转基因作物中CaMV 35S序列信号双重放大的电化学基因传感方法

Electrochemical Genosensing for Duo-Amplification Signal of CaMV 35S Sequence in Genetically Modified Organism

该文设计并制备了一种针对转基因花椰菜花叶病毒35S(CaMV 35S)启动子检测的新型电化学基因传感器。首先制备了血红素功能化的还原氧化石墨烯(Hemin-rGO),并将其修饰于玻碳电极(GCE)表面,再通过电沉积金纳米粒子(AuNP)固定巯基化的DNA捕获探针。通过设计的夹心型传感策略,当目标序列存在时,辅助DNA通过杂交形成双链并利用其巯基化5'端共价结合AuNP,在负载硫堇(Thi)后放大电流响应,可灵敏检测目标CaMV 35S启动子序列。考察了测试底液pH值、Thi浓度、AuNP富集时间以及DNA序列杂交时间对检测性能的影响。获得最优测试条件为:pH 7.4,Thi浓度为0.5 mmol/L,AuNP富集时间为40 min,DNA杂交时间为60 min。在最优检测条件下,示差伏安电流信号与目标DNA浓度的对数在1×10^(-16)~1×10^(-10)mol/L范围内呈线性相关,检出限为9.46×10^(-17)mol/L。该基因传感器显示出高选择性,较好的稳定性和重复性,用于转基因拟南芥实际样品中CaMV 35S启动子序列的检测,其检测结果与凝胶电泳法一致。方法对食品、饲料及其他工业品等原料或成品中转基因作物成分的快速检测筛查具有很好的应用前景。

A novel electrochemical genosensor was designed and fabricated for the detection of transgenic cauliflower mosaic virus 35S(CaMV 35S)promoter in this paper.Firstly,hemin functionalized reduced graphene oxide(Hemin-rGO)was prepared and modified onto the surface of a glassy carbon electrode(GCE).And then,gold nanoparticles(AuNP)were electro-deposited on the surface of the Hemin—rGO to immobilize the thiolated capture DNA.By designing a sandwich-type sensing strategy,when the target sequence was present,the auxiliary DNA hybridized with the target and capture probe to form a double-strand and used its thiolated 5’-end to covalently bind to AuNP.Meanwhile,the amplified current response for the sensitive detection target CaMV 35S promoter sequence was obtained when the former was loaded with thionine(Thi).Effects of buffer pH value,Thi concentration,soaking time of AuNP,and incubation time of DNA sequences were investigated.The optimal conditions were as follows:pH 7.4,Thi concentration:0.5 mmol/L,soaking time of AuNP:40 min,incubation time of DNA sequences:60 min.Under the optimal conditions,there was a good linear relationship between DPV peak current and logarithms of the target concentration in the range of 1×10^(-16)-1×10^(-10) mol/L,with a detection limit of 9.46×10^(-17) mol/L.The genosensor was applied to the detection of CaMV 35S promoter sequence in transgenic Arabidopsis samples with high selectivity,good stability and repeatability.The results obtained were consistent with those of the gel electrophoresis.This method has a good application prospect in the rapid detection and screening of genetically modified organism(GMO)ingredients in food,feed and other industrial products.