A Raman Spectroscopy-Based Direct Immunoassay to Detect a Specific Antigen

Begin by incubating Raman reporter dye molecules with gold nanoparticles for reporters to bind to the nanoparticles.

Add polyethylene glycol, PEG-conjugated antibodies specific to the target antigen. The antibodies covalently attach to the nanoparticles through gold-disulfide binding. Add thiol-conjugated PEGs to block the remaining nanoparticle sites and prevent nanoparticle aggregation, forming nanoparticle probes.

Coat immunoassay plate wells with the target antigen. Block the remaining plate sites with a solution containing proteins. Add the nanoparticle probes. Perform serial probe dilution across the wells. Incubate for the antibodies in the probes to bind to the antigens, forming complexes.

Use a Raman microscope to determine the probe's optical response. The incident monochromatic laser excites the gold nanoparticles' surface electrons and causes their oscillation, creating a high local electromagnetic field on the probe surface.

The incident beam and Raman reporters interact, causing enhanced inelastic light or Raman scattering with different wavelengths and frequencies due to the enhanced electromagnetic field. The inelastically emitted Raman signal is used to generate the Raman spectrum.

Plot the Raman reporter peak's spectral area against the probe concentration to find the lower limit of probe detection and facilitate more sensitive detection of the antigen.