微渦漩應用於微混合、抓取及即時單細胞檢測之微流體元件研發

A microfluidic platform for micromixing, trapping and real-time monitoring of single cells via microvortices

林建明(Cheng-Ming Lin)

指導教授 ： 胡文聰

國立臺灣大學/工學院/應用力學研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.02961

摘要

中文摘要
以細胞為基礎的分析檢驗系統中，最基本的需求就是能有效溫和地操控細胞至特定位置且不會傷害細胞原始表現。本研究中，我們提供了一個以流體動力式的穩定流場來抓取懸浮細胞，並且可以利用此流場增加流體的混合效率。其設計是利用勞倫茲力驅動一微小平板 (100 μm × 100 μm × 1.2 μm) 在液體內作平面的共振運動。如此，有兩種型態的流場產生，其中一種是相對較小尺度的二維對轉渦漩，另一個是相對較大尺度的三維循環流。
實驗結果包含應用在微混合器之混合效率、微渦漩的動力討論、生物粒子的抓取和釋放、細胞在渦漩中的存活測試和即時單細胞免疫螢光標定之監測。三維流場應用在微流體混合中，其混合效率可在1毫米內即達到X90的混合效率(X90代表達到90% 混合所需要的距離)。在二維微渦漩的動力討論中，對轉微渦漩可以在 2-9 Vpp 操作電壓範圍，產生相對旋轉頻率達0-6 Hz，在細胞抓取實驗中特定的操作電壓 (2-7 Vpp) 範圍內，抓取的生物粒子可以持續抵抗背景流速一直達到特定上限。此背景流速上限隨著渦漩旋轉速度增加而上升，如此藉由不同操作電壓可以定義出細胞抓取時所需要的力量界線。
一系列細胞抓取實驗測試中，有人類胚胎腎臟幹細胞、紅血球細胞、人類淋巴癌血球細胞和抗體IgG，均可以成功被抓取。以細胞大小的粒子為例(10 μm)，其抓取的力量為12(+-)2.0 pN 大小等級，奈米級生物粒子(抗體)則在160(+-)50 fN 範圍。此外紅血球抓取實驗證實此渦漩有抓取非球體形細胞的能力。
另外細胞存活實驗也提供了細胞可以在微渦漩中存活至少30分鐘之依據。在以多功能目標的設計下，我們將能產生三維流場的混合器的和能產生二維微渦漩的細胞抓取元件分別整合在微流道的上游和下游。
利用這個整合型平台，Jurkat懸浮細胞 (或單細胞) 可以作即時CD45抗體的螢光免疫標定之監控，實驗結果發現僅有被微渦漩抓取中的細胞能漸漸呈現紅色螢光。這個包含有一個高效率微混合器和數個多功能抓取細胞的元件，將非常適合應用在細胞大小等級和巨分子生物粒子(像是抗體、核酸)的檢測相關應用。
在未來發展中，此種流體動力式技術可應用整合在多方面的研究和使用上，像是懸浮細胞培養、單細胞與流體動力之研究、藥物篩檢、收集並增加微量生物粒子濃度的研究。

關鍵字

渦旋 ； 循環流 ； 震盪平板 ； 勞倫茲力 ； 細胞抓取 ； 微混合

並列摘要

A quantified cell-based analysis system requires the ability to locate live cells gently without changing their natural behavior. We describe a new hydrodynamic method via a secondary, steady streaming flow to trap suspended bioparticles and enhance flow mixing. Generation of the streaming flow utilized an in-plane resonating microplate (100 μm × 100 μm × 1.2 μm) actuated by Lorentz law. Either one of the two non-linear time-mean flow structures is feasible for the finite plate resonated in liquid: (1) two-dimensional (2D), small-scale, counter-rotating microvortices, or (2) three-dimensional, large-scale, recirculating flow.
Results consist of kinematics of microvortices, efficacy in mixing enhancement, trapping and release of bioparticles, cell viability study, and monitoring of single trapped cell via immuno-staining.
Mixing performance due to the 3D circulating flow was studied and characterized. The 3D recirculating flow is shown to enhance mixing – majority of mixing (X90) is reached within 1mm. The 2D counter-rotating microvortices rotate at 0-6 Hz corresponding to 2-9 Vpp (peak-to-peak) excitation. At a particular rate of rotation (2-7 Vpp tested), a bioparticle is trapped until the background flow exceeds a limit. The flow limit increases with the rate of rotation, which defines the trap/release force boundary over the range of operation.
Trapping and releasing of 10μm polystyrene beads, human embryonic kidney (HEK) cells, red blood cells (RBCs), Jurkat cells and IgG antibodies were demonstrated. The trap/release boundary is 12(+-)2.0 pN for cell-size bioparticles and 160(+-)50 fN for antibodies. Trapping of RBCs demonstrated microvortices’ ability for non-spherical cells. Cell viability was studied via HEK cells that were trapped for 30 minutes and shown to be viable.
Designedly, a platform was combined a micromixer, providing 3D circulating flow, in the upstream with four trappers in the downstream. Single Jurkat cells were tested by immunofluorescent staining with CD45 antibodies (conjugated to R-phycoerythrin) under a real-time observation. Data show only the trapped cells subjected to the immunofluorescent treatment can express red fluorescence. This hydrodynamically controlled approach to trap a wide range of bioparticles and enhance mixing should be useful as a microfluidic device for cellular and sub-cellular bioassay applications.
Future development of the technique might include various bioassay platforms, such as cell culture of suspended cells after being trapped, study on interaction between single cell and flow dynamics, drug screening, and enrichment of low volume fraction bioparticle studies.

並列關鍵字

Vortices ； Circulating flow ； Oscillatory plate ； Lorentz force ； Cell trapping ； Micromixer

參考文獻

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