臺灣博碩士論文加值系統

近年來，生物臨床檢體之前處理與濃縮萃取之程序於醫學檢驗中扮演著十分重要的角色。於傳統醫學檢測中，臨床檢體之前處理往往浪費許多的時間，且其繁複的人工操作與步驟更容易增加檢體之浪費與檢測之準確性。因此，隨著微機電製程技術之成熟，已在許多不同的領域中有顯著的發展，尤其在微小化快速生醫檢測分析晶片更扮演了十分重要的推手角色。藉由微機電製程技術所生產之微流體生醫檢測晶片，其具有高檢測效能、可拋棄式、可攜帶性、低樣品及檢體消耗量、低耗能、體積小以及成本低等優點。尤其以整合全程微流體系統包含生物樣品前處理、傳輸、偵測及分析於同一晶片上之設計，最具發展潛力以及市場價值。
本研究提出一個利用微機電製程，以免疫學之原理利用微型磁珠(Magnetic bead)結合專一性極高之抗體，以萃取特定之DNA細菌(DNA-based bacteria)或RNA病毒(RNA-based viruses)，並將以濃縮、純化、分離後，全自動進行聚合酶連鎖反應之生物晶片系統。此微型化晶片系統主要由三大模組組成，分別為用於流體傳輸以及混合之微流體模組；另一為用於產生微型磁場以吸附微型磁珠之磁分離器；最後一個為用於產生微型溫度場以進行聚合酶連鎖反應之微型溫度控制模組。首先，透過使用鍵結高專一性及靈敏性之抗體的磁珠(Antibody-conjugated magnetic bead)，利用微流體模組傳輸運送檢體進入混合反應區，流體所造成之擾流將使目標之病原菌或病毒黏附於磁珠表面。其後，再使用由二維或三維的微型線圈所組成之磁分離器，將磁珠在流體之樣品中分離進行目標檢體之純化及濃縮，藉由此步驟，檢體將被淨化且集中，再透過晶片上之微型溫度控制模組控制反應槽內溫度高低之循環，進行聚合酶連鎖反應(PCR)將目標病毒或病原菌進行檢測。此微流體整合型系統藉由主動式微幫浦與微閥門的幫助下，能全自動地進行檢測操作流程，並大幅的縮短檢測之時間及提高其效率。
由實驗資料顯示，本晶片系統之主動式微流體模組除了可以以高達180 µl/min的傳輸速率來輸送檢體外，亦可當作微型混合器使用，並以極高之混合效率進行臨床檢體與微型磁珠之混合。而由銅金屬(Copper, Cu)所電鍍製成之磁分離器可於微流體晶片表面產生微型磁場，以吸附鍵結目標病毒之微型磁珠於晶片表面，進行生物檢體之純化、萃取以及濃縮。此外，本微型線圈更可當作用於後段進行聚合酶連鎖反應之微型加熱器。此晶片系統利用鉑金屬(Platinum, Pt)所製成之微型溫度感測器，搭配塊狀(block type)及螺旋狀(spiral type)之微型加熱器進行溫度升降之循環。此微型溫度控制模組具有極快速之升溫速率(20℃/sec)、冷卻速率(10℃/sec)、均勻的溫度分佈(±0.3℃)以及微小之溫度震盪(±0.2℃)。且由於本微型晶片系統之低阻抗以及低功率，其消耗功率只需1.2W，因此僅需要一顆9V的鎳氫電池，本攜帶式全自動微流體晶片即可運行操作至少15小時以上。再者，本晶片系統已於臨床檢體中成功的純化及檢測出國內流行之登隔熱血清第二型病毒(dengue virus serotype 2)、腸病毒71型(enterovirus 71)、上呼吸道感染疾病之A群鏈球菌(Group A streptococcus)以及肺炎鏈球菌(S. pneumoniae)，並利用抗體之高專一性以及選擇性，分別檢測出不同型態之病毒及細菌。此外，本晶片系統更成功的利用磁珠濃縮極低濃度之臨床檢體，並成功純化、萃取以及偵測。本晶片系統之檢測靈敏濃度，分別為病毒檢體100 PFU/ml以及細菌檢體100 CFU/ml，且利用本晶片之高升降溫速率之優點，相對於傳統大型儀器檢測，可以節省近50%的檢測時間。相信於不久的將來，本晶片系統所提供之快速自動化檢測平台，將於基因分析、分子生物以及快速感染性疾病之偵測將有極大之助益。

