利用金奈米粒子探針搭配分子及原子光譜儀測定汞離子的分析研究

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研究生：	劉益宏 Liu, Yi-Hung
論文名稱：	利用金奈米粒子探針搭配分子及原子光譜儀測定汞離子的分析研究 Determination of Mercuric Ion Using Oligonucleotide-Gold Nanoparticle Conjugates Coupled with Molecular and Atomic Spectrometric Detection
指導教授：	孫毓璋 Sun, Yuh-Chang
口試委員:	楊末雄 Yang, Mo-Hsiung 胡焯淳 Hu, Cho-Chun 邱泰嘉 Chiu, Tai-Chia 孫毓璋 Sun, Yuh-Chang
學位類別：	碩士 Master
系所名稱：	原子科學院 - 生醫工程與環境科學系 Department of Biomedical Engineering and Environmental Sciences
論文出版年：	2011
畢業學年度：	99
語文別：	中文
論文頁數：	113
中文關鍵詞：	汞離子、金奈米粒子、原子吸收光譜法、分子吸收光譜法
外文關鍵詞：	Mercury, Gold-nanoparticle, ET-AAS, Plate reader
相關次數：	點閱：1 下載：0
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在自然界中汞存在的化學型態包含金屬、無機與有機等三種型態。汞是一種毒性極高的元素，汞不但可破壞細胞膜、使粒線體受損、並抑制細胞中DNA的複製。此外，汞也會對腦、心臟、腎臟、腸與胃造成傷害。由於汞離子的水溶性極高，因此極容易透過飲食被攝入人體中，為確認不同環境水樣中汞的濃度，開發具高靈敏度與選擇性的環境水樣中汞離子檢測方法，一直是分析化學界中非常重要的課題。
　　為建立環境水樣中汞離子的奈米偵測技術，本研究中已成功地發展出兩套具高靈敏度與選擇性的奈米偵測技術。在第一部分的研究中，本研究使用磁性微米粒子(MMP)、寡核甘酸序列與金奈米粒子之三明治結構，搭配盤式吸收螢光分析儀與電熱式原子吸收光譜儀進行汞離子的偵測。為辨識樣品中的汞離子，本研究成功地設計了一個包含T-T mismatch的DNA序列，選擇性地進行水樣中汞離子的捕獲。根據本研究的觀察，當有汞離子存在於樣品中時，三明治結構須較高的溫度才會開始崩散(disperse)；此外，不同濃度的汞離子則會有不同的崩散溫度(disperse temperature)。為提升分析系統整體的靈敏度，本研究也設計了不同T-T mismatch位置的DNA序列，期望能藉此選擇到具有最大崩散溫度變化量的DNA序列。根據實驗結果可知，當使用1078-4th的序列來形成三明治結構時，能得到最大的溫度變化值(5.3℃)。為達到方便及高靈敏分析的目的，本研究最終係利用盤式吸收螢光分析儀進行測定，在最佳化的實驗條件下，本研究所得到的方法偵測極限為0.67 nM (0.134 □g L-1)。
在第二部份的工作中，本研究使用Hg2+ apatmer搭配金奈米粒子，開發了一套操作簡單又快速的汞離子分析方法。本研究中係藉由Hg2+ aptamer ，磁性微米粒子(MMP)與金奈米粒子(AuNPs)三者間所形成的三明治結構，再搭配盤式吸收螢光分析儀與電熱式原子吸收光譜儀進行汞離子的偵測。在測定過程中，當樣品中含有一定濃度的汞離子時，本研究所設計三明治結構中的Hg2+ apatmer，會因為與汞離子作用的關係，而發生folding的現象，並造成三明治結構中的金奈米粒子脫落，此時，即可利用盤式吸收螢光分析儀或電熱式原子吸收光譜儀進行測定。為達到高靈敏分析的目的，本研究最終係利用電熱式原子吸收光譜儀進行測定，在最佳化的實驗條件下，本方法之最低可鑑別濃度為20 nM (4.0 □g L-1)。

