Tunable Resistive Pulse Sensing vs Dynamic Light Scattering

Precise nanoparticle measurement is required by many growing fields, such as nanomedicine, virology, and the study of extracellular vesicles. All of these disciplines call for the analysis of multimodal samples, i.e., samples containing particles of different sizes. Although measurement reproducibility is critical, the techniques available for nanoparticle measurement vary greatly in their accuracy and resolution capabilities. Tunable Resistive Pulse Sensing (TRPS) and Dynamic Light Scattering (DLS) are two well-known techniques that are based on very different principles.

Figure 1.

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 particle concentration and intensity-weighted mean hydrodynamic radius.

Figure 2.

Conceptual diagram of Tunable Resistive Pulse Sensing (TRPS), a single-particle technique. A current is applied across a tunable nanopore. As each particle passes through the nanopore, a resistive pulse or ‘blockade’ is created. Size, concentration, and zeta potential are derived by analysing blockade magnitude, frequency, and duration, respectively.

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 (Figure 1). By applying a scattering autocorrelation function, the average hydrodynamic diameter of particles in the sample can be calculated. Multi-angle Dynamic Light Scattering (MADLS) is a variation of DLS which assesses particle size distribution by analysing multiple scattering autocorrelation functions, usually recorded at three angles. For both DLS methods, total particle concentration is calculated from the photon count rates. The intensity-weighted particle size distribution is then transformed into particle concentration distribution.

Tunable Resistive Pulse Sensing (TRPS) monitors current flow through a tunable nanopore (Figure 2). Particles crossing the pore cause transient changes in the flow of an ionic current, the magnitude of which are proportional to particle size. Particle size is determined for individual particles. Particle concentration is calculated from the particle flow rate measured at several different applied pressures. Electrophoretic mobility and surface charge are calculated from the speed at which the particle traverses the pore.

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Applications and Suitable Samples

Dynamic Light Scattering (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. DLS and MADLS are thought to be suitable for characterising particles from 1 nm to 3 µm in diameter in a monodisperse sample. Also 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.

In comparison, Tunable Resistive Pulse Sensing (TRPS) enables highly precise and accurate measurement of particles from 40 nm to >11 µm in diameter. High resolution measurements are possible for particles even at the smallest end of the scale. TRPS measurements are not influenced by optical properties and no knowledge of the particles properties is required prior to analysis. This means that previously uncharacterised particles, including those composed of proteins, polymers, micelles, extracellular vesicles, viruses, virus-like particles, proteins, and biological cells, can be accurately measured with TRPS.

Measurement of Mixed or Multimodal Samples

Subpopulations in multimodal samples cannot be resolved by Dynamic Light Scattering (DLS). DLS cannot resolve multimodal samples when the mean diameters are similar or when particle size distributions are broad. Multi-angle Dynamic Light Scattering (MADLS) claims to offer increased resolution as particles that scatter weakly at one detection angle may be revealed using other detection angles. Recent studies have found MADLS unable to resolve multimodal sample subpopulations. Another limitation is that larger particles can distort the particle size distribution by obscuring smaller particles.

The single-particle nature of Tunable Resistive Pulse Sensing (TRPS) measurements enables characterisation of every particle in the sample. This makes TRPS suitable for measuring multimodal samples with subpopulations of particles, even those with similar mean diameters. TRPS's ability to resolve of up to four subpopulations has been shown to be very high, and resolution of up to six subpopulations may be possible with one single pore setting.

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Figure 3.

Illustration of particles passing through a nanopore during TRPS measurement.

Figure 4.

Results of DLS and tuneable resistive pulse sensing characterisation of a quadrimodal sample (CPN100/CPN150/CPN200/CPN240 at a ratio of 25/25/25/25 with a total concentration of 1010/ml). DLS shows no clear separation of subpopulations, instead reporting an averaged size distribution. For the purpose of comparison with a continuous ensemble technique, the TRPS histogram was transformed in an equivalent continuous curve by dividing histogram data by bin size. Tuneable resistive pulse sensing offers high resolution of the four subpopulations, with baseline separation of data sets.

Particle Size and Concentration Analysis

Light-scattering techniques like Dynamic Light Scattering (DLS) offer simple approaches to obtaining bulk estimates of the size and concentration of particles in solution. However, the concentration of large particles can be significantly overestimated due to the sextic dependence of light-scattering intensity, skewing the particle size distribution. Although MADLS aims to improve on the DLS measurement any inaccuracies in initial particle size determination will be propagated through the calculation of total particle concentration, resulting in inaccurate determination of this parameter as well. Furthermore, DLS only measures the hydrodynamic diameter of particles and as a result may not be an accurate representation of particle diameter.

Tunable Resistive Pulse Sensing (TRPS) measures every particle that passes through the pore to ensure that a true size distribution is reported. Concentration analysis is also based on single-particle measurements, ensuring highly accurate calculations for each size band. Particle size and concentration are determined separately, meaning that any measurements do not influence other parameters. TRPS individually measures each particle which means that the actual diameter of every particle is reported.

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