整合光介電泳技術及微流體晶片平台應用於自動化置換溶液與細胞之操作

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研究生：	吳奐均 Wu, Huan-Chun
論文名稱：	整合光介電泳技術及微流體晶片平台應用於自動化置換溶液與細胞之操作 Integration of microfluidic devices and an optically-induced dielectrophoresis device for medium replacement and cell manipulation and separation
指導教授：	李國賓 Lee, Gwo-Bin
口試委員:	李明昌楊瑞珍
學位類別：	碩士 Master
系所名稱：	工學院 - 動力機械工程學系 Department of Power Mechanical Engineering
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	73
中文關鍵詞：	光介電泳力、奈米微珠、虛擬電極、微流體、細胞收集率、細胞存活率
外文關鍵詞：	Optically-induced dielectrophoresis (ODEP), recovery rate of cell, survival rate of cell
相關次數：	點閱：2 下載：0
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光介電泳技術是藉由交流電產生均勻電場使粒子極化，再利用投影機產生的光學圖案誘發形成虛擬電極，進而產生不均勻電場來操控粒子或細胞。由於光介電泳力的產生需要低導電度與適宜介電常數的溶液，目前常用0.2M蔗糖溶液。在傳統方式裡需要使用離心機離心多次，將細胞培養液完全置換成0.2M蔗糖溶液。此為一個複雜的且費時的動作，且反覆的置換過程中易使細胞遭受汙染，此外0.2M蔗糖溶液並不適合細胞長期滯留，若滯留過久會對影響細胞存活率。在本研究中，我們設計一個整合光介電泳技術及微流體之晶片平台，使得置換溶液過程得以完全自動化，並探討置換細胞溶液時，微型泵在不同壓力及頻率下的收集效率，同時測量在整合型晶片之介電泳力層所產生移動細胞之速度，和細胞之存活率。相較於傳統的置換方式，整個自動化置換過程只需歷時25分鐘。在置換溶液過程中有高達91%之細胞收集率。此置換完之細胞在ODEP層可以產生最高109.1μm/s之拖曳速度。更重要的是，實驗完的細胞得以存活培養，可將細胞存活率提升33.3%。新型的整合型微流體晶片可以達成自動化細胞分離及更換溶液之效果，並減少污染問題，以及大量的縮減了時間以及人力資源。本研究也是第一個將光介電泳系統與微流體系統整合的晶片，可提供一個平台快速更換細胞溶液和操控細胞。本研究可作為與光介電泳相關實驗晶片的前端裝置，進行快速自動化的溶液置換，提高實驗完之後的細胞存活率，並對生醫界發展光介電泳技術操控細胞提供貢獻。

Optically-induced dielectrophoresis (ODEP) has been demonstrated to generate virtual electrodes for manipulating particles/cells by illuminating a light pattern on photoconductive materials. Because the production of the ODEP force requires a solution that has suitably low conductivity and appropriate dielectric constant; currently 0.2M sucrose is used. However, it requires a complicated medium replacement process before one could manipulate cells. We traditionally centrifuged cells several times for the complete replacement of the medium from the culture medium to 0.2 M sucrose. It is a complex and time-consuming operation, and the repeated replacement process can easily lead to cell contamination problems. Furthermore, 0.2M sucrose is not suitable for the long-term viability of cells. In this work, we demonstrated an integration of a microfluidic device with an ODEP device such that the critical medium replacement process can be automated and the cells could be subsequently manipulated by using optical images. In comparison to conventional manual processes, the automated medium replacement process only took25 minutes. There was up to a 91% recovery rate of cells, and a 109.1μm/s dragging velocity was induced by ODEP. More importantly, the survival rate of cells could be greatly enhanced due to the faster automated process. The new, integrated microfluidic chip could automate the entire process of medium replacement without the contamination issue. It is then concluded that the developed microfluidic system may provide a platform for fast replacement of cell medium. It is also the first time that an ODEP device was integrated with microfluidic devices. By achieving automated and fast medium replacement, this device can be deployed for a wide range of ODEP-based sample pre-treatment processes while improving cell viability after completing the experiment. With further developments, this device may contribute to the area of ODEP-based cell manipulation.

Table of Contents
Abstract ............................................................................................................I
摘要..............................................................................................................III
誌謝.............................................................................................................. IV
Table of contents.......................................................................................VI
List of tables.............................................................................................. IX
List of figures..............................................................................................X
Abbreviation............................................................................................XVII
Nomenclature...........................................................................................XX
Chapter 1
Introduction 
1.1     Introduction to MEMS and microfluidic technology....................................................1
1.2     Background and literature survey.................................................................................1
1.2.1    Mechanisms for cell and micro-particle manipulation..........................................1
1.2.2    Dielectrophoresis (DEP) force for separation of particles.....................................3
1.2.3    Optically-Induced dielectrophoresis (ODEP) system and application..................6
1.2.4    Optically-Induced dielectrophoresis (ODEP) force...............................................7
1.3    Motivation and objectives...........................................................................................9
1.4     The format of this dissertation....................................................................................10
Chapter 2
Theory  
2.1    Introduction..................................................................................................................17
2.2    Amorphous silicon as a photoconductive material.............................................17
2.3    Working principle of the DEP force and ODEP...........................................................19
2.3.1    Working principle of DEP force...........................................................................19
2.3.2    Working principle of the ODEP force…………………....................................20
Chapter 3
Materials and methods
3.1    Introduction…………..................................................................................................24
3.2    Chip design and operating principle…………..............................................25
3.2.1    Chip design ………………………….................................................................25
3.2.2    Working principle of an integrating microfluidic devices...................................26
3.2.3    Working principle of the micro filtering structure.......................…….………..27
3.2.4    Working principle of measuring the maximum dragging velocity and ODEP force...................................................................................................27
3.2.5    Working principle of separating the live and dead cells...........................28

3.3    Fabrication process..............................................................................29
3.3.1    Substrate preparation …………………………........................................29
3.3.2    Coat................................................................................................................29
3.3.3    Recommended program.............................................................................29
3.3.4    Soft bake...................................................................................................29
3.3.5    Exposure..................................................................................................................30
3.3.6    Post exposure bake (PEB)............................................................................31
3.3.7    Developing..........................................................................................31
3.3.8    Rinse and dry...........................................................................................31
3.3.9    Hard bake.......................................................................................31
3.4    Experimental setup......................................................................................................32
3.4.1     ODEP chip manipulation platform.....................................................32
3.4.2     Virtual electrodes generation system...............................................................32
3.4.3     Image acquisition system..................................................................................33
Chapter 4
Results and discussion
4.1    Production of the integrated microchip..................................................................40
4.2    Micro filtering structure testing........................................................41
4.3    Medium exchange for cell sample....................................................42
4.3.1    Medium exchange driven by syringe pumps………………………………......42
4.3.2    Micropump characterization...............................................................................43

4.3.3    Medium Exchange for Cell Sample....................................................................44
4.3.4    Measurement the dragging force of cells induced by ODEP…………..……45

4.4    Static separation the live cell and dead cell..........................................................46
4.5    Dynamic separation the live cell and dead cell............................................47
Chapter 5
Conclusion and future work
5.1 Conclusion ...............................................................................................................64
5.2Future Work..............................................................................................................66

References......................................................................................................67

                                

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