Protein arrays for high-throughput screening

• US 20020106702A1
• Filed: 03/29/2002
• Published: 08/08/2002
• Est. Priority Date: 07/14/1998
• Status: Abandoned Application

• Current Assignee: Christian Itin, Dana Ault-Riche, Peter Wagner, Steffen Nock
• Original Assignee: Christian Itin, Dana Ault-Riche, Peter Wagner, Steffen Nock
• Inventors: Wagner, Peter, Itin, Christian, Nock, Steffen, Ault-Riche, Dana
• Application Number:
  US10/112,840
• Publication Number:
  US 20020106702A1
• Time in Patent Office: Days
• US Class Current: 435/7.9
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00635 by reactive plasma treatment
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07K 2319/20 containing a tag with affinity for a non-protein ligand
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• CPC Class Codes: G01N 33/551 the carrier being inorganic
• CPC Class Codes: G01N 33/6845 : Methods of identifying protein-protein interactions in protein mixtures

Abstract

Protein arrays and protein-coated substrates for the parallel, in vitro screening of biomolecular activity are provided. Methods of using the protein-coated substrates and protein arrays are also disclosed. A plurality of different members of a single protein family may be immobilized on the protein-coated substrate or array. The protein-coated substrates and protein arrays are particularly useful in high-throughput drug screening and clinical diagnostics.

Claims

1. A protein-coated substrate, comprising:

(a) a substrate; and

(b) a plurality of patches arranged in discrete, known regions on said substrate, wherein each of said patches comprises an immobilized protein with a different, known sequence and wherein each of said patches is separated from neighboring patches by from about 50 nm to about 500 μ

m.

2. A biosensor comprising a protein-coated substrate of claim 1.

3. A micromachined device comprising a protein-coated substrate of claim 1.

4. A medical device comprising a protein-coated substrate of claim 1.

5. An array of proteins, comprising:

(a) a substrate; and

(1)) a plurality of patches arranged in discrete known regions on said substrate, wherein each of said patches comprises an immobilized protein with a different, known sequence and wherein each of said patches is separated from neighboring patches by from about 50 nm to about 500 μ

m.

6. An array of claim 5, wherein said proteins are functionally related or are suspected of being functionally related.

7. An array of claim 5, wherein said proteins are structurally related or are suspected of being structurally related.

8. An array of claim 5, wherein said proteins are members of the same family.

9. An array of claim 5, wherein the proteins are selected from the group consisting of growth factor receptors, catecholamine receptors, amino acid derivative receptors, cytokine receptors, extracellular matrix receptors, immunoglobulins, lectins, cytokines, serpins, proteases, kinases, phosphatases, ras-like GTPases, hydrolases, steroid hormone receptors, heat-shock transcription factors, zinc-finger proteins, leucine-zipper proteins, homeodomain proteins, hepatitis C virus (HCV) proteases and HIV proteases.

10. An array of claim 5, wherein said patches further comprise a monolayer on the surface of the substrate and wherein the proteins of said patches are immobilized on said monolayer.

11. An array of claim 10, wherein the monolayer of an individual patch comprises molecules of the formula

X-R-Y

wherein R is a spacer, X is a functional group that binds R to the surface, and Y is a functional group for binding proteins onto the monolayer.

12. An array of claim 11,

wherein said substrate is selected from the group consisting of silicon, silicon oxide, indium tin oxide, magnesium oxide, alumina, quartz, glass, and silica,

wherein X, prior to incorporation into said monolayer, is selected from the group consisting of a monohalosilane, dihalosilane, trihalosilane, trichlorosilane, trialkoxysilane, dialkoxysilane, and monoalkoxysilane,

wherein R is an alkyl from about 8 to 22 carbons long, and

wherein Y comprises a functional group selected from the group consisting of a maleimide, N-hydroxysuccinimide, nitrilotriacetic acid, activated hydroxyl, haloacetyl, bromoacetyl, iodoacetyl, activated carboxyl, hydrazide, epoxy, aziridine, trifluoromethyldiaziridine, pyridyldisulfide, N-acyl-imidazole, imidazolecarbamate, succinimidylcarbonate, arylazide, anhydride, diazoacetate, benzophenone, isothiocyanate, isocyanate, imidoester, fluorobenzene, and biotin.

