Substrates for isolating reacting and microscopically analyzing materials

• US 7,384,742 B2
• Filed: 08/18/2003
• Issued: 06/10/2008
• Est. Priority Date: 08/16/2002
• Status: Expired due to Term

• Current Assignee: Medicinal Genomics Corporation (Courtagen Life Sciences, Inc.)
• Original Assignee: Decision Biomarkers Incorporated (Courtagen Life Sciences, Inc.)
• Inventors: Root, David, Montagu, Jean I., Dowd, Roger
• Primary Examiner(s): Lam; Ann Y.
• Application Number:
  US10/524,614
• Publication Number:
  US 20060134606A1
• Time in Patent Office: 1,758 Days
• Field of Search: 435/6, 435/4, 435/7.1, 435/7.92, 435/287.1, 435/287.3, 435/287.7, 436/518, 356/244, 356/246, 422/50, 422/55
• US Class Current: 435/6.19
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/544 the carrier being organic

Abstract

An immobilizing device for biological material comprises a rigid support (12) carrying a substrate layer (20, 20′) of polymer having biological immobilizing properties, e.g. for amino and nucleic acids. Substantially solid ultra-thin substrate layers (20′) having a thickness less than about 5 micron, preferably between about 0.1 and 0.5 micron, and micro-porous, ultra-thin substrate layers (20′) having a thickness less than about 5 micron, preferably less than 3 micron, 2 or 1 micron are shown, which may be segmented by isolating moats M. The substrate layer is on a microscope slide (302), round disc (122), bio-cassette, at the bottom of a well of a multiwell plate, and as a coating inside a tube. Fluorescence or luminescence intensity and geometric calibration spots (420) are shown. Reading is enhanced by the intensity calibration spots (420) to enable normalization of readings under uneven illumination conditions, as when reading by dark field, side illumination mode. The reference spots are shown being printed simultaneously with printing an array of biological spots or with the same equipment Methods of forming layers of the device include controlled drawing from a bath of coating composition and drying, and spinning of C-D shaped substrates. Post-forming treatment is shown by corona treatment and radiation. Adherent metal oxides (14), silica-based materials and other materials are used to unite layers of the composite. In multi-well plates the oxide promotes joining of a bottom plate (95, 95′) and upper, well-defining structure (94) of dissimilar material. The oxides (14) also provide beneficial opacity to prevent light entering the glass support, for applying potential to the substrate, etc.

Claims

1. A device constructed for immobilizing bio-material capable of being associated with a fluorophore tag or luminescent tag for optically-stimulated fluorescent emission analysis or for luminescence analysis, comprising a coating of nitrocellulose polymer of thickness less than 5 micron, the coating adhered to a rigid support via one or more adherent intervening layers comprising at least an adherent metal oxide intervening layer, the nitrocellulose coating having an outer deposit-receiving surface that has enhanced binding capability for the bio-material as the result of exposure of the coating surface to corona treatment, and a deposit of the bio-material immobilized on the corona-treated surface of the nitrocellulose coating.

2. The device of claim 1 in which the nitrocellulose coating is microporous.

3. The device of claim 1 in which the nitrocellulose coating is a solid film.

4. The device of claim 3 in which the solid film is less than 3 micron in thickness.

10. The device of claim 1, 5 or 9 in which the nitrocellulose coating is a dried residue of a coating solution of nitrocellulose and a volatile solvent.

11. The device of claim 10 in which the nitrocellulose coating is the product resulting from the process of immersing the rigid support in a bath of the coating solution and progressively drawing the rigid support from the bath under conditions in which the solvent evaporates during the drawing.

13. The device of claim 1, 5 or 9 in which said intervening layer is comprised of tantalum oxide.

16. The device of claim 1, 5 or 9 in which the rigid support is substantially transparent and a said intervening layer is substantially opaque.

18. The device of claim 1, 5 or 9 in which the outer surface of the nitrocellulose coating and the immobilized bio-material thereon is arranged to be exposed from the exterior for optical stimulation of a fluorophore tag associated with the bio-material and analysis of resultant emission from the fluorophore tag, the rigid support being substantially transparent and the one or more intervening layers being collectively sufficiently opaque to substantially block, from entering the rigid support, radiation of wavelengths corresponding to the stimulating and emission wavelengths of the fluorophore tag associated with the immobilized bio-material.

