Paper-based SERS Immunoassay

Surface-enhanced Raman spectroscopy （SERS） is an analytical tool for the identificationof biological or chemical analytes based on Raman scattering. Its narrow, well-resolved bands,marked reduction in fluorescence background, low SERS intensity of water and highsensitivity make SERS widely used in surface science, material science and biomedicalscience. However, whether SERS will be developed into a technique with practicalimportance depends very much on the stability of the substrate and reproducibility of the dataobtained. The most critical aspect is the choice and/or fabrication of SERS-active substrateswith high sensitivity, good reproducibility, high stability and easy production. Thisdissertation describes the simplicity of SERS microarrays fabrication as SERS-activesubstrates, and including their applications in chemical and biomolecular detection. Thedetails are summarized as follows:In the second chapter, using self-assembly technique, silver colloid was adsorbed ontothe hierarchical fibrous structure of filter paper, which produced a nanoparticle film. Weexplored the effects of Cl-on the SERS substrates, the aggregation of silver nanoparticles（AgNPs） and the morphology of Ag film were characterized using UV-Vis spectrophotometrictechnique and scanning electron microscopy. Malachite green, residues in aquatic products,was used as SERS probe molecule. Control the aggregation of AgNPs by adding differentconcentration of Cl-to get the best SERS enhancement effect. It showed from the experimentsthat,1000counts of SERS signals could be observed from5×10^-10M malachite green solution.The lowest detection limit for malachite green was10^-10M.In the third chapter, paper-based SERS technology was extended to enzyme bioanalysis.Using an electric sample spotter, silver colloid contained Cl-was spotted on PVP-coated filterpaper to form sensing arrays for the alkaline phosphatase detection. PVP is a non-ionicsurfactant with powerful viscosity. PVP-coated paper was used as the substrate of the Ag film,which prevented silver colloid from spreading throughout the paper and defined the shapes ofAg film. The AgNPs/PVP/paper substrate possessed highly ordered and uniform SERS effect.Alkaline phosphatase hydrolyzed5-bromo-4-chloro-3-indolyl phosphate （BCIP） to theformation of BCIP dimer, which was found to be highly SERS active. Recording the SERSspectra of BCIP dimmer, the assay of alkaline phosphatase was carried out ranging from4.14×10^-9-4.14×10^-13M.In the fourth chapter, taking advantage of magnetic separation and enzymatic hydrolysis,we developed a relatively simple SERS enzyme-linked immunoassay. In the proposed system, antibody immobilized on magnetic microspheres reacted with antigen, which bound withanother antibody labeled with alkaline phosphatase. If this sandwich-type immunocomplexwas subjected to reaction with BCIP, BCIP dimmer was generated. This reaction product wasadsorbed on the AgNPs/PVP/paper SERS-active substrate described above, which gave astrong SERS effect to detect mouse IgG. The detection limit of this SERS enzyme-linkedimmunoassay method was found to be about0.33ng/mL, with a linear range from1to500ng/mL.