Gradient resolved information platform

• US 20030055233A1
• Filed: 04/18/2002
• Published: 03/20/2003
• Est. Priority Date: 04/18/2001
• Status: Active Grant

• Current Assignee: Ulrich J. Krull
• Original Assignee: Ulrich J. Krull
• Inventors: Krull, Ulrich J.
• Granted Patent: US 7,183,050 B2
• Time in Patent Office: Days
• US Class Current: 536/23.1
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00524 in the shape of fiber bundles
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00545 Colours
• CPC Class Codes: B01J 2219/00574 radioactive
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00628 Ionic
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• 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 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2527/137 : Concentration of a component of medium
• CPC Class Codes: C12Q 2527/143 : Concentration of primer or probe
• CPC Class Codes: C12Q 2527/15 : Gradients
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: Y10S 977/924 : using nanostructure as support of dna analysis

Abstract

The invention provides improved methods and devices for the detection and identification in a sample of one or more target molecules which bind to probe molecules, particularly to nucleic acid probe molecules. The improved method is based on contacting the sample with a surface that is coated with one or more gradients of probe molecules, particlarly nucleic acid or nucleic acid analog probe molecules that serve to bind target molecules in the sample, particularly nucleic acids having sequences that are complementary or partially complementary to one or more probe molecules. A probe gradient generated on the surface is formed by the variation of a physical, structural or functional property of the probes on the surface. The gradient is generated, e.g., by varying density of probe molecules bound to the surface, by varying probe sequence length, by varying probe sequence, by varying probe sequence type, by varying the orientational structure of probes, and by varying the concentration of label associated with probes. Determination of the location, speed and/or extent of hybridisation of a nucleic acid on such a gradient surface is useful to identify target molecules bound to probes and/or to quantitatively measure the amount of the target in a sample. Hybridisation of target molecules to a gradient of nucleic acid probe can be examined as a function of time and/or hybridisation conditions (e.g., temperature, salt concentration, etc.) The methods and devices of this invention employ gradient surfaces to bind to one or more target molecules, particularly nucleic acids (or target sequences) in a sample, detecting their presence in the sample and quantitating the amount of one or more of such targets in a sample.

Claims

1. A surface with an immobilized layer comprising a plurality of probe molecules bound to the surface wherein the immobilized layer has a least one spatially distributed gradient of probe molecules wherein the gradient is formed by selectively varying a physical, structural or functional property of probe molecules bound to the surface as a function of the location on the surface to which the probe molecules are bound.

2. The surface of claim 1 wherein the at least one gradient is formed by selectively varying the density of bound probe molecules.

3. The surface of claim 1 wherein the at least one gradient is formed by selectively varying the size or length of the bound probe molecules.

4. The surface of claim 1 wherein the at least one gradient is formed by selectively varying the structure of the bound probe molecules.

5. The surface of claim 1 wherein the at least one gradient is formed by selectively varying the relative orientations of probe molecules bound to the surface.

7. The surface of claim 1 wherein the probe molecules are nucleic acids or nucleic acid analogs.

8. The surface of claim 7 wherein the at least one gradient is formed by selectively varying the sequence of the bound probe molecules.

9. The surface of claim 7 wherein the at least one gradient is formed by selectively varying the density of the bound probe molecules.

10. The surface of claim 9 wherein the density gradient can span an average nearest neighbour separation of from about 2 nm to over 40 nm.

11. The surface of claim 7 wherein the gradient is formed by selectively varying the concentration of polyelectrolyte immobilized along with the probe molecules.

12. The surface of claim 7 wherein the gradient is formed by selectively varying the length of the bound nucleic acid or nucleic acid analog.

13. The surface of claim 12 wherein the gradient is formed by varying the length of nucleic acid or nucleic acid probes in single base increments.

14. The surface of claim 13 wherein the gradient spans 1,000 or more bases.

15. The surface of claim 13 wherein the gradient spans 100 or more bases.

16. The surface of claim 13 wherein the gradient spans about 10 to about 50 bases.

17. The surface of claim 7 wherein the gradient is formed by selectively varying the shape of the probe molecules.

18. The surface of claim 7 wherein the gradient is formed by selectively varying the surface charge or dielectric of the surface.

19. The surface of claim 18 wherein the gradient is formed by selectively varying the concentration of charged or ionizable species co-immobilized with the probe molecules.

20. The surface of claim 1 which is fused silica, quartz, silicon, glass, a plastic, a metal, a transparent electrode, a ceramic, a semiconductor, a conductive form of carbon, paper, or a conductive polymer.

21. The surface of claim 1 wherein the immobilized layer containing the at least one gradient is substantially continuous.

