Methods of immobilizing ligands on solid supports and apparatus and methods of use therefor

• US 20030143570A1
• Filed: 08/01/2002
• Published: 07/31/2003
• Est. Priority Date: 08/27/1999
• Status: Abandoned Application

• Current Assignee: Matrix Technologies Corporation
• Original Assignee: Matrix Technologies Corporation
• Inventors: Abrams, Ezra S., Zhang, Tianhong, Patterson, Brian C., Mielewczyk, Slawomir
• Application Number:
  US10/210,400
• Publication Number:
  US 20030143570A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393

Abstract

A method is provided for immobilizing a ligand, e.g., a nucleic acid, on a solid support. The method includes providing a solid support containing an immobilized latent thiol group, activating the thiol group, contacting the activated thiol group with a nucleic acid comprising an acrylamide functional group, and forming a covalent bond between the two groups, thereby immobilizing the nucleic acid to the solid support. Kits containing the solid supports and method of utilizing the solid supports are also provided.

Claims

1. A method of immobilizing an affinity ligand on a solid support comprising:

providing a solid support comprising an immobilized thiol group;

contacting the thiol group with a nucleic acid comprising an acrylamide functional group; and

forming a covalent bond between the two groups,

thereby immobilizing the ligand on the solid support.

2. The method of claim 1, wherein the ligand is selected from the group consisting of a nucleic acid, a modified nucleic acid and a nucleic acid analog.

3. The method of claim 2, wherein the solid support comprises a plurality of thiol groups.

4. The method of claim 3, wherein a plurality of ligands are immobilized on the solid support.

5. The method of claim 4, wherein the solid support is formed from a compound selected from the group consisting of glass, plastic and metal.

6. The method of claim 5, wherein the solid support comprises two or more spatially distinct regions, each region comprising a plurality of immobilized nucleic acids.

7. The method of claim 6, wherein the solid support further comprises a polymer layer.

8. The method of claim 7, wherein the solid support comprises a microarray.

9. The method of claim 1, wherein the thiol groups comprise reduced disulfide groups.

10. A method of immobilizing an affinity ligand on a solid support comprising the steps of:

providing a solid support comprising immobilized latent thiol groups;

activating the latent thiol groups; and

reacting the activated thiol groups with an affinity ligand having at least one acrylamide functional group, thereby immobilizing an affinity ligand on a solid support.

11. The method of claim 10, wherein the ligand is selected from the group consisting of a nucleic acid, a modified nucleic acid and a nucleic acid analog.

12. The method of claim 11, wherein the steps of activating the latent thiol groups and reacting the activated thiol groups occur essentially simultaneously.

13. The method of claim 12, wherein the solid support is formed from a compound selected from the group consisting of glass, plastic and metal.

14. The method of claim 13, wherein the solid support comprises two or more spatially distinct regions, each region comprising a plurality of immobilized nucleic acids.

15. The method of claim 14, wherein the solid support further comprises a polymer layer.

16. The method of claim 15, wherein the solid support comprises a microarray.

17. The product formed by the method of claim 10.

18. The method of claim 10, further comprising:

contacting a solid support with a silane compound represented by the formula R_nSiX<sub>(4−

n)</sub> wherein X is selected from the group consisting of a halogen, an alkoxide, an alkane, an acyloxy, and an amine, and R is a compound having at least one substituent selected from the group consisting of an α

,β

-unsaturated carbonyl and a vinyl to form a solid support having an unsaturated aliphatic surface; and

contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing a free radical initiator, disulfide bisacrylamide, and optionally an acrylamide, wherein the disulfide bisacrylamide has the formula

wherein n and m are each, independently, a positive integer, thereby forming a solid supporting comprising immobilized latent thiol groups.

19. The method of claim 18, wherein the latent thiol groups are activated by contacting the solid support with a disulfide reducing agent.

20. The method of claim 18, wherein the polymerization solution further includes alkylene bisacrylamide.

21. The method of claim 18, wherein the free radical initiator is added to the polymerization solution after the solution is in contact with the unsaturated aliphatic surface of the solid support.

