為了提升DNA分子於微渠道內之水力拉伸性能，本研究發展出二種新穎的拉伸方式。先利用加熱含有DNA分子之緩衝溶液，流經深度100 μm之漸縮漸擴微渠道(8:1:8)進行拉伸。為將拉伸渠道往奈米尺度發展，將多壁奈米碳管(MWCNT)塗覆於寬度為50 μm/400 μm與高度為10 μm之直管渠道底層，使底層增加表面粗糙度而提升拉伸性能。本研究使用電場驅動，依照流體在微渠道中的流動特性，利用不同區域產生的延展流和剪力流等不同機制拉伸DNA分子，結合高精度的共軛交雷射掃描顯微鏡(CLSM)進行渠道中央區域的延展流動拉伸量測與全內反射螢光顯微鏡(TIRFM)進行近於壁面受剪力流影響之DAN分子形變量測，並利用微質點影像測速儀(μPIV)光學技術量測微渠道之速度場與向量場分佈，結合以上光學可視化量測數據資訊，可針對DNA分子特性進行更伸入的探討。
由前期研究成果得知，加熱含有DNA分子之緩衝溶液，發現DNA分子內部的鍵結力會因溫度提升而逐漸減小，使DNA分子流經渠道縮擴變化之區域更容易改變初始纏繞形態，除了在渠道縮擴處產生延展流拉伸外，於緩衝溶液加熱後，還包含熱泳動、熱擴散等熱效應對DNA分子影響， DNA分子之拉伸性能產生加乘效果，發現熱效應對於DNA分子拉伸形變的效果大於渠道幾何造型變化帶來的影響。在環境溫度為55°C和電場強度為10 kV/m時，結合熱流場之效應，DNA分子最大拉伸率可達到DNA分子總輪廓長度之62 % (約占總數量的30%)。
在渠道奈米化的發展中，著重近於壁面區域的觀測，使用MWCNT塗覆於微渠道底層後，表面粗糙度約75 nm，驅動流體後發現DNA分子流經底部區域，壁面會有較大的速度梯度產生，再加上DNA分子與渠道表面之奈米碳管接觸，進而增加剪力效應對DNA分子拉伸的影響，部分流動的DNA分子能被定點鉗制在塗覆奈米碳管上，使DNA分子直接於定點處進行拉伸，此種設計大幅提升檢測晶片之實用性，當渠道深度縮減達到10 μm時，只需施加較低電場強度(5 kV/m)，DNA分子拉伸率即有顯著的效果，與表面未處理的狀態相較， DNA分子最大拉伸長度更能增加近二倍，其最大拉伸率可達到DNA分子總輪廓長度之78 % (約占總數量的5 %)。
藉由本研究水力拉伸實驗所得之經驗，可找尋出更有效且節省時間的DNA分子操控方式，更能以實驗數據分析得到DNA分子精確的相關參數，亦能深入了解DNA分子與生具有的特殊性質。

This study developed two novel stretching approaches to enhance the hydrodynamic stretching of DNA molecules in micro-channels. First, DNA stretching was performed flowing heated DNA-containing buffer solution through a 100 μm depth converging-diverging microchannel (8:1:8). To develop stretching channels at a nanoscale, multiwall carbon nanotubes (MWCNT) were coated on the bottom layer in 50/400 µm width and 10 µm depth straight channel to increase surface roughness, which enhances stretching performance. An electrical field driver was used in this study to generate different DNA stretching mechanisms, either elongation flow or shear flow, in different regions according to the flow characteristics of the fluid in the microchannel. The microchannel was integrated with confocal laser scanning microscopy (CLSM) to measure the amount of stretching in the central region of the channel, and with total internal reflection fluorescence microscopy (TIRFM) to measure the deformation of DNA molecules caused by the shear flow near the wall region. Next, micro particle image velocimetry (μPIV) was employed to measure the velocity field and vector field in the channel. Combining the aforementioned optical systems allowed visualization of the measurement data to extensively explore DNA characterization.
A previous study determined that DNA containing buffer solution should be heated to a temperature range of 25°C–55°C. The results revealed that the bonding force between DNA molecules diminished gradually as temperature increased. In addition to stretching in the elongation flow in the converging-diverging region of the channel, thermal effect exerted a greater influence on DNA stretching than did the changes in the geometric shape of a channel. Finally, at an ambient temperature of 55°C and electrical field of 10 kV/m, the maximum stretching ratio reached 62 % for DNA molecules contour length (30% of the total) as both thermal field and flow field influenced DNA stretching.
After MWCNT was used to coat the bottom layer of the microchannel, the surface roughness was approximately 75 nm. A greater velocity gradient was generated near the wall area because electrical field was applied. In addition, the DNA molecules came into contact with the MWCNT, magnifying the shear effects on DNA stretching. The experimental results indicated that when the depth of the channel was reduced to 10 μm, only 5 kV/m of electrical field was required to significantly enhance DNA stretching efficiency. Achieving a maximum stretching ratio reached 78 % for DNA molecules contour length (accounting for approximately 5% of the total). Experimental data analysis was conducted to obtain accurate DNA parameters and afford insights into the special characteristics of DNA molecules.

