Light-mediated method and apparatus for the regional analysis of biologic material

• US 6,159,681 A
• Filed: 05/27/1999
• Issued: 12/12/2000
• Est. Priority Date: 05/28/1997
• Status: Expired due to Term

• Current Assignee: Syntrix Biosystems Incorporated
• Original Assignee: Syntrix Biochip Incorporated
• Inventors: Zebala, John A.
• Primary Examiner(s): Chin, Christopher L.
• Assistant Examiner(s): PHAM, MINH QUAN K
• Application Number:
  US09/322,060
• Time in Patent Office: 565 Days
• Field of Search: 435/4, 435/5, 435/6, 435/7.1, 435/7.2, 435/7.21, 435/7.71, 435/40.5, 435/40.52, 435/176, 430/56, 430/60, 436/518
• US Class Current: 435/4
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00675 In-situ synthesis on the substrate
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B01J 2219/00729 Peptide nucleic acids [PNA]
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6809 : Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6841 : In situ hybridisation
• CPC Class Codes: C12Q 2521/331 : Methylation site specific nuclease
• CPC Class Codes: C12Q 2531/113 : PCR
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 50/14 : Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 33/5008 : for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5014 : for testing toxicity
• CPC Class Codes: G01N 33/502 : for testing non-proliferative effects
• CPC Class Codes: G01N 33/5044 : involving specific cell types
• CPC Class Codes: G01N 33/5047 : Cells of the immune system
• CPC Class Codes: G01N 33/5088 : of vertebrates
• CPC Class Codes: G01N 33/5091 : for testing the pathological state of an organism
• CPC Class Codes: G01N 33/544 : the carrier being organic
• CPC Class Codes: G01N 33/552 : Glass or silica
• CPC Class Codes: G03F 7/00 : Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor using photoresist structures for special production processes, see the relevant places, e.g. B44C, H01L, e.g. H01L21/00, H05K

Abstract

Compositions and methods are provided for performing regional analysis of biologic materials. The methods provided herein employ a photoresist layer that is established over a biologic material (which may be immobilized on a substrate). Regions of interest are selected and irradiated to expose specific regions of biologic material. Exposed biologic material may then be selectively analyzed using any of a variety of analytic methods.

Claims

1. A method for analyzing a discrete region of a biologic material, comprising the steps of:

(a) irradiating photoresist coated on a first region of a biologic material, wherein the biologic material is immobilized on a substrate, such that;

(i) photoresist coated on the first region is substantially removed and photoresist coated on a second region of the biologic material is not substantially removed, resulting in exposed biologic material in the first region;

or

(ii) photoresist coated on a second region is substantially removed and photoresist coated on the first region of the biologic material is not substantially removed, resulting in exposed biologic material in the second region; and

(b) determining the presence or absence of a substance of interest in the exposed biologic material; and

therefrom analyzing a discrete region of the biologic material for the presence or absence of a substance of interest.

2. A method according to claim 1, wherein the photoresist comprises a polyphenolformaldehyde polymer and a diazoquinone.

3. A method according to claim 1, wherein step (b) comprises the steps of:

(i) contacting the exposed biologic material with a detection reagent that generates a detectable response indicative of the presence or absence of a substance of interest in the exposed biologic material; and

(ii) detecting a response generated by the detection reagent.

4. A method according to claim 3, wherein the detection reagent comprises an antibody, nucleic acid sequence or enzyme.

5. A method according to claim 3, wherein the detection reagent comprises a marker.

6. A method according to claim 5, wherein the marker is a chromophor, fluorophor or radionuclide.

7. A method according to claim 1, wherein step (b) comprises an assay selected from the group consisting of electrophoresis, chromatography, mass spectrometry, DNA sequencing, peptide sequencing nucleic acid hybridization and PCR.

8. A method according to claim 1, wherein the step of irradiating further comprises contacting the photoresist with a developer.

9. A method according to claim 1, wherein photoresist is removed from at least 100 discrete regions.

10. A method according to claim 1, wherein photoresist is removed from at least 10,000 discrete regions.

11. A method according to claim 1, wherein the first and second regions each have an area of less than 10,000 square microns.

12. A method according to claim 1, wherein the first and second regions each have an area of less than 100 square microns.

