Method for attachment of silylated molecules to glass surfaces

• US 7,297,553 B2
• Filed: 05/28/2003
• Issued: 11/20/2007
• Est. Priority Date: 05/28/2002
• Status: Active Grant

• Current Assignee: Nanosphere Incorporated (DiaSorin S.p.A.)
• Original Assignee: Nanosphere Incorporated (DiaSorin S.p.A.)
• Inventors: Garimella, Viswanadham, Bernal, Yasmith
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US10/447,073
• Publication Number:
  US 20040096856A1
• Time in Patent Office: 1,637 Days
• Field of Search: 436/518, 422/61, 422/68, 528/10, 528/25, 528/28, 528/33, 514/63, 552/4, 435/6
• US Class Current: 436/518
• CPC Class Codes: C03C 17/30 with silicon-containing compounds
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: G01N 33/552 Glass or silica
• CPC Class Codes: Y10T 436/17 Nitrogen containing
• CPC Class Codes: Y10T 436/172307 Cyanide or isocyanide
• CPC Class Codes: Y10T 436/20 Oxygen containing

Abstract

A method for the efficient immobilization of silylated molecules such as silylated oligonucleotides or proteins onto unmodified surfaces such as a glass surface is provided. Also provided are compounds, devices, and kits for modifying surfaces such as glass surfaces.

Claims

1. A method for immobilizing a molecule onto a surface, said method comprising the steps of:

(a) contacting Si(NCY)₄ with an agent having a formula ii;

(R₁)(R₂)(R₃)Si—

X-Z 

 

ii

so as to form a first reactive intermediate, wherein Y represents oxygen or sulphur, R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy;

X represents linear or branched C₁-C₂₀ alkyl or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or more heteroatoms comprising oxygen, nitrogen, or sulfur; and

Z represents a hydroxy or amino group, with the proviso that at least one of R₁, R₂ or R₃ represents C₁-C₆ alkoxy;

(b) contacting the first reactive intermediate with a molecule so as to form a second reactive intermediate;

(c) contacting the second reactive Intermediate with said surface so as to immobilized the molecule onto said surface.

2. The method of claim 1 wherein the surface is a glass surface.

3. The method of claim 1 wherein the surface has at least one group that reacts with the reactive intermediate.

4. The method of claim 3 wherein the group comprises a hydroxyl or amino group.

5. The method of claim 1 wherein the agent is 4-aminophenyl trimethoxysilane, 4-aminophenyl tilethoxysilane, 3-aminophenyltrimethoxysilane, 3-amenophenyltriethoxysilane, or mixtures thereof.

6. The method of claim 5 wherein the agent and tetraisocyanatosilane is present at a 4:

1 molar ratio.

7. The method of claim 1 wherein the molecule and the first reactive intermediate is present in a 3:

1 molar ratio.

8. The method of claim 1 wherein Si(NCY)₄ and the agent are present at a 1:

1 molar ratio and the first reactive Intermediate has a formula iv;

(R₁)(R₂)(R₃)Si—

X-Z-CYNH—

Si(NCY)₃ 

 

iv

wherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy;

X represents linear or branched C₁-C₂₀ alkyl or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or more heteroatoms comprising oxygen, nitrogen, or sulfur;

Y represents oxygen or sulfur; and

Z represents oxygen or NH, with the proviso that at least one of R₁, R₂, or R₃ represents C₁-C₆ alkoxy.

9. The method of claim 8 wherein R₁, R₂ and R₃ represents methoxy, X represents phenyl, Y represents oxygen, and Z represents NH.

10. The method of claim 8 wherein the second reactive intermediate has a formula v:

(R₁)(R₂)(R₃)Si—

X-Z-CYNH—

Si(NHCYL-M)₃ 

 

v

wherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy;

L represents a linking group;

X represents linear or branched C₁-C₂₀ alkyl or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or more heteroatoms comprising oxygen, nitrogen, or sulfur;

Y represents oxygen or sulfur; and

Z represents oxygen or NH; and

M represents a molecule, with the proviso that at least one of R₁, R₂, or R₃ represents C₁-C₆ alkoxy.

11. The method of claim 10 wherein L represents a nucleophilic group from the molecule.

12. The method of claim 11 wherein the nucleophilic group comprises —

NH, —

S—

, —

O—

, or —

OOC—

.

13. The method of claim 11 wherein Si(NCY)₄ and the agent are present at a 1:

2 molar ratio and the first reactive intermediate has a formula vi;

[(R₁)(R₂)(R₃)Si—

X-Z-CYNH]₂—

Si(NCY)₂ 

 

vi

wherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy;

X represents linear or branched C₁-C₂₀ alkyl or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or more heteroatoms comprising oxygen, nitrogen, or sulfur, Y represents oxygen or sulfur; and

Z represents oxygen or NH, with the proviso that at least one of R₁, R₂, or R₃ represents C₁-C₆ alkoxy.

14. The method of claim 13 wherein R₁, R₂ and R₃ represents methoxy, X represents phenyl, Y represents oxygen, and Z represents NH.

15. The method of claim 13 wherein the second reactive intermediate has a formula vii:

[(R₁)(R₂)(R₃)Si—

X-Z-CYNH]₂Si[NHCYL-M]₂ 

 

vii

wherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy;

L represents a linking group;

X represents linear or branched C₁-C₂₀ alkyl or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or more heteroatoms comprising oxygen, nitrogen, or sulfur;

Y represents oxygen or sulfur; and

Z represents oxygen or NH; and

M represents a molecule, with the proviso that at least one of R₁, R₂, or R₃ represent C₁-C₆ alkoxy.

16. The method of claim 15 wherein L represents a nucleophilic group from the molecule.

17. The method of claim 16 wherein the nucleophilic group comprises —

NH, —

S—

, —

O—

, or —

OOC—

.