Mercury is a toxic element that exists in metallic, inorganic, and organic forms. Among which, the toxicity of Hg2+ is well-known and can cause the disruption of cell membranes, the impairment of mitochondrial function, and the inhibition of DNA replication in a cell. In addition, it also damages the organs like brain, heart, kidney, stomach, and intestines. Hg2+ is easily ingested by human beings due to its high water solubility; therefore, it should be desirable to develop a sensitive and selective method which is able to detect and quantify Hg2+ existing in environmental waters.
To explore the possibility of using nanosensing techniques for the detection of trace Hg2+ ions in aqueous samples, we have developed two independent methods in this study. In the first experiment, a sandwich structure containing magnetic microparticles (MMPs), duplex sequences, and gold nanoparticle (AuNPs) coupled with UV/Vis spectrophotometric and ET-AAS was employed to determine mercuric ion. In which, the duplex sequence was designed with one thymine-thymine (T-T) mismatch for the purpose to specific recognize the mercuric ions. Based on our observations, as higher concentration of mercuric ion is present in the samples, the melting temperature (Tm) of duplex hybrids in the sandwich structure would shift to higher temperature; moreover, different concentrations of mercuric ions could result in different Tm. To utilize this phenomenon to determine mercuric ion and improve its sensitivity, we altered the positions of the T-T mismatch within the duplex to enhance the Tm shift with the participation of Hg2+ as well. In our developed analytical procedure, we found the utility of 1078-4th double helix structure can reach the largest melting temperature shift (5.3℃). To separate the “sandwich” structures containing T-T and T-Hg2+-T, individually, a higher hybridization temperature (60℃) was used to remove through the dissociation of multiplexes containing T-T structure. Thereafter, the AuNPs- oligonucleotide sequences conjugates containing T-Hg2+-T base pairs were collected and determined by UV/Vis spectrophotometry through the AuNPs absorption. Under the optimized condition, we found that Hg2+ concentration of 0.67 nM (0.134 □g L-1) could be measured with sufficient reliability.
In the second experiment, we demonstrate that functional DNA-linked gold nanoparticles (AuNPs) can quickly, simply detect Hg2+ ions in aqueous solution. A linker DNA molecule which contains thymine residues and is complementary to the DNA sequences on the AuNPs was designed to form sandwich structures by react with magnetic microparticle probes. When Hg2+ ions were present in the sample, Hg2+ ions can cause the Hg2+ aptamer DNA sequences to fold by forming thymine-Hg2+-thymine bonds. Thereafter, the folding of Hg2+ aptamer DNA can unwind the AuNPs probe rapidly and disassemble the sandwich structure. To detection the released AuNPs probes, we tried to use UV/Vis spectrophotometer and ET-AAS as the end-determination means. Based on the experimental results, the lowest distinguishable concentration was 20 nM (4.0 □g L-1) through the use of ET-AAS.

目錄
中文摘要...........................................................................................    I
英文摘要...........................................................................................    III
目錄...................................................................................................    V
圖目錄...............................................................................................    X
表目錄...............................................................................................    XV

