Methods of detecting targets on an arrary

• US 20040219590A1
• Filed: 05/27/2004
• Published: 11/04/2004
• Est. Priority Date: 02/10/2000
• Status: Abandoned Application

• Current Assignee: Edward Carlson, Kenneth D. Coblentz, Todd Dickinson
• Original Assignee: Edward Carlson, Kenneth D. Coblentz, Todd Dickinson
• Inventors: Carlson, Edward, Coblentz, Kenneth D., Dickinson, Todd
• Application Number:
  US10/856,039
• Publication Number:
  US 20040219590A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00274 Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00511 Walls of reactor vessels
• 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 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• 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 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00662 Two-dimensional arrays within two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 : Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00702 : Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/0072 : Organic compounds
• CPC Class Codes: B01L 2300/0636 : Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0654 : Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0822 : Slides
• CPC Class Codes: B01L 3/5085 : for multiple samples, e.g. microtitration plates
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 21/6428 : Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates

Abstract

The invention relates to methods of detecting a target analyte in a biological sample using composite microsphere arrays having first and second assay locations. Preferred target analytes include nucleic acid, and more specifically, nucleic acid having one or more single nucleotide polymorphisms (SNPs).

Claims

1. A method of detecting a target analyte in a biological sample, comprising:

a) providing a composite array comprising;

a substrate having a surface;

a first and a second assay location on said surface, wherein said assay locations comprise a population of microspheres, and wherein said microspheres comprise bioactive agents;

a physical partition separating said first assay location from said second assay location;

b) adding said biological sample to said first assay location under conditions sufficient to allow said target analyte to bind to said bioactive agents; and

c) detecting the binding of said bioactive agents to said target analyte.

2. The method of claim 1, wherein the binding of said bioactive agents to said target analyte is detected by a change in an optical signature of the microspheres.

3. The method of claim 1, wherein said target analyte comprises a nucleic acid.

4. The method of claim 3, wherein said bioactive agents comprise a nucleic acid.

5. The method of claim 4, wherein said microspheres further comprise labeled single-stranded nucleic acid sequences hybridized to said bioactive agents.

6. The method of claim 1, wherein said microspheres are randomly distributed on said surface at said assay locations.

7. The method of claim 1, wherein said microspheres are separated by a distance of less than 25 μ

m.

8. The method of claim 1, wherein said microspheres are separated by a distance of less than 15 μ

m.

9. The method of claim 1, further comprising detecting a target analyte in a second biological sample by adding said second biological sample to said second assay location and thereafter detecting the binding of said bioactive agents to said target analyte.

10. The method of claim 1, wherein said substrate comprises a microscope slide.

11. The method of claim 1, wherein said first and second assay locations are separately sealed to form hybridization chambers.

12. The method of claim 11, wherein said hybridization chambers comprise flexible membranes.

13. The method of claim 1, wherein said physical partition is a non-permanent sealant.

14. The method of claim 13, wherein said non-permanent sealant is selected from the group consisting of:

rubber, silicon, petroleum jelly, wax and parafilm.

15. The method of claim 1, wherein said physical partition is a ridge or rim of sufficient width and height to prevent said bioactive agents from moving from said first assay location to said second assay location.

16. The method of claim 1, wherein said physical partition is a trough of sufficient width and depth to prevent said bioactive agents from moving from said first assay location to said second assay location.

17. The method of claim 1, wherein said physical partition is a gasket.

18. The method of claim 17, wherein said gasket is adapted to fit within an indentation or channel on the substrate.

19. The method of claim 1, wherein said target analyte comprises a detectable optical label.

20. The method of claim 19, wherein said optical label is a fluorophore.

21. The method of claim 1, wherein said biological sample comprises blood.

22. The method of claim 1, wherein said bioactive agents comprise a detectable optical label.

23. The method of claim 1, wherein said detectable label is a fluorophore.

24. A method of detecting a target nucleic acid in a biological sample, wherein said target nucleic acid comprises one or more single nucleotide polymorphisms (SNPs) at one or more predetermined positions, comprising:

a) providing a composite array comprising;

a substrate having a surface;

a population of microspheres, wherein said microspheres are linked to capture probes configured to bind to said target nucleic acid at said one or more predetermined positions;

a first and second assay location on said surface, wherein said assay locations comprise said population of microspheres;

a physical partition separating said first assay location from said second assay location;

b) adding said biological sample to said first assay location under conditions sufficient to allow said target nucleic acid to bind to said capture probes; and

c) detecting the binding of said target nucleic acid to said capture probes.

25. The method of claim 24, wherein the binding of said capture probes to said target nucleic acid is detected by a change in an optical signature of the microspheres.

26. The method of claim 24, wherein said microspheres are randomly distributed on said surface at said assay locations.

27. The method of claim 24, wherein said microspheres are separated by a distance of less than 25 μ

m.

28. The method of claim 24, wherein said microspheres are separated by a distance of less than 15 μ

m.

29. The method of claim 24, further comprising detecting a target analyte in a second biological sample by adding said second biological sample to said second assay location and thereafter detecting the binding of said capture probes to said target analyte.

30. The method of claim 24, wherein said substrate comprises a microscope slide.

31. The method of claim 24, further comprising adding a SNP allele-specific probe, configured to hybridize to said target nucleic acid, wherein said SNP allele-specific probe is labeled with a fluorophore.

32. The method of claim 24, wherein said physical partition is a non-permanent sealant.

33. The method of claim 32, wherein said non-permanent sealant is selected from the group consisting of rubber, silicon, petroleum jelly, wax and parafilm.

34. The method of claim 24, wherein said physical partition is a ridge or rim of sufficient width and height to prevent said capture probes from moving from said first assay location to said second assay location.

35. The method of claim 24, wherein said physical partition is a trough of sufficient width and depth to prevent said capture probes from moving from said first assay location to said second assay location.

36. The method of claim 24, wherein said physical partition is a gasket.

37. The method of claim 36, wherein said gasket is adapted to fit within an indentation or channel on the substrate.

38. The method of claim 24, wherein said biological sample comprises blood.

39. The method of claim 24, wherein said target nucleic acid having one or more SNPs is causatively linked to a human disease.

40. The method of claim 39, wherein said disease is Alzheimer'"'"'s disease.

41. The method of claim 24, wherein said target nucleic acid comprises a detectable optical label.

42. The method of claim 41, wherein said optical label is a fluorophore.

43. The method of claim 24, wherein said microspheres further comprise labeled single-stranded nucleic acid sequences hybridized to said capture probes.

44. The method of claim 24, wherein said capture probes comprise a detectable optical label.

45. The method of claim 44, wherein said optical label is a fluorophore.