The overall goal of this procedure is to amplify the signals of nanoparticle-based probes in assays carried out on either paper or glass supports. Using this method, you can acquire a colorimetric signal for spots that contain as few as 10 nanoparticles. The main advantage of this technique is its versatility.
It can be applied without major modification to different nanoparticles on paper, glass, and potentially other supports. To begin the procedure, prepare at least 10 milliliters of a 10 millimolar MES solution in deionized water. Adjust the pH of the solution to six with a four-molar sodium hydroxide solution.
Next, combine one milliliter of a 1.2 times 10 to the minus ninth molar suspension of 40 nanometer gold nanoparticles with one milligram of alpha mercapto omega carboxy PEG and vortex the mixture. Adjust the pH of the mixture to 12 with a four molar sodium hydroxide solution. Gently stir the mixture at room temperature overnight.
Then, centrifuge the mixture at 3, 800 times G for 15 minutes and discard the supernatant. Resuspend the pellet of PEG-modified gold nanoparticles in 500 microliters of deionized water by gentle pipetting. Next, dissolve five micromoles of EDC in 250 microliters of the 10-millimolar pH six MES buffer.
Dissolve 7.5 micromoles of sulfo-NHS in another 250 microliters of the MES buffer. Combine these solutions with the PEG-modified gold nanoparticles and incubate at 37 degrees Celsius for 30 minutes. Centrifuge the mixture for five minutes and discard the supernatant.
Resuspend the pellet in 500 microliters of the MES buffer. Add to the suspension a sufficient quantity of IgG antibody for an antibody concentration of 100 picograms per milliliter. Incubate the mixture at 37 degrees Celsius for two hours.
Centrifuge the mixture for 10 minutes and remove the supernatant. Resuspend the pellet in 500 microliters of the MES buffer and repeat the centrifugation. Then, resuspend the pellet in 500 microliters of a pH 7.5 buffer of 10 millimolar sodium phosphate and 0.3 molar sodium chloride in deionized water.
Incubate the mixture at 37 degrees Celsius for 30 minutes. Centrifuge the mixture for 10 minutes twice, as previously described. Resuspend the pellet in 500 microliters of 1%BSA in 10 millimolar pH six MES buffer and incubate the mixture at four degrees Celsius overnight.
Centrifuge the mixture for 10 minutes twice. Finally, resuspend the pellet in 500 microliters of MES buffer to obtain the IgG modified gold nanoparticles. Use a robotic printer to deposit microarrays of a protein G concentration gradient on nitrocellulose membranes.
Next, load a microarray into a stainless steel filter holder of an appropriate size. Connect a syringe containing an eight picomolar suspension of IgG modified gold nanoparticles to the inlet of the filter holder. Mount the syringe on a syringe pump.
Set up a 50-milliliter tube to collect the outlet flow from the filter holder. Then, flow the suspension through the filter holder at one milliliter per minute. Afterwards, allow the microarray to dry for 30 minutes at room temperature.
Clean and dry the filter holder with water and filter paper. Scan the dry microarray in a flatbed scanner at 4, 800 DPI and save the image as a 24-bit color TIFF file. Next, prepare both a five millimolar chloroauric acid solution and a 1.027 molar hydrogen peroxide solution in 10 millimolar pH six MES buffer.
To premix the enhancement solution, place equal values of the chloroauric acid and hydrogen peroxide solutions in a microcentrifuge tube and thoroughly mix the solutions. Apply the premixed enhancement solution to the microarray sensor surface. Alternatively, apply equal volumes of the chloroauric acid and hydrogen peroxide solutions to the sensor surface and thoroughly mix them directly on the sensor.
Incubate the sensor with the enhancement solution for up to five minutes, then submerge the microarray in deionized water for five seconds to rinse away the enhancement solution. Allow the microarray to air dry at room temperature. Repeat this process for each microarray to be enhanced.
Scan the enhanced microarrays and save the images. In a standard paper-based immunoassay, IgG modified gold, silver and iron oxide nanoparticles detected protein G concentrations at two times 10 to the fifth, two times 10 to the fourth, and two times 10 to the seventh molecules per spot, respectively. No visual signal was observed from IgG modified silicon nanoparticles.
Incubating the microarrays in enhancement solution resulted in a 100-fold signal increase across the gold, silver and iron oxide nanoparticles. In the case of silicon nanoparticles, this enhancement allowed a visual signal to be observed. This enhancement was applied to a commercial glass-based allergen component microarray immunoassay of four human serum samples.
Staining with anti-IgE labeled gold nanoparticles and enhancing the signal had comparable results to the standard fluorometric assay. The average linear correlation between the mean fluorescence intensity and the mean colorimetric intensity was 0.79. Once learned, this technique can be done in under 10 minutes if performed correctly.
