Microfluidics to produce and manipulate microcrystals

Transcription

1 Microfluidics to produce and manipulate microcrystals Micha Heymann Sathish Akella Dongshin Kim Brandeis University Sol Gruner Cornell University Bramie Lenhoff University of Delaware Nicolas Dorsaz Laura Filion Mark Miller Dwipayan Chakrabarti David Wales Daan Frenkel Cambridge University

2 McPherson Crystallization of Biological Macromolecules Crystallization = Formulation + Kinetics 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 of supersaturation 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.

3 Nucleation barrier G G r 4 r 3 2 Crystallization rate e kt G * * r 2 r Crystal nucleus size barrier height G * kt kT ( ) kT (ln S) 2

4 U(r) Attractive hard sphere b r kt fluid xtal oiling out liquid liquid Phase diagram Foffi et al, PRE 65, (2002) Kinetic trap

5 Vary Quench Depth Nucleate only one crystal Transform gel to crystal Supersaturation depth duration supersaturation nucleate grow time screen supersaturation kinetics

6 exit high salt low salt 20 mm flow 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, (2001).

7 Create and store microdrops Create isolated aqueous microdrops of protein solution in an inert oil using flow focusing nl drops, 50 micron nozzle 100 micron channel oil aqueous High speed camera: 5000 frames/sec 50x time lapse 40 drops / sec oil

8 Control temperature and concentration TEMPERATURE CONCENTRATION drop drop PDMS membrane Heat conducts through sealed chambers. reservoir Water permeates, drop swells. 8

9 Reversible Permeation PDMS leakage flux 5mm Doyle (05), Leng / Ajdari (06) flow well channel aqueous drop PDMS membrane reservoir flux reservoir oil 40 ~ 80 m 25 m glass posts data: Š. Selimović A( ) A o X X o 9 movie: J. Shim

10 Salt and lysozyme in wells 1 mm 3.6M 4M Reservoir osmolality 15 m Protein / salt PDMS membrane glass Oil PDMS reservoir

11 concentration Thermoelectric Thermoelectric temperature

12 cold hot concentration reservoir inlets high salt low salt protein temperature oil reservoir outlets concentrated dilute concentration temperature precipitate soluble initial drop temperature protein Equilibrium phase behavior

13 c 3 c 2 Control kinetics supersaturation Time 1 C 2 Time 2 First generate concentration gradient then Generate temperature gradient Time 3 1 C 3 time

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15 Control kinetics temperature high temp time low temp Vary temperature, concentration constant

16 Decoupling Nucleation and Growth (Tempereature Control) 20 C (drop filling) initial 4 C for 0.5 hrs (nucleation) temperature time 14 C (crystal growth) Measured date: Apr 29, 2009 Oil: FC-43, Surfactant: 12% Tridecafluoro-1-Octanol, Aqueous: Lysozyme 75 mg/ml, 0.05M NaAc, ph M NaCl 500 um time lapse: 20 hours final

17 Decoupling Nucleation and Growth (Composition Control) Initial NaCl 6M in Reservoir for 42hrs Time lapse: 4 days NaCl 2M in Reservoir for 96hrs Final 300 m PEG / lysozyme

18 density temperature

19 density temperature T = 0.4 T = 0.3 T = 0.28 T = 0.22 Nicolas Dorsaz T = 0.1

20 Slight Quench Deep Quench # contacts time

21 60 m 600 m supersaturation ?? 1 time small drop One crystal per drop when growth rate >> nucleation rate

22 1D crystal nucleation and growth c 2 t c c t v c

23 Lysozme. Emulsion 50 m does not crystallize at Room T, but bulk does. Optimal quench conditions

24 Shotgun Diffraction (ShDi) Lysozyme crystal-bearing drops in a thin-walled 200 m diameter glass capillary, mounted for data collection at CHESS. The central circle is 100 m across. Diffraction pattern from the lysozyme crystal above, taken with a 100 μm collimated monochromatic beam at CHESS beam line F1.

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26 PhaseChip fraden.brandeis.edu