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研究生:王裕昕
研究生(外文):Yu-Sin Wang
論文名稱:可功能化抗沾黏性雙離子自組裝單層膜於生物感測器之應用
論文名稱(外文):Functionalizable Antifouling Zwitterionic Self-Assembled Monolayer for Biosensor Application
指導教授:黃俊仁黃俊仁引用關係
指導教授(外文):Chun-Jen Huang
學位類別:碩士
校院名稱:國立中央大學
系所名稱:生醫科學與工程學系
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:2017
畢業學年度:105
語文別:中文
論文頁數:90
中文關鍵詞:非特異性吸附自組裝混和單層膜生物感測器介面兩性雙離子材料表面化學
外文關鍵詞:non-specific adsorptionself-assembled mixed monolayerbiosensor interfacezwitterionic materialsurface chemistry
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本研究目的為建立具功能化和低非特異生物分子吸附特性之生物感測器介面。減少非特異性(nonspecific)生物分子與電極之間的相互作用,是提升生物感測器靈敏度和專一性之關鍵因素。而一個分子上同時帶有正電荷及負電荷基團的兩性雙離子材料,因為其具有良好的生物相容性(biocompatibility)以及抵抗非特異性生物分子吸附之特性,所以常被應用於生醫領域上。在雙離子材料內,最特別的就是羧基甜菜鹼(carboxybetaine)材料,除了具備良好抗生物分子吸附的特性之外,羧基甜菜鹼分子上羧酸官能基(carboxylic acid)可被轉化為功能化中間體,以建立配體功能化之表面。自組裝單層(Self-assembled monolayer, SAM)是一種已被確立的表面改質材料,可選擇性地修飾表面以獲得期望的化學和物理性質。本研究是利用羧基甜菜鹼烷烴硫醇(carboxybetaine alkanethiolate)與磺酸甜菜鹼硫醇分子混合共同修飾在金表面上,以同時提供表面良好的抗非特異性吸附和功能化之特性。由高解析電子能譜儀表面分析的實驗結果發現,羧基甜菜鹼硫醇與磺酸甜菜鹼硫醇分子其溶液混合的比例與表面的組成比例相似,可藉由改變溶液的組成比例來控制表面分子的組成。表面電漿共振(Surface plasmon resonance, SPR)生物感測器結果顯示,當羧基甜菜鹼硫醇與磺酸甜菜鹼硫醇分子以1:9的莫爾比例混合修飾時,所表現出的抗沾黏能力最為優異。且在該混和比例下,進行氨基偶聯NHS/EDC化學改質能夠成功的使抗體固定化於表面上。並且將此表面應用於表面電漿共振感測器,測得此系統下的感測器最低檢測濃度為80 ng/ml。此種新型的生物感測器介面,讓生物感測器具有良好的抗非特異性吸附性質以及賦予感測器多功能化的能力,能因應目前市場所需,以利於生物感測器發展。
Reducing nonspecific interaction of biomolecules with an electrode is a key issue for increasing sensitivity and specificity for biosensors. Zwitterionic materials, carrying both positively and negatively charged moieties in a molecule, have shown excellent biocompatibility by resisting undesired biomolecules adsorption. In particular, zwitterionic carboxybetaine materials are the most attractive. Besides the high resistance to nonspecific biomolecules adsorption, the carboxylic acid group can be transformed to functionalizable intermediates to create a ligand-functionalized substrate. Self-assembled monolayers (SAMs) are well-established as a means to selectively modify surfaces to achieve desired chemical and physical properties. In this study, we used carboxybetaine and sulfobetaine terminated alkanethiol to form mixed SAMs on gold surface. The zwitterion terminated mixed SAMs can provide functionalizable antifouling properties. X-ray photoelectron spectroscopy showed the molar ratio of carboxybetaine and sulfobetaine terminated alkanethiol molecular mixing in solution was in agreement with the feed-in molecules ratio. It supported that the composition of the surface molecules can be controlled by varying feed-in molar ratio in the solution. Surface plasmon resonance biosensor showed that there were the best ability of anti-fouling when carboxybetaine and sulfobetaine terminated alkanethiol mixed SAM with a molar ratio of 1 : 9. Under this condition, we can successfully attach the antibody on the surface via amino-coupled NHS / EDC chemical modification. Then, the surface was applied to the surface plasmon resonance sensor. The limit of detection was 80 ng/ml. The new type of biosensor interface offers anti-fouling properties and functionalizable capacity. It can meet the needs of the market to facilitate the development of biological sensors
摘要 i
Abstract iii
目錄 v
表目錄 x
方案清單 xi


