This protocol offers a versatile method to detect and quantify uric acid with high sensitivity. Hence, it is a protocol for diagnostic and clinical applications. This protocol can quickly quantify biomarkers in a complex matrix, such as body fluids.
This technique can be potentially extended to diagnose diseases that use uric acid as a biomarker such as gout, hypertension, and cardiovascular diseases. It can also be used to detect and quantify other hazardous substances for environmental monitoring. When attempting the protocol for the first time, researchers may struggle to form stable aggregates of CB7 and gold nanoparticles.
Mixing the gold nanoparticles in CB7 solutions using sonication is recommended as other techniques could affect the aggregation kinetics. A video showing critical steps of adding CB7 mixing can increase the chance of success and reproducibility for researchers when new to the system. For gold seeds synthesis, dissolve 98.5 milligrams of auric in 10 milliliters of deionized water in a glass vial to obtain a 25 millimolar auric chloride solution and 64.5 milligrams of sodium citrate powder in 500 microliters of deionized water in a glass vial.
Next, dilute one milliliter of the auric chloride solution with 99 milliliters of water in a 250 milliliter blue capped bottle to obtain a 0.25 millimolar auric chloride solution and add 99.5 milliliters of the 0.25 millimolar auric chloride solution to a 250 milliliter three-necked round bottom flask equipped with a condenser. Then heat the solution at 90 degrees Celsius under vigorous stirring for 15 minutes before injecting 500 microliters of the sodium citrate solution into the flask continuing to stir until the solution turns ruby red. To initiate seeded growth of the gold nanoparticles, transfer 25 milliliters of the gold seed solution to a 50 milliliter tube and cool the solution to 70 degrees in a ThermoMixer.
While the solution is cooling, fill a three milliliter Luer lock disposable syringe with 2.5 milliliters of 25 millimolar auric chloride solution and fill another three millimeter Luer lock disposable syringe with 2.5 milliliters of 60 millimolar sodium citrate solution. Place the syringes into syringe pumps and use Luer-to-MicroTight Adapters to connect 150 micromolar internal diameter PEEK tubing to the syringes. Insert the tubing into the centrifuge tube containing the cooled gold seed solution in the ThermoMixer and set both the syringe pumps to dispense 167.5 microliters of solution over 20 minutes.
Set the ThermoMixer rotation speed to 600 rotations per minute and press Start on the syringe pump containing the 25 millimolar auric chloride solution. After two minutes, press Start on the syringe pump containing the 60 millimolar sodium citrate solution. After 30 minutes, remove an aliquot of the gold nanoparticle solution for analysis.
To prepare a 0.4 millimolar CB7 solution, add 4.65 milligrams of CB7 to a 15 milliliter glass vial and 10 milliliters of water to the vial. After tightly capping, sonicate the vial at room temperature until the CB7 solid is completely dissolved. To prepare a 0.4 millimolar uric acid solution, add 2.69 milligrams of uric acid to a 50 milliliter centrifuge tube and add 40 milliliters of water to the tube.
After tightly capping, swirl the mixture in a ThermoMixer set to 70 degrees Celsius and 800 revolutions per minute for two hours. When the solution has cooled to room temperature, dilute one milliliter of the 0.4 millimolar uric acid solution with nine milliliters of water in a 15 milliliter glass vial to obtain a 0.04 millimolar uric acid solution. After tightly capping, sonicate the vial for 30 seconds.
To prepare the CB7-uric acid complexes, add 750 microliters of the 0.4 millimolar CB7 solution and 750 microliters of the 0.4 millimolar uric acid solution to a 1.5 milliliter tube and sonicate the mixture for 30 seconds. Then hold the tube at room temperature for 30 minutes to ensure the formation of the host-guest complexes. Click the spectroscopy application wizards icon in the Raman system software and select Raman.
Start a new acquisition and set the integration time to 30 seconds, the scans to average to five and the boxcar to zero. Then store the background spectrum and enter the laser wavelength. For SERS substrate formation, add 900 microliters of the 40 nanometer gold nanoparticle solution and 100 microliters of preformed CB7-uric acid complex solution to a 1.5 milliliter tube and sonicate until the solution changes from ruby red to purple.
Transfer the sample solution to a semi-micro cuvette and place the cuvette into the Raman sample holder. Close the cover and start the measurement. Then set the auto saving to record five consecutive SERS spectra.
The ultraviolet visible spectra for the synthesized gold nanoparticles show a shift of the localized surface plasmon resonance peaks from 521 to 529 nanometers after 10 growing steps, while the dynamic light scattering data show a narrow size distribution as the size of nanoparticles increases from 25.9 to 42.8 nanometers. The average sizes of G0, G5, and G10 measured from transmission electron images are approximately 20, 33, and 40 nanometers respectively. In these representative analyses, host-guest complexes of CB7 and uric acid were formed with empty CB7 mediating the formation of precise plasmonic nanojunctions within the gold nanoparticle CB7 nanoaggregates as supported by the characteristic uric acid signals in the SERS spectrum.
Conversely, no SERS signals of uric acid can be observed in the absence of CB7, illustrating the key role of CB7 in triggering gold nanoparticle aggregation. The high sensitivity of the detection scheme presented in this protocol is demonstrated by the observation of clear SERS signals from the uric acid peaks at 640 and 1130 reciprocal centimeters down to the 0.2 micromolar detection limit. In addition, very strong correlations between the SERS intensity and log concentrations of uric acid were obtained by power law for both peaks with linear regions found in the range of 0.2 to 2 micromolar.
Uric acid has a low water solubility, so it is necessary to ensure that the uric acid powder has a soft before the next step. Advanced data analysis can be performed, making it possible to quantify multiple biomarkers present in the matrix.
