荧光核酸探针检测Pb~（2+）方法研究
摘要
随着金属制品、汽油、蓄电池、化学涂料等产品使用量的不断增加以及工农业三废的排放,铅以多种途径广泛进入到人们的生活环境中,并呈加剧趋势,不仅直接影响农作物的生长发育、产量、品质等,而且间接通过食物链对人体健康造成严重的危害,铅污染已经引起了广泛关注。传统的重金属铅检测技术已经难以满足环境与食品安全监控的需求,因此,建立方便、快速的重金属铅的检测技术十分必要。
本研究以DNAzyme与金属离子间的特异性为研究思路,以近年来新发展起来的FRET为监测手段,设计一种以DNAzyme为母体可以检测重金属Pb~(2+)的荧光核酸探针,将核酸分析法结合发光方法,用于重金属Pb~(2+)的检测,为加强我国环境和食品安全控制和监督提供技术支持。
在实验中,首先,将8-17 DNAzyme增加了两个“G-C”碱基对进行增强热稳定性的结构修饰,并标记上一个荧光基团“FAM”和两个荧光淬灭基团“Dabcyl”,设计成双淬灭Pb~(2+)荧光核酸探针P1;在探针P1的基础上去掉底物链上的一个荧光淬灭基团“Dabcyl”,设计成单淬灭Pb~(2+)荧光核酸探针P2;在探针P2的基础上去掉增加的两个“G-C”碱基对,设计成单淬灭Pb~(2+)荧光核酸探针P3。并根据实际情况,采用温度梯度法对三种核酸探针的热稳定性加以比较,探针P1的热稳定性最好,选取P1作为深入研究的材料。
随后研究了探针P1对Cd~(2+)、Zn~(2+)、Mg~(2+)、Cu~(2+)、Mn~(2+)、Pb~(2+) 6种二价金属离子的响应,结果表明探针P1对Pb~(2+)具有很强的特异性,在探针浓度为2.5×10~(-7)mol/L , Pb~(2+)浓度在8.5×10~(-8)mol/L～7.5×10~(-6)mol/L范围内和探针的荧光强度呈线性关系,最小检出限8.5×10~(-8)mol/L。
经过自来水中的添加回收率实验,探针P1检测结果和FAAS检测结果的相符率为:99.25%,该探针可用于Pb~(2+)的定性和定量检测。
With the increasing use of metal products, lead to a broad variety of ways to enter into people's living environment, and the trend was increasing, not only affects crop growth and development, yield, quality and so on, but also on human health through the food chain, causing serious hazards, lead pollution has caused widespread concern. Traditional heavy metal detection technology has been difficult to meet the environment and food safety monitoring requirments, the establishment of convenient, fast heavy metal detection technology is necessary.
This study based on the new development of the specificity bettween DNAzyme and metal ion in recent years, using FRET as a monitor tool, designed an nucleic acid fluorescent probe which can detect heavy metals. The method would be nucleic acid analysis method combined with light-emitting method for detection of heavy metal lead that to enhance our environment and food safety control and supervision of the provision of technical support.
In the test, at first ,we desinged three kinds of probe. structure of the 8-17 DNAzyme was modified with two pairs of“G-C”to enhance its secondary structure’thermal stability and a double quenching fluorescent probe P1 was designed by employing one fluorophore“FAM”and two quenchers“Dabcyl”based on the fluorescence resonance energy transfer (FRET) principle. Structure of the 8-17 DNAzyme was modified with two pairs of“G-C”to enhance its secondary structure’thermal stability and a single quenching fluorescent probe P2 was designed by employing one fluorophore“FAM”and two quenchers“Dabcyl”based on the fluorescence resonance energy transfer (FRET) principle. And another single quenching fluorescent probe P3 was designed by employing one fluorophore“FAM”and two quenchers“Dabcyl”based on the fluorescence resonance energy transfer (FRET) principle. Compared the thermal stability of the three kinds of nucleic acid fluorsence probe by temperature gradient method, and choose the most stable P1 for in-depth study.
