Porous silica substrates for polymer synthesis and assays

• US 6,824,866 B1
• Filed: 04/07/2000
• Issued: 11/30/2004
• Est. Priority Date: 04/08/1999
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Frank, Curtis W., Fidanza, Jacqueline A., Vinci, Richard, Glazer, Marc I., McGall, Glenn
• Primary Examiner(s): Morris, Terrel
• Assistant Examiner(s): Vo, Hai
• Application Number:
  US09/545,207
• Time in Patent Office: 1,698 Days
• Field of Search: 428/304.4, 428/317.9, 428/315.5, 428/315.7, 428/325, 428/312.2, 428/312.6, 428/312.8, 528/10, 536/23.1, 536/24.3, 536/25.3
• US Class Current: 428/317.9
• 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 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential 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 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B01J 2219/00731 Saccharides
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 40/12 : Libraries containing saccharides or polysaccharides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: Y10T 428/249953 : Composite having voids in a component [e.g., porous, cellular, etc.]
• CPC Class Codes: Y10T 428/249967 : Inorganic matrix in void-containing component
• CPC Class Codes: Y10T 428/249969 : Of silicon-containing material [e.g., glass, etc.]
• CPC Class Codes: Y10T 428/24997 : Of metal-containing material
• CPC Class Codes: Y10T 428/249978 : Voids specified as micro
• CPC Class Codes: Y10T 428/249979 : Specified thickness of void-containing component [absolute or relative] or numerical cell dimension
• CPC Class Codes: Y10T 428/249986 : Void-containing component contains also a solid fiber or solid particle
• CPC Class Codes: Y10T 428/252 : Glass or ceramic [i.e., fired or glazed clay, cement, etc.] [porcelain, quartz, etc.]

Abstract

Methods are provided for making and using thin films of porous silica substrates to synthesize arrays of polymers. Methods are also provided for assaying such polymers on porous silica substrates. The porous silica substrates offer an increase in array density and signal enhancement over conventional flat glass substrates. Examples of polymers that can be synthesized and assayed include biological polymers such as nucleic acids, polynucleotides, polypeptides, and polysaccharides. Arrays of nucleic acids or polynucleotides can be used for a variety of hybridization-based experiments such as nucleic acid sequence analysis, nucleic acid expression monitoring, nucleic acid mutation detection, speciation, effects of drug therapy on nucleic acid expression, among others.

Claims

1. A porous substrate for the synthesis of polymer arrays comprising:

a support region; and

a porous region on the support region, the porous region being primarily inorganic and having a surface wherein polymers are placed such that polymer arrays are formed, the porous region comprising pores of a pore size of about 2 nm-500 nm, a porosity of about 10-80%, and a thickness of about 0.01 μ

m to about 70 μ

m.

2. The porous substrate of claim 1, wherein the porous region is formed by an additive method.

3. The porous substrate of claim 2 wherein the additive method includes the application of colloidal silica on the support region.

4. The porous substrate of claim 2 wherein the additive method includes the application of alkoxysilane on the support region.

5. The porous substrate of claim 2 wherein the porous region has a thickness from about 0.1-1 microns.

6. The porous substrate of claim 2, wherein the porous region has a thickness of from about 0.1 μ

m to about 0.5 μ

m.

7. The porous substrate of claim 2, wherein the porous region has a thickness of from about 1 μ

m to about 20 μ

m.

8. The porous substrate of claim 2, wherein the porous region is formed by codepositing an organic template material with silica, followed by removing the organic template material.

9. The porous substrate of claim 8 wherein the organic template material comprises particles of about 10-100 nm and the silica comprises particles of about 7-100 nm.

10. The porous substrate of claim 9 wherein an organic template particle size is about equal to a silica particle size.

11. The porous substrate of claim 9 wherein a silica particle size is less than or equal to about ⅔

an organic template particle size.

12. The porous substrate of claim 9 wherein a silica particle size is less than about 10% of an organic template particle size.

13. The porous substrate of claim 8 wherein the organic template material is deposited in a volume ratio to the silica of about 10:

1 to 1;

10.

14. The porous substrate of claim 8 wherein the organic template material is removed using a baking process at a temperature of above about 150°

C.

15. The porous substrate of claim 14 wherein the silica is densified using an annealing process.

16. The porous substrate of claim 8 wherein the porous region has an effective surface area of about 15-40 times a flat substrate with an equivalent two dimensional structure.

17. The porous substrate of claim 1 wherein the porous region comprises silica.

18. The porous substrate of claim 17 wherein the porous region further comprises organic polymer of less than or equal to about 10% mole fraction.

19. The porous substrate of claim 18, wherein the organic polymer coats silica particles of the porous region.

20. The porous substrate of claim 17, wherein the porous region comprises a plurality of pores, each of the plurality of pores having a size of from about 2 to about 100 nm.

21. The porous substrate of claim 17, wherein the porous region comprises a plurality of pores, each of the plurality of pores having a size of from about 2 to about 50 nm.

22. The porous substrate of claim 17, wherein the porous region comprises a plurality of particles, each of the plurality of particles having a size from about 5-500 nm.

23. The porous substrate of claim 17, wherein the porous region comprises a plurality of particles, each of the plurality of particles having a size from about 5-200 nm.

24. The porous substrate of claim 17, wherein the porous region comprises a plurality of particles, each of the plurality of particles having a size from about 70-100 nm.

