Development of simple "capillary electrophoresis - chemiluminescence detection" system and its application to a gene diagnosis

简易“毛细管电泳-化学发光检测”系统的研制及其在基因诊断中的应用

• 批准号: 13650873
• 负责人: TSUKAGOSHI Kazuhiko
• 金额: $ 0.96万
• 依托单位: Doshisha University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13650873/
• 关键词: Capillary electrophoresis; Chemiluminescence; DNA; Sensitive analysis; Separation ・ detection; 遺伝子

A simple and convenient chemiluminescence detection cell was designed for capillary electrophoresis. The detection cell easily combined with capillary electrophoresis equipment. Luminol chemiluminescence was adapted for use with the detection cell. Detailed analysis and testing of the system revealed that luminol could be determined over a range of 2.5 x 10^<-10> to 6.5 x 10^<-7> M (correlation coefficient, 0.999), with a detection limit (S/N=3) of 2.5 x 10^<-10> M (7 amol). Furthermore, each component in a mixture of glycine, glycylglycine, and glycylglycylglycine, which were labeled with isoluminol isothiocyanate, was baseline separated and sensitively detected. Moreover, the stacking procedure was applied to post column detection in capillary electrophoresis. When using acetonitrile stacking under certain conditions in the present system, chemiluminescence intensities of luminol and labeled compounds were about an order of magnitude higher than those obtained without stacking. The d … More etection limit for luminol was 1.5 x 10^<-11> M (S/N=3), representing the highest sensitivity of luminol yet reported. Also, the effect of p-iodophenol as an enhancer of luminol chemiluminescence was examined under weak alkaline conditions. The chemiluminescence intensity of luminol was about two orders of magnitude higher than that in the unenhanced reaction.Next, we developed capillary electrophoresis with chemiluminescence detector using a polymer solution as separation medium for analysis of biopolymers, such as DNA and protein. Peroxyoxalate chemiluminescence reagent of bis (2,4,6-trichlorophenyl)oxalate was used together with fluorescein-labeling reagent. Fluorescein-labeled DNA was prepared through polymerase chain reaction using fluorescein-labeled deoxyadenosine triphosphate. A mixture of the labeled DNA fragments (500, 600, 700, 800, 900, and 993 bp) was successfully separated and detected by the present system. A mixture of proteins (lysozyme, cytochrome C, and ribonuclease A) which were labeled with fluoresein isothiocyanate was also separated and detected. Less

设计了一种用于毛细管电泳的简单方便的化学发光检测池，该检测池易于与鲁米诺化学发光设备结合使用，该系统的详细分析和测试表明可以在一定范围内测定鲁米诺。 2.5 x 10^<-10> 至 6.5 x 10^<-7> M（相关系数，0.999），其中检测限(S/N＝3)为2.5×10^-10M(7amol)。此外，用异硫氰酸异鲁米诺标记的甘氨酸、甘氨酰甘氨酸和甘氨酰甘氨酰甘氨酸的混合物中的各组分被基线分离并进行分析。此外，当在一定条件下使用乙腈堆叠时，该堆叠程序适用于毛细管电泳中的柱后检测。在本系统中，鲁米诺和标记化合物的化学发光强度比未叠加时获得的化学发光强度高出大约一个数量级，鲁米诺的 d … 更多腐蚀极限为 1.5 x 10^<-11> M (S/N=3)，表示。鲁米诺的化学发光强度是迄今为止报道的最高灵敏度。鲁米诺比未增强反应高约两个数量级。 接下来，我们开发了带有化学发光检测器的毛细管电泳，使用聚合物溶液作为分离介质来分析生物聚合物，例如双（2，2， 4,6-三氯苯基)草酸酯与荧光素标记试剂一起使用，制备荧光素标记的DNA。使用荧光素标记的脱氧腺苷三磷酸的聚合酶链反应，通过本系统成功分离和检测了标记的DNA片段（500、600、700、800、900和993bp）的蛋白质混合物（溶菌酶、细胞色素）。用异硫氰酸荧光素标记的C和核糖核酸酶A)也被分离和检测到。

项目成果

Kazuhiko Tsukagoshi: "Separation and Determination of Emetine Dithiocarbamate Metal Complexes by Capillary Electrophoresis with Chemiluminescence Detection of the Tris(2,2'-bipyridine) Ruthenium(II) Complex"Journal of Chromatography A. 953. 283-289 (2002)

Kazuhiko Tsukagoshi：“通过毛细管电泳和化学发光检测三(2,2-联吡啶)钌(II)络合物来分离和测定依米丁二硫代氨基甲酸盐金属络合物”Journal of Chromatography A. 953. 283-289 (2002)

Kazuhiko Tsukagoshi: "Application of Capillary Electrophoresis with Sensitive Detection to Analysis for Saccharide Molecules"Analytical Sciences. 18. 709-710 (2002)

Kazuhiko Tsukagoshi：“毛细管电泳与灵敏检测在糖分子分析中的应用”分析科学。

Kazuhiko Tsukagoshi, Takeshi Nakamura, and Riichiro Nakajima: "Batch-type Chemiluminescence Detection Cell for Sensitization and Simplification of Capillary Electrophoresis"Analytical Chemistry. 74. 4109-4116 (2002)

Kazuhiko Tsukagoshi、Takeshi Nakamura 和 Riichiro Nakajima：“用于毛细管电泳敏化和简化的批量式化学发光检测单元”分析化学。

Kazuhiko Tsukagoshi: "Influence of Silicon Membrane Interposed between Glass Plates on Microchip Capillary Elecerophoresis with a Chemiluminescence detecter"Analytical Sciences. 17. 1129-1131 (2001)

Kazuhiko Tsukagoshi：“插入玻璃板之间的硅膜对使用化学发光检测器的微芯片毛细管电泳的影响”分析科学。