Method for performing crystallization and reactions in pressure-driven fluid plugs

• US 7,901,939 B2
• Filed: 01/26/2004
• Issued: 03/08/2011
• Est. Priority Date: 05/09/2002
• Status: Active Grant

• Current Assignee: University of Chicago
• Original Assignee: University of Chicago
• Inventors: Ismagliov, Rustem F., Gerdts, Cory John, Zheng, Bo, Tice, Joshua David
• Primary Examiner(s): Gakh; Yelena G
• Application Number:
  US10/765,718
• Publication Number:
  US 20050087122A1
• Time in Patent Office: 2,598 Days
• Field of Search: None
• US Class Current: 436/4
• CPC Class Codes: B01D 2009/0086 Processes or apparatus therefor
• CPC Class Codes: B01D 9/0072 Crystallisation in microfluidic devices
• CPC Class Codes: B01F 25/433 Mixing tubes wherein the shape of the tube influences the mixing, e.g. mixing tubes with varying cross-section or provided with inwardly extending profiles
• CPC Class Codes: B01F 25/4331 Mixers with bended, curved, coiled, wounded mixing tubes or comprising elements for bending the flow
• CPC Class Codes: B01F 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01J 14/00 Chemical processes in general for reacting liquids with liquids; Apparatus specially adapted therefor B01J8/00, B01J19/08 take precedence
• 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 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00599 Solution-phase processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B01J 2219/00736 Non-biologic macromolecules, e.g. polymeric compounds
• CPC Class Codes: B01J 2219/00756 Compositions, e.g. coatings, crystals, formulations
• CPC Class Codes: B01J 2219/00783 Laminate assemblies, i.e. the reactor comprising a stack of plates
• CPC Class Codes: B01J 2219/00837 comprising coatings other than catalytically active coatings
• CPC Class Codes: B01J 2219/0086 Dimensions of the flow channels
• CPC Class Codes: B01J 2219/00869 Microreactors placed in parallel, on the same or on different supports
• CPC Class Codes: B01J 2219/00889 : Mixing
• CPC Class Codes: B01J 2219/00891 : Feeding or evacuation
• CPC Class Codes: B01J 2219/00894 : More than two inlets
• CPC Class Codes: B01J 2219/00903 : Segmented flow
• CPC Class Codes: B01J 2219/00975 : Ultraviolet light
• CPC Class Codes: B01J 2219/00977 : Infrared light
• CPC Class Codes: B01L 2200/0673 : Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2200/12 : Specific details about manufacturing devices
• CPC Class Codes: B01L 2300/0867 : Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2400/0487 : fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502715 : characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• 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 3/502784 : specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07K 14/43 : Sweetening agents, e.g. thaumatin, monellin
• CPC Class Codes: C12N 9/2462 : Lysozyme (3.2.1.17)
• CPC Class Codes: C12Q 1/6806 : Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Y 302/01017 : Lysozyme (3.2.1.17)
• CPC Class Codes: C30B 29/54 : Organic compounds
• CPC Class Codes: C30B 29/58 : Macromolecular compounds
• CPC Class Codes: C30B 7/14 : the crystallising materials being formed by chemical reactions in the solution
• CPC Class Codes: G01N 1/28 : Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Qmounting specimens on microscopic slides G02B21/34; means for supporting the objects or the materials to be analysed in electron microscopes H01J37/20 ; laboratory gas handling apparatus B01L5/00
• CPC Class Codes: G01N 35/08 : using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
• CPC Class Codes: Y10T 117/10 : Apparatus
• CPC Class Codes: Y10T 137/0318 : Processes
• CPC Class Codes: Y10T 436/117497 : with a continuously flowing sample or carrier stream
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/25 : including sample preparation

Abstract

The present invention provides microfabricated substrates and methods of conducting reactions within these substrates. The reactions occur in plugs transported in the flow of a carrier-fluid.

Claims

1. A method of crystallization, comprising the steps of:

a. providing a first carrier-fluid, a first fluid comprising a precipitant, a second fluid comprising water, and a third fluid comprising a crystallization target wherein the capillary number of an interface between the first carrier fluid and either the first fluid, the second fluid, the third fluid or a mixture thereof is calculated to be sufficient to allow a plurality of plugs to form in the first carrier-fluid, at least one of the plugs comprising the first, second and third fluids;

b. introducing the first carrier-fluid into a first microchannel of a substrate;

c. introducing into a plug-forming region of the first microchannel;

1) the first fluid comprising the precipitant;

2) the second fluid comprising water; and

3) the third fluid comprising the crystallization target;

where each of the first, second and third fluids is substantially immiscible with the first carrier-fluid;

where pressure is applied when introducing each of the first, second and third fluids; and

where the crystallization target forms a crystal in the at least one of the plugs.

