Abstract
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.
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