Substrates for isolating reacting and microscopically analyzing materials Number:7,384,742 from the United States Patent and Trademark Office (PTO) owispatent

Title: 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 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.

Patent Number: 7,384,742 Issued on 06/10/2008 to Montagu,   et al.

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Inventors:	Montagu; Jean I. (Brookline, MA), Dowd; Roger (Natick, MA), Root; David (N. Chelmsford, MA)
Assignee:	Decision Biomarkers, Inc. (Waltham, MA)
Appl. No.:	10/524,614
Filed:	August 18, 2003
PCT Filed:	August 18, 2003
PCT No.:	PCT/US03/25685
371(c)(1),(2),(4) Date:	November 02, 2005
PCT Pub. No.:	WO2004/018623
PCT Pub. Date:	March 04, 2004

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Current U.S. Class:	435/6
Field of Search:	435/6,4,7.1,7.92,287.1,287.3,287.7 436/518 356/244,246 422/50,55

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References Cited [Referenced By]

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U.S. Patent Documents

5112736	May 1992	Caldwell et al.
5310650	May 1994	McMahon et al.
5486452	January 1996	Gordon et al.
5491097	February 1996	Ribi et al.
5552272	September 1996	Bogart
5807522	September 1998	Brown et al.
5837196	November 1998	Pinkel et al.
6017496	January 2000	Nova et al.
6051388	April 2000	Bodenhamer
6197599	March 2001	Chin et al.
6207369	March 2001	Wohlstadter et al.
6210878	April 2001	Pinkel et al.
6329209	December 2001	Wagner et al.
6365418	April 2002	Wagner et al.
6381013	April 2002	Richardson
6406921	June 2002	Wagner et al.
6472224	October 2002	Wischerhoff et al.
6475808	November 2002	Wagner et al.
6630358	October 2003	Wagner et al.
6861251	March 2005	Green
6921637	July 2005	Audeh et al.
7047610	July 2006	Cozzette et al.
2002/0068157	June 2002	Wischerhoff
2002/0095073	July 2002	Jacobs et al.
2003/0228637	December 2003	Wang

Foreign Patent Documents

	0366241		Feb., 1990		EP
	0366241		May., 1990		EP
	WO 98/20353		May., 1998		WO
	WO 99/07892		Feb., 1999		WO
	WO 00/33078		Jun., 2000		WO
	WO 2004/063719		Jul., 2004		WO

Other References

"FAST Slides Protocol", pp. 104-106, www.schleicher-schuell.com/bioscience. cited by other . FAST Slides S&S nitrocellulose slides, p. 1, Product Specifications, http://www.arraying.com (2005). cited by other . Ge, S. et al. "Monitoring of Bacterial Gene Expression Using Ollgonucleotide Microarray Analysis" Abstracts, 42.sup.nd Interscience Conference on Antimicrobial Agents and Chomotherapy. American Society for Microbiology, 2002. cited by other . Grace Bio-labs Oncyte Film Slides, Nov. 12, 1998. cited by other . Kukar et al., "Protein Microarrays to Detect Protein-Protein Interactions Using Red and Green Flourescent Proteins", Analytical Biochemistry 306, 50-54 (2002). cited by other . "New Chip on the Block", Laboratory Medicine, 30(3):180. cited by other . Onyiriuka et al., "Surface Modification of Polystyrene by Gamma-Radiation", Applied Spectroscopy, vol. 44, No. 5 (1990). cited by other . Pinkel et al., "High resolution analysis of DNA copy number variation using comparative genomic hybridization to microrays", Nature Genetics, 20:207. cited by other . Schleicher & Schuell, "Fast.TM. Slides" Oct. 2001. cited by other . Steinitz et al., "An improved method to create nitrocellulose particles suitable for the immobilization of antigen and antibody", Journal of Immunological Methods, 187:171. cited by other . Grace Bio-labs, "HybriWell.TM. Seal System", Nov. 12, 1998, http://www.gracebio.com/prodhybriseal.htm. cited by other . Grace Bio-labs, "MultiWell.TM. Press-to-Seal Arrays", Nov. 12, 1998, http://www.gracebio.com/prodpress.htm. cited by other.

Primary Examiner: Lam; Ann Y.
Attorney, Agent or Firm: Fish & Richardson P.C.

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Claims

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What is claimed is:

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.

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.

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 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.