22. The surface of claim 21 wherein the at least one gradient is substantially continuous.

23. The surface of claim 1 which comprises two gradients.

24. The surface of claim 23 wherein the two gradients are formed in orthogonal dimensions on the surface.

25. The surface of claim 23 wherein at least one gradient is a gradient of density of bound probe molecules.

26. The surface of claim 25 wherein the probe molecules are nucleic acids or nucleic acid analogs and the second gradient is formed by varying the sequence of the nucleic acids of nucleic acid analogs.

27. The surface of claim 1 wherein the gradient or gradients are formed by dip-casting, through gradients of light activation, by spraying, rolling, capping, sequence annealing, sequence degradation, sequence extension or combinations thereof.

28. The surface of claim 1 wherein the probe molecules are bound to the surface by adsorption, absorption, ionic bonding, covalent bonding, avidin-biotin, or thiol-gold interactions.

29. The surface of claim 1 wherein the immobilized layer further comprises detectable labels that indicate binding of target molecules to probe molecules on the surface.

30. The surface of claim 1 further comprising detectable labels bound to probe molecules in the gradient.

31. The surface of claim 30 wherein the detectable label is a fluorescent label tethered to a probe molecule.

32. The surface of claim 1 further comprising a fluorescent label bound to the surface.

33. The surface of claim 1 further comprising a spatially distributed gradient formed by varying the density of detectable labels tethered to probe molecules.

34. The surface of claim 33 wherein the detectable label is a fluorescent molecule.

35. The surface of claim 1 which is a waveguide, operating in either the evanescent mode or direct mode of excitation.

36. The surface of claim 1 further comprising a reference region in the at least one gradient formed.

37. A biosensor for detection of one or more target molecules comprising the surface of claim 1.

38. The biosensor of claim 37 further comprising a spatially-resolved method for detection of target molecules bound in the at least one gradient.

39. A method for separating two or more target molecules in a sample which comprises the steps of:

providing a surface of claim 1;

contacting the surface with the sample under conditions that allow binding or hybridisation of the target molecules to probe molecules in the at least one gradient.

40. The method of claim 39 wherein the probe molecules are nucleic acids or nucleic acid analogs.

41. The method of claim 40 wherein the target molecules are nucleic acids or nucleic acid analogs.

42. The method of claim 39 further comprising the step of applying a spatially resolved gradient of binding or hybridisation conditions to the surface to which the target molecules are bound.

43. The method of claim 42 wherein a spatially-resolved temperature gradient is applied to the surface.

44. The method of claim 43 wherein the spatially-resolved temperature gradient is formed in a direction that is orthogonal to the gradient of probe molecules into which the target molecules are initially bound.

45. The method of claim 42 wherein a spatially resolved gradient of ionic strength is applied to the surface.

46. The method of claim 39 wherein a detectable reference is added to the sample.

47. A method for detecting one or more target molecules in a sample which comprises the steps of:

providing a surface of claim 1;

contacting the sample with the surface under conditions that allow hybridisation or binding of target molecules to probe molecules in the gradient; and

detecting binding or hybridisation of one or more target molecules at a specific location in the at least one gradient.

48. The method of claim 47 wherein the probe molecules are nucleic acids or nucleic acid analogs.

49. The method of claim 48 wherein the target molecules are nucleic acids or nucleic acid analogs.

50. The method of claim 47 wherein the at least one gradient on the surface is formed by selective variation of the density of probe molecules.

51. The method of claim 47 wherein binding or hybridisation is detected by fluorescence.

52. The method of claim 47 further comprising a step of applying a spatially-resolved gradient of binding or hybridisation conditions to the surface to which the target molecules are bound.

53. The method of claim 52 wherein a spatially-resolved gradient of temperature is applied to the surface.

54. A method for measuring the amount of one or more target molecules in a sample which comprises the steps of:

contacting a sample with a surface of claim 1 under conditions such that one or more target molecules bind or hybridize to the probe molecules in the at least one gradient; and

detecting binding or hybridisation as a function of time after contact of the sample with the surface wherein the rate of binding or hybridisation of a target molecule to the surface is indicative of the amount of that target molecule in a sample.

55. The method of claim 54 wherein the probe molecules are nucleic acids or nucleic acid analogs.

56. The method of claim 54 wherein the target molecules are nucleic acids or nucleic acid analogs.

57. The method of claim 54 further comprising the step of applying a spatially resolved gradient of binding or hybridisation conditions to the surface to which the target molecules are bound.

