磁性免疫親和管柱結合毛細管電泳-質譜法以建立線上免疫分析系統

CNU IR > College of Sustainable Environment and Recreation > Dept. of Occupational Safety > MOST Project > Item 310902800/2397

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Title:	磁性免疫親和管柱結合毛細管電泳-質譜法以建立線上免疫分析系統
Authors:	鄭淨月
Contributors:	職業安全衛生系
Keywords:	磁性免疫分析蛋白質消化毛細管電泳電噴灑質譜法基質輔助雷射脫附游離法 Magnetic immunoaffinity Capillary electrophoresis Electrospray-Mass Spectrometry Protein digestion
Date:	2005
Issue Date:	2008-07-31 14:37:49 (UTC+8)
Publisher:	台南縣：嘉南藥理科技大學職業安全衛生系
Abstract:	本計劃擬發展出一套可自動化的磁性免疫親和管柱( Magnetic Immunoaffinity Column, MIAC )結合毛細管電泳-質譜法以建立一套免疫分析系統，可以直接偵測含複雜成份的生物檢體中微量之蛋白質。磁性免疫親和管柱主要是由磁性粒子所組成，磁性奈米粒子(Magnetic Nanoparticles, MNPs)直徑約為6 nm，MNPs 核心是氧化鐵，表面以共價鍵鍵結抗體，將已修飾上抗體的MNPs 以流動注入法填充至鐵弗龍管柱中，以磁鐵在磁性免疫親和管柱的兩邊建立一個外加磁場，吸住MNPs，以做為分析系統中的固定相，抓取特定的蛋白質後，再加入修飾胰蛋白酶的奈米金粒子 (Gold Nanoparticles，GNPs)將被抓取的特定蛋白質消化，以毛細管電泳-質譜法分離偵測所產生的蛋白質胜肽碎片，進行資料庫搜尋比對。因為已被修飾的MNPs 只與特定的蛋白質鍵結，所以在此免疫分析系統中，可以注入”dirty”的樣品進行分析，而不會發生管柱阻塞情況，MNPs 又可回收重覆使用，經濟效益佳；又因為是流動系統，可注入較大量的樣品體積，進行特定的蛋白質的線上濃縮，可增加本系統的靈敏度，同時也節省免疫分析及蛋白質消化反應所需的時間，進而對這些生化樣品中微量的蛋白質建立一套快速而可靠的分析方法。 A magnetic immunoaffinity column (MIAC) coupling capillary electrophoresis-Mass Spectrometry (CE-MS) has been developed for on-line biological samples monitoring assays using two protein-coated nanoparticles. Paramagnetic nanoparticles can be used as a mobile solid phase on which an immunoaffinity assay is assembled and can easily be used in small volumes to miniaturize an analytical system. When microbeads are dispersed throughout a solution, the distance that reagents must diffuse is minimized. With these characteristics, microbeads are good candidates for use in a fluidic system, as has been demonstrated for CE-MS assay. The particles showed a high magnetic responsiveness in magnetic field, and no aggregation of the particles was observed after the particles had been treated in the magnetic field. Antibody (ex. immunoglobulin G, IgG) was coupled to the particles to prepare a magnetic affinity support for protein adsorption. A complex mixture containing a trace amount of potential protein candidates were directly injected onto MIAC operated under a flow rate sufficiently low. After target protein adsorption was completed, MNPs with target protein were directly injected to the mixer mixing with Trypsin-Gold Nanoparticles (Trypsin-GNPs) when liberating the magnetic field. The digested analysts were then eluted into CE-MS. Finally, the MNPs was recycled and reused. Meanwhile, the results of this work, supported by experimental validation, can aid in the understanding of trace level compounds of biological interest. Our results can potentially be applied to assessing the feasibility of biological monitoring.
Relation:	計畫編號：NSC94-2113-M-041-004
Appears in Collections:	[Dept. of Occupational Safety] MOST Project

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