CONTENTS
論文審定書…………………………………………………………………i
ACKNOWLEDGMENTS(誌謝)…………………………………………..ii
中文摘要…………………………………………………………………..iii
ABSTRACT...……………………………………………………………...v
CONTENTS…..………………………………………………………......vii
LIST OF TABLES………………………………………………………….x
LIST OF FIGURES……………………………………………………......xi
NOMENCLATURE………...…………………………………………....xvi
CHAPTER 1……………………………………………………………......1
INTRODUCTION………………………………………………………….1
1.1 Background………………………………………………………...1
1.2 Literature survey…………………………………………………...3
1.3 Objective………………………………………………………….22
1.4 Outline of the dissertation…………………………………….…..25
CHAPTER 2………………………………………………………...…….27
EXPERMIEMTAL EQUIPMENT..…..………………………………..….27
2.1 Microscale particle image velocimetry (μPIV) system…….….....27
2.2 Confocal laser scanning microscopy (CLSM) system……..….....29
2.3 Total internal reflection fluorescence microscope (TIRFM) system…………………………...…………….………………...30
2.4 Nanoscale particle image velocimetry (nPIV) system……...…......32
2.5 Electro driven system………………….……………………….…33
2.6 Fabrication equipment...…....…………………….………………34
2.7 Other apparatus ………..……………….……………….…..……34
CHAPTER 3………………………………………………………………50
EXPERIMENTAL METHODS AND PROCEDURES……………..…….50
3.1 Microchannel fabrication…………………………………..……..50
3.2 Sample preparation………………………...……………………..51
3.3 Measurement principles……………………………………..……51
3.4 Theory of electro-osmotic flow and electrophoresis……….…….59
3.5 Compare advantages/ disadvantages of elongation flow and shear flow………………………………………………………….…….63
3.6 One-sided MWCNT walls fabrication process……………………64
3.7 Experimental testing and flow loop………………………………66
CHAPTER 4………………………………………………………………87
THEORY ANALYSIS………………………………………………….…87
4.1 Dimensionless parameter analysis of the flow field……………...87
4.2 The definition of worm-like chain (WLC) ………………….……91
4.3 The definition of freely-jointed chain (FJC) ……………..………92
4.4 Free energy change………………………………………….……94
CHAPTER 5……………………………………………………………....98
DATA REDUCTION AND UNCERTAINTY……………………….……98
CHAPTER 6………………………………………………………..……103
RESULTS AND DISCUSSION…………………………………………103
6.1 DNA molecule stretching in a heated converging-diverging microchannel…………………………………….....…………103
6.2 DNA molecule stretching on one-sided carbon nanotube walls in microchannels…………………...…………….……….………111
CHAPTER 7…………………………………………………………..…137
CONCLUSION AND RECOMMENDATIONS…………………...……137
7.1 Conclusion………………………………...………………….…137
7.2 Recommendations for future work…………………….…..……142
REFERENCES………………………………………………………..…143
APPENDIX...……………………………………………………………177
PUBLICATION LIST……………………………………………………186


LIST OF TABLES
Table 2-1 μPIV system laser specifications…………………………..……36
Table 2-2 80C77 Hisense PIV CCD camera specifications………..………37
Table 2-3 CLSM（Olympus FV300）specifications………………..………38
Table 2-4 Continues wave laser specifications………………...…………..39
Table 2-5 TIRFM laser connector specification (FCP8)………..………….40
Table 3-1 Spectral characteristics of cyanine dimer nucleic acid stains bound to dsDNA…………………………………………………….…68
Table 3-2 Converging-diverging channel parameter and operation conditions……………………………………………………….69
Table 3-3 MWCNT channel parameter and operation conditions…..…….70
Table 5-1 Measurement variables uncertainty……………………………102
Table 6-1 Local velocity map with different heating temperatures...…….119
Table 6-2 Local velocity map with different heating temperatures and electric fields…………………………………………………..120
Table 6-3 DNA molecules average stretching ratio (near wall)……......…121
Table 6-4 Compared with DNA stretching image in different cases….…..122


