Microfluidic protein crystallography

• US 20050205005A1
• Filed: 12/06/2004
• Published: 09/22/2005
• Est. Priority Date: 04/06/2001
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Quake, Stephen R., Hansen, Carl L., Sommer, Morten
• Granted Patent: US 7,459,022 B2
• Time in Patent Office: Days
• US Class Current: 117/206
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00355 peristaltic
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00396 Membrane valves
• CPC Class Codes: B01J 2219/00398 in multiple arrangements
• CPC Class Codes: B01J 2219/00439 using micromirror arrays
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00704 integrated with the reactor apparatus
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B01J 2219/00756 Compositions, e.g. coatings, crystals, formulations
• CPC Class Codes: B01L 2200/025 Align devices or objects to ensure defined positions relative to each other
• CPC Class Codes: B01L 2200/027 : for microfluidic devices
• CPC Class Codes: B01L 2200/0605 : Metering of fluids
• CPC Class Codes: B01L 2200/0673 : Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2200/10 : Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0681 : Filter
• CPC Class Codes: B01L 2300/0861 : Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/123 : Flexible; Elastomeric
• CPC Class Codes: B01L 2300/14 : Means for pressure control
• CPC Class Codes: B01L 2300/18 : Means for temperature control
• CPC Class Codes: B01L 2400/0472 : Diffusion
• CPC Class Codes: B01L 2400/0481 : squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0655 : pinch valves
• CPC Class Codes: B01L 2400/0688 : surface tension valves, capillary stop, capillary break
• CPC Class Codes: B01L 3/06 : Crystallising dishes
• CPC Class Codes: B01L 3/5027 : by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/50273 : characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502738 : characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: B01L 7/54 : using spatial temperature gradients
• CPC Class Codes: B01L 9/527 : for microfluidic devices, e.g. used for lab-on-a-chip
• CPC Class Codes: C30B 29/58 : Macromolecular compounds
• CPC Class Codes: C30B 35/00 : Apparatus not otherwise provided for, specially adapted for the growth, production or after-treatment of single crystals or of a homogeneous polycrystalline material with defined structure
• CPC Class Codes: C30B 7/00 : Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions from molten solvents C30B9/00; by normal or gradient freezing C30B11/00; under a protective fluid C30B27/00
• CPC Class Codes: C30B 7/10 : by application of pressure, e.g. hydrothermal processes
• CPC Class Codes: C30B 7/14 : the crystallising materials being formed by chemical reactions in the solution
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: F04B 43/043 : Micropumps
• CPC Class Codes: F04B 43/14 : having plate-like flexible members
• CPC Class Codes: F16K 2099/0074 : using photolithography, e.g. etching
• CPC Class Codes: F16K 2099/0078 : using moulding or stamping
• CPC Class Codes: F16K 2099/008 : Multi-layer fabrications
• CPC Class Codes: F16K 2099/0094 : Micropumps
• CPC Class Codes: F16K 99/0001 : Microvalves microdevices B81B1/00; manufacture or treatment of devices or systems in or on a substrate B81C1/00; microfluidic structures B01L3/5027; micropumps F04B19/006
• CPC Class Codes: F16K 99/0015 : Diaphragm or membrane valves
• CPC Class Codes: F16K 99/0028 : Valves having multiple inlets or outlets
• CPC Class Codes: F16K 99/0057 : the fluid being the circulating fluid itself, e.g. check valves
• CPC Class Codes: G01N 2013/003 : Diffusion; diffusivity between liquids
• CPC Class Codes: Y10T 117/10 : Apparatus
• CPC Class Codes: Y10T 117/1004 : with means for measuring, testing, or sensing
• CPC Class Codes: Y10T 117/1008 : with responsive control means
• CPC Class Codes: Y10T 117/1024 : for crystallization from liquid or supercritical state

Abstract

The use of microfluidic structures enables high throughput screening of protein crystallization. In one embodiment, an integrated combinatoric mixing chip allows for precise metering of reagents to rapidly create a large number of potential crystallization conditions, with possible crystal formations observed on chip. In an alternative embodiment, the microfluidic structures may be utilized to explore phase space conditions of a particular protein crystallizing agent combination, thereby identifying promising conditions and allowing for subsequent focused attempts to obtain crystal growth.

Claims

1. A method of crystallization comprising:

utilizing a microfludic formulator device to generate a solubility fingerprint of a crystallization target over a range of conditions; and

utilizing the microfluidic formulator to map phase space around those conditions of the solubility fingerprint resulting in precipitation of the crystallization target.

2. The method of claim 1 wherein the solubility fingerprint is generated by combining the crystallization target with a buffer and a precipitant.

3. The method of claim 2 wherein the solubility fingerprint is generated by combining the crystallization target with the buffer, the precipitant, and an additive.

4. The method of claim 3 wherein the solubility fingerprint is generated by combining the crystallization target with the precipitant comprising polyethylene glycol, and with the additive comprising a salt.

5. The method of claim 3 wherein a mixed volume of the crystallization target and the precipitant is about 1 μ

L or less.

