Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

NOTE: All chemicals are used as received without further purification. Use ultrapure water throughout the study with a resistivity of 18 MΩ·cm. All the prepared dispersions are generally stored at 5 °C in the dark for any further characterization or future stability studies but this can vary depending on the material composition and other associated properties like dissolution. For example, Ag NPs are generally stable for some time if stored between 2 – 5 °C away from sunlight; however, dynamic changes are bound to happen within the suspensions and dispersions will re-agglomerate nevertheless and are known to sediment down with time. Analyze these materials using DLS, UV-vis, and TEM for quality verification before biological testing^4,5,13,14. A concentration of 0.02 mg/mL is used for analysis below. The sample concentration is suitable for analysis using DLS, UV-vis, disc centrifuge, zeta-potential calculations, and TEM analysis.

1. Calibration of Delivered Power by Sonicators

NOTE: The effective acoustic power delivered to the sonicated suspension is an important parameter in order to obtain reproducible dispersions. This is different from the electrical input or output power of the generator indicated by the manufacturer as this is the actual power that is deliverable to the suspension during sonication²⁶. Amongst many methods for the calculation of effective delivered power, the most commonly used method is calorimetry²⁶. This is known to be a simple and efficient way for the direct measurement of effective power delivered to a suspension⁷. In this method, the temperature increase in the liquid at a given sonicator setting is recorded over time and the effective power delivered is calculated using the following equation:

where P is the delivered acoustic power (W), T is the temperature (K), t corresponds to the time (s), C_P is the specific heat of the liquid (4.18 J/g·K for water), and M is the mass of the liquid (g).

1. Calibration of delivered power by ultrasonic probe fitted with a vial tweeter
   NOTE: The method is adapted from Taurozzi et al.⁷ and the following steps are recommended.
   1. Place an empty plastic vial on the microbalance and tare the balance.
   2. Fill the vial with 1.5 mL of DI water (resistivity 18 MΩ·cm) and record the mass of the liquid using the balance.
   3. Place the vial in one of the six vial holes in the high intensity part of the vial tweeter and immerse a temperature probe connected to a digital temperature meter using a clamp. Make sure that the probe does not touch the walls of the vial and is approximately 2 cm below the liquid surface.
   4. Set the vial tweeter setting at 24 kHz and 10 W (amplitude adjustment at 50%) and operate in the continuous mode.
      NOTE: Other amplitude adjustments tested here are 70%, 90%, and 100%.
   5. Record the increase in water temperature for the initial 5 min at an interval of 30 s and ensure that the vial or the set up does not shift position.
   6. Create a temperature versus time graph in a spreadsheet software and obtain the best linear fit for the curve using least squares regression.
   7. Obtain the slope of the graph (which is the rise of temperature over time) and calculate the power delivered using Equation 1. Repeat the experiment three times and obtain the mean value.
   8. Repeat the procedure from steps 1.1.1 – 1.1.4 for 70%, 90%, and 100% amplitude settings. The power value obtained using this procedure is reported during dispersion procedure.
2. Calibration of delivered power by an ultrasonic bath
   1. Place an empty plastic vial on the microbalance and tare the balance.
   2. Fill the vial with 1.5 mL of DI water (resistivity 18 MΩ·cm) and record the mass of the liquid using the balance.
   3. Place the vial in the middle of the ultrasonic bath half dipped in water and secure it with a clamp. Immerse a temperature probe connected to a digital temperature meter using a clamp. Make sure that the probe does not touch the walls of the vial and is approximately 2 cm below the liquid surface.
   4. Set up the ultrasonic bath at 40 KHz and 80 W and operate in the continuous mode.
   5. Record the increase in water temperature for the initial 5 min at an interval of 30 s and ensure that the vial or the set up does not shift position.
   6. Create a temperature versus time graph in excel and obtain the best linear fit for the curve using least squares regression.
   7. Obtain the slope of the graph in a spreadsheet software (which is the rise of temperature over time) and calculate the power delivered using Equation 1. Repeat the experiment three times and obtain the mean value. The power value obtained using this procedure is reported during the dispersion procedure.

