Protein arrays for high-throughput screening

Abstract

Protein arrays and protein-coated substrates for the parallel, in vitro screening of biomolecular activity are provided. Methods of using the protein-coated substrates and protein arrays are also disclosed. A plurality of different members of a single protein family may be immobilized on the protein-coated substrate or array. The protein-coated substrates and protein arrays are particularly useful in high-throughput drug screening and clinical diagnostics.

1 - 41 . (canceled)  
     
     
         42 . An array of proteins, comprising: a substrate; and a plurality of patches arranged in discrete known regions on said substrate, wherein each of said patches comprises an immobilized protein, and wherein each of said patches is separated from neighboring patches by at least about 50 nm, and wherein the array comprises 100 or more patches per 1 cm 2 .  
     
     
         43 . The array of  claim 42 , wherein the array comprises 1000 or more patches per 1 cm 2 .  
     
     
         44 . The array of  claim 42  or  43  wherein each of said patches is separated from neighboring patches by from about 50 nm to about 500 μm.  
     
     
         45 . The array of  claim 42 , wherein at least some of said patches comprise different immobilized proteins.  
     
     
         46 . The array of  claim 45  which comprises at least about 9 different immobilized proteins.  
     
     
         47 . The array of  claim 46  which comprises at least about 100 different immobilized proteins.  
     
     
         48 . The array of  claim 47  which comprises at least about 1000 different immobilized proteins.  
     
     
         49 . The array of  claim 42 , wherein the area of the substrate surface covered by each of the patches is about 100 nm 2  or more.  
     
     
         50 . The array of  claim 49 , wherein the area of the substrate surface covered by each of the patches is between about 100 nm 2  and about 40,000 μm 2 .  
     
     
         51 . The array of  claim 50 , wherein the area of the substrate surface covered by each of the patches is between about 1 μm 2  to about 10,000 μm 2 .  
     
     
         52 . The array of  claim 42 , wherein said plurality of patches further comprise functionalized surfaces formed on said substrate by microstamping or microcontact printing.  
     
     
         53 . An array of  claim 42 , wherein said proteins are functionally related or are suspected of being functionally related.  
     
     
         54 . An array of  claim 42 , wherein said proteins are structurally related or are suspected of being structurally related.  
     
     
         55 . An array of  claim 42 , wherein said proteins are members of the same family.  
     
     
         56 . An array of  claim 55 , wherein the proteins are selected from the group consisting of growth factor receptors, catecholamine receptors, amino acid derivative receptors, cytokine receptors, extracellular matrix receptors, immunoglobulins, lectins, cytokines, serpins, proteases, kinases, phosphatases, ras-like GTPases, hydrolases, steroid hormone receptors, heat-shock transcription factors, zinc-finger proteins, leucine-zipper proteins, homeodomain proteins, hepatitis C virus (HCV) proteases and HIV proteases.  
     
     
         57 . An array of  claim 42 , wherein said patches further comprise a monolayer on the surface of the substrate, and wherein the proteins of said patches are immobilized on said monolayer.  
     
     
         58 . An array of  claim 57 , wherein the monolayer of an individual patch comprises molecules of the formula X—R—Y, wherein R is a spacer, X is a functional group that binds R to the surface, and Y is a functional group for binding proteins onto the monolayer.  
     
     
         59 . An array of  claim 58 , wherein the monolayer of an individual patch comprises at least two different X—R—Y molecules.  
     
     
         60 . An array of  claim 58  or  59 , wherein said substrate is selected from the group consisting of silicon, silicon oxide, indium tin oxide, magnesium oxide, alumina, quartz, glass, and silica, wherein X, prior to incorporation into said monolayer, is selected from the group consisting of a monohalosilane, dihalosilane, trihalosilane, trichlorosilane, trialkoxysilane, dialkoxysilane, and monoalkoxysilane, wherein R is an alkyl from about 8 to 22 carbons long, and wherein Y comprises a functional group selected from the group consisting of a maleimide, N-hydroxysuccinimide, nitrilotriacetic acid, activated hydroxyl, haloacetyl, bromoacetyl, iodoacetyl, activated carboxyl, hydrazide, epoxy, aziridine, trifluoromethyldiaziridine, pyridyldisulfide, N-acyl-imidazole, imidazolecarbamate, succinimidylcarbonate, arylazide, anhydride, diazoacetate, benzophenone, isothiocyanate, isocyanate, imidoester, fluorobenzene, and biotin.  
     
     
         61 . An array of  claim 57 , further comprising a coating between said substrate and said monolayer, wherein said coating is formed on the substrate or applied to the substrate.  
     
