Rapid, Accurate Isolation and Quantitation of Extracellular Vesicles

Isolate highly pure samples of extracellular vesicles (EVs) for fundamental research or clinical purposes. Carry out complex, multi-parameter measurements easily with single-particle resolution and unmatched precision.

Our bespoke EV bioanalysis system, comprising of qEV Isolation columns and the Tunable Resistive Pulse Sensing (TRPS) characterisation instrument the Exoid, is the only streamlined and standardised method of isolating and quantifying EVs. The speed, reproducibility, and simplicity of the Exoid analysis is unparalleled, with highly accurate evaluation of EV size distribution, concentration, and surface charge in minutes. Real-time measurements can be obtained using only 35 µL of a diluted sample, giving immediate insight into the nature of the sample, enabling assessment of changes over time, and making analysis of high-value or low volume samples feasible.

The latest additions to the Izon range are the qEV Concentration Kit and the qEV RNA Extraction Kit. These kits are designed to help researchers to adjust their experiments for optimal EV results and suitable for an array of down-stream analyses.

Research

Investigation of EV biophysical/functional heterogenicity and roles in normal cellular activities and dysfunctions leading to diseases.
qEV isolation for variable sample volumes, precise analytical tools, detailed support materials, training and guidance.

Diagnostics

EVs are present in all biofluids, have different marker molecules, and allow disease detection, monitoring progression and response to treatment.
qEV automated isolation for small samples, quick and reproducible analytical tools.

Therapeutics

EVs offer efficient targeted cargo delivery, low immunogenicity and high bioavailability. E.g. drug-loaded EVs, functionalised or naïve EVs, EV-based vaccines.
qEV isolation for large volumes, no toxic chemicals, accurate and reliable analytical tools.

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Reliable and validated isolation protocol

Achieve rapid, reproducible separation of EVs from protein aggregates and other contaminants in a simple, semi-automated system. Obtain highly pure samples of EVs from different types of EV-containing matrices without additional preparative steps.

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Accurate, real-time, label-free quantification

Achieve direct and accurate quantification of EVs for research, clinical purposes, or for the development of EV-based therapeutics, rapidly and without the need for antibodies, detector molecules, or prior knowledge of optical properties.

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Simple, high-throughput, multi-parameter analysis of heterogenous samples

Simultaneously assess the particle concentration as well as size and zeta potential of every particle within a sample. Identify and individually characterise subpopulations of particles with ease.

Figure 1.

Challenges in the Extracellular Vesicles field

EVs are released by every living cell, from complex multicellular eukaryotic organisms, such as cell in the human body, to unicellular protist parasites, from bacterial cells to plants and fungi. EVs isolated from a single cellular origin may present significant heterogenicity, a reflection on the intricate cellular biology and biogenesis mechanisms generating different EV subtypes. The unique molecular composition and biological roles EVs play in each of these organisms is unimaginably diverse and lays the foundation to specialised advanced EV research. Within the plethora of EV functions (Figure 1), the biggest potential and contribution to human medicine is their application in diagnostics and as therapeutics.

Due to EVs nanoscale dimensions, concentrations, and the complexity of the fluids in which they are found in vivo or in vitro, the most critical step of EV analysis is isolation. Firstly, the EVs of interest must be reliably separated from non-vesicular components without compromising their integrity or functions. Secondly, EVs can be concentrated or remain (avoiding significant loss) in sufficient quantities suitable for an analysis tool that is accurate and reproducible.

Isolate Functional EVs With Ease and Reproducibility

Isolation of EVs is a challenging task due to their small and heterogenous size distribution, co-existence with other non-vesicular structures (e.g. proteins and lipoproteins) and susceptibility to breakage, deformation and/or aggregation. There are several techniques currently available for EVs isolation that have varying degrees of success. Some techniques are not suitable for unprocessed biofluids such as plasma/serum which means that additional laborious preparative steps are required with a high risk of introducing variability to EV sample processing. Our qEV Isolation Columns use Size Exclusion Chromatography (SEC) to isolate EVs from a range of samples with minimal sample preparation. qEV Isolation Columns enable rapid and gentle purification of EVs with almost complete (>97%) removal of contaminating proteins, including free proteins and viral-protein aggregates (Figure 2).

qEV Overview

Figure 2.

qEV column (qEV1, 35 nm) offers efficient separation of EVs from contaminating proteins in a plasma sample, which elute in later volumes than vesicles. The EV concentration was measure using a qNano and relative protein levels by absorbance at 280 nm.

