Enhanced Electrochemical DNA Sensing On Carbon Nanocomposite And Detection Of CaMV35S Sequence In Transgenic Crops

In recent years,the detection of specific DNA sequences has potential applications in environmental protection,food safety and quality monitoring,molecular diagnosis and other fields,and has become an important research field.With the development of transgenic biotechnology,its uncertainty and immaturity make the safety of genetically modified food arouse public's attention.Detection of specific nucleotide sequence of genetically modified food is also more and more important.Among different DNA detection methods,electrochemical methods have attracted a large attention.Due to its simplicity,speed and low cost,they have become new topics in the fields of medicine,biology and food science.In this paper,we use 5'-GGC TCC TAC AAA TGC CAT CAT T-3 'sequence and of Ca MV35 S promoter(5'-TCA TCA TCA GTC TGA TAA CGT A-3')as detecting sequence.The glassy carbon electrode(GCE)was modified by nanometer carbon nanomaterials,such as Thi-r GO and hemin-r GO,as well as gold nanoparticles(Au NPs).A new electrochemical DNA biosensor with convenient operation,high sensitivity and low detection limit was constructed.The main contents of this paper include two parts:1.A label-free electrochemical DNA biosensor based on thionine functionalized reduced graphene oxideDue to the outstanding electrochemical activity of Thi in Thi-r GO nanocomposites,the signal transduction induced by the redox reaction of thi is directly induced.However,the redox reaction will be hindered by the biomolecules that are subsequently fixed to the surface of the GCE.Therefore,the detection of target sequence(t DNA)can be achieved by detecting the volt-ampere response current of Thi before and after the hybridization of CP and t DNA.In this paper,a transducing interface was designed on a glassy carbon electrode(GCE)to construct a label-free DNA biosensor.The transducing interface was consisted of the gold nanoparticles(Au NPs),the capture DNA sequence(CP),and thionine functionalized reduced graphene oxide(Thi-r GO).Due to the excellent electrochemical activity of Thi in Thi-r GO nanocomposites,the signal transduction induced by the redox reaction of thi is directly induced.However,the redox reaction will be hindered by the biomolecules that fixed on the surface of the GCE.Therefore,the detection of target sequence(t DNA)can be achieved by detecting the volt-ampere response current of Thi before and after the hybridization of CP and t DNA.Under the optimum conditions,the peak current decreases linearly with the logarithm of t DNA concentration in the range from 1.0×10-17 to 1.0×10-12 M,and the detection limit is 4.28×10-19M(S/N = 3).The DNA biosensor shows the advantages of acceptable selectivity,stability and repeatability.The proposed DNA biosensor is used for t DNA detection in real biological samples,the recovery rate is higher than 95.61%.2.A sandwich-type DNA biosensor for the detection transgene Cauliflower Mosaic Virus 35S(Ca MV35S)promoter based onhemin-r GO and thionine-decoratedgold nanoparticle signal amplificationIn this work,a novel eletrochemical DNA biosensor was developed for the detection of target DNA fragment from the transgene cauliflower mosaic virus 35S(Ca MV35S)promoter.Firstly,Hemin functionalized reduced graphene oxide(hemin-r GO)was prepared according to our previous methods and then modified onto the surface of GCE,then Au NPs were eletrodeposited on the surface of the hemin-r GO to immobilize the capture DNA.On the basis of this platform,we designed a sandwich type electrochemical DNA biosensor.When the target DNA was existed,the third auxiliary DNA labeled with 5'-SH could hybridization with the target DNA,and then the Au NPs is adsorbed by the covalent binding of Au-S.After that,the electrode was soaked in the thi solution which can generate a huge signal because of the strong electrostatic adsorption between Au NPs and Thi molecules.Under the optimal conditions,the concentration of ampere signal and target DNA concentration is linear in the range of 1x10-10 ~1x10-16mol/L,and the detection limit is 99.45 a M.The proposed DNA biosensor showed a acceptable selectivity,stability and reproducibility.The sensor has been used in the detection of Ca MV35 S transgenic sequences in real samples,and the results are satisfactory.