1：Experimental Design and Method Selection:

The study designed a dual enhanced colorimetric immunoassay using in situ synthesis of gold nanoparticles (Au NPs) with TMBZ as a reducing agent and further amplification via peroxidase-like activity of Au NPs with TMBZ-H2O2. The method avoids multistep synthesis and washing steps.

2：The method avoids multistep synthesis and washing steps. Sample Selection and Data Sources:

2. Sample Selection and Data Sources: Recombinant influenza virus A (H5N1), A (H4N6), and A (H9N2) were used as samples. Blood samples were also tested for practical application. Specific antibodies and reagents were sourced from commercial suppliers.

3：List of Experimental Equipment and Materials:

Materials included HAuCl4·3H2O, TMB, H2O2, 96-well plates, antibodies (e.g., anti-H5N1 HA antibody), viruses, and buffers. Equipment included microplate reader (Cytation 5), TEM (Tecnai G2 F20), UV-vis spectrophotometer, FT-IR spectrometer (FT/IR6300), zetasizer (Zetasizer Nano ZS), and centrifuge (D3024 Micro-centrifuge).

4：Experimental Procedures and Operational Workflow:

Steps involved preparation of antibody-gold ion conjugates, virus adsorption on plates, addition of conjugates and TMBZ for color development, further amplification with TMBZ-H2O2, and absorbance measurement. Specificity and sensitivity tests were conducted with controls and comparisons to ELISA and commercial kits.

5：Data Analysis Methods:

Absorbance was measured at 450 nm using a microplate reader. Limit of detection (LOD) was calculated, and statistical analysis included standard deviations from triplicate measurements.