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.

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.

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.

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.

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.sub.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.

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.

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.sub.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.

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Description

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TECHNICAL FIELD

This invention pertains to substrates for isolating, reacting and microscopically analyzing bio-materials, especially proteins, and genetic materials. The invention also pertains to methods for making and testing the substrates, to bio-array products employing the substrates, and to methods of binding, reacting, assaying and imaging materials on the substrates.

Embodiments of the invention in particular pertain to coated glass slides, multi-well plates and similar rigid supports that receive the protein or genetic material and retain the material in precise position while an assay is performed, the array is washed, and the altered array is analyzed for fluorescent emission. The invention also pertains to multi-well plate constructions as well as to tubes, bio-cassettes, disk-form substrates and other configurations.

Embodiments of the invention pertain to techniques for isolating, binding and discriminating between different bio-molecules and for establishing conditions for reaction.

Embodiments of the invention pertain to examination of tissue, for instance biopsies of potentially malignant tissue or of contaminated materials such as potable water, e.g. to enable detection of rare events.

BACKGROUND

The search for improving and extending the capabilities of optical analysis have long involved considerations of the substrate on which the specimen is supported during the analysis.

In the case of biological material, use has been made of polymeric substrates, in particular, porous substrates also referred to as "membranes" and "matrices," to immobilize the material while the material undergoes genetic analysis or is used for cell or protein research. Historically, porous matrices were first created as filters, to separate particulates contained within a liquid. In the process, a number of porous polymeric matrices were identified to have strong binding affinity for a number of bio-polymers. These matrices became the substrates of choice for cytochemistry and bio-polymer studies, especially where radioactive labels were employed.

The ability of nitrocellulose membranes (also referred to as "cellulose nitrate") to serve as substrates to bind single stranded DNA, i.e., to immobilize DNA, was demonstrated by Nirenberg in 1965 in flow-through assays. Such membranes were commonly formed using fibrous cellulose as a starting material.

Cellulose, to which nitrocellulose is related, is formed as a chain of glucose units, which is the universal building material for living cells. Nitrocellulose membranes benefit in this regard by relationship to cellulose, and have been commonly used substrates because of their molecular binding properties. The membranes have been used to bind cells, bio-polymers, proteins, genetic material and nucleic acids, as well as serving as substrates for non-biological chemicals.

The use of micro-porous polymeric membranes, in particular, nitrocellulose, for blotting bio-molecules from electrophoretically separated molecules was developed by Southern for DNA-DNA interactions. The technique is commonly called "Southern" blotting in honor of the developer. The other compass directions have been developed. "Western" blotting is a technique that has been employed to immobilize protein on an immobilizing substrate for protein-protein interactions.

Southern's need was for a method to identify the separated zones in electrophoretic separation. The blotting method employed micro-porous nitrocellulose to specifically identify the electrophoretically separated zones.

A brief outline of the original Southern blotting technique may help understand the general function of the nitrocellulose substrate: A sample, in this case containing DNA, is separated on a gel media by electrophoresis and is denatured by treatment with sodium hydroxide. A micro-porous nitrocellulose membrane is placed over the gel. Blotter paper is placed over the membrane to absorb the water from the gel and a weight is added on top. The weight forces the water and separated molecules into the micro-porous membrane as the gel collapses beneath the membrane. This leaves an image on the membrane comprised of the separated bio-molecules. DNA from the zones is bound non-specifically to the micro-porous nitrocellulose membrane. The nitrocellulose membrane is washed and blocked by diffusional methods. For performing an assay, the nitrocellulose membrane is then incubated with a solution of a known labeled DNA. If the DNA added is an exact match to a zone of the DNA immobilized from the electrophoretic separation, the labeled DNA will bind and the zone will be labeled. After successive washings, a visual image of the labeled zones then is prepared using X ray film if a radioactive label were used, thus identifying the zones.

Following the original development of Southern's techniques, in an effort to increase throughput, a trend developed to replace radioactive tracers with fluorescent tags, with the stimulated fluorescent emissions being imaged by optics. It was noticed, however, that available porous polymeric membranes, themselves, exhibited fluorescent emission over a wide spectral range. This fluorescent emission, as background noise, limited the use of polymeric membranes in fluorescent studies of proteins. While nitrocellulose membranes have been identified as one of the least offenders, still, when used as a substrate material, nitrocellulose has been found to have objectionable fluorescence that has limited both the accuracy of detection and the throughput of assays.

