MultisHRP-DNA-coated CMWNTs as signal labels for an ultrasensitive hepatitis C virus core antigen electrochemical immunosensor

• Methylene blue embedded on graphitized mesoporous carbon as electroactive material.
• Multis horseradish peroxidases-DNA coated CMWNTs were used as secondary signal label.
• The immunosensor exhibited excellent behavior with the detection limit 0.01 pg mL−1.
• The extreme enhancement of the sensitivity was due to multienzyme amplification.

An ultrasensitive and selective electrochemical immunosensor was developed for the detection of hepatitis C virus (HCV) core antigen. The immunosensor consists of graphitized mesoporous carbon–methylene blue (GMCs–MB) nanocomposite as an electrode modified material and a horseradish peroxidase-DNA-coated carboxyl multi-wall carbon nanotubes (CMWNTs) as a secondary antibody layer. After modification of the electrode with GMCs–MB nanocomposite, Au nanoparticles were electrodeposited on to the electrode to immobilize the captured antibodies. The bridging probe and secondary antibodies linked to the CMWNTs, and DNA concatamers were obtained by hybridization of the biotin-tagged signal and auxiliary probes. Finally, streptavidin-horseradish peroxidases (HRP) were labeled on the secondary antibody layer via biotin-streptavidin system. The reduction current of MB were generated in the presence of hydrogen peroxide and monitored by square wave voltammetry. Under optimum conditions, the amperometric signal increased linearly with the core antigen concentration (0.25 pg mL−1 to 300 pg mL−1). The immunosensor exhibites the detection limit as low as 0.01 pg mL−1 and it has a high selectivity. The new protocol showed acceptable stability and reproducibility, as well as favorable recovery for HCV core antigen in human serum. The proposed immunosensor has great potential for clinical applications.