Substrates for isolating, reacting and microscopically analyzing materials

• US 20060286550A1
• Filed: 05/08/2006
• Published: 12/21/2006
• Est. Priority Date: 08/16/2002
• Status: Active Grant

• Current Assignee: Medicinal Genomics Corporation (Courtagen Life Sciences, Inc.)
• Original Assignee: Clinical MicroArrays, Inc.
• Inventors: Montagu, Jean I., Root, David, Dowd, Roger
• Granted Patent: US 7,297,497 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• 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

1-50. -50. (canceled)

51. A device constructed for immobilizing material for exposure to liquid during an assay, the device comprising a polymer layer on a rigid support, the layer having an outer deposit-receiving region that has binding properties for the material, wherein the layer comprises nitrocellulose in the form of a coating of substantially solid film.

52. The device of claim 51 in which the substantially solid film is less than about 1 micron in thickness.

53. The device of claim 51 in which the substantially solid film is between about 0.1 and 0.5 micron in thickness.

54. The device of claim 51 in which the substantially solid film is a dried residue of a coating solution of nitrocellulose and a volatile solvent.

55. The device of claim 54 having an array of spots of material applied to the substantially solid film for use in the assay.

56. The device of claim 55 in which the array of spots of 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.

57. The device of claim 55 in which the spots are between about 100 and 400 micron in diameter.

58. The device of claim 55 in which the substantially solid film transmits light without substantial scatter.

59. The device of claim 55 in which the rigid support is glass.

60. The device of claim 55 in which the rigid support is plastic.

61. The device of claim 55 constructed to expose deposits of the material on the substantially solid film for direct illumination or optical inspection from the exterior.

62. The device of claim 61 in which a portion of the device below the substantially solid film is substantially opaque to light from the solid film.

63. The device of claim 62 in which the rigid support is substantially transparent and an adherent intervening layer between the solid film and the rigid support is substantially opaque.

64. The device of claim 55 in which deposits of material on the substantially solid film are subject to illumination or inspection via the rigid support, the device being functionally transparent so that light can pass through the rigid support and the substantially solid film to the spots.

65. The device of claim 64 in which there is at least one substantially transparent intervening adherent layer between the substantially solid film and the rigid support.

66. The device of claim 51 in which the substantially solid film of nitrocellulose is adhered to the rigid support via one or more adherent intervening layers.

67. The device of claim 66 in which an intervening layer is comprised of an adherent metal oxide or a silicon-based material.

68. The device of claim 66 in which an intervening layer is comprised of tantalum oxide or silane.

69. The device of claim 66 in which the rigid support is of glass and an adherent intervening layer is an adhesion-promoting layer comprised of silane, epoxy silane, polylisine, PEI, GAP, an adherent metal oxide, colloidal silica or a soluble silicate.

70. The device of claim 51 in which the substantially solid film has its outer deposit-receiving surface in treated state for enhanced immobilization of the material.

71. The device of claim 70 in which the treated state is the result of exposure of the surface to an energetic surface-altering condition.

72. The device of claim 71 in which the treated state is the result of exposure of the surface to corona treatment.

73. The device of claim 71 in which the treated state is the result of exposure of the surface to charged particles.

74. The device of claim 71 in which the treated state is the result of exposure of the surface to gamma radiation.

75. The device of claim 71 in which the treated state 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.

76. The device of claim 51 constructed to immobilize material capable of being associated with a fluorophore tag for optically-stimulated fluorescent emission analysis.

77. The device of claim 55 combined with a reader for fluorescence from fluorescent labels associated with at least some of the spots.

78. The device of claim 77 in which the reader comprises a CCD sensor.

79. The device of claim 51 in which the polymer layer is a drawn coating.

80. The device of claim 51 in which the outer surface of the polymer layer is generally flat, arranged to receive deposit of a spotted array of bio-material.

