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研究生：	張振旻 Chang, Chen-Min
論文名稱：	微流體技術應用於粒線體DNA缺陷之檢測 Development of microfluidic systems for detection of mitochondrial DNA defects
指導教授：	李國賓 Lee, Gwo-Bin
共同指導教授:	謝達斌 Shieh, Dar-Bin
學位類別：	博士 Doctor
系所名稱：	工學院 - 工程科學系 Department of Engineering Science
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	86
中文關鍵詞：	粒線體基因、微流體、斷損、突變
外文關鍵詞：	Mitochondrial DNA, Microfluidics, Deletion, Mutation
相關次數：	點閱：109 下載：3
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粒線體是動物細胞的能量供給與新陳代謝的中心，它提供了許多能量來保持生理上的功能，並且在細胞的凋亡中，扮演著相當重要的角色。當粒線體的功能下降時，生物體內會產生過量的活性氧及造成生物分子上的氧化損傷，進而加速了粒線體基因的缺陷產生。在許多的報告中已證實粒線體的功能失調，與粒線體基因的改變有關。除了粒線體本身相關的疾病外，老化、第二型糖尿病與癌症皆是影響人類相當重要的疾病。粒線體基因因為缺乏有效率的修復系統，因此較核基因容易受到氧的攻擊而造成損傷，因此粒線體基因的損傷，是眾多粒線體疾病的原因之一。而粒線體缺陷的定性與定量在臨床疾病診斷與粒線體功能失調程度的判斷是相當重要的。這是因為相同粒線體基因的缺陷，當在不同的器官或組織中時，在臨床上的徵狀與治療是不同的，因此量化粒線體基因缺陷的程度，在疾病的診斷與治療上是相當重要的。
傳統粒線體基因改變的檢測，主要是利用微克隆、直接定序、即時定量聚合酶連鎖反應與微陣列分析等方法，但這些方法是費時且繁瑣複雜的過程。近來，微機電系統技術及微流體裝置上的發展，直接影響到了生物醫學設備的微型化。微機電系統技術能夠將多功能的微型元件整合成一晶片系統，此晶片系統具有高靈敏度、輕巧簡潔與能夠快速分析等優勢。
在這項研究過程中，自動化的粒線體基因萃取模組，採用立體結構的方式，藉由整合微幫浦、微混合器與微加熱器來達成。實驗結果呈現出萃取模組比商用套件有更佳的萃取效率，萃取模組為30分鐘,而商用套件則是需要300分鐘。粒線體基因斷損檢測系統整合基因萃取模組、微型聚合酶連鎖反應元件與微毛細管電泳元件來達成。研究數據顯示粒線體基因的斷損能夠利用此晶片系統自統偵測出。實驗結果顯示出晶片系統的聚合酶連鎖反應較傳統設備快速，而粒線體基因缺陷的程度，能夠藉由缺陷基因的光學訊號強度與總粒線體基因的光學訊號強度的比值獲得。粒線體基因突變檢測模組則是利用前述的基因萃取模組與微型聚合酶連鎖反應元件外，再將具備精密光學檢測的定量系統整合入系統中。實驗結果顯示出所開發的晶片系統能夠應用於粒線體基因的突變分析，粒線體基因突變的程度，則能夠藉著基因與酵素的反應程度而獲得。與傳統的方式比較，所開發出的晶片系統證實能夠針對粒線體基因的缺陷來進行定性與定量分析。因此所開發出的晶片系統，預期能夠成為應用於臨床診斷粒線體疾病強而有力的工具之一。

