Signal Amplification Of The JE Virus Electrochemical AC Impedance Immunosensor

Since ancient times, diseases caused by virus have been always the threat to human health. Among them, Japanese encephalitis virus (JEV), which transmits by culex, is one of the most severe virus that causes diseases. It can be the infringer of the central nervous system, resulting in viral encephalitis. In clinic, its symptoms are mainly composed of high fever, convulsions, disturbance of consciousness and meningeal stimulation, which has a high case fatality rate and many of the survivors with nerve, cognitive and mental sequelae. It is one of the serious public health problem widely raged in our country and many other countries in Asia. So far there is no effective antiviral drugs, for JEV infection encephalitis mainly support and symptommatic treatment. Therefore, to establish a rapid, sensitive JEV early diagnosis method has important application value.After JEV infect humans, itusually has low viral load in the blood and goes through a brief period of viremia into the brain tissue, therefore, it is necessary to develop a highly sensitive and rapid method for virus analysis. Electrochemical method has attracted great attention in the field of biological sensors because of its high sensitivity, easy to miniaturization and other characteristics. In a lot of biosensors, electrochemical impedance spectroscopy technology based on fast detection, high sensitivity, little interference to the system advantages, favored by the researchers. In this paper, a highly sensitive electrochemical impedance immunosensor were prepared to detect IgM and JEV, using antibody as molecular recognition material, IgM and JEV as target and HRP biomaker as signal amplification material in the whole process.This thesis consists of four chapters:Chapter 1:IntroductionFirstly, it maked a brief overview of the JEV, introducing its biological characteristics, pathogenic mechanism, prevention measures and the traditional detection methods. Secondly, it introduced the principle and classification of the biosensor and elaborated the application of biosensor in virus detection. Then, the electrochemical immunosensors were reviewed, and mainly highlighted the electrochemical impedance technique and its application in the virus detection. Subsequently, the technology of screen printing was described in brief and presented its application in the terms of virus sensors. Finally, the research background, research ideas, purpose and contents of thie paper were expounded.Chapter 2:A highiy sensitive electrochemical immunosensor for the detection of IgM based on the insulating film signal amplificationAn ultrasensitive impedimetric immuno-assay based on horseradish peroxidase (HRP) tag-initiated deposition of an insulating film on gold electrode was developed. When the target IgM existed, it will be captured on the surface of electrode using double antibody sandwich method. Then, HRP labeled on the second antibody was used to the biocatalytic oxidation of 4-chloro-l-naphthol (CN) to the insoluble product, which hindered the electronic transfer and thus played a role in signal amplification. Finally, the IgM was tested by comparing the change of impedance values. The response was linearly related to the target IgM concentration ranging from 1.6 fg/mL to 1.6 ng/mL, and the limit of detection (LOD) was 0.53 fg/mL(S/N= 3). In addition, the impedimetric immunosensor showed good selectivity.Chapter 3:Signal amplification impedimetric detection of Japanese encephalitis virus (JEV) based on a horseradish peroxidase tag-initiated deposition of an insulating film on gold electrodeThis study constructed a "sandwich-type" electrochemical virus immunosensor using double antibody sandwich method. Firstly, the gold electrode was functionalized with-COOH by a mixed self-assembly of 11-mercaptoundecanoic acid (MUA), 6-mercaptohexanol (MCH) and dithiothreitol (DTT), where MCH and DTT play a role in sealing the electrode. Secondly, the Abi was fixed by forming amide bond between-NH2 and the-COOH. Afterwards, JEV and HRP-anti-JEV were fastened on the modified electrode. The HRP-Ab2 biocatalyzed oxidation the CN in the presence of H2O2, generating an insoluble precipitation on the immunosensor, thus causing a local alteration of the conductivity. Finally, electrochemical impedance spectroscopy was adopted to detect the accumulation of precipitate on the electrode, thus achieving the detection of JEV. The method has a high sensitivy and specificity.Chapter 4:An ultrasensitive impedimetric electrochemical immunosensor for the detection of JEV on a disposable gold nanoparticles-modified screen printed carbon electrodeIn this study, a sensitive and low cost electrochemical impedance immuno-assay for the detection of Japanese encephalitis virus (JEV) was developed based on a disposable gold nanoparticles-modified screen printed carbon electrode.Firstly, Chrono potentiometry (?t>1 ms) was employed as a technique to make the electrodeposition of gold nanoparticle to SPCE. Secondly, the electrode was functionalized with the self-assembled monolayers (SAM). And then, the immunosens-or was fabricated through covalent grafting the SAM modified AuNPs-SPCE with specific antibody. Subsequently, the HRP-labeled second antibody was linked to the electrode through a specific immunity reaction after the JEV bound to the immobilized antibody. Finally, the HRP catalyzed the CN oxidation to produce an insoluble precipitation which introduced a barrier for the electron transfer between the redox probe in the electrolyte solution and the electrode. It showed a good linear relationship between the concentration of JEV and the change of impedance value caused by the amount of precipitation in tne range of 500 pfu/mL to 500000 pfu/mL and the detection limit was 167 pfu/mL (S/N= 3). The sensor provides a low-cost, high sensitivity and good selectivity method for the detection of JEV.