Polyanion/polycation multilayer film for DNA immobilization

• US 20030003272A1
• Filed: 06/21/2001
• Published: 01/02/2003
• Est. Priority Date: 06/21/2001
• Status: Active Grant

• Current Assignee: Corning Incorporated
• Original Assignee: Corning Incorporated
• Inventors: Laguitton, Bruno
• Granted Patent: US 6,689,478 B2
• Time in Patent Office: Days
• US Class Current: 428/141
• 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 2219/00628 Ionic
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• 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 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B05D 1/185 applying monomolecular layers B05D1/204 takes precedence
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: B82Y 40/00 Manufacture or treatment of nanostructures
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/00 Methods of creating libraries, e.g. combinatorial synthesis
• CPC Class Codes: Y10T 428/24355 Continuous and nonuniform or irregular surface on layer or component [e.g., roofing, etc.]
• CPC Class Codes: Y10T 428/261 In terms of molecular thickness or light wave length
• CPC Class Codes: Y10T 428/31504 Composite [nonstructural laminate]
• CPC Class Codes: Y10T 428/31645 Next to addition polymer from unsaturated monomers
• CPC Class Codes: Y10T 428/31667 Next to addition polymer from unsaturated monomers, or aldehyde or ketone condensation product
• CPC Class Codes: Y10T 428/31913 : Monoolefin polymer
• CPC Class Codes: Y10T 428/31917 : Next to polyene polymer

Abstract

A substrate with a charged surface coated with one or more bilayers of oppositely charged polyelectrolytes. Subsequent optional bilayers of polyelectrolytes must bear alternating opposite charges. The final or top layer may be activated for biomolecular attachment.

Claims

1. A substrate having a charged film comprising:

a) a substrate having a charged surface;

b) a first layer of polyelectrolyte having a charge opposite the charged surface; and

c) additional layers of polyelectrolytes, each having a charge opposite to each adjacent layer, wherein, an outermost layer of polycation contains an amine functional group.

2. The film of claim 1 wherein the substrate is an organic material.

3. The film of claim 1 wherein the substrate is an inorganic material.

4. The film of claim 3 wherein the substrate is chosen from the group consisting of mica, silicium, and an alkaline earth aluminoborosilicate glass.

5. The film of claim 1 wherein the polyelectrolytes are selected from a group consisting of polystyrene sulfonate, polyallylamine hydrochloride, sulfonated polyaniline, and a tetrahydrothiophenium precursor polyelectrolyte of polyvinylpyrole.

6. The film of claim 1 wherein the substrate having thereon 1 to 6 bilayers of polyelectrolytes.

7. The film of claim 6 wherein the bilayers have a surface roughness of between 10 and 60 root mean square.

8. The film of claim 6 wherein the substrate having thereon 5 bilayers of polyelectrolytes.

9. The film of claim 7 wherein the bilayers have a surface roughness of 40 root mean square.

10. The film of claim 1 wherein a first specific binding entity is attached to the outermost polycation layer.

11. The film of claim 10 wherein a second specific binding entity is attached to the first specific binding entity.

12. The film of claim 11 wherein said first specific binding entity is a DNA probe;

wherein said second specific binding entity is a DNA target.

14. The method of claim 13 wherein the substrate is made of an organic material.

15. The method of claim 13 wherein the substrate is made of an inorganic material.

16. The method of claim 15 wherein the substrate is chosen from the group consisting of mica, silicium, and an alkaline earth aluminoborosilicate glass.

17. The method of claim 13 wherein the salt comprises a water soluble inorganic salt.

18. The method of claim 17 wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride, and calcium chloride.

19. The method of claim 13 wherein the polyelectrolytes are selected from the group consisting of polystyrene sulfonate, polyallylamine hydrochloride, sulfonated polyaniline, and a tetrahydrothiophenium precursor polyelectrolyte of polyvinylpyrole.

20. The method of claim 13 further comprising the step of attaching a first specific binding entity to the aminated outermost polycation layer.

21. The method of claim 20 further comprising the step of attaching a second specific binding entity to the first specific binding entity.

22. The method of claim 21 wherein said first specific binding entity is a DNA probe;

wherein said second specific binding entity is a DNA target.

24. A coated substrate produced by the method of claim 23.

25. The method of claim 23 whereby steps b through d are repeated from 1 to 5 times.

26. The method of claim 25 whereby steps b through d are repeated 4 times.

27. The method of claim 23 wherein the outermost layer of polycations is aminated.

28. The method of claim 27 wherein a specific binding entity is attached to an aminated site for detection.

13. A method for the preparation of a substrate having a charged film comprising the steps of:

a) providing a substrate having a surface with a uniform surface charge;

b) mixing a first polyelectrolyte with a salt to form a first polyelectrolyte mixture having a uniform charge opposite the charge of the substrate surface;

c) applying the first polyelectrolyte mixture to the substrate surface, forming a first polyelectrolyte layer;

d) mixing a second polyelectrolyte, having a charge opposite to the first polyelectrolyte, with a salt to form a second polyelectrolyte mixture;

e) applying the second polyelectrolyte mixture to the first polyelectrolyte layer, forming a second polyelectrolyte layer;

f) applying one or more additional layers of a polyelectrolyte mixture to the second layer, each additional layer having a charge opposite each immediately adjacent layer;

g) applying an outermost polycation layer; and

h) aminating the outermost polycation layer with an amine functional group to form an aminated outermost polycation layer.

23. A method of coating a substrate for immobilization of biomolecules comprising the steps of:

a) providing a substrate having a charged surface;

b) immersing the substrate in a polycationic solution mixed with salts of high ionic strength to form a polycationic mixture, whereby said polycations from said polycationic mixture electrostatically attach to said charged substrate surface creating a polycationic coating;

c) rinsing the substrate;

d) immersing the substrate in a polyanionic solution mixed with salts of high ionic strength to form a polyanionic mixture, whereby said polyanions electrostatically attach to said polycationic coating thereby forming a polyanionic coating; and

e) immersing the substrate in the polycationic mixture to form an outermost coating of polycations.

29. A substrate having a charged film comprising:

a) a substrate;

b) five polyelectrolyte bilayers attached to said substrate surface;

c) a polycation layer functionalized with an amine group attached to an outermost bilayer;

d) a first bound entity attached to the amine functional layer; and

e) a second bound entity attached to the first bound entity.

30. A method of producing a charged film attached to a substrate for the immobilization of biomolecules comprising the steps of:

a) providing a substrate having an electrically charged surface;

b) applying a first layer of polyelectrolyte to the substrate surface having an electrical charge opposite to the electrical charge of the substrate surface;

c) rinsing the substrate with ultra pure water;

d) applying a second layer of a polyelectrolyte to the first layer of polyelectrolyte having an electrical charge opposite to the electrical charge of the first layer of polyelectrolytes;

e) repeating steps b to d to produce a charged film with 10 layers of alternatively charged polyelectrolytes;

f) applying an outermost polycation layer;

g) aminating the outermost polycation layer with an amine functional group; and

h) immobilizing a binding entity on the amine functional group.