Technology

NanoView’s mission is to facilitate the translation of EV research into diagnostic and therapeutic platforms.  We are making EV analysis simple by removing the hurdles associated with sample purification and bringing multi-parameter measurement of EVs closer to biological source.

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Label Free & Fluorescence

 
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Summary

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Key Features of Fluorescent Measurements:

 
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fluorescence

Single binding event sensitivities mean that even the smallest EVs are detectable using 3 color fluorescence

Biomarker colocalization

Measure up to 4 proteins per EV. Measure EV sub-populations through relative protein expression

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EV CARGO

Probe for luminal EV proteins and cargo in single EVs

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Sensitivity

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THREE COLOR FLUORESCENCE

Extracellular vesicles (EVs) are captured from solution without purification and bound to one of 6 different capture antibodies, plus control, on the the ExoView™ chip.

Secondary fluorescent antibodies can be added, and up to 3 colors can be measured simultaneously. The secondary antibodies are common to all 6 capture antibodies.

ExoView’s single binding event sensitivity enables detection of even poorly expressed proteins on extracellular vesicles as small as 50nm in size.

Measure 3 color fluorescence on single EVs on the ExoView™ microarray chip.

BIOMARKER COLOCALIZATION

ExoView™ provides the ability to measure up to 4 markers on a single EV.

Biomarker colocalization allows unique sub-populations of extracellular vesicles to be identified and measured. EVs are simultaneously sized and counted.


Blue Spots are vesicles that are positive for CD41a + CD63.
Green Spots are vesicles that positive for CD41a + CD81.
Red Spots are vesicles that are positive for CD41a + CD9.
Other colors are vesicles that express various combinations of R/G/B fluorescent signal and CD41a (via capture on surface).

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 DETECTING EV CARGO THROUGH FLUORESCENCE

EVs on the ExoView™ chip can be permeated and probed for luminal proteins and cargo. Three color fluorescence allows a combination of staining of three surface and/or luminal proteins on single EVs.

These experiments show the ability to detect luminal proteins such as ALIX and Sytenin through the permeabilization of bound EVs.

 SENSITIVITY

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Label Free Key Features:

 
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PURIFICATION NOT REQUIRED

Measure the changes in your sample, not the biases from your purification technique

EV SIZE

Measure the size of individual, antigen-positive EVs with high resolution

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STREAMLINED WORKFLOW

Automated measurement of 9 samples increases sample throughput

EV COUNT

Count the number of antigen positive EVs expressing specific surface markers

 

 PURIFICATION NOT REQUIRED:

Reduce the biases associated with sample purification by removing the need to purify.

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Variability and biases introduced through sample purification are commonly cited as the largest limiting factors in the progression of EV-based platforms into clinical diagnostics and therapeutics. Common nanoparticle characterization techniques, such as NTA and TRPS, are unable to discriminate EVs from particles that co-purify, and are subject to errors in data interpretation.  These co-purified particles influence EV counts and limit the ability to link clinical observations to measured data.   ExoView™ bypasses these challenges by measuring only specific EVs that exhibit target surface antigens without the need for purification. The platform works with as little as 35µL of sample and provides EV phenotype, size and count direct from biological fluids onto a functionalized array.

Key Feature include:

·         Reduction in time, cost and biases associated with sample purification.

·         A single sample workflow in which size, count and EV phenotype can be measured.

·         Specific measurement of EVs and not containment lipoproteins.

·         Small sample volume - just a few µls of sample required. 

·         Measurement of samples as close to biological source as possible - reducing the biases associated with sample preparation.

Common issues with purification

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 MEASURING EV SIZE

ExoView™ uses Single Particle Interferometric Reflectance Imaging Sensing (SP-IRIS) to measure the size of single EVs by analyzing the enhanced interferometric signal of bound EVs when attached to a layered substrate.  Using this methodology, EVs as small as 50nm can be imaged and measured. ExoView has unrivaled peak-to-peak resolution, and can more accurately resolve distinct vesicle sub-populations within a heterogeneous sample when compared to existing techniques, such as NTA.  For example, when using NTA, EVs with a size difference of 2X will result in a 2X difference in scattering, whereas ExoView achieves an 8X difference.  Furthermore, unlike NTA, the presence of larger EVs does not obscure or bias the relative count of particles within a heterogeneous sample population. 

Unlike traditional techniques such as NTA or TRPS, ExoView™ measures only EVs expressing specific surface markers, therefore excluding other particulates. Size measurements are performed without the need to purify, even from complex solutions like plasma, urine or cell culture supernatent.

Ever wonder why NTA distributions always measure around 100nm? Ever wonder if there are EVs present below 100nm?

While, NTA can measure down to 10-15nm, only materials with a high refractive index are visible. For biological samples, the limit of detection is in the 50-80nm range, depending on instrument configuration. However, when challenged with a heterogeneous sample population, the ability to see small vesicles is influenced by the presence of larger EVs that scatter significantly more signal. The limit of detection of NTA combined with a heterogeneous size distribution often manifests in size distributions of ~100nm. But is it real? Or is there material smaller than 100nm?

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In this example, EVs from L-540 Hodkin cell lines were purified using ultracentrifugation and measured by TEM, NanoSight (NTA), Zetaview (NTA), and ExoView™. EVs captured on the anti, CD9, CD63, CD81 antibody spots matched with the size of vesicles as measured by TEM. Moreover, due to the shape of the size distribution, the few larger particles measured at ~100nm in TEM and ExoView™ represented the bulk of the particles measured by NTA. This demonstrates that even small errors in size distributions can result in large miscalculations of EV counts due to the increasing concentrations of small and undetected vesicles through NTA measurements.

 MICROARRAY DESIGN

ExoView’s microarray format allows for many probes to be measured simultaneously, dramatically increasing throughput. The ExoView Tetraspanin Kit contains arrays that have been functionalized with antibodies against CD9, CD63, CD81, CD41a plus IgG negative control. Additionally, customers can work with NanoView Biosciences to design arrays with antibodies against an antigen of their choice.

 MEASURING EV COUNT

The number of EVs bound to a chip can be measured and related to a concentration of antigen-positive EVs in solution. Control tests are performed to ensure that the binding events measured are specific and not related to non-specific 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. Similarly, non-specific binding events observed on the negative control can be subtracted from experimental results. 

The graph below shows the linearity response 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.

 SPECIFICITY

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