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系統識別號 U0026-0812200912045187
論文名稱(中文) 結合奈米訊號增強技術及阻抗測定之免疫分析晶片
論文名稱(英文) Development of an Immunoassay Chip Based on Signal Enhancement by Gold Nanopatricles and on Impedance Measurement
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 94
學期 2
出版年 95
研究生(中文) 黃浩軒
研究生(英文) Hao-Hsuan Huang
電子信箱 sickman1216@yahoo.com.tw
學號 n9693154
學位類別 碩士
語文別 中文
論文頁數 113頁
口試委員 口試委員-林弘萍
口試委員-張長泉
指導教授-林裕城
中文關鍵字 微阻抗晶片  酵素聯結免疫吸附分析法  金奈米粒子  銀訊號增強反應 
英文關鍵字 silver enhancement reaction  gold nanoparticles  electro-microchip  enzyme-linked immunosorbent assay 
學科別分類
中文摘要 本研究提出新型阻抗免疫分析方法,利用微阻抗晶片偵測免疫反應,使用金奈米粒子作為抗體之標定物,並扮演催化劑的角色,催化銀析出反應,配合銀析出反應放大電性偵測訊號。有別於傳統酵素聯結免疫吸附分析法(enzyme-linked immunosorbent assay, ELISA),採用現行傳統的抗原抗體固定技術,將抗原抗體固定於晶片表面,並加入銀訊號增強技術,以增強金奈米粒子產生的反應偵測訊號。

利用電感、電容、電阻量測儀(LCR meter)偵測阻抗晶片之電性訊號,並應用在免疫分析是一個全新的偵測方法,在本研究中使用兩種不同的分析模型:(a)直接型免疫分析法(兩層式)是將抗原直接固定在晶片表面,作為定性實驗,尋求較佳的實驗參數;(b)三明治型免疫分析法(三層式)使用抗體檢測待測抗原,並對抗原作定量分析。在阻抗免疫分析法中,結合濕式與乾式兩種不同的阻抗偵測方法。

放大、偵測並分析電性訊號,利用其阻抗改變的情況,判斷免疫反應的發生,建構出全新的免疫分析方法,所需整體反應偵測時間約為30分鐘,濕式阻抗免疫分析方法之靈敏度為10-8 g/mL,而乾式阻抗免疫分析方法之靈敏度則可達10-9 g/mL。本研究設計不同的電極間距,用以討論電極間距與偵測靈敏度之間的關係。除此之外,在阻抗免疫分析方法中,將討論專一性、靈敏度等問題;利用阻抗分析晶片、奈米粒子技術與銀訊號增強方法,提供了免疫分析一個全新的方向與思維。


英文摘要 This study reports a novel immunoassay using an electro-microchip to detect the immuno-reaction signal, a gold nanoparticles (ANPs) as a label of antigen or antibody and as a catalyst for silver precipitation, and the silver enhancement reaction to magnify the detection signal. 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 nanoparticles.

The LCR meter instrument, a new detection method for immunoassay, is used to detect the electric single. There are two major formats developed in this study: (a) direct immunoassay (two-layer format), the antigen is immobilized on the chip directly to optimize the assay conditions; (b) sandwich immunoassay (sandwich format), first and secondary antibodies are used to quantify and qualify antigens. Two different electro-detection methods are used in electro-immunoassay: wet type electro-detection and dry type electro-detection.
The change of the impedance to corroborate immuno-reaction is detected, amplified and analyzed to construct this new immunoassay. The total detection time of the immunoassay is about 30 minutes. The sensitivity of the wet type immunoassay is about 10-8 g/mL for detected antigen and in dry type immunoassay the sensitivity is reached 10-9 g/mL. Different sizes of the electrodes are designed to discuss the relationship between gap size and sensitivity. In addition, the specificity and sensitivity of the electro-immunoassay are discussed. Using electro-microchip, nanoparticles and silver enhancement provided a new immunoassay method.


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 4
1-2-1 前言 4
1-2-2 傳統酵素聯結免疫吸附分析法 5
1-3 近代免疫分析法之發展 10
1-4 研究動機及目的 14
1-5 論文架構 16
第二章 材料與方法 17
2-1 研究方法與實驗架構 17
2-1-1 免疫分析模型 18
2-1-2 銀增強溶液阻抗訊號放大原理 20
2-1-3 偵測模式 22
2-2 實驗材料製備 24
2-2-1 金奈米粒子合成方法 24
2-2-2 金奈米粒子標記原理 26
2-2-3 金奈米粒子標記抗體合成方法 28
2-3 實驗偵測儀器與原理 30
2-3-1 紫外光-可見光分光光譜儀 30
2-3-2 阻抗免疫偵測系統之建立與操作準備歩驟 32
第三章 阻抗偵測晶片設計與製作 34
3-1 晶片設計 34
3-2 阻抗晶片結構 36
3-3 光罩與設計 40
3-4 晶片製程 42
3-4-1 基材清洗 43
3-4-2 金屬蒸鍍 46
3-4-3 微影 (Lithography) 49
3-4-4 金屬蝕刻 53
3-4-5 免疫反應區製作 56
3-4-6 晶片滅菌 60
第四章 阻抗免疫分析模型之測試與實驗 61
4-1 基材選擇 62
4-2 抗體與金奈米粒子接合之驗証 63
4-3 銀增強溶液訊號測試 66
4-3-1 銀增強溶液電性測試 66
4-3-2 金奈米粒子與抗體接合後之催化能力測試 68
4-3-2-1 銀離子濃度變化量測 69
4-3-2-2 金奈米粒子與抗體接合後催化能力電性實驗 70
4-4 濕式電性銀析出免疫偵測實驗 72
4-4-1 濕式電性銀析出免疫實驗步驟 72
4-4-2 電極間距與偵測靈敏度 76
4-4-3 濕式阻抗免疫分析 78
4-5 乾式電性銀析出免疫偵測實驗 83
4-5-1 乾式電性銀析出免疫實驗步驟 84
4-5-2 新型阻抗晶片與偵測靈敏度實驗 87
4-5-2-1 反應隔離物測試 87
4-5-2-2 電極間距、寬度與偵測靈敏度測試 88
4-5-3 乾式阻抗免疫分析 93
4-6 銀析出現象微觀分析 98
第五章 結論與建議 102
5-1 結論 102
5-2 建議 105
附錄 106
參考文獻 107

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