Advances in Zeta Potential Analysis Highlighted at ISEV 2021

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How is TRPS used to derive EV characteristics? How has a post-processing protocol created new possibilities for zeta potential analysis? Find out in this wrap-up of the ISEV2021 presentation by Dr. Priscila Dauros-Singorenko.

In mid-May, the EV community welcomed in ISEV 2021, the 10th annual meeting of the International Society for Extracellular Vesicles. As a conference sponsor, Izon Science shared a 10-minute presentation highlighting recent advances in zeta potential analysis and the implications for extracellular vesicle (EV) research. Click here to watch the full talk by Izon’s Priscila Dauros-Singorenko or read on to learn about key points from the presentation.

How TRPS is used to derive EV characteristics

The determination of physicochemical properties such as size, concentration, and charge is critical to advancing EV research.  

Using tunable resistive pulse sensing (TRPS), heterogenous populations can be profiled based on concentration, size, and surface zeta potential (Figure 1).  

Figure 1. Three-dimensional plot of concentration vs particle diameter and zeta potential of a trimodal sample.

In TRPS, the current across a nanopore is constantly measured. When a nanoparticle passes through the nanopore, there is a temporary increase in resistance which presents as a ‘blockade’. Certain aspects of the blockade correspond to particle characteristics:

  • The number of pulses corresponds to particle concentration
  • Pulse magnitudes are proportional to particle size
  • Pulse durations correspond to particle charge

The qNano is the original TRPS instrument, produced by Izon Science. To improve usability and efficiency, Izon launched The Exoid – a more sensitive TRPS instrument with a fully automatic pressure system and automated stretcher unit.

TRPS can not only be used to analyse monodisperse samples (e.g., liposomes or synthetic particles), but it can also resolve multimodal samples with precision – an important consideration for EV research where samples contain heterogenous subpopulations (Figure 2).

TRPS DLS and MALs comparison from Vogel et al 2021
Figure 2. Tunable resistive pulse sensing (TRPS, top), nanoparticle tracking analysis (NTA, middle) and multi-angle dynamic light scattering (MADLS, bottom) measurements of quadrimodal polystyrene standards. Adapted from Vogel et al., 2021. Journal of Extracellular Vesicles.

Zeta potential and advances in resolution

Zeta potential is a measure of a particle’s effective charge in a certain medium, and an indicator of particle stability. It is defined as the electrostatic potential at the shear plane, i.e., the interface between the stationary second layer and the mobile phase (Figure 3). Unlike many other techniques, TRPS can detect differences in zeta potential, even when particles are the same size (Figure 3).

Figure 3. A negatively charged particle surrounded by a stationary layer of positive charge (stern layer), and a second, stationary ionic layer. Zeta potential is the electrostatic potential at the shear plane, i.e., the interface between the stationary second layer and the mobile phase.
Figure 4: Zeta potential analysis of a 1:1 mixture of 400 nm bare polystyrene particles (left) and 400 nm charged carboxylated polystyrene particles (right) using tunable resistive pulse sensing (TRPS, top) and phase analysis light scattering (PALS, bottom).

Simultaneous size and zeta potential measurements can also be obtained for more complex samples. Figure 5 shows a heterogenous mixture resolved into five distinct subpopulations.  

pentamodal zeta potential plot
Figure 5. TRPS-enabled size and zeta potential measurements of a pentamodal mixture containing 70 nm and 100 nm charged carboxylated polystyrene particles (CPC 70 and CPC 100), 100 nm bare polystyrene particles (CPN 100), magnetic particles (Adem), and DNA-bound magnetic particles (Adem+DNA).

 

Post-analysis processing enables precise particle-by-particle measurement of zeta potential, size and concentration

TRPS-enabled zeta potential analysis is a relatively new development. Previously, using TRPS, it was possible to measure only two parameters at once: size and concentration, and more recently, size and zeta potential. To provide researchers with a way to concurrently measure all three parameters (size, zeta potential and concentration), Izon Science created a TRPS post-processing protocol.

As shown in Figure 6, the post-processing protocol was needed to ensure all particles were represented accurately, as highly charged particles are overestimated in counts. The correction is based purely on physical principles, rather than a fitting model.  

corrected 3d zeta potential graphs
Figure 6. The development of a post-processing protocol has made it possible to obtain concurrent measurements of zeta potential, size and concentration. Left: without the correction, it was not possible to obtain accurate, concurrent measurements of all three parameters as highly charged particles would be overrepresented. Right: using the correction, the concentration is no longer skewed and particles are accurately represented.

Applications of zeta potential analysis in EV research

The ability to obtain TPRS-enabled zeta potential measurements with high sensitivity and resolution can strengthen many areas of EV research, including studies of:  

  • EV composition under different conditions
  • EV drug loading
  • Stability of therapeutic EVs following storage
  • EVs in cancer research: general biology and therapeutic development  
  • EV-based cancer diagnostics. Most approaches are based on detecting surface markers; determining EV charge as part of the detection process could improve specificity.  
  • Method development: e.g., EV labelling or EV capture systems. Zeta potential can be altered by the binding of molecules to the EV surface, such as aptamers, proteins, antibodies, lectins, DNA, and RNA. Monitoring zeta potential can therefore aid method development by enabling researchers to assess binding capacity.

References

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