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

1. Prepare UV-Vis/Raman Probes

1. Prepare bare AuNP solution
   1. Prepare a 2 ml solution of AuNPs with a concentration of approximately 1 x 1011 particles per ml.
      1. If the AuNPs need to be concentrated, fill low-binding centrifuge tubes with 2,000 μl of stock AuNP and centrifuge at 5,000 x g for 20 min or until the supernatant is clear. Remove the supernatant by pipetting, being careful not to disturb the AuNP pellet.
      2. Combine the remaining AuNP solutions into one tube and estimate the concentration by obtaining a UV-Vis measurement and comparing values to known concentrations as this is a linear relationship.
2. Determine the appropriate Raman reporter labeling ratio
   1. Prepare a working solution of the Raman reporter dissolved in methanol. This concentration will be dependent on the reporter used. In this work, prepare 3,3′-diethylthiatricarbocyanine iodide (DTTC) at a working solution of 200 μM.
   2. Assuming a final volume of 100 μl for each well, add enough of the working reporter solution to each well of the first row of a 96-well plate such that the Raman reporter will range in concentrations from 0.2 μM to 10 μM. Add enough HPLC-grade water to each well such that the volume is 80 μl. Add 20 μl of AuNP to each well making a final volume of 100 μl for each well. An example is provided in Table 1.
   3. Measure the UV-Vis spectra from 400 to 700 nm using a plate-reading UV-Vis spectrophotometer. The appropriate concentration is the highest concentration with defined peaks for the UV-Vis spectra. Repeat step 2.2.2 at increasing concentrations until the highest concentration ratio of Raman reporters to AuNPs is found.
      NOTE: The dye and the AuNP shape, size, and manufacturer influence the appropriate concentration. Therefore, the steps listed must be evaluated and altered depending on the components used. This protocol involved the use of a positively charged dye. As such, binding between the AuNP and reporter was improved by using negatively charged AuNPs. This was done by using citrate-capped AuNPs. See the Discussion section for further details.
3. Binding Raman reporter and PEG-Ab to AuNP
   1. Prepare two 1.5 ml batches of AuNP and Raman reporter at the previously determined concentration, allowing the Raman reporter to bind to the AuNPs for 30 min at room temperature.
   2. Add the PEGylated antibody (PEG-Ab) to one batch of the AuNP and Raman reporter solution to create a 200:1 ratio of antibodies to particles. This solution will be for the test samples. In a separate microcentrifuge tube, add the PEGylated antigen to the other batch of the AuNP and Raman reporter solution at a 200:1 ratio of antibody to particles to be used as the control. Incubate the solutions for 30 min at room temperature.
      NOTE: The ratio of antibodies to particles will be specific to the AuNPs and dye used and should be optimized for each individual case. The objective here is to have the highest ratio of antibodies for the AuNP probes to bind to while preventing aggregation of the particles. Use the following equation to determine the appropriate volumes to add together:
      
      where V is volume, and C is concentration expressed in particles or antibodies per ml. The final volume should be approximately 1.5 ml.
4. Block remaining sites on the AuNP surface with mPEG-SH.
   1. Prepare mPEG-SH by dissolving solid methoxy polyethylene glycol thiol to a 200 μM concentration using water. Vortex the solution until mPEG-SH is completely dissolved.
   2. Add mPEG-SH at a 40,000:1 ratio to the AuNP-PEG-Ab solution made in step 2.3. Incubate the solution at room temperature for 10 min to ensure the remaining sites on the gold nanoparticle are blocked. Use the following equation to determine the appropriate volumes to add together:
      
      where V is volume, and C is concentration expressed in particles or antibodies per ml. The final volume should be approximately 1.5 ml.
5. Recover functionalized Raman probes.
   1. Centrifuge particles at 5,000 x g for 20 min in low-bind centrifuge tubes or until the supernatant is clear. Remove the supernatant by pipetting being careful not to disturb the AuNPs.
   2. Resuspend the particles with 1 ml of 1x PBS solution that was made previously. Estimate the AuNP concentration by taking a UV-Vis measurement of a small volume of solution (3 μl) and compare the results to measurements from a known AuNP concentration. Adjust the volume such that the final solution is at least 1 x 10¹¹ particles per ml.
   3. Store solutions at 4 °C until it is used for functionalizing the immunoassay plate. Use the solutions within one week.