一、 文獻探討 ................................................................................................... 1


1.1 生物感測器 ............................................................................................... 1


1.1.1. 生物沾黏對於感測器之影響 ................................................................... 2


1.2 蛋白質吸附 ............................................................................................... 3


1.2.1. 蛋白質與表面之間反應 ........................................................................... 3


1.2.2. 單一蛋白質溶液之蛋白質吸附模型 ....................................................... 4


1.3 自組裝單層 ............................................................................................... 8


1.3.1 硫醇在金表面之自組裝單分子膜 ........................................................... 9


1.3.2 多分子自組裝單層膜 ............................................................................. 11


1.4 抗沾黏塗層 ............................................................................................. 13


1.4.1 乙二醇材料塗層 ..................................................................................... 13


1.4.2 雙離子材料塗層 ..................................................................................... 14


1.4.3 抗沾黏塗層的功能化 ............................................................................. 16

1.5 生物識別元件 ......................................................................................... 19


1.6 表面電漿共振影像感測器 ..................................................................... 21


二、 研究目的 ................................................................................................. 23


三、 材料與方法 ............................................................................................. 24


3.1 實驗藥品 ................................................................................................. 24


3.2 材料合成 ................................................................................................. 26


3.2.1 羧基甜菜鹼硫醇 ..................................................................................... 26


3.2.2 磺酸甜菜鹼硫醇 ..................................................................................... 28


3.3 傅立葉轉換紅外光光譜鑑定 ................................................................. 30


3.4 自組裝膜的製備 ..................................................................................... 30


3.5 水接觸角測量 ......................................................................................... 30


3.6 高解析電子能譜儀分析 ......................................................................... 31


3.7 表面電漿共振影像感測器蛋白質吸附測試 ......................................... 31
3.8 循環伏安法(Cyclic voltammetry,CV)自組裝層性質分析 32


3.9 細菌貼附測試 ......................................................................................... 32


3.10 抗體於基材表面固定化程序 ................................................................. 32


3.11 測量特異性蛋白質結合 ......................................................................... 33


四、 實驗結果與討論 ..................................................................................... 34


4.1 羧基甜菜鹼硫醇材料性質鑑定 ............................................................. 34

4.1.1 羧基甜菜鹼硫醇 NMR 頻譜分析 34
4.1.2 羧基甜菜鹼硫醇質譜儀鑑定 ................................................................. 37
4.1.3 羧基甜菜鹼硫醇 FTIR 鑑定 38
4.1.4 XPS 表面元素分析 39
4.1.5 羧基甜菜鹼硫醇最佳化修飾溶劑蛋白質吸附測試 ............................. 42
4.1.6 羧基甜菜鹼硫醇與磺酸甜菜鹼硫醇差異 pH 值水接觸角之比較 43
4.1.7 羧基甜菜鹼硫醇與磺酸甜菜鹼硫醇之差異循環伏安法之比較 ......... 45
4.2 羧基甜菜鹼硫醇與磺酸甜菜鹼硫醇混合自組裝單層膜 ..................... 49
4.2.1 混合自組裝單層膜 XPS 表面元素分析 50


4.2.2 混合自組裝單層膜分子混合比例表面親水性測試 ............................. 51


4.2.3 混合自組裝單層膜分子混合比例抗非特異性吸附測試 ..................... 52


4.2.4 混合自組裝單層膜分子混合比例抗微生物吸附測試 ......................... 54


4.2.5 混合自組裝單層膜表面功能化測試 ..................................................... 56


4.2.6 SPR 感測器偵測二級抗體 58


五、 結論 ......................................................................................................... 60


六、 未來工作 ................................................................................................. 61


七、 參考資料 ................................................................................................. 63
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