Methods, systems and apparatus for separation and isolation of one or more sample components of a sample biological material

• US 20030215855A1
• Filed: 03/27/2003
• Published: 11/20/2003
• Est. Priority Date: 04/02/2002
• Status: Active Grant

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Caliper Holdings Incorporated
• Inventors: Dubrow, Robert S., Bousse, Luc J., Phan, Huan L.
• Granted Patent: US 7,419,784 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 2200/0605 Metering of fluids
• CPC Class Codes: B01L 2200/0631 Purification arrangements, e.g. solid phase extraction [SPE]
• 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 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• 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 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• 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: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

Devices, systems and methods for use in separating sample materials into different sample components and then isolating one or more of the sample components for further processing or analysis are disclosed. Devices employ configurations that optionally allow a sample material to be electrophoretically separated into sample components in a separation matrix within a separation conduit. The sample components may then be detected in a detection zone in the separation conduit, and then selected components shunted to a component collection conduit within the device downstream of the detection zone for further processing or analysis based on information received at the detection zone. Methods of using these devices, and systems that incorporate these devices are also envisioned.

Claims

1. A method of separating a sample material into a plurality of separated components and isolating one or more of said separated components comprising:

providing a microfluidic substrate including a separation conduit having a separation matrix disposed therein;

providing a detector in sensory communication with said separation conduit at a first location along a length of the separation conduit for detecting said one or more sample components in the separation conduit;

providing a sample component collection conduit in said substrate in fluid communication with said separation conduit at a second location downstream from said first location;

providing a processor which is operably coupled to said detector and to a fluid direction system for controlling movement of sample components from said separation conduit into said sample component collection conduit based on information received from said detector;

transporting a first sample material through the separation conduit to separate the first sample material into a plurality of sample components;

detecting said plurality of sample components at said first location with said detector; and

moving a selected one or more of said plurality of sample components from said separation conduit into the sample component collection conduit in response to instructions from the processor to the fluid direction system.

2. The method of claim 1, further comprising a sample loading conduit having a loading end and a waste end, the loading end being contacted with the source of the sample material, and further comprising applying a first pressure difference across the sample loading conduit to move the sample material into the loading end of the sample loading conduit and toward the waste end of the sample loading conduit.

3. The method of claim 2, wherein a negative pressure is applied to the waste end of the sample loading conduit to supply the first pressure difference across the sample loading conduit.

4. The method of claim 2, wherein the sample loading conduit and separation conduit are in fluid communication at a first fluid junction, and further comprising moving a portion of the sample material in the sample loading conduit through the first fluid junction and into the separation conduit.

5. The method of claim 4, wherein the sample material comprises one of a nucleic acid, a protein, or a carbohydrate.

6. The method of claim 4, wherein the step of moving the sample material from the sample loading conduit through the fluid junction and into the separation conduit comprises applying a voltage difference through the fluid junction to electrokinetically move the sample material from the sample loading conduit into the separation conduit.

7. The method of claim 1, wherein the step of separating the sample material comprises applying a voltage difference across the separation conduit, to electrophoretically. separate the sample material into one or more sample components.

8. The method of claim 1, wherein the sample component collection conduit is in fluid communication with at least a first agent, and wherein the method further comprises transporting an amount of the first agent into the sample component collection conduit to mix with the one or more sample components therein.

9. The method of claim 8, wherein the first agent is selected from a plasmid, a diluent, a detergent, a protein affinity label, or a ligation agent.

10. The method of claim 1, wherein the sample component collection conduit is in fluid communication with a source of at least a first ligation enzyme, and wherein the method further comprises transporting an amount of the first ligation enzyme into the sample component collection conduit to mix with the one or more sample components therein.

11. The method of claim 10 further comprising performing at least one ligation reaction between two or more sample components in the sample component collection conduit.

12. The method of claim 10 further comprising performing at least one ligation reaction between at least one sample component and a plasmid in the sample component collection conduit.

13. The method of claim 10, wherein the sample component collection conduit is in fluid communication with at least a first buffer solution containing ATP, and wherein the method further comprises transporting an amount of the first buffer solution into the sample component collection conduit to mix with the one or more sample components.

14. The method of claim 10, wherein the sample component collection conduit is in fluid communication with a source of a solution containing at least one plasmid, and wherein the method further comprises ligating said one or more sample components to said plasmid in at least a portion of the component collection conduit.

15. The method of claim 1, wherein the sample component collection conduit is in fluid communication with at least one component collection well, the method further comprising transporting said one or more sample components from said component collection conduit to said at least one collection well.

