Spectradyne's particle analysis blog

When every particle countsTM

Extracellular vesicles exosomes nanoparticles gene therapy are all done here

Welcome to Spectradyne's blog!

September 24, 2019 - Are you listening to your data?

Everyone working with nanoparticle formulations is used to using statistical measures to describe their mixtures. Readers of this blog will be familiar with the Concentration Spectral Density (CSD) that Spectradyne uses to convey the high resolution information on size and concentration captured by the nCS1, such as the plot to the right, showing a mixture of 3 calibration bead sizes (505 nm, 990 nm and 1760 nm diameter beads). Data from just a single 10 second acquisition is shown, hence the slightly noisy statistics. Followers of this blog will also be used to seeing the time domain plots as well, since of course the nCS1TM counts every particle: The second plot to the right shows the time trace of the data used to make the CSD plot above it. Expanding the vertical axis, we can see the 505 nm particle transits above the noise floor in the third plot to the right. This is all very interesting and extremely useful for characterizing all sorts of particle mixtures: extracellular vesicles, virus particles, aggregated proteins... But, at Spectradyne, we love data and we're always looking for more ways to dig into what they can tell us: Have you ever heard your formulation? We turned the time-domain data above into an audio file, take a listen, crank it up: Can you hear all three bead populations?

We also slowed it down five times, and then 50 times, for a different and richer listening experience. We feel we get an even better appreciation of the particles this way. Take a listen to the two versions. First, slowed down five times.

Next, slowed down 50 times.

Of course, these are artificial bead mixes. What does a real customer sample sound like in the raw? First, real time:

Then, slowed down 50 times:

Why do we do these things with particle sizing data? Because we can: The nCS1TM really does measure every particle that goes by. Does your current particle sizer do that? Are you listening to your data? Do you want to hear what your formulations are trying to tell you? Give us a call!

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four component mixture

A three-component mixture of beads measured with Spectradyne's nCS1TM.

NTA 208nm beads

Ten seconds' worth of time domain data for the bead mixture above.

NTA three bead diameters

Expanding the vertical scale to show the smallest (505 nm) beads.

September 10, 2019 - Nanoparticle measurements of arbitrarily polydisperse mixtures

In this blog, we often talk about the importance of orthogonal measurements, especially if all one has is metrology based on light scattering (e.g. DLS, NTA). This admittedly serves Spectradyne's purposes well because, after all, our technology is an electrical one and it's inherently orthogonal to measurements based on light scattering!

However, another critically important feature of our MRPS technology is that it measures particles individually as they pass through a sensing volume, so that a measurement of one particle does not affect that of another (within certain limits on concentration, of course). This means that Spectradyne's nCS1TM can measure an arbitrarily polydisperse sample just as easily as it can measure a monodisperse one.

To understand this better, let's back up and make sure we understand what is meant by an "arbitrarily polydisperse" mixture. It's an odd phrase, really, and one that perhaps Spectradyne invented. The meaning we are trying to convey is simply that Spectradyne's MRPS technology can measure accurately a mixture that has ANY mix of particle size and concentration. Let's illustrate this with a beautiful plot of a four-component mixture of polystyrene beads, seen at right.

We see four distinct populations in only 100 nm of size range! Moreover, the concentration is readily reported separately for each sub-population. Now, there are of course resolution limits with Spectradyne's technology, as there are with any measurement technology. If two of the peaks were, say, only 5 nm apart instead of 20 nm apart, we wouldn't fully resolve them. But this resolution limit, related to the size measurement uncertainty of each reading, can be specified and understood from measurements of a monodisperse sample, if one is so inclined. It's not caused by the polydispersity of the sample.

Let's look at what happens if an instrument does not have the core property of independence of measurements, as the nCS1TM does. At right we see a NTA measurement of some 208 nm polystyrene beads. It's a nice plot. (Note that in this and the following data presentations we'll just use normalized concentration units, referenced to the 208 nm measurement, for simplicity.)

Let's look at a couple more measurements of monodisperse bead samples, 94 nm and 150 nm, and plot those separate measurements on the same axes as the 208 nm one. This combined plot looks great too - the measurement method is clearly working well.

But what happens if we put all three bead populations in the same mixture, and try to measure that polydisperse mixture? Well, as we see in the third NTA measurement, the wheels kind of fall off the cart. We see that the concentration measured for the 150 nm beads is somewhat suppressed, maybe by 15%. But the poor 94 nm beads disappear completely!

