Methods and compositions for attachment of biomolecules to solid supports, hydrogels, and hydrogel arrays

• US 6,372,813 B1
• Filed: 06/25/1999
• Issued: 04/16/2002
• Est. Priority Date: 06/25/1999
• Status: Expired due to Term

• Current Assignee: Amersham Biosciences (General Electric Company)
• Original Assignee: Motorola, Inc. (Motorola Solutions, Inc.)
• Inventors: Johnson, Travis, Beuhler, Allyson, McGowen, John, Lajos, Robert Emil, Brush, Charles Kimball
• Primary Examiner(s): Berman, Susan W.
• Application Number:
  US09/344,620
• Time in Patent Office: 1,026 Days
• Field of Search: 435/6, 435/174, 435/177, 435/180, 435/181, 522/114, 522/116, 522/117, 522/120, 522/121, 522/149, 522/152, 430/270.1, 430/281.1, 430/287.1, 526/238.1, 526/303.1, 526/306, 526/307.7, 527/200, 527/201, 527/207, 527/300, 527/309, 527/314, 527/315, 536/25.3
• US Class Current: 522/114
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393

Abstract

The present invention provides solid supports (e.g., glass) and polymer hydrogels (particularly polymer hydrogel arrays present on a solid support) comprising one or more reactive sites for the attachment of biomolecules, as well as biomolecules comprising one or more reactive sites for attachment to solid supports and polymer hydrogels. The invention further provides novel compositions and methods for the preparation of biomolecules, solid supports, and polymer hydrogels comprising reactive sites. The invention also provides for preparation of crosslinked solid supports, polymer hydrogels, and hydrogel arrays, wherein one or more biomolecules is attached by means of the reactive sites in a photocycloaddition reaction. Advantageously, according to the invention, crosslinking of the hydrogel and attachment of biomolecules can be done in a single step.

Claims

1. A method for preparing a crosslinked polyacrylamide hydrogel or hydrogel array comprising one or more attached biomolecule(s), said method comprising:

(a) obtaining a polyacrylamide reactive prepolymer that includes one or more reactive site(s) capable of 2+2 photocycloaddition;

(b) placing said polyacrylamide reactive prepolymer on a solid support;

(c) crosslinking said polyacrylamide reactive prepolymer to obtain a crosslinked polyacrylamide hydrogel or hydrogel array;

(d) obtaining one or more biomolecule(s) that includes one or more reactive site(s) capable of 2+2 photocycloaddition; and

(e) contacting the product of step (c) with the product of step (d) and irradiating with ultraviolet light sufficient for 2+2 photocycloaddition to occur such that said crosslinked polyacrylamide hydrogel or hydrogel array comprising one or more biomolecule(s) attached by 2+2 photocycloaddition is obtained.

2. The method of claim 1, wherein the reactive site(s) present on the polyacrylamide reactive prepolymer and the reactive site(s) present on the biomolecule(s) are selected from the group consisting of dimethyl maleimide, maleimide, thymine, polythymine, acrylate, cinnamate, and citraconimide.

3. The method of claim 1, wherein said biomolecule(s) comprise a nucleic acid fragment containing less than about 1000 nucleotides, and further optionally comprise a spacer region.

4. The method of claim 1, wherein said crosslinking is done by 2+2 photo cycloaddition between one or more reactive site(s) of said polyacrylamide reactive prepolymer.

5. A method for preparing a crosslinked polyacrylamide hydrogel or hydrogel array comprising one or more attached biomolecule(s), said method comprising:

(a) obtaining a polyacrylamide reactive prepolymer that includes one or more reactive site(s) capable of 2+2 photocycloaddition;

(b) placing said polyacrylamide reactive prepolymer on a solid support;

(c) obtaining one or more biomolecule(s) that includes one or more reactive site(s) capable of 2+2 photocycloaddition; and

(d) contacting the product of step (a) with the product of step (b) and irradiating with ultraviolet light sufficient for 2+2 photocycloaddition to occur such that said crosslinked polyacrylamide hydrogel or hydrogel array comprising one or more biomolecule(s) attached by 2+2 photocycloaddition is obtained.

6. The method of claim 5, wherein the reactive site(s) present on the polyacrylamide reactive prepolymer and the reactive site(s) present on the biomolecule(s) are selected from the group consisting of dimethyl maleimide, maleimide, thymine, polythymine, acrylate, cinnamate and citraconimide.

7. The method of claim 5, wherein said biomolecule(s) comprise a nucleic acid fragment containing less than about 1000 nucleotides, and further optionally comprise a spacer region.

8. A method for preparing a solid support comprising one or more attached biomolecule(s), said method comprising:

(a) treating said solid support with a coupling agent to attach one or more amine group(s) to the surface of said solid support;

(b) attaching to said solid support by said amine group(s) one or more reactive site(s) capable of 2+2 photocycloaddition;

(c) obtaining one or more biomolecule(s) that includes one or more reactive site(s) capable of 2+2 photocycloaddition; and

(d) contacting the product of step (b) with the product of step (c) and irradiating with ultraviolet light sufficient for 2+2 photocycloaddition to occur such that said solid support comprising one or more biomolecule(s) attached by 2+2 photocyloaddition is obtained.

9. The method of claim 8, wherein the reactive site(s) present on the solid support and the reactive site(s) present on the biomolecule(s) are selected from the group consisting of dimethyl maleimide, maleimide, thymine, polythymine, acrylate, cinnamate and citraconimide.

10. The method of claim 8, wherein said biomolecule(s) comprise a nucleic acid fragment containing less than about 1000 nucleotides, and further optionally comprise a spacer region.

11. The method of claim 8, wherein the solid support is a material selected from the group consisting of nylon, polystyrene, glass, latex, polypropylene, and activated cellulose.

12. The method of claim 8, wherein the solid support is a material selected from the group consisting of a bead, membrane, microwell, centrifube tube, and slide.