臺灣博碩士論文加值系統

黏蛋白-1(mucin-1；MUC1)是人類癌症研究的重要生物標記，在常見癌症，如肺腺癌、乳癌等都會有過度表現的狀況。在臨床上以MUC1 SEA單元作為標靶治療，若能快速且精準的偵測此單元，將能對癌症的早期診斷有偌大的益處。近年來發展適體感測器用於即時檢測MUC1，其具備高靈敏度及與MUC1高結合力等優勢。為了達成高效感測的目標，除了提升適體的效能、優化感測條件外，並且可以使用抗體輔助來提升適體感測器之性能。針對實驗室已篩選出MUC1 SEA單元(從未有篩選過)的K2適體進行優化與感測應用。首先，透過表面電漿共振分析證明K2適體與MUC1 SEA單元有專一性的結合，接著使用熔解曲線分析對K2適體的結構進行穩定性分析，透過溫度和離子強度的調整，找到最適的結合條件為2 mM MgCl2 及100 mM NaCl應用於電化學適體感測器分析，而且發現K2適體和抗體各別與MUC1 SEA單元有不同的結合位點。整合K2適體的最適化條件且與抗體結合MUC1 SEA單元的位點不同，將其應用於抗體輔助電化學適體感測器檢測MUC1 SEA單元，使用電化學阻抗頻譜進行感測。實驗結果顯示在只有K2適體的電化學適體感測器中，檢測MUC1 SEA單元的濃度線性區間在29.6至66.6 nM之間且檢測極限在8.59 nM，此感測器計算得到的KD,app值為77.6 nM，R2為0.904。另外，透過使用抗體輔助電化學感測器測量到MUC1 SEA單元的濃度線性區間為1.23至11.1 nM且檢測極限為0.48 nM，此感測器計算得到的KD,app值為33.8 nM，R2為0.977，綜合兩種感測器的數據結果比較，以抗體輔助電化學適體感測器顯著地提升檢測濃度範圍及偵測極限，也證明其對MUC1 SEA 單元作為標靶檢測具有潛力。

Mucin-1 (MUC1) is an important biomarker, which is over-expressed in human cancers such as lung cancers and breast cancers. Clinically, the MUC1 SEA module has been used as therapeutic targets. If the module can be detected quickly and accurately, it will be beneficial to early diagnosis of cancers. In recent years, aptasensing has been developed for real-time detection of MUC1, which has the advantages of high sensitivity and strong affinity. In order to improve the sensing performance of MUC1 detection, in addition to enhancing the performance of the aptamer and optimizing the sensing conditions, this work used the antibody-assisted to develop the aptasensing of detecting MUC1. The K2 aptamer of MUC1 SEA module (which has never been selected before) was selected by our team for optimization and sensing. Firstly, the surface plasmon resonance (SPR) analysis results proved that the K2 aptamer specifically bound to MUC1 SEA module. In addition, the stability of aptamer’s structure was discussed by using the melting curve analysis. To find the optimal condition, such as temperature change and ion’s concentration change, the most suitable condition was 2 mM MgCl2 and 100 mM NaCl for electrochemical aptasensing. Moreover, K2 aptamer and antibody had different binding sites. The optimal conditions for K2 aptamer and antibody having different binding sites were integrated into the antibody-assisted electrochemical aptasensing for MUC1 SEA module detection. In this test, electrochemical impedance spectroscopy (EIS) was used to analyze the MUC1 module. The results showed that only the K2 aptamer in the sensing had a linear response for MUC1 SEA module in the range of 29.6-66.6 nM and the detection limit is 8.59 nM. Then, the KD, app value obtained by the aptasensing was 77.6 nM and R2 = 0.904. In addition, the MUC1 SEA module was detected in the linear range of 1.23-11.1 nM and within the detection limit of 0.48 nM by antibody-assisted adaptive detection. Finally, the KD,app value obtained by the aptasensing was 33.8 nM and R2 = 0.977. By comparing two antigen methods, the antibody-assisted method significantly improved the detection range and detection limit of MUC1 SEA module. This method proved that MUC1 SEA module has great potential in target detection.

致謝 i
摘要 ii
Abstract iii
目錄 v
圖目錄 viii
表目錄 xii
符號說明 xiii
第一章 緒論 1
1.1 研究背景 1
1.2 研究動機 3
1.3 研究目的 4
1.4 研究架構 5
第二章 文獻探討 6
2.1 MUC1 SEA單元 6
2.2 適體的結構穩定性與結合力 8
2.2.1 鹼基差異對適體結構的影響 8
2.2.2 環境因子對適體結合力的影響 12
2.3表面電漿共振分析 14
2.3.1實驗原理與反應機制 14
2.3.2 MUC1適體的動力學參數 21
2.4 MUC1適體感測回顧 24
第三章 研究方法 30
3.1 實驗儀器與藥品 30
3.1.1 實驗儀器 30
3.1.2 實驗藥品 31
3.2 實驗操作方法及步驟 32
3.2.1 酶聯寡核苷酸分析法 32
3.2.2 表面電漿共振分析 35
3.2.3 熔解曲線分析 38
3.2.4 電化學適體感測器 40
第四章 實驗結果與討論 43
4.1 適體序列優化 43
4.1.1 K2適體的核心區域鹼基亂數對MUC1 SEA單元結合力的影響 43
4.1.2 K2適體的引子截斷對MUC1 SEA單元結合力的影響 47
4.1.3 K2適體的動力學分析 51
4.1.4 K2-de5’適體的動力學分析 54
4.1.5 K2適體和K2-de5’適體的專一性比較 56
4.1.6 小結 58
4.2 適體環境優化 59
4.2.1 溫度對K2適體的結合力影響 59
4.2.2 溶液離子濃度對K2適體的結合力影響 62
4.2.3 溶液離子濃度對K2適體的結構影響 64
4.2.4 小結 66
4.3 適體與抗體競爭結合分析 67
4.3.1 SPR分析測定結合位點 67
4.3.2微盤分析儀測定結合位點 69
4.3.3 小結 71
4.4 電化學阻抗式適體感測MUC1 SEA單元 72
4.4.1靈敏度感測分析 72
4.4.2專一性感測分析 81
4.4.3 小結 87
4.5 抗體輔助電化學阻抗式適體感測MUC1 SEA單元 88
4.5.1 抗體輔助分析 88
4.5.2 動力學分析 90
4.5.3 小結 94
第五章 結論與未來展望 95
5.1 結論 95
5.2 未來展望 96
參考文獻 97
第六章 附錄 102
附錄一 MUC1 SEA單元候選適體挑選 102
附錄二 K2適體修飾後之結合力差異 107
附錄三 溶液離子對K2-de5’適體的結合力影響 108
附錄四 溶液離子對K2-de5’適體的結構影響 110
附錄五 溫度對K2適體的選擇性影響 112
附錄六 酸鹼值對K2 適體的結合力影響 114
附錄七 抗體與MUC1 SEA單元的結合力驗證 115
附錄八 K2適體與抗體以三明治方式於微盤分析儀測定 117

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