Methods and devices for performing chemical reactions on a solid support

• US 20040029258A1
• Filed: 04/11/2003
• Published: 02/12/2004
• Est. Priority Date: 04/11/2002
• Status: Active Grant

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Lin, Chao, Opalsky, David, Maczuszenko, Andrzej, Griswold, Charles, Heaney, Paul, Bruce, Phillip III, Worl, Ralf, Walker, Sheila A.
• Granted Patent: US 7,364,897 B2
• Time in Patent Office: Days
• US Class Current: 435/287.2
• CPC Class Codes: B01L 2200/025 Align devices or objects to ensure defined positions relative to each other
• CPC Class Codes: B01L 2200/0689 Sealing
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/1844 using fans
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/50851 specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• 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: B01L 9/523 for multisample carriers, e.g. used for microtitration plates
• CPC Class Codes: B01L 9/527 for microfluidic devices, e.g. used for lab-on-a-chip
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism

Abstract

Apparatus and methods for carrying out biochemical processes directly on a substrate are provided. A substrate assembly includes a cartridge that removably supports a substrate in a fixed position, and a reaction containment member that is removably located on top of the substrate. The reaction containment member includes one or more cavities that form chambers directly above one or more target locations on the surface of the substrate. The chambers can be used to conduct biochemical processes directly over the substrate, as well as to perform thermal cycling of material contained inside the chamber using a heating element disposed directly on the substrate. The substrate assembly is preferably used in combination with a processing machine that dispenses materials into the chambers and that conducts biochemical reactions of materials contained within the chambers, without requiring the substrate assembly to be moved from one location to another location during the processes.

Claims

1. An assembly for holding a substrate having at least one target location suitable for containing sample material, comprising:

a cartridge base having at least one mounting location that can support the substrate in a fixed position;

a reaction containment member that is removably located on top of the substrate such that a bottom surface of the reaction containment member is juxtaposed with a top surface of the substrate, wherein the reaction containment member and the substrate collectively form at least one chamber that is located over at least one target location on the substrate when the reaction containment member is juxtaposed on the substrate top surface, thereby permitting a chemical reaction to take place in the chamber.

2. An assembly as defined in claim 1, additionally comprising a cartridge cover that removably mates with the cartridge base to secure the substrate in the fixed position between the cartridge base and the cartridge cover and to also secure the reaction containment member in a fixed position on top of the substrate.

3. An assembly as defined in claim 1, wherein the mounting location comprises a ledge that supports an underside of the substrate along a periphery of the substrate.

4. An assembly as defined in claim 1, wherein the ledge is sized such that the cartridge base touches no more than 10% of the surface area of the substrate.

5. An assembly as defined in claim 1, wherein the cartridge base has an aperture that extends through the cartridge base such that the substrate is positioned over the aperture when the substrate is in the fixed position so that a bottom surface of the substrate is at least partially exposed through the aperture.

6. An assembly as defined in claim 1, wherein the reaction containment member includes at least one channel in the bottom surface of the reaction containment member, and wherein the channel and the top surface of the substrate collectively form the chamber.

7. An assembly as defined in claim 6, wherein the channel is elongated and is located over a row of target locations on the top surface of the substrate when the reaction containment member is juxtaposed on the substrate top surface.

8. An assembly as defined in claim 7, wherein the reaction containment member includes an inlet port that communicates with a first end of the elongate channel and an outlet port that communicates with a second end of the elongate channel.

9. An assembly as defined in claim 7, wherein the reaction containment member includes a plurality of elongate channels that are each located over a row of target locations on the top surface of the substrate when the reaction containment member is juxtaposed on the top surface of the substrate.

10. An assembly as defined in claim 1, wherein the chamber is formed by a well in the reaction containment member that forms an opening in a top surface of the reaction containment member and an opening in the bottom surface of the reaction containment member.

11. An assembly as defined in claim 10, wherein the reaction containment member includes a plurality of wells.

12. An assembly as defined in claim 2, additionally comprising a reaction containment member back plate to which the reaction containment member can be attached, and wherein the reaction containment member back plate can be attached to the cartridge cover.

