臺灣博碩士論文加值系統

在過去的十幾年中，利用微機電裝置對生物分子，如細胞、DNA、蛋白質等進行操控與分析已成為目前生物技術發展上的重要分支之一。由於尺寸上的匹配，許多技術在光學、機械、電學及其他領域上對於生物的應用，都已發展出各種不同的方法。從最基本的操控，如細胞的傳輸和分類，到更進一步的療法，如細胞分解、培養和胞膜電穿孔等，都已被廣泛的開發出來。現今，隨著技術和各類微機電基礎元件發展設計上的日趨成熟，在系統整合這一方面的研究，儼然成為目前的研究趨勢。
在本篇論文中，我們提出一個利用介電泳原理設計的細胞操控暨胞膜電穿孔的微晶片系統。它整合了以介電泳電極設計的細胞的排列、分配、固定等功能，並以單一細胞的基因穿透為設計目標。其中以模擬軟體CFDRC的數值模擬與分析來評估並最佳化設計參數，並在驗證設計概念後，以微機電製程將晶片以兩道光罩的製程加以實現。實驗結果利用HEK 293細胞配合導電度為9.2μS/cm-1的介電泳操控溶液，調整施加電壓在4Vpp至6Vpp 的條件下，成功實現了利用介電泳原理的細胞操控技術，並以染劑YOYO-1驗證胞膜電穿孔之基因轉殖功能。據筆者所知，本論文的研究，成功的整合微系統技術與生醫技術發展相當前瞻性的單一細胞電穿孔晶片。

Manipulation and analysis of biological molecules, such as cells, DNA and proteins via using MEMS devices has become an important branch of biotechnology during the past decades. Owning to the dimension match with the cells of μm in size, numerous techniques in optical, mechanical, electrical and other fields have developed various methods for biological applications. From fundamental manipulations, like cell transportation and separation to further cell treatments, like cell lysis, culture and electroporation, the biotechnology research has been driven forwards where people never thought about the two decades ago. With the mature in biotechnology and MEMS technology, the system integration will become a popular trend nowadays.
In this thesis, a cell-on-chip microsystem via the design of enhanced dielectrophresis for single cell electroporation is proposed. It integrated with the functions of cell alignment by the bow-tied shaped DEP electrodes, cell sorting by the six-array DEP electrodes and cell immobilization by quadruple DEP electrodes for the purpose of gene delivery. Numerical simulations by using the software CFDRC are carried out for the purpose of Lab chip design and evaluate the electric field distribution for the optimization of our device design. Through the simulations and analyses, our design concept is proofed, and the chip is realized via the MEMS micromachining process. The bio-experimental results of dielectrophoretic manipulations and single cell electroporation are successfully demonstrated by using HEK 293 cells in the experimental buffer (8.5% sucrose and 0.3% D-glucose, anhydrous in ddH2O, conductivity of 9.2μS/cm-1). The applied electrical potential varies from 4Vpp to 6Vpp for the DEP cell manipulation. The phenomenon of electroporation under an applied electrical potential is successfully demonstrated by YOYO-1 fluorescent dye under laser excitation. Based on our knowledge, this research demonstrated the pioneer of single-cell electroporation via microsystem integration.

1. INTRODUCTION 1
1.1 Background and Motivation 1
1.2 Literature Survey 3
1.2.1 Cell Manipulation 3
(a) Optical manipulation 3
(b) Mechanical manipulation 4
(c) Electrical manipulation 6
1.2.2 Micro-electroporation 7
2. BIOCHIP DEVELOPMENT 10
2.1 Theory Description 10
2.1.1 Theory of Dielectrophoresis 10
2.1.2 Basic Principle of Electroporation 13
2.2 Biochip Design 15
3. SIMULATIONS AND ANALYSES 20
3.1 DEP Alignment 20
3.2 DEP Sorting 26
3.3 Micro-electroporation 29
4. MICROFABRICATION 31
4.1 Process Flow 31
4.2 Fabrication Results 33
5. EXPERIMENTAL SETUP AND RESULTS 35
5.1 Experimental Setup 35
5.1.1 Material Preparations 35
5.1.2 Device Setup 36
5.2 Preliminary Testing 38
5.3 Experimental Results 39
6. CONCLUSION AND FUTURE WORK 48
6.1 Conclusion 48
6.2 Future Work 49
REFERENCE 51

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