16. The method of claim 15 further comprising exposing the one or more sample components to a wash buffer solution in the at least one collection well.

17. The method of claim 16 further comprising vortexing the one or more components in the at least one collection well to homogenize the one or more components therein to facilitate their removal from the at least one collection well.

18. The method of claim 17 further comprising performing at least one analytical operation on the one or more sample component materials following their removal from the at least one collection well.

19. The method of claim 18 wherein said performing at least one analytical operation comprises performing a mass spectroscopy based analysis of the one or more sample components.

20. The method of claim 1 wherein said processor includes a computer which includes appropriate programming for receiving a signal from the detector that is indicative of a separated component passing the detector, determining a retention time of the separated components in the separation conduit, and determining a size of the separated components by comparing the retention time of the separated components to a retention time of components of a standard reference of known size for the sample material.

21. The method of claim 20 further comprising inputting instructions to the computer to direct the fluid direction system to move a selected one or more of the separated components of interest from the separation conduit into the sample component collection conduit based on the determined size of the selected one or more sample components.

22. The method of claim 21 wherein the standard reference of known size is obtained by separating a standard sizing ladder.

23. The method of claim 22 wherein the separating a standard sizing ladder is performed prior to transporting the first sample material through the separation conduit to separate the first sample material into a plurality of first sample components.

24. The method of claim 23 wherein the reference standard sizing ladder is mixed with the sample material prior to said transporting step.

25. The method of claim 24 further comprising transporting two or more flanking size markers of a known size through the separation conduit simultaneously with said first sample material.

26. The method of claim 25 further comprising detecting said flanking size markers and separated components with the detector and comparing the known size of the flanking markers to the sizing ladder to obtain accurate sizing information of the separated components.

27. The method of claim 26 further comprising transporting the first sample material through the separation conduit at least two times prior to moving said selected one or more of said plurality of sample components from said separation conduit into the sample component collection conduit.

28. A microfluidic device for isolating one or more sample components from a sample material source comprising:

a substrate having at least one surface;

at least first and second channels fabricated into said surface of said substrate which intersect with each other at a first location along a length of the first channel, at least one of said first and second intersecting channels having at least one cross-sectional dimension in the range of from about 0.1 to about 500 μ

m;

a sample material separation system operably coupled to at least said first channel for controlling separation of the sample material into one or more sample components in the first channel in the presence of a separation matrix;

a detection zone located at a second location along the length of said first channel upstream from said first location wherein detection of said one or more sample components can occur; and

a fluid direction system operably coupled to at least said second channel for controlling movement of selected one or more sample components from said first channel into said second channel based on information received at said detection zone.

29. The device of claim 28, further comprising at least one component collection reservoir in the substrate which is fluidly coupled to said second channel.

30. The device of claim 28, further comprising two or more component collection reservoirs in the substrate which are each fluidly coupled to said second channel.

31. The device of claim 28, further comprising at least five or more component collection reservoirs in the substrate which are each fluidly coupled to said second channel.

32. The device of claim 28, further comprising a source of at least a first agent fluidly coupled to said second channel.

33. The device of claim 32, wherein said first agent is selected from one of a ligation enzyme, a diluent, a buffer, or a labeling agent.

34. The device of claim 28, further comprising a source of at least a first ligation agent which is fluidly coupled to said second channel.

35. The device of claim 34, wherein said ligation agent comprises a ligation enzyme.

36. The device of claim 34, wherein said ligation agent comprises a buffer solution which contains ATP.

37. The device of claim 34, wherein said ligation agent comprises T4 DNA ligase.

38. The device of claim 34 wherein said ligation agent comprises Topoisomerase.

39. The device of claim 34 further comprising a source of at least one plasmid which is fluidly coupled to said second channel for ligating one or more of said sample components to said plasmid.

40. The device of claim 29 further comprising a rinsing buffer solution located within said at least one component collection reservoir.

41. The device of claim 28 wherein said sample separation system comprises at least first and second fluid reservoirs fluidly coupled to said first channel, at least one electrode electrically coupled to each of said fluid reservoirs, and a control system for variably applying a voltage at said electrodes, whereby electrophoretic separation of said one or more sample components in said separation matrix within said first channel may be controlled.

42. The device of claim 28 wherein said fluid direction system includes at least first and second fluid reservoirs fluidly coupled to said second channel, at least one electrode electrically coupled to each of said fluid reservoirs, and a control system for variably applying a voltage at said electrodes, whereby electrokinetic movement of said one or more sample components in said second channel may be controlled.

