Microfluidic nucleic acid analysis

• US 20050053952A1
• Filed: 10/02/2003
• Published: 03/10/2005
• Est. Priority Date: 10/02/2002
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Hong, Jong Wook, Anderson, W. French, Quake, Stephen R., Studer, Vincent, Leadbetter, Jared
• Granted Patent: US 8,871,446 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 2300/0809 rectangular shaped
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0655 pinch valves
• 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 3/502738 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: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

Nucleic acid from cells and viruses sampled from a variety of environments may purified and expressed utilizing microfluidic techniques. In accordance with one embodiment of the present invention, individual or small groups of cells or viruses may be isolated in microfluidic chambers by dilution, sorting, and/or segmentation. The isolated cells or viruses may be lysed directly in the microfluidic chamber, and the resulting nucleic acid purified by exposure to affinity beads. Subsequent elution of the purified nucleic acid may be followed by ligation and cell transformation, all within the same microfluidic chip. In one specific application, cell isolation, lysis, and nucleic acid purification may be performed utilizing a highly parallelized microfluidic architecture to construct gDNA and cDNA libraries.

Claims

1. A microfluidic device comprising:

a first microfluidic chamber;

a first microfluidic channel configured to deliver a sample containing at least one of a virus, a bacterium, and a cell to the first microfluidic chamber; and

a second microfluidic flow channel configured to deliver a lysis chemical to the first microfluidic chamber.

2. The microfluidic device of claim 1 wherein the first microfluidic chamber comprises a rotary mixing structure.

3. The microfluidic device of claim 1 wherein the first microfluidic chamber is in selective fluid communication with the second microfluidic flow channel through a deflectable membrane.

4. The microfluidic device of claim 1 wherein:

the first microfluidic chamber comprises a plurality of first microfluidic chambers; and

the first microfluidic channel is in common fluid communication with the plurality of first microfluidic chambers.

5. The microfluidic device of claim 4 wherein the plurality of first microfluidic chambers comprise segments of the first microfluidic channel.

6. The microfluidic device of claim 4 further comprising a control structure in common operative communication with the plurality of first microfluidic chambers to define a parallel microfluidic lysing structure.

7. The microfluidic device of claim 1 further comprising a second microfluidic chamber in selective fluid communication with the first microfluidic chamber, the second microfluidic chamber containing a nucleic acid purification medium.

8. The microfluidic device of claim 7 wherein the nucleic acid purification medium comprises a coated bead exhibiting affinity to particular nucleic acids.

9. The microfluidic device of claim 7 wherein the second microfluidic chamber is defined by a junction of the first microfluidic channel and a third microfluidic channel.

10. The microfluidic device of claim 9 wherein:

the first microfluidic channel comprises a plurality of first microfluidic channels; and

the second microfluidic chamber comprises a plurality of second microfluidic chambers defined byjunctions between the plurality of first microfluidic channels and the second microfluidic channel.

11. The microfluidic device of claim 10 further comprising a control line in common operative communication to selectively permit fluid communication between the plurality of first microfluidic chambers and the plurality of second microfluidic chambers, defining a parallel nucleic acid purification structure.

12. The microfluidic device of claim 7 further comprising a first integrated microfluidic structure in fluid communication with the second microfluidic chamber and configured to amplify purified nucleic acid received therefrom.

13. The microfluidic device of claim 12 wherein the first integrated microfluidic structure comprises a rotary mixing structure having segments in thermal communication with heating elements.

14. The microfluidic device of claim 12 wherein the first integrated microfluidic structure comprises an addressable matrix of isolatable rotary mixing structures.

15. The microfluidic device of claim 7 further comprising an integrated microfluidic structure in fluid communication with the second microfluidic chamber and configured to expose purified nucleic acid received therefrom to a host vector in the presence of ligation enzymes.

16. The microfluidic device of claim 15 wherein the first integrated microfluidic structure comprises a rotary mixing structure.

17. The microfluidic device of claim 16 wherein:

the first microfluidic channel comprises a plurality of first microfluidic channels;

the second microfluidic chamber comprises a plurality of second microfluidic chambers defined by junctions between the plurality of first microfluidic channels and the second microfluidic channel; and

the first integrated microfluidic structure comprises a first set of rotary mixers in selective parallel fluid communication with respective of the plurality of second chambers.

18. The microfluidic device of claim 17 further comprising a control line in common operative communication with the set of rotary mixers to define a parallel nucleic acid ligation structure.

19. The microfluidic device of claim 15 further comprising a second integrated microfluidic structure in fluid communication with the first integrated microfluidic structure and configured to expose purified nucleic acid ligated within a host vector, to a cell for transformation and expression of the purified nucleic acid.