6. The method of claim 2 wherein solubility of the target is indicated by a change in transmission of incident light through a mixing chamber.

7. The method of claim 6 further comprising:

imaging a portion of the mixing chamber;

calculating a standard deviation of pixel intensities of light transmitted through the portion; and

comparing the standard deviation to a background value recorded in an absence of the crystallization target.

8. The method of claim 7 wherein an approximate linear dependence of pixel standard deviation on crystallization target concentration indicates precipitation of the crystallization target.

9. The method of claim 2 wherein the conditions are generated by mixing the crystallization target with a precipitant in a rotary flow microfluidic device.

10. The method of claim 1 further comprising designing an optimal screen for the crystallization target based upon the phase space map.

11. The method of claim 1 wherein the phase space map indicates a boundary of a precipitation region for the crystallization target, the method further comprising designing an optimal screen for the crystallization target so that a final condition is located on the boundary of the precipitation region.

12. The method of claim 8 wherein the final condition is achieved by mixing the crystallization target with a precipitant in a microfluidic device.

13. The method of claim 8 wherein the final condition is achieved by performing at least one of a microbatch and a hanging drop crystallization experiment.

14. The method of claim 1 further comprising:

mapping a first phase space by altering a concentration of at least one of the crystallization target and a precipitant in order to precipitate the crystallization target;

observing a condition of initial precipitation as a first precipitation threshold;

mapping a second phase space by altering the concentration of least one of the crystallization target and the precipitate to dissolve the precipitated crystallization target;

observing a condition of last remaining precipitate as a second precipitation threshold;

determining a hysteresis between the first and second precipitation thresholds.

15. The method of claim 14 wherein the hysteresis indicates a metastable region of phase space for the crystallization target, the method further comprising reproducing conditions in the metastable region.

16. The method of claim 15 further comprising reproducing a condition in the metastable region to create crystals of the crystallization target.

17. The method of claim 16 wherein the condition in the metastable region is reproduced by mixing the crystallization target with a precipitant in a microfluidic device.

18. The method of claim 16 wherein the condition in the metastable region is reproduced by performing at least one of a microbatch and a hanging drop crystallization experiment.

19. A crystallization method comprising:

empirically determining a solubility curve for a crystallization target mixed with a precipitant utilizing a microfluidic device; and

mixing the crystallization target with the precipitant at a ratio that places a final concentration of the crystallization target and the precipitant on a boundary of the solubility curve.

20. The method of claim 19 wherein the solubility curve is empirically determined by a change in transmission of light through the microfluidic device.

21. The method of claim 20 further comprising:

transmitting light through the microfluidic device;

detecting an intensity of the transmitted light received by a plurality of pixels;

calculating a standard deviation of the pixels; and

comparing the standard deviation to a background value recorded in an absence of the crystallization target.

22. The method of claim 21 wherein an approximate linear dependence of pixel standard deviation on crystallization target concentration indicates precipitation of the crystallization target.

23. The method of claim 19 wherein the solubility curve is empirically determined by mixing the crystallization target with a precipitant in a rotary flow microfluidic device.

24. The method of claim 19 further comprising designing an optimal screen for the crystallization target based upon the empirically determined solubility curve.

25. The method of claim 24 further comprising mixing the crystallization target with the precipitant so that a final condition of the optimal screen is located on a boundary of the solubility curve.

26. The method of claim 25 wherein the final condition is achieved by mixing the crystallization target with the precipitant in a microfluidic device.

27. The method of claim 26 wherein a combined volume of crystallization target and the precipitant of about 1 μ

L or less is mixed to achieve the final condition.

28. The method of claim 25 wherein the final condition is achieved by performing at least one of a microbatch and a hanging drop crystallization experiment.

29. The method of claim 19 wherein a combined volume of crystallization target and the precipitant of about 1 μ

L or less is mixed to achieve the final condition.

30. The method of claim 19 wherein a volume of crystallization target of between about 100 nL or less is mixed with the precipitant to achieve the final concentration.

31. An apparatus for investigating crystallization comprising:

a microfluidic formulator comprising a microfluidic chamber configured to receive a crystallization target and a precipitant;

a light source configured to illuminate the microfluidic chamber; and

a light detector configured to receive light transmitted through the microfluidic chamber.

32. The apparatus of claim 31 wherein the microfluidic chamber comprises a rotary flow chamber.

33. The apparatus of claim 32 further comprising a peristaltic pump actuable to flow the crystallization target and crystallizing agent through the rotary flow chamber.

34. The apparatus of claim 32 wherein the detector comprises a plurality of pixels configured to receive light transmitted through a portion of the rotary flow chamber.

35. The apparatus of claim 32 wherein the detector comprises a charge coupled device.

36. The apparatus of claim 32 further comprising a processor configured to receive an electronic signal from the light detector indicating an intensity of light transmitted through the chamber and received on the pixel, the processor comprising a computer readable storage medium having recorded instructions to calculate from the electronic signal a standard deviation of pixel intensity of light transmitted through the portion, and to compare the standard deviation to a background value recorded in an absence of the crystallization target.