Microfabricated crossflow devices and methods

• US 7,294,503 B2
• Filed: 09/14/2001
• Issued: 11/13/2007
• Est. Priority Date: 09/15/2000
• Status: Expired due to Term

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Quake, Stephen R., Thorsen, Todd
• Primary Examiner(s): Beisner; William H.
• Application Number:
  US09/953,103
• Publication Number:
  US 20020058332A1
• Time in Patent Office: 2,251 Days
• Field of Search: 435/29, 435/34, 435/287.1, 435/287.2, 435/286.2, 435/286.4, 435/286.5, 435/287.3, 435/288.5, 435/288.7, 435/293.1, 422/68.1, 422/82, 436/63, 436/53
• US Class Current: 435/288.5
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• 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 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/12 Specific details about materials
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2400/0418 electro-osmotic flow [EOF]
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0454 radiation pressure, optical tweezers
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0484 Cantilevers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0638 membrane valves, flap valves
• 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 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• 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 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: C12M 1/34 : Measuring or testing with condition measuring or sensing means, e.g. colony counters
• CPC Class Codes: C12Q 1/68 : involving nucleic acids
• CPC Class Codes: G01N 15/01 : specially adapted for biological cells, e.g. blood cells investigating sedimentation of particle suspensions in blood G01N15/05
• CPC Class Codes: G01N 15/1404 : Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/1434 : Optical arrangements
• CPC Class Codes: G01N 15/1459 : the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/1484 : microstructural devices
• CPC Class Codes: G01N 15/149 : specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 15/1492 : within droplets
• CPC Class Codes: G01N 2015/019 : Biological contaminants; Fouling
• CPC Class Codes: G01N 2015/1006 : for cytology
• CPC Class Codes: G01N 2015/1028 : Sorting particles
• CPC Class Codes: G01N 2015/1411 : Features of sheath fluids
• CPC Class Codes: G01N 2015/1481 : Optical analysis of particles within droplets sorting particles within droplets G01N15/1492
• CPC Class Codes: G01N 33/54366 : Apparatus specially adapted for solid-phase testing
• 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 436/118339 : with formation of a segmented stream
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]

Abstract

A microfluidic device for analyzing and/or sorting biological materials (e.g., molecules such as polynucleotides and polypeptides, including proteins and enzymes; viruses and cells) and methods for its use are provided. The device and methods of the invention are useful for sorting particles, e.g. virions. The invention is also useful for high throughput screening, e.g. combinatorial screening. The microfluidic device comprises a main channel and an inlet region in communication with the main channel at a droplet extrusion region. Droplets of solution containing the biological material are deposited into the main channel through the droplet extrusion region. A fluid different from and incompatible with the solution containing the biological material flows through the main channel so that the droplets containing the biological material do not diffuse or mix. Biological material within the droplets can be analyzed and/or sorted by detecting a predetermined characteristic of the biological sample in each droplet and sorting the droplet accordingly.

Claims

1. A microfluidic product comprising:

(a) a main channel with a diameter of between about 2 and 100 microns or cross-sectional dimensions in the range of 1 to 100 microns in fluidic communication with a source of extrusion fluid passing therethrough;

(b) a second microfluidic channel, wherein said channel has a diameter in the range of 2 to 100 microns or cross-sectional dimensions in the range of 1 to 100 microns and is in fluidic communication with a source of sample fluid, and said sample fluid is immiscible with the extrusion fluid; and

(c) said second microfluidic channel having at least one inlet region in communication with the main channel at a droplet extrusion region, the inlet region having a particle containing sample fluid immiscible with the extrusion fluid passing therethrough and constructed and arranged so that droplets of the sample fluid are sheared into the main channel at the extrusion region resulting in discrete sample fluid droplets containing particles in the extrusion fluid stream.

2. The product of claim 1 wherein the extrusion fluid is a non-polar solvent.

3. The product of claim 2 wherein the extrusion fluid is decane.

4. The product of claim 2 wherein the extrusion fluid is selected from the group consisting of tetradecane and hexadecane.

5. The product of claim 1 wherein the sample solution comprises an aqueous solution.

6. The product of claim 5 wherein the aqueous solution comprises ultrapure water, TE buffer, phosphate buffered saline or acetate.

