nanotech and biotech

Nanotech & BiotechTorrey DeLuca310-625-1159

torreydeluca@gmail.com

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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–

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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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