Alleviation of non-specific binding in microarray assays

• US 20060040407A1
• 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, Falcovitz-Gerassi, Yehudit
• Granted Patent: US 7,258,990 B2
• Time in Patent Office: Days
• US Class Current: 436/518
• 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 various 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 following said contacting using a liquid containing a digester which removes non-specifically bound targets or at least labeled portions thereof from said surface 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 microspheres or minute solid particles.

9. The method according to claim 1 wherein said digester is bound as a coating to 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 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. A method for the determination of the presence of one or more targets in a sample being analyzed, which method comprises:

providing a solid substrate having an upper surface,

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

immobizing binding entities within said porous hydrogel microspots,

providing different proteinaceous capture agents within the pores of various of said plurality of microspots to create a microarray, by diffusing such capture agents in an aqueous solution into said porous hydrogel microspots so as to attach to said binding entities,

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

washing said microarray following said contacting using a liquid containing a digester which removes non-specifically bound targets or at least any labeled portions thereof from said surface without affecting targets specifically bound to capture agents within said porous hydrogel microspots, and

measuring said washed microarray to detect the presence of targets bound within particular microspots.

15. The method according to claim 14 wherein said binding entities are NFkB transcription factor.

16. The method according to claim 15 wherein primary antibodies are caused to bind to said NFkB and thereafter secondary antibodies are caused to bind to said primary antibodies.

17. The method according to claim 16 wherein labels which provide fluorescent signals are bound to said secondary antibodies.

18. The method according to claim 14 wherein labels which provide detectable signals are associated with said targets either before or after said washing step if said targets do not comprise labels at the time of said binding.

19. The method according to claim 14 wherein said digester is Proteinase K.

20. The method according to claim 14 wherein said digester is bound as a coating to microspheres or minute solid particles.