1. Department of Physics, Central University of Tamil Nadu, India
Importance of Nanoparticle distribution – selection, assembly, measurements
Prof.P. Ravindran,
Department of Physics, Central University of Tamil Nadu, India
&
Center for Materials Science and Nanotechnology, University of Oslo, Norway

2. Disperse systems

3. Pharmaceutical Suspension-Definition

4. Particle Measurements
Nanoparticle General Sampling Practices
Look at outdoor concentrations for sources and variability
Ventilation system plays a role – Evaluate the effect
Background / baseline measurements
Mass Measurements - Background
Traditional workplace exposure limits are mass based
No regulations currently exist specifically for nanoparticles
Mass of one 10 µm particle
= 106 times the mass of one 100 nm particle
= 109 times the mass of one 10 nm particle
Traditional gravimetric methods are not effective for nanoparticles since toxicity data is based on large particles
It takes ~1,000,000,000 (1 billion) 10 nm particles to equal the mass of one 10 µm particle!

5. Measurement of the particles size by the PCS technique
Principle of Measurement
If the particles or molecules are illuminated with a laser, the intensity of the scattered light fluctuates at a rate that is dependent upon the size of the particles
Analysis of these intensity fluctuations yields the velocity of the Brownian motion and hence the particle size using the Stokes-Einstein relationship.
Particles, emulsions and molecules in suspension undergo Brownian motion.
This is the motion induced by the bombardment by solvent molecules that themselves are moving due to their thermal energy
Temperature and viscosity must be known
PCS – Photon correlation spectrascopy

6. Stokes-Einstein relationship
The velocity of the Brownian motion is defined by a property known as the translational diffusion coefficient (usually given the symbol, D).

8. No spherical particles
Hydrodynamic diameter is calculated based on the equivalent sphere with the same diffusion coefficient

9. He-Ne Laser
 = 633 nm
Zetasizer Nano ZS
Malvern

10. Brownian motion and scattering

11. Intensity of the scattered light fluctuates

12. Intensity of the scattered light fluctuates
Small particles- noisy curve
Large particles- smooth curve

13. Determining particle size
Determined autocorrelation function
Depend

14. Correlation function Correlograms

15. Correlogram from a sample containing large particles
Correlogram from a sample containing small particles

16. Data interpretation - Correlograms

17. turbidity is linear with concentration
Low
concentration
turbidity is linear with concentration
High
concentration
Particles are so close together that the scattered radiation is re-scattered by other particles.

18. Optical arrangement in 173°
backscatter detection

19. Directions-isotropic)
Information
Size by:
- Intensity
I  d6
Rayleigh Scattering
(For nanoparticles less than d =λ/10 or around 60nm the scattering will be equal in all
Directions-isotropic)

20. 8 nm
80 nm
This particles will scatter 106 (one million) times more light than the small particle (8 nm)
The contribution to the total light scattered by the small particles will be extremely small

21. 8 80

22. By the Mie theory it is possible to convert intensity distribution into volume
V= 4r3
r = d/2
V= 4(d/2)3 = 4d3 8
- Number
 d1

23. Two population of spherical nanoparticles :
5 nm and 50 nm
(in equal number)
Which of these distributions should I use?

24. Step by step
Size determination: PSD for particles (GSD, Grain Size Det., for polycrystalline materials)
Surface Specific Area, SSA
Z potential, hydrodynamic radius and electrophoretic mobility
Surface and 3D imaging, lattice properties

25. 1. PSD, Particle Size Distribution
Photon Correlation Spectroscopy.
Fluctuations of the light scattered from dispersed objects in suspension are due to Brownian motion and are proportional to the size of these objects.
Smaller particles move faster, causing a rapid decay of scattering
This method of measurement is standardised according to ISO

26. Dynamic Scatter Light:
In the exemple the powder contains 50% of nanparticles sized 5 nm and 50% of their aggregates, sized 50nm. The number and the volume of particles, and the intensity of the scattered light are shown.
Note that for particles of larger size the intensity is greater:
in fact, smaller particles move faster, causing a rapid decay of scattering.

27. 2. SSA, Specific Surface Area
The specific surface area, or the total surface area per gram of material, is one of the main properties characterizing nanomaterials, in which it is very larger than in bulk materials.
Measurement
The material is inserted in a closed container, under nitrogen. The gas adsorption to the surface causes a drop of the pressure of nitrogen proportional to the surface Area (B.E.T. method).

28. 3a. Z potential and hydrodynamic radius
An electrical double layer sorrounds charged particles in liquid suspensions. Around them, two regions differentiate: one (the lighter layer) where charges are diffuse, another (darker) where the charges are stricly bonds (Stern layer).
It moves together with the atoms forming the sorrounded sphere and represents the hydro-dynamic radius.
The electric potential at the boundary between Stern and diffuse radius is called Z potential.