13. An assembly as defined in claim 12, wherein the reaction containment member back plate is movable with respect to the cartridge cover when the reaction containment member is attached to the cartridge cover.

14. An assembly as defined in claim 1, additionally comprising at least one alignment pin that is positioned between the cartridge base and the cartridge cover when the cartridge base is mated with the cartridge cover, wherein the alignment pin positions the cartridge base with respect to the cartridge cover such that the chamber is located over the target location on the substrate when the substrate is in the fixed position.

15. An assembly as defined in claim 1, wherein the chamber is formed by an elongate flow channel in the reaction containment member that is positioned over a plurality of target locations on the top surface of the substrate when the substrate is in the fixed position and wherein the reaction containment member additionally includes an inlet to the flow channel and an outlet to the flow channel.

16. An assembly for holding a substrate having at least one target location suitable for containing sample material, comprising:

a cartridge having at least one mounting location on which the substrate can be located in a fixed position;

a reaction containment member that can be aligned in a juxtaposed relationship with the substrate when the substrate is in the fixed position, wherein the reaction containment member includes a channel that forms a chamber over at least one target location on the substrate when the substrate is properly aligned in the juxtaposed relationship with the substrate;

a reaction containment member back plate to which the reaction containment member can be mounted, wherein the reaction containment member back plate can be attached to the cartridge to thereby secure the reaction containment member in the juxtaposed relationship with the substrate, and wherein the reaction containment member back plate is movable with respect to the cartridge when the reaction containment member back plate is attached to the cartridge to allow the reaction containment member to be moved and aligned with the substrate when the reaction containment member back plate is attached to the cartridge.

17. An assembly as defined in claim 16, wherein the cartridge comprises a cartridge base and a cartridge cover that removably mates to the cartridge base, and wherein the mounting location is on the cartridge base.

18. An assembly as defined in claim 17, wherein the reaction containment member back plate mounts to the cartridge cover.

19. An assembly as defined in claim 16, wherein the channel is elongated and is located over a row of target locations on the top surface of the substrate when the reaction containment member is juxtaposed on the substrate top surface.

20. An assembly as defined in claim 19, wherein the reaction containment member includes an inlet port that communicates with a first end of the elongate channel and an outlet port that communicates with a second end of the elongate channel.

21. An assembly as defined in claim 19, wherein the reaction containment member includes a plurality of elongate channels that are each located over a row of target locations on the top surface of the substrate when the reaction containment member is juxtaposed on the top surface of the substrate.

22. An assembly as defined in claim 16, wherein the channel that forms the chamber comprises a well that forms an opening in a top surface of the reaction containment member and an opening in the bottom surface of the reaction containment member.

23. A device for performing chemical reactions, comprising:

a substrate having a bottom surface and a flat upper surface suitable for receiving sample material, wherein the substrate is formed of a semiconductive material and wherein the material has a thermal conductivity that is greater than the thermal conductivity of glass;

a reaction containment member located on the flat upper surface of the substrate, wherein the reaction containment member and the substrate collectively form at least one chamber over the flat upper surface, and wherein the reaction containment member is made of a thermoplastic;

a coating on the bottom surface of the substrate, wherein the coating is made of an electrically-resistive material.

24. A device as defined in claim 23, wherein the chamber is formed by an elongate flow channel in the reaction containment member and wherein the reaction containment member additionally includes an inlet to the flow channel and an outlet to the flow channel.

25. A device as defined in claim 23, wherein the chamber is formed by a well in the reaction containment member that forms an opening in the top surface of the reaction containment member and an opening in the bottom surface of the reaction containment member.

26. A device as defined in claim 23, wherein the substrate material has a thermal conductivity of approximately 0.5 W/mK to 450 W/mK.

