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研究生:簡育生
研究生(外文):Yu-sheng Chien
論文名稱:電驅動式微混合器及整合光鉗之細胞操控平台於生醫檢測之應用
論文名稱(外文):Electrokinetic Micromixer and Cell Manipulation Platform Integrated with Optical Tweezer for Bio-analytical Applications
指導教授:林哲信
指導教授(外文):Che-hsin Lin
學位類別:碩士
校院名稱:國立中山大學
系所名稱:機械與機電工程學系研究所
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:129
中文關鍵詞:微混合器微流體光鉗電滲透流生醫檢測細胞操控
外文關鍵詞:biomedical analysismicrofluidicoptical tweezerelectroosmotic flowmicromixercell manipulation
相關次數:
  • 被引用被引用:13
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近年來在眾多微機電研究領域中,將微流體元件應用於生醫檢測尤其受到重視。然而,流體於微型化之微管道中流動時,其雷諾數非常小,對於生物反應須將不同生物樣本進行均勻混合的應用,將因微流體係以層流的特性來流動,因此僅能藉由擴散作用來進行混合,而造成需要較長的微管道或較長的擴散時間來達成。
為彌補上述缺失,本研究首先提出一利用交替切換之電滲透流驅動的微型混合器,並分別提出T型及雙T型兩種微管道設計。該混合器係直接利用驅動流體之電滲透力當作液體混合之作用力。因此,該混合器不需任何可動元件,或任何外加的機械力便可以獲得良好之混合結果。此外,在T型微流體混合器方面,本研究又提出一新穎之箝位切換模式,藉此來增大流體的擺幅,以提升混合性能。該研究不但透過實驗予以驗證,並利用電腦模擬分析其混合效果。藉由實驗與模擬的驗證,本研究所發展之混合器,其混合效能在兩不同樣本液交界處下方行經1 mm時,即能獲得高達95%以上之混合效率。此研究所提倡之新方法,可容易地被運用於解決微全程分析系統領域內的混合問題。
此外,為了驗證上述微混合器於生醫反應的可行性,本研究並將其應用於一電驅動之整合型DNA/酵素反應裝置,其包含:DNA預濃縮區、DNA/酵素混合區、DNA/酵素溫控反應區及DNA純化區之整合型微流體晶片,以進行DNA之酵素切位及萃取收集之研究。在此晶片中,其濃縮部分係將DNA於瓊脂膠中進行電泳來達成。而試劑混合區,係利用交替切換電滲透流來達成液體混合的效果。另外,在混合器後端設計一長段的酵素反應區以進行DNA之酵素切位,並輔以溫度控制以提高其反應效率。研究採用λ-DNA和限制
Integrated microfluidic devices for biomedical analysis attract lots of interest in the MEMS (Micro-Electro-Mechanical-Systems) research field. However, the characteristic Reynolds number for liquids flowing in these microchannels is very small (typically less than 10). At such low Reynolds numbers, turbulent mixing does not occur and homogenization of the solutions occurs through diffusion processes alone. Hence, a satisfactory mixing performance generally requires the use of extended flow channels and takes longer to accomplish such that the practical benefits of such devices are somewhat limited. Consequently, accomplishing the goal of u–TAS requires the development of enhanced mixing techniques for microfluidic structures.
This study first presents a microfluidic mixer utilizing alternatively switching electroosmotic flow and proposes two microchannel designs of T-form and double-T-form micromixer. Switching DC field is used to generate the electroosmotic force to drive the fluid and also used for mixing of the fluids simultaneously, such that moving parts in the microfluidic device and delicate external control system are not required for the mixing purpose. Furthermore, this study also proposed a novel pinched-switching mode in the T-form microfluidic mixer, which could be effectively increase the perturbation within the fluid to promote the mixing efficiency. In this study, computer simulation for the operation conditions is used to predict the mixing outcomes and the mixing performance is also confirmed experimentally. Result shows the mixing performance can be as larger as 95% within the mixing distance of 1 mm downstream the common boundary between the different sample fluids. The novel method proposed in this study can be used for solving the mixing problem in a simple way in the field of micro-total-analysis-systems.
