Device, system, and method for depositing processed immiscible-fluid-discrete-volumes

• US 20070039866A1
• Filed: 08/22/2006
• Published: 02/22/2007
• Est. Priority Date: 08/22/2005
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Cox, David, Oldham, Mark, Schroeder, Benjamin, Wiyatno, Willy, Reel, Richard
• Granted Patent: US 9,285,297 B2
• Time in Patent Office: Days
• US Class Current: 210/265
• CPC Class Codes: B01J 2219/00353 Pumps
• CPC Class Codes: B01J 2219/0036 Nozzles
• CPC Class Codes: B01J 2219/00364 Pipettes
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00657 One-dimensional arrays
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• 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 2300/0864 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 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/0293 for liquids
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• 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 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• 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 1/6869 : Methods for sequencing
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/101 : Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: F15C 5/00 : Manufacture of fluid circuit elements; Manufacture of assemblages of such elements integrated circuits
• CPC Class Codes: F16K 2099/0084 : Chemistry or biology, e.g. "lab-on-a-chip" technology
• CPC Class Codes: F16K 99/0001 : Microvalves microdevices B81B1/00; manufacture or treatment of devices or systems in or on a substrate B81C1/00; microfluidic structures B01L3/5027; micropumps F04B19/006
• CPC Class Codes: F16K 99/0011 : Gate valves or sliding valves
• CPC Class Codes: F16K 99/0013 : Rotary valves
• CPC Class Codes: G01N 1/14 : Suction devices, e.g. pumps; Ejector devices
• CPC Class Codes: G01N 27/44743 : Introducing samples
• CPC Class Codes: G01N 27/44769 : Continuous electrophoresis, i.e. the sample being continuously introduced, e.g. free flow electrophoresis [FFE]
• 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 137/0318 : Processes
• CPC Class Codes: Y10T 137/4259 : With separate material addition
• CPC Class Codes: Y10T 137/85978 : With pump
• CPC Class Codes: Y10T 137/85986 : Pumped fluid control
• CPC Class Codes: Y10T 137/86863 : Rotary valve unit
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

Various embodiments relate to systems and/or methods for sample preparation that can be used for biochemical and/or molecular biology procedures involving small volumes, for example, micro volumes or smaller. Methods and systems that can reduce sample size requirements and increase the number of samples on a substrate are provided. Samples can be applied to a plate or other appropriate substrate and can be used for, inter alia, sequencing reactions. In some embodiments, apparatuses, systems, and/or methods for charged analyte collection are provided. Charged analytes in a sample can be electrokinetically collected or extracted from a conduit through a hole formed in a sidewall of the conduit, by application of an electric field that causes the charged analytes to migrate in a direction that is transverse to the conduit.

Claims

1. A system comprising:

at least one conduit;

a substrate separate from and spaced from the at least one conduit, the substrate comprising an electrically conductive surface; and

an apparatus for moving in a predetermined pattern relative to one another, the substrate, and the at least one conduit.

2. The system of claim 1, further comprising an apparatus for detecting a sample bound to the substrate.

3. The system of claim 1, wherein the predetermined pattern is a raster pattern.

4. The system of claim 1, wherein the electrically conductive surface comprises a gold surface.

5. The system of claim 1, wherein the electrically conductive surface comprises a gold coating on a base structure.

6. The system of claim 1, wherein the electrically conductive surface further comprises linker moieties adapted to bind to a nucleic acid.

7. The system of claim 6, wherein the linker moieties comprise a plurality of the same linker moieties.

8. The system of claim 6, wherein the linker moieties comprise a plurality of different linker moieties.

9. The system of claim 1, wherein the at least one conduit comprises an inner surface having a maximum inner cross-sectional dimension.

10. The system of claim 9, wherein the maximum inner cross-sectional dimension is a diameter.

11. The system of claim 1, further comprising a sample preparation device adapted to form a plurality of separate, spaced-apart aqueous slugs in the at least one conduit.

12. The system of claim 1, wherein the at least one conduit comprises an inner surface and the inner surface comprises an attached linker moiety.

