Measuring nanoparticle size is fundamental to biotherapeutic development. It is not only helpful for researchers formulating and optimising production, but it is also a key regulatory requirement in nanomedicine.
For a long time, Dynamic Light Scattering (DLS) has been the go-to option. But why? Well, partly because of the seven most expensive words in business: “This is how we’ve always done it”. Oftentimes though, it’s worth considering alternative ways of doing things and the potential benefits they could bring.
Tunable Resistive Pulse Sensing (TRPS) offers an alternative route, and a clearer window into your true particle size distribution. Here, we compare TRPS with DLS, highlighting key principles, strengths and limitations in the context of nanomedicine.
What is Tunable Resistive Pulse Sensing (TRPS) and how does it work?
Tunable Resistive Pulse Sensing (TRPS) is a high-resolution technique for measuring nanoparticles, specifically designed to analyse particles in the size range of 40 nm to 11 µm. Unlike traditional light scattering techniques, TRPS simultaneously measures either the size and zeta potential of individual particles, or individual particle size and concentration.
TRPS technology utilises the Coulter principle on a nanoscale and works by analysing nanoparticles suspended in electrolytes as they pass through a nanopore. Each particle causes a momentary disruption in the electrical current of the pore, forming a blockade. Specific features of the resulting blockades represent different particle characteristics:
- Blockade magnitude is directly proportional to particle size
- Blockade frequency is used to measure particle concentration
- Blockade duration represents zeta potential
TRPS is unique in that it is a true single-particle analytical technique; each particle is measured as it passes through the pore one at a time. Each particle measured is compared to a set of known calibration particles, ensuring the accuracy and repeatability of measurements. Due to the nature of this single-particle approach, TRPS provides a level of insight that is unattainable with DLS, which is a bulk analytical technique that characterises multiple particles at once (Figure 1).
Related read: analytical techniques put to the test using a four-stage, stepwise approach
What is Dynamic Light Scattering (DLS) and how does it work?
Dynamic light scattering (DLS) is an analytical technique used to measure the intensity of light scattered by particles in solution in response to a light directed at the sample. This light scattering intensity is then used to estimate the particle size distribution of colloidal particles. DLS is based on principles of Brownian motion and the interaction of light with matter.
As the name implies, DLS utilises light scattering, a phenomenon which changes dynamically. When light from a laser is directed at the sample, fluctuations in the subsequent scattering of light is measured.
DLS is based on size-dependent particle behaviour; nano-sized particles in solution are not static, therefore light scattering intensity is not constant and fluctuations can be measured. Due to the well-known principle of Brownian motion, particles display a random and rapid ‘zig-zag’ motion as they collide with other rapidly moving atoms or molecules in the sample. The intensity of Brownian motion is affected by particle size (as well as other factors such as temperature) and affects how light is scattered by the particles in solution.
Compared to large particles, small particles will diffuse more quickly in solution, causing more rapid fluctuations in the intensity of light scattering. DLS data is displayed as the distribution of light intensity, because this is what is actually measured. Light intensity of particle scattering, however, it is quite a stretch to go from light scattering to inferences about the physical characteristics of particles in a sample.
Although DLS data can be converted into a volume-based or number-based size distribution using sophisticated data processing techniques, the approach is often not sufficiently sensitive or accurate for this to be particularly insightful. This is because the presence of larger particles skews measurements towards the large side, and DLS struggles to resolve particles if they are too similar in size. Furthermore, it is important to note that volume-based and number-based distributions can tell a very different story (Figure 2).
Deeper insights from TRPS being utilised across the field of nanomedicine
DLS is used widely across many industries, largely for its ease of use. TRPS, however, offers many benefits that are particularly relevant for the nanomedicine industry, and is therefore increasingly being used to measure a range of biological particles. For instance, TRPS has aided the development of lipid nanoparticles encapsulating a small interfering RNA targeted to SARS-CoV-2, anti-cancer liposomes, and virus-like particles.
Jacobs et al. (2022)3 measured particles described as amorphous nanoparticulate drug-delivery systems, to assess the size of particles manufactured using different formulations, screw configurations of the extruder, and process parameters. As expected, in line with the tendency of DLS to overestimate particle size, DLS consistently measured particles to be much larger than was measured with TRPS. For example, in one setting, the mean particle size measured by TRPS was 105 ± 2.4 nm, much lower than the 146 ± 5.37 nm measured with DLS.
TRPS is used widely to measure the physical characteristics of extracellular vesicles (EVs), enabling subtle differences between subpopulations to detected. Such insights – unlikely to be picked up using DLS – enable new perspectives and mechanisms to be unravelled, such as the optimisation of different EV storage conditions4 and EV size/concentration profiling in the context of disease. In a slightly different setting, the Exoid (Izon’s latest TRPS instrument) represents a key part of optimising large-scale EV isolation workflows as part of Izon’s qEV PurePath for Therapeutics.
Key considerations when choosing between TRPS and DLS
Ultimately, the high resolution of TRPS allows researchers to prepare for the increasing regulatory scrutiny in the field of nanomedicine. Choosing one over the other depends on where you stand on a few key parameters:
Trusting in results: DLS experts themselves caution against avoiding blind acceptance of any result, and avoid trusting too heavily in number-based distributions. While it’s good practice to interpret all results with caution, TRPS aims to counter this in many ways. When you learn how to use TRPS, you’ll encounter all the checks and balances that exist to ensure your data is reliable, both during and after measurement. These include the use of calibration particles, taking measurements at multiple pressures, monitoring blockade size, current stability, and RMS noise, and checking particle rates increase with increasing application of pressure.
Labour required and implications on throughput: Compared to DLS, which requires only a low level of expertise5, TRPS inherently requires more time and skill to optimise and maintain parameters for measurement. This is the cost of a high-resolution technique, however – like anything – TRPS analysis can become second nature with a bit of training and practice.
Resolution and sensitivity: TRPS wins hands-down. While DLS can’t distinguish between particles with anything less than with a 3:1 ratio in size difference, and so is much more limited with interpreting polydisperse data (samples containing aggregates). TRPS allows you to catch subtle changes in particle size. Doubling the particle diameter results in an 8-fold increase in resistive pulse magnitude, which is why TRPS is very sensitive to small differences in particle diameter.
Ability to measure concentration and zeta potential: The ability of TRPS to provide information on particle concentration and zeta potential is another strong advantage. This is not possible with DLS.