13. A method according to claim 1, wherein the first and second regions each have an area of less than 1 square micron.

14. A method according to claim 1, wherein the substrate comprises a material selected from the group consisting of glass, germanium, silicon, silicon nitride, silicon oxide, gallium phosphide, gallium arsenide, polystyrene, (poly)tetrafluorethylene.

15. A method according to claim 1, wherein the biologic material is immobilized on the substrate via a linker.

16. A method according to claim 1, wherein the irradiation is selected from the group consisting of coherent light, incoherent light, x-ray light, ultraviolet light, visible light and infrared light.

17. A method according to claim 1, wherein the biologic material comprises one or more biological tissues, cells or viruses.

18. A method according to claim 1, wherein the biologic material comprises at least one compound selected from the group consisting of nucleic acid molecules, peptide nucleic acids, proteins, peptides, polysaccharides, monosaccharides, lectins and lipids.

19. A method according to claim 1, wherein the substance of interest is a cell or virus.

20. A method according to claim 1, wherein the substance of interest is a compound selected from the group consisting of nucleic acid molecules, proteins, peptides, polysaccharides, monosaccharides, lectins and lipids.

21. A method according to claim 1, wherein prior to or concurrently with the step of irradiation, the photoresist is illuminated with a light that does not substantially react with the photoresist.

22. A method according to claim 1, wherein the first region is irradiated by:

(i) placing a mask between the photoresist and a source of irradiation, wherein the mask has at least one region that is substantially transparent to the irradiation and at least one region that is substantially opaque to the irradiation; and

(ii) irradiating the mask with the irradiation such that irradiation passes through the mask to photoresist coated on the first region.

23. A method for analyzing a discrete region of a biologic material, comprising the steps of:

(a) irradiating photoresist coated on a first region of a biologic material, wherein the biologic material is immobilized on a substrate, such that;

(i) photoresist coated on the first region is substantially removed and photoresist coated on a second region of the biologic material is not substantially removed, resulting in exposed biologic material in the first region;

or

(ii) photoresist coated on a second region is substantially removed and photoresist coated on the first region of the biologic material is not substantially removed, resulting in exposed biologic material in the second region;

(b) contacting the exposed biologic material with an ablative agent, such that a substance of interest is not detectable in the exposed biologic material;

(c) substantially removing remaining photoresist, exposing remaining biologic material; and

(d) determining the presence or absence of a substance of interest in the remaining biologic material; and

therefrom analyzing a discrete region of the biologic material for the presence or absence of a substance of interest.

24. A method according to claim 23, wherein the ablative agent is selected from the group consisting of oxidants, free radicals, non-specific nucleases, non-specific ribonucleases, peptide nucleic acid clamps, high energy particles, extremes of radiation, ultraviolet irradiation and combinations thereof.

25. A method according to claim 23, wherein the photoresist comprises a polyphenolformaldehyde polymer and a diazoquinone.

26. A method according to claim 23, wherein step (b) comprises the steps of:

(i) contacting the exposed remaining biologic material with a detection reagent that generates a detectable response indicative of the presence or absence of a substance of interest in the exposed remaining biologic material; and

(ii) detecting a response generated by the detection reagent.

27. A method according to claim 26, wherein the detection reagent comprises an antibody, nucleic acid sequence or enzyme.

28. A method according to claim 26, wherein the detection reagent comprises a marker.

29. A method according to claim 28, wherein the marker is a chromophor, fluorophor or radionuclide.

30. A method according to claim 23, wherein step (b) comprises an assay selected from the group consisting of electrophoresis, chromatography, mass spectrometry, DNA sequencing, peptide sequencing, nucleic acid hybridization and PCR.

31. A method according to claim 23, wherein the step of irradiating further comprises contacting the photoresist with a developer.

32. A method according to claim 23, wherein photoresist is removed from at least 100 discrete regions.

33. A method according to claim 23, wherein photoresist is removed from at least 10,000 discrete regions.

34. A method according to claim 23, wherein the first and second regions each have an area of less than 10,000 square microns.

35. A method according to claim 23, wherein the first and second regions each have an area of less than 100 square microns.

