SUBSTRATE CHEMISTRY FOR PROTEIN IMMOBILIZATION ON A RIGID SUPPORT

• US 20080058224A1
• Filed: 10/15/2007
• Published: 03/06/2008
• Est. Priority Date: 02/27/2002
• Status: Abandoned Application

• Current Assignee: Stewart Lebrun
• Original Assignee: Stewart Lebrun
• Inventors: Lebrun, Stewart
• Application Number:
  US11/872,612
• Publication Number:
  US 20080058224A1
• Time in Patent Office: Days
• US Class Current: 506/26
• 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 2219/00385 Printing
• CPC Class Codes: B01J 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00533 essentially rectangular
• CPC Class Codes: B01J 2219/00576 fluorophore
• 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 2219/00637 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 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• 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: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 33/545 Synthetic resin
• CPC Class Codes: G01N 33/564 for pre-existing immune complex or autoimmune disease , i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9

Abstract

A device is disclosed for immobilization of proteins which includes a hydrophobic polymeric layer, preferably PVDF, attached to a rigid support. The proteins are spotted onto the dry surface of the PVDF. In preferred embodiments, the PVDF-coated rigid support advantageously provides greater ease of handling, protein immobilization in the dry state, high spot density, low background and high dynamic range. Methods of using the disclosed microarray support are also disclosed.

Claims

1. A method of preparing a protein array, comprising the steps of:

providing a substrate, comprising a rigid support and a PVDF layer attached to said rigid support by an adhesive; and

spotting a plurality of protein samples onto the PVDF layer at discrete, addressable locations, wherein the PVDF layer is in a dry state.

2. The method of claim 1, wherein the dry PVDF layer is exposed during protein spotting.

3. The method of claim 1, wherein the thickness of the PVDF layer is about 100-250 μ

m.

4. The method of claim 1, wherein the PVDF layer has a pore size of 0.2 to 0.45 μ

m.

5. The method of claim 1, wherein the PVDF layer is a sheet, a membrane, or is formed from PVDF pellets.

6. The method of claim 1, wherein the PVDF layer produces a background signal upon protein imaging using visual or fluorescent light of less than about 100 lumens.

7. The method of claim 1, wherein the PVDF layer produces a background signal upon protein imaging using visual or fluorescent light of between about 0 and 50 lumens.

8. The method of claim 1, wherein the PVDF layer produces a background signal upon protein imaging using visual or fluorescent light of between about 0 and 15 lumens.

9. The method of claim 1, wherein the rigid support is a silanated material.

10. The method of claim 1, wherein the rigid support is glass or plastic.

11. The method of claim 1, wherein the substrate further comprises a bar code.

12. The method of claim 1, wherein the substrate further comprises a removable protective film for protecting the PVDF layer prior to and/or after protein spotting.

13. The method of claim 1, wherein the substrate further comprises a template attached to the rigid support, wherein the template divides the PVDF layer into at least two distinct sections, and wherein the template is configured to allow application of different chemical reagents to the at least two distinct sections.

14. A method of making a substrate for immobilizing a plurality of proteins, comprising:

applying an adhesive layer to a rigid support;

adhering a PVDF membrane sheet to the adhesive layer on the rigid support;

allowing the adhesive layer to dry; and

cutting any excess PVDF membrane sheet away from the rigid support to form the substrate.

15. The method of claim 14, wherein the adhesive is selected from the group consisting of a double-sided inert adhesive microfilm, a silicone, a glue, a double-sided tape and direct chemical bonding.

16. The method of claim 14, wherein the rigid support is glass or plastic.

17. The method of claim 14, further comprising attaching a template to the substrate.

18. The method of claim 17, wherein the template provides from about 2-500 wells.