臺灣博碩士論文加值系統

本研究旨在開發表面電漿子共振與拉曼光譜整合之檢測系統，結合兩者優勢，包括具有即時檢測、無須標記、高靈敏度及指紋分析等，並基於表面電漿極化子會增強拉曼訊號的特性，以肝素-血小板第四因子複合物為例研究其可行性。在心血管手術、骨科手術或是侵入式手術，通常會讓患者服用肝素去避免發生血栓的情形，然而可能會出現不良的藥物反應，造成血小板減少症，稱為肝素誘導血小板減少症。目前肝素誘導血小板減少症之檢測方式會先以4T量表進行風險評估後，再進行免疫測試，其中由於抗體測試的特異性較低，可能會出現假陽性或假陰性的問題，所以為了避免誤判會再以功能測試進一步檢驗並確認。而其中的免疫測試除了要對樣品進行標記外，還需大量處理樣本及非特異性吸附等缺點。而在自行架設的SPR-Raman系統中，原先僅能量測到50 ng/mL的肝素-血小板第四因子複合物，實現至更低濃度1 ng/mL的目標物檢測，加上從不同入射偏振角度的實驗結果，皆證實於共振角處會增強拉曼散射訊號，驗證該系統的可行性。另外，還額外討論在不同入射角度下與漸逝場之穿透深度的關係，除了驗證該系統量測皆在有效範圍內之外，也發現當入射角度小於臨界角時，測量的頻譜訊號可當作一般的頻譜量測結果。最終，目標是藉由這個新型的分子檢測工具用來取代目前的免疫測試，如酵素連結免疫吸附測試，期望提高檢測品質並透過頻譜辨識去降低因非特異性吸附所造成的結果誤判，最終將提高整體檢測效率，並更精準地及早診斷和進行治療。

In this research, the purpose is to develop a detection system integrating surface plasmon resonance and Raman spectroscopy, including the advantages of real time detection, labeling free, high sensitivity and fingerprint analysis, etc. Besides, based on the fact that surface plasmon polaritons enhance the characteristics of Raman signal, the feasibility of heparin-platelet factor 4 complex was studied as an example. In cardiovascular surgery, orthopedic surgery, or invasive surgery, patients are usually given heparin to avoid thrombosis. However, it may occur adverse drug reactions and result in thrombocytopenia, which is called heparin-induced thrombocytopenia. The detection method for heparin-induced thrombocytopenia will be based on 4Ts score for risk assessment. Next, we continuously follow by immunoassays, in which the false positives or false negatives may occur due to the low specificity. Hence, in order to avoid misjudgment, functional assay will be further test and confirmation. In addition to labeling the sample, immunological assay also requires a lot of sample processing and non-specific adsorption problems. In self-installed SPR-Raman system, it enable to detect he target substance at a lower concentration of 1 ng/mL from the heparin-platelet factor IV complex, which can only be detected at 50 ng/mL without any enhancement. In addition, the experimental results from different incident polarization angles show that the Raman scattering signal will be enhanced at the resonance angle, which verifies the feasibility of the system. Furthermore, we also discuss the relationship between different incident angles and the penetration depth of the evanescent field. Bisides, to verification that the SPR-Raman system measurements are within the valid range, it is also found that when the incident angle is smaller than the critical angle, the spectrum can be regarded as a general spectrum measurement result. The goal of this research is to utilize this novel molecular detection tool to replace the current immunoassays, such as Enzyme-Linked Immunosorbent assay. It is expected to improve the detection quality and reduce the misjudgment of results caused by non-specific adsorption through spectrum identification. Ultimately, it will improve the overall detection efficiency and more accurate early diagnosis and treatment.

致謝 I
摘要 II
Abstract III
目錄 V
圖目錄 VIII
表目錄 XII
第一章 緒論 1
1.1 研究背景 1
1.2 研究動機與目的 3
1.3 論文架構 5
第二章 文獻回顧與基本原理 6
2.1 肝素簡介 6
2.1.1 肝素的歷史發展 6
2.1.2 傳統肝素與低分子量肝素 7
2.2 肝素-血小板第四因子複合物 8
2.3 肝素誘導血小板減少症之生理機制 9
2.4 表面電漿子共振 10
2.4.1 表面電漿子共振之歷史發展 10
2.4.2 表面電漿子共振之基本原理 11
2.4.3 表面電漿子共振光譜 13
2.4.4 動力學分析 14
2.5 拉曼散射 15
2.5.1 拉曼散射之發展 15
2.5.2 拉曼散射之基本原理 16
2.6 EDC/NHS活化之機制 17
2.7 乙酸胺失活羧基之機制 18
第三章 研究材料與方法 19
3.1 材料 19
3.2 儀器 20
3.3 實驗架構 20
3.4 SPR-Raman 整合量測系統 21
3.4.1 SPR光學架構系統與LabVIEW軟體 21
3.4.2 785nm拉曼探針系統與光譜量測介面 22
3.4.3 探針流道模組載台設計 22
3.5 軟體模擬 25
3.5.1 Essential Macleod模擬各角度穿透深度 25
3.6 目標物之結合量測 26
3.6.1 生物晶片備製 26
3.6.2 抗體固定化 26
3.6.3 BI-3000 SPR系統量測 27
3.6.4 SPR-Raman系統量測 28
3.7 顯微拉曼驗證 28
3.7.1 樣品備製與量測 28
3.7.2 量測介面與方式 28
第四章 目前結果與討論 30
4.1 多功能SPR-Raman系統驗證 30
4.1.1 測量不同折射率樣品的共振角 30
4.1.2 比較在共振角與非共振角的情況下所量測的拉曼光譜 31
4.1.3 各入射角度與電場穿透深度關係 33
4.2 生物樣品檢測 34
4.2.1 Anti-PF4抗體固定化 34
4.2.2 Anti-PF4/PF4免疫檢測： 35
4.2.3 Anti-PF4/ heparin-PF4 complex免疫檢測： 37
4.2.4 不同流速與相同濃度之PF4及Heparin-PF4 complex動力學分析： 38
4.2.5 運用SPR-Raman 系統檢測Heparin-PF4 complex： 40
4.3 Micro-Raman驗證 44
第五章 結論與未來展望 49
第六章 參考文獻 51

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