2. Dispersion Procedure in Aqueous Medium Using an Ultrasonic Probe Fitted with a Vial Tweeter

1. Weigh 2 mg of each of the required nanopowder using a clean spatula into three clean glass vials. Label them as vials 1, 2, and 3.
2. Pipette out 1 mL of DI water and add along the walls of each vial. Make a thick paste with the help of a clean thin glass rod, then add the rest of the water to make a final concentration of 0.2 mg/mL. In case of a hydrophobic sample, perform the pre-wetting using 1 mL of 0.5% vol/vol ethanol and add DI water to make up the required final concentration.
3. Seal each vial with its cap and shake well in the horizontal circular motion to removeany nanopowder sticking to the walls of the vial.
4. Place the three vials in the ultrasonic probe fitted with a vial tweeter and apply the first sonication treatment for 2 min at 1.1 W in pulsed mode (1 s/1 s, which means 1 s on and 1 s off). This will give a temperature rise of about 4 °C in the dispersion.
5. Take out vial 1 and pipette out a suitable amount of aliquot from the top of the vial, dilute it with DI water to a concentration of 0.02 mg/mL. Characterize the diluted dispersion for size, particle size distribution, shape, agglomeration, and zeta potential using a range of complimentary techniques such as DLS, TEM, UV-vis, and ELS (discussed in Section 4). Record and document the measurements accurately.
6. Pause for 10 min from step 2.4 to allow cooling of the sample and to avoid any abrupt temperature rise in the system. Apply a second sonication treatment to vials 2 and 3 for 4 min at the same settings of amplitude and pulsed mode. Take out vial 2, repeat step 2.5, and document the readings after 6 min of sonication.
7. Pause for 10 min, apply a third sonication treatment to vial 3 for another 4 min and then follow step 2.5. Record and document the measurements at 10 min of sonication (discussed in Section 4).
   NOTE: Lab coats, gloves and goggles must be worn when handling suspensions of nanoparticles. The sonicator must be placed in acoustic enclosure during longer experiments, and high protection ear muffs must be worn when working closer to the ultrasound source.

3. Dispersion Procedure in Aqueous Medium Using an Ultrasonic Bath

1. Weigh 2 mg of each of the required nanopowder using a clean spatula in four clean glass vials and label them as vials 4, 5, 6, and 7.
2. Pipette out a few drops of DI water and add along the walls of each vial, and make a thick paste with the help of clean thin glass rod. Then add the rest of the water to make a final concentration of 0.2 mg/mL in each vial.
   NOTE: In case of a hydrophobic sample, the pre-wetting is carried out using 1 mL of 0.5% vol/vol ethanol and then DI water is added to make up the required final concentration.
3. Seal each vial with its cap and shake well in the horizontal circular motion to remove any nanopowder sticking to the walls of the vial.
4. Place the four vials in the middle of ultrasonic bath with the vials half dipped in water and apply the first sonication treatment at 80 W for 15 min at room temperature. This would give a temperature rise of about 3 °C in the dispersion.
5. Remove vial 4 from the ultrasonic bath and pipette out a suitable amount of aliquot from the top of the vial, dilute it with DI water to a concentration of 0.02 mg/mL, and characterize the sample for size, particle size distribution, shape, agglomeration, and zeta potential using a range of complimentary techniques such as DLS, TEM, UV-vis, and ELS (discussed in Section 4). Record and document the measurements.
6. Change the water in the ultrasonic bath and apply a second sonication treatment to vials 5, 6, and 7 for another 15 min at the same settings (80 W). Remove vial 5, follow step 3.5 for characterization and document the readings at 30 min of sonication.
7. Change the water in the ultrasonic bath (to avoid any further temperature rise) and apply a third sonication treatment of another 30 min to vials 6 and 7 at the same settings with a small pause of change of water again at 15 min. Remove vial 6 and follow step 3.5. Record and document the measurements at 1 h of sonication.
8. Change the water again in the ultrasonic bath every 15 min and apply a fourth sonication treatment to vial 7 for another hour keeping the settings constant. Take out vial 7 and follow step 3.5 for complete characterization, and record the measurements at 2 h of sonication.