Mitochondria are the energy production and metabolism centres of human and animal cells, which supply most of the energy for maintaining physiological functions and play an important role in the process of cell death. Meanwhile, cellular overproduction of reactive oxygen species and oxidative damage on biological molecules occur when mitochondrial functions decline, directly accelerating the alterations of mitochondrial DNA. Alterations of mitochondrial DNA have been reported to be strongly associated with mitochondrial dysfunction, mitochondria-related diseases, aging, and many important human diseases such as diabetes and cancers. Because it lacks an effective repair system, mitochondrial DNA suffers much higher oxidative damage and usually harbours more mutations than nuclear DNA. Molecular defects in mitochondrial DNA significantly contribute to a wide variety of mitochondrial diseases. Both qualitative and quantitative mitochondrial DNA defects affect clinical presentation and the severity of the diseases due to variations in the mitochondrial dysfunction profiles. As a result, the same mitochondrial DNA defect may present different severity in different organs or tissues and cumulatively contribute the overall clinical symptoms and treatment options. Therefore, quantification of different mitochondrial DNA defects is important for the diagnosis and treatment plan of mitochondria diseases.
Traditional protocols for assess mitochondrial DNA involve micro-cloning, direct sequencing, real-time polymerase chain reaction processing and microarray detection; all of which are time-consuming and labor-intensive processes. Recently, rapid development in microfluidic devices fabricated by micro electro mechanical systems technology has made substantial impact on miniaturization of biomedical devices. The micro electro mechanical systems technology is able to integrate all functional micro components in one single chip with advantages such as compactness, high sensitivity and rapid analysis.
In this study, an extraction module of mitochondrial DNA was first designed that integrated micropumps, a micromixer and a micro temperature sensor in three-dimensional format to automate the entire process. The experimental results showed that the proposed microchip has higher extraction efficiency for mtDNA. The extraction times for the microchip and a commercial kit of mtDNA extraction were 50 minutes and 300 minutes, respectively.
A mitochondrial DNA deletion detection system integrated with a mtDNA extraction module, a micro polymerase chain reaction module and a micro capillary electrophoresis module has further been developed. The experimental results showed the PCR module could provide a comparable amplification yield when compared to a conventional instrument. The deletion rate of the mtDNA in the samples can be further quantified by measuring the percentage between the amplicon representing for the deletion and that for the total mtDNA.
Furthermore, a mitochondrial DNA mutation detection system including an extraction module, a micro polymerase chain reaction module, and a mutation detection module capable of precise quantitative measurements was developed in this study. Experimental results showed that a new quantitative detection system can be utilized for the analysis of a point mutation in mtDNA. The DNA detection module can detect the mtDNA mutation using restriction enzyme digestion. Compared to traditional methods, the new chip system demonstrates excellent mutation detection limit for small starting specimen amount and capable of both qualitative and quantitative analysis. Thus the integrated microfluidic systems harbor a great potential for fully automatic high-throughput mitochondrial DNA detection to augment future clinical diagnosis and management of mitochondria diseases.

Table of Contents
Abstract...............................................I
中文摘要...............................................IV
致謝...................................................VI
Table of Contents...................................VIII
List of Tables.......................................XII
List of Figures.....................................XIII
Abbreviations & Nomenclature........................XVII

