Method and apparatus for performing biological reactions on a substrate surface

• US 6,642,046 B1
• Filed: 12/09/1999
• Issued: 11/04/2003
• Est. Priority Date: 12/09/1999
• Status: Expired due to Term

• Current Assignee: Motorola Solutions, Inc.
• Original Assignee: Motorola, Inc. (Motorola Solutions, Inc.)
• Inventors: Kahn, Peter, Tuggle, Todd, Hawkins, George, McGarry, Mark W.
• Primary Examiner(s): REDDING, DAVID A
• Application Number:
  US09/458,534
• Time in Patent Office: 1,426 Days
• Field of Search: 435/387.1, 435/287.2, 435/288.4, 435/288.5, 435/288.7, 435/305.3, 435/305.4
• US Class Current: 435/287.2
• CPC Class Codes: B01J 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00351 Means for dispensing and evacuation of reagents
• CPC Class Codes: B01J 2219/00495 Means for heating or cooling the reaction vessels
• 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: C40B 60/14 for creating libraries
• CPC Class Codes: F28F 2260/02 having microchannels
• CPC Class Codes: G01N 2030/025 Gas chromatography
• CPC Class Codes: G01N 2035/00158 Elements containing microarrays, i.e. "biochip"
• CPC Class Codes: G01N 2035/00237 Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 30/6069 with compartments or bed substructure
• CPC Class Codes: G01N 30/6095 Micromachined or nanomachined, e.g. micro- or nanosize

Abstract

The present invention provides methods and apparatus for performing biological reactions on a substrate surface that use a low volume of sample fluid, accommodate substrates as large as or larger than a conventional microscope slide, accommodate a plurality of independent reactions, and accommodate a substrate surface having one or more hydrogel-based microarrays attached thereto. The invention further provides an apparatus that allows introduction of fluids in addition to sample fluid into each reaction chamber via standard pipet tips and associated pipettor apparatus. The invention further an apparatus that increases reaction reproducibility, increases reaction efficiency, and reduces reaction duration.

The preferred embodiment of the invention is configured to accommodate a standard microscope slide substrate having four hydrogel-based microarrays attached thereto and comprises a base plate having a well structure corresponding to each microarray and two fluid ports extending through the base plate into each well structure, a means for temporarily clamping the substrate against the base plate such that the microarrays face into the well structures, a means for sealing the perimeter around each microarray and well structure, and a means for sealing the fluid ports from the environment.

Claims

1. An apparatus for performing biological reactions on a substrate layer, comprising:

(a) a substrate having a first surface containing a plurality of biologically reactive sites disposed theron;

(b) a base plate having a first surface and a second surface, wherein the first surface further comprises a cavity comprising one or a plurality of well structures;

(c) a sealing member disposed in each well structure, wherein each sealing member defines a reaction chamber between the surface of the substrate layer containing the biologically reactive sites and the first surface of the base plate; and

(d) a first fluid port and a second fluid port extending from each reaction chamber to the lower surface of the base plate.

2. The apparatus of claim 1 comprising a multiplicity of reaction chambers in which a plurality of independently controlled biological reactions can be performed.

3. The apparatus of claim 1 further comprising:

(a) a means for delivering fluid to a reaction chamber through the first fluid port, and

(b) a means for removing fluid from a reaction chamber through the second fluid port.

4. The apparatus of claim 3 wherein the fluid delivery means comprises tubing attached to the first fluid port and the fluid removal means comprises tubing attached to the second fluid port.

5. The apparatus of claim 1 wherein the diameter of the first and second fluid ports will accommodate the outer diameter of a pipet tip.

6. The apparatus of claim 1 further comprising means for pressurizing the contents of the reaction chambers.

7. The apparatus of claim 1 further comprising a groove in the well structure, wherein the sealing member is seated in the groove.

8. The apparatus of claim 1 further comprising a heating element.

9. The apparatus of claim 8 wherein the heating element further comprises a temperature control element that permits rapid alteration of the temperature of the substrate.

10. The apparatus of claim 1 wherein the base plate is made of a thermally conductive material.

11. The apparatus of claim 8 wherein the base plate is made of titanium, copper, aluminum or ceramic.

12. The apparatus of claim 1 wherein the first surface of the base plate is coated with a biologically compatible material.

