Nanoparticle Tracking Analysis

1. Presented by
S.BHAVANA
Y18MPH312
Pharmaceutical Analysis
ANU college of Pharmaceutical Sciences
Under the Guidance of
K.E.PRAVALLIKA M.Pharm (Ph.D)
Asst.Professor
ANU college of Pharmaceutical Sciences

2. NANO PARTICLE
Nanoparticles are particles between 1 and
100 nanometres (nm) in size.
In nanotechnology, a particle is defined as a
small object that behaves as a whole unit with
respect to its transport and properties.

3. • Nanoparticle Tracking Analysis (NTA) is one of
the few methods to visualize and measure
nanoparticles in suspension ranging from 10 –
1000 nm based on the analysis of Brownian
motion. The rate of movement is related only to
the viscosity and temperature of the liquid; it is
not influenced by particle density or refractive
index. NTA allows the determination of a size
distribution profile of small particles within the
diameter range.

4. • Objects with two dimensions smaller than 100 nm are
termed nanoparticles or ultrafine particles. Fine
particles are sized between 100 and 2500 nm.
• NTA is a versatile technique capable of
multiparameter measurement for all kinds of particles,
saving time and sample amount. The powerful
capabilities and data provided by this are unique to
view particle dispersions. Optimization of the
technique and analytical design that yield the highest
quality data.

5. HISTORY
• Nanoparticle Tracking Analysis (NTA) is a
particle characterization technique that has grown
rapidly in market ten years ago, but it originated
almost 25 years ago. The commercial
implementation of this technique required the
availability of fast computer systems that are able
to cope with the computationally intensive video
analysis in reasonable time frames. The current
NanoSight instruments have become a critical
tool used in many applications to provide
information not available by other methods.

6. • Even though the NTA technology is relatively new a
detailed procedure on the practical procedure for
NTA measurement is given in the ASTM standard
for NTA measurements.
• ASTM International, formerly known
as American Society for Testing and Materials, is
an international standards organization that develops
and publishes voluntary consensus
technical standards for a wide range of materials,
products, systems, and services.

7. MEASUREMENT PRINCIPLE
• NTA technique is used in conjunction with an
ULTRAMICROSCOPE, which allows detection and tracking of
the Brownian motion of 10 to 1000 nm sized vesicles. Particles in
the sample are visualized by the illumination with a laser beam
that together allow small particles in liquid suspension to be
visualized moving under Brownian motion.
• The light scattered by the particles is captured and recorded with a
light sensitive CCD (charge-coupled device) or CMOS
(complementary metal-oxide semiconductors)camera over
multiple frames, which is arranged at a 90° angle to the irradiation
plane. Computer software is then used to track the motion of each
particle from frame to frame. The rate of particle movement is
related to a sphere as calculated through the Stokes–Einstein
equation.

8. PHYSICAL PRINCIPLE
• When small particles are dispersed in a liquid the particles move
randomly in all directions.
• The phenomenon of the random movement is termed diffusion
and is expressed by the diffusion coefficient (D).
(NTA) measure the Brownian motion of nanoparticles whose speed
of motion, or diffusion constant, Dt, is related to particle size
through the Stokes–Einstein equation.
(x,y)2 = Dt = TKb
4 3πηd
Where,
Dt - diffusion constant, a product of diffusion coefficient D and
time t
Kb - Boltzmann's constant,
T - absolute temperature,
η - viscosity
d - diameter of the spherical particle.

9. • By simultaneously tracking several particles their diameters
can be determined in parallel.
• The lower limit of the working range, i.e. the smallest
detectable particle size, depends on the scattered intensity of
the particle, the efficiency of the magnifying optics and the
sensitivity of the camera.
• Nanoparticles of silver and gold are strong scatterers due to
the comparably large refractive indices of 2-4 and can be
detected down to sizes of ~10 nm.
• The quality of an NTA result is influenced by particle
contamination. In addition, high concentrations of stabilizing
agents (e.g. surfactants) are critical as soon as they reach
their critical micellar concentration (CMC).
• Contamination in the form of particles may derive from
diluents (distilled water or buffer agents) or from chemicals
used during preparation of samples.

10. METHODOLOGY
• Brownian motion and light scattering properties are used by the
NTA technique to acquire the samples particle size distribution
in a liquid suspension.
• When passing a laser beam via the sample chamber the particles
incident on the beam path and scatter light making it possible to
view them through a 20x magnification microscope equipped
with a camera.
• The camera records a video file of the particles under Brownian
motion within the 100 µm x 80 µm x 10 µm field of view.