What happened? We hope you believe us that the 94nm particles are still there. The problem is just that the intensity of the scattered light from the 150 and 208 nm beads obscures the much weaker scattered light from the 94 nm beads. It's a phenomenon we've called a variable limit of detection, and it's something that all of you who measure protein aggregates or exosomes should be aware of, because it critically affects your results.

Now we're sure you're wondering what the same kind of experiment looks like when using Spectradyne's nCS1TM instrument, so here you go. First, let's look at the three monodisperse samples measured separately, and plotted on the same axes, at right. Very similar to the NTA result!

However, as you might expect, the nCS1TM measurement of the three-component mixture looks much different from the NTA one. With MRPS technology, the 94 nm beads are detected just as well as they were when measured individually. This is a key attribute of Spectradyne's MRPS-based nCS1TM instrument because the harsh reality of many biological mixtures is that they are highly polydisperse (if not quite "arbitrarily" polydisperse!).

In fact, we can almost guarantee you that your extracellular vesicle or protein aggregate samples have many more small particles than you think, if you are using only NTA or DLS to measure them. Let us repeat this another way in case we don't have your attention: If you are using only NTA or DLS, you aren't accurately measuring your biological nanoparticle sample. You may even have false peaks that are artifacts of your measurement limitations.

All metrologies have their limitations, and we in no way mean to imply that our MRPS technology doesn't also. But we hope that as you read these blog entries, you understand that Spectradyne tries very hard to be honest and open about our limitations. At the same time, we are scientists just like you, and it's our responsibility to expose false claims from other vendors, who perhaps don't share our commitment to such disclosure.

Good luck with your nanoparticle measurements, and please contact us if you have any questions!

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four component mixture

A four-component mixture of beads measured with Spectradyne's nCS1TM.

NTA 208nm beads

An NTA measurement of 208 nm diameter beads.

NTA three bead diameters

Separate, superposed NTA measurement of 94 nm, 150 nm, and 208 nm diameter beads.

NTA mixture measurement

Mixture of three bead diameters measured with NTA (94 nm, 150 nm, and 208 nm diameter beads).

NTA mixture measurement

Three bead diameters measured separately with Spectradyne's nCS1TM (94 nm, 150 nm, and 208 nm diameter beads).

NTA mixture measurement

Mixture of three bead diameters measured simultaneously with Spectradyne's nCS1TM.

August 23, 2019 - Large dynamic range for diverse applications

We love learning from our customers about the science of their materials, and because the nCS1TM can measure any particle type equally well, we get to learn about a very wide range of applications.

For example, scientists routinely use Spectradyne's nCS1TM to assess drug formulation stability, to quantify extracellular vesicles (EVs) and exosomes, to characterize lipid nanoparticles and emulsions, to titer viruses for gene therapy applications, and to characterize new industrial nanomaterials.

One analytical requirement shared by all of these applications is to be able to measure particles across a broad range of sizes and concentrations. In Spectradyne's experience, biological samples - be they biologic drugs, urinary vesicles, cell culture media, or serum - tend to be highly polydisperse (regardless of what DLS tells you!). Measuring concentration accurately in such samples is difficult, and we argue that Spectradyne's nCS1TM is the only technology available that does so. It's critical to have this information if a material is to be accurately characterized: At right we show the measurement of a single product that contains vesicles spanning 60 nm to 6 microns in diameter and nearly 9 orders of magnitude in concentration! Particle function depends critically on particle size, and the broad dynamic range of Spectradyne's nCS1TM allows a researcher to get a complete and accurate picture of what's in the sample to better understand how it will work.

Our customers frequently tell us how happy they are with the nCS1's dynamic range, which spans from 50 nanometers to 10 microns in diameter, and from 104/mL to 1012/mL in concentration. To see the complete picture of what's in your sample, send us samples for a free analysis, or contact us today to arrange a demonstration.

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Please continue to follow our blog as we share insights, technical details, and generally geek-out with you about nanoparticle science!

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extracellular vesicles

nCS1TM analysis of a sample containing a broad range of vesicle sizes and concentrations.

