Dynamic Light Scattering 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. Dynamic light scattering (DLS) 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.
Dynamic light scattering or DLS
Figure 1.
Diagrammatic representation of the methods underlying dynamic light scattering and tunable resistive pulse sensing. Dynamic light scattering monitors the light scattered by particles in a sample from an applied laser beam in order to calculate particle concentration and hydrodynamic radius.
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 blockage of a current applied across the pore.
Dynamic light scattering (DLS) involves monitoring fluctuations in the scattering intensity of a laser beam passing through the sample, this occurs due to the Brownian motion of the particles (Figure 1). Using this information, the average hydrodynamic diameter of particles in the sample can be calculated by applying the scattering autocorrelation function. A variation on DLS, multi-angle DLS (MADLS) determines the particle size distribution by analysing multiple scattering autocorrelation functions, usually recorded at three angles. For both DLS total particle concentration is calculated from the photon count rates. The intensity-weighted particle size distribution is then transformed into particle concentration distribution.

Tuneable resistive pulse sensing (TRPS) monitors current flow through a tuneable 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.

Applications and suitable samples

DLS and 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 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 TRPS enables highly precise and accurate measurement of particles from 40 nm to 20 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, and metal as well as 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 DLS. DLS cannot resolve multimodal samples when the mean diameters are similar or particle size distributions are broad. MALDS 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.  

The single-particle nature of TRPS measurements enables characterisation of every particle in the sample without changes to the measurement method. This makes TRPS suitable and reliable 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.  
Figure 3.
Illustration of particles passing through a nanopore.
Figure 4.
Results of dynamic light scattering 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). Dynamic light scattering shows no clear separation of subpopulations, instead reporting an averaged size distribution. For the purpose of comparison with a continuous ensemble technique, the tunable resistive pulse sensing 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 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.  

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.

Comparison table

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Single-particle technique
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Ensemble technique
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Wide size range (40 nm–20 µm)
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Smaller size range (1 nm –3 µm)
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Semi-automated operation, user-friendly system, simple protocols
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Simple protocols
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Single-particle resolution
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Limited resolution
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Number-weighted analysis
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Intensity-weighted analysis
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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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Cannot resolve multimodal samples
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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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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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