Microfluidic devices

• US 20080003142A1
• Filed: 05/11/2007
• Published: 01/03/2008
• Est. Priority Date: 05/11/2006
• Status: Abandoned Application

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Weiner, Michael, Link, Darren, Marran, David, Rothberg, Jonathan
• Application Number:
  US11/803,104
• Publication Number:
  US 20080003142A1
• Time in Patent Office: Days
• US Class Current: 422/82.08
• CPC Class Codes: B01F 23/41 Emulsifying
• CPC Class Codes: B01F 33/3011 using a sheathing stream of a fluid surrounding a central stream of a different fluid, e.g. for reducing the cross-section of the central stream or to produce droplets from the central stream
• 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: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0636 Focussing flows, e.g. to laminate flows
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• 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 2300/0636 Integrated biosensor, microarrays
• 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 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/165 Specific details about hydrophobic, oleophobic surfaces
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0424 Dielectrophoretic forces
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502746 characterised by the means for controlling flow resistance, e.g. flow controllers, baffles B01L3/502738 takes precedence
• 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: B01L 3/565 : Seals in general F16L
• CPC Class Codes: B01L 7/525 : with physical movement of samples between temperature zones
• CPC Class Codes: B01L 9/527 : for microfluidic devices, e.g. used for lab-on-a-chip
• CPC Class Codes: B03C 5/005 : Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength
• CPC Class Codes: B03C 5/026 : using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
• CPC Class Codes: C12N 15/1068 : Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12N 15/1086 : Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12Q 1/00 : Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• 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: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6844 : Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6846 : Common amplification features
• CPC Class Codes: C12Q 1/6855 : Ligating adaptors
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 : Methods for sequencing
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/191 : incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 : PCR
• CPC Class Codes: C12Q 2535/101 : Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2535/122 : Massive parallel sequencing
• CPC Class Codes: C12Q 2561/101 : Taqman
• CPC Class Codes: C12Q 2563/149 : Particles, e.g. beads
• CPC Class Codes: C12Q 2563/159 : Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2565/518 : characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/601 : being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: C12Q 2565/628 : being a surface plasmon resonance spectrometer
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: G01N 15/147 : the analysis being performed on a sample stream
• CPC Class Codes: G01N 2021/0346 : Capillary cells; Microcells
• CPC Class Codes: G01N 2035/00237 : Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 2035/00326 : Analysers with modular structure
• CPC Class Codes: G01N 21/05 : Flow-through cuvettes G01N21/09 takes precedence; handling fluid samples G01N1/10
• CPC Class Codes: G01N 21/64 : Fluorescence; Phosphorescence
• CPC Class Codes: G01N 2201/024 : Modular construction
• CPC Class Codes: G01N 27/3275 : Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
• 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 137/87571 : Multiple inlet with single outlet
• CPC Class Codes: Y10T 137/87619 : With selectively operated flow control means in inlet
• CPC Class Codes: Y10T 137/87652 : With means to promote mixing or combining of plural fluids

Abstract

The present invention provides novel microfluidic substrates and methods that are useful for performing biological, chemical and diagnostic assays. The substrates can include a plurality of electrically addressable, channel bearing fluidic modules integrally arranged such that a continuous channel is provided for flow of immiscible fluids.

Claims

1. A microfluidic substrate comprising:

a) at least one inlet module having at least one inlet channel adapted to carry at least one dispersed phase fluid; and

b) at least one main channel adapted to carry at least one continuous phase fluid, wherein said inlet channel is in fluid communication with said main channel at a junction, wherein said junction comprises a fluidic nozzle designed for flow focusing such that said dispersed phase fluid is immiscible with said continuous phase fluid and forms a plurality of highly uniform, monodisperse droplets in said continuous phase fluid.

2. The microfluidic substrate of claim 1, wherein said inlet module further comprises at least one self-aligning fluidic interconnect apparatus to connect the inlet channel to a means for introducing a sample fluid to said channel, wherein said apparatus forms a radial seal between said microfluidic substrate and said means for introducing sample.

3. The microfluidic substrate of claim 2, wherein said means is a well or reservoir.

4. The microfluidic substrate of claim 1, further comprising at least one coalescence module downstream from and in fluid communication with said inlet module via the main channel comprising a coalescence apparatus, wherein two or more droplets passing there through are coalesced to form a nanoreactor.

