Hydrogel-based microarray signal amplification methods and devices therefor
View Patent ↗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.
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.
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.
30. The method of claim 1 , wherein the probe comprises a reactive site capable of undergoing a 2+2 photocycloaddition.
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.