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系統識別號 U0026-0812200914330211
論文名稱(中文) 運用微免疫晶片搭配鉑奈米粒子與銀訊號放大技術於免疫分析之研究
論文名稱(英文) USING PLATINUM NANOPARTICLES, SILVER ENHANCEMENT FOR IMMUNOASSAY MICROCHIP
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 96
學期 2
出版年 97
研究生(中文) 王逸龍
研究生(英文) I-Long Wang
電子信箱 n9695154@ccmail.ncku.edu.tw
學號 n9695154
學位類別 碩士
語文別 中文
論文頁數 148頁
口試委員 口試委員-張長泉
口試委員-林弘萍
指導教授-林裕城
中文關鍵字 生物晶片  酵素聯結免疫吸附分析法  微機電製程  銀訊號增強反應  鉑奈米粒子 
英文關鍵字 Biochip  MEMS  silver enhance  nanoparticles  immunoassay 
學科別分類
中文摘要 金黃色葡萄球菌是造成嚴重院內感染的重要病原菌,而引起菌血症的其中一種細菌正是金黃色葡萄球菌,根據報告顯示1000位住院的病人中得到菌血症的比例約為28人,且在近年來有足見增加的趨勢,故快速鑑定的需要也更加迫切,而在臨床檢驗中,免疫分析方法是目前最常使用的檢驗技術。本研究提出新型免疫分析方法,將金黃色葡萄球菌特有的protein A與免疫球蛋白G (IgG)作為三層式ELISA免疫分析的模型,使用鉑奈米粒子作為抗體之標定物,以銀增強溶液催化銀析出反應,配合銀析出反應放大偵測之訊號,由光學式與阻抗式偵測系統來量測免疫反應訊號,建立起新型之免疫分析系統,提供另一種有效且方便的免疫分析檢測方式。在光學方面,取抗體濃度為102、10與1 μg/mL做為第一層,對抗原濃度為105到10-2 ng/mL進行量測分析,在使用銀增強溶液放大鉑奈米粒子後,奈米粒子因為銀的包覆而肉眼可見,在18分鐘內即可產生灰階顏色上的改變,並利用掃瞄器及繪圖軟體擷取出有濃度效應的灰階值。在阻抗檢測方式中,抗體濃度是採用光學實驗的最佳結果,以低抗體濃度的1 μg/mL為第一層,待測抗原濃度也取105到10-2 ng/mL進行量測分析,且阻抗晶片的設計採用兩種不同規格的設計,一種以玻璃為基材電極間距為20 μm長度為1500 μm的阻抗晶片,另一種是電極間距有15、10與5 μm長度為150、100 μm並以矽晶圓為基材的新型阻抗晶片。相較於傳統需要昂貴又耗時的檢測儀器,本研究只需簡單的光學掃瞄器與阻抗量測儀器即可完成,而所需整體反應偵測時間約為8至18分鐘,阻抗免疫之靈敏度則可達10-1 ng/mL。在光學檢測方面,將找出抗體與抗原的最低濃度極限,在阻抗式檢測方面,本研究設計不同的電極規格,以討論電極、偵測靈敏度與最快反應時間之間的關係。利用免疫分析晶片、奈米粒子技術與銀訊號增強方法,提供免疫分析一個全新的方向與思維。
英文摘要 Staphylococcus aureus is the important pathogens resulting in serious nosocomial infections. One of the bacteria caused the bacteremia is Staphylococcus aureus. The mortality rates of bacteremia ranged from 15 to 30%. There is an increasing trend year by year, which makes the rapid test necessary and urgent. Immunoassays have been commonly used in the clinical laboratories for detection of a variety of antigens and antibodies. In this study, Protein A from S. aureus and immunoglobulin G (IgG) are selected as the model immunoassay to estimate the feasibility and efficiency of the novel immunoassay. Platinum nanoparticle-labeled antibody is coupled with sliver enhance method and microchip to provide an effective and convenient immunoassay. Besides, the immunoassay has the advantages of versatile application, lower sample consumption, high sensitivity and shorter detection time. When using optical test, antibody concentration was adjusted to be 102、10 and 1 μg/mL, antigen concentration was adjusted to be 105 due to 10-2 ng/mL. The results showed that the silver precipitation phenomenon was catalyzed by Ab-PtNPs conjugates. The