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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 何佳安(Ja-An Annie Ho) | |
dc.contributor.author | Shao-Wei Sung | en |
dc.contributor.author | 宋紹瑋 | zh_TW |
dc.date.accessioned | 2021-06-08T02:21:49Z | - |
dc.date.copyright | 2020-09-14 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-08-14 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19831 | - |
dc.description.abstract | 卵巢癌佔臺灣地區婦科癌症的第二位,由於缺乏典型臨床徵狀,且當前的篩檢方法缺乏精準度,因此 75 %卵巢癌患者被診斷出來時通常已屬晚期,有遠端轉移和擴散的現象,然而患者若能被早期偵測並接受治療,則5年存活率可從10 %提升至 80 %,因此開發一種新型且非侵入性早期偵檢方法為亟欲達成的目標。先前研究指出,miR-205為典型卵巢癌之生物標誌,而miR-93被發現在CA-125出現前就有上調現象,兩者均可有效作為卵巢癌的生物標誌。於此,我們結合核酸循環放大系統 (nucleic acid circuits) 並搭配暗場顯微鏡偵測不同形狀之金奈米粒子,開發可同時偵測兩種目標物,不需酵素參與之等溫放大核酸檢測平台。在催化髮夾組裝(Catalytic Hairpin Assembly, CHA) 系統中,兩種目標 miRNA 是雜合反應中的啟動子。當目標 miRNA 被 Hairpin DNA (I) 進行專一辨認後,第二條 Hairpin DNA (II) 則啟動自發性組裝的功能,接續與前者雜合並取代目標 miRNA,使之可被循環利用。藉由暗場顯微鏡對攜有金奈米粒子之 CHA 產物進行成像,粒子會分別呈現綠色及藍色,並以程式計數作為訊號定量的基礎。本研究針對 Hairpin DNAs 序列及反應比例、緩衝溶液條件、聚乙二醇 (Polyethylene glycol, PEG) 濃度與反應時間進行實驗條件之優化,並透過電泳膠圖選定CHA 鏈置換反應的最佳條件。利用此種無需酵素參與反應的等溫放大策略,結合暗場顯微鏡計數的方法,我們建立一個新型 miRNA 的偵測平台。期望未來能應用此平台所建立好的序列設計原則,結合更多具不同散射光特性之奈米粒子,達到多因子檢測之成效,以增加早期篩檢卵巢癌或其他癌病的精準度。 | zh_TW |
dc.description.abstract | Ovarian cancer is one of the leading causes of death among gynecological cancers due to its lack of specific clinical symptom and low accuracy diagnosis tests. Previous studies reported that the five-year survival rate has increased from 10% to 80% for patients who were diagnosed and received proper treatment at early stage. Therefore, there is an urgent need to develop a non-invasive and effective method for early ovarian cancer diagnosis. MiR-205 and miR-93 have been found to be upregulated significantly in the serum of cancer patients, thus both can serve as potential biomarkers. The combination of nucleic acid circuits and plasmonic nanoparticles enables us to detect two target miRNAs simultaneously using dark-field microscopic technology. The single strand initiator (target miRNA) can interact with a toehold of the hairpin DNA (I) on the gold nanomaterials and open the structure, resulting in the formation of a liner H (I) / T duplex with a newly exposed single-stranded region. Subsequently, hairpin DNA (II) as a fuel can hybridize with the toehold region of the H (I) /T duplex to form the “H (I) / H (II)” duplex and release the target through the displacement reaction. The optimized reaction conditions for the operation of catalytic hairpin assembly, including modification of DNA sequences for various hairpins, ratio of hairpins, type of working buffer, polyethylene glycol concentration, and reaction time were investigated by polyacrylamide gel electrophoresis. The results demonstrated that the target-induced cross-opening of these hairpin reactants led to an effective generation of products, gold nanomaterials-tagged hpDNAs. With the aid of dark-field microscope, two distinct light scattering signals [green (G), and blue (B)] were exhibited. The direct counting of plasmonic particles was carried out for signal quantification. This enzyme-free, isothermal amplification strategy based on spectral-resolved dark-field images enables simultaneous detection of multiple miRNA targets in a label-free, simple and straightforward manner within 2 hours for the detection of ovarian cancer-associated miRNAs. The sensing platform was confirmed to hold great potential for early diagnosis and prognosis of ovarian cancer. