The SERS Study Of Binary Nanoparticle-metal Coupled System And Its Applications On Immunoassay

The improvement and applications of surface-enhanced Raman scattering(SERS)effect are always hot issues in the field of surface science.As the dominant contributor of SERS signals,the "hot spot"effect generated by coupled metallic nanostructures held surface plasmons(SPs)plays a critical role in improving the enhancement effect.However,as "hot spots" are usually randomly distributed on the metallic surfaces,it’s of great significance to design suitable coupling model for the fabrication of substrates with high SERS activity and also for deep understanding of SERS mechanism.For example,shell-isolated nanoparticle-enhanced Raman spectroscopy(SHINERS)can be served as an ideal model system to study the "hot spot" effect between nanostructures and metallic substrate.In this system,"hot spots" can be formed not only through the couplings of localized surface plasmon polariton(LSPP)by neighbouring shell-isolated nanoparticles(SHINs),but also couplings of LSPP of particles with propagating surface plasmon polariton(PSPP)of metallic single crystal substrates.The latter is often regarded as the main contribution of enhancement of SHINERS technique,especially that the formation of "hot spots" in the area induced by the SiO2 shells provides the theoretical basis for the preparation of nanoparticles with reasonable SERS effect.Therefore,the study of electromagnetic field distribution in the nanoparticle-substrate gap area is of great importance to understand the formation and distribution of "hot spots" as well as extend the practical applications of SERS.For example,the influence of SERS activities of nanoparticles or their coupling effects with metallic substrate are of great importance on the sensitivity in the field of SERS labeled immunoassay.Binary core-shell nanoparticles have attracted considerable interests in this field.This kind of nanoparticles has superior properties compared with naked metallic nanoparticles due to their dual properties of both inner core and outer shell,and is expected to improve the detection sensitivity of immunoassay.Based on the above two considerations,on the one hand,the p-mercaptobenzoic acid(pMBA)-embedded Au@Si02 binary core-shell nanoparticles were fabricated and their monolayer film structures were assembled at the liquid/liquid interface,and the "hot spot" coupling effect between monolayer films and metallic substrates was investigated by the dual-probes strategy,which provided the basis for the preparation of binary nanoparticles with high stability and ideal SERS effect as well as broaden the applications in immunoassay.On the other hand,binary core-shell nanoparticles of(Au-pMBA)@SiO2 and Fe3O4@Au were employed to improve the sensitivity of immunoassay by integration the advantages of binary nanoparticles,SERS and labeled immunoassay.As a consequence,the high-efficient separation and high-sensitive detection of clenbuterol(CL)were achieved.A liquid competitive magnetic immunoassay technique with superior specificity and high sensitivity was developed.The main research contents are listed as follows:(1)(Au-pMBA)@SiO2 binary core-shell nanoparticles with different shell thicknesses were prepared,and their corresponding monolayer nanoparticle films were formed by self-assembly at the liquid/liquid interface.The "hot spot" distributions of(Au-pMBA)@SiO2 films coupled with metallic or silicon wafer substrates were investigated by dual-probes strategy under the illumination of 633 nm or785 nm laser,respectively.The transfer of "hot spots" from "particle-particle gap mode" to"particle-surface gap mode" was preferred for(Au-pMBA)@SiO2 films,the thinner shells and 785 nm laser.For "particle-surface gap mode",with the increase of SiO2 shell thicknesses,the internal electromagnetic field intensities of the shell decreased slower than the external ones of the metallic substrate surface.The dissolution of SiO2 shells by NaOH solutions allowed the transfer of coupling system from "particle-surface gap mode" to "particle-particle gap mode" again.(2)By the combination of high SERS activity of Au cores,the coupling effect with substrate,the isolation and biocompatibility of SiO2 shells,binary nanoparticles(Au-pMBA)@SiO2 were explored to the sandwiched immunoassay for improving the performance of SERS-based immunoassay significantly.The limit of detection(LOD)of this approach was down to 0.1 fg·mL-1 which was improved by at least 2 orders of magnitude compared with that of the normal labeled naked Au nanoparticles in the detection of immunoglobulin(IgG).It was mainly contributed by the protection of SiO2 shells for avoiding the desorption of pMBA and improving the stability of nanoparticles.All of these facts brought the increase of the assembly efficiency of sandwiched structures which induced the supplementation of SERS coupling effect.Moreover,the protective SiO2 shells could also inhibit the laser-induced catalytic reactions of labeled molecules and replace the blocking reagent to reduce the nonspecific adsorptions,which simplified the SERS-based immunoassay protocol.(3)The magnetic separation and detection technique based on binary magnetic core-shell nanoparticles Fe3O4@Au were developed.The separations of clenbuterol(CL)and salbutamol(SAL)with high selectivity and high efficiency were achieved by combining the magnetic inner core Fe304 nanoparticles and high SERS activity of outer shell Au nanoparticles.The specific interaction between target and its corresponding antibody modified on Fe3O4@Au surfaces induced the aggregation of binary nanoparticles carried with targets.The final aggregated nanoparticles were collected by applying an external magnetic field.It resulted in the dramatical reduction of target concentration for achieving the magnetic separation of different targets.The calibration curve of competitive immunoassay based on solid substrate with the LOD and IC50 of 17.24 fg·mL-1 and 192.64 pg·mL-1 was established to evaluate the efficiency of magnetic separation.The results indicated that the detected target concentration was below the LOD of the calibration curve.It suggested that targets were removed with high efficiency.Meanwhile,the successive separations of different targets could be realized by changing the types of antibody on Fe3O4@Au surfaces,indicating the high specificity,selectivity and separation efficiency of this magnetic separation approach.It will be developed as a promising candidate for separating or retaining the hapten molecules selectively in dispersed media.(4)A novel approach of liquid competitive magnetic immunoassay with high sensitivity and specificity was constructed.The detection of targets was realized according to the inverse correlation between SERS signal intensity of labeled molecules in the magnetic immunocomplex and target concentration by the competitive reaction between target and antigen for antibody in liquid phase.The coupling effect between Au and Fe3O4@Au played the critical role in improving the performance of liquid magnetic immunoassay.By changing the modification positions of CL antibody and CL coating antigen on the two kinds of nanoparticles surfaces,i.e.Fe3O4@Au and Au,CL antibody-modified labeled Au nanoparticles/CL coating antigen-modified Fe304@Au nanoparticles or CL coating antigen-modified labeled Au nanoparticles/CL antibody-modified Fe304@Au nanoparticles were prepared,respectively.The results indicated that the former exhibited better performance in liquid competitive immunoassay with the LOD and IC50 of 0.22 fg·mL-1 and 246.31 fg·mL-1,respectively.Compared with the conventional competitive immunoassay technique,its performance was greatly improved.This approach possessed high selectivity and simplified immunoassay protocol.Due to the easy manipulation of liquid system,the SERS-based liquid magnetic immunoassay technique is developed as potential high-efficient separation and analytic technique for combining with other detection techniques.