單細胞列印系統之設計與實現

隨著生物科技的發展，在過去數十年中，每每都有許多的重大突破，也因此使人類更了解許多生物組織的分子網絡與交互作用，這些知識更廣泛應用於生物製藥產業或是進一步的基礎生物科學研究，然而，目前現有的生物技術往往是根據一整群的生物細胞的生命表現來做統計所得到的歸納結果，但是每一個生物細胞都有不同的基因組合，也造成了每個單細胞之間有不同的生命表現，因此，單一細胞的研究近來已經成為一項新興的研究領域，而提供操控與分析單細胞的實驗平台，正是目前極需各式新技術的發展與投入的研究領域。本篇論文利用了雷射脫附與微流分離的技術基礎之下，希望可以為藥物篩選或基礎生命科學研究領域提供新的研究利器。概念由微陣列檢測為基礎出發，整合了各個不同領域的技術，包括微流體力學、光學系統以及細胞生物學，這也是第一次，細胞分離與分注兩大重要功能同時被整合於單一微流元件中，且利用雷射脫附技術，微陣列中的每一滴生物樣品可被選擇性的回收做後端分析。論文內容分兩大主軸，第一個主軸著重在雷射脫附技術的設計與操作，當奈秒的極短雷射脈衝光衝擊吸收薄膜時，會產生極大的壓力波，並藉此將讓液體樣本脫離。第二個主軸著重在單細胞分離與分注元件之設計與操作，當單一顆細胞通過觀測區時，利用氣體與液體間不互溶的特性來將原本連續的液體樣品分離，並藉由持續供給的氣流將分離的液段推出微流元件外達到分注目的。論文最後部份有初步地將兩大主軸做整合，相信未來能對藥物篩選或基礎生命科學研究有一定程度的貢獻。

關鍵字

雷射脫附；微流分離；單細胞分離；單細胞分注；微陣列

並列摘要

In the past decades, extensive investments in biotechnology have yielded about many breakthrough. Therefore, human beings have gotten more knowledge about the many and complex processes involved in the interactions and molecular networks in organism. These knowledge is used extensively in drug discovery or fundamental biological research. However, current biotechnology advances are often based on expression of cell population statistic data. Because of the different gene combination of each cell, there are different expressions in each cell. Thus, single-cell analysis has become an emerging research area. No doubt, the need to provide a platform for manipulating and analyzing single cells requires any kinds of new technologies and techniques. The main works of this thesis are based on Laser-Induced-Forward-Transfer (LIFT) and Microfluidic separation technologies, and to provide a powerful tool for drug discovery or fundamental biological research. The concepts started from micro-array screening, and several different kinds of technologies have been integrated including microfluidics, optical systems and biology. This is the first time that a microfluidic device integrated single-cell separation and pipetting. By using LIFT technique, every droplet in a micro-array could be recycled for further analysis. There are two main parts in this thesis. The first part is focused on design and operation of LIFT technique. When a ~ns pulse laser beam irradiated the absorption thin film, great pressure wave was generated to transfer the droplet sample from target. The second part focused on design and operation of single-cell separation and pipetting device. When a cell was flowing through the detection area, by using the immiscible property between gas and liquid, the continuous fluid flow can be separated. Further continuous gas supply can push the separated fluid segment out of the device from pipetting tip. Preliminary integration of the two main parts were performed in the last part of the thesis. We believe this system could be useful in drug discovery or fundamental biological research.

並列關鍵字

LIFT ； microfluidic separation ； single-cell separation ； single-cell pipetting ； micro-array

參考文獻

[1]. V. Mironov, T. Boland, T. Trusk, G. Forgacs, and R. R. Markwald, “Organ printing : Computer-aided jet-based 3D tissue engineering”, Trends in Biotechnology, Vol.21 (4), 2003.

[3]. S. Baghdoyan, Y. Roupioz, A. Pitaval, D. Castel, E. Khomyakova, A. Papine, F. Soussaline, and X. Gidrol, “Quantitative Analysis of Highly Parallel Transfection in Cell Microarrays”, Nucleic Acids Res., Vol.32, e77, 2004.

[4]. D. B. Wheeler, S. N. Bailey, D. A. Guertin, A. F. Carpenter, C. O. Higgins, and D. M. Sabatini, “RNAi Living-Cell Microarrays for Loss-of-Function Screens in Drosophila melanogaster cells”, Nature Methods, Vol.1, pp. 127-132, 2004.

[5]. D. Castel, A. Pitaval, M. A. Debily, and X. Gidrol,”Cell Microarrays in Drug Discovery”, Drug Discovery Today, Vol.11 (13-14), pp. 616-622, 2006.

[6]. M. Colina, P. Serra, J. M. Fernandez-Pradas, L. Sevilla, and J. L. Morenza, “DNA Deposition through Laser Induced Forward Transfer”, Biosensors & Bioelectronics, 20, pp. 1368-1342, 2005.

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單細胞列印系統之設計與實現

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