Polymer brushes for immobilizing molecules to a surface or substrate having improved stability

• US 7,205,161 B2
• Filed: 01/10/2002
• Issued: 04/17/2007
• Est. Priority Date: 01/10/2001
• Status: Expired due to Fees

• Current Assignee: Freeslate Inc. (Unchained Labs Inc.)
• Original Assignee: Symyx Technologies Incorporated (Dassault Systemes SA)
• Inventors: Benoit, Didier, Charmot, Dominique, Klaerner, Gerrit, Mazzola, Laura T., Piotti, Marcelo Eduardo, Nomula, Srinivas
• Primary Examiner(s): CEPERLEY, MARY
• Application Number:
  US10/043,394
• Publication Number:
  US 20030108879A1
• Time in Patent Office: 1,923 Days
• Field of Search: 435/6, 436/531, 436/532, 436/527, 530/391.1, 530/402, 530/404, 530/405, 530/406
• US Class Current: 436/531
• CPC Class Codes: C08F 293/005 using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393

Abstract

Sensors for determining the presence and concentration of biomolecules in a biological sample are provided in the form of polymer brushes, which comprise a substrate having a surface modified with a hydrophobic polymer segment, attached to which is a water-dispersible or water-soluble polymer segment having functional groups that bind probes. The method of synthesis of such sensors preferably includes use of controlled free radical polymerization techniques, which allows for controlled architecture polymers to modify the surface of the substrate, and the use of monomers possessing functional groups which do not require activation prior to probe attachment. In this manner functional groups in the polymer chain are removed from the surface, which allows for solution chemistry to be more realistically reproduced with the benefits of a solid bound probe.

Claims

1. A polymer brush for binding a molecule in an aqueous sample in an assay, the brush comprising a substrate surface, a hydrophobic layer comprising hydrophobic polymer chain segments attached to the substrate surface and having a dry thickness of at least about 50 angstroms, and a hydrophilic layer attached to the hydrophobic layer containing functional groups for the attachment of a probe for binding the molecule.

2. The polymer brush of claim 1 wherein the hydrophobic layer has a dry thickness of at least about 100 angstroms.

3. The polymer brush of claim 1 wherein the hydrophobic layer has a dry thickness of at least about 1000 angstroms.

4. The polymer brush of claim 1 wherein the hydrophobic layer has a dry thickness of at least about 2000 angstroms.

5. The polymer brush of claim 1 wherein said hydrophobic polymer chain segments comprise repeat units derived from a hydrophobic monomer having a log P value of at least about 1.

6. The polymer brush of claim 1 wherein, in addition to said hydrophobic polymer chain segments, spacer molecules are also attached to the substrate surface, the ratio of said hydrophobic polymer chain segments to the sum of hydrophobic polymer chains segments and spacer molecules ranging from about 0.2:

1 to about 0.8;

1.

7. The polymer brush of claim 1 wherein at least a portion of the hydrophobic polymer chain segments are crosslinked to other hydrophobic polymer chain segments in the hydrophobic layer.

8. The polymer brush of claim 1 wherein the hydrophilic layer comprises water-soluble or water-dispersible polymer chain segments having groups for the attachment of a probe, wherein an end of each segment is attached to the hydrophobic layer, and further wherein said water-soluble or water-dispersible segments have a weight average molecular weight of at least 1,000.

9. The polymer brush of claim 8 wherein the water-soluble or water-dispersible segments have a weight average molecular weight of at least 1,000 but no more than 5,000,000.

10. The polymer brush of claim 1 wherein the hydrophilic layer comprises water-soluble or water-dispersible polymer chain segments having groups for the attachment of a probe, said segments comprising repeat units derived from a water-soluble or water-dispersible monomer having a log P value of less than about 1.

11. The polymer brush of claim 1 wherein the hydrophilic layer comprises water-soluble or water-dispersible polymer chain segments having groups for the attachment of a probe, said segments comprising repeat units derived from a water-soluble or water-dispersible monomer having a log P value of less than about 0.1.

12. The polymer brush of claim 10 wherein in an absolute difference between a log P value of the hydrophobic monomers and the water-soluble or water-dispersible monomers is at least about 1.

