A Ratiometric Coulometric Immunosensor Based On Carbon-printed Electrodes For Detection Of Japanese Encephalitis Virus

Japanese Encephalitis Virus(JEV)is a single positive-strand RNA virus which belongs to the Flaviviridae family.It can cause serious illnesses,such as Japanese Encephalitis(JE),meanwhile it has a certain impact on global viral encephalitis syndrome(AES).About 70,000 people are infected with the disease every year,the virus is transmitted to humans and livestocks through Culex mosquitoes.At present,there are no effective treatments to cure patients.Therefore,it is particularly critical to diagnose the virus early.Now,many methods of detecting viruses using electrochemical biosensors have been reported.However,it is still a tough task to detect target antigens repeatedly and reliably without any signal amplification.This dissertation solves the above mentioned problems by the electricity method which amplifies the analysis signal by only increasing the electrolysis time.In addition,environmental interference is eliminated through ratio analysis technology,so can obtain more accurate signals.Electrochemical immunosensors have attracted a great deal of researchers' attention due to their low cost,high sensitivity,easy miniaturization,portability,and in-situ analysis,but it needs complex electrode pretreatment and modification and poor selectivity.In order to simplify electrode pretreatment and achieve batch electrode modification,and to amplify electrical signals without using antibodies that bound to complex conjugates,this paper uses " Detection of Japanese Encephalitis Virus by Ratio Coulomb Immunosensor Based on Carbon Printed Electrode " as the research topic,and develops two different methods of immobilizing antibodies on the surface of carbon printed electrodes.Then,the background and total electricity is measured simultaneously by using the electricity method,the ratio of the total electricity to the background electricity is used as an electrical signal to quantify the amount of virus in the serum.First,the gold nanoparticles is electrodeposited on the surface of the carbon printed electrode to bond cysteamine,and the oxidized dextran with rich aldehyde groups,good biocompatibility,and not easily hydrolyzed was used as a cross-linking agent to fix the antibody;Secondly,we want to batch modification of carbon printed electrodes.The two electrochemical immunosensors constructed in this subject can sensitively detect JEV virus and IgM produced by early infection of JEV,and it is expected to be developed into a universal virus detection sensor,which can provide a simple detection method for clinical diagnosis.This research is supported by the funding of the National Natural Science Foundation of China,"Research on new methods for electrochemical detection of peripheral blood circulating tumor cells by signal amplification"(No.21175089)and the Central University Fundamental Research Fund(No.GK201906004 and GK201801006).This dissertation is composed of two parts:introduction and research content,which can be divided into three chapters:Chapter 1 is a general introduction.The basic principles and classification of electrochemical immunosensors are summarized.Five immobilization methods of recognition molecules(antibodies/antigens)on the electrode surface of the sensing platform are introduced.And the current research status of gold nanoparticles and their composites as well as polydopamine in electrochemical biosensors are briefly reviewed.Next,a brief introduction of the production,transmission route,affected area,patient's symptoms of JEV virus,traditional detection methods is given.Finally,it describes the research background and research content of this paper.Chapter 2 constructs a simple one-time ratio-type electrochemical immunosensor to detect Japanese Encephalitis Virus(JEV),and highly selective and sensitive quantitative detection is achieved by Coulomb detection technology.First,AuNPs are electrodeposited on the surface of a carbon printed electrode by three-pulse current method,and then cysteamine molecules are self-bonded via Au-S.In addition,in this study oxidized dextran(DPA)is used as a cross-linking agent.Compared with traditional glutaraldehyde,DPA exhibits more abundant aldehyde groups,excellent stability,difficult hydrolyzability,and excellent biocompatibility.Then,the antigen-antibody completes the immune complex through specific recognition.Finally,an electrochemical oxidation cycle is formed between the electrons generated by the HRP catalyzed H2O2 decomposition and the electroplating electron mediator PVI-Os on the electrode surface.There is a linear relationship between the electrical signals generated and the concentration of JEV.The electrochemical immunosensor proposed in this chapter has a wide linear range,low detection limit,and high selectivity,and can successfully detect JEV infection.In Chapter 3,a disposable electrochemical immunosensor based on Gp@PDA@AuNPs composite is prepared.Dopamine is polymerized on the surface of graphite powder by self-polymerization in alkaline solution.The formed polydopamine(PDA)can reduce HAuCl4 to AuNPs.By the above method,we prepare the composite material Gp@PDA@AuNPs.The surface of the freshly printed disposable carbon printed electrode has glue-like adhesion,so the composite material is firmly adhered to the electrode surface as a sensing platform,achieve batch modification of carbon printed electrodes.After that,the anti-IgM antibody is immobilized on the surface of the sensor to capture IgM in the serum,and the virus-like particles(VLP)are introduced as recognition molecules,which improved the selectivity and biosafety of the sensor.The ratio of the total charge to the background charge is used as the electrical signal,which greatly reduces the error which may be caused as sensor changes.The prolongs of the electrolysis time can achieve highly sensitive and accurate measurement of IgM which is at low concentrations.The immunosensor presented in this section provide a threshold for the diagnosis of early JEV infection.