Nanoparticle Tracking Analysis vs Tunable Resistive Pulse Sensing

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 nanoparticle tracking analysis (NTA) are two well-known techniques that are based on very different principles.
how nanoparticle tracking analysis (NTA) works
Figure 1.
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.
nanopore graphic
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.

Principles of NTA and TRPS

NTA works by tracking 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. (Figure 1). 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.  

TRPS is a true single-particle technique which uses changes in electrical current to evaluate individual particles as they move between electrodes (Figure 2). As each particle crosses through the nanopore, a transient change in ionic current is created. The magnitude of the resulting blockade is directly proportional to each individual particle volume (Figure 3). Particle concentration is precisely calculated from the particle flow rate measured at several different applied pressures. Electrophoretic mobility is calculated from the speed at which the particle traverses the pore and then zeta potential is derived from the electrophoretic mobility.

Strengths and limitations

As is the case for all analytical techniques, each have their own challenges and limitations which are important to consider when selecting a method for your application. Although NTA is straightforward to use, it struggles to properly resolve subpopulations of multimodal samples. At the lower limit of particle detection, the refractive indices of the solvent and particles play a role in determining the detectable size limit. For smaller particles, NTA requires knowledge of the optical properties of the particles being analysed. Sometimes, however, these parameters must be estimated, creating a potential source of error. Despite suboptimal resolution, NTA is commonly used to characterise particles of 30–600 nm in diameter. Unlike TRPS, NTA measurements are highly dependent on the selection of measurement parameters, such as camera settings and detection threshold.
In comparison, TRPS provides highly precise and accurate measurements with a much higher level of resolution. TRPS is not based on optical properties; instead, it requires the use of a conductive solvent. Different sized nanopores are available to facilitate a range of particle sizes, from 40 nm to >11 µm in diameter. Strategies are available to ensure conditions are correct for the analysis of the sample and standardised calibration particles. As a certain level of understanding is required to apply these strategies, a learning curve is needed for TRPS use – particularly when using the smallest nanopores. The volume of sample required for each method differs; 600 μl for NTA, compared to 35 μl for TRPS.
how TRPS works
Figure 3.
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).

Applications

TRPS and NTA are used to analyse the size, concentration and zeta potential of nanoparticles across a range of disciplines and share some common applications. Common TRPS applications include the analysis of extracellular vesicles, viruses and virus-like particles, monoclonal antibodies and other therapeutic proteins. The precision and high resolution of TRPS lends itself to applications where precision is critical, such as in nanomedicine developments. TRPS has been used for particle aggregation studies of nucleic acids, and for the monitoring of aptamer-protein interactions. TRPS can be used to analyse particles that can be dispersed in solution and are within the suitable size detection range. Despite providing a lower level of resolution, NTA is also used to analyse nanobiological particles and is used more widely in other industries such as in environmental analysis, and the development of ink and coatings.

Differences in resolution capabilities

Although both NTA and TRPS measurements are based on the analysis of individual particles, major differences in their resolution capabilities have been revealed in systematic comparative studies. When using NTA to analyse quadrimodal samples of polystyrene particles, for example, the peaks showing particle size distribution are wide and overlapping. This makes it difficult to identify all subpopulations (quadrimodal sample shown in Figure 4). In comparison, TRPS analysis of the same sample reveals four clearly distinguishable peaks, indicative of the four subpopulations known to be present. While a high level of resolution has been demonstrated for samples containing four subpopulations, it may be possible to resolve up to six subpopulations using one single pore setting.
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NTA TRPS multimodal data graph
Figure 4.
NTA (top) and TRPS (bottom) particle size distribution of a quadrimodal sample, comprised of CPN100, CPN150, CPN200, CPN240 at a ratio of 1:1:1:1 with a total concentration of 10^10 particles/mL). TRPS offers high resolution of the four subpopulations, with baseline separation of data sets. NTA shows some resolution of subpopulations, but clear separation is not achieved.

Repeatability and reliability

In addition to differences in resolution capabilities, comparative studies have identified greater variability in concentration measurements for NTA, compared to TRPS. The reproducibility and reliability of TRPS data is ensured through the use of standardised calibration particles, single-particle nature of TRPS, and the ability to optimise and maintain conditions for calibration and measurement. TRPS is further strengthened by advances of the latest TRPS system, which include in-built automation of instrument settings and a subsequent reduction in error. Unlike other techniques, TRPS measurements do not rely on prior knowledge of particle optical properties.
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Comparing TRPS to NTA

TRPS
NTA
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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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Automated data processing with user-friendly data visualisation interface
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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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Active monitoring of parameters ensures precise measurements
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Measurements depend on instrument settings and user input

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