Microdevices for high-throughput screening of biomolecules

Abstract

Methods and devices for the parallel, in vitro screening of biomolecular activity using miniaturized microfabricated devices are provided. The biomolecules that can be immobilized on the surface of the devices of the present invention include proteins, polypeptides, nucleic acids, polysaccharides, phospolipids, and related unnatural polymers of biological relevance. These devices are useful in high-throughput drug screening and clinical diagnostics and are preferably used for the parallel screening of families of related proteins.

What is claimed is:  
     
         1 . A device for processing a fluid sample, having a plurality of noncontiguous reactive sites, each of said sites comprising: 
 (a) a substrate;    (b) an immobilized biological moiety;    (b) a monolayer chemisorbed or physisorbed on a portion of a surface of the substrate, said monolayer comprising 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 said biological moiety onto the monolayer; and      (c) an affinity tag, wherein said affinity tag enhances site-specific immobilization of said biological moiety onto the monolayer.    wherein each of said sites may independently react with a component of the fluid sample and are separated from each other by a region of said substrate that is free of molecules of the formula X—R—Y.    
     
     
         2 . A device of  claim 1 , 
 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, benzophenonee, isothiocyanate, isocyanate, imidoester, fluorobenzene, and biotin.    
     
     
         3 . A device of  claim 1 , 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.  
     
     
         4 . A device of  claim 3 , 
 wherein said coating comprises a metal film,    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 carboxyl, hydrazide, epoxy, aziridine, trifluoromethyldiaziridine, pyridyldisulfide, N-acyl-imidazole, imidazolecarbamate, succinimidylcarbonate, arylazide, anhydride, diazoacetate, benzophenone, isothiocyanate, isocyanate, imidoester, fluorobenzene, and biotin.    
     
     
         5 . A device of  claim 1 , wherein the monolayer of an individual reactive site comprises at least two different X—R—Y molecules.  
     
     
         6 . A device of  claim 1 , wherein the monolayer of an individual reactive site further comprises a second molecule, wherein said second molecule is of the formula 
       X—R—V wherein R is a spacer, X is a functional group that binds R to the surface, and V is a moiety resistant to the non-specific binding of biomolecules.    
     
     
         7 . A device of  claim 1 , wherein the device further comprises at least one unreactive site, wherein said unreactive site comprises a monolayer of molecules of the formula 
       X—R—V wherein R is a spacer, X is a functional group that binds R to the surface, and V is a moiety resistant to the non-specific binding of biomolecules.    
     
     
         8 . A device of  claim 1 , further comprising crosslinking between molecules of the monolayer.  
     
     
         9 . A device of  claim 1 , wherein said affinity tag comprises a natural or unnatural amino acid.  
     
     
         10 . A device of  claim 1 , wherein said affinity tag comprises a poly(amino acid).  
     
     
         11 . A device of  claim 10 , wherein said poly(amino acid) is selected from the group consisting of poly-cysteine, poly-lysine, poly-arginine, and poly-histidine.  
     
     
         12 . A device of  claim 1 , wherein said affinity tag comprises a polypeptide or a protein.  
     
     
         13 . A device of  claim 1 , wherein said affinity tag is a component of a layer of affinity tag molecules immobilized on said monolayer.  
     
     
         14 . A device of  claim 13 , wherein said layer of affinity tag molecules comprises a hydrogel matrix.  
     
     
         15 . A device of  claim 1 , wherein said affinity tag and said biological moiety together compose a fusion protein.  
     
     
         16 . A device of  claim 1 , further comprising an adaptor molecule that links the affinity tag to the immobilized biological moiety.  
     
     
         17 . A device of  claim 16 , wherein said adaptor molecule is a membrane anchor and wherein said affinity tag is a component of a layer of affinity tag molecules, said layer being selected from the group consisting of a phospholipid bilayer and a phospholipid monolayer.  
     
     
         18 . A device of  claim 16 , wherein said adaptor molecule is a polypeptide or a protein.  
     
     
         19 . A device of  claim 18 , 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.  
     
     
         20 . A device of  claim 16 , wherein the affinity tag, adaptor molecule, and biological moiety together compose a fusion protein.  
     
     
         21 . A device of  claim 1 , wherein the biological moiety of one reactive site differs from the biological moiety of a second reactive site on the same device.  
     