July 11, 2019 - The benefits and perils of orthogonal measurements

One of the many reasons Spectradyne's customers get excited about our MRPSTM technology is that it is inherently "orthogonal" to the measurement techniques most of them use. Most scientists working with micron or submicron particles have some form of optical instrumentation at their disposal: either Dynamic Light Scattering (DLS), Multi-Angle Light Scattering (MALS), or Nanoparticle Tracking Analysis (NTA). These are standard measurement technologies in the pharma industry. Spectradyne's MRPS technology is, by contrast, a 21st century version of an electrical sensing method (the Coulter principle) that, at its core, has been around for over 60 years. Although it's been around a long time and is the gold standard method of counting blood cells, the challenges of applying the method to deep submicron particles have only recently been solved, in a robust manner, by Spectradyne's MRPS technology.

In this blog post, we'd like to review why orthogonal measurements are important, and why customers want them. This will then lead us to discuss why, in fact, some customers don't REALLY want an orthogonal measurement after all, though they may not want to admit it!

First, let's review why our customers want orthogonal measurements. Just about all of Spectradyne's customers are scientists (or managers who once were scientists!), and all scientists know that a measurement result is more powerful if the result is obtained more than once, better if obtained by different researchers/labs, and even better if obtained by a different type of instrument. Especially if one has an exciting result, a natural scientific mind-set tries to confirm that result with another method. If you don't, someone else certainly will! So, researchers want that orthogonal result because it provides confirmation and validation and is just plain good scientific practice.

But of course, therein lies a problem: what if the second method, orthogonal as it is, doesn't confirm the first result? What if Spectradyne's MRPS measurement in fact indicates that those extracellular vesicles or liposomes aren't quite as purified as you thought, based on NTA measurements? It's been known to happen, as detailed in earlier blog posts about exosomes and protein aggregates!

In fact, we can take this scenario a step further. If a new measurement method is actually independent/orthogonal to a first method, meaning that it is probing the sample through a different interaction mechanism, it's actually quite remarkable if the same result is obtained. A simple example is the sizing difference obtained from DLS and Electron Microscopy (EM) measurements. DLS probes the size of particles through their Brownian motion in liquid, thereby extracting the hydrodynamic radii of the particles. Electron Microsocopy (EM) probes a completely different aspect of a material: the material's re-emission and scattering of high energy electrons, and therefore giving information related to the core physical dimensions of particles. In this example, different information is obtained from each method; there's not one method that's right and one that's wrong. Of course, in this case the fact that the absolute sizing is different between the two methods is less important than determining if results are correlated: when DLS reports that sample A has larger particles than sample B, a similar trend in sizes should also be reported by EM.

In the case that orthogonal results are correlated, and therefore consistent with each other, a researcher is in a happy place because her measurement result has been confirmed by two methods. The results may not agree in an absolute sense, but that's to be expected. If they are at least consistent with each other, then that's confirmation enough. However, what if a first result, perhaps a desirable one, is NOT confirmed by the second, orthogonal, method?

Unfortunately, this will definitely happen sometimes! The power of a result increases when a second method confirms it exactly because there is a possibility that the second method will expose something new or different that wasn't picked up by the first method. One can't have the benefit of the affirmative measurement without the possibility of a negative result!

This is the reality that this blog post wants to illuminate: An orthogonal measurement is an independent method that may confirm a result, but it may also NOT confirm a result. And this alternate method is powerful exactly because of this possibility. It means that, in striving for an orthogonal result, a researcher may re-live the uncomfortable part of the old saying "be careful what you wish for, because you just might get it." At Spectradyne, we encounter this directly, every week, because our MRPS technology is orthogonal to most of the measurements our customers already have. Most significantly, our technology has exposed the "false peak" phenomenon that researchers commonly encounter in their NTA measurements. Many an on-site demo has become uncomfortable when we realize that a researcher didn't REALLY want an orthogonal measurement but rather just a confirmation from a second method.

If you are a scientist reading this post, ask yourself: how will you respond if confronted by an uncomfortable result that doesn't agree with your expectations? Will you admit the possibility that your understanding is incomplete and a deeper investigation is warranted? Or will you ignore the new measurement and search for another method, perhaps one not so independent, that "confirms" the first one?

Do you really want an orthogonal measurement, or just a second one? We'd advocate for courage, that more data is always better, so that having an orthogonal method is indeed the way to go.

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June 3, 2019 - Sensitivity limitations in the measurements of protein aggregates

In our last post we dealt with the thorny issue of false peaks in the measurement of extracellular vesicles, especially as exemplified by NTA-based measurements of those vesicles. These false peaks are tantalizing because they often occur at a size range where researchers might expect to see a peak in a well-isolated exosome sample.

