結合磁性奈米粒子與免疫親和毛細管電泳技術並應用於生物素之檢測

Magnetic bead based immunoaffinity capillary electrophoresis of biotin with laser-induced fluorescence detection

蔡介立(Chieh-Li Tsai)

指導教授 ： 何佳安

國立清華大學/理學院/化學系/碩士(2010年)

摘要

生物素是人體不可或缺的營養素之一，通常需由日常飲食所獲得。然而，目前定量食品中生物素的方法均需十分複雜樣品前處理方式或極高價位的儀器設備 (如質譜儀)，因此研發一個簡單、快速、便宜的定量方式實屬重要的課題。本研究以有重要生理意義的生物素為研究對象，擬研發測量生物素的感測系統。我們利用免疫親和毛細管電泳層析方法 (Immunoaffinity Capillary Electrophoretic Chromatography)為基礎，試圖發展一套定量生物素的偵測方法。免疫親和毛細管的製備方式是先將抗生物素單株抗體分子 (anti-biotin monoclonal antibody)以共價鍵的方式鍵結到表面修飾有氨基的四氧化三鐵磁性奈米粒子 (NH2-magnetic beads； 由成功大學葉晨聖老師實驗室提供)上；接著，我們可以利用置於毛細管兩側的強力磁鐵，將修飾有抗生物素抗體的磁性奈米粒子固定在毛細管中的特定區段。由於磁性奈米粒子上的抗體對生物素具有特異性的免疫親和力，因而能從樣品中辨識並捕捉生物素，藉此達到線上 (on-line)清洗及預濃縮的效果。
本系統的偵檢原理建立在樣品溶液中的生物素與微脂球表面事先修飾的生物素競爭有限的抗體位置 (antibody binding site of an antibody)。當樣品中的生物素含量少時，更多的微脂球有機會接到抗體分子上；而當樣品中的生物素含量多時，較少的微脂球會與抗體反應。當摻有介面活性劑的緩衝溶液通過該抗體磁珠區時，將破壞微脂球的脂質雙層結構，使其中包裹的carboxyfluorescein (CF)染料釋出。若以LIF（Laser-Induced Fluorescence detector）偵測CF所放射的螢光強度，則可藉此反推樣品中的生物素濃度。待單一樣品分析完成後，只消移除毛細管兩旁的磁鐵，並以高電壓促使電滲流之加速，去除管柱中的磁性奈米粒子，便可以同一支毛細管重複分析樣品。本研究初步結果顯示，修飾抗生素單株抗體的磁性奈米粒子，可有效抓取樣品溶液中的生物素，達到預濃縮的效果：若配合微脂球扮演訊號放大的角色，有潛力成為一個簡單且快速的生物素濃度定量方法。目前根據管柱外離線測試結果顯示，本偵測系統對生物素的偵測極限為11 pg，其線性範圍為10-3-10-9 M。

關鍵字

磁性奈米粒子 ； 毛細管電泳 ； 免疫分析 ； 生物素

並列摘要

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.

並列關鍵字

magnetic bead ； capillary electrophoresis ； immunoassay ； biotin

參考文獻

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