20. The microfluidic device of claim 19 wherein the second integrated microfluidic structure comprises a second rotary mixing structure.

21. The microfluidic device of claim 20 wherein:

the first microfluidic channel comprises a plurality of first microfluidic channels;

the second microfluidic chamber comprises a plurality of second microfluidic chambers defined by junctions between the plurality of first microfluidic channels and the second microfluidic channel;

the first integrated microfluidic structure comprises a first set of rotary mixers in selective parallel fluid communication with respective of the plurality of second chambers; and

the second integrated microfluidic structure comprises a second set of rotary mixers in selective parallel fluid communication with respective of the first set of rotary mixers.

22. The microfluidic device of claim 21 further comprising a control line in common operative communication with the second set of rotary mixers to define a parallel nucleic acid transformation structure.

23. A method of purifying nucleic acid comprising:

providing a sample comprising at least one of bacteria, viruses, and cells;

physically isolating a subset of at least one of bacteria, viruses, and cells within a microfluidic chamber;

lysing the at least one of the bacteria, viruses, and cells within the microfluidic chamber;

flowing nucleic acid from the at least one of the lysed bacteria, viruses, and cells through a second microfluidic chamber containing a nucleic acid purification medium.

24. The method of claim 23 further comprising diluting the sample prior to the isolation step.

25. The method of claim 23 wherein the isolation step comprises creating segments within an elongated flow channel and thereby containing bacteria, viruses, or cells therein.

26. The method of claim 23 further comprising sorting the environmental sample prior to the isolation step.

27. The method of claim 23 further comprising eluting purified nucleic acid from the nucleic acid purification medium comprising nucleic acid affinity beads.

28. The method of claim 23 wherein mRNA is eluted from the nucleic acid purification medium.

29. The method of claim 28 further comprising exposing the purified mRNA to reverse transcriptase to convert the purified mRNA into cDNA.

30. The method of claim 29 further comprising constructing a cDNA library.

31. The method of claim 28 further comprising performing gene expression analysis.

32. The method of claim 31 wherein the gene expression analysis is performed utilizing a microarray chip.

33. The method of claim 23 further comprising exposing a purified nucleic acid to a host vector and a ligation enzyme within a second microfluidic chamber to ligate the purified nucleic acid within the host vector.

34. The method of claim 33 further comprising amplifying the purified nucleic acid within a third microfluidic chamber prior to exposing the purified nucleic acid to the host vector.

35. The method of claim 34 wherein the purified nucleic acid is exposed to a host vector and a ligation enzyme by circulation within a microfluidic rotary mixing structure.

36. The method of claim 33 further comprising:

exposing the host vector containing the ligated and purified nucleic acid to a cell within a third microfluidic chamber; and

transforming the cell with the host vector.

37. The method of claim 36 wherein the host vector is exposed to the cell by circulation within a microfluidic rotary mixing structure.

38. The method of claim 37 wherein a microfluidic chip containing the first, second, and third microfluidic chambers is exposed to a heat shock to transform the cell.

39. The method of claim 36 further comprising culturing the transformed cell to express the purified nucleic acid.

40. The method of claim 36 further comprising performing the isolation, lysis, and flowing steps in parallel upon a plurality of viruses, bacteria, or cells.

41. The method of claim 40 further comprising performing the ligation and transformation steps in parallel upon the plurality of viruses, bacteria, or cells.

42. The method of claim 41 further comprising constructing one of a gDNA library and a cDNA library from the purified nucleic acid.

43. The method of claim 42 further comprising identifying the morphotype, ribotype, and genotype of a single cell type collected from an environmental sample.

44. A method of microfluidic processing comprising:

providing a sample in parallel to a plurality of microfluidic structures;

controlling processing of the sample by the plurality of microfluidic structures by manipulation of a single control structure in common communication with the plurality of microfluidic structures.

45. The method of claim 44 wherein:

the sample is processed in sequence by stages of parallel microfluidic structures; and

processing of the sample by each stage is controlled by a separate control structure in common communication with each of the parallel microfluidic structures within a stage.

46. The method of claim 45 wherein the processing stages comprise an isolation stage, a lysis stage, a nucleic acid purification stage, a purified nucleic acid ligation stage, and a cell transformation stage.

47. The method of claim 46 wherein at least one of the lysis stage, the purified nucleic acid ligation stage, and the cell transformation stage comprise parallel rotary mixing structures having circulation controlled by common sets of control structures.

48. A method of analyzing a sample comprising:

flowing a sample comprising at least one of viruses and cells down a microfluidic channel;

isolating portions of the microfluidic channel into segments containing a limited number of bacteria, viruses, or cells;

performing at least one of genetic analysis, biochemical analysis, and cloning of genomic DNA or cDNA on the limited number of bacteria, viruses, or cells.

