Method for immobilizing molecules onto surfaces

• US 20030082588A1
• Filed: 07/12/2002
• Published: 05/01/2003
• Est. Priority Date: 07/13/2001
• Status: Active Grant

• Current Assignee: Nanosphere Incorporated (DiaSorin S.p.A.)
• Original Assignee: Nanosphere Incorporated (DiaSorin S.p.A.)
• Inventors: Garimella, Viswanadham
• Granted Patent: US 7,687,437 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393

Abstract

A method for immobilizing amino-group containing molecules onto surfaces and devices having immobilized isocyanate-group containing molecules prepared by the method are disclosed. The method comprises reacting a surface (i.e., glass surface) having free hydroxyl groups with a silyl isocyanate derivatizing agent to provide immobilized reactive moieties, the agent having a formula:

(R₁O)(R₂O)(R₃O)Si—X—NCO

wherein R₁, R₂ and R₃ are independently represents 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 reacting the immobilized reactive moieties with the amino group-containing molecule so as to immobilize said molecule on the surface. Devices having a surface with immobilized molecules such as nucleic acids or proteins are useful for detection of target analytes in a sample.

Claims

1. A method for immobilizing an amino-group containing molecule on a surface having free hydroxyl groups comprising the steps of:

(a) reacting the free hydroxyl groups of said surface with a silyl isocyanate derivatizing agent to provide immobilized reactive moieties, the agent having a formula;

(R₁O)(R₂O)(R₃O)Si—

X—

NCO

wherein R₁, R₂ and R₃ independently represents 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

(b) reacting the immobilized reactive moieties with the amino-group containing molecule so as to immobilize said molecule on the surface.

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

3. The method of claim 1 wherein the agent is 3-(isocyanatopropyl) triethoxysilane.

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

5. The method of claim 4, 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.

6. The method of claim 5, wherein the lipid bound protein comprises a G-protein coupled receptor.

7. The method of claim 4, wherein the probe comprises an antibody, an antigen, a receptor, or a ligand.

8. The method of claim 1 wherein the molecule further includes one or more functional groups.

9. The method of claim 8 wherein the functional groups comprise amino, hydroxyl, carboxyl, aldehyde, vinyl, allyl or epoxide.

10. The method according to claim 1 wherein the isocyanate is present in solution at a concentration ranging between about 20 mM and about 200 mM.

11. The method of claim 8 further comprising the steps of

(c) reacting the immobilized molecule on the surface with a chemical agent that reacts with the functional group of the molecule so as to produce second immobilized reactive moieties;

and

(d) reacting the second immobilized reactive moieties with a second molecule having a functional group capable of reacting with the second immobilized reactive moieties.

12. The method of claim 11, wherein the second molecule is a probe.

13. The method of claim 12, 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.

14. The method of claim 13, wherein the lipid bound protein comprises a G-protein coupled receptor.

15. The method of claim 12, wherein the probe comprises an antibody, an antigen, a receptor, or a ligand.

16. The method of claim 12, wherein the probe has been derivatized to contain one or more amino groups.

17. A device comprising a surface with an immobilized molecule, wherein said surface is prepared by the method of claims 1 or 10.

18. The device of claim 17 wherein the surface is a glass surface.

19. The device of claim 17, wherein the molecule is a probe.

35. The method of claim 1 further comprising the step of:

(c) coupling the molecule immobilized on the surface with a second molecule, wherein the molecule immobilized on the surface and the second molecules have at least one functional group for coupling.

36. The method of claim 35 wherein the molecule immobilized on the surface has at least one carboxyl group.

37. The method of claim 36 wherein the second molecule has at least one amino group.

38. The method of claim 37 wherein the coupling occurs in the present of a coupling agent comprising EDC or EDAC.

39. The method of claim 35, wherein the second molecule is a probe.

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

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

42. The method of claim 39, wherein the probe comprises an antibody, an antigen, a receptor, or a ligand.

43. A device comprising a glass surface with an immobilized molecule, wherein said surface is prepared by the method of claim 35.

44. The device of claim 35, wherein the molecule is a probe.

20. A method for immobilizing a probe on a glass surface comprising the steps of:

(a) reacting the glass surface with (3-isocyanatopropyl) triethoxysilane to provide immobilized reactive moieties; and

(b) reacting the immobilized reactive moieties with a molecule having an amine so as to immobilize said molecule on the glass surface.

21. The method of claim 20 wherein the molecule is a probe.

22. The method of claim 21, wherein the probe comprises a protein, a peptide, a nucleic acid, a peptide nucleic acid, a linked nucleic acid, an amino acid, a nucleoside triphosphate, a carbohydrate, a lipid, a lipid bound protein, an aptamer, a virus, a cell fragment, or a whole cell.

23. The method of claim 22, wherein the lipid bound protein comprises a G-protein coupled receptor.

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

25. The method of claim 21, wherein the probe has been derivatized to contain one or more amino and/or hydroxyl groups.

26. The method according to claim 20 wherein the isocyanate is present in solution at a concentration ranging between about 20 mM and about 200 mM.

27. The method of claim 20 further comprising the steps of:

(c) reacting the immobilized molecule on the surface with a chemical agent that reacts with the functional group of the molecule so as to produce second immobilized reactive moieties; and

(d) reacting the second immobilized reactive moieties with a second molecule having a functional group capable of reacting with the second immobilized reactive moieties.

28. The method of claim 27, wherein the second molecule is a probe.

29. The method of claim 28, wherein the probe comprises a protein, a peptide, a nucleic acid, a peptide nucleic acid, a linked nucleic acid, an amino acid, a nucleoside triphosphate, a carbohydrate, a lipid, a lipid bound protein, an aptamer, a virus, a cell fragment, or a whole cell.

30. The method of claim 29, wherein the lipid bound protein comprises a G-protein coupled receptor.

31. The method of claim 28, wherein the probe comprises an antibody, an antigen, a receptor, or a ligand.

32. The method of claim 28, wherein the probe has been derivatized to contain one or more amino groups.

33. A device comprising a glass surface with an immobilized molecule, wherein said surface is prepared by the method of claim 27.

34. The device of claim 33, wherein the molecule is a probe.