13. The polymer brush of claim 10 wherein in an absolute difference between a log P value of the hydrophobic monomers and the water-soluble or water-dispersible monomers is at least about 2.

14. The polymer brush of claim 1 wherein the hydrophobic layer comprises hydrophobic polymer chain segments, one end of each of said hydrophobic segments being attached to the substrate surface, and further wherein the hydrophilic layer comprises water-soluble or water-dispersible polymer chain segments having groups for the attachment of a probe, one end of each of said water-soluble or water-dispersible segments being attached to the hydrophobic layer, the ratio of water-soluble or water-dispersible segments to hydrophobic segments being less than about 1:

1.

15. The polymer brush of claim 1 wherein the hydrophilic layer has a dry thickness ranging from about 10 angstroms to about 2000 angstroms.

16. The polymer brush of claim 1 additionally comprising a probe attached to the functional groups for binding the molecule.

17. The polymer brush of claim 16 wherein said groups for the attachment of a probe are capable of attaching said probe without first being subjected to a chemical treatment to activate said groups for probe attachment.

18. The polymer brush of claim 16 wherein the molecule is a biological molecule and the probe is selected from the group consisting of nucleic acids, polypeptides, peptide nucleic acids, markers, cells, elastin, collagen, carbohydrates, enzymes, lipids, phospholipids, hormones, drug targets, phosphates, and metal ions.

19. The polymer brush of claim 16 wherein said functionalized groups are selected from the group consisting of hydroxy groups, amino groups, carboxylic acids, carboxylic acid derivatives, and thiols.

20. The polymer brush of claim 1 wherein the hydrophilic layer is substantially free of crosslinks.

30. The polymer brush of claim 1 wherein the hydrophobic polymer chain segments are covalently bound to the substrate surface.

31. The polymer brush of claim 30 wherein the hydrophilic layer comprises water-soluble or water-dispersible polymer chain segments, and further wherein an end of each of said hydrophilic polymer chain segments is covalently bound to a hydrophobic polymer chain segment.

21. A polymer brush for binding a molecule in an aqueous sample in an assay, the brush comprising a substrate surface having a polymer layer thereon, said polymer layer comprising a first hydrophobic layer attached to the substrate surface, and a second hydrophilic layer attached to the hydrophobic layer containing groups for the attachment of a probe for binding the molecule, said brush being characterized in that said hydrophobic polymer layer has a minimum thickness such that, upon being immersed in a 10 mmolar sodium hydroxide solution for about 15 minutes, the polymer layer thickness is reduced by less than about 40%.

22. The polymer brush of claim 21 wherein the polymer layer thickness is reduced by less than about 20%.

23. The polymer brush of claim 21 wherein the polymer layer thickness is reduced by less than about 10%.

24. The polymer brush of claim 21 wherein the hydrophobic layer has a dry thickness of at least about 100 angstroms.

25. The polymer brush of claim 21 wherein the hydrophobic layer has a dry thickness of at least about 1000 angstroms.

26. The polymer brush of claim 21 wherein the hydrophilic layer has a dry thickness ranging from about 10 angstroms to about 2000 angstroms.

27. The polymer brush of claim 21 wherein the hydrophilic layer comprises water-soluble or water-dispersible intermediate segments having a weight average molecular weight of at least about 1000, and one or more functional groups capable of reacting with a probe selective for the molecule.

28. The polymer brush of claim 27 wherein said functionalized groups are selected from the group consisting of hydroxy groups, amino groups, carboxylic acids, carboxylic acid derivatives, and thiols.

29. The polymer brush of claim 28 wherein the molecule is a biological molecule and the probe is selected from the group consisting of nucleic acids, polypeptides, peptide nucleic acids, markers, cells, elastin, collagen, carbohydrates, enzymes, lipids, phospholipids, hormones, drug targets, phosphates, and metal ions.

32. The polymer brush of claim 21 wherein the hydrophobic layer comprises hydrophobic polymer chain segments, and further wherein said polymer chain segments are covalently bound to the substrate surface.

33. The polymer brush of claim 32 wherein the hydrophilic layer comprises water-soluble or water-dispersible polymer chain segments, and further wherein an end of each of said hydrophilic polymer chain segments is covalently bound to a hydrophobic polymer chain segment.