Novel DNA Biosensor Based On Functionalized Graphene And Its Research In Tumor Detection

As the industrialization process accelerates increasingly,environmental pollution becomes more and more serious,and the aging of the population quickens,the morbidity and mortality of malignant tumors are rising rapidly,and malignant tumors have become the biggest killer of human beings and the first cause of death among all.Since the patients with early-stage malignant tumors are more likely to be cured than those late-stage patients and early diagnosis can save more money,manpower and time,it has important clinical significance in increasing the cure rate of tumors and elongating the life span of the patients if the tumors can be diagnosed in an early stage.As the deepening of gene structure and function research promotes the studies on gene diagnosis and therapy,people gradually find that malignant tumors and other serious diseases are all related to genetics.Minor changes in gene sequence of DNA molecules can lead to changes in genetic information or diseases.Detecting the tumorigenesis-related gene sequence is valid and efficient for clinical application in early diagnosis,monitoring and treatment of tumors.Research on new electrochemical DNA biosensors is of great significance.In this Paper,the nanotechnology,bio-sensing technology and electrochemical analytical techniques are combined for tumor screening and early detection.Ultrasensitive electrochemical immunoassay methods were developed,which indicates a promising potential in early disease screening and diagnosis.Therefore,this research focuses on the preparation of multi-functionalized graphene nanomaterials,the construction of the sensitive interface,the development of sensitivity enhancement electrochemical DNA biosensors and the research on application of new method,bringing forward new ideas for the early detection of malignant tumors and related research.The main contents of this paper carried out as follows:(1)An electrochemical DNA biosensor based on the GO-COOH/CuO for rapid detection of lung cancer related gene CYFRA21-1Preparation of carboxyl functionalized graphene(GO-COOH)to improve the dispersion and biocompatibility of rGO.Synthesis of copper oxide nanowires(CuO NWs)and fully integrate their advantages.GO-COOH and CuO NWs intertwined to form a network structure by simply mixing.This method could avoid the complex process,and CuO NWs in the composite acts as an electron transfer "conductor",promoting the electron transfer rate of the electrode.GO-COOH/CuO NWs composite characterized by SEM,TEM,EDX、FTIR and electrochemical performance testing.The ssDNA probes were immobilized on the surface of GO-COOH/CuO NWs through the formation of amide bonds.MB as the hybridization indicator,electrochemical DNA sensors based on GO-COOH/Cu O NWs/PLLy was used to detection the synthetic sequence of CYFRA21-1 gene.Research results demonstrate that the peak current of this sensor was linear with the logarithm of target ssDNA concentration from 1.0 × 10-12 M to 1.0 × 10-6 M.And this biosensor also has excellent selectivity and stability.(2)A DNA biosensor based on NG / AuNPs for MDR1 gene detectionModification of electrochemical activity of graphene was performed by element doping method.The NG/AuNPs composites were obtained by the in situ reduction of Au3+ into NG obtained via N-doped GO,and sulfhydryl modified MDR1 probe was then fixed on surface of electrode that modified with NG/AuNPs sensors by the strong S-Au bond.The physical and chemical properties of materials herein were then characterized by SEM,TEM,XRD and XPS.Owing to the excellent electro-catalytic ability and biocompatibility of NG/AuNPs,the detection limit of the electrochemical DNA sensor obtained towards MDR1 was 3.12×10-15 mol/L.NG/AuNPs DNA biosensor obtained herein was employed for the determination of RT-PCR products of MDR1 gene in HL-60 human original myeloid leukemia cells,and the experimental results obtained were compared with that obtained by the commonly used PCR methods in molecular biology,it revealed that this electrochemical DNA sensor method established was faster and exhibited higher sensitivity,and which demonstrated potential application prospect for the determination of multi-drug resistance related MDR1 gene of leukemia in clinic.