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, phospholipids, 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.

1 - 56 . (canceled) 
     
     
         57 . A method comprising:
 providing a sample detection device, the sample detection device comprising (i) a substrate having an upper surface, (ii) a plurality of parallel channels formed in or on the upper surface of the substrate, each channel extending between a first and a second end and having a bottom wall surface, wherein the channels are linearly aligned and can be loaded with fluids containing one or more analytes, and (iii) a plurality of immobilization molecules for immobilizing proteins, peptides, small molecules or nucleic acids at immobilization regions in each channel;   (b) introducing the fluids into the channels so that the fluids contact the immobilized proteins, peptides, small molecules or nucleic acids; and   (c) monitoring the interaction between the analytes in the fluids and the immobilized proteins, peptides, small molecules or nucleic acids in the channels.   
     
     
         58 . The method of  claim 57 , wherein the plurality of parallel channels includes at least 2 parallel microchannels. 
     
     
         59 . The method of  claim 57 , wherein the plurality of parallel channels Includes at least 10 parallel microchannels. 
     
     
         60 . The method of  claim 57 , wherein the plurality of parallel channels includes at least 100 parallel microchannels. 
     
     
         61 . The method of  claim 57 , wherein each pair of adjacent channels is separated a distance of between about 10 μm and 5 mm. 
     
     
         62 . The method of  claim 57 , wherein each channel has a cross section width that is between about 10 μm and 500 μm. 
     
     
         63 . The method of  claim 57 , wherein each channel has a cross section length that is between about 1 mm and 20 mm. 
     
     
         64 . The method of  claim 57 , wherein each channel has a trapezoidal cross section. 
     
     
         65 . The method of  claim 57 , wherein each channel has a rectangular cross section. 
     
     
         66 . The method of  claim 57 , wherein each channel has a semi-circular cross section. 
     
     
         67 . The method of  claim 57 , wherein the sample detection device further comprises a cover over the channels. 
     
     
         68 . The method of  claim 57 , wherein the sample detection device further comprises a cover that is wholly or partly translucent or transparent. 
     
     
         69 . The method of  claim 57 , wherein the sample detection device further comprises a cover that is wholly or partly non-translucent or non-transparent. 
     
     
         70 . The method of  claim 57 , wherein the sample detection device further comprises a coating disposed between the bottom wall surface and the immobilization molecules 
     
     
         71 . The method of  claim 57 , wherein the immobilized proteins, peptides, small molecules or nucleic acids are chemisorbed or physisorbed to the immobilization regions. 
     
     
         72 . A method for screening a plurality of biological moieties in parallel for their ability to interact, react with or bind to a component of a fluid sample, comprising:
 (a) delivering the fluid sample to the reactive sites of a device for processing a fluid sample, said device having at least 100 noncontiguous reactive sites, each of said sites comprising:   (i) a substrate;   (ii) a different immobilized protein, peptide, or nucleic acid; and   (iii) a monolayer chemisorbed or physisorbed on the surface of said reactive sites, 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 protein, peptide, or nucleic acid onto the monolayer; and   (b) detecting, either directly or indirectly, the interaction or reaction of said component with the immobilized protein, peptide, or nucleic acid, or the binding of said component with the protein, peptide, or nucleic acid at a reactive site.   
     
     
         73 . A method according to  claim 72  wherein the device further comprises:
 (iv) an affinity tag, wherein said affinity tag enhances site-specific Immobilization of said protein, peptide, or nucleic acid onto the monolayer.   
     
     
         74 . A method according to  claim 72  wherein each of said reactive 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. 
     
     
         75 . A method according to  claim 72  in which a plurality of binding candidates is screened in parallel for their ability to bind a protein, peptide, or nucleic acid, and in which different fluid samples, each containing at least one of the binding candidates, are delivered to the reactive sites of the device, further comprising
 (c) washing the reactive sites with fluid which does not contain said binding candidate in order to elute unbound binding candidates; and   (d) detecting, either directly or indirectly, the presence of said binding candidate retained at each reactive site.   
     
     
         76 . A method according to  claim 72  in which a plurality of binding candidates is screened in parallel for their ability to bind a protein, peptide, or nucleic acid, and in which different fluid samples, each containing at least one of the binding candidates, are delivered to the reactive sites of the device, further comprising
 (c) washing the reactive sites with fluid which does not contain said binding candidate in order to elute unbound binding candidates; and   (d) detecting, either directly or indirectly, the presence of said binding candidate retained at each reactive site.   
     
     
         77 . A method according to  claim 72  in which a plurality of analytes which bind said proteins, peptides, or nucleic acids is detected, comprising:
 (a) delivering a fluid sample comprising said analytes to the reactive sites of the device;   (b) washing the reactive sites with an analyte-free fluid to remove unbound analyte; and   (c) detecting, either directly or indirectly; the presence of analyte retained at each reactive site.   
     
     
         78 . A method according to  claim 72  in which a plurality of proteins is paired with their ligands, comprising:
 (a) delivering a fluid sample comprising a ligand of a known protein family to the reactive sites of the device, 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.   
     
     
         79 . A method according to  claim 72  wherein each reactive site comprises an immobilized peptide. 
     
     
         80 . A method according to  claim 72  wherein each reactive site comprises an immobilized nucleic acid. 
     
     
         81 . A method according to  claim 72  wherein each reactive site comprises an immobilized protein. 
     
     
         82 . A method according to  claim 81  wherein the device comprises immobilized proteins of the same family. 
     
     
         83 . A method according to  claim 81  wherein the device comprises immobilized proteins that are functionally related. 
     
     
         84 . A method according to  claim 81  wherein the device comprises immobilized proteins that are structurally related. 
     
     
         85 . A method according to  claim 81  wherein the device comprises immobilized fusion proteins. 
     
     
         86 . A method according to  claim 85  wherein the device further comprises an affinity tag, wherein said affinity tag enhances site-specific immobilization of said protein onto the monolayer. 
     
     
         87 . A method according to  claim 81  wherein the device comprises immobilized recombinant proteins. 
     
     
         88 . A method according to  claim 81  wherein the device comprises immobilized kinases. 
     
     
         89 . A method according to  claim 81  wherein the device comprises immobilized antibodies. 
     
     
         90 . A method according to  claim 81  wherein the device comprises immobilized capture agents. 
     
     
         91 . A method according to  claim 72 , wherein said device comprises from about 100 to about 500 microchannels. 
     
     
         92 . A method according to  claim 72 , wherein said device comprises from about 2 to about 500 parallel microchannels per cm 2 . 
     
     
         93 . A method according to  claim 81  wherein the device comprises immobilized recombinant fusion proteins