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1、 Immunoassay using probe-labelling immunogold nanoparticles with silver staining enhancement via surface-enhanced Raman scatteringShuping Xu,aXiaohui Ji,bWeiqing Xu,*aXiaoling Li,aLianying Wang,bYubai Bai,*bBing Zhaoaand Yukihiro Ozakic aKey Laboratory for Supramolecular Structure and Material of Mi
2、nistry of Education, Jilin University, Changchun 130021, P. R. China bCollege of Chemistry, Jilin University, Changchun 130021, P. R. China. E-mail: ; Fax: +86-431-8980729; Tel: +86-431-8498912 cDepartment of Chemistry, School of Science and Technology, Kwansei-Gakuin University, Sanda, Hyogo 669-13
3、37, JapanReceived 17th October 2003, Accepted 1st December 2003 First published as an Advance Article on the web 11th December 2003This paper reports a novel immunoassay based on surface-enhanced Raman scattering (SERS) and immunogold labelling with silver staining enhancement. Immunoreactions betwe
4、en immunogold colloids modified by a Raman-active probe molecule (e.g., 4-mercaptobenzoic acid) 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 the m
5、easurements of ultraviolet-visible (UV-vis) spectra to monitor the formation of a sandwich structure onto a substrate. The immunoassay was performed by a sandwich structure consisting of three layers. The first layer was composed of immobilized antibody molecules of mouse polyclonal antibody against
6、 Hepatitis B virus surface antigen (PAb) on a silicon or quartz substrate. The second layer was the complementary Hepatitis B virus surface antigen (Antigen) molecules captured by PAb on the substrate. The third layer was composed of the probe-labelling immunogold nanoparticles, which were modified
7、by mouse monoclonal antibody against Hepatitis B virus surface antigen (MAb) and 4-mercaptobenzoic acid (MBA) as the Raman-active probe on the surface of gold colloids. After silver staining enhancement, the antigen is identified by a SERS spectrum of MBA. A working curve of the intensity of a SERS
8、signal at 1585 cm21due to the n8aaromatic ring vibration of MBA versus the concentration of analyte (Antigen) was obtained and the non-optimized detection limit for the Hepatitis B virus surface antigen was found to be as low as 0.5 mg mL21.IntroductionRaman spectroscopy is one of the most useful me
9、thods to investigate biomolecules and biological materials because it can provide rich structural information as well as quantitative and qualitative information by sharp Raman bands in a nondestructive manner. Raman spectroscopy can be almost freely applied to aqueous samples and samples under phys
10、iological conditions. However, the signals of conventional Raman spectra are too weak to be widely used for highly sensitive quantitative and qualitative analyses. Surface-enhanced Raman scattering (SERS) and surface- enhanced resonance Raman scattering (SERRS) can readily enhance Raman signals by a
11、 factor of 10314,1,2and many single molecule Raman spectra have recently been acquired from an adsorbate on a single nanoparticle.3,4This remarkable progress in SERS has broadened the applications of Raman spectroscopy in biology and biomedicine.1,5,6 Immunoassay, which is based on a specific intera
12、ction between an antigen and a complementary antibody, is a powerful analytical tool for biochemical analyses, clinical diagnosis, and environ- mental monitoring. Many analytical methods, such as surface plasmon resonance (SPR),7atomic force microscopy (AFM)8and quartz crystal microbalance (QCM),9ha
13、ve been used for a direct measurement of the antigens binding to antibody molecules immobilized on a substrate. Rapid, sensitive, and accurate methods for immunoassays have always been the goals pursued by scientists. Thus, many kinds of conventional labelling immu- noassay techniques, such as enzym
14、e immunoassay (EIA),10 fluorescence11,12and chemiluminescence,13have 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 technolo-gies because of their easily
15、controllable-size distribution, long-term stability and friendly biocompatibility with antibodies, antigen proteins, DNA and RNA.1420Many novel methods using metallic colloid nanoparticles have been developed, such as colloidal gold labelling immunoassay systems detected with transmission electron m
16、icroscopy (TEM),15,16scanning electron microscopy (SEM),17or even by naked eye,18,19imaging of gold colloidal particles by conjugating the immune complexes on conductive substrates with scanning tunneling microscope (STM),20and so on. Moreover, noble metallic colloid particles with proper size and roughness are very suitable for SERS substrates,21and there is great interest in the coupling of the nano-labelling techniques with the SERS technique. Dou et al.22developed a new
