Microarrays utilizing hydrogels

• US 20060040274A1
• Filed: 08/19/2004
• Published: 02/23/2006
• Est. Priority Date: 08/19/2004
• Status: Active Grant

• Current Assignee: Biocept, Inc.
• Original Assignee: Biocept, Inc.
• Inventors: Tsinberg, Pavel
• Granted Patent: US 7,595,157 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00527 Sheets
• 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 2219/00621 by physical means, e.g. trenches, raised areas
• 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 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• 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: Y10S 524/916 Hydrogel compositions

Abstract

A method of making a microarray by coating a flat substrate with a polymerizable hydrogel layer which contains anchoring moieties dispersed uniformly therethroughout. Following curing, a continuous layer of uniform thickness is securely attached to the upper surface of the substrate through an array region thereof. A plurality of different probes are then attached to create microspots at distinct spatial locations on the surface of this slab layer by linking the probes to the anchoring moieties in the cured hydrogel. Such anchoring moieties may employ linking systems such as organic chelators, that are activated by copper or some other metal, and complementary pairs such as avidin-biotin.

Claims

1. A method of making a microarray, which method comprises the steps of:

providing a substrate having an upper surface that is functionalized with organic molecules,

coating said surface with a polymerizable hydrogel layer so as to completely cover an array region of the surface, said layer including anchoring moieties dispersed uniformly therethroughout,

curing said coated substrate to polymerize said hydrogel layer, and

subsequently attaching a plurality of different probes to said coated surface at distinct spatial locations thereon by linking said probes to said anchoring moieties which are present within said cured hydrogel layer.

2. The method according to claim 1 wherein said anchoring moieties are organic chelators which are admixed with the components of said polymerizable layer.

3. The method according to claim 2 wherein, following said curing step, said chelators are treated with water-soluble metal ions in aqueous solution to activate said chelators.

4. The method according to claim 3 wherein said probes are molecules that have been derivatized with compounds that bind strongly to said metal ions.

5. The method according to claim 3 wherein said probes are derivitized with histidine-rich peptides.

6. The method according to claim 1 wherein said probes are derivatized with a complementary linker that binds directly to said anchoring moieties during said attaching step.

7. The method according to claim 6 wherein said linkers and said anchoring moieties are biotin and streptavidin, respectively.

8-9. -9. (canceled)

12. A microarray which comprises:

a substrate having an upper surface that is functionalized with organic molecules,

a polymerized hydrogel layer completely covering an array region of said surface, said layer including anchoring moieties dispersed uniformly therethroughout, and

a plurality of different probes attached to said hydrogel cover layer on said surface at distinct spatial locations thereon,

said probes being linked to said anchoring moieties in said polymerized hydrogel layer, and regions of said cover layer surrounding said locations being essentially free from probes.

13. The microarray according to claim 12 wherein said anchoring moieties are organic chelators which are activated with metal ions.

14. The microarray according to claim 13 wherein said probes are derivatized with histidine-rich peptides.

15. The microarray according to claim 12 wherein said probes are derivatized with a complementary linker that binds directly to said anchoring moieties.

16. The microarray according to claim 15 wherein said probes are biotinylated and said anchoring moieties are streptavidin.

17. The microarray according to claim 12 wherein said polymerized hydrogel comprises PEG or PPG or a copolymer thereof.

18. The microarray according to claim 17 wherein said polymerized hydrogel includes a polyisocyanate crosslinker.

22. The microarray according to claim 12 wherein said polymerized hydrogel layer comprises a reaction product of (a) PEG or PPG or copolymer thereof having a molecular weight between about 5,000 and about 30,000 daltons and (b) difunctional and/or polyfunctional isocyanates.

19. A microarray which comprises:

a solid, flat substrate having an upper surface that is functionalized with organic molecules,

a polymerized hydrogel layer completely covering an array region of said surface, said layer including anchoring moieties dispersed uniformly therethroughout, said hydrogel comprising (a) PEG or PPG or a copolymer thereof of a molecular weight between about 500 and about 30,000 daltons and (b) difunctional and/or polyfunctional isocyanates, and

a plurality of different probes attached at distinct spatial locations to said hydrogel layer covering said upper surface to create a plurality of microspots,

said probes being linked to said anchoring moieties in said polymerized hydrogel layer, and regions surrounding said microspots being essentially free from probes.

20. The microarray according to claim 19 wherein said anchoring moieties are organic chelators or streptavidin.

21. The microarray according to claim 19 wherein said PEG or PPG or copolymer thereof has a molecular weight of at least about 5.000 daltons.