4. Characterization of the Dispersed Samples at Different Time Points

1. Size characterization using DLS²⁷
   1. Open the DLS software. Create a size measurement file that can be individualized for a specific nanomaterial (including one for a standard) using the refractive index value from the Malvern manual. In addition, input any other data as required by the software, such as values of absorption and viscosity, and also type of dispersant.
   2. Enter in the experiment conditions for the sample, such as 2 min equilibration time, 20 °C temperature, cuvette type as low volume disposable cuvette, and experiment running in automatic mode. Press File|Save (save with the desired name).
   3. Press "File|Open new measurement" and run a DLS verification test by using standard latex beads with a nominal size of 100 nm to qualify instrument performance
      1. Use a low volume disposable cuvette. Inject 1 mL of sample using syringe or pipettes to avoid any air bubbles.
         NOTE: Clean the cuvettes with ethanol and DI water before use.
      2. Insert the cuvette into the machine. Click on the "start" button in the file measurement panel. Note that this will equilibrate the sample for 2 min and take measurements at 20 °C.
         NOTE: If samples have been previously stored in the fridge, allow them to reach room temperature prior to use.
      3. Collect at least five measurements in automatic mode and take the average of the measurements to report the size by selecting all the measurements and clicking "average" from the top panel. Export the data to excel for further analysis.
      4. Report the hydrodynamic diameter as the Z-average, with the PdI width representing the standard deviation of the Z-average in case of a monomodal distribution²⁸. In case of a significant discrepancy between the Z-average and the mean of the intensity-weighted size distribution, which is indicative of polydispersity or agglomeration, the mean of the intensity-weighted size distribution result is quoted with a comment on the sample status.
      5. Repeat step 4.1.3 for new measurements.
         NOTE: DLS is not a suitable technique for analysis of non-ideal samples. By this we mean samples which are non-spherical particles of high polydispersity, extensive agglomeration, sedimentation, etc. The repeated measurements may result in inaccurate readings due to sedimentation/settling particles. In such cases, other complimentary techniques are recommended such as disc centrifugation, which can be used to assess the dispersion in a qualitative manner.
2. Size distribution by Disc Centrifugation
   1. Open the CPS software. Select "Procedure definition", put in the sample SOP name at the top, and fill the sample parameters such as minimum and maximum diameter, particle density, refractive index, absorption, and non-sphericity factor²⁹.
      1. For example, for ZnO nanoparticles, enter 0.1 micron and 1.0 micron in the minimum and maximum diameter tabs, respectively, enter 5.61 g/mL in the particle density, 2.1 in the refractive index section, 0.001 in the particle absorption, and 1 in the non-sphericity section.
   2. Fill the calibration standard details based on the PVC standard of peak diameter 0.377 µm with a particle density of 1.385 g/mL. Also fill in the fluid parameters (sucrose, fluid density of 1.04 g/mL, and fluid refractive index of 1.35), and name and save the procedure.
   3. Chose the selected procedure (SOP saved in step 4.2.1) and inject the first gradient level, 1.6 mL of sucrose (24%) into the hole through the disc and press 'start'.
      NOTE: The role of sucrose here is to establish a density gradient within the disc while spinning at a constant speed. This automatically calculated the disc velocity depending on the size range.
   4. Wait until the software reaches the automatically calculated RPM (rotations per minute). Stabilize the sedimentation by injecting a gradient of sucrose (8% low density and 24% high density, see Table 1), 1.6 mL total volume each time starting with highest density and ending with the lowest density solution.
      NOTE: Here we mark the 8% sucrose solution as low and 24% sucrose solution as high. They are mixed in following volumes (total volume 1.6 mL each time) and injected into the disc one by one until a gradient is formed.
      1. Following this, inject 1.0 mL of dodecane cap fluid which helps maintain the gradient inside the disc for a minimum of 6 h. Allow the disc centrifuge to equilibrate for 1 h.