     
         22 . A device of  claim 21 , wherein the biological moieties are believed to be functionally related.  
     
     
         23 . A device of  claim 21 , wherein the biological moieties are believed to be structurally related.  
     
     
         24 . A device of  claim 21 , wherein the biological moieties are members of the same protein family.  
     
     
         25 . A device of  claim 24 , wherein the biological moieties 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, hepatitus C virus (HCV) proteases, and HIV proteases.  
     
     
         26 . A device of  claim 1 , wherein the biological moiety of one reactive site is identical to the biological moiety of a second reactive site on the same device.  
     
     
         27 . A device of  claim 1 , wherein said immobilized biological moiety is selected from the group consisting of a nucleic acid, a polypeptide, an antibody or fragment thereof, an epitope, a membrane protein, a hormone, and a small organic molecule which either has or is suspected of having a physiological function.  
     
     
         28 . A device of  claim 1 , wherein said device comprises a micromachined device.  
     
     
         29 . A device of  claim 1 , wherein each of said reactive sites is in a microchannel oriented parallel to microchannels of other reactive sites on the device, wherein said microchannels are microfabricated into or onto said substrate.  
     
     
         30 . A device of  claim 29 , wherein said device comprises at least 10 microchannels.  
     
     
         31 . A device of  claim 30 , wherein said device comprises from about 100 to about 500 microchannels.  
     
     
         32 . A device of  claim 29 , wherein said device comprises from about 2 to about 500 parallel microchannels per cm 2 .  
     
     
         33 . A device of  claim 29 , further comprising a cover over the microchannels.  
     
     
         34 . A device of  claim 33 , wherein the volume of said microchannel is between about 5 nanoliters and about 300 nanoliters.  
     
     
         35 . A device of  claim 34 , wherein the volume of said microchannel is between about 10 nanoliters and about 50 nanoliters.  
     
     
         36 . A device of  claim 29 , wherein the width and depth of said microchannel each are between about 10 μm and about 500 μm.  
     
     
         37 . A method for screening a plurality of biological moieties in parallel for their ability to interact with a component of a fluid sample, comprising: 
 (a) delivering the fluid sample to the reactive sites of a device of  claim 1 , wherein each reactive site of the device comprises a different biological moiety; and    (b) detecting, either directly or indirectly, the interaction of said component with the immobilized biological moiety at each reactive site.    
     
     
         38 . A method for screening a plurality of biological moieties in parallel for their ability to react with a component of a fluid sample, comprising: 
 (a) delivering the fluid sample to the reactive sites of a device of  claim 1 , wherein each reactive site of the device comprises a different biological moiety; and    (b) detecting, either directly or indirectly, formation of product of the reaction of said component with the immobilized biological moiety at each reactive site.    
     
     
         39 . A method for screening the ability of a drug candidate to inhibit the reaction of a plurality of members of a protein family with their substrate, comprising: 
 (a) combining the drug candidate and the substrate in a fluid sample;    (b) delivering the fluid sample to the reactive sites of a device of  claim 1 , wherein each reactive site of the device comprises a different member of the protein family; and    (c) detecting, either directly or indirectly, for the inhibition of product formation at each reactive site.    
     
     
         40 . A method for screening a plurality of biological moieties in parallel for their ability to bind a component of a fluid sample, comprising: 
 (a) delivering said fluid sample to the reactive sites of a device of  claim 1 , wherein each reactive site of the device comprises a different biological moiety;    (b) washing said reactive site with fluid which does not contain said component in order to elute unbound component; and    (c) detecting, either directly or indirectly, the presence of said component retained at each reactive site.    
     
     
         41 . A method for screening a plurality of biological moieties in parallel for their ability to bind a component of a fluid sample, comprising: 
 (a) adding a known ligand of said biological moieties to the fluid sample;    (b) delivering the fluid sample to the reactive sites of a device of  claim 1 , wherein each reactive site of the device comprises a different biological moiety;    (c) washing said reactive sites with fluid that does not contain either the known ligand or the component in order to elute unbound molecules of the known ligand and the component;    (d) detecting the presence of the known ligand retained at each reactive site; and    (e) comparing retention of the known ligand detected in step (d) with retention of the known ligand in the absence of said component.    
     
