www.nanosight.com
Characterisation of Nanoparticles
and Aggregates using
Nanoparticle Tracking Analysis (NTA)
LM10 Series NS300 NS500
The Instrument Range
Mai 13
As the schematic below shows, the NanoSight
technology comprises:
• a metallised optical element
• illuminated by l...
Fast and easy to use system
Loading of 200nm latex standard
Mai 13 www.nanosight.com
Titanium Dioxide
Mai 13 www.nanosight.com
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
scatte...
• Nanoparticles move under Brownian
movement due the random movement
of water molecules (red molecules in
movie) surroundi...
• Brownian motion of each particle is
followed in real-time via video
• Tracking software establishes mean
square displace...
Nanoparticle Tracking Analysis (NTA) is the gathering of unique information and
comes from assessment of individual partic...
The NanoSight NTA
2.0 (nano-particle
tracking) analysis
suite allows for
captured video
footage to be
simultaneously
track...
Minimum size limit is related to:
• Material type
• Wavelength and power of illumination
source
• Sensitivity of the camer...
True size distribution profile
Mixture of 100nm and 200nm latex microspheres
dispersed in water in 1:1 ratio
Particle dist...
Mai 13
Number count of particle in a determined volume :
Absolute concentration measurement
in 10E8 part/mL.
Particles are...
NanoSight has the unique ability to plot each particle’s size as a function of its
scattered intensity
2D size v.
number
2...
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 part...
NanoSight systems can be fitted with
• Choice of long pass or band pass filters allow specific nanoparticles to be
tracked...
Analysis of 100 nm Fluorescence standard particles suspended in FBS
Mai 13 www.nanosight.com
Modal particle size peaks: 10...
Dynamic Light Scattering (DLS or PCS)
Mai 13 www.nanosight.com
• DLS (alternatively known as Photon Correlation Spectrosco...
Comparison of different particle size
distribution
Particle by particle method Static light scattering Dynamic Light Scatt...
DLS Analysis and NanoSight equivalent
for monodisperse systems
NTA vs DLS - Monodisperse
Mai 13 www.nanosight.com
DLS NTA
With NanoSight, the analysis shows both 100nm
and 200nm peaks
Polystyrene reference spheres in water (100nm and 200nm)
NTA...
100nm particles not
seen due to intensity
bias
Mixed Sample of 100nm+200nm
particles at a ratio of 10:1 by weight
?
Mai 13...
Data reproduced from
Filipe, Hawe and Jiskoot (2010) “Critical
Evaluation of Nanoparticle Tracking Analysis
(NTA) by NanoS...
• NTA is not intensity-weighted towards larger particles, unlike DLS
• While DLS is an ensemble technique, NTA operates pa...
Comparing different Technologies
Mai 13 www.nanosight.com
Technique Strengths Weaknesses
Electronic microscope
(TEM, SEM)
...
Mai 13
Application Example : Exosomes
www.nanosight.com
•Microvesicles - typically 100nm to 1um range
•Nanovesicles or Exo...
Mai 13
Application Example : Exosomes
www.nanosight.com
Why use NanoSight in this application ?
• We COUNT particles – con...
Mai 13
Application Example : Exosomes
www.nanosight.com
Comparisons of different techniques
Flow Cytometry:
Limited lower ...
Mai 13
Application Example : Exosomes (Fluoresence)
www.nanosight.com
Mai 13
Application Example : Purified Influenza Virus
www.nanosight.com
Why Nanosight?
•Manufacturer interested in the pur...
Current methologies for counting
such as plaque assay only count
infectious particles which often
represent a small compon...
• NanoSight technology has a
unique application in the detection of
early stage aggregation in protein
therapeutics
• Prot...
• DLS analysis of aggregated protein sample generally produces a bimodal analysis.
• The protein monomer and the very larg...
Mai 13
Application Example: Drug Delivery system
www.nanosight.com
The size will affect its
1.Circulation and
residence ti...
• Multi-walled Carbon
nanotubes
• Cosmetics
• Foodstuffs
• Ink jet inks and
pigment particles
• Ceramics
• Fuel additives
...
Size
Number or
concentration
Polydispersity –
true PSD
“Relative Light
Intensity”
Fluorescence
Surface
Potential, Zeta
Pot...
• BASF, Europe
• BP Castrol, Europe
• DuPont, USA
• Epson, Japan
• Exxon Mobile, USA
• GlaxoSmithKline, Europe
• Merck, US...
NTA devices worldwilde mapping
Mai 13 www.nanosight.com
> 500 devices
≈ 800 publications
Awards
Mai 13 www.nanosight.com
of 40

