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Rustem F. Ismagilov Department of Chemistry The University of Chicago NIH, March 2004

A microfluidic system for protein crystallization using nanoliter volumes of solutions. Rustem F. Ismagilov Department of Chemistry The University of Chicago NIH, March 2004. protein solution + precipitants. Protein Crystallization is Commonly Done

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Rustem F. Ismagilov Department of Chemistry The University of Chicago NIH, March 2004

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  1. A microfluidic system for protein crystallization using nanoliter volumes of solutions Rustem F. Ismagilov Department of Chemistry The University of Chicago NIH, March 2004

  2. protein solution + precipitants Protein Crystallization is Commonly Done in 100 nL -- 1000 nL (1 mL) droplets

  3. Can We Use Nanoliter Droplets in Microfluidic Devices to Crystallize Proteins? • less sample • less labor • faster • cheaper • new approaches? • (new knobs to • turn) • potential for • integration ?

  4. Two problems of microfluidic flows: poor mixing and high dispersion

  5. Two problems of microfluidic flows: poor mixing and high dispersion OUR APPROACH: LOCALIZE REAGENTS IN DROPLETS (water in fluorcarbon)

  6. Microfluidics for screening protein crystallization conditions How to form nL droplets of protein and precipitants rapidly, reliably, and cheaply ? with control of concentrations? How to manipulate the droplets? How to control mixing inside droplets ? How to assay the protein? How to control surface chemistry of the droplet? and control adsorption of proteins to the droplet? How to analyze the results of screening of crystallization conditions?

  7. Reliable formation of aqueous droplets in oil flowing through a microchannel water oil Tice, J. D.; Lyon, A. D.; Ismagilov, R. F. "Effects of viscosity on droplet formation and mixing in microfluidic channels", Anal. Chim. Acta 2004, 507, 73-77.

  8. Forming aqueous droplets of multiple reagents WITHOUT PRIOR CONTACT spacer reagent B reagent A aqueous fluorocarbon H. Song, J. D. Tice, R. F. Ismagilov Angew. Chem.-Int. Edit. 2003, 42, 768.

  9. Control Concentrations: Vary the Flow Rates dispersion is taken care of ! H. Song and R. F. Ismagilov J. Am. Chem. Soc. 2003, 125,14613-14619.

  10. IDEA: Screening of Protein Crystallization Conditions IDEA: Form droplets with variable concentration of each component, stop the flow, wait for crystals B. Zheng, L. S. Roach, R. F. Ismagilov J. Am. Chem. Soc. 2003, 125, 11170-11171. B. Zheng, L. S. Roach, R. F. Ismagilov J. Am. Chem. Soc. 2003, 125, 11170-11171.

  11. mixing affects nucleation of protein crystals HOW TO CONTROL MIXING? INDUCE RAPID MIXING?

  12. Slow Mixing by Recirculation in Moving Droplets Movie Tice, J. D.; Song, H.; Lyon, A. D.; Ismagilov, R. F. "Formation of droplets and mixing in multiphase microfluidics at low values of the Reynolds and the capillary numbers", Langmuir 2003, 19, 9127-9133.

  13. Egypt. J. Breadmaking D, 1500 BC? H. Song, M. R. Bringer, J. D. Tice, C. J. Gerdts, R. F. Ismagilov Appl. Phys. Lett. 2003, 83, 4664-4666.

  14. H. Song, M. R. Bringer, J. D. Tice, C. J. Gerdts, R. F. Ismagilov Appl. Phys. Lett. 2003, 83, 4664-4666.

  15. Rapid Mixing in Winding Channels Slow Mixing in Straight Channels Movie Movie

  16. Time Scale of Mixing by Chaotic Advection in Plugs H. Song, M. R. Bringer, J. D. Tice, C. J. Gerdts, R. F. Ismagilov Appl. Phys. Lett. 2003, 83, 4664-4666.

