基于碳量子点-银纳米簇荧光共振能量转移纳米探针的荧光传感器检测转基因成分CaMV35S  

Detection of CaMV35S Sequence Using a Fluorescence Sensor Based on Carbon Dots-Silver Nanoclusters Fluorescence Resonance Energy Transfer Nanotags

作　　者：翟应惠 王婷婷 唐家璇 张鸿雁 操小栋[1] 叶永康[1,2] 郑海松 李云飞 ZHAI Ying-hui;WANG Ting-ting;TANG Jia-xuan;ZHANG Hong-yan;CAO Xiao-dong;YE Yong-kang;ZHENG Hai-song;LI Yun-fei(School of Food and Biological Engineering,Hefei University of Technology,Heifei 230009,China;School of Biological Science and Engineering,North Minzu University,Yinchuan 750021,China;Technology Center of Hefei Customs District,Hefei 230022,China)

机构地区：[1]合肥工业大学食品与生物工程学院,安徽合肥230009 [2]北方民族大学生物科学与工程学院,宁夏银川750021 [3]合肥海关技术中心,安徽合肥230022

出　　处：《分析测试学报》2023年第5期550-558,共9页Journal of Instrumental Analysis

基　　金：安徽省重点研究与开发计划项目(202024a07020038);宁夏自然科学基金重点项目(2022AAC02046);安徽省科技重大专项项目(202103a07020003)。

摘　　要：利用荧光共振能量转移(FRET)纳米探针结合催化发夹组装(CHA)无酶扩增信号放大途径建立了一种可用于转基因成分的荧光检测方法。首先为CaMV35S目标序列(tDNA)设计了可诱导的CHA循环的两个发夹结构序列HP1和HP2。当单链DNA标记碳点(sDNA-CDs)和DNA模板化银纳米团簇(Ts-AgNCs)杂交后,AgNCs和碳量子点(CDs)靠近,形成FRET效应,得到sDNA-CDs/Ts-AgNCs荧光猝灭的比率荧光探针。当tDNA存在时,通过杂交反应打开HP1发夹,形成HP1-tDNA双链结构;该结构可将HP2的发夹结构打开,从而形成HP1-HP2双链结构,同时释放出tDNA进入下一轮杂交,触发CHA循环。由于HP1-HP2中HP1的部分序列与Ts部分序列间的亲和性较sDNA强,因此,加入sDNA-CDs/Ts-AgNCs后,sDNA-CDs从探针中释放,使CDs(λem=464 nm)的荧光得以增强。而AgNCs仍在双链结构中,其荧光强度(λem=560 nm)基本保持不变。以IF464/IF560为检测信号,在最优条件下,该比率荧光传感器对CaMV35S检测的线性范围为0.1~50 nmol/L,检出限(LOD,S/N=3)为0.02 nmol/L。制备的传感器具有良好的选择性,将该传感器用于转基因番茄叶中CaMV35S成分的检测,结果可靠。A ratiometric fluorescent sensor for genetically modified components was constructed us⁃ing fluorescence resonance energy transfer(FRET)nanotags combined with catalytic hairpin assembly(CHA)enzyme-free amplification signal amplification pathway in this paper.First,two sequences HP1 and HP2 with hairpin structures were designed for CaMV35S(tDNA)-induced CHA,and then based on the FRET effect,AgNCs and CDs came in close proximity when single-stranded DNA la⁃beled carbon dots(sDNA-CDs)and DNA templated silver nanoclusters(Ts-AgNCs)were hybrid⁃ized,which led to the fluorescence quenching of CDs.A ratiometric fluorescent probe with sDNACDs/Ts-AgNCs fluorescence quenching is obtained.When tDNA is present,through the base com⁃plementary pairwise hybridization reaction,tDNA opens the HP1 hairpin to form an HP1-tDNA double-stranded structure.This structure can open the HP2 hairpin structure,thus forming an HP1-HP2 double-stranded structure,in which the tDNA was released into the next round of hybridization,triggering the CHA cycle.Since the affinity between the partial sequence of HP1 and the partial sequence of Ts in HP1-HP2 was stronger than that of sDNA,the fluorescence of CDs(λem=464 nm)was enhanced by the release of sDNA-CDs from the probe after the addition of sDNACDs/Ts-AgNCs.However,AgNCs were still remained in the double-chain structure,leading to the unchanged fluorescence intensity of AgNCs(λem=560 nm).Under the optimal conditions,the output signal of IF464/IF560 showed a linear relation for CaMV35S in the concentration range of 0.1-50 nmol/L,and the detection limit(LOD,S/N=3)was calculated to be 0.02 nmol/L.The pre⁃pared probe was used for the detection of CaMV35S components in transgenic tomato leaves with good selectivity and reliable results.

关 键 词：催化发夹组装 碳量子点 银纳米簇 CaMV35S序列 比率荧光检测 

分 类 号：O657.34[理学—分析化学] TP212.3[理学—化学]