LIST OF FIGURES
Figure 2-1 μPIV system (a) μPIV laser (Nd-Yag) (b)High speed camera (Dantec 80C77) (c)Microscopy for μPIV（Leica DMILM）(d) Processor……………………………………………………...41
Figure 2-2 CLSM system (a) Ar-ion/He-Ne laser (b) Scanning unit……...42
Figure 2-3 Schematic of Olympus FV300 scanning unit …………………43
Figure 2-4 (a) Inverted system microscope (Olympus IX 71) (b) Mercury lamp (Olympus BX100) ……………………………...………44
Figure 2-5 TIRFM system (a) High speed camera for TIRFM system (b)TIRFM 60× oil lens………………………………...……...45
Figure 2-6 (a) Diode laser 532 nm for TIRFM system (b)TIRFM illuminator……………………………………………...……..46
Figure 2-7 DC Power supply (Stanford Research Systems PS350)…...….47
Figure 2-8 Lithography equipment……………………………………......48
Figure 2-9 Other apparatus………………………….……………...…..…49
Figure 3-1 Microchannel fabrication process………………………...…...71
Figure 3-2 Absorption/Emission wave length of fluorescent dye [193].....72
Figure 3-3 Microscope dichroic mirror units for (a) YOYO-1 (b) JOJO-1 [194] ……………………………………………….………....73
Figure 3-4 Schematics of μPIV and TIRFM measurement region…….…...74
Figure 3-5 Working principle of CLSM………………...…………………75
Figure 3-6 Working principle of TIRFM light path………..………………76
Figure 3-7 Working principle of TIRFM system……………………..……77
Figure 3-8 Schematic of evanescent wave and light source for TIRFM system…………………………… ……………………..….…78
Figure 3-9 Schematic of EOF principle [157] …………………..…....……79
Figure 3-10 Flow profile (a) EOF flow (b) pressure driven….…..……...…80
Figure 3-11 Schematic of MWCNT coating on cover glass……………….81
Figure 3-12 Deposition MWCNT on cover glass (7.5 wt.%) …………..….82
Figure 3-13 Microchannel geometry and observed section…………...…..83
Figure 3-14 MWCNT microchannel geometry and observed section…......84
Figure 3-15 Flow loop of μPIV and CLSM system…..……………………85
Figure 3-16 Flow loop of TIRFM system……………………….……...…86
Figure 4-1 Schematic of free jointed chain model. ..........…...…...……….96
Figure 4-2 Schematic of worm-like chain……………………………...…97
Figure 6-1 DNA molecule velocity at different heating temperatures and electric strength at the channel inlet (a) Spanwise (x = 14.5 mm) and (b) transverse (x = 14.5 mm) ……………….....…………123
Figure 6-2 Velocity gradient at different electric field and at a definite channel inlet x = 14.5 mm (left column) and different channel velocity profile (right column) at y = 0 at different channel position (right column) with different heating temperature and electric strength………...…………………………...…….…124
Figure 6-3 DNA molecule mobility and diffusion coefficient distribution. (a) DNA electrophoresis velocity versus electric field and (b) relationship of diffusion coefficient and buffer solution temperatures [216, 220-222] …………………………...……125
Figure 6-4 Sample images of DNA molecule stretching with various temperatures and electric field strengths at the inlet region (x = 14.6 to 14.9 mm) via CLSM……………...…………..……..126
Figure 6-5 Average stretching length. After deducting the thermal expansion effect and coefficient of DNA thermal expansion versus temperature at the inlet region (14.6 to 14.9 mm) with the scale bar of 10 µm………………………...………………….…...127
Figure 6-6 Histogram of DNA length without electric field strength at different temperatures (a) 25°C, (b) 35°C, (c) 45°C, and (d) 55°C……………………………………………...……….…128
Figure 6-7 Histogram of the stretch ratio of DNA molecule after deducting the thermal expansion effect at Ex = 10 kV/m at different temperatures. Inlet region: (a) 25°C, (b) 35°C, (c) 45°C, and (d) 55°C. Middle region: (e) 25°C, (f) 35°C, (g) 45°C, and (h) 55°C…………………………………………………….....…129
Figure 6-8 Stretching portions of the force-extension curves as a function of temperature (a) Maximum DNA molecule hydrodynamic force versus extension after deducting the thermal expansion effect (b) Hydrodynamic force of the present study versus the force law from the WLC model………………….…………………..…130
Figure 6-9 DNA molecules stretch ratio distribution at different electric field and width in microchannel (x = 1000 μm) shows the maximum contour length in each case……………………………..……131
Figure 6-10 Surface contact angle (a) with CNT film (b) clean glass surface……….……………………………...…………..…132
Figure 6-11 DNA molecules hook at fix point of CNT and take the continuous images by TIRFM system (x = 425-575 μm, Ex = 5 kV/m) ….………………………………………..……...…133
Figure 6-12 DNA molecules local velocity at vertical position at distance of 1000 μm downstream with different electric fields in two different nanochannels. (a) width: 50 μm (b) width: 400 μm……………..…………………………...……………….134
Figure 6-13 3D velocity field distribution at different electric fields. (a) width = 50 μm (b) width = 400 μm……………...…………....……135
Figure 6-14 Maximum stretching ratio versus Wi……………..…...……136

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