藉流體集中增強橫向過濾微流道細胞分離效率之研究

細胞分離是血液分析過程中的一個重要步驟。主要應用在血液的樣本中萃取出目標細胞，以進行後端的檢測分析，常被應用在分離出循環癌症細胞 (Circulating Tumor Cells, or CTC)來研究癌症擴散的情形，或是從母體的血液中分離出其胎兒的有核紅血球(fetal Nucleated Red Blood Cells, or fNRBC) 來進行產前檢測。近年來隨著生物晶片(Lab-on-a-Chip) 快速發展，藉由製造出更接近細胞尺度的裝置，微流道晶片被有效應用在細胞分離上，可以達到更有效且敏感的細胞分離結果。一般而言，血液中帶核的細胞大小都在8 μm 以上，而紅血球為雙凹扁平狀，厚度約為2~3 μm，本研究目標在利用細胞在尺寸上的差異，來分離血液中有核紅血球(fNRBCs)。實驗設計則是利用橫向流(cross-flow) 的篩選原理，在側邊加上額外的流體(buffer)，產生流體聚集(hydrodynamic focusing)的效果，而將帶有細胞的樣本流體(sample flow)推向篩選流道(filters)，無核較小的細胞會通過篩子而收集後被移除，有核較大的細胞則無法通過篩子，沿著主要流道後被收集。此機制，可以充分利用橫向流的好處來減少細胞在流道內的阻篩，再配合流體集中的效果增加過濾的壓力、提升分離效率。在本論文裏，首先討論了實驗原理與設計，接著利用模擬分析了解流道的設計和原理，然後是微流道晶片製作以及進行利用微流道來分離不同樣本的實驗。實驗分為兩個階段，第一階段，先用2.7 μm 和10.6 μm 大小不同的微珠，模擬細胞的分離情況，驗證設計在分離尺寸上差異的微珠可行性。藉由最佳的流速結果，再進行細胞的分離，比較裝置設計對含量稀少的細胞收集的靈敏度。研究結果比較不同流速下對細胞分離、回收效率和純度的影響。本研究利用設計的微流道晶片，成功分離不同尺寸的微珠和細胞，並應用在血液的分離。關鍵詞: 微流道，非侵入式檢測，有核紅血球，細胞分離，cross-flow

關鍵字

微流道；非侵入式檢測；有核紅血球；細胞分離； cross flow

並列摘要

A microfluidic chip is proposed to separate particles using cross-flow filtration enhanced with hydrodynamic focusing. By exploiting a buffer flow from side, the particles in the sample flow are pushed on one side of the microchannels, while a larger pressure gradient in the filters is obtained to enhance separation efficiency. Our proposed mechanism has the buffer flow to create a moving virtual boundary for the sample flow to actively push all the particles to reach the filters for separation. This filtration device only requires soft lithograph fabrication to create microchannels and a novel pressurized bonding technique to make high-aspect-ratio (HAR) filtration structures. A mixture of polystyrene particles with 2.7 μm and 10.6 μm diameters are successfully separated. 96.2 ± 2.8% of the large particle are recovered with a purity of 97.9 ± 0.5%, while 97.5 ± 0.4% of the small particle are depleted with a purity of 99.2 ± 0.4% at a throughput of 10 μl/min. The experiment is also conducted with the sample solutions of spiked PC3 cells in whole blood, whole blood sample and cord blood sample. Our device offered a label-free and high separation efficiency technique with simple fabrication and integrable possibility with other components as a promising tool for continuous cell filtration and analysis.

並列關鍵字

fetal cells ； prenatal diagnosis ； microfluidics ； cross-flow ； cells separation

參考文獻

Bhagat, A. A., S. S. Kuntaegowdanahalli, and I. Papautsky, 2008 'Continuous Particle Separation in Spiral Microchannels Using Dean Flows and Differential Migration', Lab Chip, 8, 1906-14.

Chen, D. F., H. Du, and W. H. Li, 2006 'A 3d Paired Microelectrode Array for Accumulation and Separation of Microparticles', Journal of Micromechanics and Microengineering, 16, 1162-69.

Chiou, P. Y., A. T. Ohta, and M. C. Wu, 2005 'Massively Parallel Manipulation of Single Cells and Microparticles Using Optical Images', Nature, 436, 370-2.

Choi, S., and J. K. Park, 2007 'Continuous Hydrophoretic Separation and Sizing of Microparticles Using Slanted Obstacles in a Microchannel', Lab Chip, 7, 890-7.

Choi, S., S. Song, C. Choi, and J. K. Park, 2009 'Microfluidic Self-Sorting of Mammalian Cells to Achieve Cell Cycle Synchrony by Hydrophoresis', Anal Chem, 81, 1964-8.

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藉流體集中增強橫向過濾微流道細胞分離效率之研究

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