本研究發展出一套數位調變式光源系統，利用一LCD投影機取代傳統螢光顯微鏡的汞燈光源，藉由內部兩片分光鏡與多片反射鏡將汞燈光源分成RGB三原色，並配合由電腦操控作為遮光層的LCD面板來做即時的切換，並進行晶片電泳的螢光偵測，再由後端的光譜儀與電腦進行光訊號的轉換、紀錄與分析。本系統最大的優點在於可隨意切換所需之激發光波段，也捨棄使用傳統濾鏡輪（filter wheel）切換激發光的複雜機構，亦避開震動所造成的影響。利用不同激發和發射波長的螢光染劑（Atto 647N, Rhodamine B, FITC），測試此系統在不同螢光發射波段的最佳偵測波長，並利用此架構偵測一混合螢光染料，成功且有效的於單次單管的電泳實驗中將三種螢光訊號分離出來。研究中，亦利用投影機中特殊的混色光源-紫光，此光同時帶有兩種單色光的特徵波長，並進行兩種混合螢光染料的電泳偵測，實驗結果顯示此兩種螢光樣本可同時且成功的偵測與分析。最後，將本系統應用於單股DNA（single-strand DNA）的生物樣本，可成功偵測其螢光訊號並分析之。本系統成功\展示出數位調變光源的概念，未來利用即時調變技術，並配合偵測後的訊號分析，將可應用在多樣本螢光偵測的電泳技術上，並增加偵測及分析速度。

This research has successfully developed a multiple fluorescence detection method for high throughput capillary electrophoresis detection using a digitally-modulated light source and a spectrum detection system. A commercial available LCD (liquid crystal device) projector is adoped to replace the spacially-filttered light source (Hg lamp) in a conventional fluorescence microscopy. The LCD projector can be digitally controlled by a computer to create the three primary colors of RGB (red, green, and blue) for fluorescence excitation in the analytes. The emitted light from the fluorescent samples is then collected using a UV-VIS-NIR spectrometer through a ultimode fiber. Delicate optical components, such as filter wheel or acousto-optic filtering system, for filtering different excitation light sources can be excluded with this simple and novel approach. In addition, the desired wavelength for the excitation light can be selected quickly and smoothly without vibration problems come with the mechanical optical components. Three fluorescent dyes (Atto 647N, Rhodamine B, Fluorescein) with different excitation and emission wavelength has been used to demonstrate the proposed digitally-modulated light source system for high throughput CE system. The optimal operation conditions for obtaining best detection signal-to-noise ratio for different fluorescence dyes are firstly determined. In addition, the current study proposes a mixed-color light (visually in purple) composed of two specific primary lights (red and blue) to simultaneously excite a mixed sample composed of two fluorescent dyes (Atto 647N and FITC). Separation and detection of the mixed fluoresce samples using a single excitation illumination using the proposed digital-modulated CE system is successfully demonstrated. Finally, a single-strand DNA biosample is used to confirmed the proposed system is feasible of adopting in the bio-analytical applications. The technique proposed in this study has shown its potential to be a high throughput CE detection system.

