Methods of detecting targets on an array

• US 8,741,630 B2
• Filed: 12/17/2010
• Issued: 06/03/2014
• Est. Priority Date: 02/10/2000
• Status: Expired due to Term

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Dickinson, Todd, Coblentz, Kenneth D., Carlson, Edward
• Primary Examiner(s): BAUSCH, SARAE L
• Application Number:
  US12/971,996
• Publication Number:
  US 20110092389A1
• Time in Patent Office: 1,264 Days
• Field of Search: None
• US Class Current: 435/287.2
• 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 forming a fluidics chamber, said method comprising the steps of:

a) randomly distributing a population of microspheres to an array substrate having a surface comprising a plurality of depressions, wherein said population of microspheres comprises at least a first and second subpopulation of microspheres comprising a target nucleic acid, said first subpopulation having a target nucleic acid that is different from the target nucleic acid of the second subpopulation;

b) providing a population of blank microspheres and randomly distributing said population of blank microspheres to the array substrate, wherein the microspheres are present in a ratio of microspheres comprising a target nucleic acid to n² blank microspheres, wherein n is the number of blank microspheres separating the microspheres comprising target nucleic acid;

c) applying a force to said substrate, thereby moving microspheres to the bottoms of the depressions;

d) mating the substrate to a lid, said lid having at least one inlet port and one outlet port; and

e) clamping said lid to said substrate a sealant being disposed between said lid and said substrate, thereby forming a fluidics chamber.

2. The method of claim 1, wherein depressions of said substrate have a diameter that is dimensioned to accommodate not more than a single microsphere comprising a target nucleic acid.

3. The method of claim 1, wherein the target nucleic acid comprises a genome fragment or a copy of a genome fragment.

4. The method of claim 1, wherein a target nucleic acid is attached to a microsphere.

5. The method of claim 4, wherein the attachment is mediated using biotin.

6. The method of claim 1, further comprising attaching a first fluidics line to the inlet port and attaching a second fluidics line to the outlet port.

7. The method of claim 6 further comprising applying fluid through said fluidics lines.

8. The method of claim 1, wherein the depressions are regularly spaced.

9. The method of claim 1, wherein the space between depressions is greater than 5 μ

m.

10. The method of claim 1, wherein the space between depressions is less than 15 μ

m.

11. The method of claim 1, wherein said force is a magnetic force.

12. The method of claim 1, wherein said substrate is optically coupled to a CCD camera.

13. The method of claim 1, wherein said clamping step is automated.

14. The method of claim 1, wherein said clamping step comprises engaging a hook and latch mechanism.

15. The method of claim 1, wherein said clamping step comprises engaging a rotating stud and receptacle mechanism.

16. The method of claim 1, further comprising aligning said substrate with said lid using an alignment moiety.

17. The method of claim 16, wherein said alignment moiety comprises a notch.

18. The method of claim 1, wherein said sealant is selected from the group consisting of rubber and silicon.

19. The method of claim 18, wherein said sealant comprises a gasket.

20. The method of claim 1, wherein said inlet port and said outlet port are each fitted with a seal.

21. The method of claim 20, wherein said seal comprises an o-ring.

22. The method of claim 1, wherein the array substrate comprises a fiber optic bundle.

23. The method of claim 1, wherein applying force to said substrate results in filling depressions at an average density per depression of not more than one microsphere comprising a target nucleic acid.

24. The method of claim 1, wherein said surface is substantially planar.

25. The method of claim 1, wherein the microspheres are present in a ratio of at least 1:

100 microspheres comprising a target nucleic acid to blank microspheres.

26. The method of claim 25, wherein the microspheres are present in a ratio of at least 10:

1 microspheres comprising a target nucleic acid to blank microspheres.

27. The method of claim 22, wherein the fiber optic bundle is embedded within the substrate.

28. The method of claim 1, wherein the substrate is opaque.

29. The method of claim 1, further comprising combining the subpopulations of microspheres comprising nucleic acids with the population of blank microspheres prior to randomly distributing the combined population to the array substrate.