汞離子是一種具有毒性的重金屬離子，會造成非常嚴重的健康及環境的問題。當汞離子累積在生物體內時，有破壞細胞膜及粒線體的功能，一般而言，人類攝入汞離子途徑通常是經由食物或是飲用水，當暴露在高濃度汞離子下，汞離子可經由尿液排泄出人體，因此測定飲用水及尿液中汞離子的濃度，可提供短期或長期汞暴露的訊息。
感應耦合電漿質譜法(ICP-MS)及冷蒸氣原子吸收光譜法(CV-AAS)係目前常用來測定環境以及生物樣品中的汞離子的分析方法，然由於感應耦合電漿質譜儀(ICP-MS)，具有儀器操作難度高及儀器價格昂貴等缺點，因此並不普及。冷蒸氣原子吸收光譜法雖具有操作間單及儀器低廉等優點，但受限於偵測極限(sub-μg/L)，及需使用多種環境不友善試劑（如硼氫化鈉和鹽酸），因此在分析濃度極低的樣品時，仍嫌不足。
近年來，已經證實汞離子可被組成DNA核□酸中的胸腺嘧啶(T)所辨認，根據此一反應，本研究的目的旨在具功能化磁性粒子(MMP)、互補DNA與20 nm功能化金奈米粒子的三明治結構(MMP/target/AuNPs sandwich structure)中，加入T-T mismatch的設計，代此三明治結構與汞離子反應完畢後，再經由溫度選擇來分離含汞及未含汞的三明治結構，接著再利用磁性分離及加熱的方式，將含有汞離子的三明治結構分離出來，並釋出金奈米粒子，最後係藉由電熱式原子吸收光譜儀(ET-AAS)測定金的訊號來換算樣品中汞離子的濃度。根據本研究所得到的實驗數據，當利用20 nm金奈米粒子當作探針時，所得到的方法偵測極限為93 ng/L。為進一步提升所開發方法的靈敏度，本研究中已成功地使用60 nm 金奈米粒子當作探針，並藉由(1)加大含汞及未含汞的三明治結構間Tm值的差距，(2)改變其melting temperature曲線升溫區的陡峭程度，及(3) 60 nm 金奈米粒子大幅度的訊號放大的能力，使得原分析系統的偵測極限值可大幅降低至5.6 ng/L。最後，為確認所建立分析方法的實用性，本研究中亦已成功地進行了環境水樣及人體尿液中汞離子的分析。

Mercury is a highly toxic element that is found both naturally and as an introduced contaminant in the environment. In light of its severe toxicity, a variety of adverse effects, such as the disruption of cell membranes, the impairment of mitochondrial function, and the inhibition of DNA replication in a cell, are considered highly relevant to Hg2+. In addition, it can also damage the normal functions of organs, like brain, heart, kidney, stomach, and intestines. Generally, the concentration of mercury in natural waters is extremely low. Therefore, it should be desirable to develop a sensitive and selective method for the determination of Hg2+ in the environmental water.
In the present study, a new method was developed using oligonucleotide−gold nanoparticle conjugates coupled with ET-AAS to determine mercury ion in water samples. To “sandwich” structure, three complementary sequences were designed with two thymine- thymine (T-T) mismatches. Meanwhile, two particles－magnetic microparticle (MMP) and 20 nm gold nanoparticle (AuNPs)－were used as capture and reporter probes after their conjugation with certain oligonucleotide sequences. In our developed analytical procedure, mercury could be tightly bound by the thymine – thymine (T-T) mismatches by the way of the formation of T-Hg2+-T complex and the melting temperature of this double helix structure (56.6℃) could be increased significantly. To separate the “sandwich” structures containing T-T and T-Hg2+-T, individually, a higher hybridization temperature (59℃) 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 ET-AAS through the gold signal. Under the optimized condition, we found that mercury concentration of 0.5 nM(0.1 □g/L) could be measured with sufficient reliability, and a limit of detection (LOD) of 93 ng/L.
In addition, larger gold nanoparticles (60 nm) were also employed in this experiment for the purpose to enhance the analysis system sensitivity. We found out using larger gold nanoparticles not only let ∆Tm(T-T and T-Hg2+-T) largely, but also let the melting temperature curve much more sharply. The two properties could enhance the analysis system more selectively, in addition, We found out using larger gold nanoparticles add in aqua regia, we could get more gold ion signal than using 20 nm gold nanoparticles. Finally, under the optimized condition, we found out that mercury concentration of 50 pM (0.01 □g L-1) could be measured with sufficient reliability, and a limit of detection (LOD) of 5.6 ng/L.