25. The porous substrate of claim 17, wherein the porous region is silylated with a silyating agent.

26. The porous substrate of claim 25, wherein the silylating agent is selected from the group consisting of N,N-bis(hydroxyethylaminopropyl)triethoxysilane and glycidoxypropyl trimethoxy silane.

27. The porous substrate of claim 1, wherein the porous region has a porosity of from about 20-80%.

28. The porous substrate of claim 1, wherein the porous region has a porosity of from about 50-70%.

29. The porous substrate of claim 1 wherein the porous region is formed by a subtractive method.

30. The porous substrate of claim 29 wherein the porous substrate comprises phase-separable glass, a surface portion of the phase-separable glass being treated to form the porous layer.

31. The porous substrate of claim 30 wherein the phase-separable glass comprises a sodium borosilicate glass.

32. The porous substrate of claim 31 wherein the sodium borosilicate glass has been annealed and leached to provide the porous layer having a thickness of about 70 microns and comprised of a plurality of pores, at least some of the plurality of pores having a pore size greater than about 1000 Å

.

33. The porous substrate of claim 29 wherein the porous region has an effective surface area of about 50-400 times a flat substrate with an equivalent two dimensional structure.

34. The substrate of claim 1, further comprising a high density array of nucleic acids immobilized on the surface.

35. A porous substrate for the synthesis of polymer arrays comprising:

a support region; and

a porous region on the support region, the porous region being primarily inorganic and having a surface wherein polymers are placed such that polymer arrays are formed, the porous region comprising pores of a pore size of about 2 nm-500 nm, a porosity of about 10-80%, and a thickness of about 0.01 μ

m to about 70 μ

m, wherein the porous region is formed by an additive method of codepositing a latex polymer with silica, followed by removing the latex polymer.

36. A porous substrate comprising:

a support region; and

a porous region on the support region, said porous region of about 0.1-0.5 microns thick,

wherein the porous layer comprises an unsintered matrix formed from at least colloidal silica having a particle size of about 70-100 microns, the unsintered matrix defining at least a plurality of open pores having a pore size of about 10-20 nm, and

wherein the porous layer has a porosity of about 10-80%.

37. A porous substrate for the synthesis of polymer arrays comprising:

a support region;

a porous region on the support region, the porous region being primarily inorganic and having a surface including an array of immobilized polymers, the porous region comprising pores of a pore size of about 2 nm to about 500 nm, a porosity of about 10% to about 80%, and a thickness of about 0.01 μ

m to about 70 μ

m.

38. The porous substrate of claim 37 wherein the polymers include one or more of nucleic acids, polynucleotides, proteins, polypeptides, polysaccharides, polycarbamate or oligosaccharides.

39. The porous substrate of claim 37 wherein the array includes more than 100 different polymers.

40. The porous substrate of claim 39 wherein each different polymer is located on a different surface area of the surface.

41. The porous substrate of claim 39 wherein the surface area has an area between about 1 cm² to about 10⁻

10cm².

42. The porous substrate of claim 37 wherein the array has a density of greater than 60 different polymers per 1 cm².

43. The porous substrate of claim 37 wherein each polymer is at least 4 monomers in length.

44. The porous substrate of claim 37 wherein each polymer is between about 4 to about 20 monomers in length.

45. The porous substrate of claim 37 wherein the array has a density of greater than 400 different polymers per 1 cm.

54. The porous substrate of claim 37 or 46 including a plurality of surface areas, each having an area of less than about 10⁻

1 cm².

55. The porous substrate of claim 37 or 46 including a plurality of surface areas, each having an area of less than about 10⁻

2 cm².

56. The porous substrate of claim 37 or 46 including a plurality of surface areas, each having an area of less than about 10⁻

3 cm².

57. The porous substrate of claim 37 or 46 including a plurality of surface areas, each having an area of less than about 10⁻

4 cm².

58. The porous substrate of claim 37 or 46 including a plurality of surface areas, each having an area of less than about 10⁻

5 cm².

59. The porous substrate of claim 37 or 46 including a plurality of surface areas, each having an area of less than about 10⁻

6 cm².

60. The porous substrate of claim 37 or 46 including a plurality of surface areas, each having an area of less than about 10⁻

7 cm².

61. The porous substrate of claim 37 or 46 including a plurality of surface areas, each having an area of less than about 10⁻

8 cm².

62. The porous substrate of claim 37 or 46 including a plurality of surface areas, each having an area of less than about 10⁻

9 cm².

63. The porous substrate of claim 37 or 46 including a plurality of surface areas, each having an area of less than about 10⁻

10 cm².

46. A porous substrate for the synthesis of polymer arrays comprising:

a support region;

a porous region on the support region, the porous region being primarily inorganic and having a surface including an array of immobilized nucleic acids, the porous region comprising pores of a pore size of about 2 nm to about 500 nm, a porosity of about 10% to about 80%, and a thickness of about 0.01 μ

m to about 70 μ

m.

47. The porous substrate of claim 46 wherein the array includes more than 100 different nucleic acids.

48. The porous substrate of claim 46 wherein each different nucleic acid is located on a different surface area of the surface.

49. The porous substrate of claim 48 wherein the surface area has an area between about 1 cm ² to about 10⁻

10 cm².

50. The porous substrate of claim 46 wherein the array has a density of greater than 60 different nucleic acids per 1 cm².

51. The porous substrate of claim 46 wherein each nucleic acid is at least 4 monomers in length.

52. The porous substrate of claim 46 wherein each nucleic acid is between about 4 to about 20 monomers in length.

53. The porous substrate of claim 46 wherein the array has a density of greater than 400 different nucleic acids per 1 cm².