29. Measuring the Z potential.
A laser beam passes through a cell containing the nanoparticles suspension.
When an electric field is applied to the cell, the charged particles moves.
When interfering with the laser beam, they cause the laser intensity fluctuate: the recorded signal is proportional to the particle speed.
Decrease in Z potential is followed by dramatical aggregation of nanoparticles, big aggregates does not move in the beam light.
A scheme of the apparatus follows.

30. laser; 2) attenuator; 3) cell; 4) compensation optics;
5) computer

31. 3b. Electrophoretical mobility.
Uε = 2 ε ζ f (k a) / 3 η
The Henry’s equation for measuring the electrophoretical mobility (Uε) includes the following variables:
ε: dielectric constant
ζ : Z potential
η: viscosity
F (k a) : Henry’s function
Environmental variables, as pH, concentration of ions and of sufractant-acting molecules, including polymers and organics, affects the Z potential.

32. 1. Dimensional Nanometrology
SEM: Scanning Electron Microscope; SPM: Scanning Probe Microscop; AFM: Atomic Force Microscope.
The grey box displays the dimensional range of nanomaterials.

33. 4a. TEM: Transmission Electron Microscopy
Basics: The electrons interacts with the ultra thin specimen and are transmitted through that, than recorded, The image corresponding to the transmitted electrons is magnified on a screen, a photographic layer or another sensor.
The tomographic reconstruction provides 3D images, diffraction methods give informations about the crystalline state of the sample, and the cryo-vitrification shows the macromolecule assemblies inside the sample.
Resolution: depth: 200nm, lateral resolution: 2-20nm.

34. TEM: scheme

35. 4b. SEM: Scanning Electron Microscopy
Basics: SEM uses a high-energy beam of electrons. The beam is condensed and directed at the sample surface. The interactions occurring during the scanning are recorded.
Resolution: depth: 1nm-5μm, lateral
resolution: 1-20nm.

36. SEM: Scheme SEM image of Co3O4 nanoparticles in cluster

37. 4c. AFM: Atomic Force Microscopy
Basics: The tip of a probe (cantilever) is slowly scanned across the surface. A laser beam, focused on the cantilever, records on a photodetector the deflection of the cantilever, caused by the interaction of its atoms with those on the sample surface.
Resolution: depth: 0.5nm-5nm; lateral resolution: nm.

38. AFM: Scheme
AFM image of Co3O4 nanoparticles

39. AFM techniques and applications
Contact Mode (CM): The signal is the movement of the tip, or the adjustments needed to maintain the deflection constant. The stiffness of the lever must be lower than the interatomic forces at the sample surface ( nN/nm). For topological recordings.
Lateral Force Microscopy (LFM): The twisting of the cantilever is a function of the friction levels in different areas of the sample surface.
Force Modulation (FM): The tip (or the sample) is oscillated at a high frequency and pushed into the repulsive regime. The slope of the force-distance curve is correlated to the sample's surface elasticity.
Phase Imaging: The phase shift of the oscillating tip is related to specific properties of the sample, such as friction, adhesion, and viscoelasticity.

40. 4d. NMR: Nuclear Magnetic Resonance
Basics: NMR studies a magnetic nucleus by aligning it with a very powerful external magnetic field and perturbing this alignment using an electromagnetic field. The relaxation spectra is a function of nuclear identity, 3D structure of macromolecules in solution or pore dimensions.
Resolution: in the nm range.

41. 4e. SAXS: Small Angle X Ray Scattering
Basics: X ray is incident on to a sample and
scattered electrons from the sample are analyzed
at very low angles.
The lattice interplanar spacing of the crystal is
a function of the wavelength and of the incidence
angle of the x-ray.
Resolution: between 1 nm and >200 nm.

42. SAXS: Scheme (

43. SAXS: Scheme (http://pubs.usgs.gov/of/2001/of01-041/htmldocs/xrpd.htm)
BRAGG law:
2d(sinΘ) = λo
d = lattice interplanar spacing of the crystal
Θ = x-ray incidence angle (Bragg angle)
λ = wavelength of the characteristic x-ray

44. Two-dimensional small-angle X-ray scattering image.
Nanostructure of two styrene-diene-styrene triblock copolymers.
Left:  a lamella-forming triblock showing a biaxial texture (four-spot pattern).
Right:  a cylinder-forming triblock showing a single-crystal texture (six-spot pattern).
Images: Sasha Myers,

45. 4f. SANS: Small Angle Neutron Scattering
Basics: A neutron source generates a collimated beam; neutrons are scattered by the sample, placed in the beam.
A position sensitive neutron detector detects scattered neutrons with 0.05° ≤ 2θ ≤ 3°.
The scattered intensity is a function of position.
Resolution: between 0.5 nm and 500 nm.

