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

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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