Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5873
DC FieldValueLanguage
dc.contributor張書奇zh_TW
dc.contributor.author謝秀燕zh_TW
dc.contributor.authorHsieh, Hsiu-Yenen_US
dc.contributor.other環境工程學系所zh_TW
dc.date2013en_US
dc.date.accessioned2014-06-06T06:35:55Z-
dc.date.available2014-06-06T06:35:55Z-
dc.identifierU0005-2908201317424600en_US
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dc.identifier.urihttp://hdl.handle.net/11455/5873-
dc.description.abstract在目前環境微生物領域裡,當我們面對一項未知的環境樣本時,傳統培養鑑定法或是新興的分子生物檢測方式(如:螢光現址雜交法或是聚合酶連鎖反應)均無法同時快速地得知這未知樣品中包含了哪些物種、各物種之數量及各物種在該環境中之功能及其所扮演的角色。本研究為超快速高輸出微生物鑑定與定量儀器開發研究,企圖以微水珠包裹單一微生物細胞之方式進行單一細胞之特性偵測,本實驗室一系列之研究已完成有效且高度一致性之的微水珠產生方法,以雷射激發並定量經螢光染色之微水珠中螢光染劑之濃度並且進行雙螢光之同時快速偵測為本研究之研究標的。本研究採用油相作為分隔細胞之流體,其目的在於能夠針對封裝於單一水珠中之細胞進行不同之標記再進行偵測,其終極目的在於進行多螢光之單分子偵測,以達到超快速高輸出微生物鑑定與定量之目的。因為微流體生物晶片具有反應速度快、靈敏度高、再現性高、低成本等優點,可一舉解決目前流式細胞儀的缺點並可達成環境微生物檢測技術上的突破。目前之研究藉著微機電工程中之微影製程及軟微影製程進行微流體生物晶片之聚縮型微通道光罩設計、母模製作與實驗晶片開發,以雷射光激發並藉高靈敏度光學感應器(PDA-36A)擷取螢光訊號,再以類比轉數位介面卡(DAQ盒 )及電腦軟體 (LabVIEW TM )進行資料統計與分析,以完成偵測螢光染色之微水珠中雙螢光訊號之偵測並定量之研究,為下一步多螢光單分子偵測奠下良好基礎。zh_TW
dc.description.abstractTo-date, when we face unknown environmental samples, neither culturing methods nor molecular biological methods (like FISH or real time PCR) can immediately detect how many species are there, how much the amount of each species is, and what functional roles of each species is. Thus, we would like to develop a system for ultrafast microbial identification and quantification down to species level. So far we had successfully developed microfluidic chips for water droplet generation in oil-filled microchannels in the past, now to excite, detect and quantify dual fluorescent signals in droplets is the goal of this research. We use oil to separate cells for single cell detection and multi fluorescence detection, and prove the feasibility of ultrafast microbial identification and quantification of dual fluorescence detection in aqueous microdroplets. Because microfluidic chip has the advantages of high reaction rate, sensitivity and reproducibility with low cost. By using microfluidic chip, we can solve the problems of flow cytometer and conquer the technology barrier of microorganism detection. We use lithography and soft lithography to make mold and microfluidic chip. We also use a laser to excite fluorescent signals and detect them by PDA-36A photodetector as well as a DAQ card. LabVIEWTM was employed to analyze data to complete detection and quantification dual fluorescence in droplets. These approaches will provide the foundation for further detecting multi fluorescence signals generated by single molecules.en_US