12. • The size of each individually tracked particle is calculated,
thus simultaneously allowing determination of their size
distribution and concentration.
The highlights of this technique are:
• Characterization of nanoparticles in solution
• Measurement at comparably low concentration levels (down to
105 particles per cm-3)
• Single particle analysis resulting in high resolution
distributions
• Specificity with fluorescent detection

13. NTA IS A MULTI-PARAMETER TECHNIQUE:
NTA is particle characterization technique for practically all kinds
of nanoparticles suspended in media. Analysis of particles is
the determination of size and concentration.
• NTA is a fast single particle analysis tool to visualize and
quantify size, concentration, surface charge (zeta potential)
and fluorescence of the sample. These are useful for particle
characterization.
Size
• Particle size of single particles is calculated from the Brownian
motion analysis. The particle size distribution is created by
accumulation of sizes of several 100 to 1000 individual
particles.

14. Concentration
By knowing the measurement volume, the concentration is
determined by counting all objects in the field of view. The particle
concentration can be given in number of particles per cm3, as well
as area & volume, marking out NTA technique as an absolute
measurement.
Zeta potential
The zeta potential reflects the surface charge of given particles,
which is related to their stability by electrostatic forces. As charge
of nanoparticle changes when surface has different coating,
particles of same size but different surface coating can be
differentiated. For eg:conjugated and unconjugated gold
nanoparticles show difference in zeta potential.
Fluorescence detection
The detection of fluorescence, for example vesicles tagged with
specific fluorescence dye empowers NTA to specific detection for
the quantification of different subpopulations.

15. • Application of the Stokes-Einstein equation allows the
derivation of particle size and concentration.
• An interesting alternative to more typical Light Scattering
techniques such as Dynamic Light Scattering (DLS).
• NTA measures this movement through image analysis tracking
the movement of the particles on a particle-by-particle basis,
this movement can be related to particle size. DLS does not
visualize the particles. DLS observes the time dependent
fluctuations in scattering intensity caused by constructive and
destructive interference resulting from the relative Brownian
movements of the particles within a sample.

16. Characterization Nanoparticle Tracking
Analysis (NTA)
Dynamic Light Scattering
(DLS)
Size Range (nm) 10 – 1000 2 - 3000
Measurement of
Polydisperse Sample
Particle-by-particle approach
allows better resolution of
particle sizes. No intensity bias
towards larger particles
Average particle size which
is intensity biased towards
the larger/contaminant
particles within a sample
Measurement of
Monodisperse Sample
Looks at less particles than DLS
therefore repeatability is slightly
worse than DLS. Equivalent
size distribution to DLS
Slightly more reproducible
than NTA due to average
particle size from many
more particles

17. Characterization Nanoparticle Tracking
Analysis (NTA)
Dynamic Light Scattering
(DLS)
Refractive Index Requires no information
about solvent refractive
index. Relative particle
intensity can be calculated
for samples with a mixture
of particle refractive indices
Requires solvent refractive
index. In samples with a
mixture of particle refractive
indices, analysis is weighted
towards the more refractile
particles
Size Distribution Number distribution Intensity distribution which
can be converted into a
volume distribution. No
accurate information about
particle concentration can be
calculated.

18. Particle Size Determination Combined with Particle
Light Scattering
A unique feature of the NTA method is that it can determine the
quantity of light it scatters (IScat) and plot it against particle
size. This makes it possible to distinguish particles which
could have identical size yet different refractive index or
composition.

19. Applications
• NTA has been used by commercial, academic, and
government laboratories working with
1.nanoparticle toxicology
2.drug delivery
3.exosomes
4.microvesicles
5.other small biological particles, virology and vaccine
production,
6.ecotoxicology, protein aggregation,
7. orthopedic implants, inks and pigments, nanobubbles

20. Conclusion
• NTA is used to determine the size of particles in
investigations of nanoparticles metals, interactions of
them with organisms at cellular level and in the
development of methods of medication to various
diseases.
• So NTA has been very widely used now a days as it is
advantageous when compared to other processes.

21. References
• Martin Hessellov, James W. readman, James F. Ranville, Karen
Tiede. Nanoparticle analysis and characterization methodology in
environmental risk assessment of engineered nanoparticles.2008
• Reviewing the Use of Nanoparticle Tracking Analysis (NTA) for
Nanomaterial Characterization
https://www.azonano.com/article.aspx?ArticleID=4134
• Nanoparticle Tracking Analysis: Principles and Methodology
https://themedicinemaker.com/manufacture/nanoparticle-tracking-
analysis-principles-and-methodology
• Articles Nanoparticle Tracking
https://www.particle-metrix.de/en/technologies/nanoparticle-
tracking/articles-nanoparticle-tracking/introduction-to-nanoparticle-
tracking-analysis-nta.html
• Basic Principles of Nanoparticle Tracking Analysis
https://www.technologynetworks.com/analysis/webinars/basic-
principles-of-nanoparticle-tracking-analysis-290151
• Nano Particle Size Analyzer- wikipedia