Furthermore, in order to demonstrate the proposed micromixer is feasible for on-line bio-reaction, this study designs a fully integrated device for demonstration of DNA/enzyme reaction within the microfluidic chip. The microchip device contains a pre-column concentrating region, a micro mixer for DNA-enzyme mixing, an adjustable temperature control system and a post-column concentration channel. The integrated microfluidic chip has been used to implement the DNA digestion and extraction. Successfully digestion of λ-DNA using EcoRI restriction enzyme in the proposed device is demonstrated utilizing large-scale gel electrophoresis scheme. Results show that the reaction speed doubled while using the microfluidic system. In addition, on-line DNA digestion and capillary electrophoresis detection is also successfully demonstrated using a standard DNA-enzyme system of $X-174 and Hae III.
Finally, this reasearch also proposes a novel cell/microparticle manipulation platform by integrating an optical tweezer system and a micro flow cytometer. During operation, electrokinetically driven sheath flows are utilized to focus microparticles to flow in the center of the sample stream then pass through an optical manipulation area. An IR diode laser is focused to generate force gradient in the optical manipulation area to manipulate the microparticles in the microfluidic device. Moving the particles at a static condition is demonstrated to confirm the feasibility of the home-built optical tweezer. The trapping force of the optical tweezer is measured using a novel method of Stocks-drag equilibrium. The proposed system can continuously catch moving microparticles in the flowing stream or switch them to flow into another sample flow within the microchannel. Target particles can be separated from the sample particles with this high efficient approach. More importantly, the system demonstrates a continuously manipulation of microparticles using non-contact force gradient such that moving parts and delicate fabrication processes can be excluded. The proposed system is feasible of high-throughput catching, moving, manipulation and sorting specific microparticles/cells within a mixed sample and results in a simple solution for cell/microparticle manipulation in the field of micro-total-analysis-systems.
In this thesis, low-cost soda-lime glass substrates are adopted for the microchip fabrication using a simple and reliable fabrication process. Three kinds of novel microfluidic devices including an electrokinetically-driven microfluidic mixer, a high throughput DNA/enzyme reactor and an optically cell manipulation platform are successfully demonstrated. It is the author’s believes that the results of this study will give important contributions in the development of micro-total-analysis-systems in the future. With the success of this study, we have a further step approaching to the dream of lab-on-a-chip system for bio-analytical applications.
目 錄
目錄......................................................Ⅰ
圖目錄....................................................Ⅴ
表目錄...................................................XⅠ
中文摘要................................................ XⅡ
Abstract.................................................XⅣ
第一章 緒論................................................1
1-1 前言...................................................1
1-2 生醫微機電系統.........................................4
1-3 文獻回顧...............................................6
1-3-1 微型混合器...........................................6
1-3-2 微型細胞分類器......................................12
1-3-3 整合型晶片實驗室....................................17
1-4 研究動機與目的........................................23
