利用表面電漿共振影像儀驗證最適化之抗非專一性吸附場效電晶體表面於血清環境下之免疫測定

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姓名

陳欣漢(Hsin-Han Chen) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

利用表面電漿共振影像儀驗證最適化之抗非專一性吸附場效電晶體表面於血清環境下之免疫測定
(Development and verification of an optimal antifouling surface by surface plasmon resonance for immunoassay in human serum on field effect transistor)

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摘要(中)

近年來隨著醫學相關領域的發展，對於疾病的診斷和治療都需要透過儀器的檢測進行一系列的分析與探討。追求高靈敏度、反應時間快速、無須標定等的生物分子檢測平台是大家共同的目標，為了達成這樣的目的除了檢測儀器本身擁有更低的檢測極限外，檢測表面也必須避免來自非專一性吸附(Non-specific adsorption)的干擾，尤其是在臨床檢測中，檢測來自體液(血液、血清、尿液)等較複雜系統內的物質，這樣非專一性吸附，如蛋白質等所構成的汙損表面可能造成生物感測器的靈敏度下降，影響專一性和再現性，加上非專一性吸附汙染使得背景訊號的上升，總總原因導致無法清楚辨別檢測結果而造成錯誤的判斷，如此一來更體現了防汙(Antifouling)的重要。
在本研究中，選擇poly (ethylene glycol) (PEG)聚乙二醇利用自組裝單層膜(SAM)的方式修飾表面，透過EG本身藉由氫鍵抓取水分子形成緻密的水層，以此水層形成屏障抵擋來自蛋白質非專一的吸附。使用表面電漿共振影像儀(surface plasmon resonance image,SPRi)觀測非專一性物質的吸附以及在血清中的干擾測試，實驗結果中發現，使用PEG分子量800DA，SH-PEG-NH2 : SH-PEG-OH=1:20，濃度1mM的混和比例所製得的SAM，對單一蛋白質和稀釋血清可以有很好的抗非專一性吸附特性(<1 ng/cm2)，而在干擾測試中，也可以成功在血清環境中分辨不同濃度的目標物。將此最佳條件成果移轉至FET後，結果顯示不僅保有優異的抗非專一性吸附特性之外，甚至可以清楚辨識血清環境中更低濃度的目標物，因此在這項研究中，我們開發的表面提供了可以檢測生物分子並減少複雜生物介質中非特異性吸附的最佳條件，為臨床診斷平台提供了有希望的表面，可以更精準的診斷及早給予正確的治療，以此模式創造對精準醫療檢測中的平台。

摘要(英)

In recent years, with the development of medical related domain, the diagnosis and treatment of diseases require a series of analysis and discussion through instrument detection. Pursuing a high-sensitivity, short reaction time, and label-free biomolecule detection platform is a common goal for everyone. In order to achieve this goal, not only the more sensitive detection instruments, but the detection surface must also avoid the interference of non-specific adsorption. These non-specific substances are usually come from the non-specific adsorption of proteins, especially in clinical testing, detecting sample from complex systems such as physiological fluids (blood, serum, urine). The fouling surface may decrease the sensitivity of the biosensor , affecting the specificity and reproducibility, and increase the background signal. In this way, it further illustrates the importance of antifouling.
In this study, we modify poly (ethylene glycol) (PEG) to the surface by self-assembled monolayer (SAM). EG monomer can capture the water molecules by hydrogen bonds to form a dense water layer, which forms a barrier against non-specific protein adsorption. We detect the adsorption of non-specific substances and interference tests in serum by surface plasmon resonance image (SPRi). By the experimental results, the mixed SAM solution of SH-PEG-NH2 : SH-PEG-OH=1:20 (total concentration of 1mM in absolute ethanol), has an excellent antifouling ability with lower than 1 ng/cm2 nonspecific adsorption from single protein and diluted serum. Furthermore, the SPR sensor could successfully detect the biomolecule in diluted human serum in the interference test. After getting the optimal condition from SPR, we apply it to FET. We could clearly detect 0.1 nM of the target in 1% serum by FET. Therefore, in this study, the surface we developed provides the best conditions for detecting biomolecules and reducing non-specific adsorption in complex biological media, providing a promising surface for clinical diagnostic platforms, allowing for more accurate diagnosis early and give the proper treatment. To create a large platform for precision medical testing with this model.

關鍵字(中)

★ 表面電漿共振
★ 矽奈米線場效電晶體
★ 自組裝單層膜
★ 聚乙二醇

關鍵字(英)

★ surface plasmon resonance
★ Silicon nanowire field effect transistor
★ Self-assembled monolayer
★ poly (ethylene glycol)

論文目次

中文摘要 i
Abstract iii
致謝 v
目錄 vii
圖目錄 xi
表目錄 xvi
第一章緒論 1
第二章文獻回顧 3
2.1 汙損表面 3
2.1.1物理方法 4
2.1.2化學方法 5
2.1.3 聚乙二醇 6
2.1.4立體排斥 7
2.1.5水合作用 10
2.2表面電漿共振 11
2.2.1表面電漿共振原理 12
2.2.2表面電漿共振儀分類 14
2.2.3表面電漿共振儀 17
2.3矽奈米線場效電晶體生物感測器 20
2.4晶片改質 25
2.4.1自組裝單層膜表面改質技術 25
2.4.2表面分子固定化 29
2.4.3影響自組裝單層膜之因素 31
2.4.3.1反應溫度之影響 33
2.4.3.2硫醇類分子濃度之影響 36
2.4.3.3氫鍵形成之影響 37
第三章實驗藥品、儀器設備與方法 41
3.1實驗藥品 41
3.2儀器設備 44
3.3實驗方法 46
3.3.1 SPR實驗 46
3.3.1.1 SPR晶片製備 46
3.3.1.2 SPR晶片之表面改質 46
3.3.1.3緩衝溶液配置 48
3.3.1.4 Antifouling實驗 49
3.3.1.5 SPRi interference實驗 50
3.3.2 FET實驗 51
3.3.2.1 FET 晶片之表面改質 51
3.3.2.2 緩衝溶液配製 52
3.3.2.3 電性測量 53
第四章結果與討論 54
4.1表面接觸角 55
4.2 XPS表面元素分析 57
4.3 AFM 表面粗糙度分析 60
4.4表面材料抗非專一性吸附測試 63
4.4.1短鏈PEG抗非專一性吸附測試 64
4.4.1.1抗單一蛋白質吸附測試 65
4.4.1.2抗血清吸附測試 70
4.4.2長鏈PEG抗非專一性吸附測試 73
4.4.2.1抗單一蛋白質吸附測試 73
4.4.2.2抗血清吸附測試 77
4.5 免疫檢測 83
4.6最適化條件於FET之應用 85
第五章結論與未來展望 90
5.1結論 90
5.2未來展望 92
第六章參考文獻 93
第七章附件 100
7.1 不同SAMs表面於不同處理下的粗糙度 100
7.2 COB檢測 106

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指導教授

陳文逸(Wen-Yih Chen)

審核日期

2020-7-30

推文