開發以磁性奈米粒子和量子點檢測β-澱粉樣蛋白42/40比值之阿茲海默症診斷技術

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姓名

林郁璇(Yu-Hsuan Lin) 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

開發以磁性奈米粒子和量子點檢測β-澱粉樣蛋白42/40比值之阿茲海默症診斷技術
(Development of a Quantum Dot-Based Technique to Measure the Aβ42 and Aβ40 Molar Ratio as a Diagnostic for Alzheimer′s Disease)

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至系統瀏覽論文 (2029-8-31以後開放)

摘要(中)

阿茲海默症(Alzheimer′s disease, AD)是最常見的癡呆症，全世界患病人數呈上升趨勢，目前尚無實質的治療和預防方法。然而現今普遍之診斷方法因價格高昂、具有侵入性等因素而無法廣泛篩檢，導致早期檢測受到限制。研究表明血漿中的澱粉樣蛋白(amyloid beta, Aβ)之水平變化與疾病發展相關，因此已被研究作為候選生物標記物，其中 Aβ 42/40 血漿中的莫耳比隨著病程發展呈現負相關，更能作為阿茲海默症臨床試驗中的縱向分析(longitudinal analysis)依據。因此，本研究致力於開發一種早期檢測方法，使用磁性奈米粒子及量子點複合物的診斷技術。具體而言，利用 Aβ42 和 Aβ40及其與各自抗體之特異性捕獲血漿中的澱粉樣蛋白，再配合量子點高亮度和光穩定性之特性及磁性奈米粒子的順磁性執行定向分析策略(direct assay strategy, DAS)及反向分析策略(reverse assay strategy, RAS)，從而提升檢測靈敏度。我們成功地利用此檢測系統測定了 Aβ42 和 Aβ40。最低檢測濃度為0.1 μM，且在 0.4 至 2 μM 的濃度範圍內，螢光強度與濃度呈線性關係。最後，通過固定一種蛋白的濃度並改變另一種蛋白的濃度，成功分析 Aβ42/40比例的變化，驗證了系統的準確性和穩定性。此種方法不僅達到便利且低侵入性，並且為早期診斷提供一個新選擇。

摘要(英)

Alzheimer′s disease (AD) stands as the predominant form of dementia worldwide, with its prevalence on a steady rise. Despite this escalating burden, effective treatments or preventive measures remain elusive. Challenges persist in
the realm of diagnostics, where existing methods often prove inaccessible for extensive AD screening due to exorbitant costs and invasiveness. Measuring the concentrations of amyloid beta (Aβ), a protein family believed to be involved in AD pathogenesis, in the blood plasma has emerged as a promising diagnostic, owing to the correlation of the Aβ concentrations, particularly the Aβ42/Aβ40 molar ratio, with AD progression. This ratio presents an opportunity for
comprehensive longitudinal analysis in AD clinical trials. In this vein, our study sets out to develop an approach for early AD detection, leveraging the technique based on magnetic nanoparticles and quantum dot complexed with anti-Aβantibodies. By harnessing the specificity between Aβ42 and Aβ40 and their respective antibodies, our technique captures the two proteins with high precision and efficiency. Crucially, we exploit the high brightness and photostability
characteristics of quantum dots and the paramagnetism of magnetic nanoparticles to enact both directed assay strategies (DAS) and reverse assay strategies (RAS),
which enhances the detection sensitivity. We successfully utilized the detection system to measure Aβ42 and Aβ40, achieving a lowest detectable concentration of 0.1 μM. Within the 0.4 to 2 μM range, fluorescence intensity exhibited a linear relationship with concentration. In addition, effectively analyzed changes in the Aβ42/40 ratio, thereby validating the accuracy and stability of the system.
Through this integrated approach, our method not only facilitates convenient and minimally invasive diagnosis but also heralds a transformative pathway for early detection in AD.