7. The product of claim 1 wherein the particles are biological material.

8. The product of claim 7 wherein at least one of the droplets contains no more than one particle of the biological material.

9. The product of claim 8 wherein the biological material comprises viral particles and at least one of the droplets contains no more than one viral particle.

10. The product of claim 7 wherein the biological material comprises one or more cells.

11. The product of claim 7 wherein the biological material comprises one or more viral particles.

12. The product of claim 1 wherein the droplet extrusion region comprises a T-shaped junction between the inlet region and the main channel.

13. The product of claim 1 further comprising:

a detection region within or coincident with at least a portion of the main channel at or downstream from the droplet extrusion region, and a detector associated with the detection region.

14. The product of claim 13 wherein the detector is an optical detector.

15. The product of claim 13 further comprising:

a discrimination region downstream from the detection region, and a flow control system responsive to the detector and adapted to direct droplets through the discrimination region into a branch channel.

16. The product of claim 15, wherein the flow control comprises the application of radiation pressure.

17. The product of claim 15, wherein the flow control comprises an optical beam.

18. The product of claim 1 wherein the main channel resides in a layer of elastomeric material.

19. The product of claim 18 wherein the elastomeric layer is adjacent to a substrate layer.

20. The product of claim 1, further comprising:

a first inlet region in communication with the main channel at a first droplet extrusion region; and

a second inlet region in communication with the main channel at a second droplet extrusion region.

21. The product of claim 20 wherein:

a first fluid containing a first particle passes through the first inlet region, so that a droplet of the fluid containing the first particle is sheared into the main channel; and

a second fluid passes through the second inlet region, so that a droplet of the second fluid is sheared into the main channel.

22. The product of claim 21 wherein droplets containing the first particle mix with droplets of the second fluid.

23. The product of claim 22 wherein the first particle interacts with the second fluid upon said mixing.

24. The product of claim 23 wherein the interaction produces a detectable signal.

25. The product of claim 24, wherein the detectable signal is at least one of fluorescence and color change.

26. The product of claim 21, wherein the passage of each fluid through an inlet region is independently controlled.

27. The product of claim 26, wherein the passage of each fluid is controlled by at least one valve.

28. The product of claim 21, wherein droplets of the first fluid are sheared into the main channel ahead of droplets of the second fluid.

29. The product of claim 21, wherein the passage of each fluid is controlled by at least one valve.

30. The product of claim 1, wherein the sample fluid and the extrusion fluid have different refractive indexes.

31. The product of claim 1, wherein the refractive index of the sample fluid is lower than the refractive index of the extrusion fluid.

32. A product for sorting biological material comprising:

(a) a microfabricated substrate having at least one main channel with a diameter of between about 2 and 100 microns or cross-sectional dimensions in the range of 1 to 100 microns in fluidic communication with a source of extrusion fluid passing therethrough, an inlet region which meets the main channel at a droplet extrusion region in fluidic communication with a source of sample fluid, wherein said sample fluid is immiscible with the extrusion fluid and said droplet extrusion region is constructed and arranged so that droplets of the sample fluid are sheared into the main channel, and at least two branch channels meeting at a junction downstream from the droplet extrusion region;

(b) a detection region within or coincident with at least a portion of the main channel and associated with a detector; and

(c) a flow control system responding to the detector and adapted to direct biological material into a branch channel.

33. The product of claim 32 wherein:

an extrusion fluid passes through the main channel; and

a sample fluid, which is incompatible with the first fluid and contains the biological material, passes through the inlet region, so that droplets of the second fluid and containing the biological material are sheared into the main channel.

34. The product of claim 33 wherein the flow control is adapted to direct the droplets into a branch channel.

35. The product of claim 33 wherein:

the extrusion fluid is a non-polar solvent, and

the sample fluid is an aqueous solution.

36. The product of claim 35 wherein the extrusion fluid is decane.

37. The product of claim 35 wherein the extrusion fluid is selected from the group consisting of tetradecane and hexadecane.

38. The product of claim 35 wherein the aqueous solution comprises ultra pure water, TE buffer, phosphate buffered saline, or acetate buffer.

