Surface-Enhanced Raman Scattering Immunoassays Using a Rotated Capture Substrate

Abstract

A rapid, sensitive format for immunosorbent assays has been developed to meet the increasing levels of performance (i.e., reduction of incubation times and detection limits) demanded in the medical, veterinary, and bioterrorism prevention arenas. This paper introduces the concept of a rotating capture substrate as a facile means to increase the flux of antigen and label to the solid-phase surface and thereby reduce assay time. To this end, a sandwich-type assay is carried out that couples the specificity of antibody-antigen interactions with the high sensitivity of surface-enhanced Raman scattering detection. To investigate this strategy, polyclonal anti-rabbit IgG was immobilized on a gold capture substrate via a thiolate coupling agent. The capture substrate, capable of controlled rotation, was then immersed in a sample solution containing rabbit IgG, which served as a model analyte. After binding the target IgG, the substrates were immersed and rotated in an extrinsic Raman label (ERL) labeling solution, which is composed of gold nanoparticles (60 nm) coated with an aromatic moiety as the Raman scatterer and an antibody as the biospecific recognition element. The effect of substrate rotation on both the antigen binding and ERL labeling steps was investigated. Implementation of optimized rotation conditions resulted in the reduction of assay times from 24 h to 25 min and a 10-fold improvement in the limit of detection. Finally, the developed protocol was applied to the detection of rabbit IgG suspended in goat serum, which served to assess performance in a biological matrix.