At second, the qualitative assays was studied by monitoring the change of the probe fluorescent intensity after adding Cd~(2+), Zn~(2+), Mg~(2+), Cu~(2+), Mn~(2+), Pb~(2+), individually. Among these metal ions, Pb~(2+) showed a strong specificity to the probe. In addition, when the probe concentration was 2.5×10~(-7)mol/L, a good linear relationship was found between the probe fluorescent intensities and Pb~(2+) concentration in the range of 8.5×10~(-8)mol/L～7.5×10~(-6)mol/L with a detection limit 8.5×10~(-8)mol/L. The consisten rate of tap water recovery rate bettween probe P1 and FAAS is 99.25%. The results showed that the probe could be used for both qualitative and quantitative detection of Pb~(2+) at room temperature.
本研究以DNAzyme与金属离子间的特异性为研究思路,以近年来新发展起来的FRET为监测手段,设计一种以DNAzyme为母体可以检测重金属Pb~(2+)的荧光核酸探针,将核酸分析法结合发光方法,用于重金属Pb~(2+)的检测,为加强我国环境和食品安全控制和监督提供技术支持。
在实验中,首先,将8-17 DNAzyme增加了两个“G-C”碱基对进行增强热稳定性的结构修饰,并标记上一个荧光基团“FAM”和两个荧光淬灭基团“Dabcyl”,设计成双淬灭Pb~(2+)荧光核酸探针P1;在探针P1的基础上去掉底物链上的一个荧光淬灭基团“Dabcyl”,设计成单淬灭Pb~(2+)荧光核酸探针P2;在探针P2的基础上去掉增加的两个“G-C”碱基对,设计成单淬灭Pb~(2+)荧光核酸探针P3。并根据实际情况,采用温度梯度法对三种核酸探针的热稳定性加以比较,探针P1的热稳定性最好,选取P1作为深入研究的材料。
随后研究了探针P1对Cd~(2+)、Zn~(2+)、Mg~(2+)、Cu~(2+)、Mn~(2+)、Pb~(2+) 6种二价金属离子的响应,结果表明探针P1对Pb~(2+)具有很强的特异性,在探针浓度为2.5×10~(-7)mol/L , Pb~(2+)浓度在8.5×10~(-8)mol/L～7.5×10~(-6)mol/L范围内和探针的荧光强度呈线性关系,最小检出限8.5×10~(-8)mol/L。
经过自来水中的添加回收率实验,探针P1检测结果和FAAS检测结果的相符率为:99.25%,该探针可用于Pb~(2+)的定性和定量检测。
With the increasing use of metal products, lead to a broad variety of ways to enter into people's living environment, and the trend was increasing, not only affects crop growth and development, yield, quality and so on, but also on human health through the food chain, causing serious hazards, lead pollution has caused widespread concern. Traditional heavy metal detection technology has been difficult to meet the environment and food safety monitoring requirments, the establishment of convenient, fast heavy metal detection technology is necessary.
This study based on the new development of the specificity bettween DNAzyme and metal ion in recent years, using FRET as a monitor tool, designed an nucleic acid fluorescent probe which can detect heavy metals. The method would be nucleic acid analysis method combined with light-emitting method for detection of heavy metal lead that to enhance our environment and food safety control and supervision of the provision of technical support.
In the test, at first ,we desinged three kinds of probe. structure of the 8-17 DNAzyme was modified with two pairs of“G-C”to enhance its secondary structure’thermal stability and a double quenching fluorescent probe P1 was designed by employing one fluorophore“FAM”and two quenchers“Dabcyl”based on the fluorescence resonance energy transfer (FRET) principle. Structure of the 8-17 DNAzyme was modified with two pairs of“G-C”to enhance its secondary structure’thermal stability and a single quenching fluorescent probe P2 was designed by employing one fluorophore“FAM”and two quenchers“Dabcyl”based on the fluorescence resonance energy transfer (FRET) principle. And another single quenching fluorescent probe P3 was designed by employing one fluorophore“FAM”and two quenchers“Dabcyl”based on the fluorescence resonance energy transfer (FRET) principle. Compared the thermal stability of the three kinds of nucleic acid fluorsence probe by temperature gradient method, and choose the most stable P1 for in-depth study.