81. A device for immobilizing biological material for analysis based on optically-stimulated light emissions, the device comprising a polymer substrate layer having biological immobilizing properties, the substrate layer deposited on a rigid support, and having an outer deposit-receiving surface exposed to receive the biological material, wherein the substrate layer is comprised of nitrocellulose or polystyrene that is ultra-thin, having a thickness t_ut less than about 5 micron, the substrate layer comprising a substantially solid film that is substantially transparent.

82. The device of claim 81 wherein said substrate layer has binding properties for protein or nucleic acid.

83. The device of claim 81, wherein the device is constructed to enable effective light to pass in at least one direction between a deposit on said substrate layer and through said rigid support.

84. The device of claim 83, wherein said substrate layer is nitrocellulose.

85. The device of claim 84, wherein the outer surface of said substrate layer is generally flat and arranged to receive the deposit of a spotted array of bio-material.

86. The device of claim 81, wherein the deposit-receiving surface of said substantially solid substrate layer is in a treated state as the result of exposure of the surface to an energetic surface altering treatment.

87. The device of claim 81, wherein said support is selected from the group consisting of glass, fused quartz, silicon, plastic, PMMA, polystyrene, ceramic or a solid metal.

88. A method of forming the device of claims 84, 85 or 86 including applying directly or indirectly to said rigid support a fluid containing said polymer of said substrate layer under conditions to form said substrate layer, preferably by drawing the rigid support from a bath of coating composition.

89. The method of claim 88 for devices produced with a solid film substrate layer, including maintaining forming conditions to produce a solid film coating as the substrate layer.

90. The method of claim 89 including forming the substrate layer of nitrocellulose.

91. The method of claim 90 for producing a substrate support for spotted biological specimens for reaction or analysis, the steps comprising:

(a) at least partially immersing a rigid member that defines a straight support surface (e.g., planar or cylindrical) in a vat containing a coating solution comprising a biologically compatible organic film-forming composition which includes at least one volatile solvent,

(b) progressively drawing the member from the solution along a fixed path into a still environment,

(c) the fixed path being generally parallel to said straight support surface,

(d) the conditions of said still environment enabling said solvent to evaporate to leave a drawn coating of said composition adhered to said support surface, for example the rigid member being a microscope slide, or a component of a bio-cassette of the bottom member of a multiwell plate and the conditions being maintained to cause the formation of a solid thin film of nitrocellulose, for example the organic film-forming composition comprising nitrocellulose dissolved in amyl acetate or an organic solvent such as acetone, dimethylsulfoxide, ethyl acetate, or other common organic solvents.

92. A method of conducting an assay including providing a device according to claim 85 applying an array of spots of bio-material to the substrate, conducting an assay which tags at least some of the spots with a fluorescent label, and, after washing the array, reading the array by fluorescent detection, preferably the assay being based on protein-protein interaction, or involving an array comprising nucleic acid or other genetic material, or comprising viruses, peptides, antibodies, receptors, cDNA clones, DNA probes, oligonucleotides including synthetic oligonucleotides, or polymerase chain reaction (PCR) products, or the array comprising plant, animal, human, fungal or bacterial cells, or malignant cells, or cells from biopsy tissue.

93. The method of claim 92 wherein the reading is accomplished with a CCD sensor.

94. The device of claim 83 arranged to enable illumination of a deposit of biological material on said substrate layer via said rigid support.

95. The device of claim 83 or 94 arranged to enable microscopic inspection of a deposit of biological material on said substrate layer via said rigid support.

96. The device of claim 85 in which an array of bio-material spots comprises deposits on the deposit-receiving surface.

97. The device of claim 96 in which the deposits are in unreacted state.

98. The device of claim 96 in which the deposits are in a reacted state as a result of performance of an assay, at least some of the said spots being associated with a fluorescent or luminescent label.

99. The device of claim 81 wherein the deposit-receiving surface of the substantially solid substrate is in a treated state as the result of exposure to corona treatment.

100. The device of claim 81 wherein the deposit-receiving surface of the substantially solid substrate is in a treated state as the result of exposure to charged ions.

101. The device of claim 81 wherein the deposit-receiving surface of the substantially solid substrate is in a treated state as the result of exposure to gamma radiation.