We also pointed out that the "limit of detection" (LOD) issue that causes the false peaks is very much NOT limited to measurements of exosomes, specifically, or even biological nanoparticles, generally. In fact, erroneous NTA measurements are readily apparent simply by making careful measurements of polydisperse mixtures of polystyrene beads (see our tech brief for more information). The core issue is that large particles scatter far more light than small particles so the detection of smaller particles will always be obscured (to greater or lesser extent depending on user settings, which is another issue we won't discuss here) by the scatter from larger particles. It's really no different from the difficulty one has trying to see, and count, faint stars in a night sky next to a bright moon or in the presence of light "pollution" from a nearby city.

As a reminder, to the right we show the data given in the earlier blog post about exosome detection.

The cryo-TEM and Spectradyne nCS1 measurements agree extremely well from 50 nm to 400 nm whereas the NTA measurement undercounts particles below 250 nm, and dramatically so below 130 nm.

Of course, exosomes are just one type of biological nanoparticle, so let's make this more interesting. As we said earlier, similar data can be obtained for protein aggregates, as shown in the second plot to the right.

These measurements were taken on-site at a customer demo, with the NTA data collected by the customer directly. Two cartridge sizes (TS-400 and TS-2000, read more about the microfluidic cartridges here) were used for the nCS1 measurement and there is excellent agreement in the overlap region just above 200 nm. Let's set aside differences in the absolute concentration levels, since we have no independently verifiable measure of it. More important, and more striking, is the different shape in the two distributions. Whereas the nCS1 data, in blue, shows a rapidly increasing concentration of particles as their size decreases, the NTA data shows a peak in the distribution at around 130 nm.

Unfortunately, in this case we don't have a third orthogonal measurement like cryo-TEM to verify Spectradyne's measurement, but hopefully we have explained how this false peak phenomenon happens-it's exactly the same as in the EV measurement shown earlier! (For more info, you can read our tech brief.)

Another way to think about the protein aggregation result is to step back and consider why, really, there would be a peak around 120 nm in a protein formulation? The monomers, dimers, etc. are far smaller than this, and we know of no naturally occurring phenomenon that would lead to a population maximum at this size. A far more likely explanation is that the peak is an artifact of the metrology. As an aside, the reason we cut off the nCS1 data at 60 nm is that the nCS1 measurement loses sensitivity below 60 nm: as we've emphasized before, all metrologies have their limits of detection. Spectradyne is different in that we make no claims about things we know we can't measure, unlike many of our competitors!

Most scientists we talk to in the biopharma community accept this false peak interpretation when we review this kind of data with them. We've seen it many times, and our ability to educate the community on the correct interpretation of aggregation data has contributed to our success in deploying the nCS1 instrument, giving researchers an early indication of formulation instability. By making highly accurate measurements of protein aggregates, Spectradyne's Microfluidic Resistive Pulse Sensing (MRPSTM) technology saves companies time and money. With only 3 μliters of sample, one can detect aggregation far earlier than one can using conventional techniques. And, as a bonus, we'll help you understand the fundamental limitations of the measurements, unlike other instrument manufacturers.

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urinary exosomes

Urinary vesicle exosomes measured by NTA

stressed protein aggregates

Comparison of measuring a stressed protein sample with the nCS1 and with NTA

May 29, 2019 - Exosome purification: Beware of the false peaks!

In an earlier blog post (Peptalk blog), we talked a little bit about "limit of detection" (LOD) issues related to the intrinsic variability of instrumentation sensitivity, especially in instruments based on light scattering, especially Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA). It's Spectradyne's experience that this is a poorly understood topic in the life sciences, yet it's critically important to extracellular vesicle researchers who are attempting to quantify the size distribution and concentration of their vesicle populations. In fact, we have seen clear examples of prominent researchers (we won't mention any names!) believing that they have successfully isolated their EVs when, in fact, Spectradyne's metrology can clearly demonstrate otherwise. With that being said, please forgive us if this post gets a little bit technical.

A popular graph we like to show to motivate this discussion is shown to the right. It's a simple NTA size distribution from a measurement of urinary vesicles. The data was not taken by Spectradyne but by a third party. It appears to show a clear peak near 150 nm, and many exosome researchers would celebrate such a measurement since it appears to indicate a successful exosome isolation.

