Surface-enhanced Raman Scattering Applied To Label Immunoassay

A label immunoassay readout method based on Surface-enhanced Raman scattering(SERS) was developed in recent years.The method stems from the concept of forming a sandwich structure "capture antibody substrate-detection antigen-labeled antibody".The antibody covalently bound to a solid substrate specifically captures the corresponding antigen,and the captured antigen in turn binds selectively to its corresponding antibody.The antibody is conjugated with gold colloids that are labeled with Raman reporter molecules,which serve as extrinsic labels for the antibody.The presence of a specific antigen is established by the characteristic SERS spectrum of the reporter molecule. This technology has its unique superiority because of its unified high sensitivity, high selectivity of SERS as well as the specificity of biological immunity reply. This thesis has employed such technology during the conducted research and includes the following three parts:1.Taking 4,4'-dipyridyl as the reporter molecule,we combined the label molecule with certain particle size of golden sol to form the precursor of the Raman reporter-labeled nanoparticles.The factors affecting the formation of the SERS reporter-labeled immunogold colloids such as the sizes of gold particles,the amount of 4,4'-dipyridyl added to the gold colloids and the processing time were studied.We also investigated the influence incurred by the addition of the antibody on the SERS signals of reporter-labeled gold colloids.The sandwich structure of antibody-antigen-reporter-labeled immunogold nanoparticles was built by assembling solid antibody and SERS immunogold with corresponding antigen.The single or multiple type biospecific species could be identified through the detection on the SERS signal of the reporters coimmobilized with immunogold nanoparticles.2.Gold film used as immunoassay substrate was modified by 16-mercaptohexadecanoic acid(16-MHA) via the mixed self-assembled monolayer(SAM) technology.A sandwich assay which contains captured antibodies,antigens and labeled immuno-colloids was formed.By using the mixed SAM approach,detection limit ranging from 0.1 to 100 ug/mL was achieved.It is demonstrated that this technology can decrease nonspecific binding and increase specific binding efficiency.3.In the above sandwich assay containing captured antibodies,antigens and labeled immuno-colloids,we further examined the buffer solution and the utility of agitating during the incubation of Raman-reporter-labeled immuno-nanoparticles with the substrate.The study indicated that it is the PH and not the identity of the buffer influenced the incubation.It is shown that appropriate buffer can decrease nonspecific binding and increase specific binding efficiency,and the addition of agitation can decrease nonspecific binding.