In the case of DNA, despite a continuing desire to employ polymeric membranes such as nitrocellulose, a way around the fluorescence problem was found, by spotting arrays on glass or quartz slides, that have relatively little background emission, using a layer of non-polymeric silane or GAP, and other such materials as adhesion promoters to which the biopolymer is directly bound. These adhesion promoter materials, despite their own significant problems, such as difficultly in obtaining a uniform thickness, noise contribution, and reactivity, have permitted significant success with small molecules. No similar technique has existed that is as effective for protein molecules, which are approximately 1000 times larger than DNA. Resort, still, has often been made to membranes of considerable thickness of porous nitrocellulose or other self-fluorescing polymeric immobilizing material, the material either being self-supporting or backed by a support. In the case of micro-porous nitrocellulose on a backing such as a microscope slide, typically the nitrocellulose has been at least 10 micron in thickness, and its self-fluorescence has remained a limiting factor for assay throughput. The significance to biology and to the clinician of the need to conduct higher throughput, large scale assays of protein arrays is discussed for instance in Chin et al., U.S. Pat. No. 6,197,599.

New insights are presented here about the substrates on which many of the known protein assays can be conducted. These insights lead broadly to techniques that increase throughput and achieve higher accuracy imaging of fluorescently- or luminenscently-labeled proteins and other bio-materials for large scale assays for research and for clinical diagnosis.

Some Prior Techniques with Nitrocellulose

Referring specifically to the practice of depositing spots of biological fluid on a solid or micro-porous surface to create a microarray, this has been widely described. In the case for instance of using glass slides bearing porous membranes of nitrocellulose, e.g. of 12 to 15 micron thickness, the supplier, Schleicher & Schuell, has recommended creation of each spot of the biological fluid with as much as 50 nl of fluid suspension, or more.

As a different nitrocellulose approach, using less suspension, spots composed of smaller amounts of liquid mixture of bio-molecules with nitrocellulose in a colloidal form or otherwise, have been formed on a glass or other support, where the nitrocellulose is either dissolved or in suspension in a common solvent. Additional solvents are introduced to cause desiccation of the deposit, resulting in a porous matrix that serves as an immobilizing structure for the intermixed biological material. This technique has been described by Pinkel in Nature Genetics, Volume 20, October, 1998, and by Audeh et al., U.S. Patent Application Publication 2002/0015958. These publications, involving very thin deposits or spots of the mixture of the bio-material with nitrocellulose on a support, have so far failed to advance the state of the practical art. These processes appear to have an inherent source of uncertainty or error, as they do not permit spot-to-spot evaluation of the contribution of the nitrocellulose or other immobilizing substrate material to the signal detected from the spots of fluorescently labeled proteins. In the technique, the fluorescent signal from a spot itself is necessarily the sum of the emission of the glass or other support, the porous nitrocellulose or other immobilizing material and the biological material itself. As these emissions are all combined, no simple method of separation exists for the signal from the biological material from such spots of biological/nitrocellulose material.

In more common assays with the available much thicker but continuous membranes of nitrocellulose mentioned, and with other immobilizing polymers, subtraction of perturbation noise is commonly used. Owing to the general uniformity of the thickness of such substrates across the supports, standardized software can measure the emitted noise signal from the unspotted membrane and automatically subtract a value approximately representing its noise signal from the total signal derived from the spot. In the case of the spotted Pinkel or Audeh et al. mixtures, the local vicinity beyond the spot does not contain a continuation of the substrate material that contributed to the fluorescent background at the spot, so the subtraction technique cannot be used to remove the effect of the substrate material. The magnitude of fluorescent energy as measured by the level of signal detected by a confocal scanning microscope such as the Affymetrix 428 Scanner has shown that a substantial error in the measurement of sample fluorescence can be introduced by the Pinkel or Audeh et al. process, and no successful variation of the technique that allows some form of subtraction has yet been found.