36. A method according to claim 23, wherein the first and second regions each have an area of less than 1 square micron.

37. A method according to claim 23, wherein the substrate comprises a material selected from the group consisting of glass, germanium, silicon, silicon nitride, silicon oxide, gallium phosphide, gallium arsenide, polystyrene, (poly)tetrafluorethylene.

38. A method according to claim 23, wherein the biologic material is immobilized on the substrate via a linker.

39. A method according to claim 23, wherein the irradiation is selected from the group consisting of coherent light, incoherent light, x-ray light, ultraviolet light, visible light and infrared light.

40. A method according to claim 23, wherein the biologic material comprises one or more biological tissues, cells or viruses.

41. A method according to claim 23, wherein the biologic material comprises at least one compound selected from the group consisting of nucleic acid molecules, peptide nucleic acids, proteins, peptides, polysaccharides, monosaccharides, lectins and lipids.

42. A method according to claim 23, wherein the substance of interest is a cell or virus.

43. A method according to claim 23, wherein the substance of interest is a compound selected from the group consisting of nucleic acid molecules, proteins, peptides, polysaccharides, monosaccharides, lectins and lipids.

44. A method according to claim 23, wherein prior to or concurrently with the step of irradiation, the photoresist is illuminated with a light that does not substantially react with the photoresist.

45. A method according to claim 23, wherein the first region is irradiated by:

(i) placing a mask between the photoresist and a source of irradiation, wherein the mask has at least one region that is substantially transparent to the irradiation and at least one region that is substantially opaque to the irradiation; and

(ii) irradiating the mask with the irradiation such that irradiation passes through the mask to photoresist coated on the first region.

46. A method for analyzing a discrete region of a biologic material, comprising the steps of:

(a) irradiating positive photoresist coated on a first region of a biologic material, wherein the biologic material is immobilized on a substrate, such that photoresist coated on the first region is substantially removed and photoresist coated on a second region of the biologic material is not substantially removed, resulting in exposed biologic material in the first region; and

(b) determining the presence or absence of a substance of interest in the exposed biologic material; and

therefrom analyzing a discrete region of the biologic material for the presence or absence of a substance of interest.

47. A method for analyzing a discrete region of a biologic material, comprising the steps of:

(a) irradiating negative photoresist coated on a first region of a biologic material, wherein the biologic material is immobilized on a substrate, and wherein the step of irradiating comprises contacting photoresist on at least a second region of the biological material with a developer such that photoresist coated on the second region is substantially removed and photoresist coated on the first region is not substantially removed, resulting in exposed biologic material in the second region; and

(b) determining the presence or absence of a substance of interest in the exposed biologic material; and

therefrom analyzing a discrete region of the biologic material for the presence or absence of a substance of interest.

48. A method for analyzing a discrete region of a biologic material, comprising the steps of:

(a) irradiating positive photoresist coated on a first region of a biologic material, wherein the biologic material is immobilized on a substrate, such that photoresist coated on the first region is substantially removed and photoresist coated on a second region of the biologic material is not substantially removed, resulting in exposed biologic material in the first region;

(b) contacting the exposed biologic material with an ablative agent, such that a substance of interest is not detectable in the exposed biologic material;

(c) substantially removing remaining photoresist, exposing remaining biologic material; and

(d) determining the presence or absence of a substance of interest in the remaining biologic material; and

therefrom analyzing a discrete region of the biologic material for the presence or absence of a substance of interest.

49. A method for analyzing a discrete region of a biologic material, comprising the steps of:

(a) irradiating negative photoresist coated on a first region of a biologic material, wherein the biologic material is immobilized on a substrate, and wherein the step of irradiating comprises contacting photoresist on at least a second region of the biological material with a developer such that photoresist coated on the second region is substantially removed and photoresist coated on the first region is not substantially removed, resulting in exposed biologic material in the second region;

(b) contacting the exposed biologic material with an ablative agent, such that a substance of interest is not detectable in the exposed biologic material;

(c) substantially removing remaining photoresist, exposing remaining biologic material; and

(d) determining the presence or absence of a substance of interest in the remaining biologic material; and

therefrom analyzing a discrete region of the biologic material for the presence or absence of a substance of interest.