Recently, purification and enrichment of bio-samples is crucial for the analysis of biosamples with an ultra-low concentration that the performance of the detection system can be efficiently increased. Apart from that, clinical samples usually contain biological medium that would normally inhibit the subsequent ribonucleic acid (RNA)/deoxyribonucleic acid (DNA) amplification process. Moreover, there still need several tedious purification and washing steps and labor-intensive processes to complete the sample preparation. In addition, a number of large-scale equipments are usually needed in the complex pretreatment procedures and the bio-samples may also be wasted and cause some contaminations during manual operations.
To improve this, the concept of MEMS (micro-electro-mechanical-system)-based miniaturization is applied in the magnet beard-based sample purification and enrichment process. It is generally believed that the miniaturized bio-devices typically bring the advantages of shorter diagnosis times, less sample and reagent consumption, improved resolution, higher sensitivity, and lower cost. By the incorporation of the microfluidic system, the miniature bio-devices provide the powerful techniques to transport or mix the fluids in the system and make the biological diagnosis a quick and automatic process. Nevertheless, the integrated systems still need other detection chambers to perform biological diagnosis. And the on-chip microfluidic modules still need several sets of unidirectional microfluidic structures to complete the whole process. As a result, several electromagnetic valves (EMVs) are usually required to deliver the fluid in the microchannels and the manual operations are normally required since the lack of function of sample pretreatment in the micro system.
Consequently, there is a need for a sample pretreatment process by utilizing a microfluidic system to automate the nucleic acid amplification process with less human intervention and also to improve sensitivity and selectivity. The study therefore proposes three new miniature reverse transcription polymerase chain reaction (RT-PCR) systems that integrate with bio-sample pretreatment devices using superparamagnetic beads into a single chip platform. In the first system, three modules were integrated including a microfluidic nodule consists of three sets of novel pneumatic micropumps, a bead collection module with Au wires and a micro temperature control module for the polymerase chain reaction (PCR) process. For the second system proposed, the microfluidic chip integrated two functional devices for bio-samples purification and enrichment including pumping, mixing and separation by utilizing a rotary microfluidic module and a bead-collection module consists of 2-dimension/3-dimension (2-D/3-D) microcoils. The original rotary microfluidic module can provide a rapid flow pumping rate and a high mixing efficiency and can pretreat the bio-samples in a short period of time. The third microsystem uses the novel microfluidic system including a two-way serpentine-shape (s-shape) micropump requiring only one EMV to transport and to mix the bio-samples in the microchannels. In addition, new bio-separators are developed either to perform the separation of magnetic beads or to control the temperature field for the subsequent RT-PCR process. The rapid heating/cooling rate of the micro-heating chambers can significantly shorten the pre-treatment and diagnosis processes. As a whole, the developed system may provide a powerful platform integrating the functions of sample pretreatment and fast disease diagnosis.

