- 學位論文
應用奈米粒子聚集現象於微分離器之DNA檢測技術
Aggregation-based DNA Detection in Microseparators
摘要
本研究結合應用奈米粒子聚集現象與微型分離器機制，發展一創新簡易之生物分子檢測平台及方法。以檢測去氧核醣核酸(Deoxyribonucleic acid, DNA)分子為例，利用分別修飾有兩種探針DNA之螢光微粒，當兩端分別與兩種探針DNA互補之目標DNA存在時，目標DNA將探針DNA雜交反應而造成粒子聚集現象，粒子聚集大小將與目標DNA濃度有一定比例關係。將聚集後粒子通入分離器中並根據微分離器分離聚集粒子之結果，可反推直接得知目標物濃度。 粒子表面之探針DNA標定利用鏈黴抗生素蛋白(streptavidin)對生物素(biotin)的高度親和性完成，並利用動態光散射粒徑分析(dynamic light scattering, DLS)及螢光顯微鏡影像分析，探討粒子聚集程度與目標DNA濃度的關係。聚集粒子樣品通入聚二甲基矽氧烷(PDMS)材質製作之微分離器，以螢光顯微鏡及電荷耦合元件(CCD)相機拍攝長時間曝光之粒子螢光影像，分析螢光訊號峰值之位置，以定量目標物濃度與訊號峰值位置之關係。多出口之設計幫助檢測解析度，並佐以CFD-RC軟體模擬出口流速分布，驗證出口之壓阻分配計算無誤。 利用螢光分析目標DNA造成粒子聚集尺度與其在微分離器中之橫向位置，可得一劑量反應曲線，直接定量實際未知的樣品濃度；可達到之DNA最低檢測極限為33 pM，最高檢測極限10 nM，動態範圍為0.33 ~ 10 nM。雙出口的設計，幫助快速篩選目標DNA含量0.33 nM以上之樣品；多出口不對稱阻力設計則幫助快速判讀目標物濃度範圍。針對檢測專一性及基因變異檢測的表現亦經測試證實可靠與實用；以分離再結合式微反應器(SAR)測試粒子聚集效應，亦具有不錯成果。 本研究之特點是以微流體系統操控檢體與試劑迅速完成反應，並於同一平台上進行訊號偵測及分析，達到簡易、多功能、低成本、試劑用量少、無需標定目標物、亦無需複雜儀器之檢測。自評所發展之技術與系統可提供生物分子檢測、甚至化學檢測、環境監控及居家看護檢測方面更為簡便之技術；同時具有多目標物同步檢測及其他基因變異檢測之應用潛力，具備取代現有傳統技術的商機。
並列摘要
A novel aggregation-based method to execute oligonucleotide detection in a PFF (pinched flow fractionation) microseparator is developed in this thesis. The label-free target DNA hybridizes with the probe DNA on the surface of microspheres and causes the polymeric aggregate, thus enlarging the average size of the aggregated particles. Depending on the aggregated particle sizes, the microparticles were separated through a PFF microseparator, enabling subsequent concentration analysis of the target DNA . Streptavdin-coated polystyrene microspheres is conjugated respectively with two kinds of probe DNA modified with biotin, and the efficiency of labeling is analyzed by UV-Visible spectrum. The aggregation caused by mixing probe-labeled beads with the target DNA whose two ends is respectively complementary with the two DNA probes is analyzed by dynamic light scattering (DLS) particle size analysis and the fluorescence image. A PFF microseparator (20 and 1000 μm respectively for the pinched and broadening segments) is used to analyze the size of the aggregated particles. The result shows that the size of the aggregated microparticles varied with the concentration of the target DNA (Ctarget). The PFF microseparator was used to determine Ctarget, that is, the size of the aggregated microparticles. The size of aggregated particles can be evaluated by analyzing the intensity through the broadening segment, thus obtaining the concentration of target DNA. In addition, an asymmetric outlet design is used to conduct rapid screening of the DNA detection. The contribution of this study is the numerous features of this method such as simplicity, low cost and potential to detect other biochemical samples with distinct aggregation behaviors. Also, it is capable of performing SNP/mutation detection. Because of its high specificity, this method can also be used to conduct multi-target detection on a chip.
參考文獻
延伸閱讀
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