1-5 論文架構..............................................29
第二章 基本理論與數值方法.................................31
2-1 微尺度流體特性........................................31
2-2 電雙層的形成機制......................................32
2-3 電滲透流原理..........................................34
2-4 電滲流場數值分析......................................35
2-5 雷射光鉗基本原理......................................38
第三章 晶片製作及實驗架設.................................41
3-1 晶片設計..............................................41
3-1-1 微混合器............................................41
3-1-2 DNA-酵素反應及萃取裝置..............................42
3-1-3 細胞分類器..........................................43
3-2 光罩製作..............................................44
3-3 玻璃晶片製程..........................................45
3-3-1 晶片清洗............................................46
3-3-2 微影................................................47
3-3-3 微管道蝕刻..........................................48
3-3-4 玻璃鑽孔............................................48
3-3-5 晶片接合............................................49
3-4 壓克力晶片製程........................................49
3-4-1 玻璃母模製作........................................51
3-4-2 晶片熱壓程序........................................52
3-4-3 壓克力晶片化學接合..................................54
3-5 實驗架設..............................................55
3-5-1 電驅動式微型混合器..................................55
3-5-2 DNA-酵素反應及萃取系統.............................57
3-5-3 光鉗操控之細胞分類系統..............................59
第四章 結果與討論.........................................62
4-1 T型微流體混合器之混合情形.............................62
4-1-1 直接切換模式之混合結果..............................62
4-1-2 箝位切換模式之混合結果..............................66
4-1-3 混合效率最佳化之操作參數............................69
4-2 雙T型微流體混合器之混合情形...........................71
4-2-1 增加進樣側管之混合結果..............................71
4-2-2 直接切換模式之混合結果..............................73
4-2-3 混合效率最佳化之操作參數............................77
4-3 微混合器於DNA-酵素反應及萃取的應用....................78
4-3-1 DNA於微管道中被電滲透流帶動之流率分析...............78
4-3-2 DNA切位反應之膠電泳結果.............................79
4-3-3 DNA切位反應之毛細管電泳結果.........................80
4-4 光鉗於微流道中粒子分類的應用..........................83
4-4-1 樣本流聚焦情形......................................83
4-4-2 雷射光鉗搬運微尺度粒子之結果........................87
4-4-3 雷射功率與光鉗捕捉力的關係..........................89
4-4-4 光鉗於微流道粒子分類之結果..........................92
第五章 結論與未來展望.....................................99
5-1 結論..................................................99
5-2 未來展望.............................................101
參考文獻.................................................103
自述.....................................................108


圖目錄
圖1.1 整合型快速生醫檢測系統之工作流程圖..................4
圖1.2 利用週期性壁面效應變化之微型混合器..................8
圖1.3 利用交替式施加空氣壓力於側管之渾沌式混合器..........9
圖1.4 整合換能器於微管道之超音波震動混合器................9
圖1.5 埋置障礙物之渾沌式混合器...........................10
圖1.6 平面型之被動式Telsa結構混合器......................11
圖1.7 利用自旋效應之三維渦流式微混合器...................12
圖1.8 整合微加工聲波換能器之微流體切換開關...............14
圖1.9 整合磁性微奈米尖端之磁珠/細胞分類器................14
圖1.10 利用液動介電泳力之連續式細胞分類晶片...............15
圖1.11 整合光鉗及電腦迴授控制之微流體細胞分類系統.........16
圖1.12 整合光鉗及微流道之細胞分類器.......................16
圖1.13 利用光鉗操縱之微流體細胞分類器.....................17
圖1.14 整合型DNA切位及片段分析之微流體晶片................18
圖1.15 整合細胞胞解、PCR放大及電泳片段分析之微晶片裝置....19
圖1.16 整合PCR、微閥及電泳裝置之疾病檢測晶片..............20
圖1.17 整合DNA放大、電泳分離及線上光偵測之微流體晶片......21
圖1.18 整合電滲幫浦、微混合器及晶片溫控裝置之自動化微流體晶片........................................................22
圖1.19 美國Micronics公司所生產之整合型微流體晶片(ORCA chip).....................................................22
圖1.20 T型微流體混合器之工作原理示意圖,(a)一般切換模式,(b)箝位切換模式.........................................25
圖1.21 雙T型微流體混合器之工作原理示意圖..................25
