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.

In order to prepare the Raman probes, first make two 1.5-milliliter batches of gold nanoparticles and the Raman reporter at concentrations determined as described in the accompanying text protocol.

This is done by combining the Raman reporter and the gold nanoparticle solutions and allowing them to bind over 30 minutes at room temperature. Then, add the PEGylated antibody solution to one of the reporter-bound gold nanoparticle solutions to create a 200 to 1 ratio of antibodies to particles. This solution will be for the test samples.

In a separate microcentrifuge tube add a PEGylated antigen to the other reporter-bound gold nanoparticle solution at a 200 to 1 ratio of antigen to particles to be used as the control. Incubate both solutions for 30 minutes at room temperature.

Next, block the remaining sites on the nanoparticle surface. To accomplish this, first, dissolve solid methoxy polyethylene glycol thiol to a 200-micromolar concentration using HPLC water. Vortex the solution until the thiol is completely dissolved. Then, add the thiol solution at a 40,000 to 1 ratio to the nanoparticle-bound PEG-antibody solutions, and incubate the solution at room temperature for 10 minutes to ensure the remaining sites on the gold nanoparticle are blocked.

Next, recover the functionalized Raman probes by centrifuging the particles in low-bound centrifuge tubes so that the supernatant is clear. Remove the supernatant by pipetting but be careful not to disturb the nanoparticles. Then, resuspend the nanoparticles with 1 milliliter of PBS.

Estimate the gold nanoparticle concentration by taking a UV-Visible measurement from about three microliters of the solution and compare the results to those from a known gold nanoparticle concentration as this is a linear relationship.

If the nanoparticles are not sufficiently blocked, they will aggregate due to inter-particle adhesion forces suggesting the reagents were added at the incorrect concentrations. Normally, the solution is a deep red color but aggregation creates a gray hue.

Adjust the volume such that the final solutions are at least 1 x 10¹¹ particles per milliliter and store the solutions at 4 degrees Celsius until they are used for functionalizing the immunoassay plate. Use these solutions within one week. First, prepare enough of the diluted antigen to fill the polystyrene wells. Then, vortex the solution and immediately add the solution to the plates wells.

Allow the antigen to bind to the plates for 1 hour at room temperature. Then, remove the excess antigen solution by dumping the solution, and hitting the plate against a paper towel-covered tabletop. Add TBST to the wells to wash the surface. Then, remove the wash by again dumping the solution and hitting the plate against a paper towel-covered tabletop.

Next, block the remaining binding sites on the plate to prevent nonspecific binding by adding 70 microliters of HSA-blocking solution to each well of the plate, and incubating the plate at room temperature for 30 minutes. Then, remove the blocking solution and rinse the plate three times with TBST, as previously demonstrated. After removing the final rinse, cover the plate and store it dry at 4 degrees Celsius until further use.

Add 140 microliters of the probe nanoparticles previously prepared into the first column of a 96-well plate, placing the test nanoparticles in the first five rows and the control nanoparticles in the last three rows. Add 70 microliters of PBS to all other columns.

Dilute each column using a 1 to 2 serial dilution. Start by taking 70 microliters from the first column, and mixing it with the second. Then, take 70 microliters from the second column and add it to the third, and so on. Allow the plate to incubate for at least one hour.

Then, remove the unbound nanoparticle probes and wash the plate with TBST five times as previously demonstrated. After the final wash, add 70 microliters of PBS to each well and cover the plate with a plate seal. Check that the control samples are clear. If nonspecific binding has occurred, the control samples will have a similar color as the test samples.

Using an inverted Raman microscope, focus the objective onto the surface of the well that has the gold nanoparticle probes. Obtain Raman spectra of the well. Collect a spectrum ranging from 1800 inverse centimeters to 400 inverse centimeters and repeat this for each well. Finally, follow the steps in the final section of the accompanying text protocol to determine the sensitivity of the setup.