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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林啟萬(Chii-Wann Lin) | |
| dc.contributor.author | Szu-Yuan Lee | en |
| dc.contributor.author | 李思元 | zh_TW |
| dc.date.accessioned | 2021-06-15T00:37:58Z | - |
| dc.date.available | 2010-01-01 | |
| dc.date.copyright | 2008-11-25 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-11-13 | |
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Sens. Actuators B: Chem., 2007. 127(2): p. 598-605. 56. Lee, S.Y., J.G. Huang, T.L. Chuang, J.C. Sheu, Y.K. Chuang, H. Mark, D.R. Meldrum, C.N. Lee, and C.W. Lin, Compact optical diagnostic device for isothermal nucleic acids amplification. Sens. Actuators B: Chem., 2008. 133: p. 493-501. 57. Yanagida, T., M. Ueda, T. Murata, S. Esaki, and Y. Ishii, Brownian motion, fluctuation and life. Biosystems, 2007. 88(3): p. 228-42. 58. Lin, C.W., K.P. Chen, M.C. Su, T.C. Hsiao, S.S. Lee, S.M. Lin, X.J. Shi, and C.K. Lee, Admittance loci design method for multilayer surface plasmon resonance devices. Sensors and Actuators B-Chemical, 2006. 117(1): p. 219-229. 59. Lin, C.W., K.P. Chen, S.M. Lin, and C.K. Lee, Design and Fabrication of an Alternating Dielectric Multi-layer device for Surface Plasmon Resonance Sensor. Sensors and Actuators B-Chemical, 2006. 113: p. 169-176. 60. Huang, J.G., C.L. Lee, H.M. Lin, T.L. Chuang, W.S. Wang, R.H. Juang, C.H. Wang, C.K. Lee, S.M. Lin, and C.W. Lin, A miniaturized germanium-doped silicon dioxide-based surface plasmon resonance waveguide sensor for immunoassay detection. Biosens Bioelectron, 2006. 22(4): p. 519-25. 61. Lin, C.W., K.P. Chen, M.C. Su, C.K. Lee, and C.C. Yang, Bio-Plasmonics: Nano/micro Structure of Surface Plasmon Resonance Devices for Biomedicine Optical and Quantum Electronics 2005, 2005. 37: p. 1423-1437. 62. Jenison, R., S. Yang, A. Haeberli, and B. Polisky, Interference-based detection of nucleic acid targets on optically coated silicon. Nat Biotechnol, 2001. 19(1): p. 62-5. 63. Zhong, X.B., R. Reynolds, J.R. Kidd, K.K. Kidd, R. Jenison, R.A. Marlar, and D.C. Ward, Single-nucleotide polymorphism genotyping on optical thin-film biosensor chips. Proc Natl Acad Sci U S A, 2003. 100(20): p. 11559-64. 64. Jenison, R., M. Rihanek, and B. Polisky, Use of a thin film biosensor for rapid visual detection of PCR products in a multiplex format. Biosens Bioelectron, 2001. 16(9-12): p. 757-63. 65. Qiao, Y.M., Y.C. Guo, X.E. Zhang, Y.F. Zhou, Z.P. Zhang, H.P. Wei, R.F. Yang, and D.B. Wang, Loop-mediated isothermal amplification for rapid detection of Bacillus anthracis spores. Biotechnol Lett, 2007. 66. Osawa, R., A. Yoshida, Y. Masakiyo, S. Nagashima, T. Ansai, H. Watari, T. Notomi, and T. Takehara, Rapid detection of Actinobacillus actinomycetemcomitans using a loop-mediated isothermal amplification method. Oral Microbiol Immunol, 2007. 22(4): p. 252-9. 67. Ikeda, S., K. Takabe, M. Inagaki, N. Funakoshi, and K. Suzuki, Detection of gene point mutation in paraffin sections using in situ loop-mediated isothermal amplification. Pathol Int, 2007. 57(9): p. 594-9. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/41930 | - |
