Arrays of proteins and methods of use thereof

• US 20020110933A1
• Filed: 03/29/2002
• Published: 08/15/2002
• 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:
  US10/113,964
• Publication Number:
  US 20020110933A1
• Time in Patent Office: Days
• US Class Current: 436/518
• 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 for the parallel, in vitro screening of biomolecular activity are provided. Methods of using the protein arrays are also disclosed. On the arrays, a plurality of different proteins, such as different members of a single protein family, are immobilized on one or more organic thinfims on the substrate surface. The protein arrays are particularly useful in drug development, proteomics, and clinical diagnostics.

Claims

1. An array of proteins, comprising:

(a) a substrate;

(b) at least one organic thinfilm on some or all of the substrate surface; and

(c) a plurality of patches arranged in discrete, known regions on portions of the substrate surface covered by organic thinfilm, wherein each of said patches comprises a protein immobilized on the underlying organic thinfilm.

2. The array of claim 1 which comprises at least about 10 of said patches.

3. The array of claim 2 which comprises at least about 100 of said patches.

4. The array of claim 3 which comprises at least about 10³ of said patches.

5. The array of claim 1 which comprises at least about 10 different immobilized proteins.

6. The array of claim 5 which comprises at least about 100 different immobilized proteins.

7. The array of claim 6 which comprises at least about 1000 different immobilized proteins.

8. The array of claim 1, wherein the area of the substrate surface covered by each of the patches is no more than about 0.25 mm².

9. The array of claim 8, wherein the area of the substrate surface covered by each of the patches is between about 1 μ

m² and about 10,000 μ

m².

10. The array of claim 1, wherein the patches are all contained within an area of about 1 cm² or less on the surface of the substrate.

11. The array of claim 1, wherein all of the proteins immobilized on the array are functionally related.

12. The array of claim 1, wherein all of the proteins immobilized on the array are structurally related.

13. The array of claim 1, wherein all of the proteins immobilized on the array are members of the same family.

14. An array of claim 13, wherein said family is selected from the group consisting of growth factor receptors, hormone receptors, neurotransmitter receptors, catecholamine receptors, amino acid derivative receptors, cytokine receptors, extracellular matrix receptors, antibodies, lectins, cytokines, serpins, proteases, kinases, phosphatases, ras-like GTPases, hydrolases, steroid hormone receptors, transcription factors, heat-shock transcription factors, DNA-binding proteins, zinc-finger proteins, leucine-zipper proteins, homeodomain proteins, intracellular signal transduction modulators and effectors, apoptosis-related factors, DNA synthesis factors, DNA repair factors, DNA recombination factors, cell-surface antigens, hepatitis C virus (HCV) proteases and HIV proteases.

15. The array of claim 1, wherein the proteins are antibodies or antibody fragments.

16. The array of claim 1, wherein the proteins are protein-capture agents.

17. The array of claim 1, wherein the organic thinfilm on the array is less than about 20 nm thick.

18. The array of claim 1, wherein the organic thinfilm on the array comprises a monolayer.

19. The array of claim 18, wherein the monolayer comprises a self-assembled monolayer comprising molecules of the formula

(X)_aR(Y)_b

wherein R is a spacer, X is a functional group that binds R to the surface, Y is a functional group for binding the protein onto the monolayer, and a and b are, independently, integers.

20. The array of claim 19, wherein both a and b are 1.

21. The array of claim 19, wherein:

said substrate is selected from the group consisting of silicon, silicon dioxide, indium tin oxide, alumina, glass, and titania; and

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

22. The array of claim 19, wherein the substrate comprises silicon and X is an olefin.

23. The array of claim 1, wherein the substrate comprises a polymer.

24. The array of claim 19, further comprising at least one coating between said substrate and said monolayer, wherein said coating is formed on the substrate or applied to the substrate.

25. The array of claim 24, wherein:

the coating comprises a noble metal film; and

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.

26. The array of claim 24, wherein the coating comprises titania or tantalum oxide and X is a phosphate group.

27. The array of claim 1, wherein each protein is immobilized on the organic thinfilm by an affinity tag.

28. A biosensor comprising an array of proteins of claim 1.

29. A micromachined device comprising an array of proteins of claim 1.

30. A diagnostic device comprising an array of proteins of claim 1.

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

(a) delivering the sample to the array of claim 1 comprising the proteins to be screened; and

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

32. The method of claim 31, wherein the component is a protein.

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

(a) delivering said sample to the array of claim 1 comprising the proteins to be screened; and

(b) detecting, either directly or indirectly, for the presence or amount of said particular component retained at each patch.

34. The method of claim 33, wherein said particular component is a protein.

35. The method of claim 33, further comprising the step:

(d) further characterizing said particular component retained on at least one patch.

36. A method of assaying for protein-protein binding interactions, comprising:

(a) delivering a sample comprising at least one protein to be assayed for binding to the array of claim 1;

and

(b) detecting, either directly or indirectly, for the presence or amount of the protein from the sample which is retained at each patch.

37. A method of assaying in parallel for a plurality of analytes in a sample, comprising:

(a) delivering the sample to the array of claim 1, wherein at least one of the immobilized proteins of said array can react with each of said analytes; and

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

38. A method of assaying in parallel for a plurality of analytes in a sample, comprising:

(a) delivering the fluid sample to the array of claim 1, wherein at least one of the immobilized proteins of said array can bind each of said analytes; and

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

39. The method of claim 38, further comprising the step:

(d) further characterizing the analyte retained on at least one patch.