Ultra-Thin, Low-Noise Immobilizing Substrates

A new and different approach is presented to immobilizing and imaging fluorescently labeled biological materials. It employs a continuous, ultra-thin layer of a substrate of polymer that has biological binding properties, for instance, nitrocellulose. The technique is effective, using protein-immobilizing polymeric substrates, to enable imaging of fluorescently labeled proteins. The technique has other potential widespread uses, such as with proteins labeled with luminescent tags, and with other bio-materials labeled with fluorescent or luminescent tags. The technique may be used to advantage with viruses, peptides, antibodies, receptors, and other proteins; with a wide range of other labeled biological materials including plant, animal, human, fungal and bacteria cells; with nucleic acids, as a very practical substrate, with fewer problems than other materials, e.g. for cDNA clones, DNA probes, oligonucleotides including synthetic oligonucleotides and polymerase chain reaction (PCR) products; and with labeled chemicals as well. It has the attractive potential of being a low-cost, practical substrate of choice over the prior materials, such as GAP for instance, (GAP, because of high reactivity, requires costly and time-consuming precautions to avoid contamination.)

It has been found that a superior immobilizing substrate, suitable for receiving deposit of an array of spots of bio-polymers, is provided by a continuous ultra-thin layer of polymeric substrate material having biological binding properties, i.e., (a) in a 3-dimensional micro-porous form, an ultra-thin layer of thickness of less than 5 micron, down to less than a micron in thickness or approximately equal to the size of the pores, with pore size respectively from about 3 micron to 1/2 micron, or (b) as a solid surface coating, of a thickness less than 5 micron, less than 3 micron or thinner, down desirably to submicron thicknesses, e.g. 0.1 to 0.5 micron, or even as a molecular layer.

Polymer substrates of these dimensions are found useful to immobilize proteins and the broader categories of materials mentioned. Further, it is found that such continuous, ultra-thin polymer layers can readily be formed.

The uniform ultra-thin layer of biological-material-immobilizing polymer on a rigid substrate has been found to be capable of enduring the conditions of printing of spots of bio-polymers in precisely known positions, of conducting the assay, of application of successive washes, and, following handling, of being microscopically analyzed by stimulated emission. The ultra-thin layer is found to significantly reduce background noise attributable to parasitic fluorescence of the immobilizing material and to otherwise offer advantages due to considerations that will be described.

It is found also that a continuous ultra-thin layer can have such uniformity that it enables its signal contribution from its area lying beyond the spotted material to be subtracted from the measurement of the spots in a reliable manner, further increasing the quality of signals from that obtainable by prior techniques.

Those skilled with respect to protein-immobilizing membranes may have supposed that a significant depth of porous nitrocellulose or other protein-immobilizing substrate, i.e., 10 micron or more in available commercial products, would be important. Those skilled may have supposed that the significant depth of present commercial membranes was required to enable forming a uniform and durable membrane that could survive printing of spots, conducting the assay, applying successive washes and handling the unit through analysis, while still holding the deposited spots reliably in their precisely known places. Or, those skilled may have believed that current commercial thicknesses of the substrate were required to enable reliable manufacture, or to provide a liquid-receiving volume below the deposit sites to enable the carrier liquid to drain downwardly. Or, those skilled in the art may have believed a significant thickness of the immobilizing substrate was required, to provide a thickness-to-variance-in-thickness ratio sufficient to enable a reliable subtraction technique for correcting for auto-fluorescence, etc.

It has been found that no such requirements are in fact necessary. It has been found that durable, ultra-thin continuous substrates of polymer having biological binding properties can be readily fabricated of less than about 5 micron thickness, and that spot formation and precision of location is not adversely affected by the steps of spotting, assaying, washing, handling and analysis.

It has been found that, using coating techniques that are conventional for thin coatings in other contexts, the inherent variation in thickness of the polymer coating is sufficiently small, relative even to the small overall thickness of the ultra-thin substrate, that a signal from the adjoining unspotted area of the continuous coating may be used in the described subtractive techniques to enable acquisition of superior microscopy results.

It has been found that a solid (non-porous) film of 3 micron, down to thickness under 75 nm, even down to molecular thicknesses, of immobilizing polymer substrates, and in particular, of nitrocellulose, can be formed and successfully employed in high throughput protein and other bio-material assays.

It has also been realized that such ultra-thin substrates may be altered after forming as a coating or substrate, as by corona discharge, atomic particle or radiation bombardment or by controlled energy excimer laser beam treatment, to improve binding and immobilization topology or conditions.

