一种基于汞特异性DNA 和SYBR GREEN I 荧光检测Hg2+方法的建立

生物技术通报 ›› 2013, Vol. 0 ›› Issue (4): 221-224.

一种基于汞特异性DNA 和SYBR GREEN I 荧光检测Hg²⁺方法的建立

吴继魁¹, 卫碧文², 林莉², 毛芳¹, 周冬香¹, 熊振海¹

1. 上海海洋大学食品学院，上海 201306 ；2. 上海出入境检验检疫局，上海 200135

收稿日期:2013-02-17 修回日期:2013-04-22 出版日期:2013-04-22 发布日期:2013-04-22
作者简介:吴继魁，博士，讲师，研究方向：生物传感器；E-mail ：jkwu@shou.edu.cn
基金资助:
上海海洋大学博士启动基金项目（A-2400-10-0129），上海自然科学基金项目（11ZR1415400）

Highly Sensitive and Selective Detection of Hg2+ in Aqueous Solution with Mercury-specific DNA and SYBR GREEN I

Wu Jikui¹, Wei Biwen², Lin Li², Mao Fang¹, Zhou Dongxiang¹, Xiong Zhenhai¹

1. College of Food Science and Technology，Shanghai Ocean University，Shanghai 201306 ；2. Shanghai Entry-Exit Inspection and Quarantine Bureau，Shanghai 200135

Received:2013-02-17 Revised:2013-04-22 Published:2013-04-22 Online:2013-04-22

摘要/Abstract

摘要：

以富含胸腺嘧啶（thymine）的Hg²⁺ 特异性DNA 为识别元件，利用SYBR GREEN I 分子光“开关”的特性，建立了一种高灵敏、高选择性荧光均相检测Hg²⁺ 的新方法。该检测体系由两条非标记、含有5 个thymine-thymine（T-T）错配碱基对的 DNA 探针组成。T-T 错配使两条DNA 探针以单链形式存在于溶液中，而 Hg²⁺ 通过T-Hg²⁺-T 配位作用可以与两条DNA 探针结合并形成稳定的双链结构。在优化条件下，荧光强度与Hg²⁺ 浓度在0-100 nmol/L 范围呈线性，检测限为2 nmol/L（S/N=3），而其他金属离子也不会对Hg²⁺ 检测造成干扰。该方法简单快速，有望应用于水体中Hg²⁺ 的检测。

关键词: Hg²⁺, DNA, 探针, 荧光检测, SYBR, GREEN I

Abstract:

A novel “signal-on” assay for sensitive and selective detection of Hg2+ in aqueous solution based on mercury-specific DNA and a molecular light switch complex, SYBR GREEN I, was reported in the present work. This Hg²⁺ specific sensor system composed of two labelfree DNA probes with five T-T mis-matches, which could form stable DNA duplexes by thymine-Hg²⁺-thymine（T-Hg²⁺-T）in the presence of Hg²⁺. The fluorescence of SYBR GREEN I is weak in aqueous solution, and significant fluorescence can be observed when intercalating into DNA duplexes. By monitoring Hg²⁺-dependent fluorescence intensity enhancement, highly sensitive and selective determination of Hg²⁺ has been achieved. Under the optimum conditions, the fluorescence intensity was proportional to the concentration of Hg²⁺ in the range of 5-100 nmol/L. A detection limit of 2 nmol/L Hg²⁺ was achieved. The presence of other metal ions did not interfere with the detection of Hg²⁺. This approach is simple and rapid, which can be used to monitor the Hg²⁺ concentration in drinking water.

Key words: Hg²⁺, DNA probe, Fluorimetry, SYBR GREEN I

吴继魁, 卫碧文, 林莉, 毛芳, 周冬香, 熊振海. 一种基于汞特异性DNA 和SYBR GREEN I 荧光检测Hg²⁺方法的建立[J]. 生物技术通报, 2013, 0(4): 221-224.

Wu Jikui, Wei Biwen, Lin Li, Mao Fang, Zhou Dongxiang, Xiong Zhenhai. Highly Sensitive and Selective Detection of Hg2+ in Aqueous Solution with Mercury-specific DNA and SYBR GREEN I[J]. Biotechnology Bulletin, 2013, 0(4): 221-224.

参考文献

［1］ Hassett-Sipple B, Swartout J, Schoeny R. Mercury study report to Congress. Volume V ：Health effects of mercury and mercury compounds ［R］. USA ：EPA-452/R-97-007, 1997.

