Spectradyne's particle analysis blog

When every particle countsTM

Extracellular vesicles exosomes nanoparticles gene therapy are all done here

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Table of Contents

December 29, 2020 - Using positive controls to ensure particle size and concentration accuracy

Let's face it, most particle analyzers are pretty much "black boxes": you put the sample into it, and sometime later you get a result, whether it be a true particle size distribution or just an average size as in the case of Dynamic Light Scattering (DLS). Sure, the "calibration" of any of these systems can be checked by running NIST-traceable polystyrene (or silica) beads mixed at a known concentration. However, the performance on any given instrument for these beads (in water or PBS) may not be representative of how the same instrument responds to your actual samples. This is especially true for optically based systems, where the refractive index (and perhaps viscosity or other factors) of both the sample particles and suspending medium can affect the instrument response.

One of the many advantages of Spectradyne's Microfluidic Resistive Pulse Sensing (MRPS) is that there is absolutely no dependence of the measurements on particle material and optical properties such as viscosity or refractive index. In fact, MRPS directly measures particle volume: this means that a particle of volume X made of solid gold will give the same measurement as a hollow particle of equal volume. So, in the case of an MRPS system, the performance when measuring calibration beads (of any material) is directly indicative of the performance of the instrument on any other sample particle, which is not true for optical systems.

Another advantage is small sample size: MRPS technology uses microfluidic cartridges that require very small volumes of sample, just 3 uL in the case of Spectradyne's nCS1, small enough that samples of tick saliva can be reliably measured!

A far larger advantage of MRPS, though, is that each particle is measured individually, one at a time. This means that the measurement of any given particle is completely independent of the measurements of other particles in the sample. This is not true in Nanoparticle Tracking Analysis (NTA) systems, where it can be shown that these instruments have a Limit of Detection (LOD) that varies based on sample composition and can cause spurious results.

What this means is that for measurements in an MRPS system, you can actually add calibrated spheres at a known concentration directly into your sample as a positive control. By doing this, you get verification in the sample that the instrument is accurate in both particle sizing and concentration.

The figure to right shows an example of how this works in practice. An extracellular vesicle (EV) sample was run twice in Spectradyne's nCS1TM MRPS system: The first time by itself, and the second time spiked with 150 nm NIST-traceable polystyrene nanospheres diluted to be 2.0 × 109 particles/mL in concentration. As can be seen, both runs of the sample overlay perfectly, while the spiked version shows a clear peak at 150 nm with a measured concentration of 2.04 × 109 particles/mL. This type of in-measurement control cannot be done with optically based systems because the sixth-order dependence of light scattering on diameter causes the spiked-in control beads to completely "mask" the instrument's response on the sample particles!

By using in-measurement, positive controls in an MRPS system, one can be assured of each measurement's accuracy for both particle sizing and concentration. This can be especially critical in regulated environments, where proving measurement traceability is greatly simplified by having in-measurement controls.

Can your particle analyzer do this?

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EV sample measured twice in Spectradyne's nCS1TM MRPS system: Once by itself and then with 150nm beads spiked in. The graph overlay visually shows repeatability of the two, while the spiked calibration beads in the second sample quantitatively proves measurement accuracy.

November 28, 2020 - Announcing Spectradyne's Video Library!

Spectradyne has been hard at work building a library of multimedia content. The remote-friendly materials contained in this library will more easily show our readers how our technology works, and how it can be used to make quantitative nanoparticle analysis much more quickly and easily.

The fruits of our efforts in this direction can now all be accessed in the Video section of our Library webpage! Want to see a quick overview of Spectradyne's technology and how it works? Experience a virtual demo of the nCS1TM in action? Maybe you would like to learn more about the microfluidic cartridges that make our technology so powerful, or view a scientific presentation from AIChE 2020.

Now you can do all of these things! Visit our Library and scroll down past the growing list of peer reviewed journal articles to where the videos are.

Check back often for updates!

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November 3, 2020 - Nanodefence® in my floor finish? What's that?

Coronavirus lockdown has spurred many a home improvement project this year. Did you refinish your hardwood floors? In case you used Varathane Nano Defence® (Canadian product photo at right, French side), you may be wondering — what exactly is the nano part about? And just how many nanoparticles am I getting in this gallon? Well, we measured it using Spectradyne's own nCS1! (Learn more about how the technology works here).

And for the record, we undertook this test completely independently from Varathane, and received no compensation from the company — we were simply curious!

The nano part: The particle size distribution in the sample is shown below at right. We found a clear peak in the particle size distribution around 75 nm — these are the aluminum oxide particles that lend the final finish the strength and durability claimed by the manufacturer. Spectradyne can attest to the validity of both these claims after raising children from age zero to five on the floor shown: No significant wear was observed after countless indoor minihockey games.

How many nanoparticles in this gallon? The total particle concentration in the size range spanning 60 to 100 nm diameter was measured to be 5.0 × 1014 particles/mL. That's a very high concentration, but since the particles are small they don't scatter light very strongly, and the product has a mostly transparent, slightly milky appearance before application. Incidentally, that these particles are weak light scatterers makes them difficult to detect and quantify by optical methods. In any case, using a conversion factor of 3785 milliliters per gallon... carry the five... we get roughly 1.9 × 1018 particles! Wow! That's a deal for $75/gal!

This experiment is part of our ongoing EveryDayTM nanoparticles series — read about more nCS1 measurements of commonplace materials here.

Do you have an interesting sample you would like us to measure? We demonstrate our technology to scientists by measuring a couple of samples for free — send us one!

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The floor finish tested in this study.


Particle size distribution in Varathane NanoDefence® floor finish. The particles with mode centered near 75 nm are the aluminum oxide nanoparticles added to the product to impart strength and durability. The secondary population at 150 nm diameter are polystyrene control particles added to the sample before measurement.

October 1, 2020 - How does Spectradyne's nCS1 measure concentration?

Spectradyne's nCS1TM uses Microfluidic Resistive Pulse Sensing (MRPS) for detecting particles and measuring their concentration as a function of particle diameter. MRPS is a modern and microfluidic implementation of the Coulter counting method, which has been a gold standard for counting large particles and cells in the clinic for decades.

