ENZYME-IMMUNOASSAY UTILIZING SURFACE-ENHANCED RAMAN-SCATTERING OF THEENZYME REACTION-PRODUCT

We propose here a new enzyme immunoassay based on surface-enhanced Ram an scattering(SERS). In the proposed system, antibody immobilized on a solid substrate reacts with antigen, which binds with another antibod y labeled with peroxidase. If this immunocomplex,is subjected to react ion with o-phenylenediamine and hydrogen peroxide at 37 degrees C, azo aniline is generated. This azo compound is adsorbed on a silver colloi d. In this system, only the azo compound gives a strong surface-enhanc ed resonance Raman (SERRS) spectrum. The spectrum shows intense bands at 1582 and 1442 cm(-1) due to the C=C and N=N stretching modes, respe ctively. A linear relationship was observed between the peak intensity of the N=N stretching band and the concentration of antigen, revealin g that one can determine the concentration of antigen by the SERRS mea surement of the reaction product. The correlation coefficient between the peak intensity and the concentration was calculated to be 0.999 fo r the concentration range from 0.158 to 2.5 ng/mL. The detection limit of this SERS enzyme immunoassay method was found to be about 10(-15) mol/mL, which was lower by 1 order of magnitude than that found for a previously reported method employing SERRS.