A new method has been developed to improve the detection of norovirus (NoV) in complex fecal samples using nanocatalyst-based immunoassays. The method involves using multifunctional trimetallic nanoparticles, known as Ag@Fe3O4@Au NPs. These nanoparticles consist of a core of silver (Ag) and a shell of iron oxide (Fe3O4) decorated with isolated gold nanoparticles (Au NPs). The nanoparticles have enhanced catalytic activity, making them an ideal nanocatalyst for reducing 4-nitrophenol (4-NP, yellow) to 4-aminophenol (4-AP, colorless). The developed Ag@Fe3O4@Au NPs-based immunoassay achieved a limit of detection (LOD) of 1.9 pg/mL for norovirus-like particles (NoV-LP) and 6.97 RNA copy number/mL for fecal NoV. In fecal sample analysis for NoV, a heat treatment at 65°C was necessary to prevent degradation of the target protein, ensuring sensitive detection. This work successfully combined multifunctional nanocatalysts for advanced immunoassays, which could contribute to developing nano-biosensing platforms.
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