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研究生: 黃騰毅
Teng-Yi Huang
論文名稱: 石墨烯氧化物薄膜於表面電漿子共振生物感測器之研發
Development of Graphene Oxide Sheets for Surface Plasmon Resonance Biosensors
指導教授: 邱南福
Chiu, Nan-Fu
學位類別: 碩士
Master
系所名稱: 光電工程研究所
Graduate Institute of Electro-Optical Engineering
論文出版年: 2013
畢業學年度: 101
語文別: 中文
論文頁數: 106
中文關鍵詞: 表面電漿子共振分子自組裝單層膜石墨烯氧化物動力學分析肺結核桿菌
英文關鍵詞: Surface plasmon resonance, Self-assembled monolayer, Graphene oxide, Kinetic analysis, Mycobacterium tuberculosis
論文種類: 學術論文
相關次數: 點閱:152下載:11
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  • 表面電漿子共振(Surface plasmon resonance, SPR)生物感測器為利用金膜表面與生物分子交互作用所造成的微量折射率變化,進而達到免標記且高靈敏度之檢測技術,為目前光學式生物感測技術中的重要方法之一。

    本論文將石墨烯氧化物(Graphene oxide, GO)鍵結於黃金薄膜上,所得到的薄膜稱為石墨烯氧化物薄膜,並透過表面電漿子共振生物感測技術,將此薄膜應用於生物感測上。石墨烯氧化物薄膜的製作首先將胱胺二鹽酸(Cystamine, Cys)作為連接劑,並透過分子自組裝單層膜(Self-assembled monolayer, SAM)技術,將胱胺鍵結於金膜上,形成胱胺薄膜。接著將石墨烯氧化物以共價鍵結的方式固定於胱胺薄膜表面,形成石墨烯氧化物薄膜(GO sheets)。論文中利用本實驗室之SPR量測系統並透過牛血清白蛋白與肺結核桿菌等生物分子進行檢測,並比較石墨烯氧化物薄膜與傳統金膜之靈敏度、檢測極限以及動力學分析之差異。

    本論文發展之石墨烯氧化物薄膜表面電漿子共振生物感測器,與傳統金膜比較後,石墨烯氧化物薄膜之靈敏度約可提升12倍,檢測極限與動力學分析後之結果亦皆優於傳統金膜。最重要的是,石墨烯氧化物薄膜可直接透過官能基與生物分子鍵結,不必再透過抗原抗體的交互作用來執行,因此可有效地降低檢測成本。未來本系統除了可供醫學與疾病檢測外,亦可應用於製藥工業、環境檢測、農業科技等領域之分析應用,以造福人群。

    The advantage of surface plasmon resonance (SPR) biosensors includes high sensitivity, label-free, and real-time detection. These advantage leading SPR biosensors to identified as one of the most important optical detection methods for biomolecules.

    The goal of this thesis is to develop graphene oxide (GO) sheets SPR biosensor that used GO as sensing layer for biomolecules detection. First, we used cystamine as linker layer, forming Cys film by self-assembled monolayer (SAM) technique. Then, GO solution was immobilized on the surface of Cys film through covalent attachment, forming GO sheets. The GO sheets SPR biosensor was used to detect the dynamic interactions of biomolecules and antibody-antigen. For experimental verification of this system, bovine serum albumin (BSA) and LAMP DNA products were used to demonstrate by GO sheets and conventional Au films. We also compared these two films by sensitivity, detection limits and kinetic analysis.

    The sensitivity of GO sheets that can be determined by this thesis is about 12-fold higher than that obtained with the sensor based on conventional Au films. The detection limits and the results of kinetic analysis of GO sheets were also better than conventional Au films. Most important of all, GO sheets have the ability for directly detect biomolecules, skipped the immunization interactions, leading to a low cost experiment. We anticipate that graphene oxide sheet SPR biosensors will not only enable for possible applications for biomedicine and diseases but also have wide spectrum of application in pharmaceutical industry, environmental monitoring, and agriculture in the near future.