MRPS works in a simple and very straightforward way:

  • The undiluted analyte is flowed through a measurement constriction (see figure to right)
  • Every particle over a well-defined size threshold is counted as it passes through the constriction
  • For each particle, the flow rate of sample volume is measured
Voila! That is all you need to calculate concentration as directly as possible: Divide the number of particles measured by the sample volume, also measured:
Concentration = (Number of Particles (measured))/(Sample Volume (measured))
Since the volume flow rate is measured every time a particle is detected:
  • Flow rate is measured continuously, often thousands of times per measurement
  • Any variations in flow rate over time are accounted for
  • Viscous samples are be measured equally well

Microfluidics offers additional important advantages:

  • The fixed-size sensing constriction and other fluidic structures are fabricated using high precision manufacturing techniques developed for the semiconductor industry — repeatable geometry ensures consistent measurements
  • Also, microfluidic channels are small! This means only 3 microliters of your precious sample are required for analysis

Other techniques for measuring particle concentrations make significant assumptions when reporting concentration. For example, Nanoparticle Tracking Analysis (NTA), which uses a microscope to take videos of particles diffusing randomly around, must assume that no particles drift into or out of the field of view or the focal plane during a measurement. In addition, NTA has to be able to 'see' the particles to begin with, which is a well-documented challenge. Other imaging technologies require immobilization of particles on a substrate before detection. Because the diffusion dynamics and affinity for particles to be captured on the imaging surface are complex functions of the sample composition and particle size, these methods can only estimate relative concentration. Dynamic Light Scattering (DLS) is an also-ran that ASTM standards themselves state is not quantitative for concentration at all.

Accurate measurements of concentration are critical for reducing measurement variability and performing well controlled scientific experiments. Listen to two leaders in their field explain why, in this recent webinar. We have also written about this topic many times before (e.g., in this blog post, and in this application note).

Learn how the nCS1's straightforward and accurate concentration measurements can help your work by sending us a demonstration sample or two!

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Fluorescent particles passing through the sensing constriction of a Spectradyne MRPS analysis cartridge. Particles flow from left to right in the image.

September 4, 2020 - What's in this sunscreen?

We've been wondering what's in the sunscreen we keep slathering on ourselves and our kids as we practice socially-distanced outdoor activities this summer. Let's find out!

Physical sun blocks commonly use sub-micron titanium dioxide (TiO2) and zinc oxide (ZnO) particles to scatter the sun's harmful UV rays away from skin cells. Other sunscreens use chemical blockers instead, such as avobenzone, octinoxate and oxybenzone, which absorb the light before it reaches the skin. Does this mean chemical sunscreens are so-called "nano-free"? Which sunscreens give more particle-bang for your buck?

We used Spectradyne's nCS1TM to measure the particle content of four different sunscreen formulations: Physical sun blocks SheerZinc by Neutrogena (SPF 50, Sample A), Coppertone Water Babies (SPF 50, Sample B), Alba Botanica Sensitive Mineral (SPF 30, Sample C), and the chemical blocker Pure Sun Defense (SPF 50, Sample D). Being a chemical blocker, sample D did not list the popular ZnO or TiO2 in its list of active ingredients.

Figure 1 at right shows the particle size distribution of each sunscreen formulation as measured by the nCS1. Strikingly, the chemical blocker Pure Sun Defense (Sample D) contains a higher concentration of nanoparticles on the 50 — 300 nm size range than the Coppertone Water Babies (Sample C)! Also interesting is that the Alba Bonanica Sensitive Mineral sunscreen (Sample C) contains a higher nanoparticle content than the Coppertone sunscreen (B), despite holding a lower SPF.

Finally, how do the particles stack up against dollars and cents? While nanoparticle concentration does increase with increasing cost among the physical blockers, you still get the most particles for your dollar with the cheap stuff (see the Figure 2 at right).

You may ask, "Who cares?", to which Spectradyne might respond, "Who says nanoparticle analysis can't be fun?"

This experiment is part of our ongoing EveryDayTM nanoparticles series — read about more nCS1 measurements of commonplace materials here.

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sunscreen compare

Figure 1. Particle analysis of four different sunscreens. Sample D is a chemical sun block, but surprisingly contains a higher concentration of nanoparticles than other physical blockers.

sunscreen compare 2

Figure 2. Number of particles between 50-300 nm diameter per dollar spent for the sunscreens tested. While sunscreen D, Pure Sun Defense, is a chemical blocker, it is significantly cheaper than the physical blockers that overtly contain nanoparticle based ZnO or TiO2, so the number of particles per dollar is highest.

July 30, 2020 - Saké or Soju?

Your dutiful correspondent was treated to a Japanese saké-tasting event while in Kyoto for the International Society of Extracellular Vesicles meeting in 2019. While the event was thoroughly enjoyable, all sakés tasted delicious to his unqualified tongue and he was left wishing for a more precise and quantitative metric with which to evaluate them.

Fortunately, Spectradyne's applications team delivered! Three types of saké, a Japanese rice-based fermented beverage, and three types of soju, a Korean liquor that is also historically rice-based, were obtained and analyzed on the nCS1.

Personal allegiances aside, one key difference between saké and soju is that soju is traditionally distilled after fermentation, while saké is not. Based on these differences in manufacturing process, saké might reasonably be expected to contain a higher concentration of particles than soju: Distillation of soju would eliminate proteins and particles from the final product, resulting in lower particle concentration.

While a nice story, this hypothesis was NOT supported by the measurement results (see the measurement results to the right)! Liquor samples of the same type exhibited similar particle size distributions to each other, but the distilled soju samples contained 10-20 fold higher concentrations of particles than the saké. Curiously, soju sample 3 exhibited a stand-out particle size distribution from the other soju samples...

Following this confusing result, we discovered that most soju is in fact no longer distilled in the traditional fashion, instead it is made by diluting and flavoring ethanol made from sweet potatoes, thus providing a likely explanation for our surprising result! Next step: Correlating the observed differences to taste.