22. The method of claim 18, further comprising the step of derivatizing a solid support with a latent thiol group, thereby forming a solid support having immobilized latent thiol groups.

23. The method of claim 18, wherein the solid support has an amine functional group and the solid support is derivatized by contacting the solid support with a compound represented by the following structural formula:

24. The method of claim 23 wherein Y consists of a group selected from the following groups:

32. The method of claim 10, wherein the solid support is doped or undoped silica, alumina, quartz or glass, and wherein the method further comprises the steps of:

contacting the solid support with a compound comprising a silane group or a carboxylic acid and a substituted or unsubstituted alkenyl group or a group having at least one α

,β

-unsaturated carbonyl, thereby forming a solid support having an unsaturated aliphatic surface; and

contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing a free radical initiator, a disulfide bisacrylamide and optionally containing an acrylamide, wherein the disulfide bisacrylamide is represented by the following structural formula;

 

wherein;

n and m are each, independently, a positive integer, thereby

forming a solid support comprising immobilized latent thiol groups.

33. The method of claim 32 wherein the compound is represented by the formula R_nSiX₍₄₋

n) wherein X is selected from the group consisting of a halogen, an alkoxide, an alkane, an acyloxy, and an amine, and R is a compound having at least one substituent selected from the group consisting of an α

,β

-unsaturated carbonyl and a vinyl.

34. The method of claim 32, wherein the latent thiol groups are activated by contacting the solid support with a disulfide reducing agent.

35. The method of claim 32, wherein the polymerization solution further includes alkylene bisacrylamide.

36. The method of claim 32, wherein the free radical initiator is added to the polymerization solution after the solution is in contact with the unsaturated aliphatic surface of the solid support.

37. The method of claim 10, wherein the solid support is selected from the group consisting of gold, silver, copper, cadmium, zinc, palladium, platinum, mercury, lead, iron, chromium, manganese, tungsten, and alloys thereof, and wherein the method further comprises the steps of:

contacting the solid support with a compound comprising a thiol group, sulfide or disulfide group and a substituted or unsubstituted alkenyl group or a group having at least one (α

,β

-unsaturated carbonyl, thereby forming a solid support having an unsaturated aliphatic surface; and

contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing free radical initiator, a disulfide bisacrylamide and optionally containing a comonomer, wherein the disulfide bisacrylamide is represented by the following structural formula;

 

wherein;

n and m are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups.

38. The method of claim 10, wherein the solid support is selected from the group consisting of platinum or palladium, and wherein the method further comprises the steps of:

contacting the solid support with a compound comprising a nitrile or isonitrile group and a substituted or unsubstituted alkenyl group or a group having at least one α

,β

-unsaturated carbonyl, thereby forming a solid support having an unsaturated aliphatic surface; and

contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing a free radical initiator, a disulfide bisacrylamide and optionally containing an acrylamide, wherein the disulfide bisacrylamide is represented by the following structural formula;

 

wherein;

n and m in are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups.

39. The method of claim 10, wherein the solid support is copper, and wherein the method further comprises the steps of:

contacting the solid support with a compound comprising a hydroxamic acid group and a substituted or unsubstituted alkenyl group or a group having at least one α

,β

-unsaturated carbonyl, thereby forming a solid support having an unsaturated aliphatic surface; and

contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing free radical initiator and disulfide bisacrylamide and optionally containing an acrylamide, wherein the disulfide bisacrylamide is represented by the following structural formula;

wherein n and m are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups.

40. The method of claim 10, wherein the solid support is a polymer comprising a reactive functional group, and wherein the method further comprises the steps of:

contacting the solid support with a compound comprising a functional group which can react to form a bond with the reactive functional group and a substituted or unsubstituted alkenyl group or a group having at least one α

,β

-unsaturated carbonyl, thereby forming a solid support having immobilized unsaturated aliphatic groups; and

contacting the unsaturated aliphatic groups of the solid support with a polymerization solution containing a free radical initiator, a disulfide bisacrylamide and optionally containing an acrylamide, wherein the disulfide bisacrylamide is represented by the following structural formula;

 

wherein;

n and m are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups.

