Micha HeymannSathish AkellaDongshin Kim

Brandeis University

Sol GrunerCornell University

Microfluidics to produce and manipulate microcrystals

Nicolas DorsazLaura FilionMark Miller

Dwipayan ChakrabartiDavid WalesDaan Frenkel

Cambridge University

Bramie LenhoffUniversity of Delaware

Not only must a precise definition of mother liquor components and their concentration be undertaken, but the method used to grow the crystals, to produce supersaturation, should also be optimized. It is important to remember that the pathway a system follows from a regime of undersaturation to one ofsupersaturation is critical in determining the point at which nucleation takes place and the conditions under which the nuclei develop into crystals. The physical apparatus or device in which this takes place is a major determinant in this regard.

McPhersonCrystallization of Biological Macromolecules

Crystallization = Formulation + Kinetics

Nucleation barrier2

3

43

41 rrG

Crystal nucleus size

GkTG*

e

Crystallization rate

r

barrier height2

32

)(ln316

SkT

2*r

2

32

)(316*

kTkT

G

Foffi et al, PRE 65, 31407 (2002)

kT

br

U(r)Attractive hard sphere

Phase diagram

fluidxtal

liquid – liquid

Kinetic trap

oiling out

duration

supersaturation

Vary Quench DepthNucleate only one crystalTransform gel to crystal

Supe

rsat

urat

ion

time

nucleate grow

depth

screen supersaturation kinetics

low salt

high salt

flow

exit

S.K.W. Dertinger, D.T. Chiu, N.L. Jeon, and G.M. Whitesides."Generation of gradients having complex shapes using microfluidic networks,"

Analytical Chemistry 73, 1240-46 (2001).

20 mm

1 – 10 nl drops, 50 micron nozzle100 micron channel

50x time lapse 40 drops / sec

oil

oil

aqueous

High speed camera: 5000 frames/sec

Create and storemicrodrops

Create isolated aqueous microdrops of protein solution in an inert oil using flow focusing.

8

TEMPERATURE

drop

Heat conducts through sealed chambers.

CONCENTRATION

drop

PDMS membrane

reservoirWater permeates, drop swells.

Control temperature and concentration

9

Reversible Permeation

40 ~ 80 m

glass

25 m

PDMS

reservoir  flux

5mmleakage  flux

PDMS membrane

reservoir

flow channel

posts

welloilaqueous drop

XX

AA o

o

)(

movie: J. Shim

data: Š. Selimović

Doyle (05), Leng / Ajdari (06)

3.6M 4M

Salt and lysozyme in wells

Reservoir osmolality

Oil

glass

15 m

PDMS membrane

Protein / salt PDMS

reservoir

1 mm

Thermoelectric Thermoelectric

temperature

conc

entra

tion

conc

entr

atio

n

temperature

oil

protein

outletsinletshigh

low

salt

salt

reservoirreservoir conc

entr

atio

n

temperature

precipitate

solubleinitial drop

tem

pera

ture

protein

concentrated

dilute

hotcold

Equilibriumphase behavior

Control kinetics

3

4

5

6

2

1time

supe

rsat

urat

ion

C2

C3

First generate concentration gradientthen

Generate temperature gradient

Time 1

Time 2

Time 3

c3 c2

Vary temperature, concentration constant

Control kinetics

time

tem

pera

ture

high temp

low temp

Decoupling Nucleation and Growth (Tempereature Control)

500 um

initial

4°C for 0.5 hrs (nucleation)

final

20°C (drop filling)

14°C (crystal growth)

Measured date: Apr 29, 2009Oil: FC-43, Surfactant: 12% Tridecafluoro-1-Octanol, Aqueous: Lysozyme 75 mg/ml, 0.05M NaAc, pH 4.50.5M NaCl

time lapse: 20 hours

time

tem

pera

ture

Initial

NaCl 6M in Reservoir for 42hrs

NaCl 2M in Reservoir for 96hrs

Final

300 m

Time lapse: 4 days

PEG / lysozyme

Decoupling Nucleation and Growth (Composition Control)

tem

pera

ture

density

T = 0.1

T = 0.22

T = 0.28

T = 0.3

T = 0.4

tem

pera

ture

density

Nicolas Dorsaz

Slight Quench

Deep Quench

# co

ntac

ts

time

One crystal per drop when growth rate >> nucleation rate

time

3

4

5

6

2

1

supe

rsaturation

??60 m 600 m

small drop

1D crystal nucleation and growth ctc 2 cvt

c

Lysozme.Emulsion 50 m does not crystallize at Room T, but bulk does.

Optimal quench conditions

Diffraction pattern fromthe lysozyme crystal above,taken with a 100 μm collimated monochromatic beam at CHESS beam line F1.

Lysozyme crystal-bearingdrops in a thin-walled 200 m diameter glass capillary, mounted for data collectionat CHESS. The central circle is 100 m across.

Shotgun Diffraction (ShDi)

fraden.brandeis.edu

PhaseChip