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T02:21:49Z (GMT). No. of bitstreams: 1 U0001-1408202015323700.pdf: 5989961 bytes, checksum: 7ce3e4fdcfeed65494418690a54e11c3 (MD5) Previous issue date: 2020 | en |
dc.description.tableofcontents | 口委審定書 i 謝誌 ii 中文摘要 iv ABSTRACT v 目錄 vi 圖目錄 x 第1章 緒論 1 第2章 文獻回顧 3 2.1 卵巢癌 (Ovarian cancer) 3 2.1.1 卵巢癌現狀 3 2.1.2 卵巢癌之風險因子、分期、症狀與治療 3 2.1.3 卵巢癌診斷 4 2.2 微型核醣核酸 (MicroRNA) 5 2.2.1 miRNA簡介 5 2.2.2 miRNA與卵巢癌 6 2.2.3 miRNA偵測方法 7 2.3 等溫核酸放大技術 (Isothermal amplification) 8 2.3.1 Rolling Circle Amplification (RCA) 8 2.3.2 Hybridization Chain Reaction (HCR) 8 2.3.3 Catalyzed Hairpin Assembly (CHA) 9 2.4 核酸多重偵測技術 10 2.4.1 以螢光為基礎 10 2.4.2 以表面增強拉曼光譜為基礎 11 2.4.3 以電化學為基礎 11 2.4.4 以奈米電漿子 (Nanoplasmonics) 散射為基礎 12 2.5 金屬奈米電漿子與暗場顯微鏡之應用 13 2.5.1 暗場顯微鏡 (Dark-field microscope) 14 2.5.2 金屬奈米粒子與暗場顯微鏡之應用 15 第3章 實驗設計、材料與方法 16 3.1 實驗設計 16 3.2 材料 18 3.2.1 核酸序列 18 3.2.2 實驗試劑與材料 20 3.2.3 緩衝溶液 22 3.3 實驗儀器 22 3.4 實驗方法 24 3.4.1 聚丙烯醯胺膠體電泳 Polyacrylamide gel electrophoresis (PAGE) 24 3.4.2 CHA系統中Hairpin 1、2與Hairpin 3、4之序列優化 25 3.4.3 CHA反應中核酸探針比例的優化 25 3.4.4 CHA系統鹽離子濃度最佳化 25 3.4.5 探討含有Polyethylene glycol的反應溶液對CHA反應的促進效果 25 3.4.6 CHA系統反應時間最佳化 26 3.4.7 CHA系統對於目標miRNA之循環放大能力 26 3.4.8 CHA系統對於目標miRNA的專一性測試 26 3.4.9 鑑定金奈米材料與核酸探針之結合 26 3.4.10 定量金奈米材料表面之核酸分子 27 3.4.11 玻璃表面的探針修飾 27 3.4.12 輸出平台可行性測試 29 3.4.13 鑑定輸出平台表面修飾 29 3.4.14 輸出平台之條件優化 29 3.4.15 Nanoplasmonic biosensor之可行性測試 30 3.4.16 應用Nanoplasmonic platform以分析目標核酸分子 30 3.5 實驗方法之流程 32 第4章 結果與討論 33 4.1 CHA系統中Hairpin 1、2與Hairpin 3、4優化之序列 33 4.2 核酸序列之設計規則 47 4.3 CHA系統中核酸探針最佳的反應比例 48 4.4 CHA系統中最佳的鹽離子濃度 51 4.5 Polyethylene glycol溶液對CHA系統的促進效果 54 4.6 CHA系統最佳的反應時間 57 4.7 CHA最適反應條件之整理 60 4.8 CHA系統對於目標miRNA之循環放大能力 60 4.9 CHA系統對於目標miRNA的專一性 62 4.10 金奈米材料與核酸探針結合之鑑定 64 4.11 定量金奈米材料表面之核酸分子 67 4.12 輸出平台的可行性測試 69 4.13 輸出平台表面修飾的鑑定 70 4.14 輸出平台之條件優化 72 4.14.1 最佳的修飾反應時間 72 4.15 Nanoplasmonic platform分析目標核酸分子的應用 74 第5章 結論 77 參考資料 78 | |
dc.language.iso | zh-TW | |
dc.title | 利用催化髮夾組合放大結合暗場顯微鏡影像開發偵測對卵巢癌具特異性的微小核糖核酸感測器 | zh_TW |
dc.title | Catalytic Hairpin Assembly Amplification Detection of Ovarian Cancer-specific MicroRNAs Using Dark-field Imaging | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 蕭寧馨(Ning-Sing Shaw) | |
dc.contributor.oralexamcommittee | 朱士維(Shi-Wei Chu),徐士蘭(Shih-Lan Hsu),吳立真(Li-Chen Wu),邢怡銘(I-Ming Hsing),周芳如(Fang-Ju Amily Jou) | |
dc.subject.keyword | 卵巢癌,微小核糖核酸,非酵素,核酸等溫擴增技術,催化型髮夾組裝,金奈米粒子,暗場顯微鏡, | zh_TW |
dc.subject.keyword | ovarian cancer,miRNA,nucleic acid circuits,catalytic hairpin assembly,gold nanomaterials,dark-field microscope, | en |
dc.relation.page | 89 | |
dc.identifier.doi | 10.6342/NTU202003433 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2020-08-17 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
顯示於系所單位: | 生化科技學系 |
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