固定式液珠生成法結合個人醫療檢測裝置之設計與應用

Design of a Static Droplets Array Platform for In-vitro Diagnostic Testing

侯志泉(Chih-Chuan Hau)

指導教授 ： 楊鏡堂

共同指導教授 ： 王興華

國立臺灣大學/工學院/機械工程學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.10259

摘要

本研究主旨為設計個人醫療檢測系統，結合固定式液珠生成法與濃度梯度生成器，開發可利用肉眼直接觀測藥物與細胞反應之高通量矩陣形固定式液珠晶片。操作原理為利用固定式液珠生成法將包含細胞之液珠生成為陣列形態，透過流道下方的薄膜震盪以及細胞通入方式，使細胞於16×20 反應槽內分布均勻，之後通入固定體積之藥物段，利用空氣將藥物段向下游推進，使藥物段依序的與液珠進行反應，進而生成藥物梯度，流道下方的薄膜震盪加快藥物與細胞間的反應。本設計的優點在於固定式液珠提供較直接的觀測模式與反應；而本研究之濃度梯度之生成方式不必利用大量的樣本來維持所生成的梯度，故可以大量降低成本、減少細胞受損、縮短檢測時間；而流道下方的薄膜震盪不僅增加了細胞在不同液珠內之均勻度，亦加速細胞與藥物之間的反應。本實驗之細胞均勻度變異係數約為10左右，表示反應槽內的細胞數量差異不影響藥物梯度之表象，而藥物之梯度範圍目前可相差約10倍。
本研究之流道結構是利用流道與反應槽的結合使液體停留在反應槽內，將含有乳癌細胞(MDA-MB-231)的營養液進入流道中，並放入細胞培養箱中進行細胞培養，再利用空氣通入流道內即可將含有細胞之液珠生成於固定的反應槽內，生成包含細胞之固定式液珠，接著通入抗癌藥品 (Doxorubicin, DOX)並再次利用空氣推進，生成不同藥物濃度之細胞液珠，培養48小時等待細胞分化，最後檢測細胞之生存能力，進而計算出IC50。本研究已成功包覆乳癌細胞以及生成多種藥物濃度梯度，並且找出藥物為DOX、細胞密度為6 × 105、細胞培養48 h之IC50為5.81 M，期許此晶片能與其他生醫檢測晶片整合，進行多功能處理與分析，提供新世代一個方便且節能的個人醫療檢測系統。

關鍵字

個人照護 ； 液珠 ； 陣列 ； 濃度梯度 ； 氣室 ； 細胞包覆 ； 薄膜 ； IC50

並列摘要

In this study, we present a new microfluidic chip which containing both static droplet array in matrix form and steady-state concentration gradient generator for low cost cell-based drug screening. The chip contains 320 individual microreactors, in which cells can be seed and cultivated. A serial concentration gradients of drug solution are created when a drug plug passes through the each microreactors. The PDMS-based membrane in the bottom of the reactor was used to generate a gentle vibration to accelerate interaction between the cells and drug, and furthermore, can improve distribution of cells within the reactors. Cell descending in each reactors along downstream can be improved by injecting cells twice through opposite inlets so that the coefficient of deviation is approximately 14.0%.
In this thesis, we load breast cancer cells, MDA-MB-231, into the chip and incubate over night for cell to seed onto the surface of reactor. After that we inject air into chip to form 320 static droplets containing breast cancer cells, 6 l of chemo drug, Doxorubicin, plug is injected into chip and pushed by air to downstream in order to generate drug concentration gradient. After 48 hours for cell differentiation, labeling the cells with Calcein AM so that we can calculate how many cells survive after the drug dope. Then, we can obtain our final goal, IC50.
Totally 320 data points per drug compound can be quickly observed in an automatic form. The platform can potentially become a useful device for drug screening application via quantifying the half maximal inhibitory concentration (IC50) of drugs on cell models in vitro.

並列關鍵字

concentration gradient ； droplet ； array ； air chamber ； cell encapsulation ； IC50

參考文獻

Baroud, C. N., Gallaire, F. G., and Dangla, R., "Dynamics of Microfluidic Droplets," Lab Chip, 2010, 10, pp. 2032-45.

Bithi, S. S., and Vanapalli, S. A., "Behavior of a Train of Droplets in a Fluidic Network with Hydrodynamic Traps," Biomicrofluidics, 2010, 4, pp. 44110.

Boukellal, H., Selimovic, S., Jia, Y., Cristobal, G., and Fraden, S., "Simple, Robust Storage of Drops and Fluids in a Microfluidic Device," Lab Chip, 2009, 9, pp. 331-8.

Chung, B. G., Lin, F., and Jeon, N. L., "A Microfluidic Multi-Injector for Gradient Generation," Lab Chip, 2006, 6, pp. 764-8.

Cohen, D. E., Schneider, T., Wang, M., and Chiu, D. T., "Self-Digitization of Sample Volumes," Anal. Chem., 2010, 82, pp. 5707-17.