臺灣博碩士論文加值系統

血管內皮生長因子（Vascular endothelial growth factor, VEGF）是血管生成的關鍵影響因子，在癌症初期腫瘤生長時需要新生大量的血管用以輸送養分與氧氣，此時VEGF濃度就會異常增高，因此被視為癌症重要生物標的物 ( bio-marker ) 。目前檢測VEGF的技術開發也日漸成熟，並作為是否罹患癌症的重要輔助判斷，但檢測方法常需要昂貴儀器或耗材，因此本篇研究開發以螢光奈米碳點（carbon dots, CDs）搭配適體 ( aptamer, APT )組成之適體偵測器，用以針對血管內皮生長因子進行檢測。
本研究以螢光奈米碳點作為螢光探針並在aptamer上設計一個官能基--dabcyl做為螢光能量轉移淬滅物質。利用螢光共振能量轉移( Fluorescence resonance energy transfer, FRET)的方式在aptamer與CDs靜電相吸後造成螢光淬滅，而有偵測到VEGF樣品則會的透過aptamer與VEGF的特異性結合從而形成立體空間障礙使CDs無法與dabcyl結合使螢光不會下降，透過這樣的反應機制與螢光強度的高低來檢測VEGF與其對應的濃度。結果證實螢光訊號隨著VEGF的濃度的增加而上升。本研究方法的有良好的減量線線性(y = 2349.7x - 109.84，r = 0.9963) 和檢測極限低至0.08 ng/mL，且具有良好的精密度和選擇性。此外，所提出的方法已成功應用於檢測在人造血清中的樣品，人造血清樣本的檢測結果表明，回收率範圍為 85.7% 至 104.2%而相對標準偏差則落在0.9% 至2.6% 之間，具有在臨床研究和診斷中的應用潛力。

Vascular endothelial growth factor (VEGF) is a key influencing factor of angiogenesis. In the early stage of cancer, tumor growth requires a large number of new blood vessels to transport nutrients and oxygen. At this time, the concentration of VEGF will increase abnormally, so it is considered an important biomarker for cancer. At present, the development of technology for detecting VEGF is also becoming mature, and it is used as an important auxiliary judgment for cancer. However, the detection method often requires expensive equipment or consumables. Therefore, the research and development of this article use fluorescent carbon dots (CDs) with suitable the aptamer detector composed of aptamer (aptamer, APT) is used to detect VEGF.
In this study, fluorescent nanocarbon dots were used as fluorescent probes and a functional group-dabcyl was designed on aptamer as a quenching substance for fluorescent energy transfer. Fluorescence resonance energy transfer (FRET) is used to quench the fluorescence after the aptamer and CDs are electrostatically attracted, and the detected VEGF sample will be formed through the specific binding of aptamer and VEGF The three-dimensional space barrier prevents CDs from combining with dabcyl so that the fluorescence will not decrease, Through this reaction mechanism and the level of fluorescence intensity, VEGF and its corresponding concentration can be detected. The results confirmed that the fluorescence signal increased with the increase of the concentration of VEGF. The result of this study has good linearity (y = 2349.7x-109.84, r = 0.9963) and detection limit as low as 0.08 ng/mL, and has good precision and selectivity. In addition, the proposed method has been successfully applied to the detection of samples in artificial serum. The detection results of artificial serum samples show that the recovery rate ranges from 85.7% to 104.2% and the relative standard deviation fall between 0.9% and 2.6%. It has the potential for application in clinical research and diagnosis.

目錄 i
中文摘要 iv
ABSTRACT vi
圖目錄 viii
表目錄 ix
縮寫表 x
第一章、 緒論 1
第一節、 VEGF血管內皮生長因子介紹 1
第二節、 VEGF血管內皮生長因子之相關檢測方法 2
第三節、 Carbon dots螢光奈米碳點 4
第四節、 Aptamer適體 5
第五節、 實驗機轉 5
第六節、 實驗目的 8
第二章、 實驗材料及方法 9
第一節、 化學材料 9
第二節、 實驗儀器 10
第三節、 螢光奈米碳點合成方法與特徵鑑定 11
第四節、 溶液配製方式 12
第五節、 實驗方法 14
一、 螢光奈米碳點微波合成條件設定 14
二、 螢光分光光度計F-4500實驗參數設定 14
三、 螢光差距量的評估與定量 14
四、 最佳螢光奈米碳點合成比例探討 15
五、 Dabcyl修飾位置探討 16
六、 最佳適體濃度探討 17
七、 適體與VEGF-165反應時間之探討 18
八、 最佳反應酸鹼值探討 20
九、 最佳氯化鉀濃度探討 21
十、 專一性評估 23
十一、 VEGF-165減量線與檢測極限 24
十二、 實際檢體的應用 25
第三章、 結果與討論 27
第一節、 螢光奈米碳點特徵鑑定 27
第二節、 最佳螢光奈米碳點合成比例探討 31
第三節、 Dabcyl修飾位置探討 34
第四節、 最佳適體濃度探討 35
第五節、 適體與VEGF-165反應時間之探討 36
第六節、 最佳反應酸鹼值探討 38
第七節、 最佳氯化鉀濃度探討 39
第八節、 專一性評估 40
第九節、 VEGF-165減量線與檢測極限 41
第十節、 實際檢體的應用 43
第四章、 結論 44
第五章、 參考文獻 46

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