In making such developments, the importance has been recognized of the fact that bio-molecules bind to the surface of the nitrocellulose or other immobilizing polymeric material, while parasitic fluorescence is emitted from the entire volume or bulk of the material illuminated by the inspection technique. When microarrays have been spotted on commercial membranes (10 micrometer or thicker membranes), the biological material normally accumulates at the outer portion of the thickness of a membrane supported on rigid non-porous support, e.g., on only 30% or 40% of the total thickness of the membrane. This is due to the fact that flow-through conditions for the bio-molecules are obstructed when a very small volume of volatile fluid supporting the biological matter is deposited. Bio-polymers and the carrying fluids wet the surface, the necessary phenomenon, and saturate the pores to a shallow depth and block further penetration as the liquid carrier separates, migrates beyond or evaporates.

The importance has been realized of the fact that polymeric bio-immobilizing materials such as nitrocellulose behave in an approximately linear manner and emit fluorescent radiation in relationship to the volume of material exposed to the excitation beam and the level of excitation and that a layer of the material of thickness limited to less than 5 micron and preferably less than 3 micron thick and in important cases even less than 1 micron thick can provide significant advantage.

In the case of ultra-thin micro-porous substrates provided here, the percentage of the volume of porous material that actually bears the biological material, relative to the total volume of the substrate material presented to the collecting optics, may be greater than 50% and advantageously in many case, greater than 75%, unnecessary volume of the material and its deleterious fluorescence being avoided.

Experimentally it has been determined that the parasitic emission of fluorescent light increases with the thickness of a micro-porous membrane with thickness of 1, 2, 3, 4, 7 and 14 micron.

It has further been realized that the parasitic fluorescence emission of a porous membrane of a given amount of nitrocellulose or other biology-binding polymeric material per unit area of the support structure can be many times greater (measured in one case to be approximately 6.4 times greater) than that of the same material presented as a transparent solid film. The nitrocellulose in its translucent/semi-opaque 3-dimensional porous membrane form is observed to absorb excitation radiation to a much greater degree than the same material in transparent semi-crystalline form. In addition, a relatively thicker porous membrane also reflects or scatters some of the excitation energy to a much greater degree than a thinner and especially, transparent, membrane.

Furthermore, it has been observed that an ultra-thin transparent solid membrane of polymer having biology binding properties, in reflecting a minimum amount of excitation energy, minimizes the exclusion requirement of the filter that is required in a collecting system to separate the fluorescent emission energy to be detected from the excitation energy.

It has also been recognized that the strength of the fluorescent radiation signal emitted toward the collecting optics by the fluorophore-tagged bio-polymers bound to the immobilizing polymeric material is not only a function of the quantity of the bio-polymer present and of the energy of the excitation source. It is also a function of the location of the emitting bio-molecules with respect to the top surface of the immobilizing medium. Bio-molecules may be located on the outer surface or buried to varying depths of a 3-dimensional membrane structure. Fluorophores attached to the molecules located within a 3-dimensional matrix below the outer surface are twice handicapped when compared to similar molecules on the outer surface. The energy intensity penetrating a semi-opaque, diffusive material, such as a highly porous polymeric material, decreases in function with the distance traveled and the absorption characteristics of the medium. In a similar manner, the stimulated fluorescent light from excited molecules buried within the matrix is absorbed to some degree or scattered before exiting to be collected by the optical system. The deeper a particular fluorophore is in a 3-dimensional porous polymeric structure, to some degree, the less intense will be its fluorescent emission at the collecting optics.

Accordingly, the novel, solid ultra-thin polymer film of immobilizing nitrocellulose or other bio-material-immobilizing polymer material is seen to be of considerable importance. It is recognized that a surface of solid nitrocellulose or other solid immobilizing polymer may usefully provide sufficient binding sites (for bio-polymers, cells or small fragments of tissue or other material to attach to), on a single plane, in a deposited spot of useful size for an array to be assayed. A number of binding sites equal to that of a surface folded in a small pore, 3 dimensional structure (such as that of a micro-porous polymer membrane) is obtainable with a solid coating by increased spot size. It is recognized that binding sites on a general plane in some ways offers better binding opportunity, e.g. equal opportunity for attachment of bio-polymer molecules to all sites, than is possible within an equivalent surface tightly folded in a 3-dimensional form. This is especially true in comparison to micro-porous polymer structures in the case where pore size may vary, and in cases where bio-deposits are dependent upon concentration, drying conditions, etc. of the spotted fluid. Especially for large protein molecules, this consideration is believed to be obtainable from assay to assay by performing assays of the proteins or other bio-polymers on solid, or modified solid ultra-thin coatings of immobilizing polymer material.

