The ExoView technology represents an exciting leap forwards for exosome researchers and clinicians alike. The technology facilitates the characterization of Extracellular Vesicles (EVs) through their specific binding, detection and characterization on an affinity microarray platform (functionalized with antibodies, aptomers or peptides). Once bound to the surface, the ExoView measures the concentration and size of individual antigen positive EVs with a resolution down to 40nm. The array design allows for multiple antigens to be probed simultaneously.
The technology requires no labeling of the sample itself, eliminating the need for wash steps that are required in fluorescent based detection modalities. The label-free detection also removes the possibility of antibody induced sample aggregation. In addition, because the detection mechanism is specific in nature, the technology is capable of measuring directly from complex biological samples such as blood serum and cell culture medium, without the need to purify. Soluble proteins in plasma are not imaged by the ExoView platform, meaning that the technology is insensitive to their presence. The ability to measure direct from cell culture or plasma facilitates the measurement of precious samples with low volumes, removes the variability and losses associated with sample purification and accelerates the field towards clinical, point-of-care, EV based diagnostics.
Detection of EVs bound to the microarray is performed using Single Particle Interferometric Reflectance Imaging Sensing (SP-IRIS). Using brightfield microscopy, the instrument detects the enhanced scattering of a bound EVs when attached to a layered substrate. Using this methodology, EVs as small as 40nm can be imaged and measured. The experimental setup is shown below.
This video shows vesicles binding to the ExoView array in real time. The EVs bind to the surface via an antibody/antigen interaction and therefore requires no labeling and no purification of the EV sample. Once bound, the size and concentration of antigen positive EVs are measured.
The ExoView array has been designed specifically with the needs of the EV researcher in mind. The microarray format means that 100s of probes can be measured simultaneously, thus dramatically increasing throughput. As standard, the arrays have been functionalized with antibodies against CD9, CD63, CD81 and also with an IgG negative control. In addition, the array contains a lane labelled with streptavidin to allow custom designed experiments using secondary antibody labels. Alternatively, customers can work directly with NanoView Biosciences to design arrays with direct antibodies against an antigen of their choice.
Measuring EV Size
The size of an EV is known through the scattered intensity of the bound EV. The scattered intensity of the bound EVs increases according to radius^3. This provides excellent resolving capabilities when compared to techniques such as Nanoparticle Tracking Analysis (NTA), which uses EV diffusion to measure particle size. Instrument peak-to-peak resolving capabilities define how well an instrument deals with sample heterogeneity, as is often the case in EV preparations. EVs with a difference of 2X in terms of size, will result in a 8X difference in scattering in the ExoView instrument, whereas with NTA, the same change in size only results in a 2X change in EV diffusion coefficient. This means that ExoView has inherently better peak-to-peak resolving capabilities and can better resolve distinct vesicle sub-populations within a heterogeneous sample.
NIST particles (50nm, 100nm, 150nm, and 200nm) and their specified distrubtions. The solid blue line is the ExoView results for a 4 particle mixed sample.
The ExoView platform has unrivaled peak-to-peak resolving capabilities as demonstrated by its ability to resolve 5 particle populations of different sized polystyrene beads of 40nm, 70nm, 100nm, 150nm and 200nm within a single measurement. Whilst polystyrene beads are not a good analogue for EVs, the experiment serves to demonstrate the inherent resolving capabilities of the technique. Importantly unlike other techniques such as NTA, the presence of the larger EVs does not obscure or bias the relative concentrations of particles within a heterogeneous sample population.
Measuring EV Concentration
The number of EVs bound to the chip can be measured and related to a concentration of antigen positive EVs in solution, measured in EVs per mL. Control tests are performed to ensure that the binding events measured are in fact specific and not related to unspecific binding or aggregates associated with the antibody used to functionalize the chip surface. The array is measured prior to sample incubation and any contaminant events can be excluded from the analysis (fiducials are used to ensure that the exact location of contaminants are recorded and then correctly excluded from the experimental results). Similarly, non-specific binding events observed on the negative control can be subtracted from the experimental results.
The graph below shows the linearityresponse of the ExoView instrument. As a measure of comparability, a sample of EVs was measured using an NTA instrument. The sample has been linearly diluted and measured using the ExoView platform and the number of CD81 positive EVs were counted at a range of dilutions. .