In the realm of nanoparticle characterisation, the Exoid has always won the battle of resolution. Tunable Resistive Pulse Sensing is a powerful tool for those wishing to measure the size, concentration and zeta potential of extracellular vesicles, viruses and various particle types used in the field of nanomedicine. Not always an easy tool to use, we’ll grant you, but a powerful one. We’re working on the ‘making it easier’ part, starting off with a terrific little tweak to make it easier to measure smaller particles on the lower edge of a nanopore’s measurement capabilities. This will not only make the data more accurate, but will also make life easier by potentially allowing you to measure the full width of your particle population on a bigger nanopore size. That means less blocking of the nanopore and an easier run.
We have been working on this for a while, but we wanted you to have the confidence in the Exoid Control Suite update that we do. So we undertook a little research study of our own, putting the new update’s capabilities in a way that was a bit more relatable than all of the tedious development data.
How was the Exoid software update assessed?
We tested the updated software setting using the NP150, as well as CPC100s and CPC70s, which are polystyrene particles with nominal sizes (i.e., typically they are this size but they vary by batch) of 100 nm and 70 nm, respectively. These particles were chosen because they represent two levels of difficulty on an NP150. The CPC100s are generally well resolved, but likely have a small proportion of very small particles at the lower extreme of its size distribution which are not fully resolved. The size distribution of CPC70s are at or below the extremes of the NP150’s measurement range, making them very difficult to resolve.
The benefit of using CPCs is that we know the precise size of each batch of CPCs (in this case, 100 nm for the CPC100s and 61 nm for the CPC70s), meaning that we can compare the known particle size with the size measured by the Exoid. To make this comparison, we took paired recordings of the same lot of CPCs using the NP150 nanopore (i.e., the same nanopore for each paired measurement), and made these measurements using both the original and the updated Exoid software.
As is standard practice for Exoid measurements, we completed a calibration set of measurements at three pressures (using CPC200s) and then measured the CPC100s or CPC70s at the same three pressures. These measurements were made using 3 nanopores (NP150), generating an n = 12 recordings for each condition. Data are presented as mean ± standard error, and paired comparisons were calculated using the Wilcoxon Signed Rank Test.
The Exoid update improves the detection of particles towards the lower measurement capabilities of the nanopore
The particle rate tells you how many particles are being detected in your sample each minute, and is used to inform the concentration measurement of your sample. We found that the particle rate of CPC70s was significantly higher in the updated software, compared to the old software (16.3-fold increase; p<0.01, Figure 1). The CPC100 particle rate had a small but consistent trend towards an increased rate with the updated software, but this did not reach statistical significance. This may be expected due to the vast majority of CPC100s already being well resolved with the original software.
From this data, we could see that additional particles were being detected, particularly for CPC70 samples. But which particles?
Logically, this increase in rate for CPC70s as compared to CPC100s would suggest that this improved detection is for smaller particles. Unsurprisingly then, the new software update enabled the detection of smaller particles within CPC70 samples (Figure 2a). The smallest detected particles were an average of 8.83 nm smaller in the update as compared to the original software (p< 0.01). This led us to ask how many smaller particles were being detected? Of all the particles detected in each recording, the updated software detected a larger percentage of CPC70 particles smaller than 100 nm (p<0.001, Figure 2b), 90 nm (p<0.001) and 80 nm (p<0.01). There was even a trend towards increased detection of particles below 70 nm, which is below the stated limit of detection for the NP150.
The Exoid update reduces population skew
If more smaller particles are being detected, then the theoretically normally distributed monomodal CPC samples should show very little difference between their mode and mean. In other words, their populations should be less skewed due to a reduction in clipping at the smaller size range. Figure 3 shows that for both CPC70s (p<0.05) and CPC100s (p<0.05), the skew is smaller with the updated Exoid software. This higher magnitude of impact on the CPC70 recordings makes sense, as the skew in this population was already higher.
Skew and the truncation which causes it can be visualised when looking at the size histograms. To get the best visualisation of this we will look at a comparison at the highest pressure: pressure 3 in standard measurements. This is because the higher the pressure, the more likely you are to get clipping of the lower end of the size distribution. The example shown in Figure 4 shows this in action, illustrating the benefit of the Exoid update. Clearly, this will impact on the sizing accuracy of the measured particles, but to what extent?
Increased sizing accuracy with the updated Exoid software
As mentioned above, the reason for using CPCs for these experiments is that we know their mean size already. This varies slightly for every batch of CPCs, but the mean size of our CPC100s was 100 nm and for our CPC70s it was 61 nm. To determine the accuracy of the measured mean, we calculated the difference between the known and measured mean for each condition. As you can see in Figure 4, the accuracy of measurement for CPC100s – and, therefore, particles of that size – was very accurate already, being less than 3 nm off the known size with both Exoid software versions. An impressive feat!
When it came to measuring CPC70s with a mean size of 61 nm – i.e., outside of the known measurement capabilities of the NP150 – we saw, as expected, less accuracy in measurement. Despite that though, we were pleased to see the updated Exoid software did improve the sizing accuracy, with the measured particle size being 10 nm closer to the known mean size (p<0.05, Figure 5).
Whilst a population with a mean size of 61 nm (as was the case for these CPC70s) is outside of the measurement capabilities of the nanopore, the software update clearly improved their resolution. We still wouldn’t recommend that you measure CPC70s or any particle whose mean size is at or beyond the measurement capabilities of the nanopore. But it does mean that if part of your polydisperse sample lies towards this range, you can be more confident in their sizing. The reason for this increase in sizing accuracy likely comes from the increased detection of small particles towards and beyond the expected measurement capabilities of the NP150.
Discussion and conclusions
In conclusion, the latest update to the Exoid Control Suite represents a significant leap forward in the field of nanoparticle characterization, particularly for those at the lower limit of the size spectrum. By enhancing the detection of smaller particles, this update not only increases the particle rate, providing a more comprehensive understanding of nanoparticle populations, but it also significantly reduces population skew, ensuring that the data collected is a more accurate reflection of the sample being analysed. Furthermore, the increased sizing accuracy, especially notable in particles previously considered beyond the reliable measurement range, underscores the Exoid's enhanced capability to provide precise and reliable data, even at the extremities of its operational parameters. This breakthrough is particularly impactful for researchers and professionals working with nanoparticles whose size distribution takes them to the cusp of the Exoid's detection limit, as can be the case when working with extracellular vesicles, viruses, and nanomedicines, where accurate sizing is crucial for understanding biological processes or ensuring the safety and efficacy of therapeutic agents.