A surface-enhanced Raman scattering (SERS) substrate was fabricated as well ordered Au/Ag multilayered nanorod arrays (fibAu/Ag-Rn, n = 0-4) via the focused ion beam technique for the detection of the influenza A virus strain. Since the shape, thickness, space between nanorods, and dimensions affect the performance of SERS, these factors were optimized before the fabrication of the substrate by varying the thickness of the Au layer (4 variables), Ag layer (3 variables), and their corresponding Ag/Au repeated layers. Au/Ag multilayers with the optimal thickness were used to fabricate the designed SERS substrate. The results indicate that Ag layer plays an important role in the improvement of SERS mechanism by inducing the electromagnetic effect at the Au surface. The as-prepared fibAu/Ag-Rn substrate serves as an excellent SERS substrate, even verified at a very low concentration using rhodamine 6G as a reference, e.g., it exhibited an enhancement factor ranging from 2.62 × 106 to 1.74 × 107 using the proposed SERS substrate. Furthermore, the influenza A virus strains (A/WSN/33 (H1N1), A/England/12/64 (H2N2), and A/Philippine/2/82 (H3N2)) could be well distinguished at a low concentration of 106 PFU/ml.
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