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研究生:黃奕菁
研究生(外文):Huang, Yi-Ching
論文名稱:應用新型免標定光纖式粒子電漿共振生化感測平台檢測腫瘤壞死因子-α型及轉化生長因子-β1
論文名稱(外文):Fiber-Optic Particle Plasmon Resonance Biosensorsfor Detection of TNF-α and TGF-β1
指導教授:周禮君周禮君引用關係
指導教授(外文):Chau, Lai-Kwan
口試委員:王少君陳永恩
口試委員(外文):Wang, Shau-ChunMichael Chan
口試日期:2011-07-13
學位類別:碩士
校院名稱:國立中正大學
系所名稱:化學暨生物化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:119
中文關鍵詞:金奈米粒子免標記光纖式奈米生物檢測儀腫瘤壞死因子α型轉化生長因子β1退化性膝關節炎混合自組裝單層膜
外文關鍵詞:gold nanoparticlesfiber-optic particle plasmon resonance sensortumor necrosis factor-αtransforming growth factor-β1osteoarthritismixed self-assembled monolayer
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本研究首先對本實驗室所開發出來的免標記光纖式奈米生物檢測儀( Fiber-optic particle plasmon resonance sensor ;簡稱FO-PPR )進行感測能力的評估,即以濃度不同的蔗糖溶液測試感測器對折射率的感測解析度,測試結果感測解析度可達到9.13 × 10-6 RIU,感測靈敏度亦可達到4.92 AU / RIU。另外對於基礎生化分子Anti-Biotin、Anti-DNP的偵測極限分別可達到3.66 × 10-6 g / ml及2.06 × 10-8 g / ml。
憑藉著FO-PPR系統的感測能力,我們希望可以建立生化指標腫瘤壞死因子α型( Tumor necrosis factor-α, TNF-α )及轉化生長因子β1( Transforming growth factor-β1, TGF-β1 )之檢測方法,並鎖定「退化性膝關節炎」進行研究。  
在建立TNF-α感測方法的實驗中,利用混合自組裝單層膜( Mixed self-assembled monolayer ) MUA ( 11-Mercaptoundecanoic acid )與MCH ( Mercaptohexanol )當作架橋分子,修飾於奈米金粒子表面,並與Anti-TNF-α的抗體做鍵結,進行多濃度的標準品偵測。實驗結果訊號對濃度有相當良好的線性關係( R = 0.9983 ),且偵測極限( LOD )可到達 8.22 pg / ml ( 0.48 pM )。進一步我們同時使用FO-PPR系統和ELISA方法對真實樣品─退化性膝關節炎病患的內外側關節液中TNF-α含量進行定量偵測。結果兩檢測方法之數據在相關係數的分析當中是一線性關係( R=0.9838 ),且在統計上有顯著的關聯性( p-value<0.0001 )。另外也利用Wilcoxon matched-pairs signed-ranks test和 paired t test兩種統計方法評估病患內、外側關節液中TNF-α含量的差異性,其分析結果在統計上確實有明顯差異。
此外在TGF-β1的部分則分別利用MUA與MCH混合自組裝單層膜以及AUT ( 11-Amino-1-undecanethiol )與MCH的混合自組裝單層膜來進行實驗,希望能夠找到TGF-β1的最佳偵測條件。目前FO-PPR 系統應用在偵測TGF-β1偵測極限( 20.6 pg / ml, 0.82 pM )已可以到達與ELISA差不多的結果。但由於TGF-β1是一個強疏水性質的蛋白質,其可能會黏附在實驗操作過程中的任何表面上,故仍須調控實驗條件,找尋實驗最佳化,以利未來真實樣品的偵測!

關鍵詞:金奈米粒子、免標記光纖式奈米生物檢測儀、腫瘤壞死因子α型、轉化生長因子β1、退化性膝關節炎、混合自組裝單層膜
This study illustrates the feasibility of using fiber-optic particle plasmon resonance sensor (FO-PPR) with gold nanoparticles on optical fiber probe for analysis of real biological samples. FO-PPR sensor is based on gold nanoparticles-modified optical fiber where the gold nanoparticle surface was modified by a mixed self-assembled monolayer (SAM) for further conjugation of antibody and minimization of nonspecific adsorption. Upon binding of an antigen to an immobilized antibody on the gold nanoparticle surface, the absorbance of the gold nanoparticle layer on the optical fiber changes and the signal change is enhanced through multiple total internal reflections along the optical fiber.
Because the FO-PPR sensor has many advantages such as label-free, high sensitivity, real-time, and low cost, it offers a potential to monitor biomarkers of various diseases. In this thesis, we focuses on osteoarthritis concerned cytokines, tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1).
Experimental results show that the sensor response versus log concentration of TNF-α is linear (R = 0.9983) and a limit of detection (LOD) of 8.22 pg / ml (0.48 pM) is obtained, which is superior to that by fiber-optic SPR sensor and is similar to that by ELISA. Moreover, both the FOPPR sensor and the ELISA method were used to determine the concentration of TNF-α in osteoarthritis patients' knee. Results show that the detection of TNF-α in synovial fluid by FO-PPR agrees with the clinically accepted ELISA method but a much shorter analysis time is required (~10 min). Besides, not only Wilcoxon matched-pairs signed-ranks test but also paired t test were used to evaluate the difference of TNF-α content in medial and lateral compartments of Knees. According to the results, we conclude that TNF-α concentrations were significantly higher in the medial compartment of these knees.
Additionally, detection of TGF-β1 by FO-PPR sensor has a LOD of 20.6 pg / ml (0.82 pM) which is similar to that by ELISA. However, we met some troubles. Purified recombinant human TGF-β1 has been demonstrated to be extremely hydrophobic, thus it may adhere to any surfaces in the experimental process. Therefore, we still have to find the optimum experimental conditions in order to analyze real samples in future.

