Tunable Resistive Pulse Sensing vs Dynamic Light Scattering

Tunable Resistive Pulse Sensing (TRPS) and Dynamic Light Scattering (DLS) are two particle measurement techniques based on very different principles. Both TRPS and DLS are used to varying degrees to assess the physical parameters and polydispersity of particle populations across many disciplines, including the nanomedicine/pharmaceutical industry (e.g. lipid nanoparticles and liposomes), virology, and the study of exosomes and other extracellular vesicles. Compared to TRPS, DLS is a relatively crude technique when it comes to resolving the size of heterogenous particles. This is problematic for the above applications, where such limitations prevent meaningful comparisons of different samples.

Figure 1.

Comparison of Tunable Resistive Pulse Sensing (TRPS) vs Dynamic Light Scattering (DLS) characterisation of a quadrimodal sample of polystyrene particles (CPN100/CPN150/CPN200/CPN240, ratio1:1:1:1, total concentration 10^10/mL). To enable a comparison with a continuous ensemble technique, the TRPS histogram was transformed into an equivalent continuous curve by dividing histogram data by bin size.

How Does Tunable Resistive Pulse Sensing Measure Nanoparticles?

Tunable Resistive Pulse Sensing (TRPS) monitors current flow through a tunable nanopore. Particles crossing the nanopore cause transient changes in the flow of an ionic current, which can be detected and analysed via the resulting blockades. Each particle size is determined for individual particles; blockade magnitude is proportional to particle size, and particle concentration is calculated from the particle flow rate measured at several different applied pressures. Zeta potential (a measure of the effective surface charge) is derived by measuring electrophoretic mobility, which is calculated based on the speed at which the particle traverses the nanopore.

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How Does Dynamic Light Scattering Measure Nanoparticles?

Dynamic Light Scattering (DLS) involves applying a laser beam to the sample and monitoring fluctuations in the scattering intensity which results from the Brownian motion of the particles. The magnitude of the scattered intensity is a function of several parameters including particle size; therefore, by applying a scattering autocorrelation function and several assumptions, the average hydrodynamic diameter of particles in the sample can be calculated. Particle concentration cannot be measured using DLS.

Multi-Angle Dynamic Light Scattering (MADLS) is a variation of DLS which combines scattering information from multiple angles to deliver particle size distribution at a higher resolution than single-angle DLS. MADLS size measurements are obtained by analysing multiple scattering autocorrelation functions, usually recorded at three angles. The resulting intensity-weighted particle size distribution is then transformed into particle concentration distribution, using an equation which also takes into account other various factors including: the derived photon count rate from particle scattering, the derived photon count rate from a reference liquid, the instrument’s detection efficiency, and the optical properties of the particles and dispersant.

Figure 3.

Conceptual diagram of Dynamic Light Scattering (DLS), an ensemble technique. A laser beam is applied to particles in solution. The intensity of light scattered by particles is used to calculate an intensity-weighted mean hydrodynamic radius.

Figure 4.

An illustration of particles passing through a nanopore during a Tunable Resistive Pulse Sensing (TRPS) measurement.

Why Choose TRPS Over DLS?

Tunable Resistive Pulse Sensing (TRPS) enables high-resolution measurement of particles from 40nm to >11 µm in diameter. Unlike Dynamic Light Scattering (DLS), TRPS measurement of particles is independent of the particle or dispersant optical properties, meaning that you can confidently measure unknown samples and those with heterogenous optical densities.
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The most important difference between the two techniques is that TRPS measures single particles, whereas DLS follows an ensemble approach. As TRPS measures every particle that passes through the nanopore, it gives a true size distribution and measures concentration. As such, TRPS can identify small changes in size or zeta potential, allowing the identification of different subpopulations in a heterogeneous sample.

Whilst TRPS can resolve at least four subpopulations, the ensemble approach of DLS limits subpopulation identification. A recent study has shown that, at least in the researchers hands, Multi-Angle Dynamic Light Scattering (MADLS) fails to identify subpopulations within quadrimodal samples.

Furthermore, with DLS, larger particles be overestimated, distorting the particle distribution and obscuring smaller particles due to the sextic dependence of light-scattering intensity. Finally, whilst TRPS measures actual diameter, DLS can only measure hydrodynamic radius. Whilst this is not inherently inferior in many circumstances, the actual diameter may be favoured by some regulatory bodies.

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Implications for Different Applications

Given its ability to resolve samples to a high level of resolution, Tunable Resistive Pulse Sensing (TRPS) lends itself to applications where accuracy, precision and reproducibility are particularly important.

TRPS is used across a range of fields within biological and pharmaceutical research and development, including in the measurement of exosomes and other extracellular vesicles, monoclonal antibodies, lipid nanoparticles, virus-like particle vaccines, and virus preparations.

Light-scattering techniques like Dynamic Light Scattering (DLS) offer simple approaches to obtaining bulk estimates of the size and concentration of particles in solution. DLS and Multi-angle Dynamic Light Scattering (MADLS) are currently the most frequently used techniques for measuring particle size distribution in the submicron and nanometre range, respectively. However, the current frequency of use is not representative of the technique’s accuracy. Whilst DLS and MADLS are thought to be suitable for characterising particles from 1 nm to 3 µm in diameter in a monodisperse sample, the resolution of DLS is low for particles with diameters of <150 nm as the angular dependence of the light-scattering profile is low. For DLS, accurate knowledge of the optical properties of both particles and dispersant is required; therefore, unknown samples cannot be analysed in a sensible way. This is particularly important when it comes to applications such as extracellular vesicles which may vary in optical density, and in nanomedicine development where the optical density of a nanoparticle, or even a new formulation of the nanoparticle, might be unknown.