Alas, this would be a mistaken conclusion because the peak shown in the data is a phantom or, as we have taken to calling it, a false peak. How do we know? Well, fortunately this sample was measured by three orthogonal methods. We plot the NTA data along with a cryo-TEM measurement of the same sample (second plot to right).

Now, cryo-TEM is not a great method for measuring absolute concentration - so let's not worry about the absolute scale - but it is a highly accurate (and expensive) method for making relative assessments of particle counts, if one is patient about counting. It's also a non-optical method, so it's not sensitive to the optical transparency of a particle. This important orthogonal method delivers a very different conclusion from the NTA measurement. Is there a peak near 150 nm, or does the concentration of particles increase dramatically as their size decreases? What we need is a third method to break the tie. In fact, we have such a method (otherwise this would be a lame blog post), in the form of Spectradyne's nCS1 measurement, which we add to the third plot on the right (note it's plotted using a log scale).

Here we see excellent agreement between the nCS1 measurement and cryo-TEM, down to 50 nm! Now, it's important to remember that Spectradyne's nCS1 relies on an electrical method of detection called Microfluidic Resistive Pulse Sensing (MRPSTM), so it is intrinsically orthogonal both to TEM and NTA. Therefore, we have three measurement methods that rely on completely different principles of operation, and two of the three agree.

How does one explain the dramatically lower concentrations measured by NTA, especially below 130 nm, but starting as high as 250 nm? The core issue is that light-scattering intensity varies as the sixth power of particle diameter, so small particles are much harder to detect than large particles (see our tech brief for more information). The "peak" observed in the NTA data is not a peak in particle concentration but a peak indicating the limit of detection (LOD) of the NTA method. Coincidentally (and confusingly), this LOD often occurs near where researchers expect to see a purified exosome peak. The subtle nature of the phenomenon coupled with unwitting confirmation bias (present in even the best-trained scientists) leads to erroneous conclusions.

Perhaps the reader of this blog is still skeptical. After all, it would seem that Spectradyne benefits from pointing out limitations in the technology of competing methods. While this may be true, let us emphasize that ALL metrologies have detection limits. Spectradyne's nCS1 is no different: if we try to measure exosomes below 50 nm, we will fail to detect them. If we tried, anyway, to plot the measurement below 50 nm, we would show a declining population. That wouldn't mean that there are no particles with 40 nm diameters, it would just mean that we can't detect them! Our MRPS method has its limitation like any other, but Spectradyne's data presentations and interpretations are more scientifically honest.

One final note: The false peak phenomenon is exacerbated in NTA measurements, or any optical measurements, of exosomes by the low index contrast (high transparency) of the exosomes. But in fact the phenomenon can be easily demonstrated even in mixtures of high contrast particles like polystyrene beads. For a straightforward demonstration of this, check out this poster.

That's probably enough for now. We'll conclude with the simple observation that if size distribution measurements can be misleading when dealing with particles as different as exosomes and polystyrene beads, they are in fact a challenge when dealing with any type of polydisperse mixture. Our next post will illustrate this point further with a protein aggregation measurement.

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urinary esoxomes nanoparticle tracking analysis

Urinary vesicle exosomes measured by NTA

urinary exosomes TEM NTA

The same urinary vesicle exosome sample, comparing NTA to TEM results

urinary exosomes mrps

The same urinary vesicle exosome sample, comparing Spectradyne's nCS1 to NTA and TEM (note this is plotted on a log scale)

May 15, 2019 - Spectradyne at the 2019 ISEV: Kyoto, Japan


Spectradyne recently exhibited at the 2019 annual meeting of the International Society for Extracellular Vesicles (ISEV) in Kyoto, Japan. Extracellular vesicles (EVs) comprise a broad set of different biological nanoparticles including exosomes, microvesicles, and apoptotic bodies. These particles play important roles in critical biological processes of health and disease and hold exciting promise as biomarkers and therapeutic tools.