This experiment is part of our ongoing EveryDayTM nanoparticles series — read more details of the measurements here.

And if you're in the liquor business (or any other of our application areas), send us a sample!

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sake and sashimi

Enjoy some sashimi with that (cold) saké!

sake-soju particle measurement

Check out the surprising difference between particle distributions in saké and soju (click on the plot for an enlarged view). Read why we think this data looks like this in the text.

July 20, 2020 - Why Microfluidics?

Spectradyne's microfluidic resistive pulse sensing (MRPS) is a modern implementation of a well-established technique generically termed "resistive pulse sensing" (RPS), also known as the Coulter principle. The Coulter principle has been the gold standard method for cell and other particle counting for decades, although until our recent innovations, its performance has been limited to the micron regime, above the size ranges where virus, extracellular vesicles (EVs) and nanomedicines live.

So how has Spectradyne advanced this well-proven gold standard to reach 50 nm and smaller? One answer is microfluidics! Embedding the fundamental RPS technique into a disposable microfluidic cartridge enables the incorporation of key engineering features that deliver powerful benefits users love.

  • Extremely small sample volume enables completely new science. Since only 3 microliters of your precious sample are required (or even less, if you've got good pipetting hands!), the nCS1 has been used to quantify extracellular vesicles (EVs) in insect saliva — try doing that with another measurement technique!
  • Embedded filters enable direct measurements of complex biological samples — including blood plasma! As an example, see this poster from Pfizer Inc., showing quantitative analysis of nanoparticles in mouse plasma. The upstream filters embedded into each cartridge enable smooth measurements of this type of sample over long measurement times.
  • Highly consistent cartridge features like the sensing constriction itself ensure accurate, repeatable results every time. Cartridges are produced using computer chip manufacturing technology, with highly controlled features as small as a few hundred nanometers on a side. This level of control eliminates arduous calibration procedures required for other implementations of RPS: Spectradyne's microfluidic analysis cartridges are pre-calibrated at the factory.

Learn more benefits of microfluidics in this technical brief.

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mrps cartridge

Spectradyne's microfluidic cartridge enables fast and reliable measurements using only 3 microliters of your precious sample.

June 22, 2020 - Importance of concentration for EV science

Controlling for concentration is the most important and critical aspect of extracellular vesicle research. By identifying and eliminating potential sources of variability in your EV isolation process, you will enhance the quality of your EV research.

We have covered this topic of accurate EV quantification in our previous blog post and application note. You can also learn how Spectradyne's MRPS system delivers quantitatively superior EV measurements with our recent webinar, including a special presentation by Zach Troyer, Ph.D. Candidate in Molecular Virology at Case Western Reserve University, entitled "Accurate concentration enables critical improvements in isolation of extracellular vesicles (EVs)."

Check out our webinar in full by registering here!

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June 8, 2020 - Recent publications enabled by Spectradyne's nCS1TM

Researchers increasingly turn to Spectradyne's technology to gain a deeper knowledge of their particles, from extracellular vesicles (EVs) to liposomes to viruses. The high-resolution and richness of information delivered by Spectradyne's Microfluidic Pulse Sensing (MRPS) allow scientists to monitor and verify cellular behavior and process development.

Recently, several papers published by scientists at the Research Centre for Natural Sciences (Budapest, Hungary) demonstrate the varied and important roles MRPS plays in many of their studies.

In one study, the size measurements from MRPS enable determination of the thickness of the protein corona and hydration layer in extracellular vesicles and synthetic liposomes.

Z. Varga, B. Fehera, D. Kitka, A. Wacha, A. Bota, S. Berenyi, V. Pipich, J.-L. Fraikin, "Size Measurement of Extracellular Vesicles and Synthetic Liposomes: The Impact of the Hydration Shell and the Protein Corona," Colloids and Surfaces B: Biointerfaces (in press, 2020)

In the development of liposomes containing copper for in vitro and in vivo anticancer activity, the same group relied on Spectradyne's MRPS to characterize and quantify their copper complexes for the optimization of the liposomal formulation.

A. Gaal, T. M. Garay, I. Horvath, D. Mathe, D. Szollosi, D. S. Veres, J. Mbuotidem, T. Kovacs, J. Tovari, R. Bergmann, C. Streli, G. Szakacs, J. Mihaly, Z. Varga, N. Szoboszlai, "Development and In Vivo Application of a Water-Soluble Anticancer Copper Ionophore System Using a Temperature-Sensitive Liposome Formulation," Pharmaceutics 12, 466 (2020)

Most recently, the group employed MRPS as a primary tool to investigate and qualify a new method for protein quantification of extracellular vesicles by ATR-FTIR.

V. Szentirmai, A. Wacha, C. Nemeth, D. Kitka, A. Racz, K. Heberger, J. Mihaly, Z. Varga, "Reagent-free total protein quantification of intact extracellular vesicles by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy," Anal. Bioanal. Chem. (2020)

Scientists at John Hopkins Center for Nanomedicine (Baltimore, MD) studying mitochondrial function in retinal pigmented epithelial (RPE) cells used MRPS to quantify differences in EV formation in cells with diminished mitochondrial capacity. The EVs may be a diagnostic biomarker for monitoring the spread of degeneration.

J.Y. Ahn, S. Datta, E. Bandeira, M. Cano, E. Mallick, U. Rai, B. Powell, J. Tian, K.W. Witwer, J.T. Handa, M.E. Paulaitis, "Release of extracellular vesicle miR-494-3p by ARPE-19 cells with impaired mitochondria," BBA - General Subjects (in press, 2020)
doi: 10.1016/j.bbagen.2020.129598

Spectradyne's technology is affordable, easy to use, and easy to adopt. Want to learn more about how can improve your science? Contact us to arrange a demonstration.

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May 22, 2020 - Fascinating Virus Measurements with Spectradyne's nCS1TM

Virologists use Spectradyne's nCS1TM particle analyzers to gain a deeper understanding of their virus particles. The rich information delivered by the nCS1TM enables researchers to characterize and accurately quantify their virus particles, leading to significant time savings and reduced process variability.

