微流體是一種具有優勢的技術，適用於在體外模擬細胞內環境，並且也具有高效率在生物系統中觀察和測定之優化、分離、分選和檢測過程。此外，微流道之晶片也易於集成自動化操作，並保持對實驗參數準確有效的控制。然後可以通過並行和定制來提高基礎或臨床研究的效率。本論文提出利用微流道的技術製造生物晶片，重點是高通量，易於操作，可處理性和低成本。我們開發了四個自動系統的微流體生物晶片，在獨特的應用中進行了一些改進。分別是(1)一種微流體方法，其允許使用層流和擴散來生成不同軟硬度的聚丙烯酰胺基質，用於研究癌細胞的機械感測。(2)用於在單個NCI-H716細胞陣列上進行自動化鈣離子成像的微流體平台，用於研究細胞對甜味和苦味刺激的反應，(3)發展一種快速，低成本和試劑消耗，並且與生物淘洗的技術相比，成為噬菌體展示文庫親和力之分離及分選的好方法(4)高動態範圍微流道數字PCR晶片可以使用小體積或低濃度的樣品（15 L體積和10個DNA拷貝數），並且使用前無稀釋及細菌培養的步驟。這些平台可用於研究細菌，病毒或癌細胞的藥物開發，篩選和檢測機制。提出的方法的擴展還可以在檢測中找到應用，或者找出用於疾病早期診斷的生物標誌。

The microfluidics is an advantageous technique and suitable for mimicking cellular microenvironments in vitro and highly efficient optimizing, separating, sorting and detecting processes for the observation and manipulation of biological systems. Furthermore, the microfluidic chip can be amiable for easy integration and automation to control experimental parameters accurately and effectively. Therefore, microfluidic technology can improve the efficiency of both basic and clinical studies through parallel operation and customization. This thesis presents the designs and validations of microfluidic biochips with features of high-throughput, easy operation, disposability and low cost. We have developed four automatic microfluidic systems with improvements for each unique application: 1) a microfluidic approach that allows for the generation of gradient stiffness polyacrylamide substrates using laminar flow and diffusion for studying mechanical sensing of cancer cells; 2) a microfluidic platform for performing automated Ca2+ imaging on an array of single NCI-H716 cells to study cellular response to sweet and bitter taste stimulations; 3) a microfluidic platform for affinity-based sorting of phage-display libraries with low cost and reagent consumption; and 4) a high dynamic range microfluidic digital PCR chip using small volume or low concentration of samples (15 L volume and 10 DNA copies of sample) without dilution or bacterial culture before use. These platforms can be applied to the study of the mechanism of bacteria, viruses or cancer cells with drug development, screening, and detection. Extensions of the proposed method can also find applications in the detection or discovery of biomarkers for early diagnosis of diseases.