2. The method of claim 1, also comprising, before the crystallization target forms the crystal, the steps of:

a. providing a second carrier-fluid, a fourth fluid comprising a precipitant, a fifth fluid comprising water, and a sixth fluid comprising a crystallization target wherein the capillary number of an interface between the second carrier fluid and either the fourth fluid, the fifth fluid, the sixth fluid or a mixture thereof is calculated to be sufficient to allow a plurality of plugs to form in the second carrier-fluid, at least one of the plugs comprising the fourth, fifth, and sixth fluids;

b. introducing the second carrier-fluid into a second microchannel of the substrate;

c. introducing into a plug-forming region of the second microchannel;

1) the fourth fluid comprising the precipitant;

2) the fifth fluid comprising water; and

3) the sixth fluid comprising the crystallization target;

where each of the fourth, fifth and sixth fluids is substantially immiscible with second carrier-fluid;

where pressure is applied when introducing each of the fourth, fifth and sixth fluids;

where the first and second microchannels merge into a common downstream microchannel;

where at least one plug flowing from the first microchannel merges with a plug flowing from the second microchannel upon entry into the downstream microchannel.

3. The method of claim 1, where the plurality of plugs is formed in the first microchannel when the first fluid, the second fluid, and the third fluid are simultaneously introduced into the plug-forming region of the first microchannel.

4. The method of claim 1, where the first fluid, the second fluid, and the third fluid are introduced into the plug-forming region of the first microchannel to form a plurality of drops, the plurality of plugs is formed when at least two of the drops merge in the first microchannel.

5. The method of claim 1, where the capillary number is less than about 10.

6. The method of claim 5, where the capillary number is less than about 0.2.

7. The method of claim 1, wherein the first fluid is separated from the third fluid by the second fluid when the fluids are introduced into the plug-forming region.

8. The method of claim 1, wherein the fluids are simultaneously introduced into the plug-forming region via an inlet in fluid communication with the first microchannel.

10. The method of claim 1 or 9, where the crystallization target is a member of the group consisting of protein, peptide, polynucleotide, oligonucleotide, subcellular organelle, subcellular protein complex, drug, small molecule/biological macromolecule complex, virus, colloidal particle, nanoparticle and combinations thereof.

11. The method of claim 1 or 9, where the crystallization target is a protein.

12. The method of claim 1 or 9, where each plug comprises a solvent, and there is solvent transfer from one plug into another plug.

13. The method of claim 12, where the solvent is water.

19. The method of claim 1 or 9, where each of the carrier-fluids and fluids is introduced into the microchannels according to a respective flow rate under pressure, either directly into a microchannel or through an inlet feeding into a microchannel.

20. The method of claim 19, where the pressure is halted after one or more plugs are formed.

21. The method of claim 19, where at least one of the respective flow rates is varied relative to one another of the respective flow rates.

22. The method of claim 19, where at least one of the fluids is introduced into a respective carrier-fluid at a variable respective flow rate.

23. The method of claim 19, where the respective flow rates are varied resulting in a plurality of plugs exhibiting a concentration gradient among the plurality of plugs with regard to one or more of the fluids.

24. The method of claim 1 or 9, where at least one of the microchannels has a turn.

26. The method of claim 1 or 9, where at least one of the fluids comprises a salt.

32. The method of claim 1 or 9, where at least one of the carrier-fluids comprises a fluorinated compound.

34. The method of claim 1 or 9, further comprising detecting the presence of the crystal in at least one of the plugs.

35. The method of claim 1 or 9, further comprising:

removing the crystal from the microchannel;

placing the crystal in a capillary tube; and

analyzing the crystal in the capillary tube.

36. The method of claim 1 or 9, where water is evaporated from the plugs.

37. The method of claim 1 or 9, where the substrate is soaked in water prior to crystal formation.

38. The method of claim 1 or 9, where the substrate is soaked in an aqueous salt solution prior to crystal formation.

39. The method of claim 1 or 9, where each of the fluids comprises a solvent, and the solvent of each of the fluids is either the same or different.