The nCS1's ability to detect a wide variety of virus types, even in complex biological media, provides for many interesting and unique measurement results. Here we showcase a few notable virus measurement examples.

In the first example, shown to the right in Fig. 1, a myxoma virus sample was tested in its cell culture medium with little sample preparation. Myxoma viruses are brick-shaped virions of about 200 nm to 300 nm in diameter. The nCS1TM was able to detect the virus population at 260 nm above the high concentration culture media background.

The second example, shown in Fig. 2, is a comparison between a wild-type flu B virus (WT) and a mutant form. Each sample was spiked with 208 nm polystyrene beads as a positive control. The wild-type sample shows the virus particles having a mean diameter of 100 nm. The mutant virus exhibits no evidence of a peak in the distribution over the same size range, suggesting the mutation affects capsid formation. This type of data can therefore offer crucial information for scientists in studying the effects of mutations on the virus.

The third interesting example is a measurement of maraba virus, shown in Fig. 3. The maraba virus particle is reported by TEM to have a cylindrical diameter of ∼65 nm and a length of ∼180 nm.

The nCS1's non-optical electrical sensing technology directly measures particle volume — you can read more here. Optical techniques must assume a spherical shape, then infer the particle diameter from some other physical property being measured. By measuring the particle volume directly, as is done by Spectradyne's nCS1TM, the particle's equivalent spherical diameter (ESD) can be calculated.

The dimensions of the maraba virus particle give a cylindrical volume and an ESD of a sphere of same volume measuring approximately 100 nm. As seen in the graph, this is exactly what the nCS1TM measures!

In earlier posts we've described how the nCS1TM provides a fast and easy method for measuring viral titer, and how it can be used to improve viral purification processes. As the above examples further demonstrate, Spectradyne's technology also delivers unique insight into the physical characteristics of the virus, providing much richer information that cannot be obtained with other methods.

Spectradyne's technology is affordable, easy to use, and easy to adopt. Want to learn more about how it saves virologists' time? Visit our virus applications page here.

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virus fig 1

Figure 1. Measurements of a myxoma virus sample, tested in its cell culture medium with little sample preparation.

virus fig 2

Figure 2. Comparison between a wild-type flu B virus (WT) and a mutant form. Each sample was spiked with 208 nm polystyrene beads as a positive control.

virus fig 3

Figure 3. Measurement of a maraba virus. The maraba virus particle is reported by TEM to have a cylindrical diameter of ∼65 nm and a length of ∼180 nm.

May 12, 2020 - What is "Concentration Spectral Density"?

Many people look at our particle concentration graphs and ask "why is the vertical axis in units of particles/mL/nm? What does that mean?" We call these distributions Concentration Spectral Density (CSD); this represent the number of particles per unit sample volume (measured in mL) per unit particle diameter (measured in nm). By including the "per unit particle diameter" part, one creates a histogram that is already normalized by the bin size, meaning that this histogram can be easily compared with other histograms. If you want to calculate the absolute concentration of particles in a range of particle diameters, you integrate (in other words, sum) over the desired range, generating a size histogram with the bin widths you desire. That summed result would be a histogram of the number of particles per ml for the bin size you chose.

If you encounter a particle size distribution that only uses particles/mL on the vertical axis, then the data has already been binned for some defined bin size (and you may not know what that size is without further digging). If you want to compare this binned distribution with another distribution, you have to find out what bin size was used for each distribution, as the concentration at any point on the graph (the vertical axis value) depends on the bin size that was used.

We use CSD at Spectradyne so that we can quickly and easily compare results from multiple runs/samples/cartridge sizes directly, without having to worry about bin size and without having to do any conversions. Note that the CSD is in this way not only independent of bin size, but it is also independent of the cartridge parameters used for the measurement. The plot shown to the right illustrates this, showing two different samples that were each run in two different size cartridges, and yet the results are immediately comparable in the software without further calculations. Note the cartridges each cover a different nominal size range, allowing us to cover almost a 100-fold range in particle diameter (a million-fold range in particle volume!), yet the measurements overlap very well where the cartridge ranges overlap. Note also that the cartridges only use 3 μl of sample. You can read more about our cartridges here.

If your particle analyzer is not plotting CSD, ask the vendor why! You will need to understand how their data is binned in order to make valid comparisons of distributions. At Spectradyne, we take care of all of that for you, by always using CSD.

Spectradyne's technology is affordable, easy to use, and easy to adopt. Want to learn more about how it saves researchers' time? Visit our applications page here.

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CSD two examples

Figure showing measurements of two different samples, each measured in two of Spectradyne's disposable microfluidic cartridges.

April 20, 2020 - Rapid Viral Titer with Spectradyne's nCS1TM

Measuring the viral titer, the concentration of viruses in a sample, is an important procedure in virology. The modern fast pace of research and the current race to develop a vaccine for the COVID-19 pandemic highlight the necessity for a rapid and easy methodology for virus quantification.

Conventional approaches to viral titer are time-consuming, labor intensive and often suffer from poor reproducibility. Plaque titer and fluorescence focus assay (FFA) techniques require serial dilutions of the virus stock and the waiting period for cell infection and incubation can last for days. Other methods such as quantitative polymer chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) can be completed in hours but qPCR is sensitive to contamination and ELISA is highly selective and necessitates the prior knowledge of specific antibody-antigen interaction. Transmission electron microscopy (TEM) quantification is expensive and requires tedious sample preparation and a highly skilled operator.

Spectradyne's microfluidic pulse sensing (MRPS) technology delivers a complete and accurate analysis within minutes and consumes less than 3 microliters of sample. The Spectradyne's nCS1TM system utilizes disposable microfluidic cartridges, eliminating cross-contamination between samples and allowing for rapid testing of high number of samples.

With non-optical electrical sensing technology, the nCS1TM can detect and quantify virus particles directly in complex biological samples, saving substantial amount of sample preparation time. The system is practical, highly automated and easy to use.