Table 1. DTTC dilution example. Various dilutions of DTTC and the associated volumes of stock DTTC, gold nanoparticle solution, and water.

2. Immunoassay Plate Preparation

1. Bind the desired antigen to the immunoassay plate.
   1. Prepare enough diluted antigens (50 μg/ml) to fill the polystyrene wells. Vortex the solution, and immediately add the solution to the plate wells. Allow the antigen to bind to the plates for 1 hr at room temperature.
2. Wash off unbound antigens.
   1. Remove the excess antigen solution by dumping the solution into a disposal container and hitting the plate against a paper towel-covered tabletop.
   2. Add TBST to the wells to wash the surface then remove the wash in the same manner as stated previously. Repeat this step two more times.
3. Block remaining binding sites on the plate to prevent non-specific binding.
   1. Add 70 μl of HSA-blocking solution to each well of the plate and incubate at room temperature for 30 min.
   2. Remove and rinse the plate using the same procedure as specified in step 3.2. Cover the plate and store dry at 4 °C until ready for further use.
4. Functionalize the immunoassay plate.
   1. Add 70 μl of the probe nanoparticles prepared in Section 2 to the first column of a 96-well plate and dilute subsequent columns using a 1:2 serial dilution. Allow the plate to incubate for at least 1 hr. An example of how to prepare the immunoassay plate is given in Figure 1.
   2. Wash the plate with TBST five times as detailed in step 3.2, making sure to dispose of the AuNPs appropriately. After the final wash, add 70 μl of 1x PBS to each well and cover with a plate seal.
      NOTE: The control samples should be clear. If the non-specific binding has occurred, the control samples will have a similar color as the test samples.
5. Test assay sensitivity by UV-Vis and Raman spectroscopy.
   1. For each well, measure the UV-Vis spectra ranging from 400 to 700 nm using a plate-reading UV-Vis spectrophotometer.
   2. Using an inverted Raman microscope, focus the objective onto the surface of the well that has the AuNP probes. Obtain Raman spectra of the well. Collect a spectrum ranging from 1,800 cm^-1 to 400 cm^-1. Repeat this step for all wells.
   3. Using appropriate spectral software, perform an 11^th-order polynomial baseline correction for the Raman spectra and a 3^rd-order polynomial for the UV-Vis spectra.
   4. Using appropriate spectral software, normalize the Raman and UV-Vis spectra. Set the maximum value to 1 and scale all other values accordingly. To normalize the Raman spectra, select a unique polystyrene peak and set it equal to 1, and scale all other values accordingly.
   5. Using appropriate spectral software, perform peak integration for each spectrum. For Raman spectra, the peak representing the Raman reporter must be in a region absent of polystyrene peaks. To perform peak integration, specify the integral boundaries for the desired peak and record the desired peak area for all samples including the controls.
   6. Plot the average peak area of interest as a function of the log of the AuNP concentration with error bars for each point indicating its associated standard deviation. Fit these calibration points to a 4-parameter logistic curve.
   7. Determine the mean value of the blank by averaging the area of the peak of interest for a blank sample. Determine the standard deviation of these areas; this is the standard deviation of the blank.
   8. For the same peak analyzed in the previous step, find the standard deviation of that peak area for the lowest concentration.
   9. Calculate the limit of the blank and lower limit of detection as specified in the Representative results section. Use these values with the 4PL calibration curves to determine the LLOD in terms of AuNP concentration.