Design and performance of a microchip electrophoresis instrument with sensitive variable‐wavelength fluorescence detection
Design and performance of a microchip electrophoresis instrument with sensitive variable‐wavelength fluorescence detection
复制标题
灵敏可变波长荧光检测微芯片电泳仪的设计与性能
作者：
D. Belder;A. Deege;M. Maass;M. Ludwig
开发并评估了一种配备灵敏的可变波长荧光检测系统的高速微芯片电泳（MCE）模块化仪器。实验装置主要包括用于可变波长荧光检测和成像的基于灯的落射荧光显微镜和可编程的四通道双极高压源，每个通道能够提供高达+/- 10 kV的电压。光学单元配备了高灵敏度光电倍增管和可调光圈。该系统被应用于MCE分离的荧光素异硫氰酸酯（FITC）标记的胺利用蓝光（450-480 nm）的激发，以及用于分离罗丹明利用激发光在绿色光谱区域（531-560 nm）。在优化的条件下，四种FITC标记的胺的基线分离可以在小于50 s内获得，检测限为460 ppt（1 nM），信噪比为3：1。三种罗丹明可以在不到6秒的时间内基线分离，检测限为240 ppt（500 pM）。在FITC标记胺的重复MCE分离中测定的绝对迁移时间的相对标准偏差低于2.5%（n= 25）。通过应用环糊精修饰的电解质，可以在几秒钟内进行FITC标记的胺的手性分离，证明了微芯片电泳用于手性高通量筛选的潜力。
A modular instrument for high‐speed microchip electrophoresis (MCE) equipped with a sensitive variable‐wavelength fluorescence detection system was developed and evaluated. The experimental setup consists mainly of a lamp‐based epifluorescence microscope for variable‐wavelength fluorescence detection and imaging and a programmable four‐channel bipolar high‐voltage source capable of delivering up to +/– 10 kV per channel. The optical unit was equipped with a high‐sensitivity photomultiplier tube and an adjustable aperture. The system was applied to MCE separations of flurescein isothiocyanate (FITC)‐labelled amines utilizing blue light (450–480 nm) for excitation as well as for the separation of rhodamines utilizing excitation light in the green spectral region (531–560 nm). At optimized conditions baseline separation of four FITC‐labelled amines could be obtained in less than 50 s at a detection limit of 460 ppt (1 nM) with a signal‐to‐noise ratio of 3:1. Three rhodamines could be baseline‐separated in less than 6 s at a detection limit of 240 ppt (500 pM). The relative standard deviations of absolute migration times determined in repetitive MCE separations of FITC‐labelled amines were below 2.5% (n= 25). By the application of cyclodextrin‐modified electrolytes, chiral separation of FITC‐labelled amines could be performed in seconds demonstrating the potential of microchip electrophoresis for chiral high‐throughput screening.