The overall goal of this protocol is to demonstrate the development of a customizable gold nano particle based immunoassay capable of analysis using UV-Visible and Raman Spectroscopy. The use of two optical methods provides internal assay validation and increases the assay's flexibility. This method answers key questions concerning the development of a gold nano particle based immunoassay.
And explains how functionalization of the nano particle is used in this assay will influence optical absorption and light scattering. This protocol is unique because it incorporates materials found in typical research labs to fabricate an immunoassay platform that can be customized to fit individual research needs. The immunoassay is based on UV-Vis and Raman Spectroscopy.
Using Raman Spectroscopy is better than fluorescents or colorimetric methods as it has narrower bandwidths allowing for the detection of multiple biomarkers at the same time. To begin, hydrate the bi-functional crosslinking agent or the pyridyl disulfide PEG-NHS in 100 mL sodium bicarbonate. The ortho pyridyl end will bind to the gold nano particle service and the NHS end will react with the primary amine on the protein surface once activated.
Be sure to make the cross-linking solution within approximately 20 minutes of when it will be used. Next, add the bi-functional cross-linker to the antibody solution at a 2 to 1 conjugation ratio. Be sure the antibody solution is free from carrier proteins that may interact with the reaction.
In a separate microcentrifuge tube, add the bi-functional cross-linker to the antigen solution at the same ratio to be used for the control. The 2 to 1 ratio is assuming a 50%conjugation efficiency. The objective is to label each antibody with a single peg chain.
In this step, over-labeling is better than under-labeling. Finally, incubate the pegylated antibody solution at four degrees Celsius for eight hours. Store 25 microliter aliquots of the solution in low binding tubes that minus 20 degrees Celsius to limit the freeze thaw cycles.
Use each aliquot as needed. In order to prepare the Raman probes, first make two 1.5 mL batches of gold nano particles on the Raman reporter that concentrations determined as described in the accompanying text protocol. This is done by combining the Raman reporter and the gold nano particle solutions and allowing them to bind over thirty minutes at room temperature.
Then, add the pegylated antibody solution to one of the reporter bound gold nano particle 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 nano particle solution at a 200 to 1 ratio of antigen to particles to be used as the control.
Incubate both solutions for thirty minutes at room temperature. Next, block the remaining sites on the nano particle surface. To accomplish this, first dissolve solid methoxypolyethylene 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 nano particle bound peg antibody solutions and incubate the solution at room temperature for ten minutes to ensure the remaining sites on the gold nano particle are blocked. Next, recover the functionalized Raman probes by centrifuging the particles in low bind centrifuge tubes so that the supernatant is clear.
Remove the supernatant by pipetting but be careful not to disturb the nano particles. Then, re-suspend the nano particles with 1 mL of PBS. Estimate the gold nano particle concentration by taking a UV visible measurement from about 3 microliters of the solution and compare the results to those from a known gold nano particle concentration as this is a linear relationship.
If the nano particles 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 volumes such that the final solutions are at least 1 times 10 to the eleventh particles per milliliter and store the solutions at four degrees celsius until they are used for functionalizing the amino assay 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 plate's wells.
Allow the antigen to bind to the plates for one hour at room temperature. Then remove the excess antigen solution by dumping the solution and hitting the plate against a paper towel covered table top. 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 table top.
Next, block the remaining binding sites on the plate to prevent non-specific binding by adding 70 microliters of HSA blocking solution to each well of the plate and incubating the plate at room temperature for thirty 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 four degrees Celsius until further use.
Add 140 microliters of the probe nano particles previously prepared into the first column of a 96 well plate. Placing the test nano particles in the first five rows, then the control nano particles 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 nano particle 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 non-specific binding has occurred, the control samples will have a similar color as the test samples. For each well, use a plate reading UV-Visible Spectrophotometer to measure the UV-Visible spectra ranging from 400 to 700 nanometers.
Using an inverted Raman microscope, focus the objective onto the surface of the well that has the gold nano particle probes. Obtain a Raman spectra of the well. Collect a spectrum ranging from 1800th 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 set up. 60 nano meter gold particles were prepared as described in this video. They were then analyzed using UV-Visible Spectroscopy and Raman Spectroscopy.
The peak areas where each gold nano particle concentration were determined using an open source spectral analysis software, and were used to generate a logarithmic calibration curve like the one shown here. Based on this data, the lower limit of detection was found to be 3.5 picomolar for the UV-Visible detection and 1.7 picomolar when using Raman Spectroscopy. After watching this video, you should have a good understanding of how to customize nano particle probe based immunoassays using UV-Vis and Raman Spectroscopy.