5. The microfluidic substrate of claim 4, wherein the coalescence apparatus comprises one or more electrodes that generate an electric field.

6. The microfluidic substrate of claim 5, wherein the electrodes comprise electrically conductive material that is integrally contained in one or more channels and isolated from the inlet and main channels.

7. The microfluidic substrate of claim 4, wherein said coalescence module comprises an expanded portion of the main channel between the electrodes to bring successive droplets into proximity, whereby the paired droplets are coalesced within the electric field.

8. The microfluidic substrate of claim 4, wherein said coalescence module comprises a narrowed portion of the main channel to center droplets within the main channel followed by a expanded portion of the main channel between the electrodes to bring successive droplets into proximity, whereby the paired droplets are coalesced within the electric field.

9. The microfluidic substrate of claim 1, wherein said substrate further comprises at least one detection module downstream from and in fluid communication with said coalescence module, said detection module comprising a detection apparatus for evaluating the contents or characteristics of the droplet or nanoreactor.

10. The microfluidic substrate of claim 9, wherein said detection apparatus comprises an optical or electrical detector.

11. The microfluidic substrate of claim 1, wherein said substrate further comprises a sorting module in proximity to and in fluid communication with said detection module, said sorting module comprising a sorting apparatus adapted to direct said droplet or nanoreactor into or away from a collection module in response to the contents or characterization of the droplet or nanoreactor evaluated in the detection module.

12. The microfluidic substrate of claim 11, wherein the sorting apparatus comprises one or more electrodes that generate an electric field.

13. The microfluidic substrate of claim 12, wherein the electrodes comprise an electrically conductive material that is integrally contained in one or more channels and are isolated from the inlet and main channels.

14. The microfluidic substrate of claim 1, wherein said inlet and main channels are coated with an anti-wetting or blocking agent for the dispersed phase.

15. The microfluidic substrate of claim 1, wherein said channels are coated with a silica primer layer followed by a perfluoroalkylalkylsilane compound, an amorphous soluble perfluoropolymer, BSA, PEG-silane or fluorosilane.

16. The microfluidic substrate of claim 1, wherein the channels are coated with a silica primer layer followed by a perfluoroalkylalkylsilane compound.

17. The microfluidic substrate of claim 11, wherein the channels in the sorting module comprise an asymmetric bifurcation geometry.

18. The microfluidic substrate of claim 11, wherein the channels in the sorting module comprise an asymmetric flow geometry.

19. The microfluidic substrate of claim 1, wherein the channels comprise well-like indentations to slow, stop or react contents of droplets.

20. The microfluidic substrate of claim 1, wherein said substrate further comprises a delay module in fluid communication with the main channel downstream of the inlet module.

21. The microfluidic substrate of claim 20, wherein said delay module is a delay line.

22. The microfluidic substrate of claim 20, wherein the delay module further comprises heating and cooling regions.

23. The microfluidic substrate of claim 1, wherein said substrate further comprises a mixing module in fluid communication with the main channel downstream of the inlet module.

24. The microfluidic substrate of claim 1, wherein said substrate further comprises a UV-release module in fluid communication with the main channel downstream of the inlet module.

25. The microfluidic substrate of claim 1, wherein said substrate further comprises a collection module connected to a means for storing a sample from said substrate.

26. The microfluidic substrate of claim 25, wherein said means is a well or reservoir.

27. The microfluidic substrate of claim 1, wherein said substrate further comprises a waste module connected to a means for collecting a sample discarded from said substrate.

28. The microfluidic substrate of claim 27, wherein said means is a well or reservoir.

29. The microfluidic substrate of claim 1, wherein said continuous phase is a non-polar solvent.

30. The microfluidic substrate of claim 1, wherein said continuous phase is a fluorocarbon oil.

31. The microfluidic substrate of claim 1, wherein said continuous phase further comprises one or more additives.

32. The microfluidic substrate of claim 31, wherein said additive is a fluorosurfactant.

33. The microfluidic substrate of claim 32, wherein said fluorosurfactant is a perfluorinated polyether.

34. The microfluidic substrate of claim 32, wherein said fluorosurfactant stabilizes said droplets.

35. The microfluidic substrate of claim 1, wherein said dispersed phase comprises a library of droplets.

36. The microfluidic substrate of claim 35, wherein said library of droplets comprises a biological/chemical material.

37. The microfluidic substrate of claim 36, wherein said biological/chemical material is selected from the group consisting of tissues, cells, particles, proteins, antibodies, amino acids, nucleotides, small molecules, and pharmaceuticals.

