Claims
- 1. A reagent composition for attaching a target molecule to the surface of a substrate, the reagent composition comprising a polymeric backbone having one or more pendent thermochemically reactive groups adapted to form covalent bonds with corresponding functional groups on the target molecule, the reagent adapted to be coated and immobilized on a surface in a manner that permits: (a) a small sample volume of a solution containing the target molecule to be applied in the form of a discrete spot on the reagent-coated surface, (b) target molecule present in the sample volume to become attached to the bound reagent by reaction between its functional groups and the corresponding thermochemically reactive groups, and (c) substantially all unattached target molecule to be washed from the spot without undue detectable amounts of target molecule in the area surrounding the spot.
- 2. A reagent composition according to claim 1 wherein the target molecule comprises a nucleic acid and the surface comprises the surface of a plastic or organosilane-pretreated glass slide.
- 3. A reagent composition according to claim 2 wherein the nucleic acid comprises one or more functional groups selected from the group consisting of amine and sulfhydryl groups.
- 4. A reagent composition according to claim 1 wherein the sample volume is on the order of twenty nanoliters or less.
- 5. A reagent composition according to claim 1 wherein the composition further comprises one or more latent reactive groups comprising photoreactive groups for attaching the reagent composition to the surface upon application of energy from a suitable source.
- 6. A reagent composition according to claim 5 wherein the thermochemically reactive groups and photoreactive groups are pendent upon one or more hydrophilic polymeric backbones.
- 7. A reagent composition according to claim 6 wherein the photoreactive groups are selected from the group consisting of photoreactive aryl ketones.
- 8. A reagent composition according to claim 7 wherein the photoreactive aryl ketones are each, independently, selected from the group consisting of acetophenone, benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles.
- 9. A reagent composition according to claim 5 wherein the target molecule is a nucleic acid and the photoreactive groups are selected from the group consisting of photoreactive aryl ketones.
- 10. A reagent composition according to claim 5 wherein the polymeric backbone is selected from the group consisting of acrylics, vinyls, nylons, polyurethanes and polyethers, the pendent thermochemically reactive groups are selected from the group consisting of activated esters, epoxide, azlactones, activated hydroxyls and maleimide, and the backbone further comprises one or more pendent photoreactive groups selected from the group consisting of aryl ketones.
- 11. A method of attaching a target molecule to the surface of a substrate, the method comprising:
(a) providing a reagent composition comprising a polymeric backbone having one or more pendent thermochemically reactive groups adapted to form covalent bonds with corresponding functional groups on the target molecule, the reagent adapted to be coated and immobilized on a surface in a manner that permits: (i) a small sample volume of a solution containing the target molecule to be applied in the form of a discrete spot on the reagent-coated surface, (ii) target molecule present in the sample volume to become covalently attached to the bound reagent by reaction between its functional groups and the corresponding thermochemically reactive groups, and (iii) substantially all unattached target molecule to be washed from the spot without undue detectable amounts of target molecule in the area surrounding the spot, (b) coating and immobilizing the reagent composition on the substrate surface, (c) providing a solution comprising a target molecule having one or more functional groups thermochemically reactive with corresponding groups provided by the reagent composition, (d) applying one or more discrete small sample volume spots of the solution on the surface of the substrate surface, and (e) allowing the thermochemically reactive groups provided by the reagent composition to form covalent bonds with corresponding functional groups provided by the target molecule in order to attach the target molecule to the surface.
- 12. A method according to claim 11 wherein the target molecule comprises a nucleic acid and the surface comprises the surface of a plastic, silicon hydride, or organosilane-pretreated glass or silicone slide.
- 13. A method according to claim 12 wherein the nucleic acid comprises one or more functional groups selected from the group consisting of amine and sulfhydryl groups.
- 14. A method according to claim 11 wherein the sample volume is on the order of twenty nanoliters or less.
- 15. A method according to claim 11 wherein the composition further comprises one or more latent reactive groups comprising photoreactive groups for attaching the reagent composition to the surface upon application of energy from a suitable source.
- 16. A method according to claim 15 wherein the thermochemically reactive groups and photoreactive groups are pendent upon one or more hydrophilic polymeric backbones and the photoreactive groups are selected from the group consisting of photoreactive aryl ketones.
- 17. A method according to claim 16 wherein the photoreactive aryl ketones are each, independently, selected from the group consisting of acetophenone, benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles.
- 18. A method according to claim 11 wherein the polymeric backbone is selected from the group consisting of acrylics, vinyls, nylons, polyurethanes and polyethers, the pendent thermochemically reactive groups are selected from the group consisting of activated esters, epoxide, azlactones, activated hydroxyls and maleimide, and the backbone further comprises one or more pendent photoreactive groups selected from the group consisting of aryl ketones.
- 19. A method according to claim 11 wherein the method is used to prepare one or more microarrays of nucleic acids upon the surface of a plastic, silicon hydride, or organosilane-pretreated glass or silicone slide, each microarray providing at least about 100/cm2 distinct nucleic acids having a length of at least 10 nucleotides, the nucleic acids each being spotted in discrete regions and defined amounts of between about 0.1 nanomoles and about 10 nanomoles.
- 20. A method according to claim 19 wherein the regions are generally circular in shape, having a diameter of between about 10 microns and about 500 microns and separated from other regions in the array by center to center spacing of about 20 microns to about 1000 microns.
