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標題: | 迴路式熱對流聚合酶連鎖反應之PDMS-玻璃複合載具暨螢光檢測系統開發 The PDMS-Glass Compound Container and The Fluorescence Detection System for Loop Convective Polymerase Chain Reaction |
作者: | Mei-Hui Lai 賴美惠 |
指導教授: | 陳炳煇(Ping-Hei Chen) |
關鍵字: | 熱對流,聚合?連鎖反應,單一溫控,PDMS,螢光檢測, Convective,Polymerase Chain Reaction,single temperature controller,PDMS,Fluorescence detection, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 迴路式熱對流聚合酶連鎖反應(Loop Convective Polymerase Chain Reaction)為一種利用對試劑容器底部的單一溫控,使流體內部產生熱對流,並得以令試劑隨著設計的迴路流動,完成聚合酶連鎖反應所需的溫度循環,毋須繁複的加熱冷卻程序。本研究目的為承接迴路式毛細管熱對流聚合酶連鎖反應(CLCPCR)的研究,針對試劑的反應載具進行改善,並探討與本研究同時開發的即時螢光檢測系統進行定量分析的可行性。
本研究利用聚二甲基矽氧烷(PDMS)作為製作載具的基材,並配合蝕刻刀模沖壓技術對薄膜樣品進行裁切成型;再以電漿處理將PDMS與玻璃材料進行封裝,並使用聚乙二醇溶液進行表面改質等步驟,完成載具的製作流程。 在底部溫度控制在122℃時,載具內的溫度分布為適合進行聚合酶連鎖反應的範圍,且藉由觀察流場運動,此一單出口的設計確實能避免渦流的產生,整體所需試劑的用量亦減少至175 μl,流道的形狀亦較先前的玻璃毛細管樣品有穩定性。並且經由定性測試,此PDMS-玻璃複合載具至少能在反應時間二十五分鐘內擴增初始模板濃度為10^5 copies/ ml的HBV檢體。 本研究另建置一基於圖形化程式編譯平台LabVIEW的即時螢光檢測系統,對反應試劑進行激發,並利用CMOS鏡頭模組偵測螢光後記錄強度值與進行曲線擬合、反曲點判別等功能,試以將其與PDMS-玻璃複合載具結合進行定量分析。 雖目前尚未驗證其定量分析之可行性,然而由於載具的製造成本成功由每件六百元降至二十元以內,且後續實驗易於更動設計,因此代表該研究方向仍有相當的發展性與討論空間。日後載具的設計最佳化與機械強度若能有所改善的話,對於整體單一溫控反應的效率與實用性能勢必為一大進展,也期盼將來其可實際投入商業化用途,普及至民間檢驗機構。 Loop Convective Polymerase Chain Reaction complete the temperature cycle for PCR with single heating control at bottom. By heating the bottom of reagent container, the fluid would form a Rayleigh–Bénard convection cell, and force the reagent go through different temperature condition without complex procedure of heating and cooling. Following the CLCPCR research, the improvement of reagent container design and the development of real-time fluorescence detection system are investigated in this study. In this research, we take Polydimethylsiloxane (PDMS) as the material to manufacture the device, forming the shape of channel with blanking procedure, bonding the film with glass slide, and then doing the surface modification with polyethylene glycol solution to make this device. Under the bottom temperature 122℃, the temperature distribution is suitable for polymerase chain reaction. Through fluid field observation, the single outlet of channel could inhibit vortex in flow. The volume of container is reduced to 175 μl, and the shape of channel is also more stable than previous glass capillary tube. To ensure its ability, by qualitative experiment, the HBV template with initial concentration 10^5 copies/ ml could be amplified successfully in 25 minutes. A real-time fluorescence detection system based on LabVIEW is also established in this research. It includes the monitoring by CMOS camera, recording the fluorescence intensity, curve fitting and indication of inflection point. And we try investigating the possibility of combining these two project to do quantitative analysis. So far, we haven’t verify the feasibility of quantitative analysis with successful result, but with the advantage that the cost is cut down from 600 NTD to 20 NTD, and the flexibility of changing design, there should be prospects in this research. In the future, if the optimization and the mechanical strength could be adjusted completely, there should be a huge improvement in efficiency and practicality, and we look forward for the commercialization and the popularization. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67654 |
DOI: | 10.6342/NTU201702028 |
全文授權: | 有償授權 |
顯示於系所單位: | 機械工程學系 |
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