Nanoparticle Tracking Analysis (particle by particle technique)

NanoSight visualizes, measures and characterizes virtually all nanoparticles. Pls contact A&P Instrument Co.Ltd in Hong Kong for detail. Email: anson@anp.com.hk
Published on: Mar 3, 2016
Published in: Technology      Business      
Source: www.slideshare.net


Transcripts - Nanoparticle Tracking Analysis (particle by particle technique)

  • 1. www.nanosight.com Characterisation of Nanoparticles and Aggregates using Nanoparticle Tracking Analysis (NTA)
  • 2. LM10 Series NS300 NS500 The Instrument Range
  • 3. Mai 13 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 www.nanosight.com Hardware: laser illuminated sample chamber + optical microscope
  • 4. Fast and easy to use system Loading of 200nm latex standard Mai 13 www.nanosight.com
  • 5. Titanium Dioxide Mai 13 www.nanosight.com Polydisperse titanium dioxide particles as seen at standard magnification
  • 6. 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 Mai 13 www.nanosight.com
  • 7. • 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 Mai 13 www.nanosight.com
  • 8. • 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 Constant T = Temperature = viscosity KBT Stokes-Einstein equation Dt = 3 dh Sizing is an absolute method Mai 13 www.nanosight.com Dt yx 2 4 ,
  • 9. 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 Mai 13 www.nanosight.com
  • 10. 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 Mai 13 www.nanosight.com
  • 11. Minimum size limit is related to: • Material type • Wavelength and power of illumination source • Sensitivity of the camera Size Concentration Minimum 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 www.nanosight.com
  • 12. 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. Mai 13 www.nanosight.com
  • 13. Mai 13 Number count of particle in a determined volume : Absolute concentration measurement in 10E8 part/mL. Particles are Counted– By Number Count www.nanosight.com Global concentration of all populations Concentration of a population selected by user
  • 14. 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 Mai 13 www.nanosight.com
  • 15. Relative intensity scattered
  • 16. 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 Mai 13 www.nanosight.com
  • 17. 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 Mai 13 www.nanosight.com
  • 18. Analysis of 100 nm Fluorescence standard particles suspended in FBS Mai 13 www.nanosight.com 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
  • 19. Dynamic Light Scattering (DLS or PCS) Mai 13 www.nanosight.com • 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.
  • 20. Comparison of different particle size distribution Particle by particle method Static light scattering Dynamic Light Scattering
  • 21. DLS Analysis and NanoSight equivalent for monodisperse systems NTA vs DLS - Monodisperse Mai 13 www.nanosight.com DLS NTA
  • 22. With NanoSight, the analysis shows both 100nm and 200nm peaks Polystyrene reference spheres in water (100nm and 200nm) NTA vs DLS – Bimodal Sample Mai 13 www.nanosight.com
  • 23. 100nm particles not seen due to intensity bias Mixed Sample of 100nm+200nm particles at a ratio of 10:1 by weight ? Mai 13 NTA vs DLS – Bimodal Sample www.nanosight.com
  • 24. 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) Mai 13 www.nanosight.com NTA vs DLS in publications
  • 25. • 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 Mai 13 www.nanosight.com
  • 26. Comparing different Technologies Mai 13 www.nanosight.com 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
  • 27. Mai 13 Application Example : Exosomes www.nanosight.com •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
  • 28. Mai 13 Application Example : Exosomes www.nanosight.com 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.
  • 29. Mai 13 Application Example : Exosomes www.nanosight.com 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
  • 30. Mai 13 Application Example : Exosomes (Fluoresence) www.nanosight.com
  • 31. Mai 13 Application Example : Purified Influenza Virus www.nanosight.com 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.
  • 32. 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. Mai 13 Application Example : Purified Influenza Virus www.nanosight.com
  • 33. • 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 Mai 13 Application Example: Protein Aggregation at controlled temperature (15-55o C) www.nanosight.com
  • 34. • 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. Mai 13 Application Example: Protein Aggregation: NTA vs DLS www.nanosight.com
  • 35. Mai 13 Application Example: Drug Delivery system www.nanosight.com 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.
  • 36. • 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 Mai 13 The NanoSight system is widely applicable www.nanosight.com
  • 37. Size Number or concentration Polydispersity – true PSD “Relative Light Intensity” Fluorescence Surface Potential, Zeta Potential + + + + Parameters measured – simultaneously, ‘real time’, particle-by-particle... Mai 13 www.nanosight.com
  • 38. • 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
  • 39. NTA devices worldwilde mapping Mai 13 www.nanosight.com > 500 devices ≈ 800 publications
  • 40. Awards Mai 13 www.nanosight.com

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