CATALYST-FREE SURFACE FUNCTIONALIZATION AND POLYMER GRAFTING

• US 20150005447A1
• Filed: 06/26/2014
• Published: 01/01/2015
• Est. Priority Date: 07/01/2013
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Berti, Lorenzo, Brown, Andrew A., George, Wayne N.
• Granted Patent: US 9,994,687 B2
• Time in Patent Office: Days
• US Class Current: 525/54.2
• 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 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00533 essentially rectangular
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C08F 220/603 and containing oxygen in addition to the carbonamido oxygen and nitrogen
• CPC Class Codes: C08F 8/00 Chemical modification by after-treatment graft polymers, block polymers, crosslinking with unsaturated monomers or with polymers C08F251/00 - C08F299/00; of conjugated diene rubbers C08C
• CPC Class Codes: C08J 2335/00 Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical, and containing at least one other carboxyl radical in the molecule, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Derivatives of such polymers
• CPC Class Codes: C08J 7/12 Chemical modification
• CPC Class Codes: C09D 133/26 Homopolymers or copolymers of acrylamide or methacrylamide
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

Some embodiments described herein relate to a substrate with a surface comprising a silane or a silane derivative covalently attached to optionally substituted cycloalkene or optionally substituted heterocycloalkene for direct conjugation with a functionalized molecule of interest, such as a polymer, a hydrogel, an amino acid, a nucleoside, a nucleotide, a peptide, a polynucleotide, or a protein. In some embodiments, the silane or silane derivative contains optionally substituted norbornene or norbornene derivatives. Method for preparing a functionalized surface and the use in DNA sequencing and other diagnostic applications are also disclosed.

Claims

1. A substrate comprising a first surface comprising silane or a silane derivative covalently bound to a functionalized molecule through a first plurality of unsaturated moieties selected from cycloalkenes, cycloalkynes, heterocycloalkenes, heterocycloalkynes, or optionally substituted variants or combinations thereof covalently attached to silicon atoms of said silane or silane derivative.

2. The substrate of claim 1, wherein said first plurality of unsaturated moieties are selected from norbornene, heteronorbornenes, norbornene derivatives, trans-cyclooctene, trans-cyclooctene derivatives, cyclooctyne, bicycloalkynes, or optionally substituted variants or combinations thereof.

3. The substrate of claim 1, wherein said first plurality of unsaturated moieties are selected from optionally substituted norbornene, optionally substituted cyclooctyne, optionally substituted bicyclononyne, or optionally substituted bicyclo[6.1.0]non-4-yne.

4. The substrate of claim 1, further comprising linkers covalently attached between silicon atoms of said silane or silane derivative and the first plurality of unsaturated moieties.

5. The substrate of claim 4, wherein the linkers are selected from optionally substituted alkylenes, optionally substituted heteroalkylenes, optionally substituted cycloalkylenes, optionally substituted heterocyclylenes, optionally substituted arylenes, optionally substituted heteroarylenes, optionally substituted polyethylene glycols, cleavable linkers, or combinations thereof.

6. The substrate of claim 5, wherein the linkers are selected from optionally substituted alkylenes or optionally substituted heteroalkylenes.

7. The substrate of claim 1, wherein the functionalized molecule comprises a polymer, a hydrogel, an amino acid, a peptide, a nucleoside, a nucleotide, a polynucleotide, a protein, or combinations thereof.

8. The substrate of claim 7, wherein said functionalized molecule comprises functional groups selected from optionally substituted alkenyl, azido, optionally substituted amino, carboxyl, optionally substituted hydrazone, optionally substituted hydrazine, hydroxy, optionally substituted tetrazole, optionally substituted tetrazine, nitrile oxide, nitrone, or thiol, provided that the functionalized molecule is not norbornene or polymerized norbornene.

9. The substrate of claim 1, wherein the functionalized molecule comprises a recurring unit of Formula (I) and a recurring unit of Formula (II):

10. The substrate of claim 1, wherein the functionalized molecule comprises a polymer of Formula (III) or (III′

);

11. The substrate of claim 10, wherein p is 5 and R^A is azido.

12. The substrate claim 1, further comprising oligonucleotides covalently attached to the functionalized molecule through a second plurality of unsaturated moieties selected from cycloalkenes, cycloalkynes, heterocycloalkenes, heterocycloalkynes, or optionally substituted variants or combinations thereof.

13. The substrate of claim 12, wherein said second plurality of unsaturated moieties are selected from optionally substituted cyclooctyne, optionally substituted cyclooctyne, optionally substituted bicyclononyne, or optionally substituted bicyclo[6.1.0]non-4-yne.

14. The substrate of claim 1, wherein the substrate is selected from glass substrate, silica substrate, plastic substrate, quartz substrate, metal substrate, metal oxide substrate, or combinations thereof.

15. The substrate of claim 1, wherein the first surface comprises both functionalized molecules coated regions and inert regions.

16. A method of preparing a first surface of a substrate of claim 1, said method comprising:

applying silane or a silane derivative comprising a first plurality of unsaturated moieties selected from cycloalkenes, cycloalkynes, heterocycloalkenes, heterocycloalkynes, or optionally substituted variants or combinations thereof covalently attached thereto onto said first surface of the substrate; and

covalently attaching a functionalized molecule comprising functional groups to said silane or silane derivative by reacting the functional groups of said functionalized molecule with said first plurality of unsaturated moieties to form a coating layer.

