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

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.

1 . An array device comprising: 
 a substrate having a surface; and    one ore 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 ore 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 ore 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: 
 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 ore 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 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.  
     
     
         12 . A method for making an array device, comprising: 
 providing a substrate;    attaching plural hydrophobic chains having first and second ends to the substrate, the first ends being attached to the substrate and the second ends being attached to a set of first hydrophilic chains, each first hydrophilic chain having a first and a second end, where the first end is attached to the second end of a hydrophobic chain, and the second end of the first hydrophilic chains having at least one functional group;    attaching a protein capture agent to the second end of a first hydrophilic chain; and    attaching a second hydrophilic chain to the second end of a first hydrophilic chain.    
     
     
         13 . The method according to  claim 12  where a first end of a hydrophobic chain is attached to the substrate via a siloxane bond.  
     
     
         14 . The method according to  claim 12  where a first end of a hydrophobic chain is attached to the substrate via a thiol-noble metal bond.  
     
     
         15 . The method according to  claim 12  where the first hydrophilic chains are attached to the hydrophobic chains via an amide bond.  
     
     
         16 . The method according to  claim 12  where the first hydrophilic chains are attached to the hydrophobic chains via a maleimide group.  
     
     
         17 . The method according to  claim 12  where the hydrophobic chain comprises a hydrocarbon chain.  
     
     
         18 . The method according to  claim 17  where the hydrocarbon chain is coupled to an oligoethylene glycol chain.  
     
     
         19 . The method according to  claim 12  where the protein capture agent comprises an antibody, avidin or streptavidin.  
     
     
         20 . The method according to  claim 12  where the functional group comprises a biotin group.  
     
     
         21 . The method according to  claim 12  where at least about 100 protein capture agents are attached per cm 2 .  
     
     
         22 . The method according to  claim 21  where the protein capture agents are different.  
     
     
         23 . The method according to  claim 12  where the array device comprises plural patches comprising protein capture agents.  
     
     
         24 . The method according to  claim 23  where the array device comprises at least 50 patches.  
     
     
         25 . The method according to  claim 24  where the array device comprises at least about 100 patches per cm 2 .  
     
     
         26 . The method according to  claim 23  where the area of each patch is from about 100 nm 2  to about 40,000 μm 2 .  
     
     
         27 . The method according to  claim 26  where the area of each patch is from about 1 μm 2  to about 10,000 μm 2 .  
     
     
         28 . The method according to  claim 26  where the area of each patch is from about 100 μm 2  to about 2,500 μm 2 .  
     
     
         29 . A method for making an array device, comprising: 
 providing a substrate;    attaching a first reagent having at least first and second reactive functional groups to the substrate by the first functional group thereby forming a monolayer;    attaching a second reagent to the monolayer, the second reagent comprising a first functional group for association with the monolayer and at least a second functional group; and    attaching a protein resistant reagent to the monolayer.    
     
     
         30 . A method for making an array device, comprising: 
 providing a substrate;    forming an organic thinfilm on the substrate where the thinfilm comprises (a) molecules of the formula X—R—Y, where X is an alkyl siloxane, R is a linear or branched hydrocarbon chain having from about 1 to about 400 carbons and is interrupted by at least one hetero group selected from the group consisting of —O—, —CONH—, —CONHCO—, —NH—, —CSNH—, —CO—, —CS—, —S—, —SO—, —(OCH 2 CH 2)   n —, —(CF 2 ) m —, where n is from 1 to 20, m is from 1 to 22 and combinations thereof; Y is a functional group; and (b) molecules of the formula X—R—V, where X and R independently are as set forth above, and V is selected from the group consisting of hydroxyl, saccharide, oligoethylene glycol, polyethylene glycol, and combinations thereof; and    attaching a protein capture agent to the monolayer via interaction of the Y functional group with protein capture agent.    
     
     
         31 . The method according to  claim 30 , further comprising attaching an affinity tag to the protein capture agent.  
     
     
         32 . The method according to  claim 31  where the affinity tag is attached to the protein capture agent via an adaptor.  
     
     
         33 . The method according to  claim 30  where the affinity tag comprises at least one amino acid.  
     
     
         34 . The method according to  claim 31  where the affinity tag comprises an antibody fragment.  
     
     
         35 . The method according to  claim 31  where the affinity tag and protein capture agent are expressed as a fusion protein.  
     
     
         36 . The method according to  claim 30  where the array device comprises an array of plural patches comprising protein capture agents.  
     
     
         37 . The method according to  claim 36  where the array device comprises at least about 100 patches per cm 2 .  
     
     
         38 . The method according to  claim 36  where the protein capture agents of at least some of the plural patches are different from the protein capture agents of other patches.  
     
     
         39 . The method according to  claim 36  where only one type of protein capture agent is present on a patch of the array.  
     
     
         40 . The method according to  claim 36  where the area of each patch is from about 100 nm 2  to about 40,000 μm 2 .  
     
     
         41 . The method according to  claim 36  where the area of each patch is from about 1 μm 2  to about 10,000 μm 2 .  
     
     
         42 . The method according to  claim 36  where the area of each patch is from about 100 μm 2  to about 2,500 μm 2 .  
     
     
         43 . The method according to  claim 36  where the array comprises at least about 10 patches.  
     
     
         44 . The method according to  claim 36  where the array comprises at least about 100 patches.  
     
     
         45 . The method according to  claim 36  where the array comprises at least about 1000 patches.  
     
     
         46 . The method according to  claim 36  where the array comprises at least about 10 different immobilized proteins.  
     
     
         47 . The method according to  claim 36  where the array comprises at least about 100 different immobilized proteins.  
     
     
         48 . The method according to  claim 36  where the array comprises at least about 1000 different immobilized proteins.  
     
     
         49 . The method according to  claim 30  where R comprises a hydrophobic portion and a hydrophilic portion.  
     
     
         50 . The method according to  claim 43  where the hydrophilic portion comprises an oligoethylene glycol chain.