臺灣博碩士論文加值系統

本研究為提出一創新之整合微流體晶片及表面電漿共振(Surface Plasmon Resonance, SPR)感測技術於免疫分析之應用。SPR影像感測器為一具共光程移相干涉技術之相位量測系統，並針對表面電漿共振所造成之高靈敏相位變化，可以高通量即時動態量測生物分子之間的交互作用之平行檢測。
在微流體晶片製作上，係利用微機電系統(Micro-electro-mechanical-systems, MEMS)製程技術將微流體元件及微型溫度控制元件製作於玻璃與聚二甲基矽氧烷(Polydimethylsiloxane, PDMS)等基材之上。本研究所製作之流速感測器能有效校正微氣動幫浦流率之誤差，以精確地控制檢體進樣之流率；微型溫度控制器能精準地控制微陣列檢測區內之溫度變化，且誤差範圍可控制在0.1℃以內，可以藉此降低SPR系統之雜訊並提高分析訊號之噪訊比。本實驗首先利用自組單層分子(Self-assembled monolayer, SAM)技術有效地改變金膜表面的化學性質而讓兔子的免疫球蛋白抗體(anti-rabbit IgG)穩定地被固定在金表面上。再利用微流體晶片中的蠕動式微氣動幫浦與微氣動閥門將二次抗檢體透過微流道傳送到微陣列反應區域，並且同時運用SPR影像感測器進行即時性分析。
最後，本研究成功地將微流體晶片結合SPR影像感測器的高靈敏度、高解析度、動態分析及不須事先標定生物分子之特性，以陣列方式分析蛋白質資訊。所得到的結果顯示，利用此一方法能有效地檢測不同濃度的免疫球蛋白抗體及非特異性抗體(non-specific antibody)的分辨。此研究可針對醫療診斷及疾病檢測分析上提供一種便利且穩定之研究工具。

This study reports a novel microfluidic chip integrated with arrayed immunoassay for SPR (Surface plasmon resonance) phase imaging of specific bio-samples. The SPR imaging system uses a surface-sensitive optical technique to detect two-dimensional spatial phase variation caused by antibodies absorbed on a sensing surface composed of highly-specific proteins films. The developed system has a high resolution and a high-throughput screening capability and has been successfully applied to the analysis of multiple bio-molecules without the need for additional labeling in the long-term measurement.
The microfluidic chip was fabricated by using MEMS (micro-electro-mechanical-systems) technology on glass and PDMS (Polydimethylsiloxane) substrates to facilitate well-controlled and reproducible analyte delivery. A micro flow sensor was fabricated to measure the pumping rate generated by the micropumps. In addition, since SPR detection is very sensitive to temperature variation, a micromachine-based temperature control module comprised of micro-heaters and a temperature sensor was used to maintain a uniform temperature with a variation less than 0.1°C during measurement. Besides, the SAM (self-assembled monolayers) technique was used to pattern the surface chemistry of the gold to adsorb rabbit IgG and its antibody to the modified substrates. The microfluidic chip is capable of transporting a specific amount of IgG solution inside multiple microchannels using micropumps/valves to the arrayed detection areas that are locally deposited so that highly-sensitive, highly-specific bio-sensing can be achieved. The developed microfluidic chips employed in SPR imaging experiments for immune detection could successfully detect the interaction of rabbit IgG and its antibodies. Various rabbit IgG concentrations and antibody interactions have been measured using the developed method. The integrated system was also tested for nonspecific interactions. Experimental data showed that no significant binding was found.
The microfluidic chips were developed to have the potential to be widely used for bio-sensing applications. The development of microfluidic devices integrated with the SPR detecting system has several advantages, including being labeling-free, having a high sensitivity, and being capable of quantitatively analyzing nano-scale bio-molecules in real-time format. The developed system could be promising for various applications including medical diagnostics and biomedical research.

Abstract .......................................i
摘要..........................................iii
致謝........................................... v
Table of Contents ............................vii
List of Tables ................................ x
List of Figures ...............................xi
Nomenclature .................................xxi

Chapter 1 Introduction..........................1
1-1 Microelectromechanical systems .............1
1-1-1 Bio-MEMS Technology ......................1
1-1-2 Microfluidic system ......................4
1-2 Surface plasmon resonance technique.........9
1-2-1 Review of SPR.............................9
1-2-2 SPR imaging system ..................... 12
1-3 Immunoassay technique..................... 16
1-3-1 SPR based immunosensor ................. 18
1-4 Motivation and Objectives................. 23

Chapter 2: Theory and Design.................. 26
2-1 The design of microfluidic chip .......... 26
viii
2-1-1 Pneumatic Micropumps and Microvalves ... 29
2-1-2 The operation of flow sensor............ 33
2-1-3 Micro Temperature Sensors and Micro Heaters....................................... 35
2-1-4 The design of the micromachine-based temperature control system.................... 39
2-2 Surface Plasmon Resonance ................ 41
2-2-1 SPR technique theory.................... 41
2-2-2 PSI of SPR imaging system .............. 44
2-3 Immunoassay method........................ 47
2-3-1 Antibody-Antigen Interactions .......... 48
2-3-2 Surface immobilization ................. 51

Chapter 3: Materials and Fabrication.......... 53
3-1 Photomask fabricate ...................... 54
3-2 Structure cleaning........................ 55
3-2-1 Glass and silicon cleaning.............. 55
3-2-2 Slide cleaning.......................... 57
3-3 Micromachine-based temperature control system........................................ 57
3-3-1 E-beam deposition ...................... 57
3-3-2 Lift-off technique ..................... 59
3-3-3 Lithography process..................... 61
3-4 Soft lithography of microfluidic system... 63
3-4-1 SU-8 molding............................ 63
3-4-2 PDMS casting ........................... 67
3-5 Structure bonding ........................ 70
3-6 SPR used slide sputtered gold thin film ...73
3-7 Reagents preparation ..................... 74

Chapter 4: Results and Discussion............. 79
4-1 The assembled microfluidic chip .......... 79
4-1-1 Micropump and microvalve ............... 81
4-1-2 Micro temperature control module ....... 87
4-2 AFM and bioassay scanner ................. 91
4-2-1 Protein analysis by using AFM .......... 92
4-2-2 Microarray fluorescence scanner......... 94
4-3 Result of SPR sensing detection .......... 96
4-3-1 Detection of SPR imaging system......... 99

Chapter 5 Conclusions and Future Work........ 110
References .................................. 114
Curriculum vitae............................. 122
Publications ................................ 123

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