Continuous ultra-thin micro-porous polymer substrates, and solid substrates of bio-material-immobilizing polymer material, supported on glass, metal or plastic, used to immobilize fluorescently-tagged or luminenscently tagged bio-polymers, can achieve superior signal-to-noise ratio and other advantages that provide superior information or diagnostic efficacy.

Excellent analysis results have been obtained employing nitrocellulose as the ultra-thin micro-porous polymer material or as an ultra-thin solid polymer coating, while very desirable results are also realized to be obtainable with polystyrene. Other ultra-thin polymers that have biological binding properties may also be used e.g., cellulose acetate, cellulose triacetate, ethyl cellulose, activated nylon, polytetrafluoroethylene (PTFE), polyvinyl difluoride (PVDF), polyamides, polyvinylchloride, di-vinyl benzene and agarose, including copolymers and blends.

In one specific embodiment, a continuous micro-porous polymer matrix thinner than about 5 micron, and preferably as thin as 3, 2 or 1 micron or less, is provided to support bio-polymers under study. With the use of this structure, while the parasitic noise is reduced according to the thickness of the ultra-thin, polymeric substrate, it is found that the fluorescing signal is minimally reduced in comparison to use of the presently available commercial materials, the transferred volume of fluid not being appreciably altered in its course into the thickness of the ultra-thin material. Ordinarily the 3-dimensional matrix offers a larger binding surface (more bio-material binding sites) than the footprint of the same support.

Another important embodiment, however, is the ultra-thin, solid, i.e. non-porous, coating of polymer with biological binding properties, thinner than 5 micron, preferably less than 3 micron and preferably as thin as 2, 1, 0.5 or 0.1 micron, or even at molecular thicknesses, deposited on a rigid supporting medium of extremely low fluorescent properties, such as low fluorescence glass, fused quartz, ceramic, PMMA, polystyrene, other plastic or metal. The overall background-perturbing effect of such a bio-compatible substrate is preferably of the order of or even less than that of its supporting rigid structure.

In these embodiments, the number of photons necessary to obtain a statistically reliable signals dictates the spot area dedicated for attachment of the bio-polymers. Preferably, this area is approximately a spot with diameter greater than 100 micron in diameter and less than 1,000 micron, preferably less than 500 micron. In many cases, the preferred spot sizes of the bio-polymers are below about 500 micron, for instance 100 to 400 micron, 150 micron and 300 micron being common dot sizes. This enables the formation of suitable microarrays with provision for the needed sequence of dilutions and provision of process reference spots to enable large scale, high speed throughput of the assay and analysis. The system is more economical, with respect to amount of biological material required per spot, e.g. less than one nl per spot required, in comparison to prior art schemes of spotting on 10 or 12 micron or greater thickness micro-porous nitrocellulose on glass, for which a recommended amount by one supplier for an individual spot has been as high as 50 nl or higher.

Methods of manufacturing such ultra-thin, immobilizing polymer layers are provided that are found to produce particularly good substrates.

Novel methods are provided of depositing ultra-thin coatings of nitrocellulose or other immobilizing polymer, i.e. material having biological binding properties, on a suitable solid support, in many cases, a microscope glass slide having approximate dimensions of 25.times.75 mm, by about 1 mm thick, as will now be described.

Blank glass microscope slides are obtained, such as part No. 2951 from Erie Scientific Co. in Portland, N.H., with a short frosted section at one end. These are pretreated by applying a surface adhesion promoter and/or a layer permitting the application of indicia to the slide for important purposes such as identification and serialization, registration for microscopic analysis or other processes, provisional classification markings, or simply for presentation or branding. A number of choices can be made. Two preferred embodiments are: (a) A painted/covered region is applied over the frosted area with or without the addition of a 1 to 3 mm (and preferably 2 mm) wide frame tracking the outer periphery of the slide. The painted region over the frosted area may later be laser-marked with identification and serialization or other marking as desired. The process used may impart non-serialized markings. (b) A coating is applied over the entire slide on the frosted surface side by vapor deposition or sputter coating e.g. tantalum followed by air oxidation to form a thin layer of tantalum oxide in order to provide opacity ranging between 10% and 90% with respect to a nominal laser wavelength of 635 nm. The coated region over the frosted area is suitable later to be laser marked with identification and serialization or other marking including non-serialized markings. Also the region to be spotted may be divided into sub regions (or islands), e.g. by removing a circular or square moat of tantalum oxide surrounding each individual array. Such isolated region may serve to protect the spotted array portion from delaminating when an adhesive gasket is applied outside of the moat during experiments.

