胱氨酸,谷氨酸的检测限分别为0.01,0.01,0.5,0.7和O.1nM(S/N=3)。对比经典的CZE模式,本实验采用瞬时捕获结合动态pH连接在线富集模型的富集倍数为87-430。此外,该方法已成功地应用于细菌(大肠杆菌、鼠伤寒沙门氏菌和金黄色葡萄球菌)和HaCaT细胞中还原型谷胱甘肽的定量测定。第四章将毛细管电泳(CE)-激光诱导荧光(LIF)技术,联合胶束扫集-动态pH界面两种在线富集技术,对八种氨基酸进行了分离检测和在线富集。经过对分离和富集的各种参数的探究,最佳条件下,精氨酸,赖氨酸,色氨酸,组氨酸,缬氨酸,酪氨酸,丝氨酸,丙氨酸的检测限分别为0.07,0.35,0.15,0.20,0.20,0.15,0.33,0.10nM(S/N=3),富集倍数达到44-67之间。另外,将该方法应用于人体唾液中检测氨基酸,获得令人满意的回收率。
[Abstract]:Capillary electrophoresis (Capillary Electrophoresis, CE) is the separation and detection of analyte in an electric field based on the mobility difference or distribution behavior between various components in an electric field. Because of its high efficiency, low cost and small dose, these years are becoming more and more scientific research workers in the field of analysis and separation. In addition, the successful application of different detectors to capillary electrophoresis has brought more space for the application of CE detection technology. With the unremitting efforts of researchers, capillary electrophoresis has been developed and widely used in the fields of food, medicine, environment and biochemistry. The detection sensitivity of CE can not reach the requirement of trace component detection in biological system. In order to solve this problem, in addition to improving and improving all kinds of detectors, researchers are also working on various on-line enrichment techniques and combined with CE in order to solve this problem. Detection of complex samples. In this paper, capillary electrophoresis is combined with non-contact conductance detector (C4D) and laser induced fluorescence detector (LIF) respectively. By exploring and establishing different on-line enrichment systems, the on-line enrichment technology is further studied and used in different solid samples. The main contents are as follows: the first chapter is the introduction and the introduction package. In the second chapter, five kinds of artificial additives (nitrite, isoascorbic acid, sorbic acid) have been realized in the second chapter, including field amplification and capillary electrophoresis noncontact conductivity detection (FESI-CE-C4D). On line enrichment and separation detection of salt nitrate and sulfate. Under the best conditions, the detection limit of five substances is 0.05-1mgL-1 (S/N=3). In order to evaluate the application potential of the model, the model is applied to the detection of artificial additives in the ham sausage, the sample processing operation is simple, and the recovery rate is good (91.9%-106.2%). The third chapter is the hair. CE - laser induced fluorescence (LIF) combined with dynamic pH interface and instantaneous capture of two on-line enrichment techniques have successfully realized the effective enrichment and separation detection of glutathione. The experiments have explored various parameters for on-line enrichment and separation detection. Under the dynamic pH interface, the analyte will be compressed into a narrow area. The domain, at the same time, was captured by the SDS micelles at the sample zone and the micelle boundary, and finally separated. Under optimal conditions, the detection limits of glutathione, glycine, oxidized glutathione, cysteine, and glutamic acid were 0.01,0.01,0.5,0.7 and O.1nM (S/N=3) respectively. Compared with the classical CZE model, the instantaneous capture combined with the dynamic pH connection was used in this experiment. The enrichment factor of the line enrichment model is 87-430.. The method has been successfully applied to the quantitative determination of the reduced glutathione in bacteria (Escherichia coli, Salmonella typhimurium and Staphylococcus aureus) and HaCaT cells. In the fourth chapter, the technique of capillary electrophoresis (CE) laser induced fluorescence (LIF), combined micellar sweep collection dynamic pH interface On line enrichment technology, eight kinds of amino acids were separated and on-line enriched. Through the exploration of the various parameters of separation and enrichment, the detection limits of arginine, lysine, tryptophan, histidine, valine, tyrosine, serine and alanine were 0.07,0.35,0.15,0.20,0.20,0.15,0.33,0.10nM (S/N=3) and enriched times under the optimum conditions. The number reached 44-67. In addition, the method was applied to the detection of amino acids in human saliva, and a satisfactory recovery rate was obtained.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O658.9

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