     
         62 . An array of  claim 61 , wherein the mean roughness of said coating is less than about 5 angstroms for areas of at least 25 μm 2 .  
     
     
         63 . An array of  claim 62 , wherein the mean roughness of said coating is less than about 3 angstroms for areas of at least 25 μm 2 .  
     
     
         64 . An array of  claim 61 , wherein said coating is an ultraflat, template-stripped surface.  
     
     
         65 . An array of  claim 63 , wherein said coating comprises a metal film, wherein the monolayer of an individual patch comprises at least two different X—R—Y molecules, and wherein X prior to incorporation into said monolayer, is a functional group selected from the group consisting of an asymmetrical or symmetrical disulfide, sulfide, diselenide, selenide, thiol, isonitrile, selenol, trivalent phosphorus compounds, isothiocyanate, isocyanate, xanthanate, thiocarbamate, phosphines, amines, thio acid and dithio acid, wherein R is an alkyl chain from about 8 to about 22 carbons long, and wherein Y comprises a functional group selected from the group consisting of a maleimide, N-hydroxysuccinimide, nitrilotriacetic acid, activated hydroxyl, haloacetyl, bromoacetyl, iodoacetyl, activated carbokyl, hydrazide, epoxy, aziridine, trifluoromethyldiaziridine, pyridyldisulfide, N-acyl-imidazole, imidazolecarbamate, succinimidylcarbonate, arylazide, anhydride, diazoacetate, benzophenonee, isothiocyanate, isocyanate, imidoester, fluorobenzene, and biotin.  
     
     
         66 . An array of  claim 57 , wherein the monolayer of an individual patch further comprises a second molecule, wherein said second molecule is of the formula X—R-V, and wherein R is spacer, X is a functional group that binds R to the surface, and V is a moiety resistant to the non-specific binding of proteins.  
     
     
         67 . An array of  claim 57 , wherein said array further comprises at least one patch comprising a monolayer of molecules of the formula X—R-V, wherein R is a spacer, X is a functiorial group that binds R to the surface, and V is a moiety resistant to the non-specific binding of proteins.  
     
     
         68 . An array of  claim 58 , further comprising crosslinking between molecules of the monolayer of a patch.  
     
     
         69 . An array of  claim 59 , further comprising an affinity tag, wherein said affinity tag enhances site-specific immobilization of the biological moiety onto the monolayer.  
     
     
         70 . An array of  claim 69 , wherein said affinity tag comprises an unnatural amino acid.  
     
     
         71 . An array of  claim 69 , wherein said affinity tag comprises a poly(amino acid).  
     
     
         72 . An array of  claim 71 , wherein said poly(amino acid) is selected from the group consisting of poly-cysteine, polylysine, poly-arginine, and poly-histidine.  
     
     
         73 . An array of  claim 69 , wherein said affinity tag comprises a polypeptide or a protein.  
     
     
         74 . An array of  claim 67 , wherein the affinity tag and the biological moiety together compose a fusion protein.  
     
     
         75 . An array of  claim 67 , further comprising an adaptor molecule that links the affinity tag to the immobilized biological moiety.  
     
     
         76 . An array of  claim 75 , wherein said adaptor molecule is a polypeptide or a protein.  
     
     
         77 . An array of  claim 76 , wherein said adaptor molecule is selected from the group consisting of green fluorescent protein, glutathione S-transferase, maltose-binding protein chitin-binding protein, and thioredoxin.  
     
     
         78 . An array of  claim 75 , wherein the affinity tag, adaptor molecule, and biological moiety together compose a fusion protein.  
     
     
         79 . A method for screening a plurality of proteins for their ability to interact with a component of a fluid sample, comprising: delivering the fluid sample to the array of  claim 42;  and detecting, either directly or indirectly, the interaction of said component with the immobilized protein of each patch.  
     
     
         80 . A method for screening a plurality of proteins for their ability to bind a component of a fluid sample, comprising: delivering said fluid sample to the array of  claim 42;  washing the array with fluid which does not contain said component in order to elute unbound component; and detecting, either directly or indirectly, the presence of said component retained at each patch.  
     
     
         81 . A method for detecting in a fluid sample the presence of a plurality of analytes which react with said proteins, comprising: delivering the fluid sample to the array of  claim 42;  and detecting the interaction of the analyte with the immobilized protein at each patch.  
     
     
         82 . A method for detecting in a fluid sample the presence of a plurality of analytes which bind said proteins, comprising: delivering the fluid sample to the array of  claim 42;  washing said array with an analyte-free fluid to remove unbound analyte; and detecting, either directly or indirectly, the presence of analyte retained at each patch.