Alleviation of non-specific binding in microarray assays

• US 7,258,990 B2
• Filed: 08/19/2004
• Issued: 08/21/2007
• Est. Priority Date: 08/19/2004
• Status: Expired due to Fees

• Current Assignee: Biocept, Inc.
• Original Assignee: Biocept, Inc.
• Inventors: Tsinberg, Pavel, Falcovitz-Gerassi, Yehudit
• Primary Examiner(s): Le; Long V.
• Assistant Examiner(s): Yu; Melanie J.
• Application Number:
  US10/922,387
• Publication Number:
  US 20060040407A1
• Time in Patent Office: 1,097 Days
• Field of Search: 435/7.1, 435/283.1, 435/287.1, 435/287.2, 435/288.4, 435/288.7, 422/50, 422/61, 422/55, 422/68.1, 422/82.08, 436/518, 436/528, 436/535
• US Class Current: 435/7.1
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• 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

Abstract

A post-incubation treatment is employed to effectively remove targets, such as proteins/protein complexes, or other label-bearing moieties that may non-specifically bind to a microarray substrate during a binding assay. Following incubation, a one-step wash is carried out with a liquid containing digester, e.g., a digestive enzyme (protease) or lysosome, which is effective to remove non-specifically bound targets or at least labeled portions of such targets from the substrate. Proteases are bound to or coated onto large molecules or onto solid particles of such a size such that they are prohibited from entering the porous surfaces of 3-D hydrogel microspots and are unable to reach and digest labeled target-probe complexes that are disposed within such porous hydrogel microspots. Digested segments of such protein which contain labels (or of essentially the entire protein) are carried away in the wash liquid and thus are not present to create background noise during imaging.

Claims

1. A method for the determination of the presence of one or more targets in a sample being analyzed, which method comprises:

providing a substrate having an upper surface,

attaching a plurality of 3-dimensional porous microspots of hydrogel to said surface,

providing different capture agents within the pores of at least some of said plurality of microspots to create a microarray on said surface,

contacting said microarray with a solution containing targets of a size that allows binding with said capture agents within said porous hydrogel microspots,

washing said microarray with a liquid following said contacting which liquid contains minute solid particles that carry containing a digester which particles are of such size that they and said digester do not enter said 3-dimensional microspots whereby said digester removes non-specifically bound targets or at least any labeled portions thereof from said upper surface of said substrate without affecting targets specifically bound to capture agents within said porous hydrogel microspots,

associating labels with said targets either before or after said washing if said targets do not comprise labels at the time of said binding, and

measuring said washed microarray to detect signals from labeled targets bound within particular microspots.

2. The method according to claim 1 wherein said targets are proteins.

3. The method according to claim 1 wherein said targets are antibodies.

4. The method according to claim 1 wherein said targets are antigens.

5. The method according to claim 2 wherein said digester is an exopeptidase.

6. The method according to claim 2 wherein said digester is an endopeptidase.

7. The method according to claim 2 wherein said digester is proteinase K.

8. The method according to claim 1 wherein said digester is bound as a coating to said minute solid particles.

9. The method according to claim 1 wherein said digester is bound as a coating to said particles, wherein said particles are dendrimers of such size that prohibits entry into said porous hydrogel.

10. The method according to claim 1 wherein said digester is bound as a coating to said particles, wherein said particles are a protein complex greater than 1 micron in size.

11. The method according to claim 1 wherein said upper surface is a solid surface.

12. The method according to claim 1 wherein said capture agents are proteins.

13. The method according to claim 1 wherein said capture agents are antibodies.

14. The method according to claim 8 wherein said minute solid particles are microspheres.

15. The method according to claim 1 wherein said different capture agents are provided within the pores of said microspots by initially immobilizing binding entities within said hydrogel and then diffusing said capture agents in an aqueous solution into said porous hydrogel microspots so as to attach said capture agents to said binding entities.

16. The method according to claim 15 wherein said hydrogel is an isocyanate-functional hydrogel and said binding entities are covalently bound to the isocyanate moieties.

17. The method according to claim 15 wherein said digester is proteinase K.

18. A method for the determination of the presence of one or more targets in a sample being analyzed, which method comprises:

providing a microarray having a plurality of 3-dimensional porous hydrogel microspots on an upper surface of a substrate,

providing different capture agents within the pores of at least some of said plurality of microspots,

incubating said microarray with a solution containing targets of a size that they can bind with said capture agents within said porous hydrogel microspots,

associating labels with said targets that do bind to said capture agents in said microspots if said targets do not comprise labels at the time of said binding,

washing said upper surface of said microarray with a liquid following said incubating, which liquid contains a digester that is present on the outer surface of minute solid particles that are of such size that they and said digester do not enter said 3-dimensional microspots so that said digester removes non-specifically bound targets or at least any labeled portions thereof from said upper surface of said substrate without affecting said targets specifically bound to capture agents within said porous hydrogel microspots, and

measuring said washed microarray to detect signals from said labeled targets bound within particular hydrogel microspots.

19. The method according to claim 18 wherein said targets are antibodies, antigens or other proteins.

20. The method according to claim 19 wherein said digester is an endopeptidase.

21. The method according to claim 18 wherein said minute solid particles are microspheres of at least 1 micron in size and said digester is proteinase K.

22. The method according to claim 18 wherein said different capture agents are provided within the pores of said microspots by initially immobilizing binding entities within said hydrogel and then diffusing said capture agents in an aqueous solution into said porous hydrogel microspots so as to attach said capture agents to said binding entities.

23. The method according to claim 22 wherein said hydrogel is an isocyanate-functional hydrogel and said binding entities are covalently bound to the isocyanate moieties and wherein said labels provide fluorescent signals.

24. A method for the determination of the presence of one or more targets in a sample being analyzed, which method comprises:

providing a microarray having a plurality of 3-dimensional porous hydrogel microspots at least about 20 μ

m thick on an upper surface of a substrate,

providing different capture agents within the pores of at least some of said plurality of microspots,

incubating said microarray with a solution containing labeled targets of a size that they can bind with said capture agents within said porous hydrogel microspots,

washing said upper surface of said microarray with a liquid following said incubating, which liquid contains a digester that is present on the outer surface of minute solid particles at least 1 micron in size that do not enter said 3-dimensional microspots so that said digester removes non-specifically bound targets or at least any labeled portions thereof from said upper surface of said substrate without affecting said labeled targets specifically bound to capture agents within said porous hydrogel microspots, and

measuring said washed microarray to detect signals from labeled targets bound within particular hydrogel microspots.