13. The system of claim 6, wherein the linker moieties comprise at least one of steptavidin moieties, biotin moieties, and thiol moieties.

14. The system of claim 1, further comprising a capillary electrophoretic sequencer adapted to inject a sample component when a sample component is disposed on the electrically-conductive surface.

15. The system of claim 1, wherein the at least one conduit comprises a conduit having an inner diameter of about 300 microns or less.

16. The system of claim 15, wherein the inner diameter is about 50 microns or less.

17. The system of claim 1, wherein the electrically conductive surface comprises channels.

18. The system of claim 17, wherein the channels comprise linker moieties adapted to bind to a nucleic acid.

19. The system of claim 18, wherein the electrically conductive surface further comprises linker moieties adapted to bind to a nucleic acid.

20. The system of claim 19, wherein the linker moieties comprise a plurality of the same linker moiety.

21. The system of claim 1, wherein the conduit comprises a capillary tube.

22. The system of claim 1, further comprising a plurality of immiscible-fluid-discrete-volumes spaced-apart from one another by a spacing fluid that is immiscible with the immiscible-fluid-discrete-volumes, disposed in the at least one conduit.

64. The system of claim 1, wherein the substrate is covered with an insulating or dielectric material.

23. A system comprising:

an aqueous sample injection unit in fluid communication with at least one conduit comprising a maximum inner cross-sectional dimension;

a spacing fluid injection unit in fluid communication with the at least one conduit, the aqueous sample injection unit and the spacing fluid injection unit in fluid communication with the at least one conduit;

a control unit adapted to flow an aqueous sample and a spacing fluid from the aqueous sample injection unit and the spacing fluid injection unit, respectively, and adapted to inject volumes of aqueous sample and spacing fluid that respectively form slugs in the at least one conduit wherein each slug has an outer dimension that is equal to the maximum inner cross-sectional dimension of the at least one conduit;

an electrically conductive substrate; and

a capillary electrophoretic sequencer adapted to inject a sample component when a sample component is disposed on the electrically-conductive surface.

24. The system of claim 23, wherein the electrically conductive surfaces comprise at least one channel.

25. The system of claim 23, wherein the electrically conductive substrate comprises linker moieties adapted to bind to a nucleic acid.

26. The system of claim 25, wherein the linker moieties comprise a plurality of the same linker moiety.

27. The system of claim 23, wherein the electrically conductive substrate comprises at least one channel.

28. The system of claim 23, wherein the aqueous fluid injection unit and the spacing fluid injection unit comprise separate units.

29. A method comprising:

forming in a conduit a plurality of aqueous sample slugs spaced apart from one another by slugs of spacing fluid, at least one of the aqueous sample slugs comprising at least one target analyte; and

dispensing the aqueous sample slugs one-at-a-time from the conduit onto a substrate to form a pattern of spaced apart aqueous samples on the substrate, the substrate comprising an electrically conductive surface.

30. The method of claim 29, wherein the electrically conductive surface comprises at least one linker moiety bound thereto, wherein the at least one linker moiety is adapted to bind the at least one target analyte.

31. The method of claim 30, further comprising capturing the at least one target analyte with the at least one linker moiety to form at least one bound target analyte.

32. The method of claim 30, further comprising contacting the electrically conductive surface with a mixture of sequencing reaction components.

33. The method of claim 29, wherein the electrically conductive surface comprises a gold- coated surface.

34. The method of claim 30, wherein the at least one target analyte comprises at least one target nucleic acid sequence.

35. The method of claim 29, further comprising reacting the at least one bound target analyte with a reagent mixture to form at least one target analyte product.

36. The method of claim 30, further comprising reacting the at least one bound target analyte with a reagent mixture to form at least one target analyte product.

37. The method of claim 35, further comprising positioning a collection device adjacent the at least one target analyte product and injecting at least a portion of the at least one target analyte product into a collection device.

38. The method of claim 37, wherein the collection device comprises a capillary of a capillary electrophoretic analyzer.

39. The method of claim 37, wherein the injecting comprises applying a potential to the electrically conductive surface relative to the capillary.