13. The apparatus of claim 12 wherein the biologically compatible material is fluorinated ethylene propylene, polypropylene, elemental gold, or elemental platinum.

14. The apparatus of claim 12, further comprising a biologically compatible primer layer disposed between the first surface of the base plate and the biologically compatible base plate surface coating.

15. The apparatus of claim 14 wherein the primer layer is a fluoropolymer.

16. The apparatus of claim 1 wherein the sealing member is an O-ring, a gasket, a compressible washer, or a layer of grease.

17. The apparatus of claim 1 wherein the substrate is a microscope slide.

18. A method for performing biological reactions, comprising the steps of:

(a) loading a substrate having a first surface containing a plurality of biologically reactive sites into the apparatus of claim 1,

(b) loading a biological fluid sample into each reaction chamber of the apparatus,

(c) affixing the fluid port seal to the second surface of the base plate,

(d) heating the apparatus,

(e) allowing the reaction to proceed to completion,

(f) removing the fluid port seal,

(g) removing the fluid samples from the reaction chambers, and

(h) removing the substrate from the apparatus.

19. An apparatus according to claim 1, further comprising a compression plate seated on the substrate for affixing the substrate to the base plate.

20. The apparatus of claim 19 wherein the compression plate is removably affixed to the first surface of the base plate.

21. The apparatus of claim 19 wherein the base plate has a perimeter and plurality of retaining pins disposed along the perimeter, and wherein the compression plate has a perimeter and a plurality of apertures disposed along the perimeter aligned with the retaining pins on the base plate, and wherein the compression plate is removable affixed to the base plate by positioning the compression plate so that the retaining pins extend through the apertures.

22. The apparatus of claim 19 further comprising a retaining plate removably seated on the compression plate.

23. The apparatus of claim 19 further comprising a compliance layer disposed between the compression plate and the substrate.

24. The apparatus of claim 23 wherein the compliance layer comprises

(a) a first layer of low-compression material, and

(b) a second layer of pressure-sensitive adhesive.

25. The apparatus of claim 24 wherein the first layer of low-compression material is silicone sponge rubber, natural sponge rubber, or neoprene sponge rubber.

26. The apparatus of claim 23 wherein the compliance layer further comprises one or more viewing ports extending through the compliance layer at a position that corresponds to the position of each reaction chamber, and wherein the compression plate further comprises one or more viewing ports extending through the compression plate at a position on the plate that corresponds to the position of each reaction chamber and the position of each viewing port in the compliance layer.

27. The apparatus of claim 19 wherein the compression plate further comprises one or more viewing ports extending through the compression plate at a position on the plate that corresponds to the position of each reaction chamber.

28. The apparatus of claim 19 wherein the compression plate is made of fluorinated ethylene propylene, acetal resin, polyurethane, polypropylene, or acrylonitrile-butadiene-styrene.

29. An apparatus according to claim 1, further comprising a fluid port seal for temporarily closing the fluid ports and isolating the reaction chambers.

30. The apparatus of claim 29 wherein the fluid port seal comprises a layer of thermally conductive material and a layer of biologically inert adhesive.

31. A method for performing biological reactions, comprising the steps of:

(a) loading a substrate having a first surface containing a plurality of biologically reactive sites into the apparatus of claim 1,

(b) loading a biological fluid sample into at least one reaction chamber of the apparatus,

(c) affixing the fluid port seal to the second surface of the base plate,

(d) heating the apparatus, and

(e) allowing the reaction to proceed to completion.

32. A method for performing biological reactions according to claim 31, further comprising loading a first biological fluid sample into a first reaction chamber and a second biological fluid sample into a second reaction chamber.

67. A method for performing biological reactions, comprising the steps of:

(a) loading a biological fluid sample into each reaction chamber of the apparatus of claim 30,

(b) affixing the fluid port seal to the second surface of the base plate,

(c) heating the apparatus,

(d) allowing the reaction to proceed to completion,

(e) removing the fluid port seal,

(f) removing the fluid samples from the reaction chambers, and

(g) removing the substrate from the apparatus.

