Fabrication Of The Amperometric Enzyme Immunosensor For Antigen Of Avian Influenza H5N1 Virus/Antibodies Against Avian Influenza H5N1 Virus Based On 4-Spce

Avian influenza virus(AIV)infects a variety of animals including human beings,pigs,horses,marine mammals,and birds.Recently,hundreds of outbreaks of highly pathogenic avian influenza(HPAI)caused by virus subtype H5N1 occur in nations and regions one after another.AIV have had devastating effects on the poultry industry worldwide and occasionally have crossed directly the species barrier to infect humans and other animals.Therefore,it is important that rapid,sensitive and specific assays should be designed for effectively prevention and control of avian influenza.Electrochemical immunosensors,which combine the specificity of immuno-chemical systems with the advantages of electrochemical transducer, such as robustness,sensitivity and inexpensive instrumentation,have been the subject of continued research in recent years.It has been widely researched in food safety rapid detection,clinical medicine,environmental monitoring, military affairs and other areas.The researches carried out in this paper would be described as follows:1.Four-channel screen-printed carbon electrode preparation and performance testIn this paper,several multi-electrode structures were fabricated by screen printing technique which designed with carbon working electrode,carbon graphite electrode and Ag/AgCl reference electrode.Four-channel screen-printed carbon electrode was selected for the follow-up study.The reproducibility of those electrodes was assessed by morphological and electrochemical methods.Horseradish peroxidase(HRP)was immobilized on the four carbon working electrodes using physical adsorption.Cyclic voltammetry(CV)was empolyed to demonstrate the feasibility of electron transfer between peroxidase and electrode.The H₂O₂ biosensor prepared displayed a quick and sensitive response to hydrogen peroxide.Linear calibration for H₂O₂ was obtained in the range from 2.5×10^-5mol/L to 7.0×10^-4 mol/L with a detection limit of 1.5×10^-5mol/L at a signal-to-noise ratio of 3. Compared with other sensors,the immobilization of enzyme was simple and the sensor exhibited a better stability,reproducibility and accuracy.2.A disposable amperometric electrochemical immunosensor for antigen of avian influenza H5N1 virus based on chitosan and screen-printed carbon electrode A disposable electrochemical immunosensor for detection of antigen of avian influenza H5N1 virus(AIV-Ag H5N1)based on the screen-printed carbon electrode coated with chitosan membrane and HRP-anti-AIV H5N1 has been developed in the experiment.The immunosensor was incubated with the one-step immunoassay assay format involving AIV-Ag H5N1 for 10 min at 20℃.The access of the active center of HRP catalyzing the oxidation reaction of thionine by H₂O₂ was partly inhibited by VP,which connected on the surface of the immunosensor by immunoreaction.Cyclic voltammetry(CV)was used as electrochemical means to detect the products of the enzymatic reaction. According to the decrease of reduction current,AIV-Ag H5N1 could be detected. Under the optimum conditions of immunoreaction and electrochemical detection, AIV-Ag H5N1 was rapidly detected by sigmoidal curve method and form a linear calibration from 3.75 to 125 ng/mL with an associated detection limit of 0.56 ng/mL(S/N=3).The immunosensor had acceptable specificity, reproducibility(RSD＜7.1%),stability(the amperometric response was 91.46% of the initial response after two weeks),accuracy(91.2%of the results obtained by the immunosensor were in agreement with those obtained by AGP)and good consistency.Therefore,the immunosensor could be hopefully used to rapidly detect AIV-Ag H5N1.3.A disposable amperometrie electrochemical immunosensor for antibodies against avian influenza H5N1 virus based on chitosan Au colloid and four-channel screen-printed carbon electrodeA novel electrochemical immunosensor for detection of antibodies against avian influenza H5N1 virus(anti-AIV H5N1)based on the four-channel screen-printed carbon electrode(SPCE)coated with chitosan Au colloid and AIV-Ag H5N1 has been developed in the experiment.Then the immunosensor was characterized by cyclic voltammetry(CV)and laser scanning confocal microscope(LSCM).CV was used as electrochemical means to detect the products of the enzymatic reaction.HRP-labeled rabbit-anti-chicken IgG,which connected on the surface of the immunosensor by immunoreaction,could catalyze the oxidation reaction of thionine by H₂O₂.According to the increase of reduction current,anti-AIV H5N1 could be detected.Under the optimum conditions of immunoreaction and electrochemical detection,a response current increase percentage of(K)more than 30%was determined to be a statistical cut-off positive criterion to distinguish samples,namely K＜20%,negative;20%＜K＜30%,suspicious;K≥30%,positive.The immunosensor had acceptable specificity,reproducibility(RSD=2.48%),stability(the amperometric response was 89%of the initial response after 11 d)and accuracy(92.9%of the results obtained by the immunosensor were in agreement with those obtained by AGP). Therefore,effective prevention and control of avian influenza could be supplied by the immunosensor.