Methods and compositions for the target-localized anchoring of detectable label

Patent metadata

Field	Value
Publication number	US-9447455-B2
Application number	US-201213396352-A
Country	US
Kind code	B2
Filing date	Feb 14, 2012
Priority date	Feb 16, 2011
Publication date	Sep 20, 2016
Grant date	Sep 20, 2016

Abstract

Highly reactive functionalized substrates and linker molecules for use in the detection of molecular targets and other analytes of interest are provided as are kits, reaction mixtures and methods utilizing the same.

First claim

What is claimed is:

1. A reaction mixture comprising:
a functionalized substrate comprising
a supporting material;
one or more target-specific capture probes bound to the surface of the supporting material, wherein each target-specific capture probe is capable of specifically and non-covalently binding to a target molecule analyte; and
a plurality of molecules bound to the surface of the supporting material, other than the one or more target-specific capture probes, wherein each of the plurality of molecules comprises a first covalent bond-forming reactive group; and
a plurality of functionalized detectable label particles, wherein each of the functionalized detectable label particles comprises a plurality of molecules bound to the surface of a detectable label particle to provide the functionalized detectable label particle, wherein each of the plurality of molecules bound to the surface of the detectable label particle comprises a second covalent bond forming reactive group, wherein the first covalent bond-forming reactive group is capable of forming a covalent bond with the second covalent bond-forming reactive group, and wherein the plurality of functionalized detectable label particles do not have binding affinity for the target molecule analyte.

2. The reaction mixture of claim 1 , further comprising the target molecule analyte specifically bound to one of the one or more target-specific capture probes.

3. The reaction mixture of claim 2 , further comprising a label probe specifically bound to the target molecule analyte.

4. The reaction mixture of claim 3 , further comprising an enzyme conjugate specifically bound to the label probe.

5. The reaction mixture of claim 4 , wherein a functionalized detectable label particle of the plurality of functionalized detectable label particles is linked via multiple covalent bonds to the surface of the supporting material, in proximity to the target molecule analyte specifically bound to one of the one or more target-specific capture probes, via interaction of the first covalent bond forming reactive groups with the second covalent bond forming reactive groups.

6. The reaction mixture of claim 5 , comprising a plurality of spatially separated, individually detectable target loci.

7. The reaction mixture of claim 5 , wherein the target molecule-analyte is present in the reaction mixture at a concentration of less than about 100 fM.

8. The reaction mixture of claim 5 , wherein the target molecule-analyte is present in the reaction mixture at a concentration of about 0.1 fM to about 100 fM.

9. A reaction mixture comprising:
a functionalized substrate comprising
a supporting material;
one or more target-specific capture probes bound to the surface of the supporting material, wherein each target-specific capture probe is capable of specifically and non-covalently binding to a target molecule analyte; and
a plurality of molecules bound to the surface of the supporting material, other than the one or more target-specific capture probes, wherein each of the plurality of molecules comprises a first covalent bond-forming reactive group;
a plurality of functionalized detectable label particles, wherein each of the plurality of functionalized detectable label particles comprises a plurality of molecules bound to the surface of a detectable label particle to provide a functionalized detectable label particle, wherein each of the plurality of molecules bound to the surface of the detectable label particle comprises a member of a specific binding pair; and
a plurality of linker molecules wherein each linker molecule in the plurality of linker molecules is configured to non-covalently bind one of the plurality of molecules bound to the surface of one of the plurality of functionalized detectable label particles,
wherein each linker molecule comprises a plurality of second covalent bond-forming reactive groups, wherein the first covalent bond-forming reactive group is capable of forming a covalent bond with one of the second covalent bond-forming reactive groups, and wherein the plurality of functionalized detectable label particles do not have binding affinity for the target molecule analyte.

10. The reaction mixture of claim 9 , further comprising the target molecule analyte specifically bound to one of the one or more target-specific capture probes.

11. The reaction mixture of claim 10 , further comprising a label probe specifically bound to the target molecule analyte.

12. The reaction mixture of claim 11 , further comprising an enzyme conjugate specifically bound to the label probe.

13. The reaction mixture of claim 12 , wherein a functionalized detectable label particle of the plurality of functionalized detectable label particles is bound to multiple linker molecules of the plurality of linker molecules which are bound, via covalent bonds between the first covalent bond forming reactive groups and the second covalent bond forming reactive groups, to the surface of the supporting material, in proximity to the bound target molecule analyte.

14. The reaction mixture of claim 13 , comprising a plurality of spatially separated, individually detectable target loci.

15. The reaction mixture of claim 13 , wherein the target molecule-analyte is present in the reaction mixture at a concentration of less than about 100 fM.

16. The reaction mixture of claim 13 , wherein the target molecule-analyte is present in the reaction mixture at a concentration of about 0.1 fM to about 100 fM.

17. The reaction mixture of claim 1 , wherein the functionalized detectable label particles are functionalized magnetic beads.

18. The reaction mixture of claim 17 , wherein the functionalized magnetic beads have a diameter of about 100 nm to about 1 μm.

19. The reaction mixture of claim 17 , wherein the functionalized magnetic beads have a diameter of about 1 μm or greater.

20. The reaction mixture of claim 1 , wherein each of the plurality of molecules bound to the surface of the supporting material is a synthetic, non-naturally occurring molecule.

21. The reaction mixture of claim 1 , wherein the supporting material comprises silicon.

22. The reaction mixture of claim 20 , wherein each of the synthetic, non-naturally occurring molecules comprises a dendrimer structure.

23. The reaction mixture of claim 20 , wherein each of the synthetic, non-naturally occurring molecules comprises a tyrosine amino acid.

24. The reaction mixture of claim 20 , wherein each of the synthetic, non-naturally occurring molecules comprises a phenol or phenol derivative copolymer.

25. The reaction mixture of claim 20 , wherein each of the synthetic, non-naturally occurring molecules comprises lysine and tyrosine amino acids.

26. The reaction mixture of claim 20 , wherein each of the synthetic, non-naturally occurring molecules comprises tyramine or a tyramine derivative.

27. The reaction mixture of claim 20 , wherein each of the synthetic, non-naturally occurring molecules comprises thymine or a thymine derivative.

28. The reaction mixture of claim 9 , wherein the functionalized detectable label particles are functionalized magnetic beads.

29. The reaction mixture of claim 9 , wherein the supporting material comprises silicon.

30. The reaction mixture of claim 9 , wherein each of the plurality of molecules bound to the surface of th

Assignees

• Hu Celine
• Perkins Julie
• Gao Hetian
• Wang Lisen
• Headway Tech Inc

Inventors

• Hu Celine
• Perkins Julie
• Gao Hetian
• Wang Lisen

Classifications

• C07D495/04

  Ortho-condensed systems · CPC title

• C07D207/408

  Radicals containing only hydrogen and carbon atoms attached to ring carbon atoms · CPC title

• C12Q1/6816 Primary

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6834

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title

• C12Q2525/197

  incorporating a spacer/coupling moiety · CPC title