27. A reaction containment member for forming at least one well over a substrate, comprising:

a body having a top side and a bottom side, the body including;

at least one interior surface forming at least one elongate flow channel on the bottom side of the body, the elongate flow channel being open along the bottom side of the body;

an inlet port that defines an upper opening in the top side of the body and a lower opening in the interior surfaces of the body;

an outlet port that defines an upper opening in the top side of the body and a lower opening in the interior surfaces of the body;

at least one pressure relief cavity located within the body, wherein the cavity is located relative to the interior surfaces so as to define a region of reduced thickness along one of the interior surfaces that forms the elongate flow channel;

wherein the body can be positioned over a substrate such that the elongate flow channel aligns over a row of target locations on a surface of the substrate such that the surface of the substrate and the interior surfaces of the body collectively enclose the row of target locations within the elongate flow channel.

28. A reaction containment member as defined in claim 27, wherein the body includes a plurality of flow channels with each flow channel having a corresponding inlet port and a corresponding outlet port, and wherein the body can be positioned over a substrate such that each of the flow channels aligns over a corresponding row-of target locations on the substrate.

29. A reaction containment member as defined in claim 27, wherein the body is made of a thermoplastic.

30. A method of performing a chemical reaction on a substrate having at least one target location that contains a sample of biological material, comprising;

(a) locating a reaction containment member on the top surface of the substrate such that the reaction containment member and the substrate collectively form a chamber directly over the at least one target location;

(b) dispensing a fluid into the chamber to expose the at least one target location to the fluid;

(c) conducting a chemical reaction within the chamber using the fluid;

(d) heating the substrate to increase the temperature within the chamber;

wherein steps (b)-(c) are performed while the substrate remains in a stationary position.

31. A method as defined in claim 30, additionally comprising mounting the substrate on a cartridge that supports the substrate in a fixed position.

32. A method as defined in claim 30, wherein heating the substrate comprises:

forming a first set of contacts between a resistive coating on the substrate and two or more electrical conductors, wherein at least one of the electrical conductors in the first set is coupled in series to a first resistor;

forming a second set of contacts between the resistive coating on the substrate and two or more electrical conductors, wherein at least one of the electrical conductors in the second set is coupled in series to a second resistor;

applying a first electrical current to the first set of electrical conductors so that the current flows from the first set of electrical contacts to the second set of contacts through the resistive coating;

wherein the resistive value of the first and second resistors differ with respect to one another to thereby vary the current at one contact with respect to another electrical contact.

33. A method as defined in claim 30, wherein the chamber is formed by a flow channel in the reaction containment member, the flow channel having an inlet and an outlet, and additionally comprising sealing the inlet and the outlet by inserting hollow pins into the inlet and outlet.

34. A device for performing a reaction of a target biomolecule, comprising:

a work station on which a substrate containing the target biomolecule can be mounted;

at least one dispenser movably coupled to the work station such that the dispensing pin can be moved to a position wherein a material can be dispensed from the dispenser onto the target biomolecule when the substrate is mounted on the work station;

a thermal cycler coupled to the work station, the thermal cycler comprising at least one heating contact that can cause the substrate to heat when the substrate is mounted on the work station.

35. A device as defined in claim 34, wherein the substrate is mounted on the work station as part of a substrate assembly comprising:

a cartridge base having at least one mounting location that can support the substrate in a fixed position;

a reaction containment member that is removably located on top of the substrate such that a bottom surface of the reaction containment member is juxtaposed with a top surface of the substrate, wherein the reaction containment member and the substrate collectively form at least one chamber that is located over the target biomolecule on the substrate when the reaction containment member is juxtaposed on the substrate top surface, thereby permitting the reaction to take place in the chamber.

36. A device as defined in claim 34, wherein the dispenser is movable with respect to the substrate along three separate axes when the substrate is mounted on the work station.

37. A device as defined in claim 34, wherein the thermal cycler further comprises a cooler that can cool the substrate when the substrate is mounted on the work station.

38. A device as defined in claim 37, wherein the cooler comprises a fan that blows air over a bottom surface of the substrate when the substrate is mounted on the work station.

39. A device as claimed in claim 34, wherein the thermal cycler further comprises a temperature sensor that senses the temperature of the substrate when the substrate is mounted on the work station.

