Nanoparticle Tracking Analysis (particle by particle technique)

www.nanosight.com

Characterisation of Nanoparticles and Aggregates using Nanoparticle Tracking Analysis (NTA)

LM10 Series NS300 NS500

The Instrument Range

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As the schematic below shows, the NanoSight technology comprises:

• a metallised optical element• illuminated by laser beam

.

Laser as seen at low magnification

NanoSight LM20 device

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Hardware: laser illuminated sample chamber + optical microscope

Fast and easy to use system

Loading of 200nm latex standard

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Titanium Dioxide


Polydisperse titanium dioxide particles as seen at standard magnification

Particles are Visualised, not imaged

Particles are too small to be imaged by the microscope

Particles seen as point scatterers moving under Brownian motion

Larger particles scatter significantly more light

Speed of particles varies strongly with particle size

Microvesicles purified from serum by ultracentrifugation


• Nanoparticles move under Brownian movement due the random movement of water molecules (red molecules in movie) surrounding them.

• Small particle move faster than larger particles.

• Diffusion Coefficient can be calculated by tracking the movement of each particle and then through application of the Stokes-Einstein equation particle size can be calculated.

Principle of Measurement


• Brownian motion of each particle is followed in real-time via video

• Tracking software establishes mean square displacement and diffusion coefficient (Dt)

• Then from Stokes Einstein can be obtained particle (sphere equivalent hydrodynamic) diameter, dh

KB = Boltzmann ConstantT = Temperature

= viscosity

KBT

Stokes-Einstein equation

Dt = 3dh

Sizing is an absolute method


Dt

yx

2

4

,

Nanoparticle Tracking Analysis (NTA) is the gathering of unique information and comes from assessment of individual particles, rather than averaging over a bulk sample.

This provides a distinct advantage in determining particle size

analysistrackingcapture

Nanoparticle Tracking Analysis


The NanoSight NTA 2.0 (nano-particle tracking) analysis suite allows for captured video footage to be simultaneously tracked and analysed…

100+200nm nanoparticles being tracked and analysed by NanoSight NTA 2.0

Particle Sizing in action - Software Analysis


Minimum size limit is related to: • Material type • Wavelength and power of illumination

source • Sensitivity of the camera

Size ConcentrationMinimum concentration is related to:

• Poor statistics (Requiring longer analysis time)

10 – 40 nm

1 000 – 2 000

nm

Maximum Size limit is related to:

• Limited Brownian motion• Viscosity of solvent

Approx 105-6 / mL

Maximum concentration is related to:

• Inability to resolve neighboring particles• Tracks too short before crossing occurs

Approx 109 / mL

NTA Detection Limits

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True size distribution profile

Mixture of 100nm and 200nm latex microspheres dispersed in water in 1:1 ratio

Particle distribution displays a number count vs particle size.


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Number count of particle in a determined volume :

Absolute concentration measurement in 10E8 part/mL.

Particles are Counted– By Number Count

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Global concentration of all populations

Concentration of a population selected by user

NanoSight has the unique ability to plot each particle’s size as a function of its scattered intensity

2D size v. number

2D size v. intensity

3D size v. Intensity v. concentration

Relative intensity scattered


Relative intensity scattered

100 PS

60nm Au

30nm Au

In this mixture of 30 nm and 60 nm gold nanoparticles mixed with 100 nm polystyrene, the three particle types can be clearly seen in the 3D plot confirming indications of a tri-modal

given in the normal particle size distribution plot. Despite their smaller size, the 60 nm Au can be seen to scatter more than the 100 nm PS.

Resolving mixtures of different particle types and sizes


NanoSight systems can be fitted with

•Choice of long pass or band pass filters allow specific nanoparticles to be tracked in high backgrounds

Applications in:• Nanoparticle toxicity studies• Nano-rheology• Bio-diagnostics• Phenotyping specific exosomes

• Laser diode capable of exciting fluorophores and quantum dots

405nm 488nm 532nm 635nm

Fluorescent Mode available


Analysis of 100 nm Fluorescence standard particles suspended in FBS


Modal particle size peaks: 103 nm

Concentration: 9.5 x108 particles/ml

• 100 nm fluorescently labelled particles suspended in 100% FBS• Sample maintained at a constant temperature (37 oC)• Sample viscosity – 1.33 cP

• Excitation wavelength - 532 nm• Using a 565 nm Long pass optical filter

No filter 565 nm Long pass optical filter

This allows selective visualising of fluorescent/fluorescently

labelled particles

Dynamic Light Scattering (DLS or PCS)


• DLS (alternatively known as Photon Correlation Spectroscopy (PCS)) is deservedly an industry standard technique and widely used for 40 years...

