以奈米金粒子為標記物之半抗原分子檢測

Development of a high detection sensitivity method of hapten using a gold nanoparticle immunoassay

陳錫圭(Hsi-kuei Chen)

指導教授 ： 侯劭毅

國立臺北科技大學/工程學院/生物科技研究所/碩士(2006年)

摘要

半抗原分子(hapten)是一種分子量小於5kD的小分子，單獨存在時並不會引發免疫反應，但是當半抗原分子(例如生物素，Biotin)與載體(carrier)以共價鍵結結合並注射到動物體內即會引發免疫抗體反應，所產生的抗體對半抗原分子有專一性的結合，即使半抗原分子不再與載體結合。有一些環境污染物，特別是農業上常用的除草劑如digoxin、甲基巴拉松或是戴奧辛等等都是半抗原分子。常見的半抗原分子檢測方法為酵素免疫分析法(ELISA)，這種方法的缺點是需要較長的分析時間和昂貴的分析儀器。因此，我們希望能開發出一種簡單和敏感度極高的方法來作為現場即時監測系統。在我們的研究中，我們是以具有紅色色澤的奈米金粒子作為標記物並將其接在抗體上。當分析樣品中存在著我們要偵測的半抗原分子時，帶有奈米金粒子的抗體即會去認此一半抗原分子，試紙因奈米金粒子的聚集作用而呈現出紅色標記。由研究結果中我們發現，試紙上所呈現的紅色色澤強度與Biotin的濃度成一正比例關係，肉眼可觀察到紅色色澤變化的極限值約為10 femtomole。而為了提高此種檢測方法的靈敏度，我們還以銀還原方法來放大奈米金的呈色效果。經過銀還原反應後，其靈敏度可以大幅提昇到100 zeptomole，約60,000個Biotin分子。由於此種奈米金粒子免疫檢測方法靈敏度相當高，加以不需要任何貴重或是笨重的儀器，因此我們預測這種檢測方法未來在現場即時檢測上將扮演著相當重要地角色。

關鍵字

奈米金粒子 ； 半抗原 ； 免疫檢測 ； 生物素

並列摘要

The objectives of the present work are to develop an easy and inexpensive gold nanoparticle immunoassay to detect hapten in field. To immobilize samples, biotin, our model molecule for hapten, was covalent linkage to a peptide, SGQSWRPQGRFG, and the biotin-peptide bound on nitrocellulose membrane. The binding is due to that the strong dipole of the nitrate ester interacts with the strong dipole of the peptide bond. Then antibody-coated gold nanoparticles that served as a detection reagent were added to the membrane. By human eyes, red dots were observed for 10 femtomole of biotin and no dot was observed for negative control. The assay sensitivity can be increased by silver enhancement to 100 zeptomole. The assay will allow future studies for fast detection of environmental pollutants, such as DTT, dioxin and so on.

並列關鍵字

gold nanoparticle ； hapten ； immunoassay ； Biotin

參考文獻

[1] Rodriguez, A. C., A. P. Ramos, G. E. Hawkes, F. Berti and M. Resmini, “Stereoselective Synthesis of a Novel Pseudopeptide Hapten for the Generation of Hydrolytic Catalytic Antibodies”, Tetrahrdron: Asymmetry, 15, 1847-1855 (2004).

[2] Kolosova, A. Y., J. H. Park, S. A. Eremin, S. J. Park, S. J. Kang, W. B. Shim, H. S. Lee, Y. T. Lee and D. H. Chung, “Comparative Study of Three Immunoassays Based on Monoclonal Antibodies for Detection of the Pesticide Parathion-methyl in Real Samples ”, Analytica Chimica Acta, 511, 323-331 (2004).

[3] Richard, C. and Jr. Krueger, “Use of a Novel Double-sandwich Enzyme-linked Immunosorbent Assay Method for Assaying Chondroitin Sulfate Proteoglycans that Bear 3-nitrotyrosine Core Protein Modifications, a Previously Unrecognized Proteoglycan Modification in Hydrocephalus”, Analytical Biochemistry, 325, 52–61 (2004).

[4] Moore, E., M. Pravda and G. G. Guilbault, “Development of a Biosensor for the Quantitative Detection of 2,4,6-trichloroanisole Using Screen Printed Electrodes”, Analytica Chimica Acta, 484, 15-24 (2003).

[5] Giraudi, G., I. Rosso, C. Baggiani, C. Giovannoli, A. Vanni and G. Grassi, “Development of an enzyme-linked immunosorbent assay for benalaxyl and its application to the analysis of water and wine”, Analytica Chimica Acta, 392, 85-94 (1999).