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研究生:葉俊邑
研究生(外文):Yeh, Chun-Yi
論文名稱(外文):Micro-fluidic devices using non-linear electrokineticsto determine biological samples.1. Micro-concentrator using ion-exchange resin granule; 2. Induced ac electro-osmosis micro-mixer.
指導教授:王少君
指導教授(外文):Wang, Shau-Chun
口試委員:周禮君莊怡哲
口試委員(外文):Chau, Lai-KwanJuang, Yi-Je
口試日期:2011-07-15
學位類別:碩士
校院名稱:國立中正大學
系所名稱:化學暨生物化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:68
中文關鍵詞:濃縮微流體
相關次數:
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摘要
微流體系統於生化分析有許多的優點,如減少樣品與試劑的用量、縮
短分析時間與更有效的分析,而且更少的開發成本使其更容易整合成一可
攜式裝置。然而,於生醫相關領域的應用上,原先含有的分析物只有極少
量,假使沒有作濃縮的前處理步驟,要作定量有一定的難度存在。故需要
去發展一個於微流體上的微型化濃縮元件。
本實驗室先前的研究中,曾利用一個陽離子交換樹酯顆粒,使其置於
一適當的電場下(100 V/cm)來濃縮酸性的螢光染料分子。此染料分子的濃縮
倍率達105倍到106倍左右。此研究我們先探討了染料的解離度對濃縮倍率造
成的影響,發現當被濃縮的染料分子pH值達到pKa + 2或更高時已近乎完全
解離,其濃縮倍率已達到最大值。相似的,當以此濃縮器來濃縮蛋白質,
於含有較高電荷態的pH值下亦具有較高的濃縮倍率。換句話說,於適當pH
值去濃縮每一種蛋白質是與蛋白質的等電點(pI值)有關,當緩衝溶液pH值調
整適宜,伴隨著選擇性濃縮的可能性。我們以fluorescein標記的BSA與Texas
red標記的avidin於不同的pH值情況下去驗證選擇性濃縮此兩種蛋白質的可
能。於pH 8.01時,無法看到放紅光以Texas red所標記的avidin,而可清楚看
到放綠光被標記的BSA。當緩衝溶液為鹼性的pH 8.01時,只有pI為4.8的BSA
有帶高電荷,而pI為10.5的avidin是近乎中性不帶電。當緩衝溶液為pH 11.22
時兩蛋白質均有解離而帶電荷,以螢光標記的BSA與avidin均可看到有濃縮現象的產生。
由於微流體裝置中的槽道尺寸狹小,於管道中的流體無法轉動,故必
定要耗費長時間來混合。本實驗室先前已發展出一種藉由外加高頻率交流
電場來達成高效率混合的微型化混合裝置。我們使用介電材質來製作具有
曲度結構之微混合槽,透過施加高頻率交流電場,其電力線穿過具曲度之
尖角生成誘導電荷,進而產生非線性渦流電滲流動。此裝置已被證明能加
速葡萄糖與被溶膠-凝膠包埋的葡萄糖氧化酶之間的接觸而更快產生過氧
化氫,過氧化氫再與魯米諾與鐵離子反應,能比未施加電場下產生更強的
化學冷光。此研究對嘗試使用此裝置來輔助化學冷光法以定量葡萄糖進行
初步的探討。
Microfluidic systems have various bio-analytical advantages such as
minimum simple and reagent consumption, reduced analysis time, and assay
cost effectiveness, as well as less development efforts to integrate into portable
equipments. However, in biomedical applications because assayed sample often
contains only ultra-trace amount of analytes, without pretreatment steps to
concentrate sample, quantitative determination is problematic. Therefore there
remains a need to develop miniaturized concentration unit in microfluidic
device.
Our previous work using an ion exchange resin granule to concentrate
acidic fluorescence dyes when this resin granule is placed in a moderate electric
field (100 V/cm). The concentration factor of dyes using this device is
enomorous 10^5 to 10^6. In this study we firstly study the effects of dye
dissociation in concentration factor to conclude that the concentration factor
reaches the maximum when the concentrated dye becomes fully dissociated at
the pH condition of pKa + 2 or higher. Similarly when protein is trapped using
this granule device, protein has higher concentration factor at pH condition to
obtain higher charge state. In other words, the suitable pH value to concentrate
each protein is dependent on protein isoelectric point, pI value. When buffer pH
is adjusted, selective protein trapping can be accomplished.
