Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5873
標題: 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
Flow-OmicsTM:Ultrafast microbial identification and quantification – dual fluorescence detection in aqueous microdroplets
作者: 謝秀燕
Hsieh, Hsiu-Yen
關鍵字: 環境微生物;environmental microorganism;微機電系統;生物晶片;微流體;光纖;螢光;MEMs;bio-chip;microfluidic;optical fiber;fluorescence
出版社: 環境工程學系所
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摘要: 
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。

To-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.
URI: http://hdl.handle.net/11455/5873
其他識別: U0005-2908201317424600
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