Dynamic_Light_Scattering_principle_and_application.pptx

Principle and application of DLS

1. An Introduction to Dynamic Light Scattering, Static Light Scattering & LASER Doppler Velocimetry Dr Shibsekhar Roy, Dept. of Biochemistry, UCS, OU, Hyderabad

2. Why we need a new technique for biomolecular investigation? Size Issue Conventional techniques for size measurement in nanometer scale TEM (Transmission electron microscopy) SEM (Scanning electron microscopy) AFM (Atomic force microscopy) & many other sophisticated derivatives of the above Limitations: Size obtained from these methods are of the dried/ de-hydrated form of the molecule, which is majorly nonfunctional . Real picture of the active state of the molecule cannot be known

3. Conventional advanceed biophysical techniques— Surface plasmon resonance (SPR), B io-layer interferrometry (BLI ), I sothermal titration calorimetry (ITC) Why we need a new technique for biomolecular investigation ? Contd.. Limitations: Poor fits of the signals to theoretical binding curves Ambiguous determination of the appropriate association model Erratic or irreproducible data Results that vary with immobilization chemistry or chip coating Results that depend on which binding partner is immobilized or titrated Results that vary greatly from lot to lot of reagent Fouling of microfluidic channels.

4. What else we don’t know? Homogeneity of the sample Electrostatic property of the Surface Sample conductivity Aggregation propensity Molecular weight Summation of all of these properties along with the size measured in solution (aqueous) phase are known as hydrodynamic properties

5. Size Does Matter (Let’s scatter) Measurement of Size is very important for characterization of particulate matters including proteins, polymers, micelles, vesicles, carbohydrates, nanoparticles, biological cells Gels etc We will discuss the scattering mode of size measurement

6. Two approaches of Size analysis

7. LASER Diffraction

8. What is Dynamic Light Scattering

9. Other scattering Modes

10. Basic DLS Optics [Alternatively 632nm, 558nm WLs are there also]

11. Brownian Motion

12. How do the Scattered Light Waves Interact?

13. Nature of DLS Signal Typical Intensity profiles How they really look like?

14. Correlation function- the basis of size measurement

15. Knowing the Key Players Stokes-Einstein Equation

16. What is Hydrodynamic Diameter?

17. Comparing Hydrodynamic Size to the Size Values Measured Differently Normal trend of measured size by different methods: DLS > SEM > TEM > AFM SEM TEM

18. Relation between ACF and Size

19. Mixture of Particles

20. Distribution of Mixed Population (Bi-modal sample) When 20 and 500 nm particles are measured separately When 20 and 500 nm particles are measured together

21. Polydisperse sample Cumulants

22. PDI table Working samples normally stays between PDI of 0.1 to 0.25

23. Study of protein folding Folding funnel of a protein Roy et. al., Biophys Chem. 2006 Jan 1;119(1):14-22.

24. Visualization of the folding intermediates by size dynamics of Cyt -C during refolding pathway

25. Assisting protein binding study

26. Studying protein-protein interaction Fib- AuNP Thr-AgNP Nano -Fibrin Roy et. al., FEBS Lett . 2007 Nov 27;581(28):5533-42 .

27. Immunoassay for ultra sensitive detction of tumor marker protein Chem. Commun . , 2016, 52 , 7850-7853 Detection in fM level

28. Major Precaution: Dust

29. Why DLS? Some Major Advantages

30. Zeta Potential-Introduction

31. Instrument

32. Marker for solution behaviour

33. How to carry out the study?

34. Study of assembly and aggregation CdSrod CdSrod + Cyt -C Roy et. al ., Biophys Chem. 2006 Oct 20;124(1):52-61

35. Acknowledgement Horiba Instruments Malvern Instruments Washington State University Manuals

36. Thank You for the Patience