33. An apparatus for performing biological reactions on a substrate having a first surface containing a plurality of biologically reactive sites attached thereto, comprising:

thereon;

(b) a base plate having a first surface and a second surface, wherein the first surface further comprises a first cavity comprising one or a plurality of well structures and a second cavity, and wherein the substrate is removably seated in the cavity, and wherein the well structures are in direct communication with the first surface of the substrate,

(c) a groove in each well structure having an inner perimeter and a width,

(d) a sealing member disposed in the groove in each well structure, wherein each sealing member defines a reaction chamber between the first surface of the substrate layer and the first surface of the base plate,

(e) a compression plate having a cavity and one or more viewing ports extending through the compression plate and corresponding in position to the reaction chambers, wherein the compression plate is removably seated on the second surface of the substrate for removably affixing the substrate in the cavity of the base plate,

(f) a compliance layer disposed in the cavity of the compression plate having one or more viewing ports extending through the compliance layer and corresponding in position to the reaction chambers;

(g) a retaining plate removably seated on the compression plate,

(h) a plurality of retaining pins disposed around the perimeter of the base plate which are removably inserted into a corresponding plurality of apertures in the compression layer and a corresponding plurality of apertures in the retaining plate,

(i) a biologically compatible primer layer applied to the base plate and the retaining pins,

(j) a biologically compatible surface coating applied to the primer layer,

(k) a first fluid port and a second fluid port extending from each reaction chamber to the lower surface of the base plate,

(l) a fluid port seal for temporarily closing the fluid ports and isolating the reaction chambers from the environment,

(m) a heating element disposed beneath the base plate, and

(n) a thermal cycling device operatively connected to the heating element.

34. An apparatus for performing biological reactions on a substrate layer, comprising:

(a) a substrate having a first surface containing a plurality of biologically reactive sites disposed thereon and a second surface opposite the first surface,

(b) a base plate having a first surface and a second surface, wherein the first surface further comprises a cavity comprising one or a plurality of well structures,

(c) a compression plate removably seated on the substrate for removably affixing the substrate to base plate,

(d) a sealing member disposed in each well structure, wherein each sealing member defines a reaction chamber between the surface of the substrate layer containing the biologically reactive sites and the first surface of the base plate,

(e) a first fluid port and a second fluid port extending from each reaction chamber to the lower surface of the base plate, and

(f) a fluid port seal for temporarily closing the fluid ports and isolating the reaction chambers from the environment.

35. The apparatus of claim 34 comprising a multiplicity of reaction chambers in which a plurality of independently controlled biological reactions can be performed.

36. The apparatus of claim 34 further comprising:

(a) a means for delivering fluid to a reaction chamber through the first fluid port, and

(b) a means for removing fluid from a reaction chamber through the second fluid port.

37. The apparatus of claim 36 wherein the fluid delivery means comprises tubing attached to the first fluid port and the fluid removal means comprises tubing attached to the second fluid port.

38. The apparatus of claim 34 wherein the diameter of the first and second fluid ports will accommodate the outer diameter of a pipet tip.

39. The apparatus of claim 34 further comprising means for pressurizing the contents of the reaction chambers.

40. The apparatus of claim 34 wherein the first surface of the substrate further comprises an array of 3-dimensional anchoring structures for biological molecules.

41. The apparatus of claim 40 wherein the 3-dimensional anchoring structures are gel-pads.

42. The apparatus of claim 41 wherein the 3-dimensional anchoring structures are polyacrylamide gel-pads.

43. The apparatus of claim 34 wherein the compression plate is removably affixed to the first surface of the base plate.

44. The apparatus of claim 43 wherein the base plate has a perimeter and plurality of retaining pins disposed along the perimeter, and wherein the compression plate has a perimeter and a plurality of apertures disposed along the perimeter aligned with the retaining pins on the base plate, and wherein the compression plate is removable affixed to the base plate by positioning the compression plate so that the retaining pins extend through the apertures.

45. The apparatus of claim 34 further comprising a retaining plate removably seated on the compression plate.

46. The apparatus of claim 34 further comprising a compliance layer disposed between the compression plate and the substrate.

