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Development of Selective Analysis for Individual Functional Groups by High-performance Capillary Electrophoresis (HPCE) with Laser-induced Fluorescence (LIF) Detection

通过高性能毛细管电泳 (HPCE) 和激光诱导荧光 (LIF) 检测对单个官能团进行选择性分析的发展

基本信息

批准号：
09672193
负责人：
TOYO'OKA Toshimasa
金额：
$ 1.79万
依托单位：
University of Shizuoka
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

HPCE is an excellent means for separation of multi-components with high speed and separatability. This study deals with search of labeling reagents for various functional groups such as carboxyl, amine, thiol and hydroxyl, by HPCE-LIF detection. Trace analysis of biologically importances by recommended CE-LIF procedures is also carried out with the selected fluorescent labeling reagents.Fluorophors possessing fluoresceine (e.g. FITC) and 4-nitro-2, 1,3-benzoxadiazole (e.g. NBD-F) moieties were suitable for argon-ion (Ar) laser (excitation wavelength, 488 nm) detection, in terms of wavelength and sensitivity. Whereas 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole structures such as DBD-F were recommended for helium-cadmium (He-Cd) laser (excitation wavelength, 442 nm) detection. The detectability using the reagents with LIF detection increased more than two orders of magnitude, as comparing with UV detection. On the other hand, the detection sensitivity with He-Cd laser at 325 nm was lower than that at 442 nm, but higher than that of UV detection. Cyanine dyes were selected for diode (DIO) laser detection at 630 nm. However, the low stability of the reagents in solution and the low derivatization yields were important disadvantages. As the application with HPCE-LIF detection , chiral separation of DL-amino acids and bioactive thiols in rat hepatocytes were performed after labeling with DBD-PyNCS and ABD-F, respectively. The derivatives of DL-amino acids were completely separated by HPCE and detected sensitively (a few fmol level) with Ar-laser detection at 488 nm. Intracellar thiols (e.g. glutathione, cysteine, N-acetylcysteine) after labeling with ABD-F were also separated and detected by HPCE-LIF with He-Cd laser at 325 nm. The concentration of glutathione in the single cell was fmol level. The proposed procedure seems to be applicable to highly sensitive detection of biological substances in a cell.

高效毛细管电泳法是一种分离速度快、可分离性好的多组分分离方法。本研究采用高效毛细管电泳法(HPCE-LIF)对羧基、胺、硫醇、羟基等多种官能团的标记试剂进行了研究。所选择的荧光标记剂中含有荧光素(如FITC)和4-硝基-2，1，3-苯并恶二唑(如NBD-F)的荧光体在波长和灵敏度方面适合于Ar离子(激发波长，488 nm)的检测。而4-(N，N-二甲氨基磺酰基)-2，1，3-苯并恶二唑结构，如DBD-F，被推荐用于氦-镉(He-Cd)激光(激发波长，442 nm)的检测。与紫外检测相比，LIF检测试剂的可检测性提高了两个数量级以上。He-Cd激光325 nm的检测灵敏度低于442 nm，但高于紫外光检测。选择菁染料进行二极管(DIO)630 nm激光检测。然而，试剂在溶液中的稳定性低和衍生化产率低是一个重要的缺点。作为HPCE-LIF检测的应用，分别用DBD-PyNCS和Abd-F标记大鼠肝细胞中的DL-氨基酸和生物活性硫醇。用高效毛细管电泳法将DL-氨基酸的衍生物完全分离，并在488 nm处用Ar-激光检测。用ABD-F标记细胞内的硫醇(如谷胱甘肽、半胱氨酸、N-乙酰半胱氨酸)，用HPCE-LIF在325 nm处用He-Cd激光进行检测。单细胞内谷胱甘肽的浓度为fmoL水平。建议的程序似乎适用于对细胞中生物物质的高灵敏检测。