Comparison table of the time and labor cost of virus titer techniques:

Technique Method Reproducibility Time Labor Cost
MRPS (nCS1) Viral particle Excellent Minutes Low Inexpensive
Plaque titer Infectivity assay Poor Days High Inexpensive
FFA Infectivity assay Poor Days High Expensive
qPCR Viral nucleic acid Excellent Hours Moderate Expensive
ELISA Viral protein Good Hours Moderate Inexpensive
TEM Viral particle Excellent Weeks High Expensive
The virus measurement examples to the right showcase the nCS1's ability to detect a broad variety of viruses, from human adenovirus and HIV virus to murine leukemia virus. The size and concentration of the virus sample was measured accurately and rapidly within minutes.

Spectradyne's technology is affordable, easy to use, and easy to adopt. Want to learn more about how it saves virologists time? Visit our virus applications page here.

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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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human adenovirus

Human adenovirus sample characterized with Spectradyne's nCS1TM.

hiv human immunodeficiency virus

Human immunodeficiency virus (HIV, inactivated) characterized with Spectradyne's nCS1TM.

murine leukemia virus

Murine leukemia virus sample characterized with Spectradyne's nCS1TM.

April 10, 2020 - Using Spectradyne's nCS1TM for validating a virus purification process

Tight process controls are required to ensure high purity virus preparations, in an ever-increasing range of applications that include vaccines, viral vectors for gene therapy and oncolytic immunotherapy.

Existing purification methodologies, i.e. ultracentrifugation, precipitation, chromatography and density gradient, are selective and do not work for all viruses. In establishing an effective purification protocol it is therefore necessary to investigate each step to establish the most suitable scheme for isolating virus from impurities in its growth environment (e.g., culture media or cellular contaminants).

Fast and accurate quantification of virus after each purification stage enables quick feedback that informs decisions and validates the efficiency of the chosen purification approach.

Spectradyne's nCS1TM is an advanced, easy to use microfluidic technology that delivers a rapid assessment of the quality of the virus preparation. Complete and accurate sample analysis is obtained in minutes-without cell culture-and requires only 3 microliters of sample.

You can read our previous blog post detailing the value of the nCS1 for virus quantification.

The example shown to the right illustrates how the nCS1's high quality data provides crucial information to researchers in the assessment of the effectiveness of a sequential virus purification strategy.

The initial preparation exhibits no evidence of a viral population over the measured size range, 125-600 nm. The first enrichment step, Purified 1, fails to isolate the virus particles above the culture media background. However, the Purified 2 sample exhibits a clear peak at 230 nm and demonstrates that the second purification step does a dramatically better job of purifying the virus particles.

Spectradyne's nCS1TM is a perfect tool for process development of virus products — it delivers rapid results while being affordable, easy to use, and easy to adopt.

Want to learn more about how it saves virologists time? Visit our virus applications page.

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virus enrichment

Quantitative analysis of sample purification steps using the nCS1TM enable better informed decisions and speed up R&D.

April 2, 2020 - Using Spectradyne's nCS1TM for virus quantification

With the current COVID-19 outbreak, it is more critical than ever for scientists to rapidly and accurately quantify viral concentration for the research and development of vaccines, viral antigens and antiviral agents. Accurate concentration measurement of the virus particles is essential in the assessment of viral bioactivity and loading efficiency.

Conventional methods for quantifying virus such as live biological titer, fluorescent focus assay (FFA), quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) are time-consuming, labor intensive, and require large amount of sample volume. And optical particle counting techniques such as Nanoparticle Tracking Analysis (NTA) don't have the sensitivity required to accurately count virus particles, since virus are much smaller than the optical diffraction limit and have low refractive index contrast relative to the surrounding aqueous medium.

In contrast (pun intended!), Spectradyne's nCS1TM instrument uses electrical sensing (non-optical) to detect virus particles in state-of-the-art disposable microfluidic cartridges. The system is practical and easy to use-delivering total virus count in minutes-and the microfluidic implementation requires only 3 microliters of sample for complete and accurate analysis, saving precious analyte volume. The electrical measurement does not require shining light through the sample, so virus can be quantified directly in complex biological samples such as cell culture media or plasma.

The microfluidic cartridges are single-use disposables, eliminating the risk of cross-contamination between samples and the time-consuming task of cleaning in between sample runs.

The precision in concentration measurement achievable in the nCS1TM is demonstrated in a serial dilution experiment shown to the right. The 4-point dilution series was performed on a retrovirus drug formulation for a gene therapy company.

The viral population was readily detected as a prominent peak over a broad background of other particles. The viral concentration decreases linearly as the dilution factor increases, as expected: Plotting the relative concentration measured across the entire range against the dilution factor, we get a correlation coefficient of 1.00.

The nCS1TM accurately quantitates virus particles in complex biological samples and exhibits highly linear response to concentration.

Spectradyne's technology is affordable, easy to use, and easy to adopt. Want to learn more about how it saves virologists time? Visit our virus applications page.

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virus dilution series

Virus concentration measurement as a function of particle diameter, for different dilution levels.

virus dilution scaling

This plot shows the scaling of measured viral concentration as a function of dilution, showing the expected unit scaling.

March 26, 2020 - Do you measure your backgrounds?

In many analytical sciences, quantifying the background signal (or blank) before measuring the sample of interest is an important part of experimental design, and a critical requirement for ensuring meaningful results. A positive test signal must be compared to the background signal level in order to assess its significance and to exclude the contribution of the background from the interpretation of results. For example, in an enzyme-linked immunosorbent assay (ELISA) it's important to measure the signal from a negative control reaction that contains no target antigen, to obtain the baseline signal for other reactions.

Not everyone considers the importance of background measurements in particle analysis, though the same fundamental principles hold true. A common scenario where this is relevant is when the sample of interest must be diluted before measurement — in this case the particle content of the diluent itself must be measured and accounted for in subsequent analysis. The measured background level can also inform the measurement protocol: For example, if the sample of interest is at a concentration of 1×109 particles per mL and the background in the diluent contains 1×108 particles per mL in the same size range, then it's clear the sample cannot be diluted more than ten-fold before the measurements are no longer meaningful. These experimental bounds can only be obtained through careful measurements.