47. The apparatus of claim 46 wherein the compliance layer comprises

(a) a first layer of low-compression material, and

(b) a second layer of pressure-sensitive adhesive.

48. The apparatus of claim 47 wherein the first layer of low-compression material is silicone sponge rubber, natural sponge rubber, or neoprene sponge rubber.

49. The apparatus of claim 34 wherein the compression plate further comprises one or more viewing ports extending through the compression plate at a position on the plate that corresponds to the position of each reaction chamber.

50. The apparatus of claim 46 wherein the compliance layer further comprises one or more viewing ports extending through the compliance layer at a position that corresponds to the position of each reaction chamber, and wherein the compression plate further comprises one or more viewing ports extending through the compression plate at a position on the plate that corresponds to the position of each reaction chamber and the position of each viewing port in the compliance layer.

51. The apparatus of claim 34 further comprising a groove in the well structure, wherein the sealing member is seated in the groove.

52. The apparatus of claim 34 wherein the base plate further comprises a second cavity, and wherein the substrate is removably seated in the second cavity.

53. The apparatus of claim 34 further comprising a heating element.

54. The apparatus of claim 53 wherein the heating element further comprises a temperature control element that permits rapid alteration of the temperature of the substrate.

55. The apparatus of claim 34 wherein the substrate is an integral element of the base plate.

56. The apparatus of claim 34 wherein the base plate is made of a thermally conductive material.

57. The apparatus of claim 56 wherein the base plate is made of titanium, copper, aluminum or ceramic.

58. The apparatus of claim 34 wherein the first surface of the base plate is coated with a biologically compatible material.

59. The apparatus of claim 58 wherein the biologically compatible material is fluorinated ethylene propylene, polypropylene, elemental gold, or elemental platinum.

60. The apparatus of claim 58, further comprising a biologically compatible primer layer disposed between the first surface of the base plate and the biologically compatible base plate surface coating.

61. The apparatus of claim 60 wherein the primer layer is a fluoropolymer.

62. The apparatus of claim 34 wherein the sealing member is an O-ring, a gasket, a compressible washer, or a layer of grease.

63. The apparatus of claim 34 wherein the substrate is a microscope slide.

64. The apparatus of claim 34, wherein the fluid port seal comprises a layer of thermally conductive material and a layer of biologically inert adhesive.

65. The apparatus of claim 34 wherein the compression plate is made of fluorinated ethylene propylene, acetal resin, polyurethane, polypropylene, or acrylonitrile-butadiene-styrene.

66. An apparatus for performing biological reactions on a substrate surface, comprising:

(a) a substrate having a first surface containing a plurality of biologically reactive sites attached thereto and a second surface opposite the first surface,

(b) a base plate having a first surface and a second surface, wherein the first surface further comprises a cavity and one or a plurality of well structures, and wherein the substrate is removably seated in the cavity, and wherein the well structures are in direct communication with the first surface of the substrate,

(c) a groove in each well structure having an inner perimeter and a width,

(d) a sealing member disposed in the groove in each well structure, wherein each sealing member defines a reaction chamber between the first surface of the substrate layer and the first surface of the base plate,

(e) a compression plate having a cavity and one or more viewing ports extending through the compression plate and corresponding in position to the reaction chambers, wherein the compression plate is removably seated on the second surface of the substrate for removably affixing the substrate in the cavity of the base plate,

(f) a compliance layer disposed in the cavity of the compression plate having one or more viewing ports extending through the compliance layer and corresponding in position to the reaction chambers,

(g) a retaining plate removably seated on the compression plate,

(h) a plurality of retaining pins disposed around the perimeter of the base plate which are removably inserted into a corresponding plurality of apertures in the compression layer and a corresponding plurality of apertures in the retaining plate,

(i) a biologically compatible primer layer applied to the base plate and the retaining pins,

(j) a biologically compatible surface coating applied to the primer layer,

(k) a first fluid port and a second fluid port extending from each reaction chamber to the lower surface of the base plate,

(l) a fluid port seal for temporarily closing the fluid ports and isolating the reaction chambers from the environment,

(m) a heating element disposed beneath the base plate, and

(n) a thermal cycling device operatively connected to the heating element.