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研究生:張志瑋
研究生(外文):Jhih-Wei Chang
論文名稱:非標定生物檢測應用與低成本電漿子檢測平台的發展
論文名稱(外文):Applications of Label-Free Biosensing and Development of Low-Cost Plasmonic Sensing Platforms
指導教授:魏培坤
指導教授(外文):Pei-Kuen Wei
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生醫光電研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:80
中文關鍵詞:表面電漿子共振奈米壓印技術青黴素結合蛋白2α奈米金屬結構
外文關鍵詞:Surface plasmon resonancesNanoimprintingPenicillin-Binding Protein 2αNanostructures
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奈米金屬結構型態表面電漿子共振感測器的非標定、高通量檢測特性非常適合化學及生物感測應用。然而,低成本、大量製作的技術及發展可攜式、低成本檢測系統是重要的研究議題。在本論文中,我們提出利用奈米熱壓法及金屬濺鍍技術製作以環烯烴共聚合物塑膠為基板的雙層金奈米金屬狹縫結構陣列晶片並用於抗藥性金黃色葡萄球菌的檢測。在實驗中,我們檢測青黴素蛋2α來間接檢測抗藥性金黃色葡萄球菌,而最低偵測濃度可達100 ng/mL。為進一步提升感測器的檢測靈敏度,我們製作週期為1000 nm的雙層金奈米狹縫陣列感測晶片並測試其靈敏度,實驗結果顯示晶片的波長靈敏度為926 nm/RIU,而品質因素值高達272。此外,我們提出使用市售低價穿透式掃瞄器、632.8 nm雷射線濾光片及感測晶片建立一個適用於高通量檢測的低成本、可攜式檢測平台並利用此平台進行胎牛血清蛋白與其抗體的結合實驗以確認晶片的表面靈敏度。此檢測平台具有非標定、高通量檢測、操作簡易、快速檢測、低成本及可攜式的優點,將有益更廣泛的感測應用並非常適用於定點照護檢測。
Nanostructure-based surface plasmon resonance sensors are capable of sensitive and label-free detection for chemical and biological sensing applications. However, low-cost mass-production techniques and development of portable low-cost sensing platforms are the main issues which should be addressed. In this study, double-layer gold nanoslit arrays were fabricated on a cyclic olefin polymer (COP) film using hot embossing nanoimprinting lithography and metal sputtering techniques and then utilized to detect methicillin-resistant staphylococcus aureus (MRSA). In the experiment, penicillin-Binding Protein 2α present in MRSA was detected using the plasmonic biochips and the minimum detectable concentration of penicillin-Binding Protein 2α was 100 ng/mL. In order to improve the sensitivity of the biochips, double-layer gold nanoslit array with a period of 1000 nm was fabricated and tested. The result shows that the wavelength sensitivity of the chip was 926 nm/RIU and the figure of merit value was up to 272. In addition, we combined an inexpensive transmission-type scanner, a 632.8 nm laser line filter and plasmonic biochips to establish a portable low-cost sensing platform capable of high-throughput detection. An antigen-antibody interaction experiment in aqueous environment was conducted using the platform to verify the detection sensitivity in surface binding event. The proposed sensing platform has the advantages of label-free high-throughput detection, simple operation method, quick detection, low price and portable. It can benefit various sensing applications and is suitable for point-of-care detection.
誌謝 I
摘要 III
Abstract IV
目錄 V
圖目錄 VI
表目錄 IX
第一章 緒論 1
1.1 研究背景 1
1.2 研究動機與目的 8
1.3 論文架構 11
第二章 表面電漿共振理論 12
2.1 表面電漿子共振簡介 12
2.2 表面電漿子共振原理 13
2.3 狹縫表面電漿子激發與共振模態 22
第三章 實驗設備和製程流程 27
3.1 電子束微影技術 27
3.2 反應式離子蝕刻技術 29
3.3 奈米壓印技術 31
3.3.1 氣壓式奈米壓印機 31
3.3.2 直接加壓式奈米壓印機 33
3.4 光學量測系統 34
3.4.1 穿透式光譜量測系統 34
3.4.2 穿透式影像系統 35
3.4.3 商用掃瞄機量測系統 36
3.5金屬奈米結構晶片製備流程 37
第四章 實驗結果與討論 45
4.1雙層金奈米狹縫陣列檢測青黴素結合蛋白2α 45
4.1.1乾式檢測青黴素結合蛋白2α 45
4.1.2濕式微流道檢測青黴素結合蛋白2α 49
4.2近紅外光範圍雙層金奈米狹縫晶片靈敏度測試 55
4.3低成本、可攜式檢測平台之開發 59
4.3.1單層金奈米狹縫陣列晶片靈敏度測試 60
4.3.2 低成本、可攜式檢測平台之應用 63
第五章 總結 74
參考文獻 76

