High throughput screening of crystallization materials

• US 20030061687A1
• Filed: 04/05/2002
• Published: 04/03/2003
• Est. Priority Date: 06/27/2000
• Status: Active Grant

• Current Assignee: California Institute of Technology, Regents of the University of California (University of California)
• Original Assignee: California Institute of Technology
• Inventors: Quake, Stephen R., Hansen, Carl L., Berger, James M.
• Granted Patent: US 7,195,670 B2
• Time in Patent Office: Days
• US Class Current: 23/295R
• CPC Class Codes: B01J 2219/00274 Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
• 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 2200/0642 Filling fluids into wells by specific techniques
• 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 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0887 Laminated structure
• 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 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/049 vacuum
• CPC Class Codes: B01L 2400/0638 membrane valves, flap valves
• 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 3/502707 : characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• 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 3/502761 : specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• 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: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/125 : Allele specific primer extension
• CPC Class Codes: C30B 7/08 : by cooling of the solution
• CPC Class Codes: C30B 7/14 : the crystallising materials being formed by chemical reactions in the solution
• 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 2099/0084 : Chemistry or biology, e.g. "lab-on-a-chip" technology
• 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 99/0026 : Valves using channel deformation
• CPC Class Codes: F16K 99/0034 : Operating means specially adapted for microvalves
• CPC Class Codes: F16K 99/0059 : actuated by a pilot fluid
• 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 137/0318 : Processes
• CPC Class Codes: Y10T 137/0396 : Involving pressure control

Abstract

High throughput screening of crystallization of a target material is accomplished by simultaneously introducing a solution of the target material into a plurality of chambers of a microfabricated fluidic device. The microfabricated fluidic device is then manipulated to vary the solution condition in the chambers, thereby simultaneously providing a large number of crystallization environments. Control over changed solution conditions may result from a variety of techniques, including but not limited to metering volumes of crystallizing agent into the chamber by volume exclusion, by entrapment of volumes of crystallizing agent determined by the dimensions of the microfabricated structure, or by cross-channel injection of sample and crystallizing agent into an array of junctions defined by intersecting orthogonal flow channels.

Claims

1. A method for promoting interaction between two solutions, the method comprising:

defining a first microfluidic chamber;

priming the first microfluidic chamber with a first solution;

defining a second microfluidic chamber;

priming the second microfluidic chamber with a second solution;

placing the first microfluidic chamber into fluid communication with the second microfluidic chamber to define a microfluidic free interface between the first solution and the second solution; and

permitting diffusion to occur between the first solution and the second solution such that the first solution interacts with the second solution.

2. The method of claim 1 wherein:

the first solution comprises a crystallization target material solution;

the second solution comprises a crystallizing agent solution; and

the interaction comprises alteration of a solvent environment of the target material to promote crystallization of the target material.

3. The method of claim 1 wherein:

the first solution comprises a sample solution;

the second solution comprises a reagent solution; and

the interaction comprises a chemical reaction between the sample and the reagent.

4. The method of claim 1 wherein defining the first chamber and the second chamber comprises placing an elastomer block bearing a first patterned recess on a lower surface into contact with a substantially planar substrate.

5. The method of claim 1 wherein defining the first chamber and the second chamber comprises placing a substantially planar lower surface of an elastomer block into contact with a substrate bearing a first patterned recess.

6. The method of claim 1 wherein defining the first chamber and the second chamber comprises placing an elastomer block bearing a first patterned recess on a lower surface into contact with a substrate bearing a second patterned recess, the first patterned recess aligned with the second patterned recess.

7. The method of claim 1 wherein:

the elastomer material is permeable to a gas;

priming the first microfluidic chamber comprises introducing the first solution solution under pressure into the first microfluidic chamber to displace into the surrounding elastomer the gas formerly occupying the first chamber, thereby enabling substantially complete filling of the first chamber; and

priming the second microfluidic chamber comprises introducing the second solution under pressure into the second microfluidic chamber to displace into the surrounding elastomer the gas formerly occupying the second chamber, thereby enabling substantially complete filling of the second chamber.