  17. H. Song, M. R. Bringer, J. D. Tice, C. J. Gerdts, R. F. Ismagilov Appl. Phys. Lett. 2003, 83, 4664-4666.

  18. H. Song, M. R. Bringer, J. D. Tice, C. J. Gerdts, R. F. Ismagilov Appl. Phys. Lett. 2003, 83, 4664-4666.

  19. Ultimately interested in Structure/Function -- can we test for function, can we do kinetics ?

  20. H. Song and R. F. Ismagilov J. Am. Chem. Soc. 2003, 125,14613-14619.

  21. H. Song and R. F. Ismagilov J. Am. Chem. Soc. 2003, 125,14613-14619.

  22. H. Song and R. F. Ismagilov J. Am. Chem. Soc. 2003, 125,14613-14619.

  23. We can control mixing and surface chemistry and do kinetics Can we grow protein crystals?

  24. We can control mixing and surface chemistry and do kinetics Can we grow protein crystals?

  25. Screening of Protein Crystallization Conditions relative concentration volume, nL B. Zheng, L. S. Roach, R. F. Ismagilov J. Am. Chem. Soc. 2003, 125, 11170-11171.

  26. Protein Crystallization in Plugs B. Zheng, L. S. Roach, R. F. Ismagilov J. Am. Chem. Soc. 2003, 125, 11170-11171.

  27. IT WORKS ! but PROBLEM: evaporation of solution through PDMS -- can’t do long-term experiments -- hard to be quantitative

  28. Next Generation: Crystallization in Glass Capillaries Coupled to a PDMS microfluidic system Bo Zheng, Joshua Tice, Spencer Roach, Rustem Ismagilov, Angewandte Chemie, 2004, in press

  29. Next Generation: Crystallization in Glass Capillaries Coupled to a PDMS microfluidic system no evaporation for 6 months! Bo Zheng, Joshua Tice, Spencer Roach, Rustem Ismagilov, Angewandte Chemie, 2004, in press

  30. Still get crystals nL Volumes if willing to wait long enough (the system allows) Under these microbatch conditions, the rate of crystal growth depends on surface chemistry AND on mixing

  31. How to Perform Vapor Diffusion Crystallizations in Capillaries?? Movie Bo Zheng, Joshua Tice, Spencer Roach, Rustem Ismagilov, Angewandte Chemie, 2004, in press

  32. Controlled Water Diffusion Between Plugs through Water-Permeable Oil Initially 0.5 M NaCl Bo Zheng, Joshua Tice, Spencer Roach, Rustem Ismagilov, Angewandte Chemie, 2004, in press

  33. Crystallization by Vapor Diffusion in a Glass Capillary notice refractive index match Bo Zheng, Joshua Tice, Spencer Roach, Rustem Ismagilov, Angewandte Chemie, 2004, in press

  34. Analysis of crystals: optical is easy (index match) what about diffraction?

  35. CAN WE GET DIFFRACTION FROM THESE CRYSTALS DIRECTLY ON-CHIP ? Use X-ray Capillaries Coupled to a PDMS microfluidic system Bo Zheng, Joshua Tice, Spencer Roach, Rustem Ismagilov, Angewandte Chemie, 2004, in press

  36. On-Chip X-ray Diffraction (shown to 2.0 A) (Argonne) Bo Zheng, Joshua Tice, Spencer Roach, Rustem Ismagilov, Angewandte Chemie, 2004, in press

  37. Conclusions: New Microfluidic Technology: • Compartmentalization in droplets, control of concentrations • Rapid mixing by chaotic advection • Control of Surface Chemistry • Integrating Functional Characterization (kinetics) and Structural Characterization (crystallization) • Direct Diffraction with no handling To do: control nucleation (good and bad) • Understand quantitatively the effect of mixing • Use surface chemistry to control heterogeneous nucleation To do: new proteins • Can we get structures on-chip ? (Cryo?) • Membrane proteins? ??

  38. Acknowledgements Ismagilov Group in Chemistry at Chicago: Droplet-based microfluidics, kinetics, and protein crystallization: Bo Zheng, Spencer Roach, Josh Tice, Cory Gerdts, Delai Chen, Helen Song, Michelle Bringer (postdoc positions) Chicago and Argonne: Andrzej Joachimiak (MCSG), Phoebe Rice, Keith Moffatt Financial support: • NIH (NIBIB), • Beckman Young Investigator Program, • Office of Naval Research, • Searle Scholars Program,

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