Tunable Resistive Pulse Sensing (TRPS) vs Nanoparticle Tracking Analysis (NTA)

Tunable Resistive Pulse Sensing (TRPS) and Nanoparticle Tracking Analysis (NTA) are two techniques based on very different principles. Both are used to measure the physical characteristics of particles, as is required by many growing fields across biotherapeutics, nanomedicine, virology, and extracellular vesicles. While all of these disciplines call for the analysis of samples containing heterogenous particles, the techniques available for nanoparticle measurement vary greatly in their precision, accuracy and resolution capabilities.

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Differences in resolution capabilities between TRPS (top) and NTA (bottom). Particle size distribution of a quadrimodal mixture comprised of polystyrene standards (1:1:1:1 mix of CPN100, CPN150, CPN200, and CPN240). Vogel et al. 2021.

Differences in Resolution Capabilities

The extent to which a technique can be used to identify difference between sample characteristics is limited in part by its resolution capabilities. Although both Tunable Resistive Pulse Sensing (TRPS) and Nanoparticle Tracking Analysis (NTA) measurements are based on the analysis of individual particles, major differences in their resolution capabilities have been revealed in systematic comparative studies (Vogel et al. 2021, Caputo et al. 2021).

TRPS analysis of a quadrimodal sample, for example, reveals four clearly distinguishable peaks, representing the four subpopulations known to be present.

When using NTA to analyse quadrimodal samples of polystyrene particles, however, the peaks showing particle size distribution are wide and overlapping. The lower resolution makes it difficult to identify all subpopulations – and subsequently, obtain precise information about particle size distribution in the sample.

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How Does TRPS Measure Nanoparticles?

Tunable Resistive Pulse Sensing (TRPS) is a true single-particle technique which uses changes in electrical current to evaluate individual particles suspended in an electrolyte solution as they move between electrodes through a tunable nanopore. Current flow is monitored, and each particle is detected as it passes through the nanopore, due to the transient change in current that is subsequently created.

Physical characteristics of particles in the sample are obtained via the analysis of resulting blockades, which represent individual particles. Specifically, particle volume is directly proportional to blockade magnitude, and particle concentration is precisely calculated from the particle flow rate measured at several different applied pressures. Zeta potential is derived via measurements of electrophoretic mobility, which is calculated from the velocity with which the particle traverses the nanopore.

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Key features of tunable resistive pulse sensing (TRPS) showing nanoparticles passing through a nanopore (top left), signal trace with resistive pulses (top right), high-resolution particle size distribution (bottom left), and a breakdown of how each individual blockade is analysed (bottom right).

how nanoparticle tracking analysis (NTA) works

Conceptual diagram of nanoparticle tracking analysis (NTA). A laser beam is applied to particles in solution. Successive optical video images are used to track the movement of individual particles and determine a particle diffusion coefficient, which is used in the Stokes-Einstein calculation of hydrodynamic radius.

How Does NTA Measure Nanoparticles?

In contrast with the true single-particle nature of Tunable Resistive Pulse Sensing (TRPS), Nanoparticle Tracking Analysis (NTA) uses the tracking of particle motion via light scattering to assess the mean squared displacement of particles moving under Brownian motion, in a sample chamber illuminated by a laser beam. The tracking of particles enables a diffusion constant to be calculated, which is used in the Stokes-Einstein equation to calculate hydrodynamic diameters. The Stokes-Einstein equation also takes into account the temperature and viscosity of the suspension. Particle concentration calculations are based on the number of particles tracked in an estimated illumination volume.

Figure 2.

In tunable resistive pulse sensing (TRPS), nanoparticles are driven one-by-one through the nanopore by applying a combination of pressure and voltage.

Why Choose TRPS Over NTA?

Tunable Resistive Pulse Sensing (TRPS) is a single-particle measurement technique that generates high-resolution data on the physical characteristics of nanoparticles. Given the direct relationships that exist between blockade characteristics and physical properties of nanoparticles, TRPS enables true particle-by-particle analysis. The resulting high-resolution measurements enable you to gain deep insights on particle characteristics. Different sized nanopores are available to facilitate a range of particle sizes, from 40 nm to 11 µm in diameter, and only 35 μL of sample is needed for a TRPS measurement. With TRPS, the use of standardised calibration particles and ability to monitor the signal-to-noise ratio in real time ensures that conditions are optimised and maintained throughout the calibration and measurement process.

In contrast, the resolution capabilities of Nanoparticle Tracking Analysis (NTA) are relatively lower; NTA struggles to properly resolve subpopulations of multimodal samples and has been shown to overestimate particle concentration, making it more difficult to compare samples with confidence.

NTA measurements have also been shown to be very sensitive to and dependent on instrument settings, such as camera sensitivity, and also require knowledge of the optical properties of the particles being analysed. At the lower limit of particle detection, the refractive indices of the solvent and particles play a role in determining the detectable size limit. At times, these parameters must be estimated, which creates potential sources of error

Comparing TRPS to NTA

Tunable Resistive Pulse Sensing (TRPS)

Nanoparticle Tracking Analysis (NTA)

Approach to size measurement

Particle size is directly proportional to blockade magnitude; each particle is measured individually.

Brownian motion is tracked for individual particles and the resulting diffusion coefficient is used to calculate hydrodynamic diameter.

Size range capabilities

Wide size range; particles from 40 nm - 11 µm can be measured using appropriately sized nanopores.

Smaller size range (approximately 10 – 2000 nm).

Limitations of size analysis

Different sized nanopores are needed to cover the entire size range.

Size measurement limited by uncertainty of Brownian motion calculation, subject to sensitivity limitations. Difficult to set settings sensitive enough to cover particle size range.

Resolution capabilities

Can resolve subpopulations in multimodal samples.

Resolution of multimodal samples is relatively limited.

Concentration

Calculated directly from blockade frequency, using standardised calibration particles of known size and concentration.

Determined from the number of particles tracked in an estimated illumination volume. NTA generally overestimates particle concentration (Vestad et al. 2017, Barchurski et al. 2019, Vogel et al. 2021).

Ease of data processing

Automated data processing with user-friendly data visualisation interface.

Advanced data analysis required.

Influence of instrument parameters/particle properties

Parameters are actively monitored to ensure they are optimised for the measurement of calibration and sample particles.

Measurements and sensitivity are highly dependent on instrument settings. Depends on accurate knowledge of particle properties and dispersant.