Chapter 1: Introduction................................1
1.1 Functions of mitochondrion.........................1
1.2 Mitochondrial DNA damage and diseases..............1
1.3 Mitochondrial DNA 4,977 bp deletion................2
1.4 Mitochondrial DNA damage diagnosis.................3
1.5 Motivation and objective of mitochondrial DNA extraction.............................................4
1.6 Motivation and objective of mitochondrial DNA deletion detection..............................................5
1.7 Motivation and objective of mitochondrial DNA mutation detection..............................................6
Chapter 2 Materials and Methods........................10
2.1 Cell preparation...................................10
2.1.1 Culture of cells containing deleted mitochondrial DNA....................................................10
2.1.2 Culture of cells contain mutated mitochondrial DNA
.......................................................10
2.2 Mitochondrial DNA isolation........................11
2.2.1 Conventional mitochondrial DNA isolation protocol
.......................................................11
2.2.2 Mitochondrial DNA isolation using the extraction chip...................................................12
2.3 Chip manufacturing process.........................12
2.4 Nucleic acid amplification.........................13
2.4.1 Theory...........................................13
2.4.2 Conventional PCR amplification of mitochondrial DNA....................................................14
2.4.3 Preparation of the PCR reagent on the chip.......15
2.4.4 Conventional real-time PCR amplification of mitochondrial DNA......................................15
2.4.5 Preparation of real-time PCR reagents on the chip...................................................16
2.5 DNA fragment detection.............................16
2.5.1 Slab-gel electrophoresis.........................16
2.5.2 Preparation of slab-gel electrophoresis..........17
2.5.3 Theory of capillary electrophoresis..............17
2.5.4 Preparation of the reagents in the micro capillary electrophoresis module.................................18
2.6 Digestion for Mutation Identification..............19
2.6.1 Digestion of mtDNA harboring A3243G point-mutation by Apa1 enzyme............................................19
2.6.2 Preparation of Apa1 restriction enzyme...........20
Chapter 3 Development of Microfluidic Chip for Mitochondrial DNA Extraction...........................25
3.1 Chip design........................................25
3.2 Experimental process...............................27
3.3 Results and Discussion.............................28
3.3.1 Characterization of the micropump................28
3.3.2 Characterization of the micromixer...............28
3.3.3 Quantification of the extracted mitochondrial DNA....................................................29
Chapter 4 Development of Microfluidic System for DNA Deletion Detection.....................................40
4.1 Primer design for mtDNA to examine 4,977 bp deletion...............................................40
4.2 Chip design........................................40
4.3 Experimental setup.................................41
4.4 Results and Discussion.............................43
4.4.1 Characterization of the microfluidic system......43
4.4.1.1 Characterization of the micropump..............43
4.4.1.2 Characterization of the micromixer.............43
4.4.2 Mitochondrial DNA extraction.....................43
4.4.3 Mitochondrial DNA fragment amplification.........43
4.4.4 Mitochondrial DNA separation and detection.......44
Chapter 5 Development of Microfluidic System for Mutation Detection..............................................54
5.1 Chip design........................................54
5.2 Experimental process and setup.....................55
5.3 Results and discussion.............................57
5.3.1 Characterization of the peristaltic micropump/ suction-type micropump.................................57
5.3.2 Characterization of the micromixer...............58
5.3.3 Mitochondrial DNA extraction.....................58
5.3.4 Identification of mitochondrial DNA..............58
5.3.5 Point mutation detection of mitochondrial DNA....59
5.3.5.1 DNA fragment digestion by restriction enzyme of Apa1 pre-treatment.....................................59
5.3.5.2 Point mutation detection by real-time PCR and in a microfluidic chip system...............................60
Chapter 6 Conclusions..................................69
6.1 Summary of the dissertation results................69
6.2 Future work........................................70

References.............................................71
Biography..............................................83
Publication list.......................................84


List of Tables
Table 2-1..............................................22
Design of primers for mtDNA
Table 3-1..............................................32
The dimensions of the components integrated onto the micro chip
Table 4-1..............................................49
The dimensions of the components integrated onto the mtDNA extraction and analysis system.
Table 5-1..............................................63
The dimensions of the components integrated onto the microfluidic platform for point-mutation detection.


