Amplified visual immunosensor integrated with nanozyme for ultrasensitive detection of avian influenza virus

Nanomaterial-based artificial enzymes or nanozymes exhibit superior properties such as stability, cost effectiveness and ease of preparation in comparison to conventional enzymes. However, the lower catalytic activity of nanozymes limits their sensitivity and thereby practical applications in the bioanalytical field. To overcome this drawback, herein we propose a very simple but highly sensitive, specific and low-cost dual enhanced colorimetric immunoassay for avian influenza A (H5N1) virus. 3,3´,5,5´- Tetramethylbenzidine (TMBZ) was used as a reducing agent to produce gold nanoparticles (Au NPs) with blue colored solution from a viral target-specific antibody-gold ion mixture at first step. The developed blue color from the sensing design was further amplified through catalytic activity of Au NPs in presence of TMBZ-hydrogen peroxide (H₂O₂) solution in second step. Hence, the developed dual enhanced colorimetric immunosensor enables the detection of avian influenza virus A (H5N1) with a limit of detection (LOD) of 1.11 pg/mL. Our results confirmed that the developed assay has superior sensitivity than the conventional ELISA method, plasmonic-based bioassay and commercial flu diagnostic kits. Proposed sensing method further showed its capability to detect viruses, avian influenza A (H4N6) and A (H9N2) virus, in blood samples with limit of detection of 0.0269 HAU and 0.0331 HAU respectively.