Table of Contents
Abstract I
中文摘要 III
致謝 V
Table of Contents VII
List of Tables XII
List of Figures XIII
Nomenclature XXIII
Abbreviation XXVI

Chapter 1: Introduction ….1
1.1 MEMS and microfluidic technology 1
1.2 Background and literature survey 1
1.2.1 Pretreatment of the bio-samples……………………………………………..1
1.2.2 Magnetic beads in biological applications…………………………………..3
1.2.3 Microfluidic system in biological application………………………………4
1.2.4 Micro mixer in bio-reaction……………………………..…………………..5
1.2.5 MEMS-based magnetic bio-separator…………………..…………………...5
1.2.6 Nucleic acid amplification……………………………..………………..…..6
1.3 Motivation and Objectives 7

Chapter 2: Theory and Design …14
2.1 Immunology in biological application 14
2.1.1 Antibody and antigen structure 14
2.1.2 Purification utilizing antibody-conjugated magnetic beads 14
2.2 Overview of the designed miniature micro systems 16
2.2.1 Microfluidic control system 17
2.2.1.1 The principle of pneumatic micropumps and microvalves………...17
2.2.1.2 Membrane activation theory……………………………………….18
2.2.1.3 Design of the on-chip microfluidic module………………………..19
2.2.2 Magnetic bio-separator system 20
2.2.2.1 Theory of magnetic bio-separator………………………..………...21
2.2.2.2 Design of the bio-separator……………………………………..….23
2.3 Nucleic acid amplification technique 23
2.3.1 DNA, RNA and cell lysis 23
2.3.2 Reverse transcription and polymerase chain reaction 25
2.3.3 Design of the micro temperature control module 26
2.3.4 Analysis of slab-gel electrophoresis 28
Chapter 3: Materials and Methods 38
3.1 Materials 38
3.1.1 RNA-based virus preparation 38
3.1.2 DNA-based bacteria preparation 39
3.1.3 Antibody-conjugated magnetic beads preparation 40
3.1.4 Primer design and RT-PCR reagents 40
3.2 Working principle and experimental method 42
3.2.1 RNA/DNA extraction 42
3.2.1.1 Standard RNA/DNA extraction procedures………………………..42
3.2.1.2 Procedures for RNA/DNA extraction from a crude bio-sample…...42
3.2.2 Working principle of the integrated micro systems…………………..…..43
3.2.3 Experimental setup………………………………………….………..…..44
3.2.4 Sample pretreatment utilizing the microfluidic system…….………..…...45

Chapter 4: Fabrication 55
4.1 Overview of the micro fabrication techniques 55
4.2 Formation of the microfluidic control module 56
4.2.1 Substrate cleaning 56
4.2.2 SU-8 mold fabrication 57
4.2.3 PDMS casting 58
4.3 Manufacture of the bio-separator module 60
4.3.1 Glass substrate cleaning 60
4.3.2 2-D bio-separator 61
4.3.3 3-D bio-separator 62
4.4 Fabrication of the micro temperature control module 64
4.4.1 Patterning 64
4.4.2 Metal deposition and lift-off process 65

Chapter 5: Results and Discussion ……76
5.1 Characterization of the microfluidic control module 76
5.1.1 Pumping process using the pneumatic micropump 76
5.1.2 Mixing process using the pneumatic micropump 78
5.2 Characterization of the bio-separator module……………………...79
5.2.1 Magnetic beads collection 79
5.2.2 Magnetic field and temperature 81
5.2.3 Trapping effect 83
5.3 Characterization of the micro temperature control module 83
5.3.1 Thermal cycling of the micro temperature module 83
5.3.2 Temperature uniformity 85
5.4 Biological application 86
5.4.1 Incubation process utilizing the on-chip microfluidic module 86
5.4.2 Purification and detection of RNA-based virus/DNA-based bacteria 88
5.4.3 Specific detection of four serotypes of dengue virus 88
5.4.4 Selectivity of antibody-conjugated magnetic beads 89
5.4.5 Sensitivity of the miniature RT-PCR system 90
5.4.6 Enrichment of the microfluidic system 91
5.4.7 Comparison of magnetic beads with traditional method 92
5.4.8 Comparison between the proposed three types of microfluidic systems 93

Chapter 6: Conclusions and Future Work 108

References 110

Biography 120

Publication lists 121

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