Abstract
Vitamins are of the most important nutrients that could hardly be synthesized in sufficient quantities by human beings and thus are highly depended on the dietary intake of nutrient supplement. Biotin (Vitamin B7) is a water-soluble B-complex vitamin, which acts as a coenzyme in the metabolism of fatty acids and leucine, and it plays a vital role in gluconeogenesis. Biotin deficiency often disturbs the energy metabolism and various physiological functions, while immune depression and reduced collagen synthesis are also associated with biotin deficiency. Therefore it is urgently necessitated to develop a simple, rapid, inexpensive and highly sensitive quantitation method for Biotin detection and quantitation. Herein, we studied the quantitative analysis of biotin. Chromatography (IA-CEC) that actually comprises of two analytical techniques, competitive immunoassay and CEC. Such design allows the online target capturing by magnetic beads (MBs) that contain immobilized anti-biotin antibody. The analytical system is constructed as followed, (1) the magnetic nanoparticle (Fe3O4) were first functionalized with primary amine group to form (Fe3O4-NH2, kindly provided by Professor CS Yeh at NCKU), (2) the covalently immobilization of anti-biotin monoclonal antibody molecules for the fabrication of antibody-anchored MBs, (3) strong magnets were subsequently placed on both sides of capillary, allowing the confinement of the syring-introduced anti-biotin antibody modified on the MBs possess specific immunological affinity toward biotin, enabling the identification/capture of biotin, and finally the detection of biotin. The detection principle is based on competition between sample biotins and signal amplifiers, biotin-derivatized liposomes encapsulating carboxyfluorescein (CF), for a limited number of paratoped on the antibodies. After washing off, unbounded sample biotin and biotin-liposomes, the lipid bilayer structure of bound liposomes was destroyed by 0.2 % triton X-100, resulting in the release of the trapped carboxyfluorescein (CF) dye. The fluorescence emission of CF was measured by laser-induced fluorescence (LIF) detector and the concentration of biotin was therefore determined. As one single analysis was completed, magnets could be removed from the sides of capillary, leading to the wash-out of MBs and renewal of our capillary. In summary, we have demonstrated a potential analytical method, which is simple, sensitive, economical, low sample/reagent comsumption for detection of biotin. Preliminary results show that the calibration curve for biotin obtained using IA-CEC possess a linear dynamic range of 10-3-10-9 M with a detection limit (LOD) of 11 pg.