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

设计了一种简便的毛细管电泳化学发光检测池。该检测池易于与毛细管电泳设备联用。鲁米诺化学发光适用于检测池。对系统的详细分析和测试表明，鲁米诺的测定范围为2.5 x 10^M<-10>至6.5 x 10^<-7>M（相关系数，0.999），检测限（S/N=3）为2.5 x 10^<-10>M（7阿莫尔）。此外，在甘氨酸，甘氨酰甘氨酸和甘氨酰甘氨酰甘氨酸的混合物中的每种组分，用异硫氰酸异鲁米诺标记，基线分离和灵敏地检测。此外，还将该方法应用于毛细管电泳柱后检测。当在本系统中使用乙腈堆积在一定条件下，鲁米诺和标记化合物的化学发光强度约一个数量级高于那些没有堆积。的d ...更多信息 鲁米诺的检测限为1.5 × 10 ~<-11>（-1）M（S/N=3），代表了迄今报道的鲁米诺的最高灵敏度。此外，在弱碱性条件下，对碘苯酚作为鲁米诺化学发光增强剂的效果进行了研究。鲁米诺的化学发光强度比未增强的反应提高了约两个数量级。其次，我们建立了毛细管电泳化学发光检测器，以聚合物溶液为分离介质，用于分析生物大分子，如DNA和蛋白质。采用过氧化草酸二（2，4，6-三氯苯基）草酸酯化学发光试剂与荧光素标记试剂联用。荧光素标记的脱氧腺苷三磷酸通过聚合酶链反应制备荧光素标记的DNA。本系统成功地分离和检测了标记的DNA片段（500、600、700、800、900和993 bp）的混合物。还分离并检测了用异硫氰酸荧光素标记的蛋白质（溶菌酶、细胞色素C和核糖核酸酶A）的混合物。少

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。