Non-specific binding resistant protein arrays and methods for making the same

• US 20020132272A1
• Filed: 10/27/2001
• Published: 09/19/2002
• Est. Priority Date: 07/14/1998
• Status: Active Grant

• Current Assignee: Zyomyx, Inc.
• Original Assignee: Zyomyx, Inc.
• Inventors: Wagner, Peter, Kernen, Peter, Lu, Hongbo, Tran, Huu
• Granted Patent: US 6,897,073 B2
• Time in Patent Office: Days
• US Class Current: 435/7.9
• CPC Class Codes: B01J 2219/00385 Printing
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• 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 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00635 by reactive plasma treatment
• 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 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07K 2319/20 containing a tag with affinity for a non-protein ligand
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• 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: G01N 33/551 : the carrier being inorganic
• CPC Class Codes: G01N 33/6803 : General methods of protein analysis not limited to specific proteins or families of proteins
• CPC Class Codes: G01N 33/6845 : Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: Y10S 435/81 : Packaged device or kit
• CPC Class Codes: Y10S 435/973 : Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays

Abstract

Arrays of protein-capture agents useful for the simultaneous detection of a plurality of proteins which are the expression products, or fragments thereof, of a cell or population of cells in an organism are provided. A variety of antibody arrays, in particular, are described. Methods of both making and using the arrays of protein-capture agents are also disclosed. The invention arrays are particularly useful for various proteomics applications including assessing patterns of protein expression and modification in cells.

Claims

1. An array device comprising

a substrate having a surface;

and,

one or more protein immobilization regions on said surface, said protein immobilization regions each comprising;

(i) an ordered hydrophobic monolayer formed of alkyl chains having proximal ends which are chemisorbed or physisorbed to said surface within said immobilization regions, and opposite hydrophobic distal ends;

(ii) a hydrophilic monolayer attached to said ordered hydrophobic monolayer, said hydrophilic monolayer comprising a set of first hydrophilic chains, each first hydrophilic chain having a proximal end by which said first hydrophilic chain is linked to an alkyl chain distal end, and an opposite hydrophilic distal end having a functional group for covalently attaching a protein capture agent thereto;

(iii) one or more of said protein capture agents attached to a first subset of said set of first hydrophilic chains within said immobilization regions through a residue formed by reacting said protein capture agents with said functional groups of said first subset of said first hydrophilic chains; and

,

(iv) one or more second hydrophilic chains each being attached to a second subset of said set of said first hydrophilic chains through said functional group or residue thereof, wherein said second subset of first hydrophilic chains and said first subset of first hydrophilic chains are mutually exclusive of each other;

wherein said first hydrophilic chains and said second hydrophilic chains are effective to resist non-specific protein binding.

2. The device of claim 1 wherein said second hydrophilic chains are polyethylene glycol chains.

3. The device of claim 2 wherein said first hydrophilic chains are oligoethylene glycol chains.

4. The device of claim 1 wherein at least two of said protein immobilization regions are adjacent each other.

5. The device of claim 1 wherein at least two of said protein immobilization regions are separated from each other by one or more border regions.

6. A method for making an array device comprising the steps of:

a) providing a substrate having a surface; and

one or more protein immobilization regions on said surface, said protein immobilization regions each comprising;

i. an ordered hydrophobic monolayer formed of alkyl chains having proximal ends which are chemisorbed or physisorbed to said surface within said immobilization regions, and opposite hydrophobic distal ends;

ii. a hydrophilic monolayer attached to said ordered hydrophobic monolayer, said hydrophilic monolayer comprising a set of first hydrophilic chains, each first hydrophilic chain having a proximal end by which said first hydrophilic chain is linked to an alkyl chain distal end, and an opposite hydrophilic distal end;

iii. one or more functional groups, each for covalently attaching a protein capture agent thereto and each covalently attached to one or more of said hydrophilic distal ends of said first hydrophilic chains;

b) reacting one or more of said protein capture agents to one or more of said functional groups attached to a first subset of said set of first hydrophilic chains within said immobilization regions to attach said one or more protein capture agents through a residue formed by said reaction of said protein capture agents with said functional groups of said first subset of said first hydrophilic chains; and

,

c) reacting one or more second hydrophilic chains with a second subset of said set of first hydrophilic chains so that one or more of said second hydrophilic chains attach through one or more residues of one or more of said second subset of said first hydrophilic chains,

7. The method of claim 6 wherein at least one of said second hydrophilic chains is polyethylene glycol.

8. The method of claim 6 wherein at least one of said first hydrophilic chains is oligoethylene glycol.

9. A method for making an array device comprising the steps of:

a) providing a substrate having a surface; and

one or more protein immobilization regions on said surface, said protein immobilization regions each comprising;

i. an ordered hydrophobic monolayer formed from a set of alkyl chains having proximal ends which are chemisorbed or physisorbed to said surface within said immobilization regions, and opposite hydrophobic distal ends;

ii. one or more functional groups for covalently attaching said protein captures agent thereto, each functional group being attached to said hydrophobic distal ends of a subset of said set of alkyl chains;

b) reacting one or more of said protein capture agents to one or more of said functional groups attached to a first subset of said set of alkyl chains within said immobilization regions to attach said one or more protein capture agents through a residue formed by said reaction of said protein capture agents with said functional groups of said first subset of said alkyl chains; and

,

c) reacting one or more hydrophilic chains with a second subset of said set of alkyl chains so that one or more of said second hydrophilic chains attach through one or more residues of one or more of said second subset of said set of alkyl chains, wherein said first subset of said set of alkyl chains and said second subset of said set of alkyl chains are mutually exclusive.

10. The method of claim 9 wherein at least one of said hydrophilic chains is polyethylene glycol.

11. The method of claim 9 wherein at least one of said first hydrophilic chains is oligoethylene glycol.