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刘陈,刘金权,郦瑜杰,廖力夫,肖锡林.基于λ核酸外切酶辅助的级联型铜离子荧光检测体系的构建[J].南华大学学报(自然科学版),2022,(3):69~76.[LIU Chen,LIU Jinquan,LI Yujie,LIAO Lifu,XIAO Xilin.Construction of a Cascaded Copper Ion Fluorescence Detection System Based on λ Exonuclease[J].Journal of University of South China(Science and Technology),2022,(3):69~76.]

基于λ核酸外切酶辅助的级联型铜离子荧光检测体系的构建

Construction of a Cascaded Copper Ion Fluorescence Detection System Based on λ Exonuclease

投稿时间：2022-01-27

DOI：

中文关键词: 荧光核酸传感器级联反应 λ核酸外切酶催化发夹自组装铜离子

英文关键词:fluorescent DNA sensor cascade reaction λ exonuclease catalyzed hairpin self-assembly copper ions

基金项目:国家自然科学基金项目(11475079)

作者	单位
刘陈	南华大学化学化工学院,湖南衡阳421001
刘金权	南华大学衡阳医学院公共卫生学院典型环境污染与健康危害湖南省重点实验室,湖南衡阳421001
郦瑜杰	南华大学资源环境与安全工程学院,湖南衡阳421001
廖力夫	南华大学化学化工学院,湖南衡阳421001
肖锡林	南华大学化学化工学院,湖南衡阳421001

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中文摘要:

通过将一种有效的级联信号放大策略引入铜离子荧光生物传感器中,构建了一种用于超灵敏检测铜离子的荧光检测体系。这种级联型荧光检测体系结合了λ核酸外切酶辅助的靶标回收和催化发夹自组装的循环信号扩增。在铜离子的存在下,用于识别铜离子的功能核酸的底物链被特异性切割和释放。释放出的单链DNA片段(tDNA)与5′端被磷酸标记的双链DNA(TQ-DNA)杂交形成具有平末端的双链DNA。随后,该DNA分子被λ核酸外切酶识别并切割,释放出游离的tDNA和TDNA。随着λ核酸外切酶的水解,越来越多的tDNA被回收,同时放出越来越多的TDNA。此时,TDNA作为催化发夹自组装反应的触发链,启动了新一轮的靶标循环。在TDNA的辅助下,作为分子信标的发夹DNA会与另外一个发夹DNA杂交形成新的双链DNA,同时,释放出高荧光强度。利用荧光光谱法对该体系不同反应阶段进行了表征。结果表明,该级联型铜离子荧光检测体系对痕量铜离子具有很好的响应。

英文摘要:

An efficiently cascaded signal amplification strategy was constructed and introduced into a fluorescent biosensor for ultrasensitive detection of copper ions (Cu2＋). This cascaded fluorescence detection system combines λ exonuclease (λ-exo)-assisted target recovery and cyclic signal amplification of catalyzed hairpin self-assembly (CHA). In the presence of Cu2＋, the substrate strand of the functional nucleic acids for the recognition of copper ions are specifically cleaved and released. Then, these released single-stranded DNA fragments (tDNAs) hybridize to 5′ phosphate-labeled double-stranded DNA (TQ-DNA) to form the double-stranded DNA with blunt ends. Subsequently, the DNAs are acted and cleaved by λ-exo, releasing free tDNAs and TDNAs. With the generation of λ-exo hydrolysis, the TQ-DNAs are hydrolyzed, releasing more and more tDNAs and TDNAs. Then these TDNAs, serving as trigger strand of CHA, launch a new round of targeting cycles. At the same time, the hairpin DNAs that serve as the molecular beacon will have hybridized with another hairpin DNA to form a new double-stranded DNA with the help of TDNA, emitting high fluorescence intensity. The different reaction stages of the system were characterized by fluorescence spectroscopy. The result showed that the cascaded Cu2＋ fluorescence detection system has a good response to low concentrations of Cu2＋.

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