Design And Application Study Of Novel Nanomaterial-based Electrochemical DNA Biosensor For KRAS Gene Detection

As we know,detection of gene sequences related to tumor is very important.It is critical not only to early diagnosis,but also the monitoring in cancer treatment.The detection of mutation status in kirsten rat sarcoma viral oncogene?KRAS?is one of the significant research topics in kinds of cancer,such as non-small cell lung cancer,colorectal cancer research and so on.In addition,researchers have found that the mutation status of KRAS gene could affect the treatment result of cancer.Therefore,quick detection of KRAS gene mutation is very urgent and necessary.With the development of nanotechnology and the advancement of multidisciplinary research,biochemical-based sensor methods have provided a new direction for in vitro diagnosis.Electrochemical method has good advantage in molecular detection because of simplicity and high sensitivity.In this study,a series of highly sensitive electrochemical DNA biosensors for KRAS gene detection were designed,which could provide a new method for electrochemical DNA sensors and molecular diagnostics.The main contents of this work can be summarized as follows:?1?MoS₂/MWCNTs-COOH-based electrochemical sensor for the detection of KRAS geneMoS₂/MWCNTs-COOH was prepared through one-pot hydrothermal.The 5'amino-modified capture probe was covalently immobilized on the surface of MoS₂/MWCNTs-COOH modified electrode by amide bond,and methylene blue was used as electroactive indicator.The sensitive detection of the synthetic sequence of KRAS gene was realized.Furthermore,SEM,HR-TEM,FTIR,XRD and XPS were used for the characterization of MoS₂/MWCNTs-COOH.The construction process and electrochemical performance of the sensor were characterized and tested by CV and DPV.The results showed that the MWCNTs-COOH was wound around the sheet of MoS₂,which provided a larger specific surface area for the immobilization of capture probe,and improved the sensitivity and detection performance of the sensor.The linear range of this biosensor was 1.0×10^-141.0×10^-7 M,the detection limit was 3 fM.And it exhibited good selectivity,stability and reproducibility.The results of the recovery rate of standard DNA sequence in a closed normal human serum samples indicated that this sensor could have good practical application value in gene diagnosis.?2?CHA-based electrochemical biosensor for KRAS gene detectionThe gold nanoparticle was deposited on the surface of the glassy carbon electrode by electrodeposition,and then the capture probe was fixed on the electrode surface by the Au-S bond.The CHA signal amplification strategy was used to improve the sensitivity of the sensor.Gold nanoparticle was characterized by SEM.The electrode modification process and detection performance were studied by CV and SWV.The results showed that the detection limit of this electrochemical biosensor for synthetic sequence of KRAS gene was 0.06 fM,and the detection linear range was 0.5×10^-151.0×10^-88 M.The biosensor also exhibited good specificity,stability and reproducibility.The recovery rate of standard DNA sequence in a closed normal human serum sample was studied,and got a nice result.Therefore,this biosensor has certain value in practical application.?3?Electrochemical DNA sensor based on TSDR and Au/MoS₂/rGO for simultaneously detection of two mutation sequences of KRAS geneMoS₂/rGO was firstly synthesized by hydrothermal method,and then AuNPs were synthesized in situ on it.After collected and dried,Au/MoS₂/rGO nanocomposite was obtained,and the materials were modified onto glassy carbon electrodes to immobilize signal dsDNA through Au-S bond.SEM and XRD were used for characterization of Au/MoS₂/rGO.In addition,CV and SWV were were employed for modification process and electrochemical detection performance study of the biosensor.The results showed that this electrochemical biosensor was capble for the sensitive detection of two kinds of KRAS gene mutation,which are c.35G>A and c.34G>T.The detection limits were10.20 fM and 17.16 fM,respectively,and the detection linear range was 1.0×10^-131.0×10^-7 M.With good specificity,stability and reproducibility,this biosensor could have certain value in the application of multiple gene mutation status detection.