(3)A novel electrochemical DNA biosensor based on 3D NG-Fe3O4Here we integrated of 2D graphene to prepare the functionalized graphene with 3D foam structure.3D NG-Fe3O4 was synthesized by a green environmental protection,easy to operate hydrothermal method,discusses its three-dimensional structure forming mechanism,and its forming mechanism were discussed.3D NG is not only to maintain the inherent quality of graphene,and compared with 2D graphene,3D NG has more superior performance in electric catalytic reaction such as high porosity,high specific surface area,flexibility and more excellent electrical conductivity,etc.Various techniques(SEM,TEM,XPS and EDX)were used to characterize the structure and composition of the 3D NG-Fe3O4.Owing to the large specific area and good conductivity of the 3D NG-Fe3O4,this new DNA biosensor showed a wide linear range and low detection limit for detection of DNA.Moreover,the electrochemical DNA biosensor also had good detection capability in complicated leukemia human serum samples.(4)An electrochemical DNA sensors basd on 3D GF /AgNPs for detection of non-small cell lung cancer CYFRA21-1 PT-PCR enzyme digestion productThe 3D graphene materials with more uniform and spatial structure were prepared by Chemical Vapor Deposition(CVD)method with CH4 as carbon source,nickel foam as three dimensional template and catalyst,and then the physical and chemical properties of materials obtained were characterized by SEM,TEM,EDX and contact angle measurement.The 3D GF obtained hereof was high quality and almost with no defects.Then the electrodeposition method was used to obtain the composite of silver nanoparticles(Ag NPs)on three-dimensional carbon skeleton of 3D GF,which will therefore lead to the modification of hydrophilicity of 3D GF via coating a layer of Ag NPs with excellent performance on surface of the perfect three dimensions grid structure.The dynamic mechanism of the modified electrode surface was discussed via the relationship between potential scanning speed and electrochemical response of ssDNA/3D GF/AgNPs/GCE,which proved the stable and spatial inter-connective porous structure of 3D GF /AgNPs,and which led to significant increase of the effective area of electrode,besides,provided favorable conditions for the loading of ssDNA probe sequence on surface of the electrode.And 3 D GF/AgNPs was proved to be a novel and promising electrochemical DNA sensors modification material.A satisfactory result of practical sample determination of non-small cell lung cancer gene CYFRA21-1 by the electrochemical DNA sensors obtained based on 3D GF/AgNPs was obtained,which further proved the accuracy of the current method.(5)A "sandwich" biosensors with DNA markers for detection of breast cancer MCF-7 cellsIn combination with the advantages of electrochemical biosensor and DNA modified antibody,a kind of sandwich type immunosensor for the electrochemical detection of human breast cancer MC-7 cells was prepared.Firstly,the 3 D GF was used as desirable electrode substrate material,and then the Au nanocage functionalized material of MWCNT was used as the adsorption substrate of ER protein antibody of breast cancer(antibody-ER,Ab1).The physical and chemical properties of the materials obtained were characterized by UV,SEM,TEM and cytotoxicity test.The composite of Au NCs with MWCNT-NH2 exhibited low toxicity,high active specific surface area,perfect biocompatible environment and an extremely novel and unique cage structure,which can significantly enhance the electron transfer ability of the electrode surface,can severely react with the Ab1 antibody,and can lead to the solid loads of Ab1 on the electrode surface,and therefore can adsorb more breast cancer MCF-7 cells on the sensor.The bioprobes of ssDNA-Ab2 were prepared via the modification of 5’end of ssDNA of breast cancer ER protein antibody with coupling agent EDC+NHS,and the biological activity and capture efficiency were investigated by spectrum method.And finally,the breast cancer MCF-7 cells were determined with the sandwiches-type bionic interface that established by the modification with functionalized nanomaterial-antibody Ab1,breast cancer MCF-7 cells and ssDNA-Ab2 bioprobes.And the DPV peak current response exhibited a good linear relationship with breast cancer MCF-7 cell volume,the LOD of breast cancer MCF-7 cells was 1.0×102 cells mL-1.And such immunesensor also demonstrated perfect reproducibility and stability,and can also be employed as other related tumor markers.The present establishment method provided a kind of effective electrochemical detection means for tumor cell detection,and meanwhile put forward another new way for immunosensor.