46. SANS: Scheme (

47. Life Cycle of Nanomaterials
Manufacturing
Nanomaterial
manufacturing
Transportation
Nano- Intermediate
Nano-enabled product
Use
End of life
Transportation
Disposal
Sewage
Landfill
From: L. Gibbs 2006

48. Health, Safety, and Environmental Concerns Regarding NM
Human implications
NM toxicity not yet well understood; nano-size materials do not behave like their bulk counterparts
Reactivity of NM due to large surface area
Potential for bioaccumulation
Environmental implications
Contamination of water and soil from improper disposal of NM
Bio-uptake of NM and accumulation in food chain

49. Potential NM exposure routes include:
Nanotoxicology
Nanotoxicology – Science of engineered nanodevices and nanostructures that deals with their effects in living organisms (Oberdorster et al )
Potential NM exposure routes include:
Inhalation
Dermal contact
Ingestion

50. Research Approaches to Understand NM Toxicity
In vitro and in vivo approaches allow study of the mechanisms and biological effects of NM on cells and tissues under controlled conditions
In vivo models include:
Inhalation chambers
Intratracheal instillation
Nose-only inhalation
Pharyngeal aspiration

51. Human Respiratory Tract

52. Proximal Alveolar Region SWCNT Day 3
Silver-enhanced gold-labeled aggregate SWCNT, 40 ug aspiration,
perfusion fixed. Mercer - NIOSH

53. SWCNT Response 7 Days
Pharyngeal aspiration of 40ug SWCNT in C57BL/6 mice
Mercer - NIOSH

54. Translocation/Bioaccumulation of Nanomaterials
Nanoparticles can cross alveolar wall into bloodstream
Absence of alveolar macrophage response
Distribution of NM to other organs and tissues
Inhaled nanoparticles may reach brain through olfactory nerve

55. In Vitro NM Studies
Monteiro-Riviere et al Isolated porcine skin flap model and HEK
MWCNT, substituted fullerenes, and QD can penetrate intact skin
Cytotoxic and inflammatory responses
Tinkle et al Human skin flexion studies and beryllium exposures
Penetration of dermis with 0.5μm an 1μm fluorescent beads

56. In Vitro NM Studies
Fullerenes can interact with cell membranes and specifically with membrane lipids (Isakovic et al. 2006; Sayes et al. 2004; 2005; Kamat et al. 2000).
Interactions can produce lipid peroxidation and leaky cell membranes that result in the release of cellular enzymes.
Proposed mechanism of damage is that fullerenes generate superoxide anions

57. Functionalization of NM
Different chemical groups added to the surface of CNT changed CNT properties and decreased their toxicity (Sayes et al. 2006)
Addition of water-soluble functional groups can decrease the toxicity of pristine C60 (Sayes et al. 2004)
Sayes et al - Human dermal fibroblasts
Sidewalled functionanlized CNT less toxic than surfactant stabilized CNT- H2O solubility was goal
Bottini 2006

58. Ingestion Pathway
Ingestion exposures can occur through direct intake of food or materials containing NM and secondary to inhalation or dermal exposures
Some evidence suggests that ingested NM may pass through to lymphatics
Little research to date about Ingestion exposures and the potential for distribution of NM to other tissues.

59. Workplace Studies
Handling Raw SWCNT
From Maynard 2005

60. Workplace Studies
Maynard and coworkers (2004) determined that aerosol concentrations of NM during handling of unrefined NM material were low
More energetic processes likely needed to increase airborne concentrations of NM
Gloves were contaminated with NM
Results indicated importance of dermal contact as potential exposure route
Handling Raw SWCNT

61. Environmental Risk Concerns Regarding NM
What happens to NM after product use and disposal?
What is the fate of NM in the environment?
Do NM degrade?
Will NM accumulate in the food chain?
How to evaluate real world exposures to NM?
Will effects be measurable?

62. NM and Ecotoxicology
Exposures of largemouth bass to fullerenes for 48 hr produced lipid damage in brain tissues (E Oberdorster 2004)
Exposures of Daphnia to uncoated, water soluble fullerenes for 48 hr indicated an LC50 of 800 ppb (E Oberdorster 2004)
Largemouth bass
Daphnia –water flea

63. Physicochemical Properties
Chemicals
Structure
pKa
Solubility
log P
Illustrations reproduced with permission from Herr’s Carbon Fullerene Gallery
Nanomaterials
Chemical Structure
Core Particle Composition
Size
Shape
Charge
Surface Chemistry
Surface Area
Agglomeration State
Zeta Potential
3-D Molecular Structure
3-D Crystal Structure