58. The method of claim 56 wherein a spatially resolved temperature gradient is applied to the surface.

59. The method of claim 58 wherein the spatially resolved temperature gradient is formed in a direction that is orthogonal to the gradient of probe molecules into which the target molecules are initially bound.

60. The method of claim 56 wherein a spatially resolved gradient of ionic strength is applied to the surface.

61. The method of claim 56 wherein binding or hybridisation is detected by detecting a labelled target molecules, wherein the label is provided by fluorescent intercalators, fluorescent groove binders, molecular beacons, donor-acceptor energy transfer systems, radioisotopes, surface potentials, coloured products, enzyme labelled targets, antibody labelled targets, and gold particle labelled targets, or any combination thereof.

62. The method claim 56 wherein binding or hybridisation of different target molecules is detected using different fluorescent dyes.

63. The method of claim 56 wherein the sample contains a detectable reference which binds at the surface within a defined and identifiable zone or band, wherein the location of binding of the reference facilitates identification of one or more target molecules in the sample and further comprising a step of detecting binding or hybridisation of the reference as a function of time after contact of the sample with the surface to calibrate for environmental conditions of ionic strength, pH, temperature, non-selective adsorption or combinations thereof.

64. The method of claim 56 wherein the surface comprises a detectable reference which binds at the surface within a defined and identifiable zone or band, wherein the location of binding of the reference facilitates identification of one or more target molecules in the sample and further comprising a step of detecting binding or hybridisation of the reference as a function of time after contact of the sample with the surface to calibrate for environmental conditions of ionic strength, pH, temperature, non-selective adsorption or combinations thereof.

65. A method for detecting one or more target molecules in a sample which comprises the steps of:

contacting the sample with two or more surfaces of claim 1;

applying different binding and/or hybridisation conditions to the different surfaces; and

detecting binding or hybridisation of target molecules to the surfaces and

comparing the location of binding or hybridisation of target molecules under different binding and/or hybridisation conditions.

66. The method of claim 65 wherein the probe molecules and target molecules are nucleic acids or nucleic acid analogs.

67. A method for detecting the amount of one or more target molecules in a sample which comprises the steps of:

contacting the sample with two or more surfaces of claim 1;

applying different binding and/or hybridisation conditions to the different surfaces;

detecting binding or hybridisation of target molecules to the surfaces as a function of time after contact of the sample with the surfaces; and

comparing the rate of binding or hybridisation of a target molecule to surfaces under different binding and/or hybridisation conditions to determine the amount of that target molecule in a sample.

68. The method of claim 67 wherein the two or more surfaces are exposed to different temperatures during contact with the sample.

69. The method of claim 67 wherein the two or more surfaces are exposed to different ionic strengths during contact with the sample.

70. The method of claim 67 further comprising a step of applying a spatially-resolved gradient of binding or hybridisation conditions to the two or more surfaces.

71. The method of claim 70 wherein a spatially resolved temperature gradient is applied to the two or more surfaces.

72. The method of claim 70 wherein a spatially resolved gradient of ionic strength is applied to the two or more surfaces.

82. A kit for conducting an assay for detecting the amount of one or more target molecules in a sample which comprises one or more surfaces of claim 1.

83. The kit of claim 82 wherein the probe molecules are nucleic acids.

84. The kit of claim 83 further comprising one or more reagents for carrying out the assay.

6. The surface of claim I wherein the at least one gradient is formed by selectively varying the concentration of polyelectrolyte immobilized along with the probe molecules.

73. A method of making spatial distributed surface charge gradients on a surface comprising the step:

co-immobilising of the surface functionalities that support oligonucleotide attachment with ionisable functionalities wherein the proportion of ionisable functionalities is varied as a function of location on the substrate surface to generate a charge gradient.

74. The method of claim 73 wherein the gradient is formed by co-immobilising organosilanes with zwitterionic moieties.

75. A method of preparing a surface having a density gradient of bound probe molecules which comprises the steps of:

activating the surface by immobilizing a density gradient of a linker to template the density of probe molecules, and

binding probe molecules to the linker.

76. The method of claim 75 wherein the linker is GOPS.

77. The method of claim 75 wherein the linker is HEG.

78. A method of claim 75 wherein the probe molecules are nucleic acids or nucleic acid analogs.

79. The method of claim 78 wherein probe molecules are bound to the linker by automated synthesis

80. The method of claim 78 wherein the probe molecules are bound to the linker by adsorption, absorption, covalent bonding, ionic bonding, avidin-biotin, thiol-gold interactions, or combinations thereof.

81. The method of claim 78 wherein the probe molecules are bound to the linker by assembling larger oligomers from smaller sequences to create a gradient of sequence length and sequence content.