In an alternative manner laser marking and segmentation may be performed following coating.

Preferably a laser will be used with wavelength absorbed by the coating to be removed and not absorbed by the material of the support, glass or other.

Laser ablation of the coating over the frosted region may serialize the slides or add identification or registration markings for automatic optical unit or information retrieval. Ablation enhances data acquisition reliability in processes using a variety of equipment including commercial bar code readers.

Advantageously, after application of an adhesion promoting layer, at least one durable sensitivity calibration spot may be applied. The sensitivity calibration marking is provided to act as a fiduciary marking for geometrical reference, and by suitable choice of its material, serves as a standard fluorescence reference in order to determine and accommodate long-term variations in optical instrumentation.

Similar fluorescing calibration spots are applied on the outer surface of the completed substrate. In advantageous cases they are applied in a low-density pattern interspersed with high density biological spots, and used for calibration, or for normalization in instances where uneven excitation illumination may occur, e.g. when employing illumination at an angle to the normal to the plane of an array, as in imaging via dark-field reflectance mode.)

Preferably, the calibration compound is selected to have a broad fluorescence spectrum. A temporally stable material, such as polyimide polymers (Kapton), exhibiting broad band, standard fluorescence, i.e., yielding fluorescence at a wavelength in reliable manner, is selected as the reference fluorophore and deposited on a slide surface with solvent followed by solvent evaporation. Typical spot diameters may be 150 micron and 300 micron, and can be applied using commercially available biology printers (sometimes referred to as "spotters" or "microarraying instruments".) The precise amount of material deposited is unimportant since polymers such as Kapton are optically opaque, and detected fluorescent emission from the polymer occurs at or near the surface of the deposited material giving reproducible quantum yields.

The use of the calibration material applied to each slide allows for instrument self-calibration, i.e., auto calibration, per slide. A distribution of calibration spots, in number and spacing dependent on the non-uniformity of excitation illumination incident on a slide, may be employed. As few as six distributed in an array may suffice although larger numbers also are employed, depending upon the characteristics of the reader system.

A preferred process for preparing the glass microscope slides includes the removal of all particulates and most organic matter via mechanical means using solvents and detergents. The slides are subsequently left to dry in air. The active surface (as defined as the surface with frosted area) is then subjected to ozone treatment, e.g. to remove residual organic matter and enhance the adhesive properties of the surface. The ozone reactions may be activated using corona exposure or UV illumination. In a preferred embodiment, the ozone/corona treatment is induced by translating the slide at a speed between 2 and 8 cm/min (preferably 4.4 cm/min) past a corona discharge while exposing surface to be treated normal and approximately between 1 and 4 cm (preferably 2 cm) to the jet of a standard 2.5 cm. round head of a laboratory corona treater (model BD-20AC from Electro-Technic Products Inc., Chicago, Ill.) operating near its optimal level. Preferably the pressure, temperature, and humidity are held within the human comfort zone of 65.degree.-72.degree. F., one atmosphere, and humidity between 30 and 70%.

Alternately, such slides are cleaned of debris as well as of any biological products such as by washing them for approximately 30 minutes in an ultrasonic bath with a detergent and subsequently holding them in an oven at about 450.degree. C. for approximately 8 hours.

If the preferred embodiment of pretreatment (a) above is used, the slides are then coated with a less than 1 .mu. thick layer with colloidal silica or soluble silicate. For this purpose, LUDOX CL, LUDOX CL-X, or LUDOX TMA suspended in water is employed, available from Sigma-Aldrich Co. An equivalent product may be obtained from other sources. For this purpose, slides are held for 1 second to 1 hour in a bath of 1% to 10% (preferably 3.3%) colloidal silica and exhumed (drawn from the bath) preferably at a constant rate between 0.1 and 10 in/min (preferably 0.5 in/min), along a path parallel to the plane of the microscope slide to form a coating (referred to as a "drawn coating"). This is followed by drying in air. Preferably, the environmental pressure, tempera