圖目錄
圖 1-1:使用簡易光學架設量測金奈米孔洞結構生物感測晶片,量測週期性金奈米孔洞結構、表面修飾層MUA、BSA其穿透光譜變化 3
圖 1-2:三個奈米孔洞方型陣列垂直入射穿透式影像與頻譜圖 4
圖 1-3:利用週期性金屬奈米孔洞結構產生非對稱菲諾共振,由肉眼觀測當生物分子結合,其光譜紅移而影像消失變暗 5
圖1-4:週期性金屬奈米結構穿透光譜與生物分子檢測結果 6
圖1-5:奈米熱壓印製程轉印金奈米狹縫結構 6
圖1-6:穿透式影像觀測系統與由肉眼觀測BSA與anti-BSA專一性結合後光強度改變之影像 7
圖2-1:在金屬( )和介電質( )以TM Wave激發造成集體式電偶極振盪,δ為消散場環境改變量 13
圖 2-2:非輻射性表面電漿子電磁波示意圖 19
圖 2-3:輻射性表面電漿子電磁波示意圖 20
圖 2-4:金屬表面電漿子之色散關係曲線圖 21
圖 2-5:入射光通過孔徑2a之傅立葉轉換曲線 22
圖 2-6:單狹縫金屬內狹縫電漿子電場分佈示意圖 23
圖 3-1:掃描式電子顯微鏡(LEO-1530) 28
圖 3-2:薄膜橫截面圖(a)等向性(b)非等向性蝕刻後 30
圖 3-3:反應試離子蝕刻機 (Oxford Instrument, plasmalab 80plus) 30
圖 3-4:氣壓式奈米壓印機 32
圖 3-5:氣壓式奈米壓印機腔體內示意圖 32
圖 3-6:直接加壓式奈米壓印機(Engineering system,EHN-3250) 33
圖 3-7:直接加壓式奈米壓印機壓印示意圖 33
圖 3-8:穿透式光譜量系統示意圖 34
圖 3-9:穿透式影像系統示意圖 35
圖 3-10:利用掃瞄機做為檢測平台示意圖 36
圖 3-11:母模基板其奈米結構製作流程 37
圖 3-12:於矽基板上製作週期性奈米狹縫 39
圖 3-13:週期性奈米狹縫結構SEM圖 39
圖 3-14:由氣壓式奈米壓印與電子槍蒸鍍製程完成品 41
圖 3-15:由直接加壓式奈米壓印與直流式真空濺鍍製程完成品 41
圖 3-16:PDMS微流道示意圖 42
圖 3-17:修飾青黴素結合蛋白2α抗體至生物感測晶片示意圖 43
圖4-1:TM極化波正向入射條件下,週期650nm雙層金奈米狹縫在空氣環境的穿透光譜圖 46
圖4-2:週期650nm青黴素結合蛋白2α抗體晶片表面修飾光譜 47
圖4-3:青黴素結合蛋白2α抗原濃度分別為100ng/mL、1ng/mL,、100pg/mL專一性反應前後量測結果 48
圖4-4:青黴素結合蛋白2α抗原濃度分別為100ng/mL、1ng/mL,、100pg/mL專一性反應前後量測歸一化結果 48
圖4-5:比較乾式與濕式沖洗結合緩衝溶液後光譜分佈 50
圖4-6:週期595nm青黴素結合蛋白2α抗原濃度100ng/mL專一性反應前後量測歸一化光譜結果 52
圖4-7:將感測晶片再生後量測PBP2α抗原濃度10ng/mL專一性反應前後量測歸一化結果 52
圖4-8:將感測晶片分別量測三種不同濃度PBP2α抗原200ng/mL、100ng/mL及50ng/mL光譜平移量 54
圖4-9:週期1000nm雙層金奈米狹縫結構在水中穿透光譜分佈 55
圖4-10:不同濃度甘油水混合液穿透光譜量測結果 57
圖4-11:表面電漿子共振波長與折射率關係圖 57
圖4-12:表面電漿子共振波長與折射率關係圖 58
圖4-13:十五個不同週期奈米狹縫陣列分佈示意圖 59
圖4-14:不同週期奈米狹縫結構(446nm-460 nm)在水中穿透光譜 60
圖4-15:不同濃度甘油水混合液穿透光譜量測結果 61
圖4-16:表面電漿子共振波長與折射率關係圖 61
圖4-17:波長641.7nm條件下,表面電漿子共振強度變化率與折射率關係圖 63
圖4-18:白光光源與經過632.8nm帶通濾光片光譜 64
圖4-19:利用掃瞄機檢測不同濃度甘油水混合溶液 64
原始影像與經過處理過後影像 64
圖4-20:分析影像處理後的六個週期(460-455 nm)影像隨不同濃度甘油水光強度變化 65
圖4-21:分析影像處理後的六個週期(460-455 nm)影像隨不同濃度甘油水光強度變化 66
圖4-22:分析影像處理後的六個週期(460-455 nm)將紅光除以藍光背景光源強度變化 66
圖4-23:週期(446-460 nm)共振平均強度變化與折射率關係圖 67
圖4-24:分析影像處理後的週期(446-460 nm)將紅光除以藍光背景光源的不同濃度甘油水強度變化 67
圖4-25:表面電漿子共振積分響應與折射率關係 68
圖 4-26:BSA抗原抗體專一性結合流程示意圖 69
圖 4-27:利用掃瞄機檢測BSA與Anti-BSA專一性結合 70
原始影像與經過處理過後影像 70
圖4-28:分析影像處理後的六個週期(460-455 nm)影像專一性結合前後光強度變化 71
圖4-29:分析影像處理後的週期(446-460 nm) BSA抗原抗體專一性結合反應 72
圖4-30:表面電漿子共振積分響應與BSA抗原抗體專一性結合關係,週期為(446-460 nm) 73

表目錄
表 3-1:COP(ZF16-188)和PC(Lexan8010)物理特性 31




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