Development And Evaluation Of An Electrochemical Biosensor Detection System For Human Norovirus

Human noroviruses(Hu No Vs)are one of the main food-borne pathogens that cause acute gastroenteritis in people of all ages worldwide.The public health security incidents caused by Hu No Vs have resulted in a heavy economic burden all over the world.The Hu No Vs is highly infection.Until now,there are no approved vaccines and antiviral drugs for this virus.Therefore,the rapid detection and identification for Hu No Vs is of great significance to the prevention and control of Hu No Vs outbreaks.Conventional Hu No Vs detection technologies such as ELISA and RT-q PCR have cumbersome operation,time-consuming and are not time-sensitive for pathogens identification.It greatly limits the rapid response for Hu No Vs prevention and control.Biosensors based on screen-printed electrodes(SPCE)have been widely used in the detection of pathogenic microorganisms due to their high sensitivity,easy operation,and low cost.This research aims to construct a Hu No Vs electrochemical biosensor detection system based on a SPCE.The main research of this system included SPCE pretreatment,electrode modification and characterization,and using Hu No Vs genotypes GI.1 and GII.4 recombinant major capsid proteins VP1(GI.1/GII.4 VP1)as replacements of Hu No Vs to verify the feasibility and evaluate the performance of this proposed biosensor.The research contents and results are as follows:1.The optimal protein expression conditions for the GI.1/GII.4 VP1 recombinant strains were:incubated at 37?with the addition of 0.4 m M IPTG,and induced for 4 h.The expressed GI.1/GII.4 VP1 were purified and used for following research.2.The SPCE was pretreated by electrochemical methods.The results showed that the anode peak current(Ipa)of the activated electrode increased by about 87.0%compared with the un-activated electrode.The pretreated SPCE had better reversibility.The electrochemical performance was significantly improved.3.To prepare an electrochemical biosensor,the gold nanoparticles(Au NPs)were firstly constantly electrodeposited on the surface of the SPCE working electrode.Then protein A was fixed by adsorption method.The conserved S domain of VP1 monoclonal antibody H9E was further fixed using the affinity binding ability of protein A to the antibody Fc region.Finally,the non-specific binding sites of SPCE were blocked with BSA.The preparation of the biosensor was completed.The modification steps such as the time of electrodeposition for Au NPs,fixation time of protein A,and incubation time for antigen-antibody were optimized and characterized by field emission scanning electron microscope(SEM),cyclic voltammetry(CV),differential pulse voltammetry(DPV),and electrochemical impedance spectroscopy(EIS).The results showed that the optimal time for electrodeposition of Au NPs was 240 s.The deposited Au NPs had a particle size range of 22.68?225.83 nm with an average particle size of83.09±43.6 nm based on this method.The optimal time for protein A fixation,monoclonal antibody incubation,and antigen incubation was 30 min,45 min,and 45 min,respectively.4.Under optimal conditions,DPV was used to detect different concentrations of recombinant GI.1/GII.4 VP1.The results indicated that both the concentration ranges of Hu No Vs GI.1/GII.4were within 1?10 ng/m L.The DPV responded to electrical signals and VP1 concentrations showed a good linear relationship.The limit detection(LOD)of the proposed electrochemical biosensor for GI.1 VP1 was 0.37 ng/m L with a calibration curve of y=0.410x+4.808(R~2=0.988).The LOD of GII.4 VP1 was 0.22 ng/m L with a calibration curve of y=0.670x+0.166(R~2=0.997).5.The repeatability,specificity,and stability of the biosensor were evaluated.The relative standard deviation(RSD)of the electrical signals detected by the seven biosensors was 2.97%.This result indicated that the biosensor had good reproducibility.Then,mouse norovirus(MNV)and Tulane virus(TV)as potential detection interfering substances were used to evaluate the biosensor.The results showed a good specificity for detecting Hu No Vs.Moreover,the activity of the proposed biosensor was reduced to 85.5%after a sealed storage at 4°C for two weeks.This result indicated that the sensor had a good stability.Based on the previous research and immunology,an electrochemical biosensor was constructed using a monoclonal antibody H9E as a Hu No Vs captured antibody for the first time.The results demonstrated that the proposed electrochemical biosensor had application potential for the detection of different genotypes of Hu No Vs.The human-derived norovirus electrochemical biosensor constructed in this study had advantages of good analytical performance,easy-operation,low reagent consumption,and can be applied for rapid on-site detection.It provided a new technical support in the area of human-derived norovirus detection technology.