Free flow electrophoresis device for continuous on-line separation in analytical systems. An application in biochemical detection

A free now electrophoresis (FFE) device was developed for continuous electr ophoretic separation of charged compounds and implemented in a continuous n ow biochemical detection (BCD) system. These continuous separation characte ristics make FFE well suitable for online implementation in a chromatograph ic or now injection analysis system, in which an additional separation step of charged compounds is desired. In a heterogeneous biochemical now assay for the determination of biotin, an analyte zone reacts with an excess of a n affinity protein, Subsequently the free binding sites of the affinity pro tein react with an excess of fluorescein-labeled ligand, Free and affinity protein-bound label are separated on the FFE device prior to fluorescence d etection of the separated fractions. Biotin and streptavidin were chosen as , respectively, model ligand and affinity protein. Since ail the compounds that are involved possess different electrophoretic properties, quantitativ e analysis is performed after completely separating the fluorescent affinit y complex and labeled biotin in the FFE device within 2 min. Since the devi ce is optically transparent, the separated zones can be detected in the sep aration compartment, using laser-induced fluorescence, The applicability of the BCD-FFE system in combination with a HPLC separation is demonstrated i n the bioanalysis of biotin in human urine at the micromole per liter level .