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研究生： 陳泓成
Hong-Cheng Chen
論文名稱： 利用SPRi和FET探討中性DNA與一般DNA探針於生物晶片應用上之價值
The Value of Using Neutral DNA Probes on Biosensor Comparing with Regular DNA Probes Depicted by SPRi & FET
指導教授： 陳文逸
Wen-Yih Chen
口試委員:
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程與材料工程學系
Department of Chemical & Materials Engineering
畢業學年度： 100
語文別： 中文
論文頁數： 116
中文關鍵詞： 表面電漿共振影像儀 、場效電晶體生物感測器 、基因晶片 、不帶電DNA探針
外文關鍵詞： field effect transistor sensors, Neutral DNA probe, Gene chip, surface plasmon resonance imaging
相關次數： 點閱：18  下載：0 
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    • 基因晶片是應用於基因功能研究中非常有用的工具，但對使用者以及研發者而言，傳統的DNA晶片仍然有能被改善的空間。近年來，有需多研究團隊致力於改善基因晶片的性質，如檢測靈敏度、序列辨識能力、檢測速度以及操作範圍...等等。大量的研究利用檢測技術、晶片表面製備或探針固定方法來改良基因晶片，而在這當中，感測探針的性質是關鍵因素之一。
    中性DNA是一種藉由修飾後，使骨架不帶電的DNA類似物，它在與互補股雜交時因為不會有靜電排斥，所以相較於一般DNA，中性DNA和其互補股會有較強的雜交能力，且也由於不會產生靜電排斥，所以中性DNA可以在低鹽或是無鹽的條件下進行雜交。此外，也有文獻指出以中性DNA做為表面探針時，其辨識單一不互補鹼基對之能力是較強的，因此使用中性DNA做為感測器之表面探針相當具有發展潛力。
    在本研究中，我們將會利用表面電漿共振影像儀(SPRi)以及場效電晶體生物感測器(FET)驗證中性DNA表面探針之應用價值。我們藉由比較兩種不同探針的生物晶片表面和互補股之吸附的行為去比較兩種不同表面探針在應用上檢測的靈敏度，以及去了解表面探針和互補股之交互作用行為，並進一步去探討當中影響互補股吸附量的原因。
    本研究藉由表面電漿共振影像儀以及場效電晶體生物感測器觀測表面探針和互補股間之交互作用行為，成功驗證了以中性DNA作為表面探針之應用價值。並且進一步去證明了使中性DNA探針提供較佳敏感度之原因是因中性DNA的雜交效率較好以及中性DNA探針的固定量較多所造成。

    DNA biosensors based on sequence-specific DNA hybridization have been widely used in clinical diagnostics, forensic sciences and biomedical research. In recent years intensive efforts have been focused on the development of ultrasensitive DNA biosensors capable of quantitative gene expression analysis. A large number of detection techniques and sensor surface preparation or probe immobilization methods have been developed in order to maximize the sensitivity and specificity. Nature of the sensing probe is one of the key elements generally dictating the performance of the sensor.
    Neutral DNA is an uncharged DNA analogue. It’s electrically neutral is due to the backbone modification. No electrostatic repulsive during the hybridization between neutral DNA probe and regular DNA from sample which makes the hybridization efficiency of the neutral DNA better than regular one. Here in the study, we adopted SPR and FET biosensor systems for validating the value of the neutral DNA on non-labeling gene chip applications.

    中文摘要 I Abstract III 誌謝 IV 目錄 V 圖目錄 VIII 表目錄 XII 第一章 緒論 1 第二章 文獻回顧 2 2.1 生物晶片 2 2.1.1 基因晶片 2 2.1.1.1 基因檢測技術 3 2.2 去氧核醣核酸 4 2.2.1 去氧核醣核酸結構 4 2.2.2 核酸類似物 8 2.3 生物感測器 11 2.3.1 表面電漿共振儀 12 2.3.1.1 表面電漿現象原理 14 2.3.1.2 表面電漿共振儀分類 19 2.3.1.3 表面電漿共振影像儀 23 2.3.2 奈米線場效電晶體生物感測器 27 2.3.2.1 奈米線場效電晶體生物感測器檢測原理 28 2.3.2.2 鹽離子濃度對檢測之影響 33 2.3.2.3 表面配體電性對檢測之影響 36 2.4 晶片改質 40 2.4.1 表面分子之固定化 42 2.4.1.1 共價鍵結 43 2.4.1.2 生物親和性 45 第三章 實驗儀器、方法與材料 47 3.1 實驗藥品 47 3.2 儀器設備 50 3.3 實驗方法 53 3.3.1 緩衝溶液配製 53 3.3.2 CD實驗 53 3.3.3 ESCA實驗 54 3.3.4 FET實驗 54 3.3.4.1 FET晶片改質 54 3.3.4.2 FET流體檢測 55 3.3.5 SPRi實驗 56 3.3.5.1 SPRi生物晶片製備 56 3.3.5.2 SPRi晶片改質 56 3.3.5.3 SPR流體檢測 56 3.3.6 高通量SPRi實驗 57 3.3.6.1 DNA 探針之固定化 57 3.3.6.2 DNA雜交實驗 58 第四章 結果與討論 59 4.1核酸序列二級結構模擬之結果 59 4.2 不帶電DNA專一性雜交之鑑定 63 4.3 表面探針固定化之鑑定 67 4.4 中性DNA探針應用於FET上優勢之探討 71 4.5 利用SPRi探討中性DNA探針提供較佳檢測靈敏度原因 79 4.6 中性DNA探針應用於高通量SPRi實驗上優勢之探討 84 4.6.1 不同甘油含量對探針和互補股DNA雜交影響之探討 85 4.6.2 不同濃度互補股DNA下之表面吸附情況探討 89 第五章 結論 92 第六章 參考文獻 94

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