This protocol uses nanoparticle tracking analysis to perform accurate, reliable, and reproducible measurements of the hydrodynamic diameters of nanomaterials of interest. The method is quick and easy to perform facilitating measurement of the hydrodynamic spherical equivalent diameter of particles, via particle by particle real time visual analysis and minimal sample preparation. Before performing a measurement, filter the volume of ultra pure water to be used for the analysis through a 0.02 micron syringe filter.
Dilute the sample appropriately in the filtered water. For this 60 nanometers gold colloid dispersion sample, dilute volumetrically by a factor of 50. Connect the NTA instrument, syringe pump, and computer, and switch on the hardware and software.
Remove the laser module from the system and use a tissue and compressed air to completely dry the glass surfaces and the LVFC internal channels, tubing, and fluidic ports. Place the LVFC onto the laser module, then secure it with the four screws tightening them diagonally. Attach the outlet tubing to the port on the right side of the cell.
Insert the inlet tubing into the waste bottle and flush 900 microliters of cleaning solution as quickly as the back pressure allows. Replace the syringe attached to the inlet tubing with a new syringe containing one milliliter of filtered water, and connect the inlet tubing to the left port of the flow cell. Then slowly introduce approximately 500 microliters of fluid into the sample chamber, taking care that no air bubbles are introduced during loading.
To load the laser module, insert the water-filled LVFC into the instrument and lock the cell in place. Secure the syringe and the syringe pump cradle and click Start Camera in the software interface to initialize the camera. In the hardware tab, click Scatter to move the reference position and set the camera level to 16.
Adjust the focus manually to check the diluent for any particles, and left click and drag in the main viewing window to adjust the field of view position to check for any particles. To clean the system, replace the syringe with a syringe full of air only and slowly replace the water in the chamber with air. Remove the cell from the laser module from the instrument, and disconnect the pieces of tubing.
Clean the optical glass of the laser module and the glass surfaces of the LVFC with water and dry with a tissue and compressed air. After reassembling the LVFC and laser module and priming the tubing, connect a syringe containing one milliliter of the 60 nanometers gold nanoparticle suspension to the lower port, and slowly inject 750 microliters of the sample into the LVFC chamber. After loading the laser module and initializing the camera, click Scatter to move the reference focus into position confirming that the focus is set correctly to give a clear image of the particles.
Check that the field of view is set centrally with respect to the laser beam position, and run the auto setup function to automatically optimize the focus and camera level to ensure that the optimal image quality is achieved. To analyze the sample, open the Standard Measurement Standard Operating Procedure tab and set the instrument to acquire five 60 second videos under slow and constant flow. Then set the file name and location for the data and start the run.
To analyze the data, drag the slide bar in the Detection Threshold tab to set the detection threshold so that as many visible particles are detected as possible, and tracked. Make sure that no more than five blue crosses indicative of noise are observed. To automatically process the particle tracking analysis videos, click Settings, Okay, and leave all of the processing parameters on their automatic settings.
After data processing is complete, export the data as a CSV form results file. Here, round one inter laboratory comparison results obtained using various nanoparticle tracking analysis instrument configurations are shown. With the exception of lab six, the repeatability between the five capture repeats was good even though several labs recorded a higher than expected mode size.
The nanoparticle tracking analysis result accuracy from round three was improved by all of the laboratories implementing the same standard operating procedure and instrument settings with an average mode across all of the labs of 62.02 plus or minus 1.97 nanometers. The mean diameter of the particles can be calculated using transmission electron microscopy images as a complimentary technique to NTA to validate the particle size, morphology, and aspect ratio. In this analysis, the particles appear to be mostly spherical with an average aspect ratio of 1.1.
It is important to make sure that the low volume flow cell is well cleaned prior to use, that the sample is appropriately diluted, well loaded, and focused, and that the SOP is followed. This method can be used for any other nanoparticle characterization, including exosomes and viruses to support stability studies and quality controlled processes.
