Hydrogel-based microarray signal amplification methods and devices therefor

• US 6,921,638 B2
• Filed: 12/19/2001
• Issued: 07/26/2005
• Est. Priority Date: 06/25/1999
• Status: Expired due to Fees

• Current Assignee: Amersham Biosciences (General Electric Company)
• Original Assignee: Amersham Biosciences (General Electric Company)
• Inventors: Liu, Chang-Gong, Johnson, W. Travis, Brush, Charles K., Mazumder, Abhijit
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): WILDER, CYNTHIA B
• Application Number:
  US10/025,185
• Publication Number:
  US 20020146730A1
• Time in Patent Office: 1,315 Days
• Field of Search: 435/6, 435/181, 436/532, 436/531, 522/114, 522/116, 522/117, 522/120, 522/121, 522/153, 527/200, 527/201, 536/23.1
• US Class Current: 435/6.19
• 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 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• 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: C07F 9/2408 of hydroxyalkyl compounds
• CPC Class Codes: C07F 9/5532 Seven-(or more) membered rings
• CPC Class Codes: C07F 9/5537 the heteroring containing the structure -C(=O)-N-C(=O)- (both carbon atoms belong to the heteroring)
• CPC Class Codes: C07F 9/65031 having the nitrogen atoms in the positions 1 and 2
• CPC Class Codes: C07F 9/6506 having the nitrogen atoms in positions 1 and 3
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393

Abstract

Methods and devices for detecting nucleic acid and protein targets on hydrogel microarrays are disclosed. Fluorophores are incorporated into the targets and detected. A linear correlation between target concentration and signal amplitude is maintained through the elimination of active enzyme amplification.

Claims

1. A method of detecting a target nucleic acid or protein, comprising:

(a) providing a target comprising a target nucleic acid or protein and a first member of a binding pair;

(b) hybridizing the target nucleic acid or protein to a probe attached to a hydrogel matrix through a 2+2 photocycloaddition;

(c) contacting the first member of the binding pair with a second member of the binding pair, said second member comprising a fluorophore; and

(d) detecting the fluorophore additions, thereby detecting said target nucleic acid or protein.

2. The method of claim 1, wherein the first member comprises a first member selected from the group consisting of biotin, digoxygenin, and bromouridine.

3. The method of claim 2, wherein the first member comprises biotin.

4. The method of claim 1, wherein the second member is selected from the group consisting of avidin, streptavidin, biotin antibody, digoxygenin antibody, and bromouridine antibody.

5. The method of claim 4, wherein the second member comprises streptavidin.

6. The method of claim 1, wherein the nucleic acid is synthesized by producing a cDNA from a mRNA.

7. The method of claim 1, wherein the nucleic acid is synthesized by producing a cDNA or a cRNA from a DNA.

8. The method of claim 1, wherein the target is synthesized by incorporating the first member of a binding pair into the nucleic acid by polymerization.

9. The method of claim 1, wherein the protein is from a cell lysate.

10. The method of claim 1, wherein the hydrogel matrix comprises polyacrylamide.

11. The method of claim 1, wherein the hydrogel matrix comprises polyurethane.

12. The method of claim 1, wherein the probe comprises a reactive site capable of undergoing a 2+2 photocycloaddition.

13. The method of claim 1, wherein the hydrogel matrix comprises a reactive site capable of undergoing a 2+2 photocycloaddition.

14. The method of claim 5, wherein the streptavidin is attached to one or more fluorophores.

15. The method of claim 14, wherein the streptavidin is attached to between three and four fluorophores.

16. The method of claim 1, wherein said fluorophore is selected from the group consisting of cyanine dyes and ALEXA FLUOR dyes.

17. The method of claim 16, wherein the cyanine dye is Cy-3, Cy-5, or Cy-5.5.

18. The method of claim 16, wherein the ALEXA FLUOR dye is ALEXA-532, ALEXA-647, or ALEXA-633.

19. The method of claim 1, wherein said fluorophore is ALEXA-647.

20. The method of claim 1, further comprising contacting at least one of the second members of the binding pair with an antibody comprising a first member.

21. The method of claim 20, wherein the antibody is a biotinylated anti-streptavidin antibody.

22. The method of claim 20, wherein the first member comprises biotin and the second member comprises streptavidin attached to more than one fluorophore.

30. The method of claim 1, wherein the probe comprises a reactive site capable of undergoing a 2+2 photocycloaddition.

23. A method of detecting a single nucleotide polymorphism, comprising:

(a) hybridizing a target nucleic acid to a probe attached to a hydrogel matrix through a 2+2 photocycloaddition, said probe designed to terminate at the site of the single nucleotide polymorphism;

(b) extending the probe by one nucleotide, wherein the nucleotide comprises a first member of a binding pair;

(c) contacting the first member of the binding pair with a second member of the binding pair comprising a fluorophore; and

(d) detecting the fluorophore additions, thereby detecting the single nucleotide polymorphism.

24. The method of claim 23, wherein the nucleotide is a dideoxynucleotide or an acyclonucleotide.

25. The method of claim 23, wherein the nucleotide comprises the first member of the binding pair before said nucleotide is incorporated into the probe.

26. The method of claim 23, wherein the first member of the binding pair is attached to the nucleotide after incorporation of the nucleotide into the probe.

27. The method of claim 23, wherein the first member of the binding pair comprises biotin.

28. The method of claim 26, wherein the second member of the binding pair comprises streptavidin.

29. The method of claim 23, wherein the hydrogel matrix comprises polyacrylamide or polyurethane.

31. The method of claim 28, wherein the streptavidin is attached to more than one fluorophore.

32. The method of claim 23, wherein said fluorophore is selected from the group consisting of cyanine dyes and ALEXA FLUOR dyes.

33. The method of claim 32, wherein the cyanine dye is Cy-3, Cy-5, or Cy-5.5.

34. The method of claim 32, wherein the ALEXA FLUOR dye is ALEXA-532, ALEXA-647, or ALEXA-633.

35. The method of claim 23, wherein said fluorophore is ALEXA-647.

36. The method of claim 23, further comprising contacting at least one of the second members of the binding pair with an antibody comprising a first member.

37. The method of claim 36, wherein the antibody is a biotinylated anti-streptavidin antibody.

38. The method of claim 36, wherein the first member comprises biotin and the second member comprises streptavidin attached to more than one fluorophore.