13. An array of claim 10, further comprising a coating between said substrate and said monolayer, wherein said coating is formed on the substrate or applied to the substrate.

14. An array of claim 13, wherein the mean roughness of said coating is less than about 5 angstroms for areas of at least 25 μ

m².

15. An array of claim 14, wherein the mean roughness of said coating is less than about 3 angstroms for areas of at least 25 μ

m².

16. An array of claim 14, wherein said coating is an ultraflat, template-stripped surface.

17. An array of claim 13,

wherein said coating comprises a metal film,

wherein X prior to incorporation into said monolayer, is a functional group selected from the group consisting of an asymmetrical or symmetrical disulfide, sulfide, diselenide, selenide, thiol, isonitrile, selenol, trivalent phosphorus compounds, isothiocyanate, isocyanate, xanthanate, thiocarbamate, phosphines, amines, thio acid and dithio acid,

wherein R is an alkyl chain from about 8 to about 22 carbons long, and

wherein Y comprises a functional group selected from the group consisting of a maleimide, N-hydroxysuccinimide, nitrilotriacetic acid, activated hydroxyl, haloacetyl, bromoacetyl, iodoacetyl, activated carboxyl, hydrazide, epoxy, aziridine, trifluoromethyldiaziridine, pyridyldisulfide, N-acyl-imidazole, imidazolecarbamate, succinimidylcarbonate, arylazide, anhydride, diazoacetate, benzophenonee, isothiocyanate, isocyanate, imidoester, fluorobenzene, and biotin.

18. An array of claim 11, wherein the monolayer of an individual patch comprises at least two different X-R-Y molecules.

19. An array of claim 11, wherein the monolayer of an individual patch further comprises a second molecule, wherein said second molecule is of the formula

X-R-V

wherein R is spacer, X is a functional group that binds R to the surface, and V is a moiety resistant to the non-specific binding of proteins.

20. An array of claim 11, wherein said array further comprises at least one patch comprising a monolayer of molecules of the formula

X-R-V

wherein R is a spacer, X is a functional group that binds R to the surface, and V is a moiety resistant to the non-specific binding of proteins.

21. An array of claim 11, further comprising crosslinking between molecules of the monolayer of a patch.

22. An array of claim 10, further comprising an affinity tag, wherein said affinity tag enhances site-specific immobilization of the biological moiety onto the monolayer.

23. An array of claim 22, wherein said affinity tag comprises an unnatural amino acid.

24. An array of claim 22, wherein said affinity tag comprises a poly(amino acid).

25. An array of claim 24, wherein said poly(amino acid) is selected from the group consisting of poly-cysteine, polylysine, poly-arginine, and poly-histidine.

26. An array of claim 22, wherein said affinity tag comprises a polypeptide or a protein.

27. An array of claim 22, wherein the affinity tag and the biological moiety together compose a fusion protein.

28. An array of claim 22, further comprising an adaptor molecule that links the affinity tag to the immobilized biological moiety.

29. An array of claim 28, wherein said adaptor molecule is a polypeptide or a protein.

30. An array of claim 16, wherein said adaptor molecule is selected from the group consisting of green fluorescent protein, glutathione S-transferase, maltose-binding protein chitin-binding protein, and thioredoxin.

31. An array of claim 28, wherein the affinity tag, adaptor molecule, and biological moiety together compose a fusion protein.

32. A method for screening a plurality of proteins for their ability to interact with a component of a fluid sample, comprising:

(a) delivering the fluid sample to the array of claim 5; and

(b) detecting, either directly or indirectly, the interaction of said component with the immobilized protein of each patch.