changing color of reaction could easily be observed by naked eye or scanner at 18 minutes. When using impedance test, antibody concentration was just like optical low antibody concentration (1 μg/mL) and antigen concentration was adjusted to be 105 due to 10-2 ng/mL. There are two major formats developed in chip dives: one is based on the glass chip, in(on) which the electrode gap is 20 μm and the electrode length is 1500 μm; the other one is based on the silicon waver in(on) which the electrode gap are 15, 10 and 5 μm and the electrode length are 150 and 100 μm. By amplifying, detecting and analyzing the signal, we used gray rank and impedance change simultaneously to determine the immune response and then construct out a new immune analytic method. Comparing with the traditional method which requires high-priced and dawdled instrument, this investigation only demands unsophisticated scanning and LCR meter. The measuring time is approximate 8~18 minutes and the sensitivity is approximate 10-1 ng/mL. In addition, we combine MEMS technology and silver enhancement to achieve a novel immunoassay system. 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 XVI
第一章 序論 1
1-1 研究背景 1
1-2 文獻回顧 3
1-2-1 前言 3
1-2-2 免疫分析基本理論 4
1-2-3 免疫分析法之發展 7
1-2-4 傳統酵素聯結免疫吸附分析法─ELISA 16
1-3 研究目的與動機 20
1-4 實驗架構 22
1-5 研究策略 23
第二章 材料與方法 25
2-1 實驗原理與研究方法 25
2-1-1 實驗藥品介紹 25
2-2 實驗材料製備與方法 26
2-2-1 鉑奈米粒子合成技術 26
2-2-2 鉑奈米粒子與抗體接合之原理 28
2-2-3 鉑奈米粒子與抗體接合之方法 31
2-3 金屬銀析出之反應原理 33
2-4 實驗偵測儀器介紹與原理 35
2-4-1 光學式免疫偵測平台之建立與操作歩驟 35
2-4-2 電阻式免疫偵測平台之建立與操作歩驟 38
第三章 光學與免疫玻璃分析晶片之設計與製作 41
3-1 晶片結構與設計 41
3-1-1 光學掃描晶片 41
3-1-2 電性阻抗晶片 42
3-2 光罩與設計 44
3-3 晶片製程流程 45
第四章 微電極阻抗免疫分析晶片之設計與製作 46
4-1 晶片結構與設計 46
4-2 微阻抗晶片製程 48
4-2-1 微阻抗晶片製程原理 48
4-2-2 微阻抗晶片製程流程 50
4-3 PCB板製作 52
4-4 微電極阻抗晶片鋁線打線技術 55
4-5 微電極阻抗晶片封裝技術 57
4-5-1 PDMS免疫反應區製作 58
4-5-2 微電極阻抗晶片快速檢測系統 59
第五章 銀析出免疫分析模型之結果與討論 62
5-1 決定基材 63
5-2 銀增強溶液測試 64
5-2-1 銀增強溶液之灰階值測試 64
5-2-2 銀增強溶液之阻抗值測試 65
5-3 金、銀與鉑奈米粒子催化速度之比較 67
5-4 鉑奈米粒子與銀增強溶液 69
5-4-1 鉑奈米粒子於銀增強溶液灰階值測試 69
5-4-2 鉑奈米粒子於銀增強溶液之電性測試 70
5-5 鉑奈米粒子與抗體接合之結果 72
5-6 鉑奈米粒子與抗體接合後之催化能力驗證 74
5-6-1 鉑奈米粒子與抗體接合之催化能力光學灰階值實驗 74
5-6-2 鉑奈米粒子與抗體接合之催化能力電性阻抗實驗 76
5-7 光學掃描式銀析出免疫分析實驗 78
5-7-1 光學掃描式銀析出免疫分析步驟 78
5-7-2 銀增強溶液反應時間之影響 81
5-7-3 待測抗原濃度對應灰階值之關係與結果 82
5-8 電性阻抗式銀析出免疫分析實驗 88
5-8-1 微電極免疫分析晶片 89
5-8-2 電性阻抗式銀析出免疫實驗步驟 90
5-8-3 待測抗原濃度對應阻抗值之實驗與結果 92
5-8-4 電極間距、寬度與偵測靈敏度測試 103
第六章 結論與建議 111
6-1 結論. 111
6-2 建議. .115
6-3 未來展望 116
附錄……………………………………………………………………125
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