     
         42 . A method for screening a plurality of drug candidates in parallel for their ability to inhibit a reaction of an enzyme with its substrate, comprising: 
 (a) adding the substrate to a plurality of fluid samples, each of the fluid samples containing at least one of the drug candidates;    (b) delivering each of the fluid samples to a reactive site of the device of  claim 1 , wherein the reactive site comprises the immobilized enzyme;    (c) detecting, either directly or indirectly, for inhibition of product formation at each reactive site.    
     
     
         43 . A method for screening a plurality of binding candidates in parallel for their ability to bind a biological moiety, comprising: 
 (a) delivering different fluid samples, each containing at least one of the binding candidates, to the reactive sites of the device of  claim 1 , wherein the reactive sites each comprise the immobilized biological moiety;    (b) washing the reactive sites with fluid which does not contain said binding candidate in order to elute unbound binding candidates; and    (c) detecting, either directly or indirectly, the presence of said binding candidate retained at each reactive site.    
     
     
         44 . A method for screening a plurality of binding candidates in parallel for their ability to bind a biological moiety, comprising: 
 (a) adding a known ligand of the biological moiety to a plurality of fluid samples, each of the fluid samples containing at least one of the binding candidates;    (b) delivering a different fluid sample to each of the reactive sites of the device of  claim 1 , wherein the reactive sites of the device each comprise the immobilized biological moiety;    (c) washing said reactive sites with fluid that does not contain either the known ligand or a binding candidate in order to elute unbound molecules of the known ligand and the binding candidate;    (d) detecting the presence of the known ligand retained at each reactive site; and    (e) comparing retention of the known ligand detected in step (d) with retention of the known ligand in the absence of said binding candidate.    
     
     
         45 . A method for pairing a plurality of proteins with their substrates, comprising: 
 (a) delivering a fluid sample comprising a substrate of a known enzyme family to the reactive sites of a device of  claim 1 , wherein each reactive site of the device comprises a different protein; and    (b) detecting, either directly or indirectly, for product formed by the reaction of the substrate with the protein of each reactive site.    
     
     
         46 . A method for pairing a plurality of proteins with their ligands, comprising: 
 (a) delivering a fluid sample comprising a ligand of a known protein family to the reactive sites of a device of  claim 1 , wherein each reactive site of the device comprises a different protein;    (b) washing the reactive sites with fluid that does not contain said ligand to remove unbound ligand; and    (c) detecting, either directly or indirectly, the presence of the ligand retained at each reactive site.    
     
     
         47 . A method for detecting in a fluid sample the presence of a plurality of analytes which react with said biological moieties, comprising: 
 (a) delivering the fluid sample to the reactive sites of a device of  claim 1;  and    (b) detecting the interaction of the analyte with the immobilized biological moiety at each reactive site.    
     
     
         48 . A method for detecting in a fluid sample the presence of a plurality of analytes which bind said biological moieties, comprising: 
 (a) delivering the fluid sample to the reactive sites of a device of  claim 1;     (c) washing said reactive sites with an analyte-free fluid to remove unbound analyte; and    (b) detecting, either directly or indirectly, the presence of analyte retained at each reactive site.    
     
     
         49 . A device for processing a fluid sample, comprising: 
 (a) a substrate;    (b) a plurality of parallel microchannels microfabricated into or onto said substrate; and    (c) a biological moiety immobilized within at least one of said parallel microchannels, wherein said biological moiety may interact with a component of the fluid sample.    
     
     
         50 . A device of  claim 49 , wherein said device comprises at least 10 parallel microchannels.  
     
     
         51 . A device of  claim 50 , wherein said device comprises from about 100 to about 500 parallel microchannels.  
     
     
         52 . A device of  claim 49 , wherein said device comprises from about 2 to about 500 parallel microchannels per cm 2 .  
     
     
         53 . A device of  claim 49 , further comprising a cover over the microchannels.  
     
     
         54 . A device of  claim 53 , wherein the volume of each of said microchannels is between about 5 nanoliters and about 300 nanoliters.  
     
     
         55 . A device of  claim 54 , wherein the volume of said microchannel is between about 10 nanoliters and about 50 nanoliters.  
     
     
         56 . A device of  claim 49 , wherein the width and depth of said microchannels are each between about 10 μm and about 500 μm.