摘要
通过对已知的Ni2+适体的改造,发现了对Hg2+有较强结合活性的核酸适体N1,基于N1的二级结构进行改造,获得了具有较高活性的核酸适体N5,并建立了荧光检测方法。以荧光基团FAM标记核酸适体,在94℃变性5 min,室温(25℃)下复性30 min后,适配体与标记有荧光猝灭基团DABCYL的一段互补序列Q2结合(适体与Q2的浓度比为1∶3),加入系列浓度的Hg2+与互补序列竞争结合核酸适体,以荧光信号的变化定量分析Hg2+浓度。结果表明,N1与Hg2+结合呈线性,线性范围为1.25～20 mg/L;检出限为0.62 mg/L;以N1结构为基础改造合成的序列和结构均不同的适体N4,N5,N6,N7对Hg2+的识别结合活性均不同,其中适体N5与Hg2+结合活性最为灵敏,特异性高,线性范围为0.156～2.50 mg/L;检出限为78μg/L。
A RNA aptamer modified to DNA（N1） that could bind to the target of Ni2＋ was found that could bind specifically to Hg2＋ ion with typical secondary structure.Thereafter,several DNA aptamers with a certain conformation of sequence mutation and splice were designed in order to increase the property of Hg2＋ binding,and Aptamer-based fluorescence assay for the Hg2＋ ion determination was developed.The addition of the Hg2＋ to a mixture containing the duplex of a fluorophore labeled aptamer and a quencher-modified antisense nucleic acid（Q2） would force the release of Q2 from labeled aptamer,which was spontaneously accompanied by the increase of fluorescence intensity and made Hg2＋in quantitative analysis.It was shown that stable labeled aptamer DNA and Q2 hydrogen bonds formed at concentration ration of 1 ∶ 3（labeled aptamer and Q2,respectively）,under the condition of 5 min denaturation in 94 ℃ and 30 min renaturation in room temperature（25 ℃） in the fluorescence assay preparation.For aptamer N1,the fluorescence assay results showed that linear response toward Hg2＋ concentration with fluorescence quenching system ranged from 1.25 mg/L to 20 mg/L,with the limit of detection 0.625 mg/L.In order to improve the sensitivity of aptamer N1,4 oligonucleotides（N4,N5,N6,N7） were designed and synthesized based on the structure of N1.The results showed that N5 had the best affinity and specificity to Hg2＋ which displayed the linear range for the Hg2＋ concentration detection was 0.156 mg/L to 2.5 mg/L with a detection limit of 78 μg/L.
出处
《分析化学》
SCIE
EI
CAS
CSCD
北大核心
2012年第12期1827-1831,共5页
Chinese Journal of Analytical Chemistry
基金
江苏省农业科技自主创新项目(No.CX(10)236)
苏州市科技支撑项目(No.SN201129)
中国博士后基金(No.20090451177)
江苏省博士后科研资助计划(No.0802023B)资助项目
关键词
核酸适体
荧光
汞离子
检测
Aptamer
Fluorescence
Mercury（Ⅱ）
Detection
