A novel biosensing system based on graphenemediated surface-enhanced Raman scattering (G-SERS) using plasmonic/magnetic molybdenum trioxide nanocubes (mag-MoO 3 NCs) has been designed to detect norovirus (NoV) via a dual SERS nanotag/substrate platform. A novel magnetic derivative of MoO 3 NCs served as the SERS nanotag and the immunomagnetic separation material of the biosensor. Single-layer graphene oxide (SLGO) was adopted as the 2D SERS substrate/capture platform and acted as the signal reporter, with the ability to accommodate an additional Raman molecule as a coreporter. The developed SERS-based immunoassay achieved a signal amplification of up to ∼10 9 -fold resulting from the combined electromagnetic and chemical mechanisms of the dual SERS nanotag/substrate system. The developed biosensor was employed for the detection of NoV in human fecal samples collected from infected patients by capturing the virus with the aid of NoV-specific antibody-functionalized magnetic MoO 3 NCs. This approach enabled rapid signal amplification for NoV detection with this biosensing technology. The biosensor was tested and optimized using NoV-like particles within a broad linear range from 10 fg/mL to 100 ng/mL and a limit of detection (LOD) of ∼5.2 fg/mL. The practical applicability of the developed biosensor to detect clinical NoV subtypes in human fecal samples was demonstrated by effective detection with an LOD of ∼60 RNA copies/mL, which is ∼10 3 -fold lower than that of a commercial enzyme-linked immunosorbent assay kit for NoV.

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