An Immunosensor For Ultrasensitive Determination Of Influenza Virus Subtype H7N9

Objective:Influenza, a highly acute respiratory infectious disease, presents a significant threat to the public health, and poses a huge economic cost. A novel reassortant H7N9 virus is low pathogenicity for birds, while as causative pathogens being highly pathogenic to humans, making it very difficult to predict and track outbreaks. Moreover, it is difficult to control the spread, therefore, ongoing threat of human infection with H7N9 virus has emphasized the urgent needs for sensitive techniques that can rapidly assess their potential threat to humans and control the outbreaks. Influenza virus infection is initiated by virus attachment to cell-surface sialoside receptors via influenza hemagglutinin(HA).Currently, several methods are available for the identification of the influenza virus. However, these methods are yet to be suitable for point-of-care use, the main obstacles of these techniques lie in the sophisticated protocols, expensive cost or relying on advanced instruments. Hence, we report on a quadruple signal amplification strategy for use in an immunoassay for influenza virus subtype H7N9-HA.Methods:1. Preparation of Ab2/MWCNTs/S0/{S1+S2+hemin}n nanocomposite: firstly, Ab2 and S0 were added to the above activated MWCNTs. Then S1, S2 and excess amount of hemin were added to form hemin/G-quadruplex DNAzyme. Finally, MB was injected and obtained Ab2/MWCNTs/S0/{S1+S2+hemin}n composite.2. Stepwise modified electrodes: graphene sheets, AuNPs, MPTS sol-ADH composite, AuNPs, Protein A and Ab1 were successively modified onto the electrode surface step-by-step.3. Fabrication and analysis of the electrochemical immunosensor: based on a sandwich-type immunoassay, Ab2/MWCNTs/S0/{S1+S2+hemin}n bionanocomposite was dropped onto the electrode surface to form the antigen-antibody immunocomplex. Thus, a sensitive and specific immunosensor was obtained in which a quadruple signal amplification strategy is employed, viz.(a) via the use of graphene sheets,(b) via a hybridization chain reaction,(c) the use of hemin/G-quadruplex DNAzyme concatamers, and(d) the use of ADH. The current response was obtained after scanning every electrode in 0.1 M phosphate buffer(pH 7.0) containing 0.5 mM NAD+ and 1 M alcohol with DPV measurement.4. Evaluation of the electrochemical immunosensor: such feasibility, linear range, detection limit, specificity, reproducibility, stability and great potential in clinical applications of immunosensor.Results:1. The successful synthesis of Ab2/MWCNTs/S0/{S1+S2+hemin}n bionanocomposite: to demonstrate the successful conjugation of DNA and Ab2 on the MWCNTs, the nanomaterial before and after modification was monitored by AFM. The result indicating that DNA and Ab2 were successfully immobilized onto MWCNTs.2. Morphological characterization of the stepwise modified electrodes: the morphologies of the GS, depAu, and MPTS sol-ADH were characterized by SEM, indicating that biocomposite was successfully assembled on electrode surface. XPS was used to verify the successfully binding of MPTS sol-ADH and depAu layer onto the electrode surface. CV and EIS were performed for each step by step electrode modification. All the data indicated the successful fabrication of the electrochemical immunosensor.3. Analytical performance of the immunosensor: after optimized conditions, the immunosensor displayed a good linear relationship between the peak currents and the H7N9-HA antigen concentrations. And possessed linear detection range from 0.008 ng/mL to 0.06 ng/mL with the low detection limit of 0.81 pg/mL, The regression equation was I(μA)=0.2125[C]+7.1330 with a correlation coefficient(R2) of 0.9965(defined as S/N=3).4. The immunosensor exhibited good specificity, reproducibility, and stability.Conclusion:We are presenting an electrochemical sandwich immunoassay which can sensitively and specifically detect H7N9-HA antigen. The excellent performance is achieved by using quadruple signal amplification strategy as well as protein A. MWCNTs were served as the vehicle for loading substantial hemin/G-quadruplex DNAzyme concatamers. On the basis of those merits mentioned above, the electrochemical immunosensor provided a powerful and versatile tool in point-of-care test for H7N9-HA antigen with a detection limit of 0.81 pg/mL. The basic approach described here would be applicable towards developing biodetection assays against other important targets. Furthermore, the developed immunosensor exhibited a high sensitivity and a favorable specificity, which showed a great potential in clinical applications.