$Automatic fraction collector (AFC) side on left$

Automating EV Isolation

With qEV Isolation the biophysical integrity of the vesicles is unchanged, and EVs are eluted into commonly used buffers suitable for downstream analyses with user-specified volumes. qEV Isolation columns are offered in two pore sizes targeting isolation of EVs in two particle size distributions 35-350 nm and 70-1000 nm which the user can choose from depending on their EV research needs.

The qEV Isolation columns can be combined with an Automated Fraction Collector (AFC) creating a streamlined workflow to achieve reliable and reproducible isolation in under 15 minutes with minimal inter- and intra-operator variability. Purified EVs can be used for different down-stream applications.

Learn about the AFC

Simple, High-throughput, Multi-parameter Analysis of Heterogenous Samples

Correct determination of key EV physicochemical properties is technically challenging but of utmost importance as it directly influences the outcome of investigations. Accurate quantification of EVs is critical, as concentration or presence of a specific EV subtype in a biofluid can reflect health status or a condition with potential diagnostic applications, or EVs purposed for therapeutics require appropriate dosing.

Currently the field is limited by a lack of standardised, robust techniques and a historical reliance on ensemble and indirect techniques. Ensemble techniques do not provide sufficient resolution and overlook the presence of subpopulations while indirect techniques, such as detection of EV markers, are influenced by heterogeneity within the sample, leading to inaccurate EV quantification. By comparison single particle techniques such as Tunable Resisitive Pulse Sensing (TRPS) are about to provide individual particle resolution and identify subpopulations for accurate measurements.

Compare NTA and TRPS Compare DLS and TRPS

Figure 3.

TRPS, NTA and MADLS measurements of quadrimodal sample (CPN100/CPN150/CPN200/CPN240 at 25/25/25/25). TRPS, NTA and MADLS measurements were averaged over three runs. TRPS identifies all four sub populations clearly. NTA was able to identify that multiple sub populations were present. MADLS was not able to identify any sub populations.

Figure 4.

Analysis of zeta potential in a heterogenous particle population. Graph shows zeta potential vs particle size of bare polystyrene (CPN100), charged carboxylated polystyrene particles (CPC70, CPC100), magnetic particles (Adem) and magnetic particles modified with DNA (Adem+DNA). The mix of all 5 particle types resembles very well the particle distributions when particle types are measured separate.

The Growing Importance of Surface Charge in EV Research

While concentration and size are currently the most analysed EV parameters, there is growing evidence of the importance of other EV properties such as surface charge. Many of physical interactions between EVs or biological interactions between EV and target cell may depend on EV surface charge i.e zeta potential. The Exoid’s sophisticated software simulatenously measures the size and zeta potential of individual particles, ensuring that subpopulations are also characterized by their surface charge (Figure 4). Thus, changes in zeta potential or particle size in response to the binding of an antibody or detector molecule such as an aptamer can be evaluated using TRPS, which may enable the identification and quantification of specific surface markers from different EV subpopulations. Where other techniques would require incredibly complex protocols and data analysis to evaluate these multiple factors, the semi-automated system enables pre-programmed, multi-step experiments to be run with minimal user input. Altogether, full EV characterization by the Exoid holds significant utility in many fields of EV research and applications, such as studies of EV drug loading or EV functionalization for therapeutics, EV composition and biological roles and development EV-based diagnostic tools.

Therefore, multi-parameter EV measurement techniques are becoming critical for research as well as regulatory bodies recognising the necessity of reliable, reproducible, and comparable EV multi-parameter characterization. The Exoid offers the only standardisable particle-by-particle method of characterising EV size, concentration, and zeta potential, whilst calibrated with NIST-traceable particles, ensuring accuracy that allows inter- and intra-laboratory comparisons.