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研究生:陳建州
研究生(外文):Jian-Jhou Chen
論文名稱:應用光機電整合技術發展自動化生物分子電泳純化平台
論文名稱(外文):Development of an Automated Platform for the Electrophoresis-based Purification of a Biomolecule by Application of Optomechatronic Techniques
指導教授:陳林祈
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生物產業機電工程學研究所
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:2007
畢業學年度:95
語文別:中文
論文頁數:116
中文關鍵詞:電泳分離:光學偵測:自動化純化
外文關鍵詞:electrophoresis isolation:optical detection:automated purification
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生物分子純化是進行生化實驗的第一步,此步驟在生物科技領域中扮演著舉足輕重的角色,但其操作過程需要花費許多時間與人力,並且需要依賴熟練的技術與經驗,因此自動化的技術在純化應用上有相當大的發展空間。本研究嘗試應用管柱膠體電泳、光電偵測以及自動化液體處理裝置三大光機電技術,發展出一種即時性、高準確性及節省工作時間之自動化電泳純化平台。
在管柱膠體電泳研究中,以不同的實驗參數與條件進行分析以得到最佳電泳解析度,除此之外,以不同的元件規格設計電泳裝置機構使樣本集中泳動,繼而得到高解析度之電泳分離條件,而可與光電偵測系統整合。
光電偵測系統中以光二極體為感測器,紫外光為光源,並先以追蹤染劑(bromophenol blue)為模擬樣本進行電泳即時偵測實驗。在實驗過程中待追蹤染劑泳動至偵測區域,追蹤染劑會吸收紫外光導致光源送入光二極體的光訊號減少,因此可在偵測程式中產生一峰值曲線。後續實驗證明,此系統亦能有效地同步偵測混入追蹤染劑訊號之DNA markers的訊號,並擁有足夠的解析度。所以上述電泳及偵測兩系統可再與自動化液體處理裝置結合,發展出自動化生物分子電泳純化平台。
在追蹤染劑自動純化實驗中,藉由峰值偵測及追蹤染劑遷移率推算自動純化時間點,並以實驗佐證自動純化時間點的準確度。經實驗證明此自動純化系統之回收率可達到13.8%以上,並以40~45分鐘的時間達成自動純化的目的。
綜言之,本研究實證以光機電整合技術所發展之平台,可大量處理生物分子樣本電泳純化,並以自動化的方式實現。若此一技術成熟,人們只要將需純化的樣本置於自動化平台,再以程式設定所須之操作參數,便可啟動系統讓純化自動進行。這不僅可節省實驗時間與人力,更能促進生物產業效率。未來的後續研究工作者若能以本研究為基礎發展多通道或是微小化純化平台,將更能發揮本系統高通量與減少實驗樣本體積之功效。
Biomolecule purification is an essential step for molecular biology experiments. It is usually a time-consuming and work-consuming process, and the yield of purification is subject to an operator’s experience. Therefore, automation that features labor-saving property and high reproducibility becomes more and more important for biotech industry.
By the application of optomechatronic techniques, this study integrates tubular gel electrophoresis, optical sensor, and automated liquid handling system to develop a real-time, automated platform for the electrophoresis-based purification of a target biomolecule.
For tubular gel electrophoresis, different electrophoresis conditions and device designs were compared and studied to achieve the best electrophoresis resolution. With optimal device design and operating conditions that minimize the sample-diffusion problem, high resolution of the tubular gel electrophoresis for both tracking dye and DNA markers was attained and could thus be integrated with an optical detection system. The optical detection system used a UV lamp as the light source, a photodiode as the detector of light absorption by biomolecules, and an amplifying circuit to process the detected optical signal. In the beginning, a tracking dye (bromophenol blue) was used to determine an appropriate electrophoresis condition for real-time optical detection. In practice, when a tracking dye migrates into the detection window, it absorbs ultraviolet light thus decreases the optical signal output of the photodiode. The experiment of results show that the optical detection system is able to detect each individual DNA marker during electrophoresis separation. Both the tubular gel electrophoresis and optical detection module are well-integrated and well-suited to develop the automated platform for electrophoresis-based purification of target biomolecules with an automated liquid handling system.
For automated tracking dye purification, the aspiration time is estimated by the peak position and mobility of the dye. The accuracy of this estimation has been verified by different experiments. It has been found that the automated purification system can provide a recovery rate of ca. 13.8 % and take 40~45 minutes to accomplish the purification.
The system developed in this thesis work is to be compatible with multi-channel electrophoresis or miniaturized device. Therefore, it can serve as a basis of high-throughput purification of samples and is thus beneficial to both acedemic workers and biotech-orientated industries.
口試委員審定書
誌謝
中文摘要………………………………………………………… i
Abstract………………………………………………………… iii
目錄 v
圖目錄…………………………………………………………… vii
表目錄…………………………………………………………… xiii
符號說明………………………………………………………… xiv
第一章 緒論…………………………………………………… 1
1.1 研究背景……………………………………………… 1
1.2 研究方法及架構……………………………………… 2
1.3 貢獻及本文大綱……………………………………… 4
第二章 原理與文獻回顧……………………………………… 5
2.1 生物分子電泳分析技術……………………………… 5
2.2 生物分子偵測………………………………………… 11
2.3 生物分子純化………………………………………… 20
2.4 自動化液體處理裝置簡介…………………………… 22
第三章 研究方法……………………………………………… 25
3.1 實驗藥品與材料……………………………………… 25
3.2 實驗儀器……………………………………………… 26
3.3 實驗方法……………………………………………… 27
第四章 管柱膠體電泳實驗與電泳裝置設計………………… 32
4.1 前言…………………………………………………… 32
4.2 電泳膠體製備………………………………………… 32
4.3 追蹤染劑電泳實驗…………………………………… 33
4.4 DNA marker電泳實驗………………………………… 39
4.5 小結…………………………………………………… 46
第五章 生物分子電泳即時光電偵測系統…………………… 49
5.1 光學感測元件介紹與偵測理論……………………… 49
5.2 光電即時偵測系統實驗……………………………… 52
5.3 小結……………..…………………………………… 69
第六章 系統整合與自動電泳純化…………………………… 71
6.1 前言…………………………………………………… 71
6.2 自動化液體處理裝置工作表設計…………………… 71
6.3 追蹤染劑汲取實驗..………………………………… 82
6.4 小結…………………………………………………… 93
第七章 總結…………………………………………………… 94
7.1 總結…………………………………………………… 94
7.2 未來展望……………………………………………… 96
參考文獻………………………………………………………… 97
附錄一 裝置設計圖……………………………………………… 99
附錄二 程式碼…………………………………………………… 101
附錄三 核酸分子自動純化……………………………………… 115
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