nanotech and biotech
DESCRIPTION
Dynamic Light Scattering as an application tool in Nanotch and biotech. Applications of importance in proteTRANSCRIPT
Nanotechnology & Biotech
Micelles
Liposome
Proteins
Gold Nano particles
DLS
Zeta Potential
Polymers
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Why Light Scattering?Light scattering techniques are sensitive to
the presence of small amounts of aggregate
The velocity of a particle under an appliedelectric field is proportional to the charge
The scattering intensity is a function of themolecular weight and concentration
Non-invasive technique for size, molecularweight, and charge measurements
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Gold Nanoparticles
Gustav Mie, Ann. Physik25, 377(1908)
Surface PlasmonResonance
Gold Nano-ParticlesUseful Biomarker
Color Changes w/ Size
Polymers and Proteinseasily bind to gold
Gold is chemically inert
Used in proteinscreening
Mie studied
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Gold Colloids
SEM (above) andTEM (below)
images for RM8011
Dynamic Light Scattering
QELS – Quasi Elastic Light Scattering
PCS – Photon Correlation Spectroscopy
Light Scattering
Incident momochromatic light
Light Scattered from moving particles
Wavelength shifted scattered light measured at a stationarydetector
Particle Size is calculated from the information contained inthe fluctuating scattered light signal
Dynamic Light ScatteringWhy should one consider Dynamic Light
Scattering?
Non-invasive measurementCan Measure Low quantities of materialCan Measure Concentrated SamplesGood for detecting trace amounts of aggregateGood technique for macro-molecular sizing
Cost of Materials
Must characterize using small quantities
DLS useful here
Final product cost drives analysis tool
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Light Scattering ReturnHydrodynamic Radius
Distribution & Polydispersity
Solution Composition
Molecular Weight
2nd Virial Coefficient
Conformation
Shape Estimates
Zeta Potential
pI & Charge Estimates
Formulation Stability
Brownian MotionParticles in suspension undergo Brownian motiondue to solvent molecule bombardment in random
thermal motion.
RandomRelated to SizeRelated to viscosityRelated to temperature
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Brownian Motion
DiffusionParticle is randomly diffusing
Larger particles will diffuse more slowly
Larger particles have more Inertia
Scatter light off this diffusing particle
Measure the signal fluctuation of the signal
Laser
Det
ecto
r
Stokes-Einstein
Dynamic Light Scattering
As the particle diffuses, the scattered lightintensity randomly fluctuates
The fluctuation is statistically auto-correlated,the decay of the Auto-Correlation function isproportional to the size
The Diffusion Coefficient is determined fromG, the Correlation Coefficient
The Diffusion Coefficient is Inverselyproportional to the Radius (RH)
Intensity Signal
Auto-Correlation Curve
Intensity BasedParticle SizeDistribution
G
time
time
sizeIn
tens
ity
PCS: Photon Correlation Spectroscopy
Hydrodynamic RadiusShape Information
Particles with shape
Diffuse More slowly
Over estimation of size
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Comparison DLS90o
Narrow Concentrationlimits
Classical DLS system
Large Aggregatesdegrade measurement
Back Scatter
8 times more sensitiveat the center of the cell
Adjust Scattering Areato increase
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Range of SizesTwo particles 1nm and 1µm
Volume of the 1µm particle is
1,000,000,000nm3
Volume of the 1nm particle is 1nm3
Mixed SamplesYou need 1 Billion 1nm particles to equal the
scattering from One 1µm particle!
DLS is useful for detecting these aggregates
Electron Microscopy would miss these
aggregates: AFM,
TEM, SEM, etc…
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You get the ideaSo Light Scattering is an excellent
technique for uncovering that single large
outlier in a distribution!!! I’moverhere!
The difference between 1nm and 1µm in scale is the same as the difference
between a mosquito and an elephant
Don’t Believe Me?African elephants weigh on average 3000kg
An unfed Mosquito weighs 0.0016g
A Well fed Mosquito can weigh 0.003g
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There is a 1 billion times difference in size
The same difference between 1µm and 1nm
What happens?Say we don’t care
about the aggregates
We want to knowthe size of oursmallest particles
That is like saying wewant to know the sizeour mosquitoes in aherd of elephants
Even if we only careabout the smallestparticles, can we useDLS?
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Filter for AggregationA filter will remove
our aggregates
Filters available insizes 20nm to 2µm
We can alsocentrifuge the sampleand extract thesupernatant
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Filtration in ActionFiltered Aggregated insulin with 20nm filter
Temperature ramp
up to 60oC
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•Aggregated InsulinFiltered Insulin - monomericUnstable Insulin – HighTemperature
How does Concentration AffectAnalysisDiffusion Drag
Measured Alcholoic Emulsion
Multiple Scattering
Concentration limit of technique
Aggregation Equilibrium
Concentration limit of material
Filtration has no affects
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Diffusion DragBulk Viscosity Change
Particles appear todiffuse together
Apparent Increase inparticle size
No Change indistribution width
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•Dilute Concentrated
Size
Inte
nsity
DataMexican Mudslide
Milk Emulsion
Alcoholic Beverage off the Shelfat the Grocery Store
Well understood sample
200nm size with a high zetapotential at pH 7
Extremely stable sample
Diffusion DragUse bulk viscosity for Concentrated sampleApparent size shift upwards with concentrationPolydespersity- distribution width is constant
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1.00002.00004.00006.00008.000010.00001.00001.0000
2.00002.0000
4.00004.0000
6.00006.0000
8.00008.0000
10.000010.0000
0.010.020.030.040.00.0
0.0
10.0
10.0
20.0
20.0
30.0
30.0
40.0
40.0
Size Intensity Distribution Overlay
Size Intensity Distribution Overlay
Size (nm) Size (nm)Frequency %
Frequency %
Mudslide neat Viscosity CorrectedMudslide diluteMudslide neatMudslide neat Viscosity Corrected Mudslide dilute Mudslide neat
Tabular DataFilename 200807171548077New Visc 200807171601079 200807171548077
Sample NameMudslide neat Corrected
Viscosity Mudslide dilute Mudslide neat
Viscosity (mPa s) 5 0.952 0.952
Median (nm) 228.1 220.9 1201.5
Mean (nm) 231.5 226.7 1218.9
CV 21.604 25.083 21.517
Polydespersity Index 0.093 0.126 0.093
Diffusion Coefficient (m2/s) 2.8174E-15 (m2/s) 1.4831E-14 (m2/s) 1.4798E-14 (m2/s)
Adjust viscosity parameterNo change in distribution width Apparent change in size is viscosity
dependent
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Multiple ScatteringIncident Light Scatters
off of more than oneparticle
Particles appear smallerin size
Distribution is widerthan dilute analysis
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•Dilute
Concentrated
Size
Inte
nsity
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