Fluorescence detection in capillary zone electrophoresis using a charge-coupled device with time-delayed integration.
Fluorescence detection in capillary zone electrophoresis using a charge-coupled device with time-delayed integration.
复制标题
使用具有延时积分的电荷耦合装置进行毛细管区带电泳中的荧光检测。
DOI：
10.1021/ac00005a022
复制
发表时间：
1991
影响因子：
7.4
通讯作者：
Scheller,RH
中科院分区：
文献类型：
--
作者：
Sweedler,JV;Shear,JB;Fishman,HA;Zare,RN;Scheller,RH
生物化学中的许多研究领域依赖于分析微量核酸、氨基酸和肽的能力，然而目前许多检测方案的灵敏度不足。例如单细胞和亚细胞区室的分析。毛细管区带电泳（CZE）是少数几种能够成功测定单个细胞内容物的技术之一（1-4），这是一种用于分析小样品体积的强大分离技术。分离效率通常超过几十万块理论塔板，典型的进样体积为10 nL或更少。最近的几篇综述详细描述了CZE的能力和性能（5-9）。对于CZE，通道直径通常在25至100 μ m范围内;因此，设计检测这些小直径毛细管中低浓度的方法是一个挑战。激光诱导荧光（LIF）是目前CZE最灵敏的检测方法，检测限在低阿摩尔范围（10-12）。在这些系统中，毛细管被用作“流动池”，激光照射垂直于毛细管，并且光电倍增管（PMT）监测荧光。Dovichi及其同事（13-15）开发了一种更灵敏的方法，该方法具有相同的激发几何结构，但使用鞘流比色皿作为样品池，从而消除了熔融石英毛细管的大部分散射光和发光。利用这种技术，已经报道了荧光标记的氨基酸在低zeptomole范围内的检测限。[Zepto（z）和yocto（y）已分别由Comite Consultatif des Unites提议作为10'21和1CT 24的新SI前缀;最终批准有待于Comite General des Poids et Mesures的下一次会议。
INTRODUCTION Numerous research areas in biochemistry depend on the ability to analyze minute quantities of nucleic acids, amino acids, and peptides, yet many present detection schemes have inadequate sensitivity. Examples are the analyses of single cells andsubcellular compartments. One of the few techniques to permit successful assays of the contents of a single cell (1-4) is capillary zone electrophoresis (CZE), a powerful separation technique for the analysis of small sample volumes. Sepa-ration efficiencies routinely exceed several hundred thousand theoretical plates, and typical injection volumes are 10 nL or less. Several recent reviews describe the capabilities and performance of CZE in detail (5-9). For CZE, channel diameters usually range from 25 to 100 am; therefore, designing methods to detect low concentrations in these small-diameter capillaries is a challenge. Laser-in-duced fluorescence (LIF) is currently the most sensitive de-tection method for CZE; detection limits are in the lowattomole range (10-12). In these systems, thecapillary is used as the “flow cell”, the laser illumination is perpendicular to the capillary, and a photomultiplier tube (PMT) monitors the fluorescence. Dovichi and co-workers (13-15) developed a more sensitive method that has the same excitation geometry but uses a sheath flow cuvette as the sample cell and thereby eliminates much of the scattered light and luminescence from the fused silica capillary. With this technique, detection limits in the low zeptomole range for fluorescently tagged amino acids have been reported.[Zepto (z) and yocto (y) have been proposed as the new SI prefixes for 10'21 and 1CT24, respec-tively, by the Comite Consultatif des Unites; final approval is pending the next meeting of the Comite General des Poids et Mesures.]