Research On The Construction Of Nano-sensing Interface And Detection Of DNA Components In Genetically Modified Food

Genetically modified organisms（GMO）can increase crop yields,enhance crop resistance to diseases and insect pests,and improve the storability of agricultural products.However,its safety and potential threats to the environment have aroused widespread concern in the world.Therefore,many countries have issued relevant policies/rules to strengthen the supervision of GMO and GM food.Thus,the simple and rapid qualitative and quantitative detection methods of GM products are indispensable tools to guarantee the supervision.Cauliflower Mosaic Virus 35S（Ca MV35S）promoter and terminator nopaline synthase（NOS）are the most DNA elements in regulating expression in GM crops.Here,we selected Ca MV35S promoter and NOS terminator as target genes,and constructed nanosensing interfaces to fabricated colorimetric and electrochemical biosensors for determination of these targets in GM crops.1. Construction of Fe₃O₄@Au NP@c DNA@H-GN nanocomposite-based sensing interface and its application in colorimetric detection of NOS in GM tomatoes.We developed a simple and label-free colorimetric biosensor for detection NOS in GM plant.We have prepared a nanocomposite material to build a biosensing interface,which consisted with gold nanoparticles doped magnetic Fe₃O₄nanoparticles（Fe₃O₄@Au NPs）,thiolated capture probe DNA（c DNA）and hemin functionalized reduced graphene oxide nanosheets（H-GN）.The c DNA acted as bridges of Fe₃O₄@Au NPs and H-GN in the nanocomposite.Based on the Au-S bond and the strongπ-πinteraction between single-stranded DNA（ss DNA）sequence and H-GN,a colorimetric sensing interface was successfully constructed.In the presence of target NOS,the c DNA in the nanocomposite will hybridize with its complementary sequence,the target NOS,to form a double-stranded DNA（ds DNA）structure.Due to the weak interaction between ds DNA and H-GN,a portion of H-GN released from the surface of Fe₃O₄@Au NPs and transferred into solution.Performing magnetic separation to separate the nanocomposite from the solution,the supernatant was used to incubate with 3,3′,5,5′-tetramethylbenzidine（TMB）in presence of H₂O₂.Because of the excellent mimic peroxidase activity of H-GN,the supernatant with the released H-GN can catalyze the oxidation reaction of TMB in presence of H₂O₂,turning the colorless solution into blue.This“signal-on”colorimetric biosensor showed a broad linear range of 0.5–100 n M for target NOS with a detection limit of 0.19 n M（S/N=3）.The biosensor has good selectivity and stability,and successfully detected the NOS sequence in transgenic tomato samples,and the recovery rate was between 93.59%and 94.71%.2. Hemin-modified polypyrrole nanospheres sensitized point-triggered chain displacement reaction signal amplification for electrochemical determination of Ca MV35S gene sequences.This work proposes a method for constructing a sensitive electrochemical biosensor determination of Ca MV35S gene sequences.We prepared hemin functionalized carboxylated polypyrrole（c PPy-hemin）nanospheres by one-pot method.It was then biofunctionalized with probe DNA to form nanotag of DNA-c PPy-hemin though covalently bond between amino group and carboxyl group.In order to construct the nanosensing interface,Au NP was electrodeposited onto the surface of the glassy carbon electrode（Au NP/GCE）,the capture probe with–SH was then immobilized onto the surface of the electrode（c DNA/Au NP/GCE）via Au-S bond.The electrochemical sensing interface was fabricated by blocking c DNA/Au NP/GCE with mercaptoethanol.In the absence of the Ca MV35S,the DNA-c PPy-hemin was association with the template DNA strand（Ts）.However,in the presence of the Ca MV35S,toehold-mediated strand displacement amplification reaction was triggered,leading to the association of plenty of fuel DNA strand（Fs）with Ts.The DNA-c PPy-hemin was released and further bound with the capture probe on the electrode surface.The current signal was generated by catalyzing H₂O₂through the excellent peroxidase activity of c PPy-hemin.Under optimal conditions,The prepared biosensor showed a good linear relationship for the determination of Ca MV35S gene sequence in the range of 10 f M–1 n M with a detection limit of 1.939f M（S/N=3）.It has good selectivity that makes it a potential method in screening and detection of GM food.