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 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 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 .- 50 . (canceled) 
     
     
         51 . An arrangement of members that provides multiple wells for reacting bio-materials and enables their analysis, the arrangement comprising: a bottom member comprised of a rigid support, a polymer substrate layer lying over an upwardly directed side of the rigid support, the substrate layer having thickness less than about 5 micron and having an upwardly exposed surface that has biological immobilizing properties, the polymer substrate layer being of dissimilar material to that of the rigid support, there being between the polymer substrate layer and the rigid support at least one adhesion-promoting intervening layer adherently joined at its oppositely directed sides to substance of said bottom member; and a bottomless well-defining member at least temporarily engaged upon the bottom member to define multiple wells for reacting fluids in respective wells with bio-material immobilized on the polymer substrate layer at the bottoms of the wells. 
     
     
         52 . The arrangement of  claim 51  in the form of a multi-well plate, the bottomless well-defining member being permanently adhered to the bottom member; the rigid support, the adherently joined intervening layer and the substrate layer of the bottom member being sufficiently transparent to enable transmission of fluorescence excitation light across the bottom member to fluorescently labeled bio-material immobilized on the surface of the substrate layer and transmission of fluorescent radiation from the labeled bio-material across the bottom member to an objective of a detection system. 
     
     
         53 . The arrangement of  claim 52  in which the bottomless well-defining member of the multi-well plate is of substance different from that of the rigid support, the bottomless well-defining member being adhered to portions of the substrate layer that lie directly beneath it. 
     
     
         54 . The arrangement of  claim 52  in which the bottomless well-defining member of the multi-well plate is of substance different from that of the rigid support, the bottomless well-defining member being adhered to portions of an intermediate adhesion-promoting layer that lie directly beneath it. 
     
     
         55 . The arrangement of  claim 52 ,  53  or  54  in which the rigid support of the multi-well plate is comprised of glass, fused quartz, or polycarbonate. 
     
     
         56 . The arrangement of  claim 51  in which the rigid planar support comprises a microscope slide and the bottomless well-defining member is removably sealed to the bottom member in manner forming the wells. 
     
     
         57 . The arrangement of  claim 56  in which the bottom-less well-defining member is a fluid-tight elastomeric gasket in the form of grid of upstanding walls pressed against the face of the microscope slide. 
     
     
         58 . The arrangement of  claim 56  in which the bottom-less well-defining member is engaged upon portions of the substrate layer that lie directly beneath it. 
     
     
         59 . The arrangement of  claim 58  in which the substrate layer includes portions at the bottoms of the wells for immobilizing the biomaterial and portions directly beneath the well-defining member. 
     
     
         60 . The arrangement of  claim 59  in which moats separate the portions of the coating at the bottoms of the wells from the portions of the coating directly beneath the well-defining member. 
     
     
         61 . The arrangement of  claims 56 ,  57  or  58  in which the at least one intervening layer is substantially opaque. 
     
     
         62 . The arrangement of  claim 51 ,  52  or  56  in which the substrate layer comprises nitrocellulose. 
     
     
         63 . The arrangement of  claim 51 ,  52  or  56  in which the substrate layer comprises polystyrene. 
     
     
         64 . The arrangement of  claim 51 ,  52  or  56  in which the intervening layer is an adherent metal oxide. 
     
     
         65 . The arrangement of  claim 64  in which the intervening layer is tantalum oxide. 
     
     
         66 . The arrangement of  claim 51 ,  52  or  56  in which arrays of spots of biomaterial are immobilized at the bottoms of respective wells. 
     
     
         67 . A multi-well plate device for immobilizing biological material in which the bottom portion of the device comprises a polymer substrate layer having biological immobilizing properties for protein or nucleic acid, the substrate layer supported on a rigid support and having an outer deposit-receiving region exposed to receive the biological materiel, wherein the substrate layer has thickness less than about 5 micron, the bottom portion of the multi-well plate device being sufficiently transparent to enable transmission of fluorescence excitation light across the bottom portion to fluorescently labeled bio-material immobilized on the surface of the substrate layer and transmission of fluorescent radiation from the labeled bio-material across the bottom portion to an objective of a detection system. 
     
     
         68 . The multi-well plate device of  claim 67  wherein at least one intervening layer lies between said rigid support and said ultra-thin polymer substrate layer, said intervening layer adherently joined at its oppositely directed sides to substance of said device. 
     
     
         69 . A bottom member for a multi-well plate, the bottom member comprised of a rigid support, a polymer substrate layer lying over an upwardly directed side of the rigid support, the substrate layer being ultra-thin with a thickness less than about 5 micron, and having an upwardly exposed surface that has biological immobilizing properties, the polymer substrate layer being of dissimilar material to that of the rigid support, there being between the polymer substrate layer and the rigid support at least one adhesion-promoting intervening layer adherently joined at its oppositely directed sides to substance of said bottom member, the rigid support, the adherently joined intervening layer and the substrate layer of the bottom member being sufficiently transparent to enable transmission of fluorescence excitation light across the bottom member to fluorescently labeled bio-material immobilized on the surface of the substrate layer and transmission of fluorescent radiation from the labeled bio-material across the bottom member to an objective of a detection system. 
     
     
         70 . The multi-well plate device of  claim 67  or the bottom member for a multi-well plate of  claim 69  in which the deposit-receiving region of the substrate layer is in a surface-treated state for enhanced adhesion of deposits of biological material thereon. 
     
     
         71 . The multi-well plate or the bottom member for a multi-well plate of  claim 70  in which the surface treatment is that provided by corona treatment. 
     
     
         72 . The multi-well plate device of  claim 68  or the bottom member for a multi-well plate of  claim 69  wherein said intervening layer comprises an adherent metal oxide. 
     
     
         73 . The multi-well plate device or the bottom member for a multi-well plate of  claim 72  in which the intervening layer is tantalum oxide.