Mazereeuw M, de Best C M, Tjaden U R, Irth H, van der Greef J
Division of Analytical Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden University, The Netherlands.
Anal Chem. 2000 Aug 15;72(16):3881-6. doi: 10.1021/ac991202k.
A free flow electrophoresis (FFE) device was developed for continuous electrophoretic separation of charged compounds and implemented in a continuous flow biochemical detection (BCD) system. These continuous separation characteristics make FFE well suitable for online implementation in a chromatographic or flow injection analysis system, in which an additional separation step of charged compounds is desired. In a heterogeneous biochemical flow assay for the determination of biotin, an analyte zone reacts with an excess of an affinity protein. Subsequently, the free binding sites of the affinity protein react with an excess of fluorescein-labeled ligand. Free and affinity protein-bound label are separated on the FFE device prior to fluorescence detection of the separated fractions. Biotin and streptavidin were chosen as, respectively, model ligand and affinity protein. Since all the compounds that are involved possess different electrophoretic properties, quantitative analysis is performed after completely separating the fluorescent affinity complex and labeled biotin in the FFE device within 2 min. Since the device is optically transparent, the separated zones can be detected in the separation compartment, using laser-induced fluorescence. The applicability of the BCD-FFE system in combination with a HPLC separation is demonstrated in the bioanalysis of biotin in human urine at the micromole per liter level.
开发了一种用于带电化合物连续电泳分离的自由流动电泳（FFE）装置，并将其应用于连续流动生化检测（BCD）系统中。这些连续分离特性使FFE非常适合在色谱或流动注射分析系统中在线应用，在这些系统中需要对带电化合物进行额外的分离步骤。在用于测定生物素的异质生化流动分析中，分析物区与过量的亲和蛋白反应。随后，亲和蛋白的游离结合位点与过量的荧光素标记配体反应。在对分离的馏分进行荧光检测之前，在FFE装置上分离游离的和与亲和蛋白结合的标记物。分别选择生物素和链霉亲和素作为模型配体和亲和蛋白。由于所有涉及的化合物都具有不同的电泳性质，因此在2分钟内在FFE装置中完全分离荧光亲和复合物和标记的生物素后进行定量分析。由于该装置是光学透明的，因此可以在分离隔室中使用激光诱导荧光检测分离的区带。在人尿中生物素的微摩尔每升水平的生物分析中证明了BCD-FFE系统与HPLC分离相结合的适用性。