40. The method of claim 29, wherein forming the pattern of spaced apart aqueous samples comprises forming a plurality of rows of aqueous samples.

41. A method comprising:

forming in a conduit a plurality of aqueous sample slugs, at least one of the aqueous sample slugs comprising at least one target analyte comprising at least one respective linkage group; and

dispensing the aqueous sample slugs one-at-a-time from the conduit onto a substrate to form a pattern of aqueous samples on the substrate, the substrate comprising an electrically conductive surface adapted to bind the at least one respective linkage group to form an attached analyte.

42. The method of claim 41, further comprising bonding the at least one linkage group to the electrically conductive surface to form at least one captured target analyte.

43. The method of claim 42, further comprising contacting the electrically conductive surface with a mixture of sequencing reaction components after the bonding.

44. The method of claim 42, further comprising reacting the at least one captured target analyte with a reagent mixture to form at least one target analyte product.

45. The method of claim 44, further comprising positioning a collection device adjacent the at least one target analyte product and injecting at least a portion of the at least one target analyte product into the collection device.

46. The method of claim 45, wherein the collection device comprises a capillary of a capillary electrophoretic analyzer.

47. The method of claim 46, wherein the injecting comprises applying a potential to the electrically conductive surface relative to the capillary.

48. The method of claim 41, wherein the electrically conductive surface comprises a metal coated surface.

49. The method of claim 41, wherein the electrically conductive surface comprises a gold-coated surface.

50. The method of claim 41, wherein the at least one target analyte comprises at least one target nucleic acid sequence.

51. The method of claim 41, wherein forming the pattern of aqueous samples comprises forming a plurality of rows of aqueous samples.

52. A method comprising:

amplifying DNA in a plurality of aqueous sample slugs in a conduit to form amplicons, each aqueous slug separated from an adjacent aqueous slug by at least one oil slug;

moving the conduit comprising the amplicons over a substrate and depositing the amplicons from the conduit onto the substrate, the substrate comprising an electrically conductive surface;

attaching the amplicons to the electrically conductive surface;

contacting the substrate with a sequencing reaction mixture to form at least one dye-labeled spot;

positioning a capillary of a capillary electrophoretic analyzer over the at least one dye-labeled spot;

electrically contacting the dye-labeled spot with the capillary; and

injecting one or more components from the dye-labeled spot into the capillary.

53. The method of claim 52, wherein the amplifying comprises incorporating a 5′

-sulfhydryl group into a DNA molecule.

54. The method of claim 52, wherein the surface comprises an electrically conductive surface.

55. The method of claim 54, wherein the electrically conductive surface comprises a gold surface.

56. The method of claim 52, wherein the surface comprises a binding moiety.

57. The method of claim 52, further comprising washing the electrically conductive surface with a denaturing solution after depositing the amplicons from the conduit onto the electrically conductive surface.

58. The method of claim 52, wherein the sequencing reaction mixture comprises a primer and a dye-labeled terminator and the method comprises reacting the amplicons with the primer and the dye-labeled terminator to form the dye-labeled spot.

59. The method of claim 52, further comprising imaging the electrically conductive surface and determining a location of the dye-labeled spot.

60. The method of claim 52, further comprising analyzing the one or more components in the capillary electrophoretic analyzer.

61. The method of claim 52, wherein the electrically conductive surface comprises reactive groups.

62. The method of claim 61, wherein the reactive groups comprise one or more of carboxy groups, amino groups, and hydroxyl groups.

63. A method comprising:

dispensing DNA into a plurality of aqueous sample slugs in a conduit, each aqueous slug separated from an adjacent aqueous slug by at least one non-aqueous slug;

moving the conduit comprising the DNA over a substrate and depositing the DNA from the conduit onto the substrate, the substrate comprising an electrically conductive surface;

attaching the DNA to the electrically conductive surface;

contacting the electrically conductive surface with a reaction mixture;

positioning a conduit over the attached DNA;

electrically contacting the DNA with the conduit; and

injecting the DNA into the conduit.