38. The microfluidic substrate of claim 36, wherein said biological/chemical material comprises a label.

39. The microfluidic substrate of claim 35, wherein said library of droplets comprises a label.

40. The microfluidic substrate of claim 38, wherein said label is a protein, a DNA tag, a dye, a quantum dot a radio frequency identification tag.

41. The microfluidic substrate of claim 39, wherein said label is a change in viscosity, a change in opacity, a change in volume, a change in density, a change in pH, a change in temperature, a change in dielectric constant, a change in conductivity, a change in the amount of beads present in the droplets, a change in the amount of flocculent in the droplets, a change in the amount of a selected solvent within the droplets or the change in the amount of any measurable entity within the droplets, or combinations thereof.

42. The microfluidic substrate of claim 38, wherein said label can be detected by fluorescence polarization, fluorescence intensity, fluorescence lifetime, fluorescence energy transfer, pH, ionic content, temperature or combinations thereof.

43. The microfluidic substrate of claim 1, wherein the flow of said dispersed phase fluid and said continuous phase fluid is pressure driven.

44. The microfluidic substrate of claim 4, wherein the droplets to be coalesced have no charge.

45. The microfluidic substrate of claim 11, wherein the nanoreactors to be sorted have no charge.

46. A microfluidic substrate comprising:

a) at least one inlet module having at least one inlet channel adapted to carry at least one dispersed phase fluid;

b) at least one main channel adapted to carry at least one continuous phase fluid, wherein said inlet channel is in fluid communication with said main channel at a junction, wherein said junction comprises a fluidic nozzle designed for flow focusing such that said dispersed phase fluid is immiscible with said continuous phase fluid and forms a plurality of highly uniform, monodisperse droplets in said continuous phase fluid;

c) at least one nanoreactor division module downstream from the inlet module wherein the main channel is divided into at least two division channels and the nanoreactor is split into at least two daughter nanoreactors;

d) at least one second inlet channel adapted to carry at least one second dispersed phase fluid wherein said inlet channel is in fluid communication with at least one of the divisional channels at a junction, wherein said junction comprises a fluidic nozzle designed for flow focusing such that said second dispersed phase fluid is immiscible with said continuous phase fluid and forms a plurality of highly uniform, monodisperse droplets in said continuous phase fluid

e) at least one coalescence module downstream from and in fluid communication with said inlet module via the main channel comprising a coalescence apparatus, wherein at least one droplet from step (b) and at least one droplet from step (d) passing there through are coalesced;

f) at least one reorder module downstream from said dividing module such that the daughter nanoreactors from the division channel are reordered in proximity but not coalesced; and

g) at least one detection module downstream from said reorder module, said detection module comprising a detection apparatus for evaluating the contents or characteristics of at least one of nanoreactors or droplets in proximity.

47. The method of claim 46, further comprising a sorting module in proximity to and in fluid communication with said detection module, said sorting module comprising a sorting apparatus adapted to direct said droplet or nanoreactor into or away from a collection module in response to the contents or characterization of the droplet or nanoreactor evaluated in the detection module.

48. The method of claim 47, wherein the detection module evaluates the contents of two nanoreactors or droplets in proximity and the sorting module directs said droplets or nanoreactors into or away from a collection module in response to the ratio of the contents or characterization of the droplets or nanoreactors evaluated in the detection module.

49. A microfluidic substrate produced by the process of

a) providing a base plate, wherein the base plate comprises a flat surface;

b) providing a master comprising the pattern of the channels and electrodes of a microfluidic substrate;

c) providing a molding cavity, wherein the molding cavity comprises an opening for molding an elastomeric substrate;

d) assembling the base plate, master and molding cavity, such that said master is placed between the base plate and molding cavity and wherein said master pattern is located directly under and aligned to the opening for molding an elastomeric substrate;

e) providing a top plate containing one or more sliding molding pins used to form one or more fluid and/or electrical interconnects;

f) assembling the top plate onto the molding cavity of step d, such that the sliding molding pins contact points on the pattern of channels and electrodes on the master;

g) introducing a liquid elastomeric polymer into the opening on the molding cavity such that it contacts the master;

h) solidifying the elastomeric polymer within the molding cavity;

i) removing the solidified elastomeric polymer substrate from the top plate, bottom plate and molding cavity assembly; and

j) bonding the solidified elastomeric polymer substrate to compatible polymeric or non-polymeric media, thereby forming a microfluidic substrate with fluidic and/or electrical interconnects.