39. The product of claim 33 wherein at least one droplet contains no more than one particle of the biological material.

40. The product of claim 39 wherein the biological material comprises viral, cell, cell component, organelle, nucleic acid, protein, enzyme, or bead particles and at least one droplet contains no more than one viral, cell, cell component, organelle, nucleic acid, protein, enzyme or bead particle.

41. The product of claim 33 wherein the biological material comprises one or more viral, cell, cell component, organelle, nucleic acid, protein, enzyme or bead particles.

42. The product of claim 33, wherein the sample fluid and the extrusion fluid have different refractive indexes.

43. The product of claim 33, wherein the refractive index of the sample fluid is lower than the refractive index of the extrusion fluid.

44. The product of claim 32, wherein the flow control comprises the application of radiation pressure.

45. The product of claim 32, wherein the flow control comprises an optical beam.

46. A microfluidic product comprising:

(a) a main channel with a diameter of between about 2 and 100 microns or cross-sectional dimensions in the range of 1 to 100 microns in fluid communication with a source of extrusion fluid and having a stream of extrusion fluid passing therethrough, and

(b) at least one inlet region in communication with the main channel at a droplet extrusion region, the inlet region in fluid communication with a source of particle containing sample fluid and having a particle containing sample fluid immiscible with the extrusion fluid passing therethrough and constructed and arranged so that droplets of the sample fluid are sheared into the main channel at the extrusion region resulting in discrete sample fluid droplets containing particles in the extrusion fluid stream, wherein the main channel resides in a layer of elastomeric material.

47. The product of claim 46 wherein the elastomeric layer is adjacent to a substrate layer.

48. A microfluidic product comprising:

(a) a main channel having a stream of extrusion fluid passing therethrough, wherein said channel has a diameter in the range of 2 to 100 microns or cross-sectional dimensions in the range of 1 to 100 microns;

(b) a source of extrusion fluid provided in communication with the main channel;

(c) an inlet region in communication with the main channel at a droplet extrusion region, the inlet region having a particle containing sample fluid immiscible with the extrusion fluid passing therethrough and constructed and arranged so that droplets of the sample fluid are sheared into the main channel at the extrusion region resulting in discrete sample fluid droplets containing particles in the extrusion fluid stream;

(d) a source of particle containing sample fluid provided in communication with the inlet region;

wherein the particle containing sample fluid comprises (i) viral particles or (ii) animal cells.

49. A microfluidic product comprising:

(a) a first microfluidic channel, wherein said channel, wherein said channel has a diameter in the range of 2 to 100 microns or cross-sectional dimensions in the range of 1 to 100 microns and is in fluidic communication with a source of extrusion fluid;

(b) a second microfluidic channel, wherein said channel has a diameter in the range of 2 to 100 microns or cross-sectional dimensions in the range of 1 to 100 microns and is in fluidic communication with a source of sample fluid, and said sample fluid is immiscible with the extrusion fluid;

(c) a junction at which the first channel is in fluidic communication with the second channel and said junction is constructed and arranged so that droplets of the sample fluid are sheared into the first microfludic channel;

wherein when the extrusion fluid is flowed through the first channel at a first pressure and the sample fluid is flowed through the second channel at a second pressure lower than the first pressure, monodisperse droplets of aqueous solution are formed at the junction, said droplets having a diameter less than 60 microns.

50. A microfluidic product comprising:

(a) a first input channel in communication with a source of an aqueous dispersed phase containing reagents to be dispersed;

(b) a second input channel in communication with a source of a continuous phase that is immiscible with the dispersed phase;

(c) a junction at which the first channel is in fluidic communication with the second channel; and

,

(d) an output channel in communication with the junction, wherein said junction is constructed and arranged so that droplets of the aqueous dispersed phase are sheared into the output channel, and

wherein said first and second input channels and said output channel have a diameter in the range of 2 to 100 microns or cross-sectional dimensions in the range of 1 to 100 microns.

51. The microfluidic product of claim 50 wherein when the continuous phase is flowed through the first channel at a first pressure and the discontinuous phase is flowed through the second channel at a second pressure lower than the first pressure, monodisperse droplets of the dispersed phase having a diameter less than 60 microns are formed at the junction and flow through the output channel.

52. The microfluidic product of claim 50 wherein said first and second input channels and said output channel are from 50-100 microns wide and from 2-10 microns deep.