64. Diversity of Zinc Oxide Nanoparticles
Photos adapted: Dr. Z Wang, Georgia Tech

65. Size Dependent Bandgap in Quantum Dots

66. Size Dependent Bandgap in Quantum Dots

67. Size Dependent Bandgap in Quantum Dots
SIZE DEPENDENT OPTICAL ABSORPTION SPECTRA OF CAPPED CdSe NANOCLUSTERS, SYNTHESIS AND CHARACTERIZATION OF NEARLY MONODISPERSE CdE (E = S, Se, Te) SEMICONDUCTOR NANOCRYSTALLITES, MURRAY CB, NORRIS DJ, BAWENDI MG, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 115 (19): SEP )

68. Size Dependent Bandgap in Quantum Dots
SIZE AND COMPOSITION DEPENDENCE OF THE OPTICAL EMISSION SPECTRA OF CAPPED InAs (RED), InP (GREEN) AND CdSe (BLUE), BRUCHEZ, M.JR; MORONNE, M.; GIN, P.; WEISS, S.; ALIVISATOS, A.P. SEMICONDUCTOR NANOCRYSTALS AS FLUORESCENT BIOLOGICAL LABELS, SCIENCE 1998, 281, 2013

69. Size dependence of Plasmonics – Metal Nano particles
GOLD ATOMIC DISCRETE STATES
GOLD CLUSTER DISCRETE MOLECULE STATES
GOLD QUANTUM DOT CARRIER SPATIAL AND QUANTUM CONFINEMENT
GOLD COLLOIDAL PARTICLE SURFACE PLASMON – 1850 MICHAEL FARADAY ROYAL INSTITUTION GB PIONEER OF NANO!!!
BULK GOLD PLASMON

70. Plasmonics Basics – Size Effects

71. Plasmonics Basics – Size Effects
What is surface plasmon resonance of gold nanostructures?. On the top left corner is shown how the electron cloud of free-electrons in the gold respond to an oscillating electromagnetic field, depending on the shape and orientation of the particle. The formation of a dipole causes the emergence of a resonance at a specific wavelength, as shown on the right by the representative absorbance spectra. In the case of spherical particles the plasmon resonance occur at a single frequency, while for elongated nanocrystals you can have two resonance frequencies related with the two dipole oscillation modes (longitudinal or transverse).
In the bottom part of the Figure is shown the origin of the absorbance features according to the Mie theory. The absorbance A is expressed as the product of two terms. The first term is scattering-related and has a 1/l dependence, while the second term is exclusively dependent on the dielectric constants of the metal and the surrounding medium. This last term represent the resonant plasmon mode which is shown as a peak centered at the surface plasmon resonance wavelength lSPR. The product of the two terms is the spectrum observed experimentally.

72. SURFACE PLASMON RESONANCE MIE THEORY
Extinction coefficient from Mie theory is the exact solution to Maxwell’s electromagnetic field equations for a plane wave interacting with a homogenous sphere of radius R with the same dielectric constant as bulk metal (scattering and absorption contributions).
em is the dielectric constant of the surrounding medium – sensitive to environment
e = e1 + ie2 is the complex dielectric constant of the particle
Resonance peak occurs whenever the condition e1 = -2em is satisfied – sensitive to change in em of environment hence use as a surface plasmon sensor
This is the SPR peak which accounts for the brilliant colors of various metal nanoparticles – form factors can be introduced to account for non-spherical shape – Gans modification of Mie theory.

73. Extinction spectra calculated using Mie theory for gold nanospheres with diameters varying from 5 nm to 100 nm.

74. Detecting Biomolecules with Gold Nanocrystals Self Assembly and Plasmon Coupling

75. Detecting Biomolecules with Gold Nanocrystals Self Assembly and Plasmon Coupling
The coupling of plasmons can be used for the detection of oligonucleotides in solution. Gold nanocrystals can be produced with thiol-functionalized oligonucleotides bound to their surface – a construct which we call the probe. The oligonucleotides on the nanocrystals are synthesized to be complementary to the ones one wants to detect. The ultraspecific binding of oligonucleotides for their complementary strand allows the particles to bind very efficiently to the analytes in solution. Such binding of two nanocrystals to the same analyte brings the nanocrystals very close together thus enabling the coupling of the plasmons.
As shown in the diagram below, once the nanocrystals are close the dipole can extend over the ensemble of the two nanocrystals (as in resonance r2) while for single isolated particle the dipole is confined to the particle itself (resonance r1). The simultaneous presence of r1 and r2 resonances leads to an effective red shift of the absorbance peak of the nanocrystals thus changing their color, as shown in the photos thereby enabling detection of a specific oligonucleotide which shows complementary Watson-Crick base pairing.