65. A system comprising:

at least one transport conduit, the at least one transport conduit comprising at least one sidewall;

a through hole formed through the at least one sidewall;

a charged analyte collection chamber in fluid communication with the through hole;

a positive electrode;

a negative electrode; and

a power source, wherein the positive electrode, the negative electrode, and the power source are connected together and configured to form an electric field extending from the transport conduit, through the through hole, and to the charged analyte collection chamber, and of sufficient strength to cause a charged analyte in the transport conduit to electrokinetically migrate through the through hole toward the charged analyte collection chamber.

66. The system of claim 65, wherein the at least one sidewall comprises a top sidewall and a bottom sidewall, the through hole is formed through the top sidewall, the bottom sidewall has an inner surface, and one of the positive electrode and the negative electrode is formed in or on the inner surface.

67. The system of claim 65, wherein the at least one sidewall comprises a top sidewall and a bottom sidewall, the through hole is formed through the top sidewall, a second through hole is formed through the bottom sidewall, and the electric field extends through both the through hole and the second through hole.

68. The system of claim 67, further comprising an electrode housing chamber in fluid communication with the second through hole and housing one of the positive electrode and the negative electrode.

69. The system of claim 68, wherein the charged analyte collection chamber houses the other one of the positive electrode and the negative electrode that is not present in the electrode housing chamber.

70. The system of claim 65, further comprising aqueous sample immiscible-fluid-discrete-volumes within the at least one transport conduit and separated from one another by spacer immiscible-fluid-discrete-volumes comprising a fluid that is immiscible with the aqueous sample immiscible-fluid-discrete-volumes.

71. The system of claim 65, further comprising one or more additional charged analyte collection chambers, each in fluid communication with the through hole, and one or more additional electrodes, wherein each of the additional electrodes is interruptably electrically connected to the power source and disposed in a respective one of the one or more additional charged analyte collection chambers.

72. A method comprising:

moving a sample comprising a charged analyte through a transport conduit and into alignment with a portion of the transport conduit comprising a through hole formed in a sidewall thereof;

generating an electric field that extends through the through hole; and

electrokinetically migrating the charged analyte from the transport conduit and through the through hole.

73. The method of claim 72, further comprising electrokinetically migrating the charged analyte into a capillary electrophoresis apparatus.

74. A system comprising:

an immersion plate, and a layer of a first fluid retained by the immersion plate; and

a conduit comprising a tip and an interior, the interior comprising a plurality of immiscible-fluid-discrete-volumes of a second fluid, spaced apart by a spacing fluid that is immiscible with each of the immiscible-fluid-discrete-volumes, disposed in the interior, wherein the tip is positioned such that when the immiscible-fluid-discrete-volumes exit the tip, the immiscible-fluid-discrete-volumes contact the first fluid retained by the immersion plate, and the second fluid has a different density than the first fluid.

75. A method comprising:

discharging immiscible-fluid-discrete-volumes from inside a conduit having a discharge tip, through the discharge tip, and into a first fluid retained by an immersion plate, wherein the first fluid is immiscible with the immiscible-fluid-discrete-volumes and the density of the first fluid is different than the densities of each of the immiscible-fluid-discrete-volumes.

76. The method of claim 75, wherein the densities of the immiscible fluids of the immiscible-fluid-discrete-volumes are each greater than the density of the first fluid.

77. The method of claim 75, wherein the first fluid retained by the immersion plate has a surface, and the discharge tip is disposed below the surface.

78. The method of claim 75, wherein the immiscible-fluid-discrete-volumes are spaced apart from one another in the conduit by a spacing fluid that is immiscible with the immiscible-fluid-discrete-volumes.

79. The method of claim 78, wherein the spacing fluid is the same fluid as the first fluid.

80. The method of claim 75, wherein the first fluid comprises an oil.

81. The method of claim 80, wherein the immersion plate comprises hydrophilic features covered by the first fluid, at least one of the hydrophilic features comprises a gold surface, and one or more of the immiscible-fluid-discrete-volumes discharged from the conduit is in contact with one or more of the hydrophilic features and comprises a sulfhydryl-labeled primer.

