Two new characterization platforms reveal that there’s more than
one way to size up
a nanoparticle.
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The microfluidic resonator in Affinity Bioscience's Archimedes particle characterizer is so small it can detect the frequency change caused by particles moving through the channel. Image: Affinity Biosciences
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Once upon a time, when particles were measured in micrometers and an optical microscope
could do the heavy lifting, the size and distribution of particles in a solution were
discovered through the strength of a researcher’s eyesight.
To see the truly small particles, however, we needed to invent less direct solutions. After
the invention of the laser, researchers determined that Rayleigh scattering could accomplish
this task. As long as a particle is relatively small when compared to a given wavelength of
light, it will scatter light in all directions. If it’s highly-collimated light—a laser—it can
be measured accurately, and the particle characteristics can be determined by watching how the
intensity of reflect light changes over time. It’s guaranteed to change, too, because all such
small molecules are constantly moving about in Brownian motion.
This method, dynamic light scattering (DLS), has become the gold standard for
characterization of very small particles since its invention not long after the laser. It is
not, of course, the only way to analyze particles. Attenuators, sieves, filters, and
centrifuges are all employed for larger particle sizes. But for analyzing samples containing
broad distributions of species of widely differing molecular masses (such as proteins and
aggregates), DLS is reliably the cost-effective choice.
But the demands of R&D have pushed DLS to its diffraction limit. While excellent for
collecting data on the dynamic properties of softer materials, DLS is often unable to cope with
the scattering properties of large particles or those with a large refractive index (such as
gold). Such particles cause multiple scattering, especially in high concentration, clouding the
data.
Compensatory techniques like cross-correlation light scattering, or 3-D dynamic light
scattering, can isolate the scattering of interest. Additional technologies have allowed DLS to
accommodate high-throughput analysis, or even obtain information about Zeta potential. And
newer variations, such as photo-correlation spectroscopy (PCS), use Stokes-Einstein equations
and diluted sample solutions to enhance DLS sensitivity. However, new methods have hit the
market that may soon give DLS a run for its money.
In Brownian motion
Robert Brown first observed the seemingly random motion of small particles in a solution more
than a century ago. His long-accepted theory as to why such particles constantly move has
frequently been described by envisioning particles as large balloons moving over a stadium
crowd. Multiple people push up on the balloon at once and direct it in seemingly random ways.
This continuous-time stochastic process is detected in ensemble fashion by DLS, either with a
photomultiplier or CCD detector.
However, researchers also want to know the size and location of individual nanoparticles in
solution. Nanoparticle tracking analysis (NTA), invented by Bob Carr, now the CTO of
NanoSight, Amesbury, UK, is the first technology that can directly track the
Brownian motion that makes DLS work.
“The big difference between the two methods is that dynamic light scattering is an ensemble
technique and nanoparticle tracking works on a particle-by-particle basis,” says Jeremy Warren,
CEO of NanoSight. With DLS results, he says, one piece of data that describes particle size
distribution must be used to draw out a much more complicated situation.
“What you end up with is something like an average that is biased toward larger particles,”
he says.
NTA, however, builds data by tracking the speed of individual particles through the
solution. Carr, who is now NanoSight’s CTO, accomplished this by sending a laser light through
a cell of suspended liquids. That laser light produces a scatter pattern.
“We’re not really producing a resolved image where you can see the shape of each particle.
Instead we a producing a visualization of the scatter that’s produced,” he says. This is shown
by way of a series of bright spots that are dictated by the size of the particle and its
refractive index. Big particles look bigger, and are easily visualized. Under the Brownian
motion that is being exhibited, small molecules move quickly, and large molecules move slowly.
NanoSight’s NTA is able to measure the speed of the particle by taking several tens of video
frames. The speed is directly proportional to the presented particle size, and these are then
are presented as a particle size distribution.
The technology relies on a 635-nm laser beam passed through a prism-edged optical unit that
causes the laser to refract at the interface between the flat optic and a liquid layer placed
above it. This compresses the beam within the liquid film that contains the sample. A 20x
magnification microscope objective is fitted to an otherwise conventional microscope, which is
then mounted on a 30 fps CCD.
Each particle is simultaneously, but separately, visualized and tracked by software. The
average distance each particle moves in x and y in the image is automatically calculated. From
this value, the particle diffusion coefficient can be obtained and—knowing the sample
temperature and solvent viscosity—the particle’s hydrodynamic diameter identified. The use of
Stokes-Einstein equations reveals 3-D Brownian movement even through it is tracked in only two
dimensions.
The range of particle sizes that can be analyzed by NTA depends on the particle type. For
very high refractive index (Ri) particles, such as colloidal gold, accurate determination of
size can be achieved down to 10 nm diameter. For lower refractive index particles, such as
those of biological origin, the smallest detectable size might only be between 25 to 35 nm,
about the size of a virus. Upper size limits are approached when the Brownian motion of a
particle becomes too limited to track accurately, typically 1 to 2 µm diameter.
Work by graduate student, Iker Montes-Burgos of Professor Kenneth Dawson’s group at
University College Dublin has shown the utility of the NTA approach to
characterize silica nanoparticles before studying how they enter cells and decrease cell
viability. Monomodal particle sizes obtained using NTA broadly agreed with sizes obtained with
PCS. And several universities are now using the recently introduced NS500 to determine the
concentration of metallic or polymer nanoparticles produced through the wear-and-tear of
artificial joints. The NS500 has also added fluorescence capability.
