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

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结合使用。鲁米诺化学发光适用于检测池。详细分析和测试表明，该系统可在2.5 × 10^<-10> ~ 6.5 × 10^<-7 >m范围内测定鲁米诺（相关系数为0.999），检出限（S/N=3）为2.5 × 10^<-10 >m （7 amol）。此外，在甘氨酸、甘氨酸和甘氨酸的混合物中，用异硫氰酸异亮醇标记，对每种成分进行基线分离和敏感检测。此外，将堆积法应用于毛细管电泳柱后检测。当本体系在一定条件下使用乙腈叠层时，鲁米诺和标记化合物的化学发光强度比未叠层的化学发光强度高一个数量级。鲁米诺的检测限为1.5 × 10^<-11> M (S/N=3)，是目前报道的灵敏度最高的鲁米诺。在弱碱性条件下，考察了对碘酚作为发光胺化学发光增强剂的作用。鲁米诺的化学发光强度比未增强反应提高了约2个数量级。接下来，我们开发了毛细管电泳化学发光检测器，使用聚合物溶液作为分离介质，用于分析生物聚合物，如DNA和蛋白质。采用双（2,4,6-三氯苯基）草酸过氧草酸化学发光试剂与荧光标记试剂配合使用。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。标记的DNA片段（500、600、700、800、900和993 bp）的混合物通过本系统成功分离和检测。用异硫氰酸氟蛋白标记的蛋白质混合物（溶菌酶、细胞色素C和核糖核酸酶A）也被分离和检测。少

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