The ISEV 2019 meeting was excellent. We were pleased to see that the emphasis on scientific rigor and repeatability in this maturing field continues - as more and more researchers are aware, light scattering-based techniques for quantifying EVs cannot keep pace with the field's requirements for increased accuracy and dependability. As a non-optical technique, Spectradyne's nCS1 is a truly orthogonal method and delivers the most practical and accurate EV quantification currently available (learn more here). Because of its advantages, the nCS1 was featured in multiple oral presentations and two posters (one and two) at the meeting, including by John Nolan (Cellarcus Biosciences) and by Drs. Varga and Beke-Somfai (Hungarian Academy of Sciences). The posters covered important topics: (1) the role that NTA "limit of detection" issues play in generating misleading size distributions, and (2) the quantification of exosome lability in the presence of the detergent Triton X-100, and (3) the development of liposome concentration standards for EV quantification.

We met many new faces at ISEV 2019, reconnected with existing customers and collaborators, and introduced our Japanese distributor, Sanyo-Trading Co., Ltd., to Japanese EV researchers. Spectradyne would like to give special thanks to System Biosciences, who hosted a fantastic Japanese-food themed reception during the conference to highlight their new EV isolation products, and the ISEV leadership committee for organizing and executing a well-run conference.

Welcoming people - A beautiful cityscape - Strong EV science!

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extracellular vesicles EVs EV

Spectradyne's booth at ISEV 2019 - Kyoto

May 7, 2019 - Spectradyne at the 15th Annual PEGS Summit

Spectradyne recently exhibited at the 15th Annual PEGS Boston Conference, a conference focused on protein engineering, with topics as diverse as Antibodies for Cancer Therapy to Protein Expression System Engineering. A major component of all of the technical sessions focused on analytical support for drug product development, with many people attending involved in looking at protein aggregation specifically. Thus, this was a natural fit for Spectradyne, as we offer a unique solution to bridging the "measurement gap" between chromatographic techniques for small (up to 50nm) size aggregates up to compendial methods for large (greater than 10 μm) sized aggregates.

Several nCS1 customers have purchased their systems specifically for studying protein aggregation, and in fact two of these have published their findings in refereed journal articles (see our Library page for more details). As referenced in our previous blog post on Takeaways from PepTalk 2019, there are many reasons why proper metrology of submicron protein aggregates is becoming increasingly important in the industry.

We continue to demonstrate an industry-leading solution for this need, with significant advantages over competing techniques (predominantly optical). We had many significant interactions with existing customers and new prospects at the booth, and presented a poster detailing our advantages versus optical techniques for measuring protein aggregation.

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circulating biomarkers gene therapy

Spectradyne's booth at the PEGS Summit 2019

April 12, 2019 - Spectradyne at Circulating Biomarkers 2019

Spectradyne recently exhibited at the Circulating Biomarkers World Congress in San Diego, a conference focused on using measurements of extracellular vesicles (EVs) for diagnosis of various health issues. This was a small but great, focused, show that confirmed how hot the EV area is!

Research into EVs (or exosomes, or nanovesicles) as biomarkers is rapidly expanding, and has become an important area of the more general discipline of liquid biopsy. Health areas being investigated with exosomes biomarkers include cancer, cardiovascular disease, reproductive issues, and neurodegenerative disease, among others.

Of course, any exosome-based diagnostic technique must be able to differentiate healthy from unhealthy patients by quantifying the presence of the exosome biomarker or biomarkers in question. This could be as "simple" as quantifying the concentration of one population of exosomes, or more complex, for example in comparing the concentrations of multiple types of nanovesicles (or perhaps vesicles compared to non-vesicles), to establish a "fingerprint" of a disease.

In our conversations at the conference, it was clear that that while most researchers have an approach to determine whether a target EV (or related DNA or microRNA) is present in a liquid biopsy, there is much less expertise in accurate quantification of biomarker concentration.

Spectradyne's technology is well suited to this kind of exosome measurement challenge (see some examples here). Our microfluidic resistive pulse sensing (MRPS) technology counts vesicles (or other particles) individually and therefore builds up highly accurate statistics on nanoparticle concentration across any size range from 50 nm to 10 microns. For our technology, high levels of polydispersity are handled easily. This allows the researcher to clearly establish the level of background nanoparticles relative to the exosome population in any biological fluid (we measure serum, plasma, urine, and cell culture media routinely), which is important because the relative measure of exosome "signal" compared to background can be critical to the quality of the diagnostic conclusion.

Finally, importantly, MRPS is a non-optical technology and therefore provides a key check on the findings of other metrology methods, most of which are based on light scattering. Biases related to the optical properties of the exosomes or nanovesicles are inconsequential to our MRPS method, and this can lead to important insights that are otherwise missed if only optical analyses are done (see this article for an example).

