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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 | Yi-Shuang Peng | en |
dc.contributor.author | 彭宜萱 | zh_TW |
dc.date.accessioned | 2021-06-08T02:39:02Z | - |
dc.date.copyright | 2020-11-03 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-10-28 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/20032 | - |
dc.description.abstract | 子癲前症 (pre-eclampsia) 是一種以高血壓為特徵的妊娠相關疾病,根據世界衛生組織 (World Health Organization WHO) 統計約有3~8% 的孕產婦受到子癲前症的影響而造成母體損傷及胎兒發育不完全,並且在中低收入的國家中,子癲前症更是導致胎兒早產及死亡的主要原因之一。雖然子癲前症的患者大多在孕期20週後才出現不適的症狀,但許多研究也指出子癲前症可能在懷孕的初期就已發生,因此開發能夠早期偵測並診斷子癲前症的檢測方法是必要的。在本實驗中,我們選擇 miR-210 作為偵測目標並且結合雙股專一性核酸酶 (duplex specific nuclease, DSN)以及核酸增幅技術 (DNA amplification method) 並以96孔盤作為反應平台設計一種恆溫且類酵素連結免疫分析法 (Enzyme-linked immunoassay)的檢測方法。首先,目標 miRNA會被修飾在96孔盤底部的髮夾狀探針 (Hairpin probe) 辨識形成RNA-DNA雙股,接著透過DSN的專一性作用來產生附著於盤底的單股DNA,而此DNA可作為引子進而啟動下一階段的滾環式擴增法 (Rolling circle amplification, RCA) 使訊號得以被放大。在此RCA的反應中,核酸聚合酶可利用環狀探針 ( circular probe) 為模板合成出G-四聯體 (G-quadruplex) ,並且在加入氯化血紅素 (Hemin) 時形成具有辣根過氧化物酶 (Horseradish peroxidase, HRP) 活性的 DNAzyme。當環境中有3,3',5,5' –四甲基聯苯 (3,3',5,5'-Tetramethyl benzidine, TMB) 時,具HRP 活性的DNAzyme 便可催化過氧化氫的還原,進而使溶液由透明轉為藍色,透過分析顏色變化便可計算出樣品中 miR-210的含量。利用此設計,我們期望針對 miR-210開發出一套恆溫、高專一性、高靈敏性且操作門檻較低並有潛力診斷子癲前症的檢測平台。 | zh_TW |
dc.description.abstract | Pre-eclampsia is a pregnancy-related disease characterized by high blood pressure, affecting 3-8% of pregnancies. It is also one of the leading causes to higher risks of maternal damage and fetal growth restriction. In low- and middle-income countries, pre-eclampsia is a common cause of preterm delivery and fetal death. The symptoms of pre-eclampsia are often apparent at late second to early third trimester, but the abnormal interaction occurred much earlier in pregnancy. Therefore, a early detection of pre-eclampsia is an urgent need. We herein choose miR-210 as a target, designed an Enzyme-linked immunoassay (ELISA)-like detection assay coupled with isothermal signal amplification methods that combined duplex specific nuclease (DSN) specific miRNA cleavage and nucleic acid amplification. We first modified the hairpin probe on the wells of ELISA microplate. It was followed by the addition of the sample containing miR-210. After proper mixing, the hybridization occurred between the hairpin probe and the target to form RNA-DNA duplex, which can be further recognized by DSN. After the specific cleavage with DSN, a residual hairpin probe remained attached to the bottom of the well them served as the primer for subsequent rolling circle amplification (RCA). In the RCA, the G-quadruplex was generated via nucleic acid polymerase using a circular probe as a template. Horseradish peroxidase (HRP)-mimicking DNAzyme was formed, upon addition of hemin. With the presence of 3,3’,5,5’-Tetramethyl benzidine (TMB), Hemin/G-quadruplex HRP-mimicking DNAzyme catalyzed the reduction of hydrogen peroxide, leading to the colorimetric detection of miRNA. This sensing platform was anticipated to achieve a sensitive identification of miR-210 in blood samples collected from patients with pre-eclampsia. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T02:39:02Z (GMT). No. of bitstreams: 1 U0001-2810202011494600.pdf: 5204113 bytes, checksum: 28911dc98ae0bcbdf59ce4c8e61525c9 (MD5) Previous issue date: 2020 | en |
