Substrates for Isolating, Reacting and Microscopically Analyzing Materials

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 microporous, 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 multiwell 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.

1 . A device suitable for immobilizing a bio-material, the bio-material being capable of becoming associated with a fluorophore tag or luminescent tag for optically-stimulated fluorescent emission analysis or for luminescence analysis, said device 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. 
     
     
         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. 
     
     
         5 . A device suitable for immobilizing a bio-material, the bio-material being capable of becoming 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. 
     
     
         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. 
     
     
         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. 
     
     
         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. 
     
     
         13 . The device of  claim 1 ,  5  or  9  in which said intervening layer is comprised of tantalum oxide. 
     
     
         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. 
     
     
         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. 
     
     
         17 . The device of  claim 6  in which the substantially opaque intervening layer is comprised of tantalum oxide. 
     
     
         18 . The device of  claim 1 ,  5  or  9  in which the outer surface of the nitrocellulose coating is arranged to be exposed from the exterior for optical stimulation of an optional fluorophore, 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 when said fluorophore is present in the vicinity of said coating. 
     
     
         19 . The device of  claim 18 , further including a deposit of bio-material immobilized on said nitrocellulose coating and 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 fluoropone tag. 
     
     
         20 . 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 an optional fluorophore when present in the vicinity of 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 an optional fluorophore when said fluorophore is present in the vicinity of said coating, or to enable both. 
     
     
         21 . The device of  claim 20 , further including a deposit of bio-material immobilized on said nitrocellulose coating and 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 fluoropone tag. 
     
     
         22 . The device of  claim 20  in which a said intervening layer is functionally transparent silane or functionally transparent tantalum oxide. 
     
     
         23 . 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. 
     
     
         24 . The device of  claim 1 ,  5  or  9  in which the outer surface of the nitrocellulose coating on the rigid support is generally flat. 
     
     
         25 . The device of  claim 24 , further including an array of spots of bio-material deposited on the nitrocellulose coating. 
     
     
         26 . The device of  claim 25  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. 
     
     
         27 . The device of  claim 1 ,  5  or  9  wherein the rigid support is in the form of a microscope slide. 
     
     
         28 . 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. 
     
     
         29 . The device of  claim 28  in which the nitrocellulose coating is a substantially solid film. 
     
     
         30 . The device of  claim 28  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. 
     
     
         31 . A device suitable for immobilizing a bio-material, the 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. 
     
     
         32 . The device of  claim 31  in which the polymer is selected to immobilize protein material or cellular bio-material. 
     
     
         33 . The device of  claim 31 , further including a deposit of immobilized protein material or cellular bio-material on the coating. 
     
     
         34 . The device of  claim 31  in which the treated surface is the result of exposure of the outer surface of the polymer coating to corona treatment. 
     
     
         35 . The device of  claim 31  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. 
     
     
         36 . The device of  claim 31 ,  34  or  35  in which a said intervening adherent layer between the coating of polymer and the rigid support is comprised of tantalum oxide or silane. 
     
     
         37 . The device of  claim 31 ,  34  or  35  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. 
     
     
         38 . The device of  claim 31 ,  34  or  35  in which the polymer coating is a dried residue of a coating solution of the polymer and a solvent. 
     
     
         39 . The device of  claim 38  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. 
     
     
         40 . The device of  claim 31 ,  34  or  35  in which the polymer coating is of thickness less than three micron. 
     
     
         41 . The device of  claim 31 ,  34  or  35  in which the coating of polymer is nitrocellulose or polystyrene. 
     
     
         42 . The device of  claim 31 ,  34  or  35  in which the outer surface of the coating of polymer on the rigid support is generally flat. 
     
     
         43 . The device of  claim 42 , further including a deposit of immobilized bio-material on the coating. 
     
     
         44 . The device of  claim 43  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. 
     
     
         45 . The device of  claim 42  further including an array of spots of bio-material deposited on the layer. 
     
     
         46 . The device of  claim 45  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. 
     
     
         47 . The device of  claim 31 ,  34  or  35  wherein the rigid support is in the form of a microscope slide. 
     
     
         48 . The device of  claim 31 ,  34  or  35  in which the outer surface of the coating of polymer is arranged to be exposed from the exterior for optical stimulation of an optional fluorophore, 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. 
     
     
         49 . The device of  claim 48  in which the intervening layer is comprised of a substantially opaque layer of tantalum oxide. 
     
     
         50 . The device of  claim 31 ,  34  or  35  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 an optional fluorophore by excitation radiation passing through said rigid support, or to enable microscopic analysis through said rigid support of emissions from an optional fluorophore or luminescent tag on said coating, or to enable both. 
     
     
         51 . The device of  claim 50  in which a said intervening layer is functionally transparent silane or functionally transparent tantalum oxide. 
     
     
         52 . The device of  claim 31  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, and 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. 
     
     
         53 . The device of  claim 52  wherein said coating is a substantially transparent solid film. 
     
     
         54 . The device of  claim 1 ,  31  or  53  further including an array of spotted deposits of the bio-material disposed on the deposit-receiving surface for use in the performance of an assay. 
     
     
         55 . The device of  claim 52  or  53  in which the coating is a dried residue of a coating solution of nitrocellulose or polystyrene and a volatile solvent. 
     
     
         56 . The device of  claim 55  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. 
     
     
         57 . The device of  claim 52  or  53  wherein the deposit-receiving surface of said coating is in a corona-treated state. 
     
     
         58 . The device of  claim 52  or  53  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. 
     
     
         59 . The device of  claim 52  or  53  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. 
     
     
         60 . The device of  claim 59 , wherein a said intervening layer is silane. 
     
     
         61 . The device of  claim 59 , wherein a said intervening layer is tantalum oxide. 
     
     
         62 . The device of  claim 59 , 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. 
     
     
         63 . The device of  claim 52  or  53  in which the coating has thickness less than about 1 micron. 
     
     
         64 . The device of  claim 63  in which the coating is a substantially solid, substantially transparent film of thickness t uts  between about 0.1 and 0.5 micron. 
     
     
         65 . The device of  claim 63  in which the coating of polymer is polystyrene. 
     
     
         66 . A method of forming the device of  claim 1 ,  5  or  31 , comprising providing the rigid support with the one or more adherent intervening layers and forming thereon the polymer coating. 
     
     
         67 . The method of  claim 66  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. 
     
     
         68 . The method of  claim 67  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. 
     
     
         69 . The method of  claim 66  followed by subjecting the exposed surface of the coating to an energetic surface-altering treatment to enhance the binding capability of the coating for a bio-material. 
     
     
         70 . The method of  claim 69  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.