9. A method of crystallization, comprising the steps of:

a. providing a first carrier-fluid and one or more first fluids wherein the capillary number of an interface between the first carrier fluid and either one or a mixture of the first fluids is calculated to be sufficient to allow a plurality of plugs to form in the first carrier-fluid, the plugs comprising the first fluids;

b. introducing the first carrier-fluid into a first microchannel of a substrate;

c. introducing the one or more first fluids into a first plug-forming region of the first microchannel to form a plug of a first plug type;

where the one or more first fluids comprise a precipitant and a crystallization target; and

where at least one plug of the first plug type comprises the precipitant and the crystallization target;

d. introducing one or more second fluids into either one of;

1) the first plug-forming region;

2) a second plug-forming region of the first microchannel;

or

3) a second plug-forming region of a second microchannel;

where each of the fluids is substantially immiscible with the first carrier-fluid;

where the one or more second fluids form a plug of a second plug type at either one of the first plug-forming region, the second plug-forming region of the first microchannel, or the second plug-forming region of a second microchannel;

where the one or more second fluids comprise a precipitant;

where at least one of the plugs of the second plug type comprises the precipitant;

where pressure is applied to the first and second microchannels when introducing the one or more first fluids and the one or more second fluids;

where at least one or more plugs of the first plug type alternate in sequence with one or more plugs of the second plug type; and

where the crystallization target forms a crystal in at least one of the at least one or more plugs of the first plug.

14. The method of claim 9, where the one or more second fluids is introduced into at least one of the first plug-forming region and the second plug-forming region.

15. The method of claim 14, where a second carrier-fluid is introduced into the first plug-forming region of the first microchannel so that it separates the one or more first fluids from the one or more second fluids prior to formation of any plug at the first plug-forming region.

16. The method of claim 15, where the second carrier-fluid is introduced through an inlet positioned between a first set of one or more inlets used for introducing one or more of the first fluids and a second set of one or more inlets used for introducing one or more of the second fluids.

17. The method of claim 9, where the one or more second fluids is introduced into the second microchannel of the second plug-forming region.

18. The method of claim 17, where the first and second microchannels merge into a common downstream microchannel, such that one or more plugs flowing from the first microchannel alternate with one or more plugs of the second plug type flowing from the second microchannel upon entry into the downstream microchannel.

25. The method of claim 9, where each of the carrier-fluids and fluids is introduced into the microchannels according to a respective flow rate under pressure, either directly into a microchannel or through an inlet feeding into a microchannel;

and where one or more of the respective flow rates are varied so that the concentration of a first component in at least one of the plugs of one plug-type provides a correlative and quantitative measure of a second component in an adjacent plug.

27. The method of claim 9, where the salt concentration in the plug of the first plug type at the first plug-forming region is lower than the salt concentration in the plug of the second plug type at the plug-forming region where the plug of the second type is formed.

28. The method of claim 9, where the salt concentration in the plug of the first type at the first plug-forming region is at least 2 times lower than the salt concentration in the plug of the second plug type at the plug-forming region where the plug of the second type is formed.

29. The method of claim 9, where the salt concentration in the plug of the first type at the first plug-forming region is at least 5 times lower than the salt concentration in the plug of the second plug type at the plug-forming region where the plug of the second type is formed.

30. The method of claim 9, where the salt concentration in the plug of the first type at the first plug-forming region is at least 10 times lower than the salt concentration in the plug of the second plug type at the plug-forming region where the plug of the second type is formed.

31. The method of claim 9, where a plug flowing pattern is obtained in which each of the plugs of the first plug type is adjacent to one plug of the second plug type.

33. The method of claim 9, where the first and second carrier-fluids are the same.

40. A method of crystallization, comprising the steps of:

a. providing a first carrier-fluid, a first fluid comprising a precipitant, and a second fluid comprising a crystallization target wherein the capillary number of an interface between the first carrier fluid and either the first fluid, the second fluid or a mixture of the first and second fluids is calculated to be sufficient to allow a plurality of plugs to form in the first carrier-fluid, at least one of the plugs comprising the first and second fluids;

b. introducing the first carrier-fluid into a first microchannel of a substrate;

c. introducing into a first inlet in fluid communication with the first microchannel;

1) the first fluid comprising the precipitant; and

2) the second fluid comprising the crystallization target;

where each of the first and second fluids is substantially immiscible with the first carrier-fluid;

where pressure is applied when introducing each of the first and second fluids; and

where the crystallization target forms a crystal in the at least one of the plugs.

41. The method of claim 40, where the first and second fluids form a laminar flow in the first inlet before the fluids contact the first carrier-fluid.

42. A method of conducting a reaction, comprising the steps of:

a. providing a first carrier-fluid, a first fluid comprising a first reagent and a second fluid comprising a second reagent wherein the capillary number of an interface between the first carrier fluid and either the first fluid, the second fluid or a mixture of the first and second fluids is calculated to be sufficient to allow a plurality of plugs to form in the first carrier-fluid, at least one of the plugs comprising the first and second fluids;

b. introducing the first carrier-fluid into a first microchannel of a substrate;

c. introducing into a first inlet in fluid communication with the first microchannel;

1) the first fluid comprising the first reagent; and

2) the second fluid comprising the second reagent;

where each of the first and second fluids is substantially immiscible with the first carrier-fluid;

where pressure is applied when introducing each of the first and second fluids; and

where the first and second reagent undergo a reaction in the at least one of the plugs.