43. The device of claim 28 wherein said fluid direction system includes at least first and second fluid reservoirs which are fluidly coupled to said second channel, a vacuum source coupled to at least one of said reservoirs, and a control system for variably applying a vacuum at said at least one reservoir, whereby movement of said one or more sample components in said second channel may be controlled.

44. The device of claim 28 wherein said fluid direction system includes at least first and second fluid reservoirs which are fluidly coupled to said second channel, a pressure source coupled to at least one of said reservoirs, and a control system for variably applying a pressure force at said at least one reservoir, whereby movement of said one or more sample components in said second channel may be controlled.

45. The device of claim 28 wherein said sample material comprises a protein sample.

46. The device of claim 28 wherein said sample material comprises a nucleic acid sample.

47. A system for isolating one or more sample components of a sample material following separation of said sample material into a plurality of sample components, comprising:

a microfluidic substrate having at least one surface;

a separation conduit fabricated into said surface and having a separation matrix disposed therein;

a detector in sensory communication with said separation conduit at a first location along a length of the separation conduit for detecting said one or more sample components in the separation conduit;

a sample component collection conduit fabricated into said surface and being in fluid communication with said separation conduit at a second location downstream from said first location;

a fluid direction system for controlling movement of at least one or more of said sample components within at least said sample component collection conduit; and

a processor which is operably coupled to said detector and to said fluid direction system for instructing said fluid direction system to direct movement of one or more sample components from said separation conduit into said sample component collection conduit based on information received from said detector.

48. The system of claim 47, further comprising at least one component collection reservoir in the substrate which is fluidly coupled to said sample component collection conduit.

49. The system of claim 47, further comprising two or more component collection reservoirs in the substrate which are each fluidly coupled to said sample component collection conduit.

50. The system of claim 47, further comprising at least five or more component collection reservoirs in the substrate which are each fluidly coupled to said sample component collection conduit.

51. The system of claim 47, further comprising a source of at least a first agent fluidly coupled to said sample component collection conduit.

52. The system of claim 51, wherein said first agent is selected from one of a ligation enzyme, a diluent, a buffer, or a labeling agent.

53. The system of claim 47, further comprising a source of at least a first ligation agent which is fluidly coupled to said sample component collection conduit.

54. The system of claim 53, wherein said ligation agent comprises a ligation enzyme.

55. The system of claim 53, wherein said ligation agent comprises a buffer solution which contains ATP.

56. The system of claim 53, wherein said ligation agent comprises T4 DNA ligase.

57. The system of claim 53 wherein said ligation agent comprises Topoisomerase.

58. The system of claim 53 further comprising a source of at least one plasmid which is fluidly coupled to said sample component collection conduit for ligating one or more of said sample components to said plasmid.

59. The system of claim 48 further comprising a rinsing buffer solution located within said at least one component collection reservoir.

60. The system of claim 47 further comprising a sample separation system operably coupled to said separation conduit, said sample separation system comprising at least first and second fluid reservoirs fluidly coupled to said separation conduit, at least one electrode electrically coupled to each of said fluid reservoirs, and a control system for variably applying a voltage at said electrodes, whereby electrophoretic separation of said one or more sample components in said separation matrix within said separation conduit may be controlled.

61. The system of claim 47 wherein said fluid direction system includes at least first and second fluid reservoirs fluidly coupled to said sample component collection conduit, at least one electrode electrically coupled to each of said fluid reservoirs, and a control system for variably applying a voltage at said electrodes, whereby electrokinetic movement of said one or more sample components in said sample component collection conduit may be controlled.

62. The system of claim 47 wherein said fluid direction system includes at least first and second fluid reservoirs which are fluidly coupled to said sample component collection conduit, a vacuum source coupled to at least one of said reservoirs, and a control system for variably applying a vacuum at said at least one reservoir, whereby movement of said one or more sample components in said sample component collection conduit may be controlled.

63. The system of claim 47 wherein said fluid direction system includes at least first and second fluid reservoirs which are fluidly coupled to said sample component collection conduit, a pressure source coupled to at least one of said reservoirs, and a control system for variably applying a pressure force at said at least one reservoir, whereby movement of said one or more sample components in said sample component collection conduit may be controlled.

64. The system of claim 47 wherein said sample material comprises a protein sample.

65. The system of claim 47 wherein said sample material comprises a nucleic acid sample.

66. The system of claim 47 wherein the processor comprises a computer.

67. The system of claim 66 wherein the computer includes appropriate programming for receiving a signal from the detector that is indicative of a separated component passing the detector, determining a retention time of the separated components in the separation conduit, and determining a size of the separated components by comparing the retention time of the separated components to a retention time of components of a standard reference of known size for the sample material.

