Experimental Studies On The Electrochemical DNA Biosensor Based On Au Microelectrode For Detection Of HFMD Gene

Hand,foot and mouth disease(HFMD)is a common childhood infections.Its clinical symptoms,characterized by fever,and hands,feet,mouth and hip skin rash are mild.A few patients have severe encephalitis,myocarditis,pulmonary infection,and even death.Nowdays,methods in clinical diagnosis and monitoring of foot and mouth disease genes include ring mediated isothermal amplification technology,real-time fluorescent quantitative PCR,space-time scan analysis.However,these methods have certain limitation,such as time-consuming,expensive and low sensitivity.Therefore,the development of specific,rapid and easy detection methods for the disease of hand and foot disease has broad application prospect.As the electrochemical DNA has advantages of low price,fast detection,easy miniaturization,and simplity,it has been widely used in genetic diseases diagnosis.In recent years,with the genetic engineering and nanotechnology advanced,the role of microelectrode is crucial.It has the advantage,such as,small polarization current,high signal-to-noise ratio,high mass transfer rate,and high steady state current density,which become a important research direction in the field of electrochemical DNA biosensors.According to the requirement of detection of clinical HFMD disease,this study designs DNA probes specific for the HFMD disease,and constructs the electrochemical DNA biosensor based on microelectrode.Through the investigation on the properties of sensors,such as the sensitivity and specificity,an electrochemical DNA biosensor technology to detect the HFMD disease was established.Electrochemical biosensors based on microelectrode and nanomaterials modified microelectrode were used in the detection of HFMD gene,respectively.The sensitivity,specificity of these two DNA biosensors were been investigated.The main content of this article is as follows:Chapter one: the DNA electrochemical biosensor based on microelectrode for detection of HFMD geneIn this section,a "sandwich" model of DNA biosensor for detection of HFMD gene based on the combination of microelectrodes and electrochemical biological sensing technology was constructed.The sensitivity,specificity of this DNA biosensor was investigated.The results showed this proposed sensor had good specificity and could distinguish the complementary sequence from different mismatched sequences.The relationship between the current and the concentration of HFMD was linear in the range of 0.1 pM ~ 10 nM and the detection limit was 13.3 fM(S/N=3).Gene of foot and mouth disease was detected under the simulated real sample with the serum.The results show that the sensor was able to detect the target sequence and lay the foundation for the analysis of blood samples.Chapter two: the DNA electrochemical biosensor based on nanomaterials modified microelectrode for detection of HFMD geneIn the second part of the experiment,gold nanoparticles were deposited on the surface of the microelectrode by cyclic voltammetry to form AuNPs modified microelectrode,which increased the real surface area of the microelectrode by 10.9 times,thus increasing the loading of the capture probe leading the amplification of signal.This experiment combined of the microelectrode technology,nanotechnology and the electrochemical sensing technology,establishing a sensitive and rapid method to detect hand,foot and mouth disease gene.Herein,SEM and EIS were used to characterize the modified microelectrode and the preparation process,respectively.The relationship between the current and the concentration of HFMD was linear in the range of 0.001 pM ~ 1000 pM and the detection limit was 0.3 fM(S/N=3).In the PCR amplification sample detection study for hand,foot and mouth disease genes,the sensor can realize the detection of PCR amplification products,and the product is consistent with the results of gel electrophoresis of PCR amplified,which lays the foundation for the detection of clinical samples.