    致謝.....................................................i 中文摘要................................................iii Abstract................................................iv 目錄....................................................vi 圖目錄..................................................xii 表目錄...................................................xv 第一章 緒論...............................................1 1.1 前言...............................................1 1.2 研究動機與目標.......................................1 1.3 論文架構............................................2 第二章 基本原理與文獻回顧....................................4 2.1 表面電漿子共振原理....................................4 2.1.1 表面電漿波......................................4 2.1.2 表面電漿子共振...................................5 2.1.3 表面電漿子共振之激發..............................7 2.1.4 表面電漿子共振之光學感測...........................8 2.1.5 表面電漿子共振生物感測器..........................10 2.2 表面電漿子共振金膜之固定化............................11 2.2.1 金膜之表面修飾..................................11 2.2.2 分子固定化技術..................................14 2.3 石墨烯衍生物與生物感測器上之應用........................18 2.3.1 石墨烯(Graphene)...............................18 2.3.2 石墨烯氧化物(Graphene Oxide, GO)................20 2.3.3 還原石墨烯氧化物(reduced Graphene Oxide, rGO)....22 2.3.4 以石墨烯氧化物為基礎之表面電漿子共振生物感測器........24 2.4 肺結核的診斷與恆溫環狀擴增法...........................26 2.4.1 肺結核的診斷....................................26 2.4.2 恆溫環狀擴增法..................................26 2.4.3 肺結核桿菌增幅方法比較............................28 2.4.4 肺結核桿菌DNA序列IS6110.........................30 2.5 分子動力學分析......................................30 2.5.1 分子動力學方程式.................................30 2.5.2 質傳效應.......................................34 第三章 實驗材料、儀器設備與研究方法...........................36 3.1 實驗材料...........................................36 3.2 儀器設備...........................................38 3.3 多功能電漿量測系統架構................................40 3.4 表面電漿子共振感測晶片製作............................43 第四章 石墨烯氧化物薄膜透過表面電漿子共振技術檢測生物樣本.........45 4.1 實驗目的...........................................45 4.2 實驗方法...........................................45 4.2.1 實驗溶液配製....................................45 4.2.2 金膜固定化之檢測.................................46 4.2.2.1 穿透光譜系統實驗.............................46 4.2.2.2 數位顯微鏡系統實驗...........................46 4.2.2.3 掃描電子顯微鏡(SEM)系統實驗...................47 4.2.3 多功能電漿量測系統(MPS)實驗.......................47 4.2.4 表面電漿子共振(SPR)微流道系統實驗..................47 4.2.4.1 分子自組裝單層膜(SAM)時間最佳化...............48 4.2.4.2 胱胺與石墨烯氧化物之濃度最佳化.................49 4.2.4.3 石墨烯氧化物薄膜表面官能基之影響................49 4.2.4.4 表面電漿子共振系統流速最佳化...................50 4.2.4.5 表面電漿子共振即時檢測........................50 4.2.4.6 動力學分析.................................51 4.2.4.7 牛血清白蛋白免疫反應分析......................51 4.3 實驗結果與討論......................................52 4.3.1 金膜固定化之檢測結果.............................52 4.3.1.1 穿透光譜系統實驗結果.........................52 4.3.1.2 數位顯微鏡系統實驗結果........................53 4.3.1.3 掃描電子顯微鏡(SEM)系統實驗結果...............53 4.3.2 多功能電漿量測系統(MPS)實驗結果...................54 4.3.3 表面電漿子共振(SPR)微流道系統實驗結果...............56 4.3.3.1 分子自組裝單層膜(SAM)時間最佳化結果............56 4.3.3.2 胱胺與石墨烯氧化物之濃度最佳化結果..............57 4.3.3.3 石墨烯氧化物薄膜表面官能基之影響結果............58 4.3.3.4 表面電漿子共振系統流速最佳化結果................61 4.3.3.5 表面電漿子共振即時檢測結果....................63 4.3.3.6 動力學分析結果..............................67 4.3.3.7 牛血清白蛋白免疫反應分析結果...................69 第五章 石墨烯氧化物薄膜透過表面電漿子共振技術檢測肺結核桿菌.......72 5.1 肺結核桿菌簡介......................................72 5.2 實驗目的...........................................72 5.3 實驗方法...........................................73 5.3.1 實驗溶液配製....................................73 5.3.2 實驗樣品設計條件.................................74 5.3.3 凝膠電泳對TB-DNA樣本之檢測.......................75 5.3.4圓漬點墨法對地高辛精(Dig)之免疫反應檢測..............76 5.3.5 表面電漿子共振(SPR)微流道系統實驗..................77 5.3.5.1 透過Digoxigenin抗體進行免疫分析法.............77 5.3.5.2 設計不同LAMP產物與GO薄膜交互作用..............80 5.4 實驗結果與討論......................................82 5.4.1 凝膠電泳對TB-DNA樣本之檢測結果....................82 5.4.2圓漬點墨法對地高辛精(Dig)之免疫反應檢測結果...........85 5.4.3 表面電漿子共振(SPR)微流道系統實驗結果...............86 5.4.3.1 透過Digoxigenin抗體進行免疫分析法之結果........86 5.4.3.2 設計不同LAMP產物與GO薄膜交互作用之結果..........90 第六章 結論與未來展望......................................93 6.1 結論..............................................93 6.2 未來展望...........................................94 參考文獻.................................................95

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