| dc.description.abstract | 著眼於後基因體時代的來臨，人類積極追求於基因體的功能研究，且在經歷SARS風暴之後，對於快速篩檢病原體，亦有著更為迫切的需求；生物晶片的發展於是刻不容緩。
本論文研究著手開發之「恆溫核酸增幅之光學檢測器」，為整合微型化工程製作技術與恆溫核酸增幅生物化學反應之基因檢測平台。在核酸增幅生化反應部份，採用loop-mediated amplification method (LAMP) 核酸增幅反應，針對B型肝炎病毒之聚合酶酵素基因設計引子，一小時內於單一溫度 (65℃) 下，快速連續增幅目標序列。在檢測系統部份，包含微反應晶片與控制、量測系統兩部份，前者為拋棄式設計，由聚甲基丙烯酸甲酯 (PMMA) 與蓋玻片組成，可提供核酸增幅反應之進行與量測所需之光通徑；後者由溫度控制器、加熱與光偵測系統組成，提供適當之反應與偵測環境，以量測核酸增幅反應之副產物-焦磷酸鎂所造成之混濁變化。實驗結果呈現，透過對引子設計理論之探究，所建立之HBV LAMP反應，具有良好之特異性與敏感性 (50 copies/25μl)。此外，採用化學合成方式模擬焦磷酸鎂沈澱，有效協助檢測系統之確效。爾後，建立了標準曲線 (R2 = 0.9605)，以利進行定量分析，再以7個臨床血清檢體，進行先導性測試，取反應後30分鐘量測值為基準，區分病毒量是否超過10,000 copies/ml。 結果顯示透過整合新型之恆溫增幅核酸LAMP反應，可以在1小時內順利增幅HBV DNA。此微型化裝置結合LAMP反應，可在單一反應溫度下，高敏感度、高特異性地放大HBV DNA量；它也提供適當的反應條件與連續的光學偵測系統，不需使用螢光染劑，就可偵測因焦磷酸鎂形成而致的混濁度改變。故此恆溫增幅核酸之光學檢測器對於慢性B型肝炎的早期診斷與病程監控有很大的助益；它不僅可因此改善病人的預後，也可節省龐大的醫療費用。未來將朝向研發多通道、可攜帶式、不須標定、可即時監測的個人化醫療檢測儀器，能快速鑑別病原體並進行定量，亦更能貼近未來重點照護與個人化醫療的使用需求。 | zh_TW |
| dc.description.abstract | In the post-genomic era, the field of genomics now faces the challenge of cataloging gene function. After prevalence of SARS, the demands of fast screening of pathogens grow dramatically. Therefore, it is urgent that develop the biochip for such requirement.
As a gene testing platform, the integrated isothermal device which is combined microfabricated technology and isothermal amplification. We adapt loop-mediated amplification method to amplify Hepatitis B Virus (HBV) polymerase gene under 65℃ within one hour. Our device can be partitioned into two components, one is the disposable micro-reactor chip (part A component) and the other one is the control and measurement system (part B component). To provide a reaction chamber with optical path, our design of the part A is made with polymethyl methacrylate (PMMA) and a cover glass slide. To create an appropriate condition for the LAMP reaction, part B includes an external temperature controller and an optical detection unit that yields real time output of by-product, magnesium pyrophosphate, turbidity signal. In results, we summarize the optimal rule of primer design for LAMP reaction and use LAMP reaction to amplify HBV DNA efficiently (in 60 minutes) with high specificity (12 different viral DNA and RNA) and sensitivity (50 copies/25μl). In addition, we adapt simulated reaction to mimic the precipitate of magnesium pyrophosphate. For quantitative analysis, the standard curve (R2 = 0.9605) is established and seven clinical serum specimens with different amounts of HBV DNA can be successfully detected by using this integrated isothermal device in 30 minutes with threshold level of 10,000 copies/ml. We have successfully demonstrated the feasibility of the LAMP reaction for HBV DNA amplification and detection in this novel integrated isothermal device within one hour. The compact device can amplify HBV DNA with high specificity and efficiency under isothermal conditions by using LAMP reaction. It also provides appropriate reaction conditions and a steady optical detection system for detecting turbidity