［2］ Aiyer, HN, Kawazoe T, Lim J, et al. Mercury treatment of optical nearfield fibre probes for smoother tips with reduced light leakage[J].

Nanotechnology, 2001, 12 ：368-371.

［3］ Yoon S, Miller EW, He Q, et al. A bright and specific fluorescent sensor for mercury in water, cells, and tissue[J]. Angew Chem, Int Ed, 2007, 46 ：6658-6661.

［4］ Nolan EM, Lippard SJ. A “turn-on” fluorescent sensor for the selective detection of mercuric ion in aqueous media[J]. J Am Chem Soc, 2003, 125 ：14270-14271.

［5］ Yoon S, Albers AE, Wong AP, et al. Screening mercury levels in fish with a selective fluorescent chemosensor[J].J Am Chem Soc, 2005, 127 ：16030-16031.

［6］ Yang YK, Yook KJ, Tae J. A rhodamine-based fluorescent and colorimetric chemodosimeter for the rapid detection of Hg²⁺ ions in aqueous media[J]. J Am Chem Soc, 2005, 127 ：16760-16761.

［7］ Zheng H, Qian ZH, Xu L, et al. Switching the recognition preference of rhodamine B spirolactam by replacing one atom ： design of rhodamine B thiohydrazide for recognition of Hg（II）in aqueous solution[J].Org Lett, 2006, 8 ：859-861.

［8］ Huang CC, Chang HT. Selective gold-nanoparticle-based “turnon” fluorescent sensors for detection of mercury（II）in aqueous solution[J]. Anal Chem, 2006, 78 ：8332-8338.

［9］ Lee JS, Han MS, Mirkin CA. Colorimetric detection of mercuric ion（Hg²⁺）in aqueous media using DNA-functionalized gold nanoparticles[J]. Angew Chem In Ed, 2007, 46 ：4093-4096.

［10］ Xue XJ, Wang F, Liu XG. One-step, room temperature, colorimetric detection of mercury（Hg²⁺）using DNA/nanoparticle conjugates[J]. J Am Chem Soc, 2008, 130 ：3244-3245.

［11］ Liu XF, Tang YL, Wang LH, et al. Optical detection of mercury（II） in aqueous solutions by using conjugated polymers and label-free oligonucleotides[J]. Adv Mate, 2007, 19 ：1471-1474.

［12］ Liu J, Lu Y. Rational design of “turn-on” allosteric DNAzyme catalytic beacons for aqueous mercury ions with ultrahigh sensitivity and selectivity[J]. Angew Chem Int Ed Engl, 2007, 46（40）: 7587-7590.

［13］ Kim IB, Bunz UHF. Modulating the sensory response of a conjugated polymer by proteins ： an agglutination assay for mercury ions in water[J]. J Am Chem Soc, 2006, 128（9）：2818-2819.

［14］ Miyake Y, Togashi H, Tashiro M, et al. MercuryII-mediated formation of thymine-HgII-thymine base pairs in DNA duplexes[J]. J Am Chem Soc, 2006, 128 ：2172-2173.

［15］ Wu JK, Li LY, Zhu D, et al. Colorimetric assay for mercury （II）based on mercury-specific DNA-functionalized gold nanoparticles[J]. Anal Chim Acta, 2011, 694 ：115-119.

［16］ Wu JK, Li LY, Shen BJ, et al. Ploythymine oligonucleotide modified gold electrode for voltammetric determination of mercury（II）in aqueous solution[J]. Electroanalysis, 2010, 22 ：479-482.

［17］ Ono A, Togashi H. Highly selective oligonucleotide-based sensor for mercury（II）in aqueous solutions[J]. Angew Chem Int Ed, 2004, 43 ：4300-4302.

［18］ Wang ZD, Lee JH, Lu Y. Highly sensitive "turn-on" fluorescent sensor for Hg²⁺ in aqueous solution based on structure-switching DNA[J]. Chem Commun, 2008 ：6005-6007.

［19］ Zipper H, Brunner H, Bernhagen J, et al. Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications[J]. Nucleic Acids Research, 2004, 32：e103.

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一种基于汞特异性DNA 和SYBR GREEN I 荧光检测Hg²⁺方法的建立

Highly Sensitive and Selective Detection of Hg2+ in Aqueous Solution with Mercury-specific DNA and SYBR GREEN I

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