Control experiments like these are not often considered in particle analysis because of the limitations of commonly-used technology. Most techniques, including optical ones like Nanoparticle Tracking Analysis (NTA) and Dynamic Light Scattering (DLS), are not additive. For these techniques, measurement of the combined sample A + B is not equal to the sum of the measurement of sample A and the measurement of sample B. Most researchers understand why Dynamic Light Scattering is not additive — it's because DLS only reports a skewed average of all the particles in the sample. More surprisingly though, since it's billed as a single-particle measurement technique, Nanoparticle Tracking Analysis isn't additive either. We and others have shown this pretty clearly, and you can easily test for yourself the next time you're in the lab (or maybe even working from home if you're so equipped!).

Spectradyne's nCS1 measures particles one-by-one, each particle truly independent from the others. As a result, Microfluidic Resistive Pulse Sensing (MRPS) is a true additive measurement technique, and thus holds enormous power for quantifying particles in complex media such as plasma, urine, or cell culture medium. Shown on the right is a case in point: a nanoparticle therapeutic needed to be quantified directly in plasma; subtracting the plasma background from the total sample yielded a direct and accurate measurement of the therapeutic particles themselves. Direct measurements in such complex media are fast and easy with MRPS — read about a pilot study we performed with Pfizer Inc., here.

Food for thought while working from home during COVID-19 isolation. Take care!

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A 96-well ELISA plate. An antibody linked to an enzyme will recognize a target protein in each well. When a colorimetric substrate is added to the sample, an enzymatic reaction causes a color change. Photo credit: Shenzhen Boomingshing Medical Device Co., LTD


This plot shows differential measurements made using Spectradyne's nCS1TM of blood plasma with and without a nanoparticle therapeutic.

March 10, 2020 - Spectradyne's nCS1TM and the virus that causes COVID-19 (aka "coronavirus")

Spectradyne, and Spectradyne's staff, are very concerned about the COVID-19 outbreak and its implications for people's health world-wide. While none of our staff are currently ill or have tested positive for exposure to the virus, we are watchful and encourage all to follow best practices to avoid contracting this disease and spreading it to others.

As a scientific problem, the virus that causes this disease, known as "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2), or alternatively "2019 novel coronavirus" (2019-nCoV), presents an interesting challenge. It was first sequenced by scientists in China, and later by other laboratories around the world. It is a single-strand RNA encapsulated virus closely related to the virus that caused SARS, a disease that was detected in humans in 2002-2004 but has not reappeared since. TEM images of the virus can be found on the web, such as the one shown to the right, showing SARS-CoV-2 virus emerging from the cells of a human patient.

From a physical characterization standpoint, SARS-type coronaviruses are interesting: Coronavirus is enveloped and pleomorphic (variable size), with a "corona" of spike glycoprotein structures on its surface. The result is a polydisperse particle size distribution that spans approximately 80-120 nm in diameter, well within the size range of detection accessible by Spectradyne's nCS1. Researchers have used the nCS1 to quantify a broad array of virus, including influenza, HIV, lentivirus, adenovirus, adeno-associated virus (AAV), human simplex virus (HSV), murine leukemia virus (MLV) and many others including bacteriophage.

In one of the first demonstrations performed with the MRPS technology, we demonstrated the detection of T7 bacteriophage, a bacterial virus commonly used for phage display of random peptides, with a diameter of 55-65 nm in diameter, comparable to that of non-enveloped small viruses such as Hepatitis C (with a diameter of 55 nm). Bacteriophage concentrations are typically determined after amplification, concentration and purification of the phage, using biological titer, a process which typically requires a few hours. MRPS was used for the direct, rapid (few seconds) measurement of the size and concentration of T7 phage, an all-electronic analysis that does not require infectivity-based titration.

Spectradyne's nCS1TM could therefore easily be applied to the measurement of the diameter and concentration of SARS-CoV-2, although of course this should only be done in a properly equipped, BSL3 laboratory. We'd encourage researchers with these capabilities to consider the nCS1 as a rapid means for quantifying this virus.

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This scanning electron microscope image shows SARS-CoV-2 in yellow (also known as 2019-nCoV, the virus that causes COVID-19) isolated from a patient in the U.S., emerging from the surface of cells (blue/pink) cultured in the lab.

March 5, 2020 - Spectradyne's nCS1TM Adds Value to Flow Cytometry

Recently Spectradyne has heard positive feedback from its customers that also use flow cytometry to quantify extracellular vesicles (EVs) or other particles in biological fluids such as plasma. These customers find value in Spectradyne's Microfluidic Resistive Pulse Sensing (MRPS) for three main reasons worth highlighting:

First, the non-optical detection method used by the nCS1TM delivers particle concentration and size information that can't be obtained from flow cytometry. Accurately measuring the concentration of biomarkers and therapeutics such as extracellular vesicles, liposomes or synthetic nanoparticles is critical for reducing variability and drawing clear experimental conclusions (see more discussion here). As we've written elsewhere, the nCS1TM provides more accurate concentration and size measurements of small particles in heterogenous biological samples like serum, cell culture media, and most EV preparations. Using both fluorescence-based flow cytometry and MRPS provides a unique and complete sample analysis that cannot be obtained with flow cytometry alone.

Second, the nCS1TM provides a fast and easy method for quantifying sample purity. While fluorescence-based flow cytometry can be a powerful technique for counting specifically tagged particles in complex samples, it has difficulty measuring the un-stained particles that may also be present as contaminants in the sample. Orthogonal measurements are important! Measuring just 3 microliters of the same sample with the nCS1TM delivers the total particle count and size-the missing piece for determining sample purity.

Finally, Spectradyne's technology saves significant time and cost in preparing samples for flow cytometry analysis. Flow cytometry measurements are sensitive to total particle concentration: Coincidence of multiple particles in the beam (known as "swarming") is difficult to detect and can generate incorrect and misleading results. A quick analysis with Spectradyne's nCS1TM provides the total concentration of particles in the sample-critical information required to ensure the correct dilution factor is chosen for flow cytometry the first time around.

