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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 onλExonuclease
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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。 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 combinesλexonuclease(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(TQDNA)to form the double-stranded DNA with blunt ends.Subsequently,the DNAs are acted and cleaved byλ-exo,releasing free tDNAs and TDNAs.With the generation ofλ-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+).
作者 刘陈 刘金权 郦瑜杰 廖力夫 肖锡林 LIU Chen;LIU Jinquan;LI Yujie;LIAO Lifu;XIAO Xilin(School of Chemistry and Chemical Engineering,University of South China,Hengyang,Hunan 421001,China;Hunan Key Laboratory of Typical Environmental Pollution and Health Hazards,School of Public Health,Hengyang Medical School,University of South China,Hengyang,Hunan 421001,China;School of Resource Environment and Safety Engineering,University of South China,Hengyang,Hunan 421001,China)
出处 《南华大学学报(自然科学版)》 2022年第3期69-76,共8页 Journal of University of South China:Science and Technology
基金 国家自然科学基金项目(11475079)。
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 fluorescent DNA sensor cascade reaction λexonuclease catalyzed hairpin self-assembly copper ions
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