microfluidics to produce and manipulate microcrystals...microfluidics to produce and manipulate...
TRANSCRIPT
![Page 1: Microfluidics to produce and manipulate microcrystals...Microfluidics to produce and manipulate microcrystals Nicolas Dorsaz Laura Filion Mark Miller Dwipayan Chakrabarti David Wales](https://reader030.vdocument.in/reader030/viewer/2022040616/5f105e4b7e708231d448c3e6/html5/thumbnails/1.jpg)
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
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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
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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
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Foffi et al, PRE 65, 31407 (2002)
kT
br
U(r)Attractive hard sphere
Phase diagram
fluidxtal
liquid – liquid
Kinetic trap
oiling out
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duration
supersaturation
Vary Quench DepthNucleate only one crystalTransform gel to crystal
Supe
rsat
urat
ion
time
nucleate grow
depth
screen supersaturation kinetics
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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
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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.
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8
TEMPERATURE
drop
Heat conducts through sealed chambers.
CONCENTRATION
drop
PDMS membrane
reservoirWater permeates, drop swells.
Control temperature and concentration
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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)
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3.6M 4M
Salt and lysozyme in wells
Reservoir osmolality
Oil
glass
15 m
PDMS membrane
Protein / salt PDMS
reservoir
1 mm
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Thermoelectric Thermoelectric
temperature
conc
entra
tion
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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
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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
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Vary temperature, concentration constant
Control kinetics
time
tem
pera
ture
high temp
low temp
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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
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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)
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tem
pera
ture
density
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T = 0.1
T = 0.22
T = 0.28
T = 0.3
T = 0.4
tem
pera
ture
density
Nicolas Dorsaz
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Slight Quench
Deep Quench
# co
ntac
ts
time
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One crystal per drop when growth rate >> nucleation rate
time
3
4
5
6
2
1
supe
rsaturation
??60 m 600 m
small drop
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1D crystal nucleation and growth ctc 2 cvt
c
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Lysozme.Emulsion 50 m does not crystallize at Room T, but bulk does.
Optimal quench conditions
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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)
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fraden.brandeis.edu
PhaseChip