關鍵字(中)

★ 阿茲海默症
★ β-澱粉樣蛋白
★ 磁性奈米粒子
★ 量子點

關鍵字(英)

★ Alzheimer′s disease
★ Amyloid-beta
★ Magnetic nanoparticle
★ Quantum dot

論文目次

摘要 I
Abstract II
誌謝 III
目錄 IV
圖目錄 IX
表目錄 XV
第一章緒論 1
1-1 簡介 1
1-2 研究動機與目的 2
第二章文獻回顧 3
2-1 阿茲海默症 (Alzheimer′s disease) 3
2-1-1 阿茲海默症病程發展 3
2-1-2 阿茲海默症成因假說 4
2-1-3 阿茲海默症病理學 9
2-1-4 阿茲海默症診斷及其優缺點 12
2-2 磁性奈米粒子 (Magnetic nanoparticles) 16
2-2-1 磁性奈米粒子介紹及應用 16
2-2-2 磁性奈米粒子合成 17
2-3 量子點 (Quantum Dots) 18
2-3-1 量子點光學特性及發光原理 18
2-3-2 量子點製備方法 20
2-3-3 量子點應用 21
第三章材料與方法 23
3-1 實驗材料與藥品 23
3-2 實驗架構 26
3-3 樣品製備 27
3-3-1 磁性奈米粒子合成 27
3-3-2 磁性奈米粒子-抗體偶聯物 29
3-3-3 碲化鎘(CdTe) 量子點製備 30
3-3-4 硒化鋅(ZnSe) 量子點製備 32
3-3-5 量子點-抗體偶聯物 34
3-3-6 β類澱粉蛋白質製備 35
3-3-7 生物傳感器系統整合 37
3-4 實驗儀器 38
3-5 儀器原理介紹 40
3-5-1 小角度 X-ray 散射 (small-angle X-ray scattering, SAXS) 40
3-5-2 動態光散射儀 (dynamic light scattering, DLS) 43
3-5-3 X-ray 粉末繞射儀 (X-ray diffractometer, XRD) 46
3-5-4 穿透式電子顯微鏡 (transmission electron microscope, TEM) 49
3-5-5 傅立葉轉換紅外光譜 (Fourier transform infrared spectroscopy, FTIR) 51
3-5-6 微量盤酵素免疫分析儀 (microplate reader) 53
3-5-7 紫外-可見光分光光譜儀 (ultraviolet-visible spectroscopy) 55
3-5-8 螢光光譜儀 (spectrofluorometer) 57
第四章結果與討論 60
4-1 磁性奈米粒子性質分析 60
4-1-1 SAXS 分析 60
4-1-2 DLS 分析 62
4-1-3 XRD 分析 64
4-1-4 TEM 分析 65
4-1-5 FTIR 分析 71
4-2 量子點性質分析 73
4-2-1 XRD 分析 73
4-2-2 TEM 分析 75
4-2-3 吸收光譜分析 77
4-2-4 CdTe螢光光譜分析 78
4-2-5 ZnSe螢光光譜分析 79
4-2-6 CdTe與ZnSe混合螢光光譜分析 80
4-3 磁性奈米粒子-抗體複合物性質分析 84
4-3-1 吸收光譜分析 84
4-3-2 BCA Assay分析 85
4-4 量子點-抗體複合物性質分析 87
4-4-1 吸收光譜分析 87
4-4-2 螢光光譜分析 89
4-4-3 BCA Assay分析 91
4-5 β類澱粉蛋白質狀態 94
4-6 β類澱粉蛋白質濃度檢測 96
4-6-1 Aβ42及Aβ40檢測 96
4-6-2 Aβ42/40 ratio檢測,固定Aβ40濃度並改變Aβ42濃度 105
4-6-3 Aβ42/40 ratio檢測,固定Aβ42濃度並改變Aβ40濃度 108
第五章結論 111
參考資料 113
附錄 123
附錄一磁性奈米粒子-電子顯微鏡 (MNP-TEM) 123
附錄二綠色碲化鎘量子點-高解析電子顯微鏡 (CdTe HR-TEM) 127
附錄三黃色碲化鎘量子點-高解析電子顯微鏡 (CdTe HR-TEM) 128
附錄四 BCA assay分析之標準檢量線 129
附錄五磁性奈米粒子吸收光譜 130
附錄六 Aβ42檢測,濃度0.4至2μM檢測-三獨立樣品重複實驗 131
附錄七 Aβ40檢測,濃度0.4至2μM檢測-三獨立樣品重複實驗 133

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指導教授

陳儀帆(Yi-Fan Chen)

審核日期

2024-8-15

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