derived from magnesium pyrophosphate formation without fluorescence labeling. Thus, using this integrated isothermal device has great assistance for early diagnosing and monitoring the progress of chronic hepatitis B. It can improve the prognosis of patients and save medical expense enormously. In the future, we hope to provide a multi-channel, portable, label-free, real-time monitoring personalized medical device for rapid identification and quantification of pathogenic organisms and point-of-care applications. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T00:37:58Z (GMT). No. of bitstreams: 1 ntu-97-D92548007-1.pdf: 2055949 bytes, checksum: c398a84f342f762cd802f679d52e199c (MD5) Previous issue date: 2008 | en |
| dc.description.tableofcontents | 口試委員會審定書…………………………………………………… i
致謝…………………………………………………………………… ii 中文摘要……………………………………………………………… iii 英文摘要……………………………………………………………… iv 第一章 緒論………………………………………………………… 1 1.1 生物科技的發展………………………………………………… 1 1.2 生物晶片………………………………………………………… 1 1.3 核酸增幅反應………………………………………………… 10 1.4 肝炎的成因與醫療支出……………………………………… 13 1.5 B型肝炎專論…………………………………………………… 15 1.6 研究動機與目的……………………………………………… 19 第二章 生化反應之設計、材料與方法…………………………… 20 2.1 B型肝炎病毒核酸之取得……………………………………… 20 2.2 製備B型肝炎病毒聚合酶部分基因片段之標準品…………… 20 2.3 基因選殖與標準品之製備…………………………………… 23 2.4 HBV LAMP反應之建立與特性瞭解…………………………… 29 2.5 模擬焦磷酸鎂之沈澱實驗…………………………………… 31 第三章 恆溫核酸增幅量測系統之設計、材料與方法………… 33 3.1 微反應晶片之設計、製作…………………………………… 33 3.2 量測系統之設計、組裝與製作……………………………… 33 3.3 恆溫核酸增幅量測系統之確效……………………………… 35 3.4 HBV LAMP反應於恆溫核酸增幅量測系統之檢測…………… 36 第四章 研究結果…………………………………………………… 37 4.1 HBV標準品之建立…………………………………………… 37 4.2 LAMP反應之引子設計………………………………………… 37 4.3 HBV LAMP反應之建立………………………………………… 39 4.4 確認核酸增幅產量與光密度檢測之相關性………………… 40 4.5 恆溫核酸增幅量測系統之建立與應用……………………… 40 第五章 討論與展望………………………………………………… 44 圖……………………………………………………………………… 49 表……………………………………………………………………… 74 參考文獻……………………………………………………………… 80 | |
| dc.language.iso | zh-TW | |
| dc.subject | 微反應器 | zh_TW |
| dc.subject | 生物晶片 | zh_TW |
| dc.subject | 核酸增幅反應 | zh_TW |
| dc.subject | 恆溫聚合酶 | zh_TW |
| dc.subject | B型肝炎病毒 | zh_TW |
| dc.subject | Loop-mediated amplification method | en |
| dc.subject | Microreactor | en |
| dc.subject | Hepatitis B virus | en |
| dc.subject | Biochip | en |
| dc.title | 微型化、恆溫增幅核酸之光學檢測器-應用於B型肝炎病毒核酸之快速偵測 | zh_TW |
| dc.title | Compact, Isothermal, Optical Diagnostic Device： For Rapid Detection of Hepatitis B Virus DNA | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.coadvisor | 李君男(Chun-Nan Lee) | |
| dc.contributor.oralexamcommittee | 李世光(Chih-Kung Lee),賴信志(Hsin-Chih Lai),楊龍杰(Lung-Jieh Yang),林玉娟(Yuh-Jiuan Lin) | |
| dc.subject.keyword | 生物晶片,微反應器,恆溫聚合酶,核酸增幅反應,B型肝炎病毒, | zh_TW |
| dc.subject.keyword | Biochip,Microreactor,Loop-mediated amplification method,Hepatitis B virus, | en |
| dc.relation.page | 86 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2008-11-13 | |
| dc.contributor.author-college | 工學院 | zh_TW |
| dc.contributor.author-dept | 醫學工程學研究所 | zh_TW |
| 顯示於系所單位： | 醫學工程學研究所 | |
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| ntu-97-1.pdf 未授權公開取用 | 2.01 MB | Adobe PDF |
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