68. The system of claim 67 wherein the computer is programmable to control the fluid direction system to move a selected one or more of the separated components of interest from the separation conduit into the sample component collection conduit based on the determined size of the selected one or more sample components.

69. The system of claim 47 further comprising a set of molecular weight standard polypeptides.

70. The system of claim 47 further comprising a set of DNA reference sizing ladders.

71. A method of separating a sample material into a plurality of separated components and isolating one or more of said separated components in a microfluidic device comprising separating the sample components in a separation conduit located within the device in the presence of a separation matrix disposed therein, detecting the sample components at a detection zone in the separation conduit, and transporting selected one or more of the sample components to a sample component collection conduit in the device based on information received at said detection zone.

72. The method of claim 71 wherein said sample component collection conduit intersects said separation conduit downstream of said detection zone.

73. The method of claim 71 further comprising performing a ligation reaction in the sample component collection conduit.

74. The method of claim 71 further comprising ligating one or more of said sample components to a plasmid within said sample component collection conduit.

75. The method of claim 71 further comprising performing a cloning operation on one or more of said sample components in the sample component collection conduit.

76. The method of claim 71 further comprising transporting said one or more sample components from said sample component collection conduit to at least one component collection reservoir in the device which contains a wash buffer solution.

77. The method of claim 71 wherein said transporting selected one or more sample components to said sample component collection conduit comprises electrokinetically moving said one or more component materials into said collection conduit.

78. The method of claim 71 wherein said transporting selected one or more sample components to said sample component collection conduit comprises using a pressure-driven force to move said one or more component materials into said collection conduit.

79. The method of claim 71 further comprising mixing said one or more sample components in said sample component collection conduit with a MALDI matrix material to form a first mixture.

80. The method of claim 79 further comprising transporting the first mixture from said sample component collection conduit to at least one component collection reservoir in the device.

81. The method of claim 80 further comprising removing the first mixture from the at least one component reservoir in the device in preparation of a mass spectroscopy analysis procedure.

82. The method of claim 71 wherein said one or more sample components comprise one or more proteins.

83. The method of claim 71 wherein said one or more sample components comprise one or more nucleic acids.

84. The method of claim 71 further comprising prior to said transporting step determining a retention time of the separated components in the separation conduit, and calculating a size of the separated components by comparing the retention time of the separated components to a retention time of components of a standard reference of known size for the sample material.

85. The method of claim 84 wherein said transporting comprises transporting said selected one or more separated components into the sample collection conduit based on the calculated size of the selected one or more separated components.

86. A method of separating a sample material into a plurality of separated components and isolating one or more of said separated components comprising:

providing a microfluidic substrate including a separation conduit having a separation matrix disposed therein;

providing a detector in sensory communication with said separation conduit at a first location along a length of the separation conduit for detecting said one or more sample components in the separation conduit;

providing a sample component collection conduit in said substrate in fluid communication with said separation conduit at a second location downstream from said first location;

providing a user-programmable processor which is operably coupled to said detector and to a fluid direction system, the processor including appropriate programming for receiving a signal from the detector that is indicative of a separated component passing the detector, determining a retention time of the separated components in the separation conduit, and determining a size of the separated components by comparing the retention time of the separated components to a retention time of components of a standard reference of known size for the sample material;

inputting instructions to the processor to direct the fluid direction system to move a selected one or more of the separated components of interest from the separation conduit into the sample component collection conduit based on the determined size of the selected one or more sample components;

transporting a first sample material through the separation conduit to separate the first sample material into a plurality of first sample components;

detecting said plurality of first sample components at said first location with said detector; and

moving said selected one or more of said plurality of first sample components from said separation conduit into the sample component collection conduit in response to said instructions from the processor to the fluid direction system.

87. The method of claim 86 wherein the standard reference of known size is obtained by separating a standard sizing ladder.

88. The method of claim 87 wherein the separating a standard sizing ladder is performed prior to transporting the first sample material through the separation conduit to separate the first sample material into a plurality of first sample components.

89. The method of claim 87 wherein the reference standard sizing ladder is mixed with the sample material prior to said transporting step.

90. The method of claim 86 further comprising transporting two or more flanking size markers of a known size through the separation conduit simultaneously with said first sample material.

91. The method of claim 90 further comprising detecting said flanking size markers and separated components with the detector and comparing the known size of the flanking markers to the sizing ladder to obtain accurate sizing information of the separated components.

92. The method of claim 91 further comprising transporting the first sample material through the separation conduit at least two times prior to moving said selected one or more of said plurality of sample components from said separation conduit into the sample component collection conduit.