基于羧基化聚吡咯-氯化血红素和点触发链置换反应信号放大的电化学生物传感器检测转基因作物中CaMV35S序列

Detection of CaMV35S Gene Sequence in Transgenic Crops Using an Electrochemical Biosensor Based on Carboxylated Polypyrrole-Hemin and Point-triggered Strand Displacement Reaction Signal Amplification

为适应快速增长的转基因生物(GMOs)安全评价与管理的需求,高效、可靠的转基因检测技术研究与应用的重要性日益凸显。该文采用一步法合成了羧基化聚吡咯(cPPy)与氯化血红素(Hemin)的纳米复合物(cPPy-hemin),并以其为信号标签合成了一种DNA双链结构功能化的纳米信标(DNA-cPPy-hemin)。利用cPPy-hemin增强的模拟酶催化活性,结合点触发链置换反应(TSDR)信号放大策略,制备了一种新型电化学基因传感器用于转基因成分的灵敏检测。通过信号探针(Ps)与模板链(Ts)、辅助链(As)结合形成DNA双链体结构中的-NH2功能化cPPy-hemin,制备Ts/As/Ps-cPPy-hemin双链DNA结构的纳米信标。在目标花椰菜花叶病毒35S启动子(CaMV35S)序列和燃料链(Fs)存在下,TSDR过程释放出的Ps-cPPy-hemin与固定在电化学沉积金纳米粒子修饰GCE表面上巯基化的DNA捕获探针(Cs)相结合,利用cPPy-hemin对H2O2的模拟酶催化产生的电信号,可实现对CaMV35S的定量检测。在最优条件下,该传感器对目标基因序列的检测范围为1.0×10^(-14)~1.0×10^(-9)mol/L,检出限(S/N=3)为3.2×10^(-15) mol/L。制备的传感器具有良好的选择性、重现性与稳定性,可用于转基因大豆中总DNA提取物的CaMV35S转基因片段的检测。

In order to meet the rapidly growing demands for safety evaluation and management on the genetically modified organisms(GMOs),the research and application of efficient and reliable GMOs detection technology has become more and more important.A novel electrochemical genosensor was prepared for sensitive detection of GMO components by utilizing the enhanced mimic peroxidase activity of cPPy-hemin to H2O2 and signal amplification strategy of point-triggered strand displacement reaction(TSDR) in this paper.Carboxylated polypyrrole(cPPy) and hemin(cPPy-hemin) nanocomposites were synthesized by a one-step method,which was used as signal tags to immobilize doublestranded DNA to form DNA-cPPy-hemin signal nanotags.Using the-NH2 groups in DNA duplex structure composed of signal probe(Ps),template strand(Ts) and auxiliary strand(As),the prepared cPPy-hemin was biofunctionalized to form Ts/As/Ps-cPPy-hemin. In presence of target transgenic cauliflower mosaic virus 35S promoter(CaMV35S) and fuel strand(Fs),the TSDR was triggered by the target CaMV35S,the released Ps-cPPy-hemin was captured by the immobilized capture probes(Cs) on the surface of GCE through hybridization.The quantitative detection of the target CaMV35S was realized by recording the catalytic reduction signal of H2O2by cPPy-hemin. Under the optimal conditions,the fabricated biosensor had a good linear range of 1. 0 × 10^(-14)-1. 0 × 10^(-9)mol/L,with a detection limit(S/N = 3) of 3. 2 × 10^(-15)mol/L.The biosensor possessed good selectivity,reproducibility and stability,which could be used for the detection of the CaMV35S segments in total DNA extracts from genetically modified soybeans.