8. The method of claim 1 wherein the first chamber and the second chamber are placed into fluid communication through a microfluidic flow channel defined between the elastomer block and the substrate.

9. The method of claim 8 wherein the chambers are placed into fluidic communication through actuation of a valve present in the microfluidic flow channel.

10. The method of claim 9 wherein the chambers are placed into fluidic communication through deactuation of a normally-open valve structure, such that application of a reduced pressure to a control recess crossing over the flow channel in the elastomer block causes an elastomer membrane defined between the control recess and the flow channel to relax out of the flow channel to define the microfluidic free interface.

11. The method of claim 9 wherein the chambers are placed into fluidic communication through actuation of a normally-closed valve structure, such that application of a reduced pressure to a control recess crossing over the flow channel in the elastomer block causes an elastomer membrane defined between the control recess and the flow channel to be deflected out of the flow channel into the control channel to define the microfluidic free interface.

12. A microfluidic structure facilitating crystal growth and analysis, the structure comprising:

an elastomer block;

a substrate in contact with the lower surface of the elastomer block to define a first microfluidic chamber, a second microfluidic chamber, and a third microfluidic chamber, the first, second, and third microfluidic chambers in fluid communication through a flow channel defined between elastomer block and the substrate, whereby the first chamber may be primed with a target material solution, the second chamber may be primed with a crystallizing agent, and the third chamber may be primed with a cryogen, such that crystals formed in the structure by diffusion of the crystallizing agent and the target solution may be preserved through a reduction in temperature afforded by introduction of the cryogen.

13. The microfluidic structure of claim 12 wherein the first chamber, second chamber, and third chamber are arranged in a line such that the first chamber is in fluid communication with the second chamber through a first portion of the flow channel, and the second chamber is in fluid communication with the third chamber through a second portion of the flow channel.

14. The microfluidic structure of claim 13 further comprising:

a first valve controlling fluidic communication through the first flow channel portion, the first valve defined by a first control line crossing over the first flow channel portion, a first elastomer membrane between the first control line and the first flow channel portion actuable into the first flow channel portion; and

a second valve controlling fluidic communication through the second flow channel portion, the second valve defined by a second control line crossing over the second flow channel portion, a second elastomer membrane between the second control line and the second flow channel portion actuable into the second flow channel portion, the first and second valves independently actuable to first allow diffusion between the target material solution and the crystallizing agent to form a crystal, and then to allow diffusion of the cryogen.

15. The microfluidic structure of claim 12 wherein the first chamber, second chamber, and third chamber are arranged in a triangle, such that the first chamber is in fluid communication with the second chamber through a first portion of the flow channel, the second chamber is in fluid communication with the third chamber through a second portion of the flow channel, and first chamber is in fluid communication with the third chamber through a third portion of the flow channel.

16. The microfluidic structure of claim 12 further comprising:

a first valve controlling fluidic communication through the first flow channel portion, the first valve defined by a first control line crossing over the first flow channel portion, a first elastomer membrane between the first control line and the first flow channel portion actuable into the first flow channel portion;

a second valve controlling fluidic communication through the second flow channel portion, the second valve defined by a second control line crossing over the second flow channel portion, a second elastomer membrane between the second control line and the second flow channel portion actuable into the second flow channel portion; and

a third valve controlling fluidic communication through the third flow channel portion, the third valve defined by a third control line crossing over the third flow channel portion, a third elastomer membrane between the third control line and the third flow channel portion actuable into the third flow channel portion, the first, second, and third valves independently actuable to first allow diffusion between the target material solution and the crystallizing agent to form a crystal, and then to allow diffusion of the cryogen.

17. The microfluidic structure of claim 12 wherein a thickness of at least one of the elastomer material and the substrate is reduced proximate to the chambers to permit direct interrogation of the contents of the chambers by an x-ray beam.