Keywords:gold nanoparticles; fiber-optic particle plasmon resonance sensor; tumor necrosis factor-α; transforming growth factor-β1; osteoarthritis; mixed self-assembled monolayer

中文摘要 i
Abstract iii
目次 v
圖次 ix
第一章 緒論 12
1-1 研究動機 12
1-2 奈米科技與材料 14
1-2-1 奈米科技 14
1-2-2 奈米材料之特性 16
1-3 金奈米粒子的表面電漿共振 19
1-3-1 金奈米粒子的簡介 19
1-3-2 金屬奈米粒子的表面電漿共振 20
1-3-3 金奈米粒子在生醫上的應用 21
1-4 免疫分析方法 23
1-5 生物感測器 24
1-6 免標記光纖式奈米生物檢測儀 25
1-6-1 光纖原理 25
1-6-2 漸逝波現象 28
1-6-3 免標記光纖式奈米生物檢測儀 30
1-7 生物標誌 32
1-7-1 腫瘤壞死因子-α型 33
1-7-2 轉化生長因子-β1 34
第二章 實驗 35
2-1 實驗用藥品 35
2-1-1 化學藥品 35
2-1-2 生化藥品 37
2-2 實驗用儀器及工具 39
2-2-1 實驗用儀器 39
2-2-2 實驗用器具與材料 40
2-3 金奈米粒子的合成 41
2-4 玻璃光波導基材製備 42
2-4-1 玻璃光波導基材的製備 42
2-4-2 光波導基材的清洗 43
2-5 金奈米粒子的自組裝固定化 45
2-6 免標記光纖式奈米生物檢測儀 46
2-6-1 免標記光纖式奈米生物檢測儀之建構 46
2-6-2 感測晶片封裝 47
2-7 免標記光纖式奈米生物檢測儀基礎生化分子檢測能力 48
2-7-1 檢測Anti-Biotin 48
2-7-2 檢測Anti-DNP 49
2-8 對退化性膝關節炎相關之人類腫瘤壞死因子α型( Human TNF-α )進行檢測分析 51
2-8-1 以免標記光纖式奈米生物檢測儀對Human TNF-α進行檢測 51
2-8-2 ELISA方法對Human TNF-α進行檢測 53
2-8-3 關節液真實樣品前處理方法 55
2-9 對退化性膝關節炎相關之人類轉化生長因子-β1 ( Human TGF-β1 )進行檢測分析 56
2-9-1 以免標記光纖式奈米生物檢測儀對Human TGF-β1進行檢測 56
2-9-2 ELISA方法對Human TGF-β1進行檢測 60
第三章 結果與討論 62
3-1 金奈米粒子的合成與鑑定 62
3-1-1 有機相圓球型金奈米粒子的合成 62
3-1-2 有機相圓球型金奈米粒子的鑑定 63
3-2 金奈米粒子的固定化與其鑑定 64
3-3 免標記光纖式奈米生物檢測儀之感測靈敏度測試 67
3-4 免標記光纖式奈米生物檢測儀基礎生化分子檢測能力 71
3-5 對退化性膝關節炎相關之人類腫瘤壞死因子α型( Human TNF-α )進行檢測分析結果討論 78
3-5-2以酵素免疫分析方法對Human TNF-α進行檢測 85
3-5-3對關節液真實樣品進行Human TNF-α的偵測 86
3-5-4免標記光纖式奈米生物檢測儀和酵素免疫分析方法偵測結果之統計分析 90
3-6 以免標記光纖式奈米生物檢測儀對轉化生長因子-β1 ( Human TGF-β1 )進行檢測 94
3-6-1 以免標記光纖式奈米生物檢測儀對Human TGF-β1進行檢測測試 94
3-6-2 以酵素免疫分析方法對Human TGF-β1進行檢測 104
第四章 結論 106
參考文獻 108
附錄 111
A. Linear regression and linear correlation 111
B. Wilcoxon matched-pairs signed-ranks test 113
C. Paired t test 117

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