Amperometric bioaffinity sensing platform for avian influenza virus proteins with aptamer modified gold nanoparticles on carbon chips

• A new aptamer-antibody sandwich assay pairing for the selective and sensitive detection of H5N1 viral protein.
• A new electrochemical biosensor utilizing the formation of a surface sandwich complex of DNA aptamer/H5N1 protein/antiH5N1-ALP.
• A lowest detectable concentration of 100 fM H5N1 protein with a linear dynamic range of 100 fM to 10 pM.
• A cost-effective, convenient and reproducible sensing platform composed of Au nanoparticles deposited on a carbon electrode.
• Analysis of H5N1 protein spiked human serum samples highlighting potential clinical diagnostic applications.

A sandwich assay platform involving a surface formed aptamer–protein–antibody complex was developed to obtain the highly selective and sensitive amperometric detection of H5N1 viral proteins using a gold nanoparticle (NP) modified electrode. This is the first aptamer–antibody pairing reported for the selective detection of H5N1. Nanoparticle deposited screen-printed carbon electrodes were first functionalized by the covalent immobilization of a DNA aptamer specific to H5N1 followed by the adsorption of H5N1 protein. Alkaline phosphatase (ALP) conjugated monoclonal antibody was then adsorbed to form a surface bound Au NPs-aptamer/H5N1/antiH5N1-ALP sandwich complex which was further reacted with the enzyme substrate, 4-amino phenyl phosphate (APP). The current associated with the electrocatalytic reaction of the surface bound ALP with APP increased as the H5N1 concentration increased. A lowest detectable concentration of 100 fM was obtained with a linear dynamic range of 100 fM to 10 pM using differential pulse voltammetry. As an example, the biosensor was applied to the detection of H5N1 protein in diluted human serum samples spiked with different concentrations of the viral protein target.

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