We use fluorescein-tagged BSA and Texas red-tagged avidin to
demonstrate selective trapping of these two proteins at different pH
conditions. The red emission of tagged avidin cannot be seen at pH 8.01 while
clear concentration observed with the intensified green emission of labeled BSA.
Under this buffer basicity at pH 8.01, only BSA of which pI is 4.8 is highly
charged but avidin of which pI is 10.5 is almost neutral. When buffer is adjusted
to pH 11 to fully protonated both proteins, the emission of both tagged
fluorescence labels of BSA and advidin is clearly intensified to confirm
successful concentration effects.
Inevitable long mixing time in micro-channel due to fluid irrotationality in
small scale conduits also constrains the utility of micro-fluidic devices. Our
group previously has developed a miniaturized mixing device applying external
high frequency AC field to achieve highly efficient mixing. We use the
dielectric materials to fabricate micro-mixing reservoir with the sharp curvature
at the corners, and we apply the high-frequency ac electric field to generate the
nonlinear vortex electro-osmotic flow via building up induced charges when the
field lines penetrate through the sharp corners. This device has been proven to
speed up the contacts of glucose with the glucose oxidase-entrapped sol-gel
particles to accelerate the formation of hydrogen peroxide. Under the mixing
acceleration, more hydrogen peroxide products react with luminol and ferric
ions than the amount of hydrogen peroxide produced without mixing. As a
consequence, chemiluminescence emitted with lumino reactions becomes more
intense under mixing. In this study we conduct preliminary studies using this
device to develop chemiluminescence-based miniaturized enzymatic assays to
determine glucose.
目錄
摘要 ...................................................................................................................... I
Abstract ............................................................................................................. III
目錄 ..................................................................................................................... V
圖目錄 ............................................................................................................. VIII
表目錄 ................................................................................................................. X
第一章 緒論.....................................................................................................1
1-1 前言 ...............................................................................................................1
1-2 原理 ...............................................................................................................3
1-3 前言(混合器部分) .........................................................................................9
1-4 原理(混合器部分) ...................................................................................... 10
第二章 導電性奈米孔洞離子交換樹酯對蛋白質做選擇性濃縮實驗 ..... 14
2-1 實驗藥品與設備 ........................................................................................ 14
2-1-1 藥品 .................................................................................................. 14
2-1-2 實驗器材 .......................................................................................... 14
2-2 濃縮實驗之塑膠晶片製作與裝置架設 .................................................... 16
2-2-1 晶片製作 .......................................................................................... 16
VI
2-2-2 輸出電場確認 .................................................................................. 17
2-2-3 濃縮現象的觀測裝置 ...................................................................... 17
2-2-4 藥品或溶液配製 .............................................................................. 18
2-3 實驗步驟 .................................................................................................... 21
2-3-1 活化離子交換樹酯 .......................................................................... 21
2-3-2 Fluorescein 濃縮倍率實驗............................................................... 21
2-3-3 ND Filter 測試實驗 .......................................................................... 22
2-3-4 濃縮有效範圍 .................................................................................. 22
2-3-5 單獨對蛋白質作濃縮實驗 .............................................................. 23
2-3-6 對蛋白質作選擇性濃縮實驗 .......................................................... 23
2-3-7 數據處理 .......................................................................................... 24
2-4 結果與討論 ................................................................................................ 25
2-4-1 Fluorescein 濃縮倍率與解離度的關係 .......................................... 25
