Nanoparticle Tracking Analysis vs Tunable Resistive Pulse Sensing

Precise nanoparticle measurement is being used more and more widely in the scientific community. However, the techniques available for accurate and reliable nanoparticle measurement vary greatly. Nanoparticle tracking analysis (NTA) and Tunable resistive pulse sensing (TRPS) are both well-known techniques for nanoparticle analysis that use very different techniques. In order to ensure scientific accuracy of future investigations, these techniques need to be able to perform multimodal sample measurement, particle size and concentration analysis in a reliable and repeatable fashion.
how nanoparticle tracking analysis (NTA) works
Figure 1.
Diagrammatic representation of the methods underlying nanoparticle tracking analysis and tunable resistive pulse sensing. Nanoparticle tracking analysis monitors the light scattered by particles in a sample from an applied beam.
baseline TRPS reading
Figure 2.
Diagrammatic representation of the methods underlying nanoparticle tracking analysis and tunable resistive pulse sensing. Tunable resistive pulse sensing characterises every particle that passes through a nanopore by analysing the blockade of a current applied across the pore.
NTA tracks the Brownian motion of individual particles within a sample by monitoring the scattering of applied light  (Figure 1). The diffusion constant is related to particle size by the Stokes-Einstein equation. Application of this theory can be used to calculate the hydrodynamic diameter of the particles. Particle concentration is calculated from the number of particles that are tracked in an estimated illumination volume.

TRPS monitors current flow through a tunable nanopore. Particles crossing the pore cause transient changes in the flow of an ionic current, the magnitude of which are proportional to particle size (Figure 2). Particle size is determined for individual particles. Particle concentration is precisely 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.

Applications and suitable samples

Currently NTA is often used to characterise nanoparticles with diameters of 30–600 nm (although the upper and lower limits of detection are sample dependent). However, resolution and accuracy are low for particles with diameters <250 nm as multi-Gaussian fittings are often not obvious, meaning that the size range of NTA is limited. In comparison, TRPS enables highly precise and accurate measurement of particles from 40 nm to 20 µm in diameter, with high resolution measurements possible for particles even at the smallest end of the scale.   
Particles such as those composed of proteins, polymers, and metal as well as micelles, extracellular vesicles, viruses, virus-like particles, proteins, and biological cells—including previously uncharacterised particles with unknown properties—can be accurately characterised.

NTA is often not suitable for early research or high-value samples due to the fairly large sample volume (600 µl). TRPS requires only a small sample volume (35 µl), which can be recovered for further analyses, making this technique appropriate for various samples.
Figure 3.
Results of nanoparticle tracking analysis and tunable resistive pulse sensing characterisation of a quadrimodal sample (CPN100/CPN150/CPN200/CPN240 at a ratio of 1/1/1/1 with a total concentration of 1010/ml). Tunable resistive pulse sensing offers high resolution of the four subpopulations, with baseline separation of data sets. Nanoparticle tracking analysis shows some resolution of subpopulations, but clear separation is not achieved.

Heterogenous and multimodal samples

Both NTA and TRPS are single-particle techniques, meaning that in theory the resolution of subpopulations within multimodal samples is possible with both techniques. However, recent comparison studies show that NTA can only adequately resolve subpopulations within bimodal samples when the mean diameters are sufficiently different. Furthermore, the resolution of tri- or quadrimodal samples is very limited by NTA and the total concentration may be overestimated in multimodal samples. TRPS offers accurate measurement of both size and concentration of all particles in multimodal samples, even when the mean diameters of subpopulations are similar. Resolution of up to four subpopulations has been shown to be very high for TRPS, and resolution of up to six subpopulations may be possible at one single pore setting.

Particle size and concentration analysis

TRPS accurately measures the actual diameter of every particle that passes through the pore to ensure that a true size distribution is reported. Concentration analysis is also highly accurate for each size band and for various particle types as well as multimodal mixtures. NTA only offers accuracy of concentration for standard particles in simple, monomodal preparations. Although the technique measures the hydrodynamic diameter of particles (which may not be a true representation of particle size), this has been shown to accurately reflect the known diameters of polystyrene beads.
Figure 4.
The Exoid is capable of measuring heterogenous samples over a wide size distribution, without the need for multiple measurements or adjustment of settings. Subpopulations can be identified with high resolution and accurately quantified individually.

Repeatability and reliability

The data obtained by TRPS are highly repeatable and reproducible. Semi-automation of the latest TRPS system along with automated pre-analytical processing further increase the reliability of measurements. Automated optimisation of instrument settings ensure that errors will not influence measurements, and knowledge of the optical properties of the particles is not required, reducing the potential of introducing inaccuracies through user error. In contrast, measured concentrations and size distributions vary between different NTA platforms, meaning that the repeatability and reproducibility of NTA is low and measurements may be influenced by instrument settings. Furthermore, NTA instrument settings must be carefully selected according to the size and optical properties of the particles that are being measured, introducing the potential for user error.
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Comparison table

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Single-particle technique
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Single-particle technique
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Wide size range (40 nm–20 µm)
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Smaller size range (30–600 nm, sample dependent)
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Semi-automated operation, user-friendly system, simple protocols
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Simple protocols but advanced data analysis required
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Single-particle resolution
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High resolution possible with suitable samples
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Measures actual diameter
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Measures hydrodynamic diameter
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Can resolve populations in multimodal samples
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Adequate resolution of bimodal samples. Resolution of multimodal samples is limited
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No knowledge of particle properties required
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Accurate knowledge of optical properties of particles and dispersant is required
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Small sample volume (35 µl)
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Large sample volume (600 µl)
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Measurements do not rely on user inputs or instrument settings
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Measurements depend on instrument settings and user input
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Introducing the Exoid

The Exoid is the next generation of TRPS device. The Exoid has the proven quality TRPS technology developed with the previous generation, qNano, but significantly improves the user experience making TRPS measurement easier than ever before.
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