In addition, gold nanoparticles are a rich field for research with NTA because they are
convenient for use in biological systems as way to deliver molecules to specific locations in
the body. Non-toxic and featuring a high refractive index, they are an ideal object for study
by NTA.
One constraint of the system is that a sufficient number of particles must be analyzed with
a certain time period so that a statistically meaningful and reproducible particle size
distribution profile can be obtained. A sample dilution can be used to achieve this
concentration.
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NanoSight's nanoparticle tracking analysis uses Brownian motion to locate and follow individual particles in solution. Image: NanoSight
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The benefit of being able to simultaneously measure two independent parameters, such as
particle scattering intensity and particle diameter (from dynamic behavior), can prove valuable
in resolving mixtures of different particle types. Similarly, small differences in particle
size within a heterogeneous population can be resolved with far higher accuracy than would be
achieved by other ensemble light scattering techniques.
“We also provide a measure of concentration. You don’t need to know the density or the
refractive index of the particles because the diffusion rate of the particles is independent of
density,” says Warren.
Particle characterization without photons
At Pittcon 2010, Archimedes , the award-winning product of the vendor conference was tucked
away in a small booth next to Particle Sizing Systems, Willow Grove, Pa.
(which serves as its distributor).
It resembled an atomic force microscope. Launched in 2009, the Archimedes particle metrology
system from Affinity Biosensors, Santa Barbara, Calif., takes advantage of the
precision of a cantilever-based system without needing optical properties. But instead of
reading a surface, it reads the change of resonance in a microfluidic system. By adopting a
non-optical, quantitative approach, the Affinity team has brought strong nanoscale resolution
to both particle sizing and particle density measurements.
Cantilever-based sensing platforms have long been used to profile surface characteristics,
and Affiinity Biosensor’s CEO, Ken Babcock, appreciated the accuracy of such systems enough to
want to apply it to other types of samples. Babcock is formerly a technologist at
Digital Instruments (DI) and Veeco, which is well known for
its AFMs. Starting around 2001, Veeco tried to develop a cantilever sensing platform for use in
fluid, but the sensitivity was never very good.
However, the work at Veeco caught the attention of Scott Manalis at MIT,
who had been an intern at DI when Babcock was starting his career. “He called me one day and
told me “You’re doing it wrong. Don’t put the cantilever in the fluid; put the fluid in the
cantilever,” says Babcock. Manalis and his post doc, Thomas Burg, had realized that enclosing
the fluid inside the cantilever, while placing the exterior in a vacuum environment, would
produce the high quality factor needed for high resolution frequency measurements.
Manalis and Burg began designing a solution. But their breakthrough required outside help.
Together, Babcock and Manalis successfully applied for a grant from the Institute for
Collaborative Biotechnologies in conjunction with a U.S. Army project on food safety, and with
Innovative Micro Technologies Inc., Santa Barbara, Calif., a leading MEMS
company, were able to build the complex but tiny chip.
What they developed is a microchannel resonator that was a departure from previous MEMS
chips in that it is designed to both resonate mechanically and to handle samples in solution.
The embedded sensors were sensitive to detect masses as small as 1 femtogram.
“The way I like to describe it is to imagine a tuning fork that is hollow. Imagine that the
fork is vibrating at a certain frequency. If, say, a fly lands on the tuning fork, the added
mass will cause a change in the resonant frequency. We’re using that same principle to detect
particles by suspending them in fluid and flowing them through the hollow tuning fork. That’s
how we use resonant mass detection,” says Babcock, to weigh particles one-by-one at high
resolution.
In addition to enabling ultra-high resolution measurements, the microfluidic channel is a
built-in way to deliver a sample to the resonator. And the architecture of chip allows for
high-throughput tasks.
Frequency readings are taken 1,000 times per second with a resolution of 40 ppb, allowing
the particles to be weighed as they pass through the resonator in a few milliseconds.
According to Babcock, particle size as small as 50 nm and up to 5 µm have been regularly
detected by Archimedes, and diverse samples have been measured, from nanoparticles to single
bacterial cells.
Obvious strengths of the system are resolution and accuracy, particle-by-particle, but the
system is also well-suited for distinguishing between particles close in size and determining
particle concentration through control of the fluid flow rate.
This ability can help the user determine, for example, how many millions of bacteria are in
a vial. An additional benefit, Babcock says, is that Archimedes is calibrated to within 1% and
measurements are NIST-traceable.
“There’s a future here. We’re just starting out,” says Babcock. A recent paper in Nature
Methods by the Manalis group discussed a method to use this technology to trap an
individual cell and observe its growth. He anticipates Archimedes eventually being used for
mass-based flow cytometry and direct detection of viruses, in addition to the sizing and mass
density measurement of particles.
But how will Archimedes fare in a mature DLS marketplace? With the first production systems
now in the field, Babcock says the system will occupy a niche for applications demanding very
high resolution and accuracy, and has received strong interest from a diverse set of potential
customers. One early application is in helping to quantify protein aggregation for a number of
biotech firms. In the future, even smaller resonators will help push detection limits even
further.
But how will Archimedes fare in a mature DLS marketplace? Accustomed to using a
photonics-based system, some users may be reluctant to transition immediately. Which is why
Babcock says that, for now, the system will occupy a niche for applications that need the level
of resolution and repeatability that Archimedes can provide. But applications are definitely
being found. Archimedes is now being used to help several biotech firms quantify protein
aggregation. In the future, smaller resonators could help push detection limits even
further.
Published in R & D magazine: Vol. 52, No. 4, August, 2010, pp.
14-17.