The Circulating Biomarkers conference was exciting in terms of the high quality of science on display as well as the energy of the researchers. We look forward to seeing more of this at our next big conference, ISEV, in Kyoto in just a couple weeks!

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circulating biomarkers gene therapy

Spectradyne's booth at Circulating Biomarkers 2019

March 26, 2019 - A brief history of resistive pulse sensing (RPS)

Spectradyne's nanoparticle analysis systems are based upon a long-time, proven technology, resistive pulse sensing (RPS). RPS uses electrical signal detection as its basis, which yields numerous advantages over the optically-based detection used in the majority of particle analyzers.

In the late 1940's, Wallace Coulter was studying ways to do rapid screening for diseases by blood cell analysis. Prior to this, analysis of blood cells required laborious examination under a microscope. Coulter was interested in automating this process to make it widely available as a tool for rapid screening of large populations, and eventually received a grant from the Office of Naval Research which partially funded the research. Initially, he and his brother Joseph were looking at an approach using a light beam focused through a narrow capillary tube to detect and count the cells but found that the electrical contrast created by the cells produced a 10X higher signal than photoelectric signals [M. Don, "The Coulter Principle: Foundation of an Industry," JALA: Journal of the Association for Laboratory Automation 8(6), 72-81 (2003)].

In 1953 Coulter was awarded US Patent 2,656,508, "Means for counting particles suspended in a fluid". The method is widely known as the Coulter principle, in which a particle passing through a small pore will produce a change in the electrical resistance of the pore, proportional to the volume of the particle passing through. The Coulter principle is more generically referred to as resistive pulse sensing (RPS), as shown in the figure to the right). Unlike optically-based measurements, the signal measured is independent of the particle's material properties (including optical contrast) and shape. This is a key advantage of RPS.

Since the early 1960's, Coulter counters have been the gold standard for doing whole cell blood counts. Therefore, instruments based on RPS are using technology that has a proven track record, with literally millions of samples being run every week. However, up until recently, RPS devices were generally limited to 1 micron and larger diameter particles.

Attempts to scale Coulter principle instruments to smaller sizes have found limitations caused largely by thermal noise in very small apertures. While there have been some published reports dating back to 1970 on RPS devices for sub-micron particles [R.W. DeBlois and P.C. Bean, "Counting and sizing of submicron particles by the resistive pulse technique", Rev. Sci. Inst. 41(7), 909-916 (1970)], these have been largely limited to academic, "one-off" devices that were not commercialized.

However, technological advances in seemingly unrelated disciplines have created methods that overcome these size limitations. In particular, advances in semiconductor fabrication techniques have been applied to the burgeoning field of microfluidics, which have enabled commercialization of RPS instruments that can size and measure particle concentrations to particle diameters as small as 35 nm.

Spectradyne's RPS implementation is designated microfluidic resistive pulse sensing (MRPS), implementing the fundamental RPS implementation in a microfluidic cartridge that only requires 3 μL of sample for measurement. A description of this implementation was first published in 2011, and became the basis for the incorporation of Spectradyne in 2012, and the commercial introduction of the nCS1TM instrument in 2014.

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microfluidic resistive pulse sensing

An animation of how microfluidic resistive pulse sensing (MRPS) works, with particles flowing through a nanoconstriction (left) and generating a time-dependent change in the electrical resistance of the nanochannel. microfluidic RPS cartridge

A microfluidic cartridge for resistive pulse sensing

February 26, 2019 - Takeaways from PepTalk 2019

A few weeks ago, Spectradyne exhibited at PepTalk 2019 in San Diego CA. The conference was notable for the high quality of the presentations and the deep scientific knowledge of the researchers - as a result, we had many informative conversations at Spectradyne's booth (which we think looked quite sharp, see image!).

A common theme throughout these conversations was the need for better, more efficient detection of protein aggregation. A few years ago, when we brought up Spectradyne's ability to measure submicron protein aggregates, we were often asked, "Why would we want to do that?". The discussion in these cases centered around the fact that the FDA and USP had no clear requirements on nanoscale particle measurements.

Since then, due to our efforts and those of others in the industry, the importance of nanoscale measurements for detecting protein instabilities earlier is more widely accepted. (Plus, of course, people are more aware of FDA guidance on nanomaterials.)