17. The method of claim 16, further comprising

providing oligonucleotides comprising a second plurality of unsaturated moieties selected from cycloalkenes, cycloalkynes, heterocycloalkenes, heterocycloalkynes, or optionally substituted variants or combinations thereof;

reacting said second plurality of unsaturated moieties of said oligonucleotides with said functional groups of the functionalized molecule to form covalent bonding.

18. The method of claim 16, wherein said first and second plurality of unsaturated moieties are independently selected from norbornene, heteronorbornenes, norbornene derivatives, trans-cyclooctene, trans-cyclooctene derivatives, cyclooctyne, bicycloalkynes, or optionally substituted variants or combinations thereof.

19. The method of claim 16, wherein said first plurality of unsaturated moieties of said silane or silane derivative are optionally substituted norbornene.

20. The method of claim 17, wherein the second plurality of unsaturated moieties of said oligonucleotides are selected from optionally substituted cyclooctyne, optionally substituted bicyclononyne, or optionally substituted bicyclo[6.1.0]non-4-yne.

21. The method of claim 16, wherein said silane or silane derivative further comprise linkers covalently attached between silicon atoms and said first plurality of unsaturated moieties.

22. The method of claim 21, wherein the linkers are selected from optionally substituted alkylenes, optionally substituted heteroalkylenes, optionally substituted cycloalkylenes, optionally substituted heterocyclylenes, optionally substituted arylenes, optionally substituted heteroarylenes, optionally substituted polyethylene glycols, cleavable linkers, or combinations thereof.

23. The method of claim 21, wherein the linkers are selected from optionally substituted alkylenes or optionally substituted heteroalkylenes.

24. The method of claim 16, wherein said silane or silane derivative are applied onto the first surface by chemical vapor deposition or Yield Engineering Systems (YES) method.

25. The method of claim 16, wherein the functionalized molecule comprises a recurring unit of Formula (I) and a recurring unit of Formula (II):

26. The method of claim 16, wherein the functionalized molecule comprises a polymer comprising Formula (III) or (III′

);

27. The method of claim 26, wherein p is 5 and R^A is azido.

28. The method of claim 16, further comprising a washing step to remove excess unbounded functionalized molecules.

29. The method of claim 16, further comprising a drying step.

30. A method of grafting primers on a first surface of a substrate, said method comprising:

providing a substrate having a first surface comprising silane or a silane derivative, wherein the silane or silane derivative comprises a first plurality of unsaturated moieties selected from cycloalkenes, cycloalkynes, heterocycloalkenes, heterocycloalkynes, or optionally substituted variants or combination thereof covalently attached thereto onto said first surface of the substrate;

providing pre-conjugated primers comprising oligonucleotides covalently bonded to a functionalized molecule, wherein said functionalized molecule comprises functional groups; and

contacting said pre-conjugated primers with said silane or silane derivative such that said pre-conjugated primers are covalently attached to said first surface of the substrate by reacting the functional groups of said functionalized molecule with said first plurality of unsaturated moieties of the silane or silane derivative to form covalent bonding.

31. The method of claim 30, wherein the pre-conjugated primers are prepared by reacting the functional groups of said functionalized molecule with a second plurality of unsaturated moieties of said oligonucleotides to form covalent bonds, wherein said second plurality of unsaturated moieties of said oligonucleotide are selected from cycloalkenes, cycloalkynes, heterocycloalkenes, heterocycloalkynes, or optionally substituted variants or combinations thereof.

32. The method of claim 30, wherein the first plurality of unsaturated moieties of said silane or silane derivative are selected from norbornene, heteronorbornenes, norbornene derivatives, trans-cyclooctene, trans-cyclooctene derivatives, cyclooctyne, bicycloalkynes, or optionally substituted variants or combinations thereof.

33. The method of claim 30, wherein the first plurality of unsaturated moieties of said silane or silane derivative are optionally substituted norbornene.

34. The method of claim 31, wherein said second plurality of unsaturated moieties of said oligonucleotides are selected from optionally substituted cyclooctyne, optionally substituted bicyclononynes, or optionally substituted bicyclo[6.1.0]non-4-yne.

35. The method of claim 30, wherein said silane or silane derivative are applied onto the first surface by chemical vapor deposition or Yield Engineering Systems (YES) method.

37. The method of claim 30, wherein the functionalized molecule comprises a recurring unit of Formula (I) and a recurring unit of Formula (II):

38. The method of claim 30, wherein the functionalized molecule comprises a polymer comprising Formula (III) or (III′

);

39. The method of claim 38, wherein p is 5 and R^A is azido.

40. The method of claim 30, further comprising a washing step to remove excess unbounded functionalized molecules.

41. The method of claim 30, further comprising a washing step to remove excess unbounded oligonucleotides.

42. The method of claim 30, further comprising a drying step.

36. The method of 30, wherein the functional groups of the functionalized molecule comprise azido, optionally substituted amino, optionally substituted alkenyl, optionally substituted hydrazone, optionally substituted hydrazine, carboxyl, hydroxy, optionally substituted tetrazole, optionally substituted tetrazine, nitrile oxide, nitrone, or thiol, or combinations thereof.