Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

Here, we present a step-wise protocol for the dispersion of nanomaterials in aqueous media with real-time characterization to identify the optimal sonication conditions, intensity, and duration for improved stability and uniformity of nanoparticle dispersions without impacting the sample integrity.

The overall goal of this procedure is to use real-time characterization to identify the optimal sonication conditions, intensity, and duration for obtaining stable and uniform nanomaterial dispersions in aqueous media. This method can help answer key questions in nanoscience, particularly in the field of nanotoxicology, about how to optimize nanoparticle dispersion without impacting sample integrity. The main advantage of this optimization strategy is that it improves control over the final dispersion quality, which is instrumental in insuring repeatability.

To begin the procedure, calibrate the probe sonicator fitted with a vial block sonitrobe. Then use a clean metal spatula to measure two milligrams of the chosen nanopowder into each of three clean 10 or 20 milliliter glass vials, numbered one through three. Pipette one milliliter of deionized water along the inner walls of each vial.

For hydrophobic samples, instead use one milliliter of 0.5 percent by volume ethanol in deionized water. Mix each sample into a thick paste. Then, add sufficient deionized water to the paste to achieve a final sample concentration of 0.2 milligrams per milliliter.

Horizontally swirl the vials to dislodge any nanopowder adhering to the inner walls. Transfer 1.5 milliliters of each nanopowder dispersion to clean microcentrifuge tubes labeled with the corresponding numbers. Firmly close the tubes and swirl them to dislodge nanopowder from inner walls.

Then, place the samples in the sonitrobe. Sonicate the samples at 1.1 watts, pulse for one second on and one second off for two minutes. Then, remove sample one from the block.

Transfer one milliliter from the top of the dispersion to another clean microcentrifuge tube. Add deionized water to the tube to attain a sample concentration of 0.02 milligrams per milliliter. Immediately begin sample characterization.

10 minutes after the end of the first sonication treatment, sonicate the remaining samples for four minutes, using the same amplitude and pulse settings. Dilute sample two to 0.02 milligrams per milliliter with deionized water and begin characterization. 10 minutes after the end of the second sonication treatment, sonicate sample three for another four minutes at the same settings.

Dilute sample three to 0.02 milligrams per milliliter and begin characterization. First, callibrate an ultrasonic bath with respect to the bath sensor. Next, use a clean spatula to place two milligrams of the chosen nanopowder in each of four clean glass vials, numbered four through seven.

Pipette one milliliter of deionized water along the walls of each vial and mix the powders into thick pastes. Add deionized water to achieve a sample concentration of 0.2 milligrams per milliliter in each vial. Cap the vials and swirl them to dislodge any nanopowder adhering to the inner surfaces.

Place the vials in the center of the calibrated ultrasonic bath. Ensure that the water level is halfway up the vials. Sonicate the sample at 80 watts for 15 minutes at room temperature.

Then, transfer an aliquot of sample four to a clean vial. Dilute the aliquot with deionized water to 0.02 milligrams per milliliter and begin characterization. Replace the ultrasonic bath water with fresh room temperature water to avoid heat buildup that could affect the dispersions.

To begin assessing particle size with dynamic light scattering, open the instrument software and create a size measurement file. Fill in the equilibration time, temperature, cuvette type, and experiment mode. Save the size measurement file.

Then, run a DLS verification measurement on a sample of standard latex beads with a nominal size of 100 nanometers. Confirm that the instrument performance meets the appropriate standards. Next, slowly pipette one milliliter of a dilute nanopowder dispersion into a clean, low volume disposable cuvette, being careful to avoid forming air bubbles.

Insert the cuvette into the instrument and start data collection. Take at least five measurements per sample. Then, select all measurements and calculate the average for each sample.

Export the measurement data to a spreadsheet program for further analysis. Next, to begin sample characterization with UV vis-spectroscopy, open the instrument software and prepare a new skin. Pipette two to three milliliters of a dilute nanopowder dispersion into a clean standard quartz cuvette.

Then, set the instrument wavelength range to be from 700 nanometers to 200 nanometers. Acquire a solvent blank for background subtraction. Acquire at least three spectra per sample and export the data for further analysis.

Next, to begin sample characterization with transition electron microscopy, place a drop of a dilute nanopowder dispersion on a clean 300 mesh holey carbon film. Allow the sample to air-dry under ambient conditions while protected from airborne contamination. Then, wash the grid with ultra pure water to remove effects from uneven drying.

Acquire TEM images and export them for further analysis. Prior to dispersion, zinc oxide nanomaterials with either hydrophilic or hydrophobic surface profiles showed large particle mean sizes and high polydispersity. The particle size and polydispersity of the hydrophilic zinc oxide decreased after 15 minutes of sonication in an ultrasonic bath, but increased as sonication continued.

TEM images confirm that the particles reagglomerated with continued sonication. Ultrasonic bath treatment of the hydrophobic zinc oxide particles resulted in a decrease in particle size in polydispersity, which plateaued at 30 minutes. Drying effects were observed in TEM images of the hydrophobic zinc oxides, indicating that pre-wetting with ethanol led to difficulty in immobilizing the powder on the carbon grid.

Ultrasonic probe treatment of the hydrophilic powder resulted in a homogeneous, stable dispersion after two minutes. Continuing sonication led to rapid reagglomeration. Similar behavior was observed with the hydrophobic powder.

Based on polydispersity values, the optimal dispersion conditions for the hydrophilic and hydrophobic zinc oxide powders were found to be unltrasonic bath treatment for 60 and 30 minutes respectively. Sonication is commonly used for the agglomerating and dispersing nanomaterials in aqueous media. However, optimization strategy must be reoptimized for any change in nanomaterial type or dispersion medium.

While attempting this procedure, remember to calibrate the sonicators to determine their effective acoustic energy delivered to the suspension. Be sure to record all the sonication parameters and time points evaluated. Once mastered, this technique can be used for the dispersion of nanomaterials in water, or other media, by individually adjusting the sonication type, time, and power, while taking into account the temperature rises during the process.