List of Figures
Figure 1-1..............................................8
Illustration of how ROS production results in an increased rate of mtDNA alterations, thus causing a vicious cycle of exponentially increasing oxidative damage and dysfunction.
Figure 1-2..............................................9
Illustration of how mtDNA deletion occurs during replication.
Figure 2-1..............................................23
Manufacturing process of the chip. (a) Si wafer cleaning. (b-1) PR coated on the wafer. (b-2) PDMS coated on the glass. (c) The standard lithography process. (d) Obtained a SU-8 master mold. (e) Peeling of PDMS structure. (f) Bonding of PDMS layers.
Figure 2-2..............................................24
The recognition site for the restriction enzyme for Apa1. (a) The normal mtDNA will not be cut by the enzyme. (b) The A3243G mutated mtDNA will be cut in half by the enzyme.
Figure 3-1..............................................33
(a) Schematic illustration of the microfluidic chip for the extraction of mtDNA. (b) An exploded view of the chip.
Figure 3-2..............................................34
(a) Illustration for the operating principle of the micromixer (top view). (b) Sample and magnetic beads are transported from individual wells. (c) Compressed air is applied to deflect the PDMS membrane so that a swirling flow is induced. (d) Compressed air is released which restores the PDMS membranes to their original position.
Figure 3-3..............................................35
Illustration of the procedure for using magnetic beads to extract mtDNA from cells.
Figure 3-4..............................................36
The pumping rate of the micropump operated at different EMV frequencies at a pressure of 10 and 20 psi, respectively.
Figure 3-5..............................................37
(a) Concentration profiles before and after mixing at a driving frequency of 4Hz. (b) The relationship between the cycle threshold, Ct, and the mixing time for mtDNA extraction.
Figure 3-6..............................................38
Slab-gel electropherograms of the amplified mtDNA (a) extracted by a commercial kit and by the microchip, (b) extracted by the microchip for non-deleted (lane ND) and Δ4977 bps mtDNA (lane D), (c) tested again for non-specific production by the chip protocol, which may come from the higher produced concentration, then from the kit.
Figure 3-7..............................................39
Real-time PCR analysis; (a) The relationship between the fluorescent intensity and the cycle number for mtDNA extracted by the commercial kit and by the microchip at various serial dilutions. (b) The sample DNA copy number for mtDNA extracted by a commercial kit and by the developed microchip. (c) A comparison of the mtDNA extraction efficiency shows that the performance of the chip is better than the commercial kit.
Figure 4-1..............................................47
The designed DNA primers are close to the endpoints of the 4,977 bp deletion.
Figure 4-2..............................................48
(a) Schematic illustration of the microfluidic system for mtDNA extraction and analysis. (b) An exploded view of the mtDNA extraction module. (c) An exploded view of the micro PCR module. (d) An exploded view of the MCE module.
Figure 4-3..............................................50
Illustration of the procedures for extraction and analysis of mtDNA in a microfluidic system.
Figure 4-4..............................................51
The experimental setup for the mtDNA extraction module, the micro PCR module, and the MCE module.
Figure 4-5..............................................52
(a) Slab-gel electropherograms for the amplified mtDNA and DNA fragments from a conventional PCR machine and from the developed micro PCR module. (b) The electropherogram for the normal mtDNA from the developed MCE module. (c) The electropherogram for mtDNA with 4,977 bp deletion. The presence of the 123-bp fragment indicates that the mtDNA has a 4,977 bp deletion.
Figure 4-6..............................................53
(a) Electropherograms for mtDNA samples containing different percentages of 4,977 bp deletion when separated and detected by the developed MCE module. (b) The relationship between the deletion mtDNA percentage and the signal intensity percentage (123 bp / 279 bp).
Figure 5-1..............................................62
(a) Schematic illustration of the mtDNA mutation detection system. (b) An exploded view of the mtDNA extraction module. (c) An exploded view of the mutation detection module.
Figure 5-2..............................................64
Illustration of the protocol for the extraction and detection of mtDNA mutations in a microfluidic system. This process includes mtDNA purification, identification, mutation detection and interpretation of the expected result.
Figure 5-3..............................................65
Various determination methods for mtDNA in purified samples from (a) slab-gel electrophoresis, (b) real-time PCR results, (c) results before PCR using the microfluidic system, and (d) results after PCR using the microfluidic system.
Figure 5-4..............................................66
DNA gel electrophoresis of PCR products after and before the enzymatic treatment.
Figure 5-5..............................................67
(a) The fluorescent signal from the enzyme undigested mtDNA, as analyzed by real-time quantitative PCR. (b) The fluorescent signal from the undigested mtDNA using the microfluidic system. (c) The fluorescent signal from the enzyme digested mtDNA, as detected by real-time quantitative PCR. (d) The fluorescent signal from the undigested mtDNA analyzed by the microfluidic system.
Figure 5-6..............................................68
The relationship between the mtDNA mutation ratio and the results from real-time PCR, compared to the on-chip system.

                                    

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