- 21. An activated slide comprising a flat support surface coated with the bound residue of a reagent composition comprising a polymeric backbone having one or more pendent thermochemically reactive groups adapted to form covalent bonds with corresponding functional groups on the target molecule, the reagent adapted to be coated and immobilized on the surface in a manner that permits: (a) a small sample volume of a solution containing the target molecule to be applied in the form of a discrete spot on the reagent-coated surface, (b) target molecule present in the sample volume to become attached to the bound reagent by reaction between its functional groups and the corresponding thermochemically reactive groups, and (c) substantially all unattached target molecule to be washed from the spot without undue detectable amounts of target molecule in the area surrounding the spot.
- 22. An activated slide according to claim 21 wherein the slide is adapted for fabricating a microarray wherein the target molecule comprises a nucleic acid and the surface comprises the surface of a plastic, silicon hydride, or organosilane-pretreated glass or silicone slide.
- 23. An activated slide according to claim 22 wherein the nucleic acid comprises one or more functional groups selected from the group consisting of amine and sulfhydryl groups.
- 24. An activated slide according to claim 21 wherein the sample volume is on the order of twenty nanoliters or less.
- 25. An activated slide according to claim 21 wherein the composition further comprises one or more latent reactive groups comprising photoreactive groups for attaching the reagent composition to the surface upon application of energy from a suitable source.
- 26. An activated slide according to claim 25 wherein the thermochemically reactive groups and photoreactive groups are pendent upon one or more hydrophilic polymeric backbones and the photoreactive groups are selected from the group consisting of photoreactive aryl ketones.
- 27. An activated slide according to claim 26 wherein the photoreactive aryl ketones are each, independently, selected from the group consisting of acetophenone, benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles.
- 28. An activated slide according to claim 21 wherein the polymeric backbone is selected from the group consisting of acrylics, vinyls, nylons, polyurethanes and polyethers, the pendent thermochemically reactive groups are selected from the group consisting of activated esters, epoxide, azlactones, activated hydroxyls and maleimide, and the backbone further comprises one or more pendent photoreactive groups selected from the group consisting of aryl ketones.
- 29. A microarray prepared by a method comprising:
(a) providing a reagent composition comprising a polymeric backbone having one or more pendent thermochemically reactive groups adapted to form covalent bonds with corresponding functional groups on the target molecule, the reagent adapted to be coated and immobilized on a substantially flat surface in a manner that permits: (i) a small sample volume of a solution containing the target molecule to be applied in the form of a discrete spot on the reagent-coated surface, (ii) target molecule present in the sample volume to become attached to the bound reagent by reaction between its functional groups and the corresponding thermochemically reactive groups, and (iii) substantially all unattached target molecule to be washed from the spot without undue detectable amounts of target molecule in the area surrounding the spot, (b) coating and immobilizing the reagent composition on the substrate surface, (c) providing a solution comprising a target molecule having one or more functional groups thermochemically reactive with corresponding groups provided by the reagent composition, (d) applying one or more discrete small sample volume spots of the solution on the surface of the substrate surface, and (e) allowing the thermochemically reactive groups provided by the reagent composition to form covalent bonds with corresponding functional groups provided by the target molecule in order to attach the target molecule to the surface.
- 30. A microarray according to claim 29 wherein the target molecule comprises a nucleic acid and the surface comprises the surface of a plastic, silicon hydride, or organosilane-pretreated glass or silicone slide.
- 31. A microarray according to claim 30 wherein the nucleic acid comprises one or more functional groups selected from the group consisting of amine and sulfhydryl groups.
- 32. A microarray according to claim 29 wherein the sample volume is on the order of twenty nanoliters or less.
- 33. A microarray according to claim 29 wherein the composition further comprises one or more latent reactive groups comprising photoreactive groups for attaching the reagent composition to the surface upon application of energy from a suitable source.
- 34. A microarray according to claim 33 wherein the thermochemically reactive groups and photoreactive groups are pendent upon one or more hydrophilic polymeric backbones and the photoreactive groups are selected from the group consisting of photoreactive aryl ketones.
- 35. A microarray according to claim 34 wherein the photoreactive aryl ketones are each, independently, selected from the group consisting of acetophenone, benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles.
- 36. A microarray according to claim 29 wherein the polymeric backbone is selected from the group consisting of acrylics, vinyls, nylons, polyurethanes and polyethers, the pendent thermochemically reactive groups are selected from the group consisting of activated esters, epoxide, azlactones, activated hydroxyls and maleimide, and the backbone further comprises one or more pendent photoreactive groups selected from the group consisting of aryl ketones.
- 37. A microarray according to claim 29 wherein the method is used to prepare one or more microarrays of nucleic acids upon the surface of a plastic, silicon hydride, or organosilane-pretreated glass or silicone slide, each microarray providing at least about 100/cm2 distinct nucleic acids having a length of at least 10 nucleotides, the nucleic acids each being spotted in discrete regions and defined amounts of between about 0.1 femtomoles and about 10 nanomoles.
- 38. A microarray according to claim 37 wherein the regions are generally circular in shape, having a diameter of between about 10 microns and about 500 microns and separated from other regions in the array by center to center spacing of about 20 microns to about 1000 microns.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of U.S. patent application Ser. No. 08/940,213, for “Reagent and Method for Attaching Target Molecules to a Surface”, filed Sep. 30, 1997, the entire disclosure of which is incorporated herein by reference.
Continuation in Parts (1)
|
Number |
Date |
Country |
Parent |
08940213 |
Sep 1997 |
US |
Child |
09227913 |
Jan 1999 |
US |