50. The top plate of claim 49, wherein the sliding molding pins are surrounded by an elastomeric sleeve.

51. The microfluidic substrate of claim 49, wherein said master is generated by photolithography.

52. The microfluidic substrate of claim 49, wherein said master is generated by photolithography and converted to a durable metal master.

53. The microfluidic substrate of claim 49, wherein said master is generated by micromachining.

54. The microfluidic substrate of claim 49, wherein said master is generated by rapid prototyping methods such as stereolithography.

55. The microfluidic substrate of claim 49, wherein said master is a silicon or glass substrate patterned with photoresist.

56. The microfluidic substrate of claim 49, wherein said master is a silicon or glass substrate patterned with SU-8.

57. The microfluidic substrate of claim 49, wherein said elastomeric polymer is a silicone elastomeric polymer.

58. The microfluidic substrate of claim 57, wherein said silicone elastomeric polymer is polydimethylsiloxane.

59. The microfluidic substrate of claim 49, wherein said elastomeric polymer is solidified by curing.

60. The microfluidic substrate of claim 49, wherein said elastomeric polymer is treated with high intensity oxygen or air plasma to permit bonding to the compatible polymeric or non-polymeric media.

61. The microfluidic substrate of claim 49, wherein said polymeric and non-polymeric media is glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, or epoxy polymers.

62. A method of producing microfluidic substrate comprising

a) providing a base plate, wherein the base plate comprises a flat surface;

b) providing a master comprising the pattern of the channels and electrodes of a microfluidic substrate;

c) providing a molding cavity, wherein the molding cavity comprises an opening for molding an elastomeric substrate;

d) assembling the base plate, master and molding cavity, such that said master is placed between the base plate and molding cavity and wherein said master pattern is located directly under and aligned to the opening for molding an elastomeric substrate;

e) providing a top plate containing one or more sliding molding pins used to form one or more fluid and/or electrical interconnects;

f) assembling the top plate onto the molding cavity of step d, such that the sliding molding pins contact points on the pattern of channels and electrodes on the master;

g) introducing a liquid elastomeric polymer into the opening on the molding cavity such that it contacts the master;

h) solidifying the elastomeric polymer within the molding cavity;

i) removing the solidified elastomeric polymer substrate from the top plate, bottom plate and molding cavity assembly; and

j) bonding the solidified elastomeric polymer substrate to compatible polymeric or non-polymeric media,

thereby producing a microfluidic substrate with fluidic and/or electrical interconnects.

63. The top plate of claim 62, wherein the sliding molding pins are surrounded by an elastomeric sleeve.

64. The microfluidic substrate of claim 62, wherein said master is generated by photolithography.

65. The microfluidic substrate of claim 62, wherein said master is generated by photolithography and converted to a durable metal master.

66. The microfluidic substrate of claim 62, wherein said master is generated by micromachining.

67. The microfluidic substrate of claim 62, wherein said master is generated by rapid prototyping methods such as stereolithography.

68. The microfluidic substrate of claim 62, wherein said master is a silicon or glass substrate patterned with photoresist.

69. The microfluidic substrate of claim 62, wherein said master is a silicon or glass substrate patterned with SU-8.

70. The microfluidic substrate of claim 62, wherein said elastomeric polymer is a silicone elastomeric polymer.

71. The microfluidic substrate of claim 70, wherein said silicone elastomeric polymer is polydimethylsiloxane.

72. The microfluidic substrate of claim 62, wherein said elastomeric polymer is solidified by curing.

73. The microfluidic substrate of claim 62, wherein said elastomeric polymer is treated with high intensity oxygen or air plasma to permit bonding to the compatible polymeric or non-polymeric media.

74. The microfluidic substrate of claim 62, wherein said polymeric and non-polymeric media is glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, or epoxy polymers.