19. The device of claim 1, 5 or 9 in which the rigid support, the one or more intervening layers and the nitrocellulose coating collectively are functionally transparent to light to enable optical excitation of a fluorophore taa associated with the deposit of bio-material on said coating by excitation radiation passing through said rigid support, or to enable microscopic analysis through said rigid support of optically-stimulated fluorescent emissions passing from a fluorophore tag associated with the deposit of bio-material on said coating, or to enable both.

20. The device of claim 19 in which a said intervening layer is functionally transparent silane or functionally transparent tantalum oxide.

22. The device of claim 1, 5 or 9 in which the outer surface of the nitrocellulose coating on the rigid support is generally flat, arranged to receive deposit of a spotted array of the bio-material.

23. The device of claim 22, including an array of spots of bio-material deposited on the nitrocellulose coating.

24. The device of claim 23 in which the array of spots of bio-material comprises protein, peptides, antibodies, viruses, or nucleic acid or other genetic material, receptors, eDNA clones, DNA probes, oligonucleotides including synthetic oligonuceleotides, or polymerase chain reaction (PCR) products, or plant, animal, human, fungal or bacterial cells, or malignant cells or cells from biopsy tissue.

25. The device of claim 1, 5 or 9 wherein the rigid support is in the form of a microscope slide.

47. A method of forming the device of claims 1, 5 or 29, comprising providing the rigid support with the one or more adherent intervening layers and forming thereon the polymer coating.

48. The method of claim 47 in which the coating is formed by applying a coating solution of the polymer and a volatile solvent to an adherent intervening layer on the rigid support, and evaporating the solvent to form the coating layer as a dried residue of the polymer.

49. The method of claim 48 in which the coating is applied to the support by immersing the rigid support in a bath of the coating solution and progressively drawing the support from the bath of the coating solution under conditions in which the solvent evaporates during the drawing.

50. The method of claim 47 followed by subjecting the exposed surface of the coating to an energetic surface-altering treatment to enhance the binding capability of the coating for the bio-material.

51. The method of claim 50 in which the treatment is corona treatment, flame treatment, bombardment by charged particles comprising electrons, ions or sub-atomic particles, or electromagnetic radiation of ultraviolet, gamma or X-ray wavelength.

52. A method of emission analysis comprising providing the device of claim 1, 5 or 29, including applying said bio-material as an array of spots of material to the outer deposit-receiving surface of the polymer coating, conducting an assay which tags at least some of the spots with a fluorescent or luminescent label, and, after washing the array, reading the array by optical detection.

53. The method of claim 52 in which reading is accomplished by a CCD sensor.

56. The device of claim 1, 29 or 54 wherein an array of spotted deposits of the bio-material is disposed on the deposit-receiving surface for use in the performance of an assay.

5. A device constructed for immobilizing a bio-material capable of being associated with a fluorophore tag or luminescent tag for optically-stimulated fluorescent emission analysis or for luminescence analysis, comprising a coating of nitrocellulose polymer of thickness less than 5 micron, the coating adhered to a rigid support via one or more adherent intervening layers, the nitrocellulose coating having an outer deposit-receiving surface that has enhanced binding capability for the bio-material as the result of exposure of the coating surface to an energetic surface-altering treatment, and a deposit of the bio-material immobilized on the treated nitrocellulose surface.

6. The device of claim 5 in which the treated surface of the nitrocellulose coating is the result of exposure of the surface to corona treatment.

7. The device of claim 5 in which the treated surface of the nitrocellulose coating is the result of exposure of the surface to charged particles.

8. The device of claim 5 in which the treated surface of the nitrocellulose coating is the result of exposure of the surface to gamma radiation.

9. The device of claim 5 in which the treated surface of the nitrocellulose coating is the result of exposure of the outer surface to at least one of corona treatment, flame treatment, bombardment by charged particles comprising electrons, ions or sub-atomic particles, or electromagnetic radiation of ultraviolet, gamma or X-ray wavelength.

12. The device of claim 5 or 9 in which said intervening layer is comprised of an adherent metal oxide or a silicon-based material.

14. The device of claim 5 or 9 in which said intervening layer is comprised of silane.

15. The device of claim 5 or 9 in which the rigid support is of glass and said adherent intervening layer is comprised of silane, epoxy silane, polylisine, PEI, GAP, an adherent metal oxide, colloidal silica or a soluble silicate.

