Enzyme-amplified SERS immunoassay with Ag-Au bimetallic SERS hot spots

Surface-enhanced Raman scattering (SERS) enables rapid detection of single molecules with high specificity. However, quantitative and sensitive SERS analysis has been a challenge due to the lack of reliable SERS-active materials. In this study, we developed a quantitative SERS-based immunoassay using enzyme-guided Ag growth on Raman labeling compound (RLC)-immobilized gold nanoparticle (Au NP)-assembled silica NPs (SiO₂@Au-RLC@Ag). The enzyme amplified Ag⁺ reduction as well as Ag growth on the RLC-immobilized Au NP-assembled silica NPs (SiO₂@Au-RLC), which resulted in a significant increase in SERS signal. In the presence of target antigens such as immunoglobulinG (IgG) or prostate-specific antigen (PSA), Ab₁-Antigen-Ab₂ immune complex with alkaline phosphatase triggered an enzyme-catalyzed reaction to convert 2-phospho-L-ascorbic acid (2-phospho-L-AA) to ascorbic acid (AA). As produced AA reduced Ag⁺ to Ag, forming an Ag hot spot on the surface of SiO₂@Au-RLC, which enhanced the SERS signal of SiO₂@Au-RLC@Ag in a solution with a target antigen concentration. The plasmonic immunoassay for IgG detection showed a high linearity of SERS intensity in the range of 0.6 to 9.0 ng/mL with a detection limit (LOD) of 0.09 ng/mL, while an LOD of 0.006 ng/mL was obtained for PSA. The results indicate that the sensitivity of our novel SERS-based immunoassay is higher than that of conventional enzyme-based colorimetric immunoassays.