40. A method for performing a reaction of a target biomolecule, comprising:

(a) performing one or more reactions of the target biomolecule in the presence of the surface of a substrate, wherein the reaction(s) is performed substantially in solution and the substrate comprises one or more target detection location(s) in contact with the solution;

(b) capturing one or more reaction products of on the surface of the substrate at the target detection location, wherein the capture is achieved through a non-covalent interaction between the reaction product(s) and the surface of the substrate or a moiety attached to the surface of the substrate; and

(c) detecting the reaction product or products at the target detection location.

41. The method of claim 40, wherein capture is achieved through ionic interactions, Van der Waals forces or hydrogen bonds.

42. The method of claim 40, wherein capture is achieved through hybridization of the reaction product(s) with a capture oligonucleotide attached to the surface of the substrate.

43. The method of claim 40, wherein the reaction product(s) is detected by mass spectrometry.

44. The method of claim 40, wherein the surface of the substrate forms an interior surface of a chamber.

45. The method of claim 40, wherein the biomolecule is a nucleic acid.

46. The method of claim 45, wherein at least one reaction of the nucleic acid comprises amplification of at least a portion of the nucleic acid molecule.

47. The method of claim 46, wherein the nucleic acid is amplified by an amplification procedure selected from the group consisting of:

polymerase chain reaction (PCR), ligase chain reaction (LCR) and strand displacement amplification (SDA).

48. The method of claim 46, wherein at least one reaction undergoes at least one temperature thermocycle comprising heating and cooling.

49. The method of claim 48, wherein the rate of heating is selected from the range of about 3°

C./second up to about 100°

C./second.

50. The method of claim 48, wherein the rate of heating is selected from the group consisting of rates of heating of at least about:

3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95 and at least about 100°

C./second.

51. The method of claim 48, wherein the heating is achieved using thermal conductivity or electrical conductivity.

52. The method of claim 48, wherein the rate of cooling is selected from the range of about 3°

C./second up to about 100°

C./second.

53. The method of claim 48, wherein the rate of cooling is selected from the group consisting of rates of cooling of at least about:

3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95 and at least about 100°

C./second.

54. The method of claim 48, wherein the cooling is achieved using water, air or a cold block.

55. The method of claim 45, wherein at least one reaction of the nucleic acid comprises extension of an oligonucleotide primer hybridized to the nucleic acid.

56. The method of claim 55, wherein the extended primer is captured on the surface of the substrate.

57. The method of claim 55, wherein at least one reaction undergoes at least one temperature thermocycle comprising heating and cooling.

58. The method of claim 57, wherein the rate of heating is selected from the range of about 3°

C./second up to about 100°

C./second.

59. The method of claim 57, wherein the rate of heating is selected from the group consisting of rates of heating of at least about:

3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95 and at least about 100°

C./second.

60. The method of claim 57, wherein the heating is achieved using thermal conductivity or electrical conductivity.

61. The method of claim 57, wherein the rate of cooling is selected from the range of about 3°

C./second up to about 100°

C./second.

62. The method of claim 57, wherein the rate of cooling is selected from the group consisting of rates of cooling of at least about:

3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95 and at least about 100°

C./second.

63. The method of claim 57, wherein the cooling is achieved using water, air or a cold block.

64. The method of claim 40, wherein prior to step (c), matrix is deposited at the target detection location and detection is performed by MALDI mass spectrometry.

65. The method of claim 40, wherein the surface of the substrate is enclosed by one or more reaction chambers within a removable reaction containment member.

66. The method of claim 65, wherein the temperature difference from within a single reaction chamber is selected from ≦

;

2.0°

C., 1.5°

C., 1.0°

C., 0.5°

C. and ≦

0.3°

C.

67. The method of claim 65, wherein the temperature difference between different reaction chambers is selected from ≦

;

1.0°

C., 0.5°

C., 0.4°

C., 0.3°

C., 0.2°

C. andy ≦

0.1°

C.

68. A method for determining the identity of a nucleotide in a target nucleic acid molecule, comprising:

hybridizing a single-stranded portion of the target nucleic acid molecule with an oligonucleotide that is complementary to a region of the single-stranded portion of the target nucleic acid in solution and in the presence of a capture moiety at a discrete target detection location on a substrate;

exposing the hybridized nucleic acid and oligonucleotide to conditions that permit extension of the oligonucleotide;

capturing the product(s) of the extension reaction on the surface of the substrate at the discrete target detection location, wherein the capture is achieved through a non-covalent interaction between the product(s) and the surface of the substrate or a moiety attached to the surface of the substrate.

