Study On Rapid Detection Of Influenza A Virus By Electrochemical Immunosensor

Influenza viruses present a long-term threat to human health. They can causeseasonal epidemics annually, exhibit continuous variations, and sometimes lead tomajor outbreak and pandemics. For its prevention and control, one of the keystrategies is the rapid and accurate screening and diagnosis of suspected cases.Nowadays, there have been many conventional detection methods, such as virusisolation and culture, enzyme-linked immunosorbent assay (ELISA) and real-timereverse transcriptase polymerase chain reaction(rRT-PCR) recommended by WHO,but these techniques still cannot meet the demands in sensitivity, time-saving,specificity, cost-efficiency, and etc.In this study, we choose the A/Beijing/501/2009influenza virus as targetanalyte, and develop an electrochemical Immuno-sensor (EIS) for the detection ofA(H1N1) influenza virus on Screen-printed Carbon Electrode(SPCE), throughexploring the mechanism of the electrochemical immunosensor, developing thetechnology and method of the detection of A(H1N1) influenza virus based on thiselectrochemical immunoassay biosensor. The main results are as follows:(1) We cultivated, collected and characterized the experimental materials,including influenza viruses, the corresponding monoclonal antibody and HRP labelledantibody. The results show that the propagated chicken embryo allantoic fluidcontains influenza A (H1N1) virus,which is verified by colloidal gold-based rapidimmunochromatographic strip assay for influenza A and B subtype virus andrRT-PCR with the WHO recommended primers, and its titers are128HA Unit, whichis determined by haemagglutination assay. It is proved that the A/Beijing/501/2009(H1N1) virus in chicken embryo allantoic fluid can be used for subsequentexperimental study. The affinity between A1-24antibody, influenza A (H1N1) virusand HRP labeling A1-6monoclonal antibody is examined by ELISA, and the resultsshow that these two kinds of antibody material can form high affinity immunesandwich composites with influenza A(H1N1) influenza virus. This immuno-model can be used to build influenza A (H1N1) virus electrochemical immune biosensor(EIS).(2) The electrochemical characteristics of different immobilized methods andmaterials were compared, and the simple physical adsorption of capture antibody onelectrodes was choosen as the optimal capture antibody immobilized method toconstruct the electrochemical immune biosensor for influenza A (H1N1) virus,because this method not only has high signal/background value but also present moresimple and low-cost without expensive nano materials. And then characterization ofelectrochemical behavior of this novel immunobiosensor was manifested by CyclicVoltammetry (CV), and the results demonstrate that the electric active substance onthe surface of the sensor produces current signals under diffusion control, and thecurrent response signal of the sensor is positively proportional to the concentration ofinfluenza A (H1N1) virus, which can be used to quantitative assay of influenzaA(H1N1) virus.(3) The main factors including the amounts of antibody immobilized on SPCE,the reaction temperature between antibody and antigen, the reaction time, and etc.,were studied to investigate their influences on the detection performances of thebiosensors. The optimization concentration of the capture antibody H1-24anddetection antibody of A1-6-HRP are400μg/ml and80μg/ml, respectively, to producestrong detected signals reaching more than80%of the maximum signal. The optimalincubation temperature are37℃, under which the antigen-antibody reaction could beof the highest activity and quickly reach equilibrium state to improve the reactionefficiency. Based on the above conditions, the optimized incubation time of two stepsfor antigen and antibody are45and30minutes respectively, to obtain high sensitivityas well as short detection time. And the whole detection process can be completedwithin2hours and save1-2hours compared with ELISA. Therefore, this novel sensorcan realize rapid detection of A(H1N1) influenza virus.(4) Under the above optimal detection conditions, we established the standardcurve of influenza A (H1N1) virus in the method of EIS, and the standard curve ofELISA and qPCR were also established to investgate the detection performance ofEIS. The statistical analysis of detection results of influenza A(H1N1) and H7N9,H3N2virus, influenzaB virus, EV71and adeno virus as negative control, shows that EIS is of good specificity. RSD values of reproducibility are less than10%while therecovery rates are80%~110%, showing good accuracy and precision. The stability ofthe EIS biosensor can be reliable within10days of storage, and the signal canmaintain over85%after30days, indicating good stability. The detection limit of EIScan reach0.41HA Unit, which is comparative with ELISA, with the detection linearrange is4~64HA Unit. The overall false positive rate of EIS is10%, which is betterthan ELISA. Furthermore, the quantitative accuracy between EIS and qPCR is ofcomparability as the correlation coefficient of the line between the results of EIS andqPCR is0.9517.(5) Based on miniaturized Electrochemical Emstat and the array of portableelectrode, an electrochemical immunosensor array is applied to detect severalinfluenza viruses based on the technology and method of EIS developed in this study.The results show that this novel biosensor has the prospect of application insimultaneous detection of a variety of pathogens since the miniaturized equipmentsare easy to carry in field.