Enzyme Immunoassay Utilizing Surface-Enhanced Raman Scattering of the Enzyme Reaction Product

We propose here a new enzyme immunoassay based on surface-enhanced Raman scattering (SERS). In the proposed system, antibody immobilized on a solid substrate reacts with antigen, which binds with another antibody labeled with peroxidase. If this immunocomplex is subjected to reaction with o-phenylenediamine and hydrogen peroxide at 37 °C, azoaniline is generated. This azo compound is adsorbed on a silver colloid. In this system, only the azo compound gives a strong surface-enhanced resonance Raman (SERRS) spectrum. The spectrum shows intense bands at 1582 and 1442 cm-1 due to the CC and NN stretching modes, respectively. A linear relationship was observed between the peak intensity of the NN stretching band and the concentration of antigen, revealing that one can determine the concentration of antigen by the SERRS measurement of the reaction product. The correlation coefficient between the peak intensity and the concentration was calculated to be 0.999 for the concentration range from 0.158 to 2.5 ng/mL. ...