49. The method of claim 48 wherein isolating comprises causing deflection of a valve into the microfluidic channel to define adjacent segments.

50. The method of claim 49 wherein the deflecting the valve comprises deflecting a membrane defined between the microfluidic channel and an adjacent control channel.

51. The method of claim 48 wherein isolating comprises isolating less than fifty bacteria, cells, or viruses within a segment.

52. The method of claim 49 wherein performing comprises:

lysing the contents of the segment to expose material present within the cell or virus; and

purifying the exposed contents of the segment.

53. The method of claim 52 wherein the purified contents comprise nucleic acid, the method further comprising incorporating the purified nucleic acid into a cell for expression.

54. The method of claim 53 further comprising amplifying the purified nucleic acid prior to incorporation into the cell for expression.

55. A method of processing nucleic acid comprising:

providing a sample comprising multiple biological entities;

physically isolating an individual biological entity or a subset of the biological entities within a microfluidic chamber;

lysing the individual biological entity or the subset of biological entities; and

obtaining experimentally manipulatable nucleic acid preparations from the individual biological entity or the subset of biological entities.

56. The method of claim 55 wherein obtaining comprises purifying a mixture resulting from lysis of the individual biological entity or the subset of biological entities.

57. The method of claim 56 wherein purification is accomplished by flowing the mixture past an affinity bead, and then eluting the purified nucleic acid from the affinity bead.

58. The method of claim 55 wherein physical isolation is accomplished by flowing the sample through a microfluidic channel, and then transforming the microfluidic channel into isolated segments.

59. The method of claim 55 further comprising amplifying a portion of the nucleic acid.

60. The method of claim 55 further comprising purifying the nucleic acid preparation, and amplifying a portion of the purified nucleic acid.

61. The method of claim 60 wherein substantially the entire purified nucleic acid is amplified to create a library.

62. The method of claim 60 wherein an individual gene or gene fragment of the purified nucleic acid is amplified by a specific primer set.

63. The method of claim 62 wherein the gene or gene fragment of the purified nucleic acid is amplified by reverse transcriptase polymerase chain reaction (RT-PCR) to amplify only an expressed gene or gene fragment.

64. The method of claim 60 further comprising analyzing the sequence of the amplified portion of the purified nucleic acid.

65. The method of claim 60 further comprising transforming an organism with the amplified portion of the purified nucleic acid.

66. The method of claim 65 further comprising culturing the transformed organism to express the amplified portion of the purified nucleic acid.

67. A method of obtaining nucleic acid from a sample of heterogenous biological elements, the method comprising:

providing a sample comprising heterogenous biological elements;

physically isolating an individual biological element or a subset of the biological elements of the sample within a microfluidic chamber;

lysing the individual biological element or the subset of biological elements;

purifying nucleic acid from the individual biological entity or the subset of biological entities; and

amplifying at least a portion of the purified nucleic acid.

68. The method of claim 67 wherein substantially the entire purified nucleic acid is amplified to allow reproduction of the individual biological element or subset of biological elements.

69. The method of claim 67 wherein only a portion of the entire purified nucleic acid is amplified to allow reproduction of a gene or gene fragment associated with the individual biological element or subset of biological elements.

70. A method of characterizing phylogenetic, gene, and functional diversity exhibited by a specific environment comprising a plurality of biological elements, the method comprising:

providing a sample from the environment comprising heterogenous biological elements;

physically isolating within a microfluidic chamber an individual biological element or a subset of the biological elements of the sample;

lysing the individual biological element or the subset of biological elements to expose nucleic acid present therein;

purifying the exposed nucleic acid from the individual biological entity or the subset of biological entities;

amplifying at least a portion of the purified nucleic acid; and

identifying at least one of phylogenetic, gene, and functional diversity of the amplified portion of the purified nucleic acid.

71. The method of claim 70 wherein genetic diversity is identified by sequence analysis.

72. The method of claim 70 further comprising:

observing morphology of the individual biological element or subset of biological elements prior to lysis; and

correlating the genetic diversity with the observed morphology.

73. A method of obtaining genetic information regarding an individual biological element of a complex environmental sample, the method comprising:

providing an environmental sample comprising heterogenous biological elements;

physically isolating within a microfluidic chamber an individual biological element or a subset of the biological elements of the sample;

lysing the individual biological element or the subset of biological elements to expose nucleic acid present therein;

purifying the exposed nucleic acid; and

amplifying at least a portion of the purified nucleic acid.

74. The method of claim 73 further comprising identifying genetic information represented by the amplified and purified nucleic acid.

75. The method of claim 73 further comprising:

transforming a cell with the amplified portion of the purified nucleic acid ligated into an appropriate vector; and

culturing the transformed cell to clone the amplified sequence