- 學位論文
DNA濃縮暨分離之液珠式微流體晶片
A Droplet-Based Micro Chip for DNA Enrichment and Separation
摘要
本研究之主旨為利用界面活性劑的構想研發被動式DNA濃縮技術，並用以開發DNA濃縮暨分離之液珠式微流體晶片，在無電、磁等外力場輔助下，於數秒內將晶片內的DNA濃縮、將高濃度區段分離並取出。本研究亦詳細探討DNA濃縮的機制與各種可能影響濃縮成果的狀況。 研發概念係將原為親水性的寡核苷酸 (oligonucleotide，或稱single-stranded DNA；ssDNA) 修飾上親油性的螢光分子TAMRA，並將修飾過後的ssDNA溶液通入液珠式流道中，形成離散的液珠群；液珠中之ssDNA將受親油端之極性，自身比重與流場剪應力的合併作用下聚集於液珠末端，而達成無外力場、無結構輔助下，於自由流動的液珠內濃縮ssDNA之目的。為收集液珠末端的高濃度ssDNA，本研究藉十字型流道、以對稱的剪切力分割母液珠，形成含低濃度ssDNA的前端液珠 (front droplet)，與尺寸較小而高濃度的尾端液珠 (rear droplet)。爾後再採背壓 (back pressure) 控制，或表面能量井控制法，使尺寸不一的前、尾端液珠分別進入不同流道，而達到分離與收集高濃度ssDNA 之目的。目前液珠移動速度為5.96 mm/s 時，可濃縮2.5 倍。ssDNA上所修飾之分子親油性越強、液珠移動速度越快、所使用之油體與ssDNA溶液黏性差異越大，皆會使濃縮成效會提高。由於ssDNA濃度越高，其所釋出的光訊號則越強，此一設計除了濃縮便利的優點外，還可提升DNA檢測極限至新低濃度。未來可應用於DNA雜交檢測，迅速將特定檢測訊號濃縮取出。此晶片除了流體的輸入、不需外加任何驅動裝置，相當容易與其他生醫檢測晶片整合，進行多功能處理與分析。期許此元件可廣泛應用於其他生醫檢體，例如蛋白質或細菌等的濃縮、反應及檢測，提供新世代的人們一個快速的即時檢測系統。
並列摘要
The condensed DNA reveals several interesting properties and plays an essential role in the fields of biology, biophysics and biochemistry. However, the investigation of enrichment of DNA in droplet-based biomedical chip is scarce. To fully utilize the advantages droplet-based microfluidic offer and to further complete the function of biomedical chip, a new method to concentrate DNA on-chip is demanded. This thesis proposes a novel droplet-based microfluidic device to enrich and to separate hydrophobically functionalized single-stranded DNA (ssDNA) in free-flow microdroplets without complicated design and fabrication, and without external field. The enrichment ability depends on several factors including the hydrophobicity of the fluorescent label, oil viscosity and flow rate. The mechanism of the enrichment process is also detail investigated and analyzed by micro particle image velocimetry (micro-PIV). A droplet called mother droplet is generated and undergoes enrichment in which the ssDNA aggregates at the end of the droplet through a combined effect of hydrodynamic repulsion, ssDNA specific gravity and affinity attraction. On passing through the cross junction, the mother droplet was divided into a front droplet and a rear droplet. The rear droplets are smaller but contain greater ssDNA concentration. Based on a correlation between pressure resistance, surface energy and droplet size, the rear droplets are separated into different channels and collected to obtain a solution with highly concentrated ssDNA. The fluorescent intensity of droplets is analyzed and shows that the rear droplet is approximately 3 times the original concentration. Three vortices are found after performing the micro-PIV experiment. It is the interaction of ssDNA specific gravity with the recirculating microvortices that concentrate ssDNA in the rear of the plug. The hydrophobic label greatly enhances the performance since it reduces the mobility of the ssDNA. This thesis realized ssDNA enrichment and separation in free-flow microdroplets without additional medium or external addition. This is a novel approach to purify and extract ssDNA from a dilute solution, and to improve the detection limit of dilute DNA solution during hybridization. Hence it is advantageous to biomedical chips.