Studies On Spectral Properties Of Colloidal Gold & Its Application For Immunoassay

Immunoassay, which is based on a specific interaction between an antigen and a complementary antibody, is a powerful analytical tool for biochemical analyses, clinical diagnosis, and environmental monitoring. Many analysis methods, such as surface plasmon resonance (SPR), atomic force microscopy (AFM) and quartz crystal microbalance (QCM), have been developed for a direct measurement of the antigens binding to antibody molecules immobilized on a substrate. The rapid, sensitive, and accurate methods of immunoassay have always been the goals pursued by scientists. Thus, many kinds of conventional labeling immunoassay techniques, e.g., enzyme immunoassay (EIA), fluorescence and chemiluminescence, have widely been applied to increase the detection sensitivity of analytes. Recently, metallic colloid nanoparticles (e.g., gold and silver colloids) have also been successfully applied to the label technologies because of their easily controllable-size distribution, long-term stability, and friendlybiocompatibility with antibodies, antigen proteins, DNA, and RNA. Many novel methods using metallic colloid nanoparticles have been developed, such as colloidal gold labeling immunoassay systems detected by transmission electron microscopy (TEM), or scanning electron microscopy (SEM), even by naked-eyes, imaging of gold colloidal particles by conjugating the immune complexes on conductive substrates with scanning tunneling microscope (STM), and so on. Moreover, noble metallic colloid particles with proper size and roughness are very suitable as surface-enhanced Raman scattering (SERS) substrates, and great interests have been paid to the coupling of the nano labels technique with the SERS technique.On the basis of the above facts, a new immunoassay method, which combined the immunogold nano-labeling and silver staining enhancement method with SERS has been developed. This method can be used for the quantitative detection of Hepatitis B virus surface antigen (Antigen) by means of a sort of self-assembled sandwich structure immobilized on a substrate. All the relative studies are outlined as follows.1. Extinction properties of gold nanoparticles.Gold colloids prepared by trisodium citrate reduction according to Frens method present a uniform spherical shape and long-term stability superior to other colloids. In order to effectively learn the extinction(extinction=absorption + scattering) properties of spherical gold nanoparticles before use, Mie theory has been employed to calculate the extinction spectra of a series of gold nanoparticles with the diameter of 1 -160 nm. The size dependences of peak position of extinction spectra and the extinction efficiency have been showed. The most calculated data are in agreement with the experimental results except for the too small or too big gold nanoparticles. We also apply the calculated results for two aspects. One is for the determining the density of gold nanoparticles in a colloid solution. The other is for the exploring the relationship between the SERS enhancement and the size of gold nanoparticles.2. Immuno-Identification by SPR with immunogold enhancement.SPR is a general method for the detection of chemical changes occurring at the surface of a thin noble metal film. Colloidal Gold nanoparticles have widely compatibility and stability with biomolecules. Various immune gold nanoparticles as labels have been merchandised and applied to many fields such as biologic quarantine and medicinal research. In this part, SPR biosensing using colloidal gold enhancement has been shown here. The incorporation of colloidal gold into SPR biosensing results in increased SPR sensitivity to immuno-indentification when a gold film immobilized antigen conjugate with immunogold colloid. A tremendous signal amplification is observed, as addition of the immunogold nanoparticles, and results in an over 6-fold larger signal than the direct detection of free antibody.3. Immunoassay by SERS with SERS-active immunogold labels and silver staining enhancementA novel immunoassay based on SERS and immunogold labeling with silver staining enhancement is described. Immunoreactions between immunogold colloids modified by a Raman-active probe molecule and antigens, which were captured by antibody-assembled chips such as silicon or quartz, were detected via SERS signals of Raman-active probe molecule. All the self-assembled steps were subjected to ultraviolet-visible (UV-vis) spectrometry, XPS, AFM to monitor the formation of the sandwich structure onto the substrate. The qualitative and quantitative immunoassays were performed by IgG-anti-IgG and PAb-Antigen-MAb(Immunogold), respectively. After silver staining enhancement, Antigen is identified by a SERS spectrum of MBA. A working curve of the intensity of a SERS signal of MBA versus the concentration of analyte was obtained and the unoptimized detecting limit for the Hepatitis B virus surface antigen (Antigen) is as low as 0.5 pg/mL4. Immunoassay by SERS with SERS-active immuno Au/Ag coreshell labels.Au/Ag coreshell nanoparticles labeled by monoclonal antibody and SERS-active probe were used for immunoassay analysis. PAb werecovalently immobilized on the silicon chips modified by a self-assembled monolayer of (3-amino-propyl) trimethoxysilane via glutaraldehyde (GA) activation. The Au/Ag core-shell nanoparticles were labeled by MAb and the probe molecule(MBA). Immunoreactions between the labeled Au/Ag core-shell nanoparticles and the corresponding antigens on the silicon chips were investigated by SERS of MBA. As a SERS substrate, the Au/Ag coreshell nanoparticles can enhance Raman signals of the label molecule greatly as strong as that of gold nanoparticles.5. Preparation of Au/Ag coreshell nanoparticle and studies on the SERS mechanism of Au/MBA/Ag sandwich structure.Ag-coating Au colloidal nanoparticles were prepared by deposition of Ag via chemical reduction on Au core. The thickness of Ag shell depended on the molar ratio of Au/Ag and the reaction time, which were submitted to UV-Vis spectrometry and TEM. A sort of bimetallic sandwich structure, which was formed by the analyte adsorbed on the surface of 16 nm-diameter Au core that was then coated by 3 nm-thick Ag shell, enhanced the Raman signal of the analyte remarkably. Due to the SERS electromagtic the mechanism of Au/MBA/Ag sandwich structure can be possibly explain by the electromagnetic coupling of local surface plasma (LSP) from Au core and evanescent field from Ag shell.