69. The method of claim 68, wherein capture is achieved through ionic interactions, Van der Waals forces or hydrogen bonds.

70. The method of claim 68, wherein capture is achieved through hybridization of the reaction product(s) with a capture oligonucleotide attached to the surface of the substrate.

71. The method of claim 68, further comprising detecting the captured reaction product(s).

72. The method of claim 71, wherein the reaction product(s) is detected by mass spectrometry.

73. The method of claim 68, wherein the surface of the substrate forms an interior surface of a chamber.

74. The method of claim 68, wherein the extended primer is captured through hybridization of the extended primer to an oligonucleotide immobilized on the surface of the substrate.

75. The method of claim 74, wherein hybridization occurs between an additional region of nucleotides in the primer portion of the extended primer that hybridizes to a complementary sequence of nucleotides in the immobilized oligonucleotide, wherein the additional region of nucleotides in the primer portion of the extended primer does not occur in the target biomolecule.

76. The method of claim 75, wherein the primer contains a site that can be selectively cleaved.

77. The method of claim 71, wherein prior to detection, matrix is deposited at the target detection location and detection is performed by MALDI mass spectrometry.

78. The method of claim 68, wherein the conditions that permit extension of the oligonucleotide comprise at least one temperature thermocycle comprising heating and cooling.

79. The method of claim 78, wherein the rate of heating is selected from the range of about 3°

C./second up to about 100°

C./second.

80. The method of claim 78, wherein the rate of heating is selected from the group consisting of rates of heating of at least about:

3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95 and at least about 100°

C./second.

81. The method of claim 78, wherein the heating is achieved using thermal conductivity or electrical conductivity.

82. The method of claim 78, wherein the rate of cooling is selected from the range of about 3°

C./second up to about 100°

C./second.

83. The method of claim 78, wherein the rate of cooling is selected from the group consisting of rates of cooling of at least about:

3, 4, 5, 6, 7, 8, 9, 10;

15, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95 and at least about 100°

C./second.

84. The method of claim 78, wherein the cooling is achieved using water, air or a cold block.

85. The method of claim 68, wherein the surface of the substrate is enclosed by one or more reaction chambers within a removable reaction containment member.

86. The method of claim 85, wherein the temperature difference from within a single reaction chamber is selected from ≦

;

2.0°

C., 1.5°

C., 1.0°

C., 0.5°

C. and ≦

0.3°

C.

87. The method of claim 85, wherein the temperature difference between different reaction chambers is selected from ≦

;

1.0°

C., 0.5°

C., 0.4°

C., 0.3°

C., 0.2°

C. andy ≦

0.1°

C.

88. A method for performing a reaction of a target biomolecule, comprising:

(a) performing one or more reactions of the target biomolecule in the presence of the surface of a substrate, wherein the reaction(s) is performed substantially in solution and the substrate comprises one or more target detection location(s) in contact with the solution;

(b) capturing one or more products of the reaction or reactions on the surface of the substrate at the target detection location; and

(c) detecting the reaction product or products at the target detection location.

89. The method of claim 88, wherein the capture is achieved through a non-covalent interaction between the product(s) and the surface of the substrate or a moiety attached to the surface of the substrate.

90. A method for determining the identity of a nucleotide in a target nucleic acid molecule, comprising:

hybridizing a single-stranded portion of the target nucleic acid molecule with an oligonucleotide that is complementary to a region of the single-stranded portion of the target nucleic acid in solution and in the presence of a capture moiety at a discrete target detection location;

exposing the hybridized nucleic acid and oligonucleotide to conditions that permit extension of the oligonucleotide;

capturing the product(s) of the extension reaction on the surface of the substrate at the discrete target detection location

91. The method of claim 90, wherein the capture is achieved through a non-covalent interaction between the product(s) and the surface of the substrate or a moiety attached to the surface of the substrate.