The standard measurement mode for Malvern’s NanoSight Nanoparticle Tracking Analysis (NTA) technique takes place with the sample static. In that case, the only motion in the analysis is the Brownian motion of the particles. Since NTA is a single-particle technique, the statistical representation of the analysis is increased as more particles are included in the measurement. Precision and repeatability of concentration measurements are significantly improved when a slow flow is imparted on the sample to ensure that a greater number of new particles flow through the measurement zone and are analyzed during an experiment.
The Malvern NanoSight NTA instruments provide both submicron particle size (10 nm – 1 µm) and concentration (particles/mL) measurements. NTA is used to measure a plethora of sample types, such as extracellular vesicles (including exosomes), viruses, protein aggregates, drug delivery particles (including liposomes and polymeric particles), as well as environmental and nanotoxicology samples. The degree to which each sample type will benefit from better statistical data depends on the nature of the sample (polydispersity), the particle concentration, and the number of repeat measures obtained (i.e. number of videos captured).
By making the sample flow during video capture using a syringe pump, a user can:
Many factors and combination of factors affect repeatability. For instance, the more polydisperse a sample is, the longer each measurement (video) needs to be to ensure fully representative sampling. Currently, the best practice recommendation is for users to capture 5 x 60s videos for each sample.1Capturing multiple videos for each sample provides the ability to generate a standard error of the data for further statistical analysis (t-test, etc.).
Additionally, by applying a constant, smooth flow of the sample during video capture, one can achieve better sampling per experiment since the flow ensures that new particles will be brought into the viewing area throughout the course of a measurement. In this technical note, 100 nm PLS standard beads are used to demonstrate the accuracy and repeatability of NanoSight NTA size and concentration measurements with varying syringe pump speeds. Similar experiments should be done with the user’s actual samples to verify the robustness of the analytical conditions.
NTA size and concentration measurements were performed on 100 nm polystyrene latex (PSL) standard (102 nm +/- 3 nm, ThermoScientific) diluted 100,000X in MilliQ water to achieve 1.7x108 (+/-10 %) particles per mL. Samples were run (5 x 60s videos) on an NS500 instrument with a 488 nm laser source and a NanoSight syringe pump with 1 mL Terumo Syringes. Data were analyzed using NTA 3.1 Build 48 software. Samples were run at flow rates ranging from 0-100 AU (arbitrary units) in the NTA software.
For a closer look at both repeatability in regards to both size and concentration, the 100 nm PSL standard sample of a calculated concentration was given to a user who ran ten different aliquots both with (flow) and without (static) the syringe pump (Figure 1). The use of the syringe pump improved the precision of the concentration measurement. Repeatability improves from 15% CV without the syringe pump, to 3% CV with the syringe pump. Meanwhile, size precision, accuracy and repeatability remains unaffected at 2% CV either with or without the syringe pump.
Figure 1. Concentration data from 10 consecutive experiments on the same sample with standard static sample loading and when under constant syringe pump flow. Run represents 14 x 60s videos.
These improvements are for a monodisperse latex that is stable in solution. For a normal, polydisperse sample, the potential variability could be significantly greater to start with and the potential benefits of flow mode correspondingly large. Optimum flow rates were determined by the following series of experiments and the users should reproduce these experiments when designing methods for other materials.
Malvern recommends syringe pump flow rates be set so that particles in the sample take approximately 10 seconds to cross the field of view (FOV) so that the inherent Brownian motion of the particle is what is measured, not the added motion of the diluent. For example, if the particles are crossing the FOV in a time shorter than 5 seconds, then the speed of the diluent becomes the dominant motion measured. To the software, this would be interpreted as the particles moving faster than their inherent Brownian motion, which means that the particles in the sample will be undersized. To prove this point, 100 nm PLS beads were tested at different flow rates to observe the effect on size with increasing sample speed.
With NanoSight syringe pump speeds ranging from 0 (static) to 50 AU, the measured particle size is within the range stated on the certificate (102 nm +/- 3 nm), as well as statistically the same (Figure 2). Once speeds reach above 50 AU (i.e. particles are crossing the FOV in less than the ideal approximately 10 seconds), the measured size drops below the certified range.
Figure 2. Size (nm) versus Flow Rate (AU) on 100 nm PSL standard (certified 102 nm +/-3 nm). Each point consists of 5 x 60s videos.
This experiment shows how the maximum recommended flow rates for each top plate type were derived. The following pump speeds are a general guide to recommended flow rate settings for the different sample chamber setups across NanoSight models:
Most likely, there will be some difference in the pump speed setting depending on the sample type and size range to be measured. As always, each user should test and verify the appropriate syringe pump setting with their specific instrument configuration and sample type for best repeatability. Additionally, it is recommended that, in the literature, users report the syringe pump speed and syringe type that was used for data collection on their samples to help ensure good reproducibility of results on similar samples.
The NTA concentration (with the concentration measurement upgrade2) provided on the 100 nm PSL standard shows good precision and accuracy at flow rates from 0-60 AU (Figure 3). Above 60 AU, the concentration drops as the sample is moving too quickly for the software to track some of the particles for the required number of video frames, resulting in a decrease in accuracy.
Figure 3. NTA concentration data on 100 nm PSL beads of a known concentration (1.7x108 particles/mL +/-10%) WITH the patented concentration upgrade.
The syringe pump can be easily installed on any NanoSight system and controlled by the NTA software. The units for the flow rate are arbitrary units (AU) and vary depending on the type of syringe used, laser module type (LM12 or LM14) and top plate types (i.e. flow channel, low volume flow channel and O-ring). Overall, the working flow rate of the syringe pump with the NTA software is about 4 µL/min., meaning less sample is used during an experiment compared to advancing the sample by hand between videos (‘static’ capture).
The direction the sample flows will depend on the type of laser module that is used (Figure 4). Either setup will work, as the drift correction (nm/s) in the x and y directions in the NTA software corrects for any constant, directional flow while leaving the Brownian motion of the particle to be analyzed. This means that the motion of the particle (and its size) will still be due to the Brownian motion of the particles in the sample, not to the flow rate of the sample.
Figure 4. a) Sample flows in vertical direction when syringe pump is used with an LM12 module. b) With an LM14 module the sample flows in the horizontal direction.
NTA size and concentration measurements were examined on a 100 nm PSL standard of a known concentration while capturing data by setting the NanoSight syringe pump at different rates of speed. The use of the syringe pump improves precision and repeatability of concentration measurements either with or without the concentration upgrade. By staying within the recommended syringe pump rates for the flow channel top plate (20-50 AU for the NS500 flow channel top plate and/or approximately 10 seconds for a particle to cross the FOV), collecting multiple videos from 30-60s and implementing the concentration upgrade, precision, accuracy and repeatability on both size and concentration can be obtained.
