Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21462
標題: The Development Hybrid Microfluidic Biochip for Rapid Hybridization and It''s Applications in Non-Small Cell Lung Cancer
快速微流體雜合晶片之開發暨其於非小型肺腺癌上的應用
作者: 耿碧蓮
Keng, Pi-Lien
關鍵字: microfluidic
微流體
出版社: 分子生物學研究所
摘要: Microfluidic biochip has been extensively used in biomedical research in recent years. Micro-system technology integrated with microarray may improve the gene expression quality and the efficiency of nucleic acid hybridization. In this study, we utilize a droplet-based microfluidic system to increase the DNA microarray mixing efficiency. With this device, the slow diffusion kinetics of the biomolecules reaction is expedited. Microfluidics with mixing effect enhances the hybridization efficiency and reduces the hybridization time to 15 minutes compared with conventional flat glass microarray, which takes from 4h to over night for hybridization. The miniaturization also reduces the hybridization volumes to 5 μL. To profile gene expression patterns, human lung adenocarcinoma cell lines with different invasion capabilities were employed to analyze and screen the differential gene expression for metastasis-associated genes. 891 EST clones associated with adenocarcinoma cell were amplified by polymerase chain reactions and spotted onto a 16 mm x 23 mm glass support, which is integrated with PMMA microchannels fabricated by CO2 laser machine system. The microchannel is 100μm deep and 150 μm wide. The rapid nucleic acid hybridization time is reduced to 15 min with higher signal to noise ratio and detection limit is 0.103 μg/μL. The microfluidic system allows faster and easier gene expression profiling than conventional flat glass microarray system. In the future, the microfluidic system could provide potential applications in other cancer research.
微陣列晶片在過去十幾年來已經廣泛的被應用,無論基材是薄膜、玻璃或是高分子聚合物,都是將成千上萬的生物分子為探針,用高密度的方式精確的佈放在基材上,由細胞或組織中萃取出來並加以標定螢光分子的標的物與探針進行18至24小時的雜合反應,但是,以平板玻璃為載體的cDNA microarray為例,此雜合方式卻受限於核酸分子緩慢的擴散作用以及大量的樣品需反覆製作才能因應微陣列晶片所需。因此,本研究結合了微陣列平台技術以及微機電製程技術,利用二氧化碳雷射雕刻機在低成本的PMMA聚合物上刻出微小通道,藉由夾具結合未經蝕刻的玻璃晶片與PMMA晶片,形成深度以及寬度分別為100 μm和150 μm的微流通道,使微流體於微通道中產生離散型液滴混合特性 ( Discrete Drops Mixing ),在無需要外加幫浦下,利用具有不同癌轉移能力的人類肺腺癌細胞株 ( Human Lung Adenocarcinoma Cell Lines ),CL1-0以及CL1-5為實驗材料,對891個與癌轉移相關的已知部份序列基因 ( Expressed Sequence Tags, ESTs )進行微流體晶片核酸雜合反應,並與傳統平板式玻璃晶片互相比較,由傳統玻璃晶片技術的18小時縮短到只需15分鐘即可達到反應平衡、樣品試劑量只需5 μL以及達到最低有效偵測極限0.103 μg/μL、於十五分鐘時的訊號雜訊比值比傳統平板式玻璃晶片高出15倍、於平衡時間十五分鐘時的訊號雜訊比值為傳統平板式玻璃晶片於十八小時的1.2倍,並且無發生樣品交叉汙染,加上這種塑膠基材具有透光性佳、易處理、價格低廉等特性,符合微流體生醫晶片的製作成本低廉。比起傳統的生物晶片,其訊號雜訊比 ( S/N ratio )較高,使微流體生醫晶片更進一步提高應用性,未來可望達到臨床診斷上的快速應用。
URI: http://hdl.handle.net/11455/21462
Appears in Collections:分子生物學研究所

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