Study On Surface-enhanced Raman Scattering Immunoassays Based On The Hot Spot Effect And Their Applications

Surface-enhanced Raman scattering(SERS), a powerful vibrational spectroscopic technique, can provide nondestructive and ultrasensitive characterization of molecules on or near the surface of plasmonic nanostructures. Because of its single molecule-level sensitivity and finger-printing capability, SERS has been widely applied in surface science, food safety, and the detection of chemicals, proteins, bacteria, and even cells. According to the SERS enhancement mechanism, the electric field near the sharp tips or nanometer gap junction of noble metallic nanoparticles would be plasmonic coupled and significantly magnify the near-field enhancements, which can significantly enhance the SERS intensity of molecules located in the hot spot junction. The hot spot effect makes a major contribution to the SERS enhancement phenomenon.The hot spot effect on SERS enhancement was studied by experimental methods and three-dimensional finite-difference time domain simulations. The Au film substrate, silica-coated magnetic nanoparticles(MNPs) and silver-coated MNPs were utilized as the SERS immunoassay capture substrate to realize hot spots-based high-sensitive SERS immunoassay. Some key issues of the SERS immunoassay substrate would be settled in this study. The main research contents are as follows:(1) Plasmonic Ag core-satellite nanostructures with hot spots were synthesized and their SERS performance was characterized.The Ag nanostructures with hot spots were synthesized by utilizing the ultrathin silica shell as a spacer to generate a tunable nanogap between the Ag core and satellites. To synthesize the nanoparticles, Ag nanoparticles(Ag NPs) with a diameter of ~60 nm were synthesized as cores, on which Raman dyes were adsorbed and then tunable ultrathin silica shells from 2.0 to 6.5 nm were coated, followed by the deposition of Ag NPs as satellites onto the silica surface. The relationships between the SERS signal and the important parameters, including the satellite diameter and the nanogap distance, were studied by experimental methods and three-dimensional finite-difference time domain simulations. The maximum SERS intensity of the core-satellite nanoparticles was over 14.6 times stronger than that of the isolated Raman-encoded Ag/PATP@SiO2 NP. The theoretical calculations indicated that the local maximum calculated enhancement factor(EF) of the hot spots with a 2.0 nm nanogap was 9.5 × 105.(2) A SERS immunoassay based on Au film substrate was developed.Antibody-conjugated Au film substrate and Au@AgNPs were utilized as the SERS immunoassay substrate and the SERS tags, respectively. In the presence of HBsAg in the solution, Au film substrate and Au@AgNPs formed the sandwich immunecomplexes, which would induce the formation of hot spots through the local plasmonic coupling and enhance the local electric field, thus enhancing the SERS intensity. This SERS immunoassay was able to detect HBsAg with a low limit of detection of 66.87 pg/mL and a dynamic linear range from 250 pg/m L to 150 ng/m L.(3) A SERS immunoassay based on silica-coated MNPs was developed.The MNPs were synthesized through a solvothermal reaction, coated with a silica shell by hydrolyzing TEOS and then conjugated with antibody as the SERS immunoassay substrate. The antibody conjugated silica-coated MNPs were utilized to immunomagnetic separate and enrich the analytes from the solution, and then formed the sandwich immunecomplexes with SERS tags. This SERS immunoassay was able to detect HBsAg with a low limit of detection of 4.72 pg/mL and a dynamic linear range from 10 pg/mL to 25 ng/m L.(4) A SERS immunoassay based on silver-coated MNPs was developed.Capture antibody-coated silver shell magnetic nanoparticles(Fe3O4@Ag MNPs) were utilized as the target protein enrichment platform and the SERS signal amplification substrate. Gold nanorods(AuNRs) were coated with a thin silver shell to be in resonance with the resonant Raman dye diethylthiatricarbocyanine iodide(DTTC) and the excitation wavelength at 785 nm. The silver-coated AuNRs(Au@Ag NRs) were then modified with detection antibody as the SERS tags. The Au@Ag NRs and Fe3O4@Ag MNPs formed the hot spot nanostructures in the immune complexes. Thus, the SERS signal of DTTC molecules located in the coupled plasmonic nanostructures was significantly enhanced. As a result, the proposed SERS immunoassay was able to detect CEA with a low limit of detection of 4.75 fg/m L and a wide dynamic linear range from 10 fg/m L to 100 ng/mL.The Au film, silica-coated magnetic nanoparticles and silver-coated magnetic nanoparticles substrates were used to develop the SERS immunoassays based on the hot spot effect. HBsAg and CEA were ultilized as the target analytes to verify the feasibility and advantage of the immunoassays. This study would provide a new strategy for disease diagnosis.