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).
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).
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.
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.
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.
