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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 | Amily Fang-Ju Jou | en |
dc.contributor.author | 周芳如 | zh_TW |
dc.date.accessioned | 2021-06-08T01:48:45Z | - |
dc.date.copyright | 2016-08-23 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-01 | |
dc.identifier.citation | References
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19204 | - |
dc.description.abstract | 有別於傳統DNA技術在探索如何解密序列中編碼之遺傳訊息,DNA 奈米科技賦予了DNA新穎應用價值,做為多功能奈米材料用以構建各種可操控之奈米結構體及奈米裝置。本篇論文目的即為開發更多DNA奈米科技潛能,開發新穎訊號放大檢測平台。有鑑於人體內生物核酸分子之基因變異及其不正常表現量已被證實與許多疾病和癌症劃上等號,因此,科學家們致力於找尋與其相對應之具指標性核酸生物分子。僅管聚合酶連鎖反應技術廣泛應用於傳統上檢測核酸分子,然而,污染源於反應過程一併放大及需特定可執行變溫操控之儀器等等之缺點促使更多取代此技術的相關研究蓬勃發展。本論文中第一個研究將DNA奈米科技結合電化學系統應用於檢驗阿茲海默症之基因點突變。此系統利用立足點鏈替代反應來區分點突變,配合限制酶-聚合酶協同放大反應來擴增DNA分子的產出,進而引發下游電化學反應。實驗結果確定了此平台的可行性,並展現其對於同時存在的目標突變DNA及未具突變之正常DNA序列有良好選擇性。有鑑於最新研究指出微小去氧核糖核酸分子(microRNA)對於引發阿茲海默症扮演重要角色,同時microRNA也被指出與許多疾病甚至癌症的發展相關,故microRNA也成為一新興生物指標分子。特別是存在血液中的穩定的循環microRNA更是許多相關技術發展的重點。第二個研究則針對檢測microRNA的重要性來發展攝護癌的檢測平台。此平台利用量子點修飾一段含有兩功能DNA序列,其一是能辨識目標microRNA之互補序列,另一則是一序列引子,用以引發端粒酶作用延長序列以形成具催化光學受質之G-四面體結構DNA分子。藉由雙股辨識酶協助目標miR-141循環反應,使引子裸露引發訊號催化反應,如此二階段放大反應成功偵測miR-141,並有效區分同家族內或是不同家族間的相似序列之microRNAs。更甚者,此平台並實際應用於檢測攝護腺病人的血清並與健康男性做對比。實驗結果發現我們的系統優於現有之酵素連結免疫吸附分析法測量攝護腺抗原之方法,具有其臨床應用價值。第三個研究進一步將DNA 奈米技術應用於癌細胞成像及治療。此平台利用癌細胞表面大量表現之核仁素(nucleolin)作為標鈀,使其設計的DNA探針能以雜合鍵反應自組裝形成G四面體在癌細胞表面。利用DAPI螢光染劑染色共同培養之乳癌細胞(MDA-MB-231)與乳房正常上皮細胞(M10),發現乳癌細胞能被專一性的染記標記,顯示DNA探針能成功自組裝於癌細胞上。利用具有細胞毒殺性之鋅紫原紫質(zinc protoporphyrin, ZnPP)可嵌入G-四面體之平面內的特性,分別處理癌細胞及與常細胞,發現在同時有DNA探針預先作用及ZnPP處理後的之癌細胞有顯著且協同細胞毒殺性。此結果展現此平台做為針對癌細胞同時檢測及治療的潛在應用能力。 | zh_TW |
dc.description.abstract | DNA nanotechnology, instead of exploring the genetic information that determine the biological meanings of every living organism, has been endowed with a revolutionary non-biological use that make DNA a versatile nanomaterial for engineering diverse static nanostructures and dynamic nanodevices. The aim of this work is to exploit the DNA nanotechnology for construction of novel amplified detection platforms based on nucleic acids. DNA alternation and aberrant expression of nucleic acids have been correlated with diseases and even cancers, such that tremendous efforts in finding reliable nucleic acid-based biomarkers as an index of a specific disease or cancer continue to devote. In spite of widespread application of PCR, the disadvantages, such as compromised sensitivity from contamination along with special apparatus for operating thermal cycling, motivate the development of alternative methods. The first work described an integrated detection system for identifying Alzheimer’s disease-related single nucleotide polymorphism (SNP). The system involved the utilization of toehold-mediated strand displacement reaction to differentiate SNP site, a nicking enzyme amplification reaction to amplify and translate the target input to an universal DNA translator, and a universal read-out electrochemical module to output a readable electro-response by a portable electro-analyzer. The results confirmed the feasibility of the platform for remarkable selective detection of target in discriminating the concurrence of target and interfering non-target. We substantiated the integrated system for the application in the determination of a single nucleotide change as an indicator of risk for Alzheimer's disease. Recent studies have suggested the emerging role of microRNA in Alzheimer’s disease, and the roles of microRNA in regulating gene expression have also become promising disease-/cancer-associated biomarkers. Circulating microRNA in blood, in particular, prioritizes the construction of sensing systems in the recent trend. The second work focuses on the development of an amplified sensing platform for the detection of prostate cancer-related microRNA. A “turn-on” optical sensor of a quantum dot (QD) modified with a DNA sequence consisting of a probe sequence for the target microRNA, along with a protected signal-initiating primer for telomerase-assisted formation of DNAzyme-mimicking G-quadruplexes in the catalyzation of chemiluminescent output. The target recycling by duplex-specific enzyme (DSN) enabled the digestion of DNA