Abstract i
中文摘要 ii
誌謝 iii
Table of Contents iv
List of Tables viii
List of Figures ix
Abbreviations and nomenclature xii
Chapter 1 Overview of the microfluidics and microfluidic chip 1
1.1 Fundamentals of microfluidics and microfluidic chip 1
1.1.1 Microfluidic technology 1
1.1.2 Microfluidic chip 1
1.2 Microfluidics chip for bio-systems applications 2
1.2.1 Advantages of microfluidic biochip 2
1.2.2 Applications of cellular systems on microfluidic biochip 3
1.3 Thesis overview 4
References 6
Chapter 2 Microfluidic autogenerator of stiffness-tunable polyacrylamide substrates for studying mechanical regulation of cell culture substrate on Cancer cells 8
2.1 Introduction 8
2.2 Materials and methods 10
2.2.1 Microfabrication 10
2.2.2 Polyacrylamide gel fabrication 10
2.2.3 Simulation of stiffness-tunable device 11
2.2.4 Measurement of polyacrylamide gel elasticity 11
2.2.5 Cell culture 12
2.2.6 Cell spreading and migration analysis 12
2.2.7 Cell staining 13
2.2.8 Microscopic protein quantification 13
2.2.9 Statistics 14
2.3 Results and discussions 14
2.3.1 Design and fabrication of the microfluidic device 14
2.3.2 Generation of substrates with different degrees of stiffness by using tuning flow rates and measurement of substrate elastic moduli using a rheometer 16
2.3.3 Different elastic moduli of cell fates on different PA gel substrates 19
2.3.4 Differential response of cancer cell line invasion to changes in substrate stiffness 22
2.4 Conclusions 24
References 25
Chapter 3 A high-throughput automated microfluidic platform for calcium imaging of taste sensing 28
3.1 Introduction 28
3.2 Materials and methods 29
3.2.1 Device fabrication and operation 29
3.2.2 Culture of NCI-H716 cells 30
3.2.3 Stimulation of tastants and calcium imaging on NCI-H716 cells 30
3.3 Results and discussions 31
3.3.1 Trapping and perfusion of NCI-H716 cells 31
3.3.2 Stimulation of NCI-H716 cells with sweet tastants 33
3.3.3 Order of application of tastant Stimuli 35
3.3.4 Calcium responses to glucose and denatonium benzoate after Gymnema Sylvestre (GS) treatment 37
3.4 Conclusions 38
References 40
Chapter 4 Continuous microfluidic assortment of interactive ligands 47
4.1 Introduction 47
4.2 Materials and methods 49
4.2.1 Microfluidic device design and fabrication. 49
4.2.2 Preparation and modification of agarose hydrogels 49
4.2.3 Microfluidic sorting of phage clones 50
4.2.4 Nanoparticle tracking analysis (NTA) 50
4.2.5 Bacterial amplification 50
4.2.6 DNA sequencing 51
4.2.7 Enzyme-linked immunosorbent assay (ELISA) 51
4.2.8 Biopanning of phage library 52
4.2.9 Materials and reagents 53
4.3 Results and discussions 53
4.3.1 Immobilization of antigen molecules to the agarose gel is stable under the electric field 53
4.3.2 A stronger electric field is needed to electrophorese phages of higher affinity for the antigen 54
4.3.3 Phage numbers may be evaluated using nanoparticle tracking analysis (NTA) 55
4.3.4 Phage clones are sorted using CMAIL 57
4.3.5 More than 80% antigen-interactive phages are recovered when the occurrence is above 1/10,000 62
4.3.6 Phages were enriched at different outlets based on their affinity 65
4.3.7 Five repeated CMAIL operation cycles are possible using the same device 68
4.3.8 CMAIL sorting of a phage library is of favorable recovery rate and purity 69
4.4 Discussions and conclusions 71
References 75
Chapter 5 Digital PCR on a high dynamic range microfluidic chip for complex microbial detection 80
5.1 Introduction 80
5.2 Materials and methods 81
5.2.1 Microfluidic design and fabrication of high dynamic range microfluidic chip 81
5.2.2 Installing and loading of the high dynamic range microfluidic chip 82
5.2.3 Measurement of temperature within the high dynamic range microfluidic chip 82
5.2.4 Pure and complex microbial samples collection and genomic DNA extraction 82
5.2.5 PCR amplification 83
5.2.6 Quantification of fluorescence image in DNA level on high dynamic range microfluidic
chip 83
5.3 Results and discussions 84
5.3.1 Principle and operation of the high dynamic range microfluidic chip 84
5.3.2 Examination of the high dynamic range microfluidic chip 85
5.3.3 Quantitative detection of DNA copies on the high dynamic range microfluidic chip 89
5.3.4 Real samples detection of DNA copies on the high dynamic range microfluidic digital
PCR chip 91
5.4 Conclusions 93
Reference 94
Chapter 6 Conclusions and Future perspectives 96
6.1 Summarize conclusions 96
6.2 Future perspectives 97
References 99
Publication lists 102

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