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    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Pb2+的含量

    Determination of Pb2+ in Water by Fluorescence Sensing System Based on Amplification Strategy of Hairpin DNA Dynamic Self-Assembled Dendrimer

    • 摘要: 基于GR-5脱氧核酶(GR-5 DNAzyme)对Pb2+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Pb2+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Pb2+时,GR-5E-S识别Pb2+,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;Pb2+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%。

       

      Abstract: A novel enzyme free and highly sensitive fluorescence sensing system for detecting Pb2+ was constructed based on the specific recognition of GR-5 deoxyribozyme (GR-5 DNAzyme) for Pb2+ 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(4 μL)was added into 96 μL 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 Pb2+ was present in the water sample, GR-5E-S recognized Pb2+, 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 Pb2+ 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%.

       

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