probe, and followed by the exposure of primer for DNAzyme generation, led to a two-layer amplification for the sensitive detection of the miR-141. Excellent selectivity in discriminating the inter-, intra-family microRNA was verified. We also evaluated the clinical applicability of the optical genosensors in the detection of miR-141 in the sera sample of prostate cancer patients and healthy subjects, and the results were also compared with that using a commercial ELISA kit which is based on the detection of prostate-specific antigen (PSA). The optical genosensors demonstrated better differentiation ability relative to the gold standard (ELISA), showing potential as a reliable clinical diagnostics for prostate cancer screening. In the third work, we further explored the DNA nanotechnology to design a platform for cancer cell imaging and therapy by utilizing a cancer cell-specific surface nucleolin-triggered, cell surface-directed self-assembly of G-quadruplex by hybridization chain reaction. Specific targeted imaging based on DAPI staining was observed for MDA-MB-231 breast cancer cells rather than M10 normal epithelial cells. MDA-MB-231 breast cancer cells were found to be more susceptive to treatment of a model therapeutic agent for cancer cell, zinc protoporphyrin (ZnPP), as indicated by a significant decline in the number of viable cells. The co-treatment of ZnPP and the nucleolin-directed DNA remodeling resulted in a synergistic therapeutic outcome, confirming the importance of the surface remodeling as the result of enhanced therapeutic effect. This unique theranostic paradigm based on the engineering of the molecular hairpin probes holds great promise for simultaneous diagnosis of disease, and targeted drug delivery. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T01:48:45Z (GMT). No. of bitstreams: 1 ntu-105-D01b22002-1.pdf: 31110993 bytes, checksum: f9e8dc851c4721475dff87159879a583 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | TABLE OF CONTENTS
中文摘要 I ABSTRACT III TABLE OF CONTENTS VI TABLE OF FIGURES VIII LIST OF TABLES X CHAPTER 1 INTRODUCTION 1 1.1 Background of DNA-based Biosensors 3 1.2 Principle of DNA Duplex Hybridization 8 1.3 Structure of single-stranded DNA as a functional unit 11 1.3.1 Two-dimensional DNA structures – hairpin 11 1.3.2 Two-dimensional DNA structures – triplex 18 1.3.3 Three-dimensional DNA Structure - G-quadruplex 20 1.4 Amplification strategies for the Improvement of DNA-based Biosensors 25 1.4.1 Enzyme-assisted amplification strategies 27 1.4.1.1 Strand Displacement Amplification (SDA) 27 1.4.1.2 Rolling Circle Amplification (RCA) 32 1.4.1.3 Nuclease-assisted Amplification (NAA) 34 1.4.1.4 Helicase-dependent Amplification (HDA) 36 1.4.1.5 Recombinase Polymerase Amplification (RPA) 38 1.4.2 DNA structure-switching-triggered self-catalytic amplification strategies 40 1.5 Motivation 46 CHAPTER 2 AN INTEGRATED ELECTROCHEMICAL SENSOR BASED ON DNA-STRAND-DISPLACEMENT CIRCUITRY 48 2.1 Abstract 48 2.2 Introduction 49 2.3 Methodology 52 2.4 Results and Discussion 58 2.5 Conclusions 74 CHAPTER 3 DEVELOPMENT OF OPTICAL GENOSENSORS FOR THE DETECTION OF PROSTATE CANCER-ASSOCIATED MICRORNA 75 3.1 Abstract 75 3.2 Introduction 77 3.3 Methodology 79 3.4 Results and Discussion 85 3.5 Conclusions 101 CHAPTER 4 A NOVEL TUMOR-SPECIFIC, MULTIFUNCTIONAL OLIGONUCLEOTIDE-INITIATIVE THERANOSTICS 102 4.1 Abstract 102 4.2 Introduction 104 4.3 Methodology 106 4.4 Results and Discussion 112 4.5 Conclusions 130 REFERENCES 131 | |
dc.language.iso | en | |
dc.title | 以操控核酸分子技術為工具用以發展訊號放大之檢測平台 | zh_TW |
dc.title | Engineered Nucleic Acids as Tools to Develop Amplified Detection Platforms | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 邢怡銘(I-Ming Hsing),鄭建中(Chien-Chung Cheng),徐士蘭(Shih-Lan Hsu),吳立真(Li-Chen Wu),陳平(Richard Ping Cheng) | |
dc.subject.keyword | 核酸分子,工具,訊號放大,檢測,平台, | zh_TW |
dc.subject.keyword | Nucleic acids,Tools,Amplified,Detection,Platforms, | en |
dc.relation.page | 139 | |
dc.identifier.doi | 10.6342/NTU201601681 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2016-08-02 | |
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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