18. The microfluidic structure of claim 12 wherein the substrate is formed from a material different from the elastomer block and includes a first recess, a second recess, and a third recess aligned with the first, second, and third recesses of the elastomer portion, respectively, to define volumes of the chambers.

19. A structure for applying pressure to a elastomeric microfluidic device, the structure comprising:

a first holder portion including a continuous raised rim on a lower surface thereof configured to contact a top surface of the microfluidic device and surround a plurality of material wells located therein, contact between the raised rim and the top surface of the microfluidic device defining a chamber over the material wells, an orifice in communication with the chamber enabling application of positive pressure to the chamber to drive contents of the wells into an active area of the microfluidic device.

20. The structure of claim 19 further comprising a second portion including a recess configured to receive the microfluidic device and to leave exposed a portion of at least one of an upper and a lower surface of the microfluidic device.

21. The structure of claim 20 wherein the first portion and the second portion include screw holes aligned to permit mateable engagement of the first and second portion utilizing screws.

22. The structure of claim 19 wherein the first portion includes a second continuous raised rim configured to contact the surface of the microfluidic device surrounding a second plurality of material wells located in the top surface of the microfluidic device, contact between the second raised rim and the top surface of the elastomer defining a second chamber over the second plurality of material wells, a second orifice in the second portion in communication with the second chamber enabling application of positive pressure to the second chamber.

23. The structure of claim 20 further comprising a third portion configured to mateably engage with the first portion, an upper surface of the third portion including a continuous raised rim configured to press into the surface of the elastomer surrounding a plurality of openings located in a bottom surface of the microfluidic device, contact between the raised rim and the top surface of the microfluidic device defining a second chamber over the openings, an orifice in the third portion in communication with the second chamber enabling application of positive pressure to the second chamber.

24. The structure of claim 19 further comprising a biasing member in contact with the first portion and configured to press the first portion into contact with the upper surface of the microfluidic device.

25. The structure of claim 19 wherein the raised rim comprises a flexible elastomer material and a surface of the microfludic device is rigid.

26. The structure of claim 19 wherein the raised rim comprises a rigid material and a surface of the microfludic device is flexible.

27. A method of priming a microfluidic device with a liquid material, the method comprising:

loading a plurality of wells on an upper surface of a microfluidic device with a liquid material;

biasing a holder piece against the upper surface such that a continuous raised rim of the holder piece presses against the upper surface surrounding the wells, such that a chamber is created over the wells; and

applying a positive pressure to the chamber to drive the material from the wells into an active area of the elastomeric microfluidic structure.

28. The method of claim 27 wherein the raised rim deforms to engage the upper surface.

29. The method of claim 27 wherein upper surface deforms to engage the raised rim.

30. A method of actuating a valve within a microfluidic elastomer device, the method comprising:

applying a holder piece having a continuous raised rim against a surface of a microfluidic device having a plurality of control line outlets to create a chamber over the outlets; and

applying a positive or negative pressure to the chamber to control a pressure within the control line and thereby actuate a elastomeric valve membrane of the microfluidic device that is in communication with the control line.

31. The method of claim 30 wherein the raised rim is elastic and deforms to engage the surface.

32. The method of claim 30 wherein the surface is elastic and deforms to engage the raised rim.

33. A method of exercising temporal control over diffusion between two fluids, the method comprising:

providing a microfluidic flow channel in an elastomer material, a membrane portion of the elastomer material positioned within the flow channel to define a valve;

priming a first portion of the flow channel on one side of the membrane with a first fluid;

priming a second portion of the flow channel on the opposite side of the flow channel with a second fluid; and

repeatedly moving the elastomer membrane into and out of the flow channel over time to allow diffusion between the first fluid and the second fluid across the valve.

34. The method of claim 33 wherein the elastomer membrane is moved into and out of the flow channel in response to a reduced pressure within a control recess positioned in the elastomer material adjacent to the membrane.