33. A method for screening a plurality of proteins for their ability to bind a component of a fluid sample, comprising:

(a) delivering said fluid sample to the array of claim 5;

(b) washing the array with fluid which does not contain said component in order to elute unbound component; and

(c) detecting, either directly or indirectly, the presence of said component retained at each patch.

34. A method for detecting in a fluid sample the presence of a plurality of analytes which react with said proteins, comprising:

(a) delivering the fluid sample to the array of claim 5; and

(b) detecting the interaction of the analyte with the immobilized protein at each patch.

35. A method for detecting in a fluid sample the presence of a plurality of analytes which bind said proteins, comprising:

(a) delivering the fluid sample to the array of claim 5;

(c) washing said array with an analyte-free fluid to remove unbound analyte; and

(b) detecting, either directly or indirectly, the presence of analyte retained at each patch.

37. A protein-coated substrate of claim 36,

wherein said substrate is selected from the group consisting of silicon, silicon oxide, indium tin oxide, magnesium oxide, alumina, quartz, glass, and silica,

wherein X, prior to incorporation into said monolayer, is selected from the group consisting of a monohalosilane, dihalosilane, trihalosilane, trichlorosilane, trialkoxysilane, dialkoxysilane, and monoalkoxysilane,

wherein R is an alkyl from about 8 to 22 carbons long, and

wherein Y comprises a functional group selected from the group consisting of a maleimide, N-hydroxysuccinimide, nitrilotriacetic acid, activated hydroxyl, haloacetyl, bromoacetyl, iodoacetyl activated carboxyl, hydrazide, epoxy, aziridine, trifluoromethyldiaziridine, pyridyldisulfide, N-acyl-imidazole, imidazolecarbamate, succinimidylcarbonate, arylazide, anhydride, diazoacetate, benzophenone, isothiocyanate, isocyanate, imidoester, fluorobenzene, and biotin.

38. A protein-coated substrate of claim 36, further comprising a coating between said substrate and said monolayer, wherein said coating is formed on the substrate or applied to the substrate.

39. A protein-coated substrate of claim 38,

wherein said coating comprises a metal film,

wherein X prior to incorporation into said monolayer, is a functional group selected from the group consisting of an asymmetrical or symmetrical disulfide, sulfide, diselenide, selenide, thiol, isonitrile, selenol, trivalent phosphorus compounds, isothiocyanate, isocyanate, xanthanate, thiocarbamate, phosphines, amines, thio acid and dithio acid,

wherein R is an alkyl chain from about 8 to about 22 carbons long, and

wherein Y comprises a functional group selected from the group consisting of a maleimide, N-hydroxysuccinimide, nitrilotriacetic acid, activated hydroxyl, haloacetyl, bromoacetyl, iodoacetyl, activated carboxyl, hydrazide, epoxy, aziridine, trifluoromethyldiaziridine, pyridyldisulfide, N-acyl-imidazole, imidazolecarbamate, succinimidylcarbonate, arylazide, anhydride, diazoacetate, benzophenone, isothiocyanate, isocyanate, imidoester, fluorobenzene, and biotin.

40. A protein-coated substrate of claim 36,

wherein said affinity tag comprises a poly(amino acid), and

wherein said fusion protein further comprises said affinity tag.

41. A protein-coated substrate of claim 36, wherein said adaptor polypeptide is selected from the group consisting of green fluorescent protein, glutathione S-transferase, maltose-binding protein, chitin-binding protein, and thioredoxin.

36. A protein-coated substrate comprising:

(a) a substrate;

(b) a monolayer on a portion of a surface of the substrate, said monolayer comprising molecules of the formula

X-R-Y

wherein R is a spacer, X is a functional group that binds R to the surface, and Y is a functional group for binding a fusion protein onto the monolayer;

(c) an affinity tag, wherein said affinity tag enhances site-specific immobilization of a fusion protein onto the monolayer; and

(d) a fusion protein immobilized on said monolayer by means of the affinity tag, wherein the fusion protein comprises a first polypeptide linked to a second, adaptor, polypeptide and wherein the second, adaptor, polypeptide is linked to the affinity tag.