第一章    前言................................................................................    1
1    研究背景........................................................................    1
1.1    汞物種的物理及化學特性............................................    1
1.2    汞的來源、健康效應與環境之影響..............................    2
2    微量元素分析技術........................................................    10
2.1    傳統汞分析技術發展現況............................................    11
3    汞離子生物感測器發展現況........................................    12
3.1    汞離子之寡聚核苷酸分子感測器發展現況................    13
4    研究目的與方法............................................................    23
第二章    儀器分析及原理............................................................    27
1    紫外光可見光吸收光譜儀............................................    27
2    電熱式原子吸收光譜儀................................................    29
3    盤式吸收螢光分析儀....................................................    42
3.1    吸收分析儀....................................................................    43
第三章    實驗部分........................................................................    45
1    試劑................................................................................    45
2    藥品配製........................................................................    46
3１    寡聚核苷酸探針(Oligonucleotide probes)和標的寡聚核苷酸(Target oligonucleotide)的設計.....................    47
4    DNA修飾之磁性微米粒子製備..................................    49
5    金奈米粒子探針的製備................................................    51
6    MMP/Bridge/Au-NPs最佳化條件之探討....................    54
6.1    MMP/ Bridge /Au-NPs雜和條件..................................    54
6.2    不同序列Melting temperature (Tm)值測定..................    55
6.3    汞離子反應時間對訊號之影響....................................    57
6.4    加熱時間對金奈米粒子訊號分離之影響....................    58
6.5    清洗次數對背景訊號之影響........................................    58
6.6    使用20 nm金奈米粒子之分析效能測試....................    58
6.7    T-T mismatch 對於各種金屬離子之選擇性測試.......    59
6.8    濃縮次數對訊號之影響................................................    60
6.9    濃縮對20 nm金奈米粒子分析效能測試....................    60
6.10    環境樣品之測定............................................................    60
7    MMP/ Aptamer/Au-NPs最佳化條件之探討................    61
7.1    20 nm金奈米粒子Hg2+ aptamer接合條數之探討…..    62
7.2我    20 nm 金奈米粒子Hg2+ aptamer接合條數對清洗次數之影響........................................................................    63
8    盤式吸收螢光分析儀之分析效能測試........................    63
8.1１    汞離子反應時間對訊號之探討....................................    63
8.2１    20 nm金奈米粒子Hg2+ aptamer之分析效能測試......    65
9    電熱式原子吸收光譜儀之分析效能測試....................    65
9.1１    汞離子反應時間對訊號之探討....................................    65
9.2１    20 nm金奈米粒子Hg2+ aptamer之分析效能測試......    67
9.3    Hg2+ aptamer對於各種金屬離子之選擇性測試..........    67
9.4    Hg2+ aptamer對環境樣品之測定..................................    68
9.5    Hg2+ aptamer對鎂離子與氯離子訊號抑制之測定......    68
10我    電熱式原子吸收光譜儀之最佳操作條件以及溫度程式...................................................................................    69
第四章    結果與討論....................................................................    71
第一部份    利用金奈米粒子探針搭配DNA序列設計及光學感測器測定汞離子的分析研究........................................    71
1    MMP/Bridge/Au-NPs最佳化條件之探討....................    72
1.1    MMP/Bridge/Au-NPs雜合最佳化條件之探討............    72
1.2    不同DNA序列Melting temperature (Tm)值的測定….    74
1.3    汞離子反應時間對訊號之影響....................................    76
1.4    不含汞情況下，加熱時間對訊號之影響......................    78
1.5    清洗次數對背景訊號之影響………………………....    79
1.6    分析效能測試................................................................    81
1.7    T-T mismatch 對於各種金屬離子之選擇性測試…...    83
1.8    訊號放大次數對靈敏度之影響....................................    84
1.9    多次反應對分析效能的影響........................................    85
1.10    真實樣品測定................................................................    87
第二部份    利用金奈米粒子搭配Hg2+ aptamer及電熱式原子吸收光譜儀測定汞離子的分析研究................................    89
2    MMP/Hg2+ aptamer/Au-NPs最佳化條件之探討.........    90
2.1    20 nm金奈米粒子Hg2+ aptamer接合條數之探討......    90
2.2    清洗次數對背景訊號之影響........................................    92
3    盤式吸收螢光分析儀之分析效能測試........................    94
3.1    汞離子反應時間對訊號之影響....................................    94
3.2    分析效能測試................................................................    96
4    電熱式原子吸收光譜儀之分析效能測試....................    98
4.1    汞離子反應時間對訊號之影響....................................    98
4.2    分析效能測試................................................................    99
4.3    Hg2+ aptamer對於各種金屬離子之選擇性測試..........    100
4.4    Hg2+ aptamer對環境樣品之測定..................................    101
4.5    Hg2+ aptamer對鎂離子與氯離子訊號抑制之測定......    103
第五章    結論................................................................................    105
第六章    參考文獻........................................................................    107
第七章    附錄................................................................................    113

                                

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