Spectradyne's nCS1TM delivers accurate concentration and size of particles in diverse biological samples, and by virtue of its non-optical detection method provides new insights into sample composition that cannot be obtained by optical methods such as flow cytometry alone. Learn how you can enhance your flow cytometry analysis here.

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February 19, 2020 - Spectradyne at PepTalk 2020

Spectradyne once again participated in the annual PepTalk: The Protein Science Week. This conference is held each year in San Diego, and brings together experts from global pharma, biotech, academic, and government institutions. It features several interrelated technical tracks, including "formulation & stability" and "analytics & impurities", which were of particular interest to Spectradyne customers.

Even though protein aggregation has been a hot topic for several years, it is surprising that many researchers still have a "measurement gap" in their laboratories, between very small aggregates (monomer to 50 nm) characterized by chromatographic methods, and very large aggregates (larger than 2 μm) characterized by flow imaging or light obscuration. This is the exact size range that the Spectradyne nCS1TM fills perfectly, so we continue to be well-received by people who realize that measurements in this size range are important to get right. At our booth and with our poster, "Submicron Protein Aggregation Measurements for Early Assessment of Formulation Instability", we continued to stress that detecting aggregation earlier can save formulation scientists considerable time and money early in the formulation process. This message continues to be well-received, especially as the FDA is now emphasizing the importance of these characterizations in both the IND (Investigational New Drug) and NDA (New Drug Application) application processes. The fact that the nCS1TM only requires 3 μL of sample is also critical, as in many cases of the early formulation process, sample volumes can be as small as 20 μL total.

What seemed new at this year's event was that there was quite a bit of discussion on the topic of alternative delivery methods for biologics. In particular, many people were talking about using viral vectors (AAV, lentivirus, etc.) and engineered extracellular vesicles (EVs) as delivery mechanisms for biologics. There is much interest in more effective treatment of diseases of the central nervous system (CNS), which require a mechanism to cross the blood-brain barrier, which viral vectors and EVs are capable of. The nCS1TM is also well-suited to characterizing viral vectors and extracellular vesicles, so can help researchers in this area as well. Of particular importance is correctly characterizing sample concentration, as this corresponds to dosage. The nCS1's electrical detection method provides superior concentration measurements versus other (especially optical) methods, in minutes as opposed to hours for classical assay-based titers.

We saw many existing customers, and spoke to many new prospects as well at this event. Participation in events like this are an excellent opportunity for us to learn what the current and future needs of this market are, so that we may better tailor our technology to aid in the advancement of this important science.

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January 13, 2020 - Spectradyne attends Applied Pharmaceutical Nanotechnology 2019

Spectradyne was pleased to sponsor (for the second year in a row) the one-day Applied Pharmaceutical Nanotechnology event held in late October at Pfizer in Cambridge, MA. This annual event, put on by the non-profit, The Boston Society, brings together experts from industry, academia and regulatory to discuss topical issues related to uses of nanotechnology in pharmaceuticals.

Spectradyne jointly presented a poster with Pfizer, entitled "Simulation of Label-Free PK Evaluation of Nanoparticles in Complex Media". The poster detailed work done jointly between Spectradyne and Pfizer to look at the potential of using Spectradyne's nCS1 as a way of quantifying nanoparticle drug products in a complex biological matrix such as plasma, enabling label-free PK evaluation. The poster can be downloaded from Spectradyne's website here.

Greg Troiano, VP of Technology and Chief Engineer from Seer, Inc., once again gave the initial plenary talk, updating us on the progress of this company over the past year. Seer is bringing to market some very exciting technology to analyze the proteome "to enable novel insights and breakthroughs in the understanding of biology and disease." They design multiple nanoparticles to sample the proteome, enabling close monitoring of the presence and progression of specific disease markers, which can be used to drive treatment decisions.

One of the speakers also gave a sneak peek at an announcement (officially made a month later) of a public/private consortium committing to build a new center for advanced biological innovation and manufacturing in the Metro Boston area. The center, slated to be completed in late 2021, will further cement the greater Boston's leading position in cell and gene therapies, by providing production facilities to address the current shortage of raw materials necessary for research efforts. Currently, "Researchers have to wait up to 18 months for overburdened commercial manufacturers to produce engineered cells and viral vectors needed for their work, slowing the pace of knowledge development."

Overall, this event was information rich, and provided an excellent forum for free discussion of many of the challenges facing people working in Applied Pharmaceutical Nanotechnology. Interesting and exciting new work was presented, showing that the future for these technologies is indeed bright!

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November 19, 2019 - Accurate EV Quantification

Spectradyne was pleased to participate in the inaugural event of the Extracellular Vesicle (EV) Working Group on October 29, 2019 at the Research Institute of the McGill University Health Centre (RI-MUHC) in Montreal, Canada. We would like to thank our hosts, Dr. Janusz Rak and Dr. Peter Metrakos of McGill for the invitation to sponsor, and for assembling a group of scientists advancing such exciting work in the field of extracellular vesicles from across Quebec.

One special aspect of this meeting was that the organizers intentionally and successfully created a we're-all-still-learning atmosphere that allowed scientists that are new to extracellular vesicle research to present their work to an open-minded audience. This environment allowed for creative and supportive discussion between the more experienced EV researchers and the newly minted ones, and also provided a safe space for discussing areas of disagreement.

In fact, a 30-minute panel discussion chaired by Dr. Rak was included in the morning agenda to discuss controversies in the field-and as a new and rapidly expanding field, extracellular vesicle research turned out to have more than 30 minutes' worth of controversies to talk about!

One fire-starting topic that Spectradyne has spoken on at many venues, including in this blog, and is always happy to discuss more, is the importance of using orthogonal methods to quantify extracellular vesicles. Now, because the Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018) guidelines clearly require using at least two techniques to quantify vesicles, you might not think this would be such a hot-button issue. However, relying on a single method such as Nanoparticle Tracking Analysis (NTA) to quantify extracellular vesicles is still commonplace in the field. And despite a growing awareness of its critical limitations, many researchers are still surprised when they find out that Nanoparticle Tracking Analysis is in fact grossly misrepresenting the content of their samples.

