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

设计了一种简便易行的毛细管电泳法化学发光检测池。该检测池可方便地与毛细管电泳仪联用。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2.5×10~(-7)~6.5×10~(-7)M(相关系数为0.999)，检出限(S/N=3)为2.5×10~(-10)~10~(-5)M(7 Amol)。此外，用异硫氰酸酯标记的甘氨酸、甘氨酰甘氨酸和甘氨酰甘氨酸混合物中的每个组分都被基线分离并灵敏地检测。此外，将堆积过程应用于毛细管电泳柱后检测。在本体系中，在一定条件下使用乙腈堆积时，鲁米诺和标记化合物的化学发光强度比未堆积时高出一个数量级左右。The d…鲁米诺的更高检出限为1.5×10~(-11)M(S/N=3)，代表了鲁米诺的最高灵敏度。在弱碱性条件下，考察了对碘苯酚作为鲁米诺化学发光增强剂的作用。鲁米诺的化学发光强度比未增强的反应高出约两个数量级。其次，我们建立了以聚合物溶液为分离介质的毛细管电泳联用化学发光检测器，用于DNA和蛋白质等生物大分子的分析。双(2，4，6-三氯苯基)草酸根过氧草酸盐化学发光剂与荧光素标记剂联用。用荧光素标记的脱氧腺苷三磷酸通过聚合酶链式反应制备荧光素标记的DNA。通过本系统成功地分离和检测了标记的DNA片段的混合物(500、600、700、800、900和993bp)。还分离和检测了用异硫氰酸荧光素标记的蛋白质混合物(溶菌酶、细胞色素C和核糖核酸酶A)。较少

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