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系統識別號 U0026-0812200911444349
論文名稱(中文) 應用奈米粒子標記與銀析出偵測法於微免疫分析之研究
論文名稱(英文) Silver-precipitation Immunoassay Using Nanoparticles as a Label
校院名稱 成功大學
系所名稱(中) 工程科學系碩博士班
系所名稱(英) Department of Engineering Science
學年度 93
學期 2
出版年 94
研究生(中文) 洪青元
研究生(英文) Ching-Yuan Hung
電子信箱 n9692159@ccmail.ncku.edu.tw
學號 N9692159
學位類別 碩士
語文別 中文
論文頁數 87頁
口試委員 指導教授-林裕城
召集委員-張長泉
口試委員-林弘萍
中文關鍵字 生物晶片  微機電  酵素免疫分析  銀析出現象  奈米粒子 
英文關鍵字 biochip  MEMS  immunoassay  silver precipitation  nanoparticle 
學科別分類
中文摘要   本研究之概念設計與實驗執行在於結合奈米粒子及微晶片技術,以建立新型之免疫分析系統。在此研究中,我們利用奈米粒子標記技術並配合銀析出反應,提供另一種有效且方便的免疫分析檢測方式。此外,在微晶片上執行免疫分析程序具有改良多樣性、低樣本用量與高靈敏度等特性,且可以縮短偵測反應的時間。

  有別於傳統酵素標記的免疫分析(enzyme-linked immunosorbent assay, ELISA),我們採用現行傳統抗原抗體在晶片表面之固定化技術,並加入銀析出增強溶液,以增強金奈米粒子產生的反應偵測訊號。本研究利用protein A與免疫球蛋白G (IgG) 作為免疫分析模型,以探討新型免疫分析之檢驗可行性與效能。此外,實驗的規劃與架構將朝兩個方向來探討:(一)兩層分析模式,直接檢驗固定在基材上的抗原,並調整實驗偵測的最佳條件;(二)三明治分析模式,利用兩層抗體包夾方式以對抗原作定性與定量之分析。

  研究結果顯示,本研究策略可以完成免疫分析的步驟與目標,且使用的兩種偵測方式也可配合銀析出現象訊號產生可供檢測之訊號。基於結合微晶片、奈米應用技術、化學反應訊號增強技術,與提供光學與電性偵測成果,本研究建立全新、快速、靈敏之免疫分析系統。相信本研究之高應用性、高生化反應效能及高靈敏度,將對免疫分析系統提供嶄新的思維,和立下新的里程碑。
英文摘要  The design and experiment of this research lie in combining the microchip and nanoparticles to set up a new silver-precipitation immunoassay system. In this study, gold nanoparticle-labeled antibody is coupled with silver enhancement method to provide an alternative of effective and convenient immunoassay. In addition, the immunoassay carried out on the microchip has the advantages of versatile applications, lower sample consumption, shorter detection time and high sensitivity.

 Compared with traditional enzyme-linked immunosorbent assay (ELISA), we adopt the immobilization of antigen or antibody on the chip surface, and introduce silver enhancement method to magnify the detection signal generated by the gold nanoparticle. Protein A and immunoglobulin G (IgG) are selected as the model immunoassay to estimate the feasibility and efficiency of the silver-precipitation immunoassay. There are two major formats developed in this study: 1. direct immunoassay (two-layer format), the antigen is immobilized on the chip directly to optimize the assay conditions; 2. sandwich immunoassay (sandwich format), primary and secondary antibodies are used to quantify and qualify antigens.

 The results show that the silver-precipitation immunoassay can be measured with optical scanning and electro-signal detection methods. The relationship between concentration and signal is established and the antigen detection limit is 1 ng/mL. The high applicability and biochemical efficiency of this study can provide an alternative for rapid, sensitive and convenient immunoassay.
論文目次 中文摘要 I
Abstract III
誌謝 V
目錄 VI
圖目錄 X
表目錄 XIII
第一章 緒論 1
1.1 研究背景 1
1.2 文獻回顧 3
1.2.1 前言 3
1.2.2 傳統酵素聯結免疫吸附分析法─ELISA 3
1.2.3 近代免疫分析法之發展 7
1.3 研究動機及目的 10
1.4 實驗架構 12
1.5 研究策略 13
第二章 材料與方法 15
2.1 實驗原理與研究方法 15
2.1.1 金屬銀析出呈色反應原理 15
2.1.2 金奈米粒子合成方法 16
2.1.3 金奈米粒子標記原理 18
2.1.4 奈米金標記抗體合成方法 19
2.2 實驗偵測儀器與原理 23
2.2.1 紫外光─可見光吸收光譜儀 23
2.2.2 光學式免疫分析偵測系統之建立與操作準備歩驟 24
2.2.3 電性免疫分析偵測系統之建立與操作準備歩驟 28
第三章 偵測晶片設計與製作 30
3.1 晶片結構 30
3.1.1 光學掃描晶片 30
3.1.2 電性偵測晶片 31
3.2 光罩與設計 34
3.3 晶片製程 36
3.3.1 基材清洗 37
3.3.2 金屬蒸鍍 40
3.3.3 微影 (Lithography) 43
3.3.4 金屬蝕刻 47
3.3.5 PDMS井狀反應區製作 50
3.3.6 晶片滅菌 55
第四章 銀析出免疫分析模型之測試與實驗 56
4.1 基材選擇 57
4.2 銀析出偵測訊號測試 58
4.2.1 銀增強溶液測試 58
4.2.2 水相金奈米粒子之銀析出呈色反應 59
4.2.3 水相奈米金標記抗體之銀析出呈色反應 60
4.2.4 銀離子濃度量測 62
4.3 光學掃描銀析出免疫分析實驗 64
4.3.1 銀析出免疫分析步驟 64
4.3.2 銀增強溶液反應時間之影響 68
4.3.3 試樣抗原濃度與灰階之關係 68
4.4 電性銀析出偵測訊號測試與實驗 75
4.4.1 微電極免疫分析晶片 75
4.4.2 電極間距效應 75
4.4.3 試樣抗原濃度與電阻之關係 77
4.5 微觀銀析出現象分析 78
第五章 結論與建議 80
5.1 結論 80
5.2 建議 82
附錄 83
參考資料 84
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