At PepTalk, there were multiple occasions where scientists told us that the ability to detect smaller aggregates would save them time. In these cases we didn't really even have to explain the benefits of detecting aggregates earlier with our nCS1 instrument! These scientists already understood that all micron-sized particles were once submicron particles, so why waste time waiting for big particles to form, if you can detect them earlier when they're smaller?

Increasingly, biopharma companies know that it is a competitive advantage to be more efficient in sorting winning from losing formulations. This means earlier detection of protein aggregation is good for business, regardless of a lack of hard regulations on nanoscale aggregates. The fact that Spectradyne's nCS1 instrument only requires 3 microliters of sample to make a measurement makes this process even more efficient.

So, PepTalk was great for Spectradyne in that we saw broader acceptance of the benefits of detecting protein aggregates at the submicron scale. However, for an instrumentation company like us, that's only halfway to our goal.

The other half is demonstrating that our technology is the best way to achieve those beneficial nanoscale measurements. There are, of course, other methods of detecting nano-aggregates. Most of these utilize light scattering and suffer severe sensitivity limitations in detecting submicron biological particles, which tend to be relatively transparent. More insidious, the variable detection limits of these techniques are not always obvious to a non-expert, so incorrect conclusions about particle concentration levels can easily be reached. This is a big topic, though, so we'll reserve it for another blog post.

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peptalk booth

Spectradyne exhibits at PepTalk 2019 in San Diego CA

February 7, 2019 - Overview of key applications

This first post will introduce you to the key applications of Spectradyne's nCS1TM, a cutting-edge technology for measuring the concentration and size of particles in a liquid using Microfluidic Resistive Pulse Sensing (MRPS). As an electrical (non-optical) technique that counts and sizes particles one-by-one, MRPS accurately measures the concentration of any particle material, even in the most polydisperse mixtures-that is, in real-world samples. Best of all, sample analysis requires only 3 microliters.

Detect protein aggregation earlier

Aggregated protein samples are highly polydisperse and confound light-scattering based technologies such as nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS). Note that DLS only measures a weighted average size, not a particle size distribution. Because Spectradyne's nCS1TM quantifies smaller particles more accurately, researchers are using it to quantify aggregates dramatically earlier in the process of their formation, without having to wait for the particles to grow large enough to be detected by other methods.
  • Save time and materials cost by detecting protein aggregation earlier
  • Consider new experiments made possible by the extremely small sample volume required for analysis (3 microliters)

Quantify extracellular vesicles accurately and quickly

As extracellular vesicle (EV) research has matured, conventional technologies for their quantification such as NTA and TRPS have not kept pace to meet the increasing standards of rigor. Spectradyne's nCS1 is a fast and practical method that accurately measures the concentration and size of EVs. The microfluidic implementation of RPS avoids the operational difficulties and variability inherent in other, more simplistic embodiments of the technique, making the nCS1 robust and easy to use. As a result, Spectradyne's technology is quickly being adopted by EV researchers around the world.
  • Add rigor to your bioactivity assays by accurately quantifying your EVs
  • Assess the quality of your EV isolates at all stages of purification

Quantify gene therapy vectors and nanomedicines

Accurate quantification of gene therapy vectors and nanomedicines is critical for evaluating the bioactivity of these therapeutics. However, nanoparticle-based therapeutics often scatter light weakly, making accurate quantification by conventional light-based particle sizing techniques impossible. Assessing the concentration of viral vectors by biological titer takes significant time and resources. Spectradyne's nCS1 measures low-index-contrast particles such as these accurately, quickly and easily-obtain the viral titer or quantify dosing of your favorite nanomedicine in just a few minutes.
  • Characterize the gene therapy product that cannot be produced in large enough quantities for other techniques using the nCS1-just 3 microliters required
  • Skip the plating required for live biological titer, and get accurate concentration and size in minutes

Obtain detailed size distributions of industrial nanoparticles

Nanoparticles serve as functional ingredients in a wide variety of products, and their size distribution is a critical parameter that governs their performance. Spectradyne's nCS1 measures particles one-by-one and delivers quantitative, high-resolution size distributions that cannot be obtained with other technologies.
  • Learn what fraction of your nanomaterial is present as smaller 'fines' that are undetectable by light scattering-based metrologies
  • Directly count how many large, scratch-producing particles are in your polishing slurry

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Please continue to follow our blog as we share insights, technical details, and generally geek-out with you about nanoparticle science!

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