目錄...........................................................................................................I
圖目錄......................................................................................................IV
表目錄...................................................................................................VII
中文摘要……………………………………………………….……VIII
Abstract……………………………………………………………….IX
第一章 緒論…………………………………………………………1
1-1 微全分析系統 （Micro Total Analysis System, μ-TAS）…1
1-2 毛細管電泳的發展………………………………………...2
1-3 平行化偵測（Parallel Detection）毛細管電泳的發展……5
1-4 研究動機與目的…………………………………………...7
1-5 論文架構…………………………………………….……10
第二章 理論基礎與文獻回顧………………………..……………12
2-1 毛細管電泳的原理…………………………………….....12
2-1-1 電泳現象 （Electrophoresis Phenomenon）……..13
2-1-2 電滲現象 （Electroosmosis Phenomenon）………15
2-2 螢光的理論與應用…………………………….…………20
2-3 現有的多樣本螢光偵測技術…………………………….24
2-4 調變式濾鏡應用於多重螢光偵測之優點……………….40
第三章 實驗架構與方法…………………………………….…….42
3-1 晶片製作………………………………………………….42
3-1-1 光罩製作……………………………………...43
3-1-2 晶片清洗……………………………………...43
3-1-3 微影…………………………………………...44
3-1-4 蝕刻…………………………………………...45
3-1-5 鑽孔…………………………………………...45
3-1-6 晶片接合……………………………………...46
3-2 實驗藥品………………………………………………..47
3-3 實驗架設………………………………………………..48
3-4 實驗參數………………………………………………..49
第四章 結果與討論………………………………………………..54
4-1 訊號與雜訊比（Signal to Noise Ratio, SNR）的分析…54
4-2 混合染料測試………………………………………….…64
4-3 混合光源測試…………………………………………….65
4-4 生物樣本測試…………………………………………….67
第五章 結論與未來展望………………………………..…………69
5-1 結論………………………………………………….……69
5-2 未來展望………………………………………….………70
參考文獻…………………………..……………………………………73
圖目錄
圖2.1 毛細管電泳裝置圖…………..………………………….………13
圖2.2 矽醇基解離示意圖………………..…………………….………15
圖2.3 電雙層與電位分佈示意圖…………………………...…………16
圖2.4 電解液受電場影響所產生之流動……………………...………17
圖2.5 (A)由壓力流推動的拋物線流動剖面 (B)由電滲透流推動的平
面流動剖面..…………………………....………………………..18
圖2.6 電泳及電滲流對帶電荷或中性粒子在毛細管電泳的影響…...20
圖2.7 激發光與發射光強弱之對照圖………….………….………….22
圖2.8 螢光顯微鏡之元件與光路圖……………………..……………..24
圖2.9 利用多管光纖導入激發光之多管道毛細管電泳偵測架構…...26
圖2.10 利用雷射光源結合菱鏡組之多管道毛細管電泳偵測架構…..26
圖2.11 利用馬達旋轉掃描之多管道毛細管偵測架構………………..27
圖2.12 利用兩組平凸透鏡之多管道毛細管電泳偵測架構………..…27
圖2.13 共焦掃描式多管道毛細管電泳偵測架構…..……………..…..28
圖2.14 利用邊鞘流結合微細加工之多管道毛細管電泳偵測架構…..28
圖2.15 利用光纖結合PDMS 製程之多波長毛細管電泳系統架構….30
圖2.16 利用多波長脈衝激發之毛細管電泳系統架構…………..……30
圖2.17 市售商業化濾鏡輪實體圖…………………………………….32
圖2.18 利用濾鏡輪篩選激發光之多樣本螢光偵測架構圖….......…..32
圖2.19 利用濾鏡輪篩選發射光之多樣本螢光偵測架構圖........….…33
圖2.20 AOTF 剖面圖與繞射過程……..……..………………...………35
圖2.21 利用AOD於多管道多波長的偵測實驗架構圖…….....……..37
圖2.22 利用AOTF 於單管道多波長的偵測架構圖…………....…….37
圖3.1 晶片製程示意圖……………………….………………………..42
圖3.2 晶片實體圖…………………………………….………………..46
圖3.3 系統架設與光路示意圖……………………….………………..49
圖3.4 RGB 三色16 段色階選用圖……………………..………………50
圖3.5 RGB 16 色階與光強度關係圖…………………….…………….51
圖3.6 實驗所選用的色階與光強度關係圖……………….…………..52
圖3.7 紫光光譜與光強度關係圖………………………….…………..52
圖3.8 double-L type….……...……….…………………………………53
圖4.1 Atto 647N 於濃度10-4 M 時各波段的電泳圖………….……….55
圖4.2 Atto 647N 於濃度10-4 M時各波段的細部電泳圖及SNR值…..57
圖4.3 Rhodamine B 於濃度5×10-5 M時各波段的電泳圖………….…58
圖4.4 Rhodamine B 於濃度5×10-5 M時各波段的細部電泳圖及SNR 值
..………..………………………………………………………..59
圖4.5 Fluorescein 在濃度5×10-5 M時各波段的電泳圖………………60
圖4.6 Fluorescein 於濃度5×10-5 M 時各波段的細部電泳圖及SNR 值
…………………………………………………………………61
圖4.7 三染料於各波段的SNR 比較圖……….………..………...…….62
圖4.8 單管單次偵測三種染料之電泳圖………………………………65
圖4.9 紫光同時偵測Atto 647N 與Fluorescein 之電泳圖…..…………66
圖4.10 ssDNA（Fluorescein 標定）樣本於530 nm 收光波長之電泳圖
……………………………………………………….…………68
表目錄
表格一 螢光發射於各階段之概要時間範圍…………………………23

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