At second, the qualitative assays was studied by monitoring the change of the probe fluorescent intensity after adding Cd~(2+), Zn~(2+), Mg~(2+), Cu~(2+), Mn~(2+), Pb~(2+), individually. Among these metal ions, Pb~(2+) showed a strong specificity to the probe. In addition, when the probe concentration was 2.5×10~(-7)mol/L, a good linear relationship was found between the probe fluorescent intensities and Pb~(2+) concentration in the range of 8.5×10~(-8)mol/L～7.5×10~(-6)mol/L with a detection limit 8.5×10~(-8)mol/L. The consisten rate of tap water recovery rate bettween probe P1 and FAAS is 99.25%. The results showed that the probe could be used for both qualitative and quantitative detection of Pb~(2+) at room temperature.
引文
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[2]张正洁,李东红,许增贵。我国铅污染现状、原因及对策[J].环境保护科学,2005,130(31):41～42,47
[3]黄萍.大气中铅烟及铅尘的污染及研究现状[J].环境科学,2009,8:109～110
[4]霍霞.蔬菜中重金属铅污染现状及对策[J].六盘水师范高等专科学校学报,2009, 21(3):27～31
[5] Péter N,Károly B,LászlóG,ect.Characterization of the stimulating efect of low—dose stressors in maize and bean seedlings[J].Plant Physiol,2003,160(3): 1175～1183
[6] Kupper H,Kupper F,Spiller M.Environmental relevance of heavy metal—substituted chlorophylls using the example of water plant[J].Exp.Bot,1996, 47(4):259～266
[7]张红萍.铅对植物的毒害及植物对铅的抗性机制[J].农业装备技术,2007,33(3):19～20
[8]孙琴,王超.植物外源Cd和Pb复合污染对小麦根系植物络合素和谷胱甘肽合成的影响[J].生态环境,2008,17(5):1833-1838.
[9] JIANG Xing Yu,ZHAO Ke Fu.Mechanism of heavy metal injury and resistance of plants[J].Chinese Journal of Applied & Environmental Biology,2001,7:92～99
[10]郝伟,姜勇,李琪等.重金属铅、镉对矮小拟丽突线虫(Acrobeloides nanus)的亚急性毒性作用[J].生态毒理学报,2008,3 (2):139～143
[11]张换敬,乔波,汪安泰,等。铜、镉、铅离子对水螅摄食行为的抑制作用[J].水资源保护,2008,24(3):17～20
[12]高明奇,陈航,周彬.慢性铅暴露小鼠海马细胞凋亡相关基因的表达[J].中国公共卫生,2005,21(6):668～669
[13]高明奇,孙黎光,文涛,等.慢性铅暴露小鼠海马NMDAR亚单位基因表达[J].中国公共卫生,2006,22(5):574～575
[14]文涛,孙黎光,刘阳,等。慢性铅暴露对小鼠CaMKⅡ蛋白表达的影响[J].中国公共卫生,2006,22(10):1211～1212
[15]文涛,孙黎光,宗志宏,等.铅对小鼠海马c-jun表达及学习记忆功能影响[J].中国公共卫生,2005,21(2):183～184
[16]万双秀,王俊东.铅污染的危害及防治[J].微量元素与健康研究,2005:22(1): 63～65
[17] Werner, C.; Krebs, B.; Keith, G .; Dirheimer, G.. Specific cleavages of pure tRNAs by plumbous ions [J].Biochimica et Biophysica Acta, Nucleic Acids and Protein Synthesis, 1976, 432(2):161～175
[18]李宏卫,曹建劲,苏志华,等.铅检测方法研究进展[J].安徽农业科学,2008,36(19):8255～8256,8262
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