Arrays of proteins and methods of use thereof

Abstract

Protein arrays for the parallel, in vitro screening of biomolecular activity are provided. Methods of using the protein arrays are also disclosed. On the arrays, a plurality of different proteins, such as different members of a single protein family, are immobilized on one or more organic thinfims on the substrate surface. The protein arrays are particularly useful in drug development, proteomics, and clinical diagnostics.

What is claimed is:  
     
         1 . An array of proteins, comprising: 
 (a) a substrate;    (b) at least one organic thinfilm on some or all of the substrate surface; and    (c) a plurality of patches arranged in discrete, known regions on portions of the substrate surface covered by organic thinfilm, wherein each of said patches comprises a protein immobilized on the underlying organic thinfilm.    
     
     
         2 . The array of  claim 1  which comprises at least about 10 of said patches.  
     
     
         3 . The array of  claim 2  which comprises at least about 100 of said patches.  
     
     
         4 . The array of  claim 3  which comprises at least about 10 3  of said patches.  
     
     
         5 . The array of  claim 1  which comprises at least about 10 different immobilized proteins.  
     
     
         6 . The array of  claim 5  which comprises at least about 100 different immobilized proteins.  
     
     
         7 . The array of  claim 6  which comprises at least about 1000 different immobilized proteins.  
     
     
         8 . The array of  claim 1 , wherein the area of the substrate surface covered by each of the patches is no more than about 0.25 mm 2 .  
     
     
         9 . The array of  claim 8 , wherein the area of the substrate surface covered by each of the patches is between about 1 μm 2  and about 10,000 μm 2 .  
     
     
         10 . The array of  claim 1 , wherein the patches are all contained within an area of about 1 cm 2  or less on the surface of the substrate.  
     
     
         11 . The array of  claim 1 , wherein all of the proteins immobilized on the array are functionally related.  
     
     
         12 . The array of  claim 1 , wherein all of the proteins immobilized on the array are structurally related.  
     
     
         13 . The array of  claim 1 , wherein all of the proteins immobilized on the array are members of the same family.  
     
     
         14 . An array of  claim 13 , wherein said family is selected from the group consisting of growth factor receptors, hormone receptors, neurotransmitter receptors, catecholamine receptors, amino acid derivative receptors, cytokine receptors, extracellular matrix receptors, antibodies, lectins, cytokines, serpins, proteases, kinases, phosphatases, ras-like GTPases, hydrolases, steroid hormone receptors, transcription factors, heat-shock transcription factors, DNA-binding proteins, zinc-finger proteins, leucine-zipper proteins, homeodomain proteins, intracellular signal transduction modulators and effectors, apoptosis-related factors, DNA synthesis factors, DNA repair factors, DNA recombination factors, cell-surface antigens, hepatitis C virus (HCV) proteases and HIV proteases.  
     
     
         15 . The array of  claim 1 , wherein the proteins are antibodies or antibody fragments.  
     
     
         16 . The array of  claim 1 , wherein the proteins are protein-capture agents.  
     
     
         17 . The array of  claim 1 , wherein the organic thinfilm on the array is less than about 20 nm thick.  
     
     
         18 . The array of  claim 1 , wherein the organic thinfilm on the array comprises a monolayer.  
     
     
         19 . The array of  claim 18 , wherein the monolayer comprises a self-assembled monolayer comprising molecules of the formula  
       (X) a R(Y) b    wherein R is a spacer, X is a functional group that binds R to the surface, Y is a functional group for binding the protein onto the monolayer, and a and b are, independently, integers.    
     
     
         20 . The array of  claim 19 , wherein both a and b are 1.  
     
     
         21 . The array of  claim 19 , wherein: 
 said substrate is selected from the group consisting of silicon, silicon dioxide, indium tin oxide, alumina, glass, and titania; and    X, prior to incorporation into said monolayer, is selected from the group consisting of a monohalosilane, dihalosilane, trihalosilane, trichlorosilane, trialkoxysilane, dialkoxysilane, monoalkoxysilane, carboxylic acids, and phosphates.    
     
     
         22 . The array of  claim 19 , wherein the substrate comprises silicon and X is an olefin.  
     
     
         23 . The array of  claim 1 , wherein the substrate comprises a polymer.  
     
     
         24 . The array of  claim 19 , further comprising at least one coating between said substrate and said monolayer, wherein said coating is formed on the substrate or applied to the substrate.  
     
     
         25 . The array of  claim 24 , wherein: 
 the coating comprises a noble metal film; and    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.    
     
     
         26 . The array of  claim 24 , wherein the coating comprises titania or tantalum oxide and X is a phosphate group.  
     
     
         27 . The array of  claim 1 , wherein each protein is immobilized on the organic thinfilm by an affinity tag.  
     
     
         28 . A biosensor comprising an array of proteins of  claim 1 .  
     
     
         29 . A micromachined device comprising an array of proteins of  claim 1 .  
     
     
         30 . A diagnostic device comprising an array of proteins of  claim 1 .  
     
     
         31 . A method for screening a plurality of proteins for their ability to interact with a component of a sample, comprising: 
 (a) delivering the sample to the array of  claim 1  comprising the proteins to be screened; and    (b) detecting, either directly or indirectly, for the interaction of said component with the immobilized protein of each patch.    
     
     
         32 . The method of  claim 31 , wherein the component is a protein.  
     
     
         33 . A method for screening a plurality of proteins for their ability to bind a particular component of a sample, comprising: 
 (a) delivering said sample to the array of  claim 1  comprising the proteins to be screened; and    (b) detecting, either directly or indirectly, for the presence or amount of said particular component retained at each patch.    
     
     
         34 . The method of  claim 33 , wherein said particular component is a protein.  
     
     
         35 . The method of  claim 33 , further comprising the step: 
 (d) further characterizing said particular component retained on at least one patch.    
     
     
         36 . A method of assaying for protein-protein binding interactions, comprising: 
 (a) delivering a sample comprising at least one protein to be assayed for binding to the array of  claim 1;  and    (b) detecting, either directly or indirectly, for the presence or amount of the protein from the sample which is retained at each patch.    
     
     
         37 . A method of assaying in parallel for a plurality of analytes in a sample, comprising: 
 (a) delivering the sample to the array of  claim 1 , wherein at least one of the immobilized proteins of said array can react with each of said analytes; and    (b) detecting for the interaction of the analytes with the immobilized protein at each patch.    
     
     
         38 . A method of assaying in parallel for a plurality of analytes in a sample, comprising: 
 (a) delivering the fluid sample to the array of  claim 1 , wherein at least one of the immobilized proteins of said array can bind each of said analytes; and    (b) detecting, either directly or indirectly, for the presence or amount of analyte retained at each patch.    
     
     
         39 . The method of  claim 38 , further comprising the step: 
 (d) further characterizing the analyte retained on at least one patch.