dc.description.tableofcontents | 第一章 緒論 1 第二章 文獻回顧 2 2.1 子癲前症 (PRE-ECLAMPSIA) 2 2.1.1子癲前症的現狀 2 2.1.2 子癲前症的成因、症狀及治療 3 2.1.3 子癲前症的診斷 4 2.2 微型核醣核酸 (MICRORNA) 4 2.2.1 miRNA 的生成與作用機制 4 2.2.2 miRNA 與壓力訊號 (Stress signal) 的關係 6 2.2.3 miRNA 與子癲前症之間的關係 7 2.2.4 循環性 miRNA (Circulating miRNA) 7 2.2.5 miRNA 的偵測方式 8 2.3 DNA 生化感測器 (DNA BIOSENSOR) 12 2.3.1 Biosensor 的簡介及應用 12 2.3.2 DNA Hybridization biosensors 12 2.3.3 SPR-DNA biosensor 12 2.3.4 Lateral-flow DNA systems 13 2.4 等溫核酸放大技術 (ISOTHERMAL AMPLIFICATION) 13 2.4.1 指數擴增反應 (Exponential Isothermal Amplification, EXPAR) 14 2.4.2 恆溫環狀擴增法 15 (Loop-mediated Isothermal Amplification, LAMP) 15 2.4.3 滾輪式擴增法 (Rolling Circle Amplification, RCA) 16 2.4.3.1 指數型滾輪式擴增法 (Exponential RCA amplification) 16 2.5 G-四聯體 (G-QUADRUPLEX) 18 2.5.1 G-quadruplex 分子結構 18 2.5.2 G-quadruplex 的結構多樣性 18 2.5.3 G-quadruplex 與 Biosensor 19 第三章 實驗材料與方法 20 3.1 核酸序列 20 3.2 實驗試劑與材料 23 3.3 實驗儀器 26 3.4 緩衝溶液 28 3.5 聚丙烯醯胺膠體電泳POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) 29 3.6 髮夾型探針的設計與DSN產物之探討 31 3.7 DSN 反應緩衝溶液最佳化及使用量之最佳化 32 3.8 DSN 反應溫度最佳化 32 3.9 DSN 酶切之專一性測試 33 3.10滾環式擴增法環狀模板之合成方法 33 3.11滾環式擴增法環狀模板大小最佳化 34 3.12 DSN 目標產物與環型模板MELTING CURVE與TM值測定 34 3.12滾環式擴增法反應溫度最佳化 35 3.12 DSN核酸酶辨識結合滾輪式擴增法之檢測系統可行性測試 35 3.13 修飾髮夾型探針至微量多孔盤之條件優化 36 3.14 DSN核酸酶辨識結合滾輪式擴增法之檢測平台可行性測試 37 第四章 結果與討論 38 4.1核酸酶辨識並結合滾輪式擴增法之恆溫檢測平台的建立 38 4.2 DSN 輔助目標分子之循環反應 41 4.2.1 髮夾型探針的設計 41 4.2.2 DSN產物之探討 45 4.2.3 DSN 反應緩衝溶液最佳化及使用量之最佳化 47 4.2.4 DSN 酶切之專一性測試 51 4.3 滾輪式擴增法 (ROLLING CIRCLE AMPLIFICATION, RCA) 之優化 53 4.3.1 滾環式擴增法環狀模板大小最佳化 56 4.3.3 Hemin/G-Quadruplex HRP-Mimicking DNAzyme 可行性測試與條件優化 58 4.4 恆溫檢測平台之建立 61 4.4.1 DSN 反應溫度之優化 61 4.4.2 滾輪式擴增法反應溫度之優化 63 4.5 DSN核酸酶辨識結合滾輪式擴增法之檢測系統可行性測試 65 4.6 修飾髮夾型探針至微量多孔盤之條件優化 67 4.7 DSN核酸酶辨識結合滾輪式擴增法之檢測平台可行性測試 71 4.8 DSN 核酸酶辨識結合滾環式擴增法之檢測平台可行性測試 73 4.9 應用恆溫檢測平台以分析目標核酸分子 (MIR-210) 75 4.10 DSN 反應效率之分析模型 77 第五章 結論 80 參考文獻 81 | |
dc.language.iso | zh-TW | |
dc.title | 利用核酸酶結合滾輪式擴增法開發針對具子癲前症特異性的微型核糖核酸之恆溫篩選平台 | zh_TW |
dc.title | A Highly Specific Isothermal Screening Platform for Pre-eclampsia-associated microRNA Based on Duplex Specific Nuclease and Rolling Circle Amplification | en |
dc.type | Thesis | |
dc.date.schoolyear | 109-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 吳立真(Li-chen Wu),徐士蘭(Shih-Lan Hsu),邢怡銘(I-ming HSING),周芳如(Amily Fang-Ju Jou),童寶玲(Pao-Ling Torng) | |
dc.subject.keyword | 子癲前症,miR-210,恆溫檢測平台,雙股專一性核酸酶,滾環式擴增法,G-四聯體,去氧核醣核酸酶,3,3',5,5' –四甲基聯苯, | zh_TW |
dc.subject.keyword | Pre-eclampsia,miR-210,Isothermal amplification,Duplex specific nuclease (DSN),RCA,G-quadruplex,DNAzyme,3,3’,5,5’-Tetramethyl benzidine, | en |
dc.relation.page | 88 | |
dc.identifier.doi | 10.6342/NTU202004312 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2020-10-29 | |
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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