17. The device of claim 6 in which the substantially opaque intervening layer is comprised of tantalum oxide.

21. The device of claim 5 or 9 in which an intervening layer is a coating comprising the product resulting from the process of immersing the rigid support in a bath of a coating solution comprising the material of the intervening layer and a solvent and progressively drawing the rigid support from the bath under conditions in which the solvent evaporates during the drawing.

26. The device of claim 5 or 9 comprising a coating of nitrocellulose of thickness less than about 3 micron, the rigid support comprising glass, the nitrocellulose coating being adhered to the rigid glass support via an intervening layer comprised of tantalum oxide or silane.

27. The device of claim 26 in which the nitrocellulose coating is a substantially solid film.

28. The device of claim 26 in which the nitrocellulose coating is the product resulting from the process of immersing the rigid support in a bath of a coating solution of nitrocellulose and a solvent and progressively drawing the rigid support from the bath under conditions in which the solvent evaporates during the drawing.

29. A device constructed for immobilizing a bio-material capable of becoming associated with a fluorophore tag or luminescent tag for optical emission analysis, comprising a coating of a polymer capable of binding with the bio-material, the coating having a thickness less than about 5 micron, the coating of polymer adhered to a rigid support via one or more adherent intervening layers, the coating of polymer having an outer deposit-receiving surface that has enhanced binding capability for the bio-material as the result of exposure of the surface to an energetic surface-altering treatment, and a deposit of the bio-material immobilized on the treated surface of the polymer coating.

30. The device of claim 29 in which the polymer is selected to immobilize protein material or cellular bio-material and the deposit on the coating is comprised of the respective material.

31. The device of claim 29 in which the treated surface is the result of exposure of the outer surface of the polymer coating to corona treatment.

32. The device of claim 29 in which the treated surface is the result of exposure of the outer surface of the polymer coating to at least one of corona treatment, flame treatment, bombardment by charged particles comprising electrons, ions or sub-atomic particles, or electromagnetic radiation of ultraviolet, gamma or X-ray wavelength.

33. The device of claim 29, 31 or 32 in which a said intervening adherent layer between the coating of polymer and the rigid support is comprised of tantalum oxide or silane.

34. The device of claim 29, 31 or 32 in which the rigid support is of glass and said intervening adherent layer between the coating of polymer and the rigid support is comprised of silane, epoxy silane, polylisine, PEI, GAP, an adherent metal oxide, colloidal silica or a soluble silicate.

35. The device of claim 29, 31 or 32 in which the polymer coating is a dried residue of a coating solution of the polymer and a solvent.

36. The device of claim 35 in which the polymer coating is the product resulting from the process of immersing the rigid support in a bath of the coating solution and progressively drawing the rigid support from the bath under conditions in which the solvent evaporates during the drawing.

37. The device of claim 29, 31 or 32 in which the polymer coating is of thickness less than three micron.

38. The device of claim 29, 31 or 32 in which the coating of polymer is nitrocellulose or polystyrene.

39. The device of claim 29, 31 or 32 in which the outer surface of the coating of polymer on the rigid support is generally flat, arranged to receive deposit of a spotted array of bio-material.

40. The device of claim 39 including an array of spots of bio-material deposited on the layer.

41. The device of claim 40 in which the array of deposited spots of bio-material comprises protein, peptides, antibodies, viruses, or nucleic acid or other genetic material, receptors, cDNA clones, DNA probes, oligonucleotides including synthetic oligonuceleotides, or polymerase chain reaction (PCR) products, or plant, animal, human, fungal or bacterial cells, or malignant cells or cells from biopsy tissue or other bio-material.

42. The device of claim 29, 31 or 32 wherein the rigid support is in the form of a microscope slide.

43. The device of claim 29, 31 or 32 in which the outer surface of the coating of polymer and the immobilized bio-material thereon are arranged to be exposed from the exterior for optical stimulation of a fluorophore tag associated with the bio-material and analysis, the rigid support being substantially transparent and the one or more intervening layers being collectively sufficiently opaque to substantially block light from the rigid support.

44. The device of claim 43 in which the intervening layer is comprised of a substantially opaque layer of tantalum oxide.

45. The device of claim 29, 31 or 32 in which the rigid support, the one or more intervening layers, and the coating of polymer are collectively functionally transparent to light to enable optical excitation of a fluorophore tag associated with the deposit of bio-material on said coating by excitation radiation passing through said rigid support, or to enable microscopic analysis through said rigid support of emissions from a fluorophore tag or luminescent tag associated with the deposit of bio-material on said coating, or to enable both.