• Also relies on Brownian motion and relates the random motion of particles in liquid suspension to a hydrodynamic radius. Technique relates the rate of change of the intensity of the speckle to particle size using the auto-correlation function.

• Very effective technique for measuring particle size for mono-dispersed samples from a few nm up to microns .

• Widely used, big installed base.

Comparison of different particle size distribution

Particle by particle method Static light scattering Dynamic Light Scattering

Nanosight DLS

DLS Analysis and NanoSight equivalent for monodisperse systems

NTA vs DLS - Monodisperse


DLS NTA

NanosightDLS

With NanoSight, the analysis shows both 100nm and 200nm peaks

Polystyrene reference spheres in water (100nm and 200nm)

NTA vs DLS – Bimodal Sample


Nanosight

100nm particles not seen due to intensity

bias

Mixed Sample of 100nm+200nm particles at a ratio of 10:1 by weight

?

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NTA vs DLS – Bimodal Sample

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DLS

Data reproduced from Filipe, Hawe and Jiskoot (2010) “Critical Evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates”,

Pharmaceutical Research, DOI: 10.1007/s11095-010-0073-2

NTA (red profiles)

Mixtures of polystyrene of different sizes

DLS (blue bars)


NTA vs DLS in publications

• NTA is not intensity-weighted towards larger particles, unlike DLS

• While DLS is an ensemble technique, NTA operates particle-by-particle

• NTA has much higher resolving power with respect to multimodal and polydisperse samples and heterogenous/mixed sample types

• NanoSight unique and direct view visually validates the particle size distribution data

• Number vs. Intensity vs. Size is provided for each particle size class

• NTA provides particle concentration information

• Fluorescence mode

• Zeta Potential, particle by particle

• NTA requires no information about collection angle, wavelength or solvent refractive index , unlike DLS

NanoSight’s Advantages Vs. DLS/PCS


Comparing different Technologies


Technique Strengths Weaknesses

Electronic microscope(TEM, SEM)

-Higher magnification, greater resolution, compositional information, topography and morphology

-High costs- Complex sample preparation procedure

Analytical Ultra Centrifugation -High resolution -High cost , complex sample preparation & data evaluation , -Low sample throughput

Coulter Counters (i.e IZON) -Very low cost.- Small size

-Need calibration-Surface charge of particle influences result-Clogging of aperture is common

Flow Cytometers -High Throughput-Sorting of different size of particle-Fluoresence

-Not suitable for vesicles sizing due to no accurate refractive index to calibrate-Lower limit is about 300nm

Dynamic Light Scattering -suited to mono-disperse -samples from a few nm to a few microns

-Intensity-biased Plot-No Fluorescence and no counting ability

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Application Example : Exosomes

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•Microvesicles - typically 100nm to 1um range

•Nanovesicles or Exosomes - 30-100nm

•Originally thought to be cellular debris

•Now appreciated that these cell-derived particles are of

significant importance and play an important role both in

cellular communication and signalling

•Recently found to be present at significantly elevated

levels in a number of diseases conditions

Exosomes and Nanovesicles

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Why use NanoSight in this application ?

• We COUNT particles – concentration is key to this application. Researchers can not get good concentration information any other way – this is KEY. Remember an increase in the number of exosomes could be related to the onset of disease.

•We can work in FLUORESCENCE. This means they can fluorescently label these particles and therefore SPECIATE – i.e. They know which exosomes they are looking at.

•High resolution SIZE.

•All of these parameters in a SINGLE MEASUREMENT – allows them to determine the size, concentration and type of exosome population.