Why does accurate quantification of extracellular vesicles matter?

Here's an example: Suppose a researcher would like to compare the therapeutic effect of EVs or other nanoparticles such as liposomes or gene therapy vectors that have been prepared with different methods. Each therapeutic agent is to be applied to a biological replicate of a test system (e.g., cultured cells) and the response of that system is to be quantified by measuring certain parameters such as viability, cell morphology, differentiation activity, or other indicator. What scientist, given the technology to do so, would consider performing such an experiment without first measuring the dose of each product that was applied? Without controlling for the quantity of therapeutics agent applied to the test system, important differences between the effectiveness of the different preparations may remain hidden by excess variability.

No matter the downstream measurement, extracellular vesicle concentration is always a critical experimental variable and must be carefully controlled to ensure clear experimental outcomes. Learn more about how our technology delivers this ability here.

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October 18, 2019 - Observations from ASEMV 2019

Last week Spectradyne attended ASEMV 2019 in Monterey, California, both as an exhibitor and as a presenter. We were truly impressed by the enthusiasm of the EV researchers and by how fast the field is advancing. Scientists described their work on all manner of diseases including brain cancer, Alzheimer's, and atherosclerosis (to name a few), with extracellular vesicles being studied both as key biomarkers and as therapeutic agents.

One thing we noticed at the meeting was a less dogmatic approach to what is an "EV" vs. an "exosome". At past events we've heard scientists talk about stringent definitions and the importance of knowing the origin of different vesicle types. While those topics were still present, and while it's certainly ideal to know more details about one's vesicles, there seemed to be an acknowledgement that this detailed information is often not obtainable, and probably wouldn't be for some time. Biology is exceedingly complicated, and this was a practical admission that certain details didn't need to be the focus of research. At least not yet.

With so many brilliant scientists working to make a positive impact on some of humanity's biggest health challenges, Spectradyne was gratified to have a small part in the ASEMV conference. Our talk focused on the importance of orthogonal techniques in characterizing vesicle mixtures, a topic that has been addressed in this blog before: see "The benefits and perils of orthogonal measurements."

This topic is truly crucial for the EV community because, unfortunately, we continue to see spurious data in the talks of both new and established researchers alike. In many of the talks at ASEMV 2019, scientists presented information about the detection of protein markers through various assays. On a subsequent slide they would then show an image of a vesicle size distribution with a nice well-defined peak, usually measured by NTA, with a comment similar to "and NTA confirms an exosome size of xx nm". This statement was voiced as tacit confirmation that not only were the vesicles in question present (probably true), but that they were well-purified in the formulation (probably not).

As followers of Spectradyne's MRPS technology know, these peaks are not infrequently false peaks and without some extra characterization, such as an orthogonal measurement, the purification claim must be greeted with skepticism.

In the presence of the hard-working group of scientists at ASEMV 2019, Spectradyne is justifiably humble - we aren't experts in any of the biology being presented! However, the purification question is an important one to get right. Many researchers are essentially drawing correlations between an observation (perhaps a therapeutic outcome) and an input (the presence of a certain type of EV). Without a clear understanding of the quality of the input, such a correlation can be easily mis-attributed. If that happens, then improvement of the formulation, and of the scientific outcome, cannot be well controlled (though a researcher can always get lucky), and advancement of scientific knowledge can be delayed.

Thankfully, this message truly resonated with the researchers who heard our talk. We know better than most people how difficult measurements of biological nanoparticles can be, and we know how tempting it can be to believe a nice well-behaved peak in a size distribution. However, we can all agree that however tempting that pretty peak is in a publication, it's more important in the end to get the science right.

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October 6, 2019 - Are you really measuring your particle sizes? With MRPS, the answer is "yes"

One of the major benefits of Microfluidic Resistive Pule Sensing (MRPS) is that MRPS directly measures particle volume. This sizing method is quite different from light-scattering based techniques, which first assume that all particles are spherical in shape, and then infer particle diameter from some other physical property being measured (e.g., mean squared displacement of diffusion).

In MRPS, the measured electrical response is directly proportional to the volume of the particle occluding the nanoconstriction. In order to easily display particle size results, however, the volume of each particle is converted to an Equivalent Spherical Diameter (ESD). Particle concentration is then plotted against ESD, as is typical for all particle analysis systems. The important thing to remember is that in RPS, the particle size (volume) is measured directly and stored, whereas in light scattering systems particle size is calculated from diffusion behavior assuming particles are spherical.

To show how this characteristic of MRPS can manifest, the following example may be useful. A customer-supplied virus sample was measured in the nCS1 MRPS system. A clear peak in the distribution is seen for the monomer at 136 nm (see figure to right). A small secondary "hump" can also be seen at around ~170 nm, as shown to the right in the volume-weighted particle size distribution.

The hump in the data seen at 170 nm indicates the presence of virus dimers. Remembering that the nCS1 measures volume, and that ESD scales as the cube root of volume, a dimer will have twice the volume of the monomer, corresponding to ~1.25 times the ESD of the monomer. In this case, with a monomer at 136 nm ESD, the dimer appears right where it is expected, at 170 nm.

Another interesting example is provided by a customer-supplied virus that was cylindrical in shape. The customer had TEM data showing the virus to be a cylinder of diameter ~65 nm and length of ~180 nm. Given these dimensions, we calculate the volume of the cylinder, and then the ESD of a sphere of same volume, which predicts an ESD of approximately 100 nm for the virus. As seen in the second graph to the right, this is exactly what the nCS1 measures!

The nCS1 MRPS system offers higher resolution to begin with, but the fact that it measures volume directly also yields more valuable insight into particle size distributions as well!

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monomer and dimer virus particles

A virus sample measured with Spectradyne's nCS1TM, showing monomer and dimer virus particles.

cylindrical virus

Cylindrical virus particles plotted in terms of ESD.

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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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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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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