圖1.22 快速DNA切位之T型微混合器工作原理示意圖.............27
圖1.23 DNA-酵素反應及萃取系統之工作流程圖................27
圖1.24 DNA-酵素反應及萃取系統之晶片實體影像圖............27
圖1.25 微粒子於微管道中之作動示意圖.......................29
圖2.1 電雙層及電位勢之離子分佈示意圖.....................33
圖2.2 電滲流場速度分佈示意圖.............................35
圖2.3 光鉗之工作原理示意圖...............................40
圖3.1 T型微流體混合器之尺寸大小示意圖...................41
圖3.2 雙T型微流體混合器之尺寸大小示意圖..................42
圖3.3 DNA-酵素反應及萃取裝置之尺寸大小示意圖............42
圖3.4 細胞分類器之尺寸大小示意圖.........................43
圖3.5 玻璃基材之微流體晶片製程示意圖.....................46
圖3.6 微流體混合器之晶片實體圖...........................49
圖3.7 細胞分類器之晶片實體圖.............................49
圖3.8 壓克力晶片之熱壓製程流程圖.........................51
圖3.9 空白玻璃光罩之熱壓母模製程示意圖...................52
圖3.10 壓克力晶片之熱壓成型示意圖.........................53
圖3.11 熱壓成型參數示意圖.................................53
圖3.12 壓克力晶片之化學接合製程示意圖.....................55
圖3.13 電驅動式微混合器之實驗量測架設示意圖...............56
圖3.14 晶片毛細管電泳偵測之實驗架設示意圖.................59
圖3.15 運用雷射光鉗系統操縱微粒子之實驗架設示意圖.........60
圖3.16 光鉗顯微鏡系統圖...................................61
圖4.1 T型微流體混合器,在穩態注射下之模擬及實驗結果,(a)電場強度分佈,(b)流線圖,(c)模擬之濃度影像圖,(d)實驗之濃度影像圖..............................................62
圖4.2 T型微流體混合器,在一般切換模式於兩段切換下之電場強度分佈及流線圖.............................................64
圖4.3 T型微流體混合器於一般切換模式下,藉由數值模擬及實驗測試其於固定驅動電場強度90 V/cm及各種切換頻率下之濃度分佈影像圖.......................................................65
圖4.4 T型微流體混合器於一般切換模式下,在不同切換頻率於T型端下方1000 μm處之管道橫斷面的正規化濃度亮度值............65
圖4.5 T型微流體混合器於箝位切換模式,其兩段切換下之電場強度分佈及流線圖..............................................67
圖4.6 T型微流體混合器於箝位切換模式下,藉由數值模擬及實驗測試其於固定驅動電場強度90 V/cm及各種切換頻率下之濃度分佈影像圖........................................................68
圖4.7 T型微流體混合器於箝位切換模式下,在不同切換頻率下之混合效率....................................................68
圖4.8 T型微流體混合器,其利用數值模擬及實驗評估在不同切換頻率及驅動電壓下於T型端下方1000 μm處之混合效率比較圖.......70
圖4.9 T型微流體混合器於箝位切換模式下,藉由數值模擬及實驗測試其於低驅動電壓及高驅動電壓時,該混合效率最佳化之濃度分佈影像圖......................................................71
圖4.10 雙T型微流體混合器,以數值模擬在施加100 V/cm之電場下的電場強度分佈圖(a)及兩種平行注射模式之流線,分別表示同邊注射模式(b)及交錯注射模式(c)............................72
圖4.11 雙T型微流體混合器,在平行流注射下之數值模擬及實驗所得之濃度分佈影像圖..........................................73
圖4.12 雙T型微流體混合器,於平行流注射模式在次T型下方2500 μm長之路徑上的混合效率變化圖...............................73
圖4.13 雙T型微流體混合器,兩段切換模式之電場強度分佈及流線圖........................................................75
圖4.14 雙T型微流體混合器,在藉由數值模擬及實驗測試其於固定驅動電場強度100 V/cm及各種切換頻率下之濃度分佈影像圖........76
圖4.15 雙T型微流體混合器,其在不同切換頻率下之混合效率....76
圖4.16 雙T型微流體混合器,其利用數值模擬及實驗評估在不同切換頻率及驅動電壓下於次T型端下方1200 μm處之混合效率比較圖...77
圖4.17 微流道中DNA的濃度隨時間變化圖......................79
圖4.18 λ-DNA於傳統大系統及微晶片裝置切位反應之膠電泳比較圖........................................................80
圖4.19 X-174 DNA與HaeⅢ切位酵素持續進行切位反應時,於不同時間進行DNA分離之產物,分別為:(a)6 min,(b)12 min及(c)18 min.......................................................82
圖4.20 X-174 DNA與HaeⅢ酵素進行切位反應25分鐘所得的結果..83
圖4.21 不同聚焦寬度之流體影像圖,(a)90 um,(b)75 um,(c)60 um,(d)45 um,(e)30 um,(f)15 um............84
圖4.22 在施加不同電壓下之聚焦寬度統計結果.................85
圖4.23 圖4.23 樣品流中心偏移之影像圖,(a)示意圖,(b)下偏45 μm,(c)下偏30 μm,(d)下偏15 μm,(e)正中央,(f)上偏15 μm..................................................85
圖4.24 微流體聚焦之粒子流動連續影像圖.....................86
圖4.25 微粒子於顯微鏡載玻片上靜態搬運之連續影像圖.........88
圖4.26 微粒子於微流道內搬運之連續影像圖...................89
圖4.27 微流道中流動粒子捕捉之連續影像圖...................90
圖4.28 光鉗捕捉力之黏滯拉力法測試示意圖...................91
圖4.29 流體流速與捕捉粒子所需之雷射輸出功率關係圖.........91
圖4.30 雷射輸出功率與橫向捕捉力之關係圖...................92
圖4.31 微粒子於微流道中受到光鉗橫向吸引之路徑變化連續影像圖........................................................93
圖4.32 10 um微粒子於微流道中進行分類之連續影像圖.........95
圖4.33 5 um微粒子於微流道中進行分類之連續影像圖..........96
圖4.34 5 um及10 um微粒子於微流道中進行分類之連續影像圖...98


表目錄
表3.1 PMMA之材料性質及熱壓成型參數.......................50
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