目錄
中文摘要………………………………………………………… I
英文摘要………………………………………………………… III
目錄……………………………………………………………… V
圖目錄…………………………………………………………… IX
表目錄…………………………………………………………… XIII
第一章 前言…………………………………………………… 1
1.1 研究背景……………………………………………… 1
1.1.1 汞的種類、特性、來源、生理機制及健康效應…… 1
1.1.2 檢測微量汞元素遭遇的困難………………………… 8
1.2 微量元素分析技術…………………………………… 9
1.2.1 傳統汞分析技術發展現況…………………………… 10
1.3 利用生物感測器分析汞離子之技術發展現況……… 11
1.4 研究目的與方法……………………………………… 19
第二章 儀器分析及原理……………………………………… 22
2.1 紫外光可見光吸收光譜儀…………………………… 22
2.2 電熱式原子吸收光譜儀……………………………… 24


第三章 實驗部分……………………………………………… 38
3.1 試劑…………………………………………………… 38
3.2 藥品配製……………………………………………… 39
3.3 寡聚核□酸探針(Oligonucleotide probes)和標的寡聚核□酸(Target oligonucleotide)的設計……………… 40
3.4 DNA修飾之磁性微米粒子製備…………………… 41
3.5 金奈米粒子探針的製備……………………………… 44
3.6 MMP/Target/Au-NPs雜合最佳化條件之探討……… 46
3.6.1 Tm值測定…………………………………………… 46
3.6.2 反應時間對訊號之影響探討………………………… 48
3.6.3 加熱時間對訊號之影響探討………………………… 49
3.6.4 清洗次數對訊號之影響探討………………………… 49
3.6.5 使用20 nm 金奈米粒子之分析效能測試…………… 50
3.6.6 T-T mismatch 對於各種金屬離子之選擇性測試…… 52
3.6.7 T-T mismatch 與汞離子結合後穩定性之探討……… 52
3.7 使用60 nm金奈米粒子改進偵測系統靈敏度……… 53
3.7.1 60 nm金奈米粒子之Tm值測定…………………… 54
3.7.2 探討60 nm金奈米粒子之訊號放大效果…………… 55
3.7.3 使用60 nm 金奈米粒子之分析效能測試…………… 56
3.7.4 提升使用60 nm 金奈米粒子之系統線性範圍……… 57
3.8 真實樣品之測定……………………………………… 58
3.8.1 環境樣品之測定……………………………………… 58
3.8.2 真實尿液樣品之測定………………………………… 58
3.8.3 SRM尿液樣品之測定……………………………… 59
3.9 電熱式原子吸光儀之最佳操作條件以及溫度程式… 60

第四章 結果與討論…………………………………………… 61
4.1 利用二硫蘇糖醇(Dithiothreitol, DTT)還原寡聚核□酸序列……………………………………………… 61
4.2 MMP/Target/Au-NPs雜合最佳化條件之探討……… 64
4.2.1 Melting temperature (Tm)值的測定………………… 64
4.2.2 反應時間對於訊號之影響探討……………………… 66
4.2.3 加熱時間對訊號之影響探討………………………… 67
4.2.4 清洗次數對於訊號之影響探討……………………… 68
4.2.5 使用20 nm 金奈米粒子之分析效能測試…………… 69
4.2.6 T-T mismatch 對於各種金屬離子之選擇性測試…… 71
4.2.7 T-T mismatch 對於汞離子結合力的探討…………… 72
4.3 使用60 nm金奈米粒子改進偵測系統的靈敏度…… 73
4.3.1 60 nm金奈米粒子對Tm值的影響…………………… 76
4.3.2 60 nm金奈米粒子訊號放大效果的探討…………… 78
4.3.3 使用60 nm 金奈米粒子之分析效能測試…………… 79
4.3.4 提升使用60 nm 金奈米粒子之系統線性範圍……… 81
4.4 真實樣品之測定……………………………………… 86

第五章 結論…………………………………………………… 89

第六章 參考文獻……………………………………………… 90

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