運用影像介電泳能量井陣列化單一細胞｜國立清華大學博碩士論文庫

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研究生：	黃遠鵬 Yuan-Peng Huang
論文名稱：	運用影像介電泳能量井陣列化單一細胞 Single Cell Arrayed by Image Dielectrophoretic Energy Wells
指導教授：	葉哲良 J. Andrew Yeh
口試委員:
學位類別：	碩士 Master
系所名稱：	工學院 - 奈米工程與微系統研究所 Institute of NanoEngineering and MicroSystems
論文出版年：	2006
畢業學年度：	94
語文別：	英文
論文頁數：	117
中文關鍵詞：	介電泳、能量井、實驗室晶片、微陣列、單一細胞分析
外文關鍵詞：	Dielectrophoresis, Energy well, Lab-on-a-chip, Microarrays, Single cell analysis
相關次數：	點閱：1 下載：0
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單一細胞微陣列是一種在細胞檢測上極具潛力的工具，它擁有可進行高通量分析的優點並能促進在單一細胞尺度下的相關研究。本研究提出一種新穎的元件與方法來排列單一細胞微陣列。結構式介電泳力和影像式介電泳力被用來控制細胞在晶片上的空間分佈。用介電材料製作出的微凹槽陣列當作實體的屏障來增加細胞的固定性。利用此介電微結構可以將細胞固定的很好來抵抗微流體流動所產生的拉力。

介電泳產生能量井的概念第一次被用來解釋單一細胞微陣列的分佈情況。懸浮在液體中的細胞處於不穩定的能量態，會往能量穩定的位置移動。最後細胞會穩定地停留在微凹槽邊緣底部因為該處是能量最低的位置。

利用這個新方法，單一細胞在微陣列上的填滿率在較低的細胞濃度下，可以增加至45%±1.5%相對於只靠重力的28%±2.0%。而在較高的細胞濃度下，單一細胞的填滿率可達72%±2.3%相對於只靠重力沉澱的35%±1.9%。在熟練的操作下最大的單一細胞微陣列可以接近100*100的矩陣。實驗中發現有一個可能適用於本研究的最佳細胞濃度介於7.3×10^7 to 1.5×10^8 (cells/mL)之間。

麵包酵母菌在本研究中被用來當作樣本。細胞在5 uS/cm的人工緩衝溶液中被操控在電壓10 Vpp和100 kHz ~ 1 MkHz的頻率範圍之下。許多重要的實驗參數被探討，包括光源的照度、非結晶型矽的光電效能、晶片溫度、細胞濃度、細胞死活。這項新穎的技術能促進基礎細胞生物學的研究和生物科技的發展。

Single cell microarrays is a potential tool for cellular assay. It has an advantage of high throughput screening and promotes the study at the single cell level. This research proposed a novel device and methodology to pattern single cell microarrays. Structural DEP force and image-based DEP force were produced to control the spatial distribution of cells on the chip. Microwell arrays made by dielectric material were as physical barriers for enhancement of cell immobilization. Cells can immobilize well against the hydrodynamic drag force by using dielectric microstructures.

The concept of DEP energy wells was used to explain the distribution of single cell microarrays for the first time. Cells which suspended in the medium were located at unstable energy state and moved toward stable energy state. Finally, cells could stay stably in the bottom edge of the microwells owing to the lowest energy positions.

Using this new approach, single cell occupancy was enhanced to 45%±1.5% than only 28%±2.0% by gravitational force in lower cell seeding density. In higher cell seeding density, single cell occupancy was enhanced to 72%±2.3% than 35%±1.9% by naturally gravitational sedimentation. The maximum matrix of single cell microarrays will be close to 100*100 under proficient handling. There was a possible optimized cell seeding density for this research in the range from 7.3×10^7 to 1.5×10^8 (cells/mL).

Saccharomyces cerevisiae (yeast) cell was used for the model in this research. Cells were manipulated under 10 Vpp at 100 kHz ~ 1 MkHz in 5 uS/cm artificial buffer solution. Many important parameters were characterized in the experiment including illumination of light source, photoconductive performance of amorphous silicon, chip temperature, cell seeding density and cell viability. This novel technique would promote the study of basic cell biology and biotechnology.

Chapter I    Introduction    1
1 Background    1
2 Motivation    2
3 Objective    2
4 Organization of this thesis    2
Chapter II    Literature Review    4
1 Passive single cell microarrays forming method    5
2 Active single cell microarrays forming method    9
2.1 Centrifugal force    9
2.2 Microfluidic force    10
2.3 Magnetic force    11
2.4 Negative DEP force    12
2.5 Positive DEP force    13
3 Comparison    14
Chapter III    Theory, Design and Simulation    16
1 Dielectrophoresis Theory    16
1.1 History    16
1.2 Mathematical model    20
2 Chip design    23
3 Simulation    26
3.1 Body force analysis    26
3.2 Simulation results    29
Chapter IV    Fabrication and System Setup    38
1 Fabrication    38
1.1 Mask design    38
1.2 Process flow    40
1.3 Fabrication result    42
2 System setup    47
2.1 Monitoring system    48
2.2 Programmable light source    48
2.3 Chip control system    49
2.3.1 3-D chip holder    49
2.3.2 AC frequency control    51
3 Biological sample preparations    52
Chapter V    Important Parameters Characterizations    54
1 System performance consideration    54
1.1 Transmitted light frequency spectrum    54
1.2 Illumination of light source    58
1.3 Photoconductive performance    61
2 Micro-environment consideration    63
2.1 Cell size    63
2.2 Conductivity and pH of buffer medium    65
2.3 Chip temperature    71
2.4 Cell seeding density    74
2.5 Cell Viability    76
Chapter VI    Experiments and Discussion    78
1 Operating sequences    78
2 Experimental results    80
2.1 Cell Microarrays    80
2.2 Single cell occupancy analysis    89
3 Future Work    97
3.1 Optical system improvement    97
3.2 DEP response testing of general cell types    99
3.3 Stabilization of experimental parameters    100
Chapter VII     Conclusions    101
References    102
Appendix A    107
Appendix B    108
Curriculum Vitae    114

                                

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