Research And Application Of Immunoassay And Immunochromatographic Technology Based On Surface Enhanced Raman Scattering (SERS)

Surface enhanced Raman spectroscopy（SERS） has been widely used in the chemical and biological detection and analysis because of its high sensitivity and other advantages, while the immunoassay（IA） which is based on the specific reaction of antigen and antibody has the advantages of good specificity. A new immunoassay which combines SERS with IA（SERS-IA） displays the features of high sensitivity and specificity. SERS-IA has been widely used in many areas such as food safety and environmental monitoring. In the SERS-IA, the substrate for supporting the immunoreaction is one of the topics and has been studied for a long time. There are many substrates such as gold film, glass slide, etc. used in SERS-IA, but every substrate has its drawback such as high cost or difficult fabrication. One of the main aims of this study is to explore a solid substrate which has the advantage of supporting the immunereaction, the homogeneous signals, easy to operation and low cost. With a long time experiment, nitrate cellulose membrane（e.g. NC membrane） was found to have all of these advantages. Based on NC membrane, the multi-channel SERS-IA were developed and applied for the detection of a new β-Agonist brombuterol. On the other hand, using GNPs as the substrate for preparation of SERS immmunoprobe, another aim of this study is establish a SERS based immunochromatographic technology（ICT） for the detection of Hg²⁺ in water, urine and serum samples.The thesis cludes three parts:In part 1, the research background and significance of the thesis are introduced. The basic theory of SERS and immunoassay are discussed. SERS substrate, SERS-IA and the immunochromatography technology（ICT） based on SERS（SERS-ICT） are mainly emphasized. The environment and food safety issues are also described.In part 2, different materials used as the immunoreaction supports such as silver film, silver nano particles or nano gold particles immobilized on the surface a glass, etc. have been studied. It is found that the all these materials（silver film, silver nano particles and nano gold particles） not only display the complicated preparation process but also difficulty binding with the protein firmly and the poor reproducibility of the signals. However, the nitrate cellulose membrane（e.g. NC membrane） which has been widely applied in ICT has been eventually selected as the suitable substrate for supporting immunoreaction employed in SERS-IA. NC membrane displays strong and homogeneous binding with protein, and the fabrication of the NC membrane is very convenient. A novel core-shell nanoparticles（e.g. GNPs@Ag） has been prepared and used for the prepaeation of SERS immunoprobe. Based on NC membrane and the SERS immunoprobe, the SERS-IA for the detection of brombuterol has been successfully developed. The IC50 and limit of detection（LOD） are 0.02 ng mL^-1 and 0.16 pg m L^-1, respectively, showing high sensitivity. The cross-reactivity（CR %） of the SERS-IA with clenbuterol is 40.8% due to the high similarity of the molecular structure between brombuteroland clenbuterol. The CR of the SERS-IA with other compounds is negligible. The SERS-IA has been applied for the detection of brombuterol from spiked samples and the satisfied results are obtained.In part 3, although the surface enhanced Raman spectroscopy based immunochromatographic technology（SERS-ICT） has been previously developed in our group, it is still required for further study. Using AuNPs as the substrate, the immunoprobe has been prepared and applied to SERS-ICT for the detection of Hg²⁺ in water（river water, lake water, tap water）, urine and blood samples. Under optimal experimental conditions, the SERS-ICT has the high sensitivity for Hg²⁺(lower than ng g^-1 level). The IC50 and the LOD are 0.12 ng m L^-1 and 0.45 pgm L^-1, respectively. The CR values of the SERS-ICT with another 19 anions and cations are less than 0.01%, showing the high specificity of the SERS-ICT. Also the proposed ICT has been applied for the detection of Hg²⁺ in urine samples collected from Occupational Disease Hospital and the results are confirmed by cold-vapor atomic fluorescence spectroscopy（CV-AFS）. The obtained results are satisfied.