35. The method of claim 33 wherein the first fluid is a crystallizing agent and the second fluid is a crystallization target solution, such that the elastomer membrane is repeatedly displaced to alter a solution environment of the flow channel to promote crystallization of a target material.

36. The method of claim 33 wherein a combined volume of the first fluid and the second fluid is 50 nL or less.

37. A method of capturing a concentration gradient between two fluids, the method comprising:

providing a first fluid on a first side of an elastomer membrane present within a microfluidic flow channel;

providing a second fluid on a second side of the elastomer membrane;

displacing the elastomer membrane from the microfluidic flow channel to define a microfluidic free interface between the first fluid and the second fluid;

allowing the first fluid and the second fluid to diffuse across the microfluidic free interface; and

actuating a group of elastomer valves positioned along the flow channel at increasing distances from the microfluidic free interface to define a succession of chambers whose relative concentration of the first fluid and the second fluid reflects a time of diffusion.

38. The method of claim 37 wherein the first fluid is a crystallizing agent and the second fluid is a crystallization target solution, such that a plurality of crystallizing conditions is created in the succession of chambers.

39. A method of fabricating a microfluidic device, the method comprising:

forming a plurality of wells in a top surface of a substrate;

molding an elastomer block such that a bottom surface bears a patterned recess; and

placing a bottom surface of the molded elastomer block into contact with the top surface of the substrate, such that the patterned recess is aligned with the wells to form a microfluidic flow channel between the wells.

40. The method of claim 39 further comprising:

molding a second elastomer block such that a bottom surface of the second elastomer block bears a second patterned recess; and

placing the bottom surface of the second elastomer block into contact with a top surface of the first elastomer block, such that the second patterned recess crosses over the first patterned recess to thereby define a plurality of intervening elastomer membrane valves.

41. The method of claim 39 wherein the recesses are formed in the substrate by isotropic or anisotropic etching of a silicon or glass substrate.

42. The method of claim 39 wherein the recesses are formed in the substrate by injection molding or hot embossing of a plastic substrate.

43. A method of fabricating a microfluidic device, the method comprising:

molding an elastomer block such that a bottom surface bears a patterned recess; and

placing a bottom surface of the molded elastomer block into contact with a substantially planar substrate such that the patterned recess defines a pluarlity of chambers in fluid communication through a pluarlity of flow channels.

44. A method of fabricating a microfluidic device, the method comprising:

forming a plurality of wells connected by recessed channels in a top surface of a substrate; and

placing a bottom surface of an elastomer block into contact with the top surface of the substrate to define a plurality of enclosed microfluidic chambers connected by flow channels.

45. The method of claim 44 wherein the wells and recesses are formed in the substrate by isotropic or anisotropic etching of a silicon or glass substrate.

46. The method of claim 44 wherein the wells and recesses are formed in the substrate by injection molding or hot embossing of a plastic substrate.

47. A method for forming crystals of a target material comprising:

priming a first chamber of an elastomeric microfluidic device with a first predetermined volume of a target material solution;

priming a second chamber of an elastomer microfluidic device with a second predetermined volume of a crystallizing agent; and

placing the first chamber into fluidic contact with the second chamber to allow diffusion between the target material and the crystallizing agent, such that an environment of the target material is changed to cause formation of crystal.

48. The method of claim 47 wherein the first and second volumes are predetermined by dimensions of the first chamber and the second chamber.

49. The method of claim 47 further comprising harvesting the crystal from the microfluidic device to use as a seed crystal for additional crystallization screening.

50. The method of claim 47 further comprising irradiating the crystal within the microfluidic device with x-ray radiation to determine a three dimensional structure of the crystal.

51. The method of claim 47 further comprising delivering a cryogen to the microfluidic device prior to irradiating the crystal.

52. The method of claim 47 wherein:

a concentration and volume of material within the first and second chambers represents one of a plurality of crystallization conditions within the microfluidic device; and

the method further comprises performing a second crystallization screening on a second microfluidic chip under a set of conditions related to the one of the plurality of crystallization conditions.