82. The method of claim 81, wherein the immersion plate further comprises a hydrophobic coating that surrounds one or more of the hydrophilic features.

83. A system comprising:

a dispensing conduit comprising a discharge tip;

a set of immiscible-fluid-discrete-volumes spaced-apart from one another by a spacing fluid that is immiscible with the immiscible-fluid-discrete-volumes, disposed in the conduit; and

a negative pressure source disposed adjacent the discharge tip.

84. The system of claim 83, further comprising:

a waste removal block comprising a through-hole and a waste removal conduit; and

a positioning unit operatively connected to at least one of the dispensing conduit and the waste removal block;

wherein the positioning unit is configured to move at least one of the dispensing conduit and the waste removal block relative to the other such that the dispensing conduit is moved into and through the through-hole.

85. The system of claim 83, wherein the waste removal block surrounds the dispensing conduit.

86. The system of claim 83, further comprising:

a detector disposed adjacent the dispensing conduit.

87. The system of claim 84, further comprising:

a sample tray; and

a second positioning unit;

wherein the second positioning unit is configured to position at least one of the dispensing conduit and the sample tray relative to the other with the sample tray below the dispensing conduit.

88. A method of dispensing immiscible-fluid-discrete-volumes, comprising:

providing a dispensing conduit comprising a dispensing tip, and a set of immiscible-fluid-discrete-volumes spaced-apart from one another by a spacing fluid that is immiscible with the immiscible-fluid-discrete-volumes;

providing a waste removal conduit adjacent the dispensing tip;

applying a vacuum to the waste removal conduit;

detecting a first immiscible-fluid-discrete-volume, that contains an analyte of interest, from the set of immiscible-fluid-discrete-volumes;

removing the vacuum; and

dispensing the first immiscible-fluid-discrete-volume.

89. The method of claim 88, further comprising:

reapplying the vacuum to the waste conduit;

removing a portion of the spacing fluid;

detecting a second immiscible-fluid-discrete-volume, that contains an analyte of interest, from the set of immiscible-fluid-discrete-volumes;

removing the vacuum; and

dispensing the second immiscible-fluid-discrete-volume.

90. The method of claim 88, further comprising:

providing a sample tray comprising wells; and

positioning at least one of the sample tray and the dispensing conduit relative to the other;

wherein dispensing the first immiscible-fluid-discrete-volume comprises dispensing the first immiscible-fluid-discrete-volume into the sample tray.

91. The method of claim 90, wherein positioning the sample tray under the dispensing conduit comprises positioning an empty well of the sample tray under the dispensing conduit.

92. The method of claim 88, further comprising:

positioning the dispensing tip in the waste removal block while the vacuum is applied; and

positioning the dispensing tip under the waste removal block after the vacuum is removed.

93. A system comprising:

a conduit comprising a tip;

a plurality of immiscible-fluid-discrete-volumes disposed in the conduit and spaced apart from one another by a spacing fluid that is immiscible with each of the plurality of immiscible-fluid-discrete-volumes; and

a capillary electrophoresis capillary, positioned adjacent to the tip of the conduit, the capillary electrophoresis capillary having an injection tip, wherein the capillary electrophoresis capillary and the conduit are axially aligned with one another.

94. The system of claim 93, wherein the injection tip of the capillary electrophoresis capillary comprises an electrode, and the electrode is operatively connected to a power source.

95. A method comprising:

discharging immiscible-fluid-discrete-volumes from a tip of a conduit directly into a capillary electrophoresis capillary, the conduit comprising a plurality of immiscible-fluid-discrete volumes spaced apart from one another by a spacing fluid that is immiscible with each of the immiscible-fluid-discrete-volumes.

96. A method comprising:

discharging immiscible-fluid-discrete-volumes from conduit and into an injector of a capillary electrophoresis apparatus, wherein at an interface between the conduit and the injector one or more components in the immiscible-fluid-discrete-volumes is concentrated by dielectrophoresis before entering the injector.