dc.description.tableofcontents中文摘要 i Abstract ii 目錄 iii 圖目錄 vi 表目錄 x 附錄目錄 xi 第一章 緒論 1 1.1 研究背景 1 1.2 研究目的 2 第二章 文獻回顧 4 2.1 微生物鑑別 4 2.1.1 分子生物技術之微生物鑑定方法 4 2.1.1.1 實時聚合酶連鎖反應 5 2.1.1.2 16S r RNA為目標之鑑定技術 7 2.1.1.3 流式細胞術 8 2.1.1.4 生物晶片 10 2.1.1.4.1 DNA晶片 10 2.1.1.4.2 微流體生物晶片 12 2.2 螢光分子及偵測技術 13 2.2.1 核酸染劑 15 2.2.2 分子探針 20 2.2.2.1 一般探針 21 2.2.2.2光標分子探針 21 2.2.2.3 抗原與抗體結合 23 2.2.2.4 微流體生物晶片中螢光訊號偵測方法 24 2.2.2.4.1 半導體雷射 25 2.2.2.4.2 螢光顯微鏡 26 2.3 微流體晶片 30 2.3.1 母模製作 30 2.3.1.1 光微影製程 31 2.3.1.2 蝕刻製程 32 2.3.1.2.1 濕式蝕刻 33 2.3.1.2.2 乾式蝕刻 34 2.3.2 晶片製作 34 2.3.3 微水珠之形成 35 2.3.3.1 空氣壓縮式微水珠產生法 36 2.3.3.2 抽吸導式微水珠產生法 37 2.3.3.3 以十字形微流道產生微水珠 37 2.3.4 雷射經光纖導入微流體晶片方法 38 2.3.4.1 光纖 38 2.3.4.2 光纖通道 40 第三章 材料與方法 45 3.1 實驗材料 45 3.2實驗儀器 47 3.3 實驗架構 49 3.3.1 光罩之繪製 49 3.3.2 母模製作 50 3.3.2.1 玻璃基板清洗步驟 50 3.3.2.2 光微影製程 51 3.3.3 微流體生物晶片製作 54 3.3.3.1 PDMS 翻模 55 3.3.3.2 PDMS 與玻璃基板接合 56 3.3.3.3 氧氣電漿處理 56 3.3.4 油相流體及螢光染色 58 3.3.4.1 油相流體 58 3.3.4.2 單螢光染劑樣品配製 59 3.3.4.3雙螢光染劑樣品配製 59 3.3.4.3.1 E.coli樣品前處理 59 3.3.4.3.2 E.coli樣品染上雙螢光 60 3.3.5 光路架設 60 3.3.5.1 雷射光源架設 61 3.3.5.2 收光光路架設 62 3.3.5.2.1 單螢光收光光路架設 62 3.3.5.2.2 雙螢光收光光路架設 63 3.3.5.3 螢光訊號之擷取 63 第四章 結果與討論 65 4.1 光纖置入晶片方法實驗 65 4.1.1 光纖翻模法製作之光纖通道 65 4.1.2 濕蝕刻法製作之光纖通道 69 4.1.3濕蝕刻法蝕刻光纖外殼層 71 4.1.4 以切割機製作之光纖通道 73 4.2雷射光激發微水珠螢光訊號之偵測與擷取 75 4.2.1 系統背景值訊號 80 4.2.2 AO微水珠螢光訊號偵測 80 4.2.3 使用LabVIEW™軟體降低背景雜訊 84 4.3以雙螢光染色後之E.coli樣品之雙螢光訊號 90 第五章 結論與建議 94 5.1 結論 94 5.2建議 95 附錄 97 參考文獻 99zh_TW
dc.language.isozh_TWen_US
dc.publisher環境工程學系所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2908201317424600en_US
dc.subject環境微生物zh_TW
dc.subjectenvironmental microorganismen_US
dc.subject微機電系統zh_TW
dc.subject生物晶片zh_TW
dc.subject微流體zh_TW
dc.subject光纖zh_TW
dc.subject螢光zh_TW
dc.subjectMEMsen_US
dc.subjectbio-chipen_US
dc.subjectmicrofluidicen_US
dc.subjectoptical fiberen_US
dc.subjectfluorescenceen_US
dc.title流式質體儀:超高速之微生物鑑定定量研究─微水珠中雙螢光偵測研究zh_TW
dc.titleFlow-OmicsTM:Ultrafast microbial identification and quantification – dual fluorescence detection in aqueous microdropletsen_US
dc.typeThesis and Dissertationzh_TW
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypeThesis and Dissertation-
item.cerifentitytypePublications-
item.fulltextno fulltext-
item.languageiso639-1zh_TW-
item.grantfulltextnone-
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