   5. Select "Operate analyzer" and introduce the sample ID, and press start. Inject 0.2 mL of standard with a 1 mL syringe in the disc and press the space bar at the same time. Then inject 0.2 mL of the sample and press the space bar at the same time. Wait for the measurement to finish and then click on next sample.
   6. Use the disc centrifuge control system software to acquire and process the data. For this, click "retrieve distribution" and click on the sample name; this opens the size distribution graph for the sample. Export the data to a spreadsheet manager.
3. Dispersion stability study using UV-vis spectroscopy
   NOTE: UV-vis spectroscopy is often used to understand the suspension stability and aggregation by carefully observing the changes in the peak intensity, spectral skewness, spectral shape as well the wavelength shift in the absorption spectrum⁴. The detailed steps are as follows.
   1. Open the UV-vis spectrophotometer software and click "spectrum scan"³⁰.
   2. Use a standard quartz cuvette (semi microrectangular quartz cell 100 mm, 190 – 2,700 nm). Inject 2 – 3 mL of sample using a pipette.
      1. Before use, wash the cuvettes with 50% nitric acid for 10 min and then wash three times with purified water. Then rinse with acetone, remove excess, and air dry.
   3. Preset the instrument setting range to 700 nm to 200 nm wavelength from the wavelength tab by clicking "Instrument" at the top of the software panel and clicking 'set wavelength'.
   4. Click "baseline". Background subtract each spectrum using a corresponding 'blank' i.e., a cuvette filled with only dispersing medium, which in this case is water.
      NOTE: In case of hydrophobic samples, a similar ratio of ethanol:water is used as the dispersing medium.
   5. Collect at least three individual spectra on each sample by clicking "Instrument| Property", and enter '3' in the number of spectra. Take average values for analysis. Save the data and export the data for further analysis.
4. Zeta potential measurements using ELS
   1. Open the DLS software. Create a zeta potential measurement file that can be individualized for a specific nanomaterial using the refractive index value from the Malvern manual. Input other information that can be plugged into the software e.g., absorption, viscosity, and type of dispersant as found in the sample setting tab. Click "File|Save" and save with the desired name.
   2. Click "File|Open new measurement" and verify the instrument performance by using a reference standard DTS 1235 (zeta potential standard). This is a polystyrene latex standard in aqueous buffer at pH 9 and has a zeta potential of -42 ± 4.2 mV.
   3. Prepare the sample in a syringe of at least 1 mL capacity. Use a disposable folded capillary cell fitted with an electrode on each side for the zeta potential measurements. Carefully inject the sample into the capillary cell through one of the ports on the capillary cell checking that there are no bubbles.
      1. Once the sample starts to emerge from the other end, insert the stoppers and remove any liquid which may have spilt onto the electrodes. Clean the cuvettes thoroughly with ethanol and DI water.
   4. Insert the folded capillary cell into the machine. Equilibrate for 2 min and acquire the measurements at 200 °C unless specified. If dispersion samples have been previously stored in the fridge, allow the dispersion samples to reach room temperature prior to use.
   5. Collect at least five measurements in automatic mode and report the averaged zeta potential value. Export the data, analyze^17,18 (typically, zeta potential value below -25 mV and above 25 mV is considered stable), and interpret online or offline.
5. Morphological characterization using TEM
   1. Use grids (300 mesh) holey carbon films for the sample preparation. Put a drop of the dispersion sample (approximately 0.1 mL, 0.02 mg/mL) on a clean grid.
   2. Allow sample to air dry in ambient conditions whilst keeping the grids covered to prevent airborne contamination.
   3. Wash the grids with ultra-pure water to remove any drying effects, and subject to TEM imaging.
      NOTE: The addition of the dispersion drop on the grid increases the concentration of particles on the grid surface thus leading to attractive inter-particle forces. Uneven drying can lead to artifacts. A small rinse with ultra-pure water eliminates this risk and is helpful for a uniform drying of the grids³¹.
   4. Acquire the images in a dm3 format and later examine them offline using TEM software.
      NOTE: Images can be used to deduce complementary information surrounding particle size, structure and shape. Files are converted to tiff, in which quantification on properties such as shape and size can be carried out.