With purity key to scientific research, diagnostic and therapeutic applications, the most important step in extracellular vesicle (EV) research is their isolation. EVs exist in vivo or in vitro in highly complex samples, be they biofluids or conditioned culture media. This makes isolation a tricky challenge that we at Izon Science are dedicated to surmounting. Our 35 nm and 70 nm qEV columns set a benchmark in the field for their efficiency, reliability, and purity, fundamentally changing the way scientists isolate and study EVs. But that doesn’t mean that the job is done. As the field develops, so do isolation needs.
Researchers in the EV field have become increasingly interested in the newly identified exomeres and supermeres – non-vesicular particles in the sub-EV size range. Alongside this, research into the smallest viruses such as adeno-associated viruses (AAVs) for gene therapy is gaining significant traction. Both of these applications revolve around particles which lie below the current ideal isolation range of our qEV columns. That is why we have developed a new range of columns: the qEV 20 nm series. These columns are not just an incremental improvement; they mark the entrance into a realm previously uncharted. As their name suggests, they are optimised to isolate particles down to the tiny size of 20 nm, opening up a whole new world of particle isolation.
Small particle isolation with the qEV 20 nm series
Of most interest is the ability of the 20 nm series of columns to isolate smaller particles more efficiently than the existing 35 nm series of columns. To look at this, we used carboxylated polystyrene particles (CPCs) of known sizes as a sample and isolated them into the full purified collection volumes (PCVs) of the respective column types. True to their name, the 20 nm series of columns isolated significantly more particles in the 20 nm size range (CPC24; p<0.05). This was also true for particles with a mean size of 50 nm (CPC50s; p<0.05) and 70 nm (CPC70s; p<0.05) but not 100 nm (CP100s), suggesting improved recovery of particles less than 100 nm in size.
As such, whilst qEV 20 nm series columns are particularly aimed at those needing to isolate particles in the 20-35 nm size range such as exomeres and AAVs, they will also be helpful for those wishing to enrich small EVs more generally.
The 20 nm series isolate more particles than other qEV series
As shown in Figure 2, 20 nm columns isolate more than double the number of particles isolated by the 35 nm (p<0.05) and 70 nm (p<0.01) series of columns, at least when comparing default PCVs (the collection volume programmed on the AFC). This means that the 20 nm columns are a great choice not only if you want to isolate smaller particles with greater efficiency, but also if EV yield is your priority. But does this increased yield come at a cost?
The ‘hump’
To introduce you to the column and its elution profile, we plotted the qEVoriginal 20 nm elution profile alongside the existing 35 nm and 70 nm series columns (Figure 3). Here you can see that, compared to the 35 nm and 70 nm columns, that there is a new ‘hump’ of protein in the PCV of the 20 nm columns. This is present in all qEV column sizes. It is possible that this ‘hump’ represents the very exomeres and supermeres that this series of columns was in part designed to isolate. We are currently working to see whether this is indeed the case.
Whatever the identity of the protein hump, Figure 4 shows that this does indeed result in an increase in protein in the default PCV as compared to the 35 nm (p<0.001) and 70 nm (p<0.0001) series. Whilst this is highly significant, it is also modest. Just 6% of the total protein from the original plasma samples were present in the default PCV.
The qEV 20 nm columns compromise EV purity for yield, but are still purer than other methodologies
The increase in particle yield with the 20 nm columns does result in a decrease in purity as compared to the 35 nm (p<0.01) and 70 nm (p<0.01) series of columns (Figure 5). However, this decrease in purity still puts the qEV 20 nm series ahead of ultracentrifugation, density gradient ultracentrifugation and precipitation, where the purity from plasma/serum literature comparison studies was on average in the 1e+08 range.1-5 The 20 nm columns are also purer than our Legacy columns, especially the 35 nm series of Legacy columns. As such, whilst purity is compromised as compared to Gen 2 / 35 nm and 70 nm series columns, the 20 nm series columns still outperform other methodologies.
Empowering nanoscale discovery: the impact of qEV 20 nm series columns
The qEV 20 nm series columns from Izon Science mark a significant advancement in the field of particle isolation, particularly for small particles like the smallest extracellular vesicles, exomeres, and small viruses such as adeno-associated viruses. This innovation not only extends the boundaries of what's possible in nanoparticle research but also upholds the high purity standards that the qEV series is known for. With these columns, researchers can now explore previously inaccessible realms of the nanoscale world, enhancing the potential for new discoveries in diagnostics and therapeutics.
qEV 20 nm series columns are now available in qEVsingle, qEVoriginal, qEV1, qEV2 and qEV10 sizes, with the qEV100 soon to follow. Please note that at first, different sized columns will have different lead times as production ramps up to meet demand.