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Comparisons of different techniques

Flow Cytometry:Limited lower limit of detection – can’t detect the

smaller exosomes DLS:

Samples are often not very homogenous – therefore not suited for measurement with DLS.

DLS cannot count and also can’t phenotype the exosomes – meaning it has limited value.

EM:Time consuming and expensive

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Application Example : Exosomes (Fluoresence)

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Application Example : Purified Influenza Virus

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Why Nanosight?

•Manufacturer interested in the purity of their product – i.e. The size distribution of particles within a sample.

•Manufacturer interested in the dosage of the vaccine i.e. How many viruses are present?

•Manufacturers need to know the ratio of infective to non-infective viruses in their sample.

•Manufacturer interested in detecting specific viruses as the samples are purified – therefore fluorescence is of interest to them.

•COUNTING aspect is the most important feature as manufacturers often use long winded infectivity assays to characterise their samples.

Current methologies for counting such as plaque assay only count infectious particles which often represent a small component in attenuated vaccines i.e. perhaps only 1% of product remains infective.

The ability to count viruses in liquid suspension is essential for those working in vaccine development.

Particle aggregation and yield quality are factors which need to be understood when developing these viral vaccines.

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Application Example : Purified Influenza Virus

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•NanoSight technology has a unique application in the detection of early stage aggregation in protein therapeutics

•Protein monomer is too small to be individually resolved by this technique, but early stage aggregates are readily detected

•Protein monomer at high concentration causes high background noise in image, with the aggregate forming the resolvable particles

•Both size and number of aggregates can be calculated and studied, providing insight into product stability.

Data reproduced from Filipe, Hawe and Jiskoot (2010) Pharmaceutical Research, DOI: 10.1007/s11095-010-0073-2

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Application Example: Protein Aggregation at controlled temperature (15-55o C)

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•DLS analysis of aggregated protein sample generally produces a bimodal analysis.

•The protein monomer and the very large aggregates get picked up through DLS as these are the regions which scatter most light. The monomer scatters a lot of light by virtue of its high concentration and the larger particles by virtue of their size.

•Clearly a protein sample cannot aggregate from monomer to large micron sized aggregates with nothing in between. Whilst NanoSight cannot measure the monomer it can provide valuable information in the 30nm and above range which is typically the region which is poorly served by alternative techniques.

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Application Example: Protein Aggregation: NTA vs DLS

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Application Example: Drug Delivery system

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The size will affect its 1.Circulation and residence time in the blood, 2. the efficacy of the targeting,3. the rate of cell absorption

Advantage of using Iiposome as a drug deliver• Drugs can be targeted to specific areas by attaching ligands to the liposome• Readily absorbed by cells• The rate of drug release can be controlled• Allows potentially lower doses of drug to be used, reducing toxicity and side-

effects.

• Multi-walled Carbon nanotubes

• Cosmetics

• Foodstuffs

• Ink jet inks and pigment particles

• Ceramics

• Fuel additives

• Liposomes and other drug delivery vehicles

• Virus and VLP samples

• Exosomes and nanovesicles

• Nanobubbles

• Magnetic Nanoparticles

• Precursor chemical for wafer fabrication.

• Quantum dots

• Polymers and colloids

• CMP Slurries

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The NanoSight system is widely applicable

www.nanosight.com

Size

Number or concentration

Polydispersity – true PSD

“Relative Light Intensity”

FluorescenceSurface Potential, Zeta Potential

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+

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Parameters measured – simultaneously, ‘real time’, particle-by-particle...


• BASF, Europe• BP Castrol, Europe• DuPont, USA• Epson, Japan• Exxon Mobile, USA• GlaxoSmithKline, Europe• Merck, USA• Medimmune• Nestle, Europe• NIST, USA

Selected Users

• Novartis, Europe• Proctor & Gamble, USA• Roche, Europe• Smith & Nephew, Europe• Solvay, Europe• Toshiba, Japan• Unilever, Europe• US EPA • US Forces• Wyeth Biopharma, USA

NTA devices worldwilde mapping


> 500 devices≈ 800 publications

Awards


Nanoparticle Tracking Analysis (particle by particle technique)

Technology

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