|
Table of Contents
Abstract I
摘要 IV
致謝 VI
Table of Contents VIII
List of Tables XII
List of Figures XIII
Abbreviation XXIV
Nomenclature XXVII
Chapter 1 Introduction 1
1.1 MEMS and Microfluidic Technology 1
1.2 Systematic Evolution of Ligands by Exponential Enrichment (SELEX) 1
1.3 Aptamers in Biological Applications 2
1.4 Background and Literature Survey 3
1.4.1 Isolation of aptamer 3
1.4.2 Microfluidic Components – Micropumps and Micromixers 5
1.4.3 Aptamer Amplification by Using Polymerase Chain Reaction (PCR) 6
1.4.4 C-Reactive Protein (CRP) and Its Clinical Application 6
1.4.5 Alpha-Fetoprotein (AFP) and Its Clinical Application 7
1.5 Motivation and Objectives 8
Chapter 2 Methods, Theory and Design 17
2.1 Selection of High Affinity Aptamers using SELEX 17
2.2 Overview of the Aptamer Selection Process 18
2.3 Experimental Processes of the Magnetic Bead-based Microfluidic Systems 19
2.3.1 The SELEX protocol 19
2.3.2 The Competitive Assay Protocol 20
2.3.3 Procedure for Detection of the Target Protein 21
2.4 Design of the Magnetic Bead-Based Microfluidic Systems 23
2.4.1 Design of the SELEX Chips 24
2.4.2 Design of the Competitive Assay Chip 25
2.4.3 Design of the Protein Detection Chip 26
2.5 Working Principle of Microfluidic Systems 26
2.5.1 Microfluidic Control Module 26
2.5.1.1 Membrane Activation Theory 27
2.5.1.2 Working principle of Circular Micropump/Micromixer 27
2.5.1.3 Working principle of Suction-Type Micromixer 28
2.5.2 Nucleic Acid Amplification Module 29
2.5.2.1 Design of the Micro Temperature Control Module 30
2.6 Analysis of Polyacrylamide Gel Electrophoresis (PAGE) 31
Chapter 3 Materials and Fabrication 53
3.1 Preparation of the Target Protein-Conjugated Magnetic Beads 53
3.1.1 Preparation of the CRP-Conjugated Magnetic Beads 53
3.1.2 Preparation of the AFP-Conjugated Magnetic Beads 53
3.2 Preparation of the BSA-Conjugated Magnetic Beads 54
3.3 Preparation of the DNA aptamer- Conjugated Magnetic Beads 55
3.3.1 Preparation of the CRP-Specific aptamer-Conjugated Magnetic Beads 55
3.3.2 Preparation of the AFP-Specific aptamer-Conjugated Magnetic Beads 55
3.4 Polymerase Chain Reaction Process 56
3.5 Procedure of TA-cloning 57
3.6 Fabrication of the Microfluidic Systems 58
3.6.1 Overview of the Microfabrication Techniques 58
3.6.2 Fabrication of the Micro Temperature Control Module 59
3.6.3 Formation of the Microfluidic Control Module 61
3.6.3.1 PDMS Casting 62
3.7 Experimental Setup 63
3.7.1 Experimental Setup for SELEX 63
3.7.2 Experimental Setup for Competitive Assay 64
3.7.3 Experimental Setup for Protein Detection 64
3.7.4 Custom-made optical and control systems 65
Chapter 4 Results and Discussion 75
4.1 Characterization of the Microfluidic Control Module 75
4.1.1 Characterization of the Pneumatic Micropump 75
4.1.2 Characterization of the Pneumatic Micromixer 76
4.1.3 Characterization of the Suction-Type Micromixer 78
4.2 Characterization of the Micro temperature Control Module 79
4.2.1 Temperature Uniformity of the Block-Type Microheater 79
4.2.2 Temperature Uniformity of Array-Type Microheater 80
4.3 Screening of CRP-Specific Aptamers 80
4.3.1 TA Cloning of the CRP-Specific DNA 81
4.3.2 Competitive Assays of the CRP-Specific Aptamers Utilizing a Tradition Method 81
4.3.3 The Dissociation Constant of the CRP-Specific Aptamer 83
4.3.3.1 Detection Chip Modifications 83
4.3.3.2 Measurement of CRP-specific aptamer dissociation constant 83
4.4 Screening of AFP-Specific Aptamers 84
4.4.1 TA cloning of the AFP-Specific DNA 84
4.4.2 Competitive Assays of the AFP-Specific Aptamers 84
4.4.3 The Dissociation Constant of the AFP-Specific Aptamer 85
4.4.3.1 Detection Chip Modifications 85
4.4.3.2 Measurement of AF-specific aptamer dissociation constant 85
4.5 Application of the CRP-Specific Aptamer in a Clinical Samples 86
4.6 Application of the AFP-Specific Aptamer in a Clinical Samples 87
4.7 Comparison of the Prototype Microfluidic Systems with the Traditional Method 88
Chapter 5 Conclusions and Future Works 103
5.1 Review of the Dissertation 103
5.2 Future Works 104
References 106
Curriculum Vitae 120
Publication List 121
|