46. The device of claim 45 in which a said intervening layer is functionally transparent silane or functionally transparent tantalum oxide.

54. The device of claim 29 constructed for immobilizing bio-material in the form of protein bio-material or cellular bio-material, wherein the coating is comprised of nitrocellulose polymer or polystyrene polymer that is ultra-thin, having a thickness t_ut less than about 3 micron, the treated surface is the result of exposure of the outer surface of the polymer coating to at least one of corona treatment, flame treatment, bombardment by charged particles comprising electrons, ions or sub-atomic particles, or electromagnetic radiation of ultraviolet, gamma or X-ray wavelength, and the coating carrying a deposit of protein bio-material or cellular bio-material.

55. The device of claim 54 wherein said coating is a substantially transparent solid film.

57. The device of claim 54 or 55 in which the coating is a dried residue of a coating solution of nitrocellulose or polystyrene and a volatile solvent.

58. The device of claim 57 wherein the coating is the product resulting from the process of immersing the rigid support in a bath of the coating solution and progressively drawing the rigid support from the bath under conditions in which the solvent evaporates during the drawing.

59. The device of claim 54 or 55 wherein the deposit-receiving surface of said coating is in a corona-treated state.

60. The device of claim 54 or 55 wherein the deposit-receiving surface of said substrate layer is in a treated state produced by an energetic surface-altering treatment comprising exposure of the outer surface to electromagnetic radiation of gamma wavelength.

61. The device of claims 54 or 55 wherein a said intervening layer is of substance selected from the group consisting of silane. epoxy silane, polylisine, PEI, GAP, an adherent metal oxide, colloidal silica and soluble silicates.

62. The device of claim 61, wherein a said intervening layer is silane.

63. The device of claim 61, wherein a said intervening layer is tantalum oxide.

64. The device of claim 61, wherein the rigid support has characteristic luminescence or fluorescence in response to incident stimulating radiation, a said intervening layer being sufficiently opaque to be effective to at least substantially limit passage of light between the rigid support and the coating of polymer.

65. The device of claim 54 or 55 in which the coating has thickness less than about 1 micron.

66. The device of claim 65 in which the coating is a substantially solid, substantially transparent film of thickness t_uts between about 0.1 and 0.5 micron.

67. The device of claim 65 in which the coating of polymer is polystyrene.

68. A device constructed for immobilizing bio-material capable of becoming associated with a fluorophore tag or luminescent tag for optical emission analysis, comprising a deposit-receiving coating of substantially solid nitrocellulose of thickness less than 5 micron adhered to a rigid support via one or more adherent intervening layers at least one of which is an adherent metal oxide, the adherent intervening layer, or layers collectively being substantially opaque, the coating of nitrocellulose having an outer deposit-receiving surface in corona-treated state for enhanced immobilization of the bio-material and a deposit of the bio-material immobilized on the corona-treated nitrocellulose deposit-receiving surface.

69. A device constructed for immobilizing bio-material capable of becoming associated with a fluorophore tag or luminescent tag for optical emission analysis, comprising a deposit-receiving coating of substantially solid nitrocellulose of thickness less than 5 micron adhered to a rigid support via one or more adherent intervening layers at least one of which is an adherent metal oxide, the adherent intervening layer, or layers collectively, being substantially opaque, the coating of nitrocellulose having an outer deposit-receiving surface in surface-treated state for enhanced immobilization of the bio-material, the surface-treated state being the result of exposure of the outer surface of the coating of nitrocellulose to at least one of corona treatment, flame treatment, bombardment by charged particles comprising electrons, ions or sub-atomic particles, or by electromagnetic radiation of ultraviolet, gamma or X-ray wavelength, and a deposit of the bio-material immobilized on the surface-treated nitrocellulose deposit-receiving surface.

70. A device constructed for immobilizing bio-material capable of becoming associated with a fluorophore tag or luminescent tag for optical emission analysis, comprising a deposit-receiving coating of substantially solid nitrocellulose of thickness less than 5 micron adhered to a rigid support via one or more adherent intervening layers, the coating of nitrocellulose having an outer deposit-receiving surface in treated state for enhanced immobilization of the bio-material, the surface-treated state being the result of exposure of the outer surface to at least one of corona treatment, flame treatment, bombardment by charged particles comprising electrons, ions or sub-atomic particles, or electromagnetic radiation of ultraviolet, gamma or X-ray wavelength, and a deposit of the bio-material immobilized on the surface-treated nitrocellulose deposit-receiving surface.