2-4-2 ND Filter 測試結果 .......................................................................... 28
2-4-3 濃縮有效濃度範圍 .......................................................................... 30
2-4-4 單獨對蛋白質作濃縮 ...................................................................... 32
2-4-5 對蛋白質作選擇性濃縮 .................................................................. 35
VII
2-5 結論 ............................................................................................................ 40
第三章 利用微混合器輔助化學冷光法定量葡萄糖實驗 ......................... 41
3-1 實驗藥品與設備 ........................................................................................ 41
3-1-1 藥品 .................................................................................................. 41
3-1-2 實驗器材 .......................................................................................... 41
3-2 濃縮實驗之塑膠晶片製作與裝置架設 ................................................... 43
3-2-1 晶片製作 .......................................................................................... 43
3-2-2 輸出電場確認 .................................................................................. 44
3-2-3 混合現象的觀測裝置 ...................................................................... 45
3-2-4 藥品或溶液配製 .............................................................................. 45
3-3 實驗步驟 .................................................................................................... 48
3-3-1 利用微混合器提高訊號實驗 .......................................................... 48
3-4 結果與討論 ................................................................................................ 49
3-4-1 利用微混合器提高訊號實驗 .......................................................... 49
3-5 結論與未來展望 ........................................................................................ 52
第四章 參考文獻 ......................................................................................... 53
圖目錄
圖1-1 表面帶電材質引起溶液中電荷分佈與zeta 電位示意圖。 ............... 3
圖1-2 陽離子交換樹酯於均勻電場下的濃縮機制示意圖 ............................ 8
圖1-3 不對稱槽道尖角示意圖 ...................................................................... 11
圖2-1 共聚酯塑膠晶片之設計 ...................................................................... 17
圖2-2 觀測裝置示意圖 .................................................................................. 18
圖2-3 Fluorescein 濃縮實驗數據處理示意圖 ............................................... 24
圖2-4 Fluorescein 於pH 6.59 時的濃縮情形(10-8 M) ................................... 26
圖2-5 Fluorescein 於pH 8 時的濃縮情形(2 × 10-6 M) .................................. 26
圖2-6(A) pH 值與fluorescein 亮度的關係圖( 2×10-6 M ) ........................... 27
圖2-6(B) 濃縮倍率與pH 值的關係圖 .......................................................... 27
圖2-7 有加濾片的校正曲線 ........................................................................... 29
圖2-8 未加濾片的校正曲線 ........................................................................... 29
圖2-9 pH 8.1 時各濃度fluorescein 的濃縮倍率 ........................................... 30
圖2-10 BSA 於pH 8.01 時的濃縮情形(1.89×10-6 M) ................................... 32
圖2-11 BSA 於pH 8.01 時的校正曲線 .......................................................... 33
圖2-12 Avidin 於pH 11.22 時的濃縮情形(1.25×10-6 M) .............................. 33
圖2-13 Avidin 於pH 11.22 時的校正曲線 ..................................................... 34
IX
圖2-14 Avidin 於pH 8.01 時的濃縮情形(1.25×10-6 M) ................................ 34
圖2-15 Avidin 於pH 8.01 時的校正曲線 ....................................................... 34
圖2-16 BSA 於pH 7.92 時的濃縮情形(約為0.5×10-6 M) ............................ 37
圖2-17 Avidin 於pH 7.92 時的濃縮情形(約為1.56×10-6 M) ....................... 37
圖2-18 BSA 於pH 11.74 時的濃縮情形(約為0.5×10-6 M) .......................... 38
圖2-19 Avidin 於pH 11.74 時的濃縮情形(約為1.56×10-6 M) ..................... 38
圖2-20 Avidin 於pH 8.01 時的校正曲線。(Avidin:BSA = 3:1) ............. 39
圖2-21 BSA 於pH 8.01 時的校正曲線。(Avidin:BSA = 3:1) ................ 39
圖2-22 Avidin 於pH 11.41 時的校正曲線。(Avidin:BSA = 3:1) ........... 39
圖2-23 BSA 於pH 11.41 時的校正曲線。(Avidin:BSA = 3:1) .............. 39
圖3-1 共聚酯塑膠晶片之設計 ...................................................................... 44
圖3-2 實驗裝置架設示意圖 .......................................................................... 45
圖3-3 未施加電場下,溶液靜置五分鐘後才加入鐵氰化鉀產生的冷光訊號
........................................................................................................................... 50
圖3-4 施加電場(30 V/cm)下,溶液輔助混合五分鐘後才加入鐵氰化鉀後產
生的冷光訊號 ................................................................................................... 50
圖3-5 luminol 受鐵氰化鉀催化反應機制圖 .................................................. 51
X
表目錄
表2-1 各pH 值下的濃縮倍率 ..................................................................... 28
表2-2 有效濃縮的濃度範圍其結果 ............................................................ 31
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