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

Rustem F. Ismagilov

Department of ChemistryThe University of Chicago

NIH, March 2004

A microfluidic system for protein crystallization

using nanoliter volumes of solutions

protein solution + precipitants

Protein Crystallization is Commonly Done in 100 nL -- 1000 nL (1 L) droplets

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 ?

Two problems of microfluidic flows:poor mixing and high dispersion

OUR APPROACH:

LOCALIZE REAGENTS IN DROPLETS

(water in fluorcarbon)

Two problems of microfluidic flows:poor mixing and high dispersion

How to form nL droplets of protein and precipitantsrapidly, 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?

Microfluidics for screening protein crystallization conditions

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.

water

oil

Reliable formation of aqueous droplets in oil flowingthrough a microchannel

reagent Areagent B

spacerWITHOUT PRIOR CONTACTForming aqueous droplets of multiple reagents

H. Song, J. D. Tice, R. F. Ismagilov Angew. Chem.-Int. Edit. 2003, 42, 768.

aqueous

fluorocarbon

Control Concentrations: Vary the Flow Rates

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

dispersion is taken care of !

IDEA: Screening of Protein Crystallization Conditions

B. Zheng, L. S. Roach, R. F. Ismagilov J. Am. Chem. Soc. 2003, 125, 11170-11171.


IDEA: Form droplets with variable concentration of each component, stop the flow, wait for crystals

mixing affects nucleation of protein crystals

HOW TO CONTROL MIXING?

INDUCE RAPID MIXING?

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.

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.

Slow Mixing in Straight Channels

Movie Movie

Rapid Mixing in Winding Channels


Time Scale of Mixing by Chaotic Advection in Plugs

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

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

We can control mixing and surface chemistry and do kinetics

Can we grow protein crystals?

Screening of Protein Crystallization Conditions


volume, nL

rela

tive

co

nce

ntr

atio

n

Protein Crystallization in Plugs


IT WORKS ! but

PROBLEM: evaporation of solution through PDMS -- can’t do long-term experiments

-- hard to be quantitative

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

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

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

How to Perform Vapor Diffusion Crystallizations in Capillaries??

Movie


Controlled Water Diffusion Between Plugs through Water-Permeable Oil

Initially 0.5 M NaCl


Crystallization by Vapor Diffusion in a Glass Capillary

notice refractive index match


Analysis of crystals: optical is easy (index match)

what about diffraction?

CAN WE GET DIFFRACTION FROM THESE CRYSTALS

DIRECTLY ON-CHIP ?

Use X-ray Capillaries Coupled to a PDMS microfluidic system


On-Chip X-ray Diffraction (shown to 2.0 A) (Argonne)


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 nucleationTo do: new proteins• Can we get structures on-chip ? (Cryo?)• Membrane proteins? ??

Conclusions:

Financial support:• NIH (NIBIB), • Beckman Young Investigator Program, • Office of Naval Research, • Searle Scholars Program,

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

Acknowledgements

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

Documents

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