基于发夹DNA动态自组装树枝状聚合物放大策略的荧光传感体系测定水中Pb^(2+)的含量  

Determination of Pb^(2+)in Water by Fluorescence Sensing System Based on Amplification Strategy of Hairpin DNA Dynamic Self-Assembled Dendrimer

作　　者：陈贺 郑洋 蒋旭燕 吴继魁[1,2,3] CHEN He;ZHENG Yang;JIANG Xuyan;WU Jikui(School of Food Science,Shanghai Ocean University,Shanghai 201306,China;Laboratory of Quality and Safety Risk Assessment for Aquatic Product on Storage and Preservation(Shanghai),Ministry of Agriculture and Rural Affairs,Shanghai 201306,China;Shanghai Engineering Research Center of Aquatic-Product Processing&Preservation,Shanghai 201306,China)

机构地区：[1]上海海洋大学食品学院,上海201306 [2]农业部水产品贮藏保鲜质量安全风险评估实验室(上海),上海201306 [3]上海水产品加工及贮藏工程技术研究中心,上海201306

出　　处：《理化检验（化学分册）》2024年第2期155-160,共6页Physical Testing and Chemical Analysis(Part B:Chemical Analysis)

基　　金：国家自然科学基金(31972772);上海自然科学基金(11ZR415400)。

摘　　要：基于GR-5脱氧核酶(GR-5 DNAzyme)对Pb^(2+)的特异性识别和发夹DNA动态自组装树枝状聚合物信号放大策略,构建了一种新型无酶、高灵敏检测Pb^(2+)的荧光传感体系。分别于95℃恒温孵育合成序列扩展GR-5 DNAzyme复合物(GR-5E-S,由GR-5 DNAzyme酶链GR-5E和底物链GR-5S合成)以及Y型骨架(由骨架链Y1、Y2、Y3合成),于25℃恒温孵育合成发夹三聚体(由Y型骨架和发夹链H1、H2、H3合成,其中发夹链H1上修饰有荧光团和猝灭团)。将水样4μL添加到96μL含400 nmol·L^(-1)GR-5E-S,400 nmol·L^(-1)发夹三聚体的反应溶液中,于25℃孵育40 min。当水样中存在Pb^(2+)时,GR-5E-S识别Pb^(2+),底物链被切割,酶链和底物链分开并释放出目标链T,目标链T与发夹链H1杂交触发动态自组装过程,生成树枝状聚合物,于520 nm处测量荧光强度。结果显示:建立的荧光传感体系可在40 min内完成检测;Pb^(2+)的浓度在0.1~10.0 nmol·L^(-1)内与对应的传感体系的荧光强度呈线性关系,检出限(3s/k)为19 pmol·L^(-1);按照标准加入法进行回收试验,回收率为98.0%~108%,测定值的相对标准偏差(n=5)不大于15%。A novel enzyme free and highly sensitive fluorescence sensing system for detecting Pb^(2+)was constructed based on the specific recognition of GR-5 deoxyribozyme(GR-5 DNAzyme)for Pb^(2+)and the signal amplification strategy of hairpin DNA dynamic self-assembled dendrimer.The sequence extended GR-5 DNAzyme complex(GR-5E-S,synthesized from GR-5 DNAzyme enzyme chain GR-5E and substrate chain GR-5S)and Y-shaped skeleton(synthesized from skeleton chains Y1,Y2,and Y3)were synthesized by incubating at a constant temperature of 95℃.Hairpin trimers(synthesized from Y-shaped skeleton and hairpin chains H1,H2 and H3,in which fluorescent and quenching groups modified on hairpin H1)were synthesized by incubating at a constant temperature of 25℃.Water sample(4μL)was added into 96μL of the reaction solution containing 400 nmol·L^(-1)GR-5E-S and 400 nmol·L^(-1)hairpin trimers,and the mixed solution was incubated at 25℃for 40 min.When Pb^(2+)was present in the water sample,GR-5E-S recognized Pb^(2+),and the substrate chain was cleaved.The enzyme chain and substrate chain were separated,and the target chain T was released.Target chain T hybridized with hairpin chain H1 to trigger a dynamic self-assembly process,generating the dendrimer.Fluorescence intensity was measured at 520 nm.It was shown that the established fluorescence sensing system could complete detection within 40 min.Linear relationship between values of the concentration of Pb^(2+)and the fluorescence intensity of the corresponding sensing system was kept in the range of 0.1-10.0 nmol·L^(-1),with detection limit(3s/k)of 19 pmol·L^(-1).Test for recovery was made according to the standard addition method,giving recoveries in the range of 98.0%-108%,and RSDs(n=5)of the determined values was not more than 15%.

关 键 词：GR-5脱氧核酶 催化发夹自组装等温扩增技术 Pb^(2+) 荧光传感体系 

分 类 号：O657.32[理学—分析化学]