Dynamic Light Scattering as an application tool in Nanotch and biotech. Applications of importance in prote

Published on: **Mar 3, 2016**

Source: www.slideshare.net

- 1. Nanotech & Biotech Torrey DeLuca 310-625-1159 torreydeluca@gmail.com
- 2. Nanotechnology & Biotech• Micelles• Liposome• Proteins• Gold Nano particles• DLS• Zeta Potential• Polymers
- 3. Why Light Scattering?• Light scattering techniques are sensitive to the presence of small amounts of aggregate• The velocity of a particle under an applied electric field is proportional to the charge• The scattering intensity is a function of the molecular weight and concentration• Non-invasive technique for size, molecular weight, and charge measurements
- 4. Gold Nanoparticles Surface Plasmon ResonanceGustav Mie, Ann. Physik25, 377 (1908)
- 5. Gold Nano-Particles• Useful Biomarker• Color Changes w/ Size• Polymers and Proteins easily bind to gold• Gold is chemically inert• Used in protein screening• Mie studied
- 6. Gold Colloids SEM (above) and TEM (below) images for RM 8011
- 7. 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 stationary detector Particle Size is calculated from the information contained in the fluctuating scattered light signal
- 8. Dynamic Light Scattering Why should one consider Dynamic Light Scattering? Non-invasive measurement Can Measure Low quantities of material Can Measure Concentrated Samples Good for detecting trace amounts of aggregate Good technique for macro-molecular sizing
- 9. Cost of Materials• Must characterize using small quantities• DLS useful here• Final product cost drives analysis tool
- 10. Light Scattering Return Hydrodynamic Radius Distribution & Polydispersity Solution Composition Molecular Weight 2nd Virial Coefficient Conformation Shape Estimates Zeta Potential pI & Charge Estimates
- 11. Brownian MotionParticles in suspension undergo Brownian motiondue to solvent molecule bombardment in random thermal motion. – Random – Related to Size – Related to viscosity – Related to temperature
- 12. Brownian Motion
- 13. Diffusion Particle 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 r ecto D et
- 14. Dynamic Light ScatteringPCS: Photon Correlation Spectroscopy Intensity Signal As the particle diffuses, the scattered light intensity randomly fluctuates time The fluctuation is statistically auto-correlated, the decay of the Auto-Correlation function is Auto-Correlation Curve proportional to the size G The Diffusion Coefficient is determined from time G, the Correlation Coefficient Intensity Based Particle Size Distribution Intensity The Diffusion Coefficient is Inversely proportional to the Radius (RH) Stokes-Einstein size
- 15. Hydrodynamic Radius • Shape Information • Particles with shape • Diffuse More slowly • Over estimation of size
- 16. Comparison DLS• 90o • Narrow Concentration limits • Classical DLS system • Large Aggregates degrade measurement• Back Scatter • 8 times more sensitive at the center of the cell • Adjust Scattering Area
- 17. Range of SizesTwo particles 1nm and 1µm 3Volume of the 1nm particle is 1nm Volume of the 1µm particle is 3 1,000,000,000nm
- 18. Mixed Samples• You need 1 Billion 1nm particles to equal the scattering from One 1µm particle!• DLS is useful for detecting these aggregates• Electron Microscopy would miss theseaggregates: AFM,TEM, SEM, etc…
- 19. You get the ideaSo Light Scattering is an excellenttechnique for uncovering that single largeoutlier in a distribution!!! I’m over here!
- 20. The difference between 1nm and 1µm in scale is the same as the difference between a mosquito and an elephant
- 21. Don’t Believe Me?• African elephants weigh on average 3000kg• An unfed Mosquito weighs 0.0016g• A Well fed Mosquito can weigh 0.003g There is a 1 billion times difference in size The same difference between 1µm and 1nm
- 22. What happens?• Say we don’t care about the aggregates• We want to know the size of our smallest particles• That is like saying we want to know the size our mosquitoes in a herd of elephants• Even if we only care about the smallest particles, can we use DLS?
- 23. Filter for Aggregation• A filter will remove our aggregates• Filters available in sizes 20nm to 2µm• We can also centrifuge the sample and extract the supernatant
- 24. Filtration in Action• Filtered Aggregated insulin with 20nm filter• Temperature ramp• o up to 60 C Aggregated Insulin Filtered Insulin - monomeric Unstable Insulin – High Temperature
- 25. How does Concentration Affect Analysis Diffusion Drag – Measured Alcholoic Emulsion Multiple Scattering – Concentration limit of technique Aggregation Equilibrium – Concentration limit of material – Filtration has no affects
- 26. • Diffusion Drag Bulk Viscosity Change• Particles appear to diffuse together• Apparent Increase in particle size• No Change in distribution width Dilute ConcentratedIntensity Size
- 27. Data Mexican Mudslide Milk Emulsion Alcoholic Beverage off the Shelf at the Grocery Store Well understood sample 200nm size with a high zeta potential at pH 7 Extremely stable sample
- 28. Diffusion Drag– Use bulk viscosity for Concentrated sample– Apparent size shift upwards with concentration– Polydespersity- distribution width is constant Frequency % Size Intensity Distribution Overlay 40.0 30.0 20.0 10.0 0.0 2.0000 1.0000 4.0000 6.0000 8.0000 10.0000 Size (nm) Size 1.0000 Intensity Frequency Size 40.0 30.0 20.0 10.0 0.0 (nm) 10.0000 % 8.0000 6.0000 4.0000 2.0000 Distribution OverlayMudslide neat Viscosity Corrected Mudslide dilute Mudslide neat Mudslide neat Viscosity Corrected dilute
- 29. Tabular Data Filename 200807171548077New Visc 200807171601079 200807171548077 Mudslide neat Corrected Sample Name 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 parameter• No change in distribution width• Apparent change in size is viscosity dependent
- 30. Multiple Scattering• Incident Light Scatters off of more than one particle• Particles appear smaller in size• Distribution is wider than dilute analysis Dilute Intensity Concentrated Size
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