Femtomolar detection of prostate-specific antigen: An immunoassay based on surface-enhanced Raman scattering and immunogold labels

A novel reagent for low-level detection in immunoadsorbent assays is described. The reagent consists of gold nanoparticles modified to integrate bioselective species (e.g., antibodies) with molecular labels for the generation of intense, biolyte-selective surface-enhanced Raman scattering (SERS) responses in immunoassays and other bioanalytical applications. The reagent is constructed by coating gold nanoparticles (30 nm) with a monolayer of an intrinsically strong Raman scatterer. These monolayer-level labels are bifunctional by design and contain disulfides for chemisorption to the nanoparticle surface and succinimides for coupling to the bioselective species. There are two important elements in this label design; it both minimizes the separation between label and particle surface and maximizes the number of labels on each particle. This approach to labeling also exploits several other advantages of SERS-based labels: narrow spectral bandwidth, resistance to photobleaching and quenching, and long-wavelength excitation of multiple labels with a single excitation source. The strengths of this strategy are demonstrated in the detection of free prostate-specific antigen (PSA) using a sandwich assay format based on monoclonal antibodies. Detection limits of similar to1 pg/mL in human serum and similar to4 pg/mL in bovine serum albumin have been achieved with a spectrometer readout time of 60 s. The extension of the method to multianalyte assays (e.g., the simultaneous determination of the many complexed forms of PSA) is discussed.