Metallic alloy nanoparticles are synthesized by combining two or more different metals. Bimetallic or trimetallic nanoparticles are considered more effective than monometallic nanoparticles because of their synergistic characteristics. In this review, we outline the structure, synthesis method, properties, and biological applications of metallic alloy nanoparticles based on their plasmonic, catalytic, and magnetic characteristics.

In this study, silica-coated magnetic iron oxide nanoparticles (MNPs@SiO₂) were covalently conjugated onto graphene oxide (GO/MNP@SiO₂) for protein isolation. First, MNPs were precisely coated with a silica layer on the surface by using the reverse microemulsion method, followed by incubation with 3-glycidyloxypropyltrimethoxysilane (GPTS) to produce the GPTS-functionalized MNPs@SiO₂ (GPTS-coated MNPs@SiO₂) that display epoxy groups on the surface. The silica shell on the MNPs was optimized at 300 µL of Igepal^®CO-520, 5 mg of MNP, 100 µL of TEOS, 100 µL of NH₄OH and 3% of 3-glycidyloxypropyltrimethoxysilane (GPTS). Simultaneously, polyethyleneimine (PEI) was covalently conjugated to GO to enhance the stability of GO in aqueous solutions and create the reaction sites with epoxy groups on the surface of GPTS-coated MNP@SiO₂. The ratio of PEI grafted GO and GPTS-coated MNP@SiO₂ (GO/MNP ratio) was investigated to produce GO/MNPs@SiO₂ with highly saturated magnetization without aggregation. As a result, the GO/MNP ratio of 5 was the best condition to produce the GO/MNP@SiO₂ with 9.53 emu/g of saturation superparamagnetization at a magnetic field of 2.0 (T). Finally, the GO/MNPs@SiO₂ were used to separate bovine serum albumin (BSA) to investigate its protein isolation ability. The quantity of BSA adsorbed onto 1 mg of GO/MNP@SiO₂ increased sharply over time to reach 628 ± 9.3 µg/mg after 15 min, which was 3.5-fold-higher than that of GPTS-coated MNP@SiO₂. This result suggests that the GO/MNP@SiO₂ nanostructure can be used for protein isolation.

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.