利用金奈米粒子探針搭配電熱式原子吸收光譜儀測定汞離子的分析研究

Determination of Mercuric Ion Using Oligonucleotide−Gold Nanoparticle Conjugates Coupled with ET-AAS Detection

許敦傑

指導教授 ： 孫毓璋

國立清華大學/原子科學院/生醫工程與環境科學系/碩士(2010年)

摘要

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。

關鍵字

汞離子 ； 金奈米粒子 ； 原子吸收光譜法

並列摘要

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.

並列關鍵字

mercury ； nanoparticle ； ET-AAS

參考文獻

3 Clarkson, T.W. N, Engl J Med. 2003, 349, 1731–1737.

7 Halliwell, B.; Gutteridge, J. Free radicals in biology and medicine. 1989, Oxford, UK: Claredon Press.

9 Zalups, R.K.; Lash, L.H. Toxicology of Metals. 1996, 145–163.

11 Zalups, R.K. Pharmacol. Rew. 2000, 52, 113–143.

13 莊青青，生藥中重金屬元素-鋅、鐵、錳、銅、鉛之測定分析，高雄醫學大學碩士論文, 1997, p6.