41. The method of claim 40, wherein the polymeric solid support is polystyrene.

42. The method of claim 40, wherein the reactive functional group of the polymeric solid support is an amine group or a hydroxyl group and the compound is represented by the following structural formula:

43. The compound of claim 42, wherein Y is selected from the group consisting of:

44. The method of claim 10, further comprising the step of derivatizing a solid support with a latent thiol group, thereby forming a solid support having immobilized latent thiol groups.

45. The method of claim 44, wherein the solid support is selected from the group consisting of doped or undoped silica, alumina, quartz or glass, and the solid support is derivatized by contacting it with a compound comprising a silane group or a carboxylic acid group and a latent thiol group.

46. The method of claim 44, wherein the solid support is selected from the group consisting of platinum or palladium, and the solid support is derivatized by contacting it with a compound comprising a nitrile or isonitrile group and a latent thiol group.

47. The method of claim 44, wherein the solid support is a polymer comprising reactive functional groups, and the solid support is derivatized by contacting it with a compound comprising a functional group which can react to form a bond with the reactive functional group and a latent thiol group.

48. The method of claim 44, wherein the polymeric solid support is polystyrene.

49. The method of claim 47, wherein the reactive functional group of the polymeric solid support is an amine or a hydroxyl group and the solid support is derivatized by contacting the solid support with a compound represented by the following structural formula:

50. The method of claim 49, wherein Y is selected from the group consisting of:

51. The method of claim 49, wherein R₄ is selected from the group consisting of:

26. A method of forming an array of nucleic acids immobilized on a solid support comprising:

forming an amine-derivatized region on the support;

treating the amine-derivatized region with a thiolating agent such that latent thiol groups immobilized on the support are formed;

activating the latent thiol groups;

contacting the activated thiol groups with a plurality of nucleic acids comprising acrylamide functional groups; and

forming a covalent bond between the two groups, thereby forming an array of nucleic acids immobilized on the solid support.

25. The method of claim 26, wherein R₄ is selected from the group consisting of:

27. The method of claim 26, wherein each nucleic acid comprises a nucleotide sequence substantially identical to the nucleotide sequence of the other nucleic acids of the array.

28. The method of claim 26, wherein nucleic acids with a plurality of nucleotide sequences are contained in the array.

29. The method of claim 26 comprising a plurality of amine-derivatized regions.

30. The method of claim 26 further comprising a step of blocking unbonded reactive thiol groups remaining following the binding of the nucleic acids to the thiol groups.

31. The microarray prepared by the method of claim 26.

52. A method of preparing a solid support having immobilized thiol groups comprising contacting a solid support with a silane compound represented by the formula

R_nSiX₍₄₋

n)

wherein X is selected from the group consisting of a halogen, an alkoxide, an alkane, an acyloxy, and an amine, and R is a compound having at least one substituent selected from the group consisting of an a,P-unsaturated carbonyl and a vinyl.

53. The method of claim 52 wherein the halogen is selected from the group consisting of Cl and Br.

54. The method of claim 52 wherein the alkoxide is selected from the group consisting of —

O—

CH₃ and —

O—

CH₂—

CH₃.

55. The method of claim 52 wherein the alkane is selected from the group consisting of —

CH₃ and —

CH₂—

CH₃.

56. The method of claim 52 wherein the acyloxy is —

O—

CO—

CH₃.

57. The method of claim 52 wherein the amine is selected from the group consisting of —

N(CH₃)₂ and —

N(CH₂—

CH₃)₂.

58. The method of claim 52 wherein R is selected from the group consisting of —

CH₂—

NH—

CO—

C(CH₃)═

CH₂, —

CH₂—

O—

CO—

CH═

CH₂, and —

CH₂—

CH═

CH₂.