18. The method of claim 1 wherein the molecule comprises a probe.

19. The method of claim 18, wherein the probe comprises a protein, a peptide, a nucleic acid, a peptide nucleic acid, an amino acid, a linked nucleic acid, a nucleoside triphosphate, a carbohydrate, a lipid, a lipid bound protein, an aptamer, a virus, a cell fragment, or a whole cell.

20. The method of claim 19, wherein the lipid bound protein comprises a G-protein coupled receptor.

21. The method of claim 18, wherein the probe comprises an antibody, an antigen, a receptor, or a ligand.

22. A method for immobilizing a molecule onto a glass surface, said method comprising the steps of:

(a) contacting Si(NCY)₄ with an agent having a formula ii;

(R₁)(R₂)(R₃)Si—

X-Z 

 

ii

so as to form a first reactive intermediate, wherein Y represents oxygen or sulfur;

R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy;

X represents linear or branched C₁-C₂₀ alkyl or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or more heteroatoms comprising oxygen, nitrogen, and sulfur; and

Z represents a hydroxy or amino group, with the provision that at least of one R₁, R₂, or R₃ represents C₁-C₆ alkoxy;

(b) contacting the first reactive intermediate with a molecule so as to form a second reactive intermediate;

(c) contacting the second reactive intermediate with said surface so as to immobilized the molecule onto said surface.

23. The method of claim 22 wherein the surface has at least one group that reacts with the reactive intermediate.

24. The method of claim 23 wherein the group comprises a hydroxyl or amino group.

25. The method of claim 22 wherein the agent is 4-aminophenyl trimethoxysilane, 4-aminophenyl trimethoxysilane, 3-aminophenyltriethoxysilane, 3-aminophenyltriethoxysilane, or mixture thereof.

26. The method of claim 22 wherein the agent and tetraisocyanatosilane is present at a 1:

1 molar ratio.

27. The method of claim 22 wherein the molecule and the first reactive intermediate is present in a 3:

1 molar ratio.

28. The method of claim 22 wherein Si(NCY)₄ and the agent are present at a 1:

1 molar ratio and the first reactive intermediate has a formula iv;

(R₁)(R₂)(R₃)Si—

X-Z-CYNH—

Si(NCY)₃ 

 

iv

wherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy;

X represents linear or branched C₁-C₂₀ alkyl or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or more heteroatoms comprising oxygen, nitrogen, or sulfur;

Y represents oxygen or sulfur; and

Z represents oxygen or NH, with the proviso that at least one of R₁, R₂, or R₃ represents C₁-C₆ alkoxy.

29. The method of claim 28 wherein R₁, R₂ and R₃ represents methoxy, X represents phenyl, Y represents oxygen, and Z represents NH.

30. The method of claim 28 wherein the second reactive intermediate has a formula v:

(R₁)(R₂)(R₃)Si—

X-Z-CYNH—

Si(NHCYL-M)₃ 

 

v

wherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy;

L represents a linking group;

X represents linear or branched C₁-C₂₀ alkyl or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or more heteroatoms comprising oxygen, nitrogen, or sulfur;

Y represents oxygen or sulphur, and Z represents oxygen or NH; and

M represents a molecule, with the proviso that at least one of R₁, R₂, or R₃ represents C₁-C₆ alkoxy.

31. The method of claim 30 wherein L represents a nucleophilic group from the molecule.

32. The method of claim 31 wherein the nucleophilic group comprises —

NH, —

S—

, —

O—

, or —

OCC—

.

33. The method of claim 22 wherein Si(NCY)₄ and the agent are present at a 1:

2 molar ratio and the first reactive intermediate has a formula vi;

[(R₁)(R₂)(R₃)Si—

X-Z-CYNH]₂—

SI(NCY)₂ 

 

vi

wherein R₁, R₂ and R₃ Independently represents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy;

X represents linear or branched C₁-C₂₀ alkyl or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or more heteroatoms comprising oxygen, nitrogen, or sulfur;

Y represents oxygen or sulfur; and

Z represents oxygen or NH, with the proviso that at least one of R₁, R₂, or R₃ represents C₁-C₆ alkoxy.

34. The method of claim 33 wherein R₁, R₂ and R₃ represents methoxy, X represents phenyl, Y represents oxygen, and Z represents NH.

35. The method of claim 33 wherein the second reactive intermediate has a formula vii:

[(R₁)(R₂)(R₃)Si—

X-Z-CYNH]₂Si(NHCYL-M)₂ 

 

vii

wherein R₁, R₂ and R₃ independently represents C₁-C₆ alkoxy, C₁-C₆ alkyl, phenyl, or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy;

L represents a linking group;

X represents linear or branched C₁-C₂₀ alkyl or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or more heteroatoms comprising oxygen, nitrogen, or sulfur;

Y represents oxygen or sulfur and Z represents oxygen or NH; and

M represents a molecule, with the proviso that at least one of R₁, R₂, or R₃ represents C₁-C₆ alkoxy.

36. The method of claim 35 wherein L represents a nucleophilic group from the molecule.

37. The method of claim 36 wherein the nucleophilic group comprises —

NH, —

S—

, —

O—

, or —

OOC—

.

38. The method of claim 22 wherein the molecule comprises a probe.

39. The method of claim 38, wherein the probe comprises a protein, a peptide, a nucleic acid, a peptide nucleic acid, an amino acid, a linked nucleic acid, a nucleoside triphosphate, a carbohydrate, a lipid, a lipid bound protein, an aptamer, a virus, a cell fragment, or a whole cell.

40. The method of claim 39, wherein the lipid bound protein comprises a G-protein coupled receptor.

41. The method of claim 22, wherein the probe comprises an antibody, an antigen, a receptor, or a ligand.