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研究生:吳靜玫
研究生(外文):Ching-Mei Wu
論文名稱:利用蛋白質感應片及表面電漿共振技術發展偵測金屬離子之系統
論文名稱(外文):Application of Protein-based Biosensor Chips and Surface Plasmon Resonance Technology for the Detection and Quantification of Metal Ions
指導教授:林立元林立元引用關係
指導教授(外文):Lih-Yuan Lin
學位類別:碩士
校院名稱:國立清華大學
系所名稱:分子與細胞生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:76
中文關鍵詞:金屬硫蛋白血清白蛋白重金屬生物感應器表面電漿共振
外文關鍵詞:metallothioneinserum albuminheavy metalbiosensorsurface plasmon resonance
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金屬硫蛋白與血清白蛋白是生物體內能與重金屬結合的兩種主要蛋白質,在重金屬的傳送、儲存及解毒等功能上扮演著重要的角色。近年來,重金屬污染已成為一種全球性的危害,因此,一個快速、有效的偵測方法是迫切需要的。在本實驗中,我們嘗試建立蛋白質生物感應片偵測系統,配合表面電漿共振技術來偵測並定量重金屬離子。本實驗分兩個部分,第一部分我們先將金屬硫蛋白吸附在CM5感應片上,其最佳吸附能力之pH值為4。金屬硫蛋白感應片在30℃時,與金屬有較好的結合情形,而流速則對結合力沒有影響。此外,感應片對不同金屬也有區別性,與鎘、鋅和鎳等金屬具結合能力,但對錳、鎂和鈣則沒有。而鎘、鋅和鎳等金屬在不同濃度範圍內與金屬硫蛋白感應片的結合能力有很好的相關性,其中,鎘的偵測極限可達1 �嵱。另外,緩衝溶液中的NaCl、pH值及 Tween 20等成分都會影響金屬和金屬硫蛋白感應片的結合情形,當NaCl濃度將低至1 mM時,只能和鎘結合,而無法與鋅和鎳結合。此外,在分子之間的結合動力學研究中發現金屬硫蛋白感應片對金屬的親和力為鎘>鋅>鎳。因此金屬硫蛋白感應片不但可以評估和金屬結合的特性,也適合發展成為偵測及定量重金屬的生物感應器。
在第二部份,我們改將血清白蛋白吸附在CM5感應片上,其最佳吸附能力之pH值為5。系統的溫度及緩衝溶液中的成分都會影響金屬和感應片的結合情形。在25℃、1 mM NaCl環境中,感應片與鎘、鋅和鎳等金屬有最佳結合能力,而與錳、鎂和鈣結合能力較差。血清白蛋白感應片可以偵測不同濃度範圍的鎘、鋅和鎳等金屬,其濃度與結合能力有很好的相關性。其中,對鎘、鋅和鎳等金屬的偵測極限可達0.01 uM∼0.1 uM。在競爭實驗中,我們發現鋅和鎳會降低20∼30%鎘的結合能力,而鋅和鎳彼此則無競爭現象發生。同時,在動力學研究發現血清白蛋白感應片對金屬的親和力為鎳>鋅>鎘。此外,血清白蛋白感應片也可以經由簡單的管柱分離而順利的偵測到血清樣品中鎘的濃度。因此血清白蛋白感應片除了可以評估和金屬結合的特性,也適合發展成為偵測及定量溶液中及生物體內重金屬的生物感應器。
Metallothionein (MT) and serum albumin are two important metal-binding proteins, play roles in metal transport, storage and detoxification in organism. In this study, we attempt to establish protein-based biosensors for the detection of metal ions by surface plasmon resonance. In the first part, rabbit MT was immobilized onto a carboxymethylated dextran matrix (CM5 chip). The optimal pH for the immobilization was determined at 4, and the highest interaction temperature was observed at 30oC. The sensor chip binds cadmium, zinc or nickel, but not magnesium, manganese and calcium. Calibration curves for the quantification of metal ions showed excellent linearity. The sensitivity for metal detection was at the micromolar level. The interaction between the metal ions and the sensor chip was significantly influenced by the presence of NaCl, Tween 20 and the pH of the reaction buffer. In the environment containing 1 mM NaCl, the MT chip effectively differentiated cadmium from zinc and nickel. In displacement analysis, zinc and nickel did not affect cadmium-MT interaction. The binding affinity between the metal ions and the immobilized MT follows the order of cadmium > zinc > nickel.
In the second part, bovine serum albumin was immobilized onto a CM5 chip. The optimal pH for the immobilization was determined at 5, and the highest interaction temperature was found at 25oC. The interaction between the metal ions and the sensor chip was significantly influenced by the reaction buffer. The optimal buffer condition used for the analysis contains 1 mM NaCl, 0.005% Tween-20 and 0.01 M HEPES, pH 7.4. Using this condition, a linear calibration curve can be established within the range of 10-8 to 10-4 M for metals. The sensitivity for cadmium, zinc and nickel detection were at the 0.01~0.1 uM. When measuring the solution containing two species of metal ions by the albumin chip, zinc and nickel were able to reduce cadmium-albumin interaction about 20~30%. However, zinc and nickel show an additive in binding to the chip when both metals are mixed together. The binding affinity between the metal ions and the immobilized albumin follows the order of nickel > zinc > cadmium. A procedure was also developed to analyze cadmium content in the serum using the albumin-based sensor chip. Results from our studies show that the protein-based biosensor can be effectively used for the detection and measurement of metal ions in the solution and in biological sample.
目次
中文摘要...............................................1
英文摘要...............................................3

第一部份 以金屬硫蛋白感應片偵測金屬離子
序言................................................5
材料與方法..........................................12
結果................................................16
討論................................................22
參考資料............................................27
附圖................................................32
附表................................................42

第二部份 以血清白蛋白感應片偵測金屬離子
序言................................................43
材料與方法..........................................47
結果................................................51
討論................................................56
參考資料............................................61
附圖................................................65
附表................................................76
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