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研究生： 黄莉雅
Li-Ya Huang
論文名稱： 利用表面電漿共振影像儀探討核酸共軛之蛋白質晶片最佳化研究
Optimization of DNA-Conjugate Protein Chip Studies by Surface Plasmon Resonance Image
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： 99
語文別： 中文
論文頁數： 98
中文關鍵詞： 生物感測器 、最佳化 、DNA蛋白質共軛物
外文關鍵詞： DNA-conjugate antibody, optimization, biosensor
相關次數： 點閱：5  下載：0 
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    • 近年來，蛋白質生物感測器廣泛的應用於環境監測、生物檢測或疾病診斷方面，但是因為蛋白質的不穩定性和不易保存，可能會隨著使用次數的增加而失去活性，進而影響檢測的準確性。為了改善這些問題，本研究加入了穩定性較高的去氧核醣核酸(deoxyribonucleic, DNA)作為生物感測器的元件之一，並使用表面電漿共振影像儀(surface plasmon resonance image, SPRi)觀測晶片表面蛋白質吸附情形來做最佳化研究之探討。
    利用乙烯乙二醇(ethylene glycol, EG)可抵抗蛋白質吸附的特性，製作寡聚乙烯乙二醇(oligo ethylene glycol-thiol, OEG-thiol)與終端為羧基的硫醇類EG分子(COOH-thiol)組成的混合自組裝單分子層膜(mixed SAMs)，接著將單股DNA修飾於mixed SAMs表面，再利用雜交的方式固定上修飾有互補股DNA的抗體，形成DNA共軛蛋白質的蛋白質晶片。藉由調控晶片上單股DNA的比例與鹼基數的多寡，減少蛋白質非專一性的吸附；調整再生溶劑的濃度或種類，使晶片能有良好的再現性；再改變緩衝溶液的酸鹼值，增加目標物的吸附，以降低偵測極限。研究發現：單股DNA佔表面2 %以及鹼基數為20個時，晶片表面的抗非專一性吸附效果及專一性結合能力都有很好的表面，1 N氫氧化鈉溶液可使晶片達92 %左右的再現性。這些結果證實最佳化研究的確可使蛋白質晶片擁有良好的精確性，且此種檢測方式可用於各種檢測上，可說具有相當的發展潛力。

    Protein-based biosensors are widely used for disease diagnoses, environmental monitoring, drug screening and etc... . However, the fabrication, stability, and accuracy of protein-based biosensors are usually affected by the decreasing biological activity of the immobilized protein. With the knowledge of DNA-based biosensors, in this study we utilized the ssDNA-protein conjugates to immobilize onto the complementary ssDNA-modified self-assembled monolayers (SAMs) through the help of the surface plasmon resonance image (SPRi) measurement. Thereby a DNA-based biosensor can be transformed into protein-based biosensor.
    The specificity and sensitivity of DNA-conjugate anti-HSA with its target were measured to demonstrate the feasibility of this platform. The mixed SAMs composed of oligo(ethylene glycol) (OEG) terminated thiols and carboxylic-functionalized OEG (COOH-OEG)termiated thiols, and DNA length were examined to obtain the optimized detection efficiency. Moreover, the regeneration conditions of protein-based biosensor were also discussed. The results revealed that the DNA length with 20 based pairs immobilized onto OEG:COOH-OEG = 50:1 mixed SAMs owns the high sensitivity and low non-specific binding. It could be also found that the using of 1N NaOH is able to dehybridize the immobilized protein-ssDNA, and the regeneration efficiency of protein-ssDNA biochip is about 90 %. Consequently, the optimized stability of protein biosensors seems to be feasible under the DNA mediation; and our approach provides important information on high sensitive protein-based biosensors.

    中文摘要 i Abstract ii 誌謝 iii 目錄 iv 圖目錄 vii 表目錄 xi 第一章 緒論 1 第二章 文獻回顧 3 2.1 表面電漿共振(Surface Plasmon Resonance，SPR) 3 2.1.1 表面電漿共振原理 4 2.2 生物感測器 10 2.2.1 生物感測器簡介 10 2.2.2 生物感測器分類 11 2.2.2.1 Immunosensor晶片 11 2.2.2.2 Enzyme晶片 13 2.2.2.3 Receptor-protein晶片 14 2.2.2.4 Whole cells晶片 16 2.2.2.5 DNA晶片 16 2.3 晶片改質 18 2.3.1 Langmuir-Blodgett薄膜 18 2.3.2 電漿處理 20 2.3.3 自組裝單層膜 22 2.3.4 影響自組裝單層膜之因素 25 2.3.4.1 反應溫度之影響 27 2.3.4.2 飽和碳鏈段長短之影響 29 2.3.4.3 硫醇類分子濃度之影響 29 2.3.4.4氫鍵形成之影響 30 2.3.5 表面分子之固定化 35 2.3.5.1 共價鍵結 35 2.3.5.2 生物親和性 (biochemical affinity) 38 第三章 實驗儀器、方法與材料 40 3.1 實驗藥品 40 3.2 儀器設備 43 3.3 實驗方法 44 3.3.1 緩衝溶液的配製 44 3.3.2 人類血清白蛋白與溶菌酶簡介 45 3.3.3 核酸蛋白質共軛物之配製 46 3.3.4 晶片改質 47 3.3.4.1 混合SAMs分子的選擇 47 3.3.4.2 SAMs溶劑的選擇 52 3.3.4.3 去氧核醣核酸固定化 54 3.3.4.4 晶片改質步驟 56 3.3.5 最佳化條件測試 57 3.3.5.1 DNA鹼基數多寡 58 3.3.5.2 Mixed SAMs比例 58 3.3.5.3 緩衝溶液條件 59 3.3.5.4 再生溶劑選擇 59 第四章 結果與討論 60 4.1 ESCA表面改質之鑑定 60 4.2 DNA鹼基數多寡對正電荷分子吸附之影響 65 4.3 Mixed SAM比例對非特異性吸附之影響 69 4.4 緩衝溶液條件 71 4.5 再生溶劑條件找尋 74 第五章 結論 77 第六章 參考文獻 79

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