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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張建成 | |
dc.contributor.author | Zong-Ying Li | en |
dc.contributor.author | 李宗穎 | zh_TW |
dc.date.accessioned | 2021-06-17T06:59:27Z | - |
dc.date.available | 2024-08-12 | |
dc.date.copyright | 2019-08-12 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-05 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72459 | - |
dc.description.abstract | 無法簡單、快速的檢測是結核病傳播的主要原因。為此,本研究將銅奈米粒子之特性結合分子生物技術,利用其螢光性質取代傳統核酸擴增試驗修飾螢光標定物質之檢測方法,並應用於結核病快速檢測。除了聚合酶連鎖反應(Polymerase chain reaction, PCR)放大後,接著使用環滾式擴增法(Rolling circle amplification, RCA)對DNA片段進行二次放大,並以特定單股DNA做為模板,將銅離子還原形成螢光銅奈米粒子。在DNA擴增實驗中,調整分子倒位探針(Molecular inverse probes, MIP)循環數、RCA加熱時間及酵素使用量的參數測試,在生成銅奈米粒子的實驗中對不同濃度還原劑、銅離子溶液及反應時間做探討,選擇最適化的條件參數,最後利用真實檢體進行臨床試驗。研究結果顯示,本系統能有效率且精準地放大結核菌目標DNA片段之訊號,採用最適化條件參數製作R2值為0.9998之檢量線,DNA濃度檢測極限可達5 fg/μL,且臨床檢體的試驗結果顯示具有87.5 %的敏感度及100 %的特異度,證明此方法在實際臨床應用之可靠性。由以上實驗結果,本系統能在保有高靈敏度、精確度情形下,大幅降低檢測分子生物技術放大訊號之專業人力、金錢、時間成本,在檢測部分也開發可攜式螢光檢測平台,能夠在一分鐘內將以DNA為模板的銅奈米粒子螢光訊號轉為數值供使用者讀取,判斷DNA標準品濃度在20 fg/μL以上為陽性結果。期許未來結合可攜式核酸擴增裝置,將放大、檢測訊號步驟簡化且合併於系統化裝置中,使低資源地區的居民能更有效率檢測結核病,以實踐及早發現、及早治療的願景。 | zh_TW |
dc.description.abstract | Detection without easily and rapidly testing is the primary reason for Tuberculosis spreading. For this purpose, the study combines molecular biological techniques and features of copper nanoparticles (CuNPs). The method of labeling fluorescent group for detection in Nucleic acid amplification techniques (NAAT) is substituted by optical properties of CuNPs and applied for rapid TB diagnosis. In addition to polymerase chain reaction (PCR), rolling circle amplification (RCA) is then performed to amplify the DNA fragmentation two times. And these specific single-stranded DNA (ssDNA) are used as templates for reduction of fluorescent CuNPs from Cu ions. In the part of DNA amplification experiment, the parameters of thermal cycles in Molecular inversion probes (MIP), heat time in RCA and unit of phi29 DNA polymerase were adjusted. In the other part experiment about the productivity of CuNPs, we discussed the various concentration of reductant and copper ion solution and reaction time to select optimized conditions and finally conducted clinical trials with real sample. The results show that we can efficiently and accurately amplify the signals of TB specific DNA fragment. We use the optimization parameters to construct the calibration curve with R2 value of 0.9998, LOD of standard DNA concentration 5 fg/μL, and the result of clinical tests shows detection rate with 87.5 % sensitivity and 100 % specificity, which demonstrates reliability of practical clinical applications. As mentioned above, the system keeps high sensitivity and specificity while greatly reducing professional labor, money and time cost of detecting signal amplificated by molecular biology. In term of detecting, we develop portable fluorescent detection platform converting optical signals from DNA-templated CuNPs to digital number for user reading in one minute and judge the concentration of DNA standard above 20 fg/μL as a positive result. In the future, we expect to combine this platform with portable NAAT device to simplify procedures and integrate amplification and detection DNA signal into a system device. And we look forward to helping the resident living in the low-resource area more efficiently detecting TB to realize the vision for early detection and treatment. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T06:59:27Z (GMT). No. of bitstreams: 1 ntu-108-R06543061-1.pdf: 1897715 bytes, checksum: 7f7462dcf4f8dde226cb94adaf2293ae (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 致謝 i
摘要 ii Abstract iii 圖目錄 vii 表目錄 viii 第一章:前言與文獻回顧 1 1.1 結核病對世界公衛影響 1 1.2 傳統結核病檢測方式 1 1.3 核酸擴增反應(Nucleic acid amplification test, NAAT) 2 1.4 生物感測器 3 1.5 奈米技術應用於生物感測器檢測結核病 4 1.6 以DNA為模板形成之銅奈米粒子 6 1.7 開發低成本電子元件應用於化學分析 9 1.8 本研究提出之檢測系統 10 第二章:實驗部分 12 2.1 實驗材料 12 2.1.1 實驗試劑與耗材 12 2.1.2 實驗儀器 13 2.2 溶液、樣品製備 15 2.2.1 TB IS6110標準品配製 15 2.2.2 NaCl、MOPS溶液配製 15 2.2.3 MOPS buffer溶液配製 15 2.2.4 Sodium ascorbate溶液配製 15 2.2.5 CuSO4溶液配製 15 2.3 螢光檢測裝置製造 16 2.3.1 Arduino系統架設 16 2.3.2 3D列印裝置結構 17 2.4 最適化實驗測試 18 2.4.1 DNA訊號放大部分 19 2.4.2 螢光檢測部分 22 2.4.3 螢光檢測裝置測試 22 2.5 應用於臨床檢體檢測 23 第三章:結果與討論 24 3.1 穿透式電子顯微鏡驗證銅奈米粒子結構 25 3.2 全波長吸收光暨螢光複合分析系統驗證銅奈米粒子之螢光性質 26 3.3 加熱循環數、加熱時間、酵素使用量參數對TB檢測系統訊號放大之影響 27 3.4 銅離子、還原劑濃度對TB檢測系統螢光銅奈米粒子生成之影響 30 3.5 利用最佳化參數進行螢光檢測裝置測試 34 3.6 利用最佳化參數進行臨床樣品試驗 35 第四章:結論與展望 39 參考文獻 40 | |
dc.language.iso | zh-TW | |
dc.title | 結合銅奈米粒子與螢光檢測裝置應用於結核病分子診斷 | zh_TW |
dc.title | The Development of Arduino-Based Fluorescent Detection Device and Copper Nanoparticles for Tuberculosis Molecular Diagnosis | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 陳建甫 | |
dc.contributor.oralexamcommittee | 林真真,朱錦洲,黃執中,崔博翔 | |
dc.subject.keyword | 結核病,生物感測器,核酸擴增試驗,螢光銅奈米粒子,Arduino系統, | zh_TW |
dc.subject.keyword | Tuberculosis,biosensor,NAAT,fluorescent CuNPs,Arduino system, | en |
dc.relation.page | 43 | |
dc.identifier.doi | 10.6342/NTU201902565 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-05 | |
dc.contributor.author-college | 工學院 | zh_TW |
dc.contributor.author-dept | 應用力學研究所 | zh_TW |
顯示於系所單位: | 應用力學研究所 |
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