Nanoparticle Tracking Analysis (NTA) with NanoSight equipment. NTA can be used in a variety of research areas including the following: extracellular vesicle characterization, viral vaccine research and development, development of drug delivery systems, protein aggregation studies, and more!

1. Nanoparticle Tracking
Analysis
Dr. Santosh Kumar Yadav
Cardeza Center for Hemostasis and Vascular Biology,
Cardeza Foundation for Hematologic Research,
Department of Medicine.

2. What is Nanoparticle?

3. Exosomes
 Secreted by all mammalian cells
 Cell-to-cell communication and signaling
 Diagnostics & Biomarkers
 Therapeutics

4. • Liposomes and other drug delivery vehicles
• Virus samples
• Protein aggregation
• Ink jet inks and pigment particles
• Magnetic Nanoparticles
• Multi-walled Carbon nanotubes
• Cosmetics
• Foodstuffs
• Ceramics
• Fuel additives
• Metal oxides in magnetic storage media
• Precursor chemicals for wafer fabrication.
• Quantum dots
• Polymers and colloids
• CMP Slurries
• Nanobubbles
Application of Nanoparticle Tracking Analysis

5. Nanoparticle Tracking Technology
NTA technologies were developed by Bob Carr along with John Knowles in 2003
 Proprietary optical element
 Specially configured laser beam
 Microscope

6. Load sample
Nanoparticle Tracking Analysis in Action
60 nm to 800 nm
Titanium Dioxide (in water)
Pass laser beam Visualize the particles

7. Improvements over the years in NPT
LM10
LM20
Boxed-up' LM10
• Multiple automated features, including computer-controlled peristaltic pumps and stage
positioning.
• A fluidics system is used to inject samples into a small viewing chamber,
• Sample temperature control is also programmable.
• Fluorescence measurement
NS500
NS300

8. Nanoparticle Tracking Analysis
 Nanoparticle in suspension.
 Tracking many individual particles simultaneously.
 Analysis
• Particle size, concentration and distribution
• Relative light scattering intensity and
• Fluorescence
Nanoparticle Tracking Analysis

9. 1. Nanoparticles move under Brownian motion
2. Small particles move faster than larger particles
3. Diffusion Coefficient is calculated by tracking and analyzing the movement
of each particle separately but simultaneously
Principle of Measurement

10. • Brownian motion of each particle is followed in real-time via video
• Video analysis software measures mean square displacement in two dimensions
• Particle diameter(sphere equivalent hydrodynamic) d is then obtained from the Stokes-
Einstein equation
• NTA being the absolute method does not require calibration.
Particle Size and Size distribution
100+200nm polystyrene in water
Larger particles scatter
significantly more light
Small particles as
point scatterers

11. Particle Shape
 Spherical particles exhibit constant scatter
signals with NanoSight
 Images of non-spherical particles flash
under NanoSight.
 Filamentous and disc-shaped particles
exhibits most pronounced flashing

12. 3D Plots (size v. intensity v. number)
Size
(size v. intensity v. number)
(size v. intensity)
(size v. number)
Intensity
Size
Number
3D Plots resolve the particle that can not be detected by 2D Plots
Size

13. Identification of Type of Particle
Higher scattering but smaller
size of gold v. polystyrene
50nm Gold + 100nm Latex
Plotting size v relative scattering intensity = differences in particle composition
100nm + 200nm Latex
Scaling of size to intensity
for two sizes of polystyrene

14. Resolving Mixtures of Different Particle Types and Sizes
100 PS
60nm Au
30nm Au
The three particle types can be clearly seen in
the 3D plot
Despite their smaller size, the 60nm Au can
be seen to scatter more than the 100nm
polystyrene
Mixture of 30nm and 60nm gold nanoparticles mixed with 100nm polystyrene

15. Differentiating Virus in Complex Background
Current methodologies 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 two populations are similar sizes but are differentiated because the virus scatters more light

16. Protein Aggregation at 500C
•The protein monomer is too small to
be individually resolved by this
technique,
•Early-stage aggregates are readily
detected
•Both size and number of aggregates
can be calculated and studied,
providing insight into product stability.

17. Detection of Fluorescent Particles in Mixture
Mixture of 50nm fluorescent particles and 200nm non-fluorescent particles analyzed
under light scatter mode

18. Same mixture when scatter removed by fluorescence filter
Detection of Fluorescent Particles in Mixture

19. Fluorescence Detection in Biological Fluids
Analysis of cellular micro- and nano-vesicles labelled with an appropriate fluorescent antibody
Light scatter Antibody Fluorescence Light scatter and Fluorescent

20. Size
 Minimum Size limit (10 - 40nm)
• Material type
• Wavelength and power of illumination source
• Sensitivity of the camera
 Maximum Size limit (1000-2000nm)
• Limited Brownian motion
Concentration
 Minimum concentration (approx 106/ml)
• Poor statistics (Requiring longer analysis time)
 Maximum concentration (approx 1010/ml)
• Inability to resolve neighboring particles
• Tracks too short before crossing occurs
NTA Detection Limits

21. 1.User and sample details
2.Capture settings
3.Analysis settings
4.Histogram data
5.Graphs
6.Tables
7.Excel compatible raw data files
Auto-generated Report contains

22. Other techniques to analyze nanoparticles
Electron Microscope: Time consuming and expensive
Flow Cytometry: Limited lower limit of detection
Dynamic Light Scattering: Can not differentiate lower and higher scatter signal

23. Materials and Equipment Needed
Materials needed:
• Insulin syringe
• Water and
• Ethanol to wash the tubing
Equipment needed:
• NS300
• Peristaltic pump

24. • Low sample volumes
• Little sample preparation
• Minimal instrument consumables
• Non-destructive and the sample can be recovered
• High resolution particle size distribution through single particle
detection and analysis
• Visualization of particles down to 10nm, dependent on material
• Count and concentration measurement
• Simultaneous scattered light and fluorescence measurement
• User-programmable Batch Measurement for Time-Based Study
Advantage of the Nanoparticle Tracking Analysis

25. Platelet derived Exosomes
alpha-granule
Resting
Platelets
Activated
Platelets
Exosomes

26. Thank You