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A surface-enhanced Raman scattering (SERS) based biosensor using a direct immunoassay platform is demonstrated for influenza A detection. The nucleoprotein of influenza A virus, which is one of the most conserved and abundant structural proteins on the virion, was used as a target. In this study, highly sensitive biosensors were realized by combining specific recognition of antibody–antigen interactions and high signal enhancement of the SERS effect. SERS probes were fabricated by decorating PEGylated, 4,4′-thiobisbenzenethiol (TBBT)-labeled gold nanoparticles (NPs) with influenza A antibodies. To improve the sensitivity, a SERS immunoassay was performed on two-dimensional (2D) arrays of gold@silver core–shell (Au@Ag) NPs, which work as SERS substrates. The SERS signal of TBBT was utilized to detect the selective nucleoprotein–antibody recognition. The SERS signal was enhanced ∼4 times by using the SERS substrates instead of a flat Au film. These results indicate that using a well-tuned Au@Ag 2D array as a SERS substrate is an effective way of improving sensitivity of SERS-based biosensors. Our SERS immunoassay system revealed high selectivity and good reproducibility with a sample-to-sample variation of 4.6% (relative standard deviation). To demonstrate the applicability of our SERS immunoassay system to real biological samples, the detection of influenza A using infected allantoic fluid was also performed. The linear relation between the concentration of infected allantoic fluid and the SERS signal was obtained in the range of 5 to 56 TCID50 per mL (R2 = 0.96 for the TBBT Raman bands at 1565 cm−1) with the lowest detection limit of 6 TCID50 per mL. These findings demonstrated the potential of this SERS immunosensor platform for the highly sensitive and specific detection of target molecules in a complex matrix commonly found in clinical specimens.

Extrinsic surface-enhanced Raman scattering detection of influenza A virus enhanced by two-dimensional gold@silver core–shell nanoparticle arrays