Methods of immobilizing ligands on solid supports and apparatus and methods of use therefor

Abstract

A method is provided for immobilizing a ligand, e.g., a nucleic acid, on a solid support. The method includes providing a solid support containing an immobilized latent thiol group, activating the thiol group, contacting the activated thiol group with a nucleic acid comprising an acrylamide functional group, and forming a covalent bond between the two groups, thereby immobilizing the nucleic acid to the solid support. Kits containing the solid supports and method of utilizing the solid supports are also provided.

What is claimed is:  
     
         1 . A method of immobilizing an affinity ligand on a solid support comprising: 
 providing a solid support comprising an immobilized thiol group;    contacting the thiol group with a nucleic acid comprising an acrylamide functional group; and    forming a covalent bond between the two groups,    thereby immobilizing the ligand on the solid support.    
     
     
         2 . The method of  claim 1 , wherein the ligand is selected from the group consisting of a nucleic acid, a modified nucleic acid and a nucleic acid analog.  
     
     
         3 . The method of  claim 2 , wherein the solid support comprises a plurality of thiol groups  
     
     
         4 . The method of  claim 3 , wherein a plurality of ligands are immobilized on the solid support.  
     
     
         5 . The method of  claim 4 , wherein the solid support is formed from a compound selected from the group consisting of class, plastic and metal.  
     
     
         6 . The method of  claim 5 , wherein the solid support comprises two or more spatially distinct regions, each region comprising a plurality of immobilized nucleic acids.  
     
     
         7 . The method of  claim 6 , wherein the solid support further comprises a polymer layer.  
     
     
         8 . The method of  claim 7 , wherein the solid support comprises a microarray.  
     
     
         9 . The method of  claim 1 , wherein the thiol groups comprise reduced disulfide groups.  
     
     
         10 . A method of immobilizing an affinity ligand on a solid support comprising the steps of: 
 providing a solid support comprising immobilized latent thiol groups;    activating the latent thiol groups; and    reacting the activated thiol groups with an affinity ligand having at least one acrylamide functional group, thereby immobilizing an affinity ligand on a solid support.    
     
     
         11 . The method of  claim 10 , wherein the ligand is selected from the group consisting of a nucleic acid, a modified nucleic acid and a nucleic acid analog.  
     
     
         12 . The method of  claim 11 , wherein the steps of activating the latent thiol groups and reacting the activated thiol groups occur essentially simultaneously.  
     
     
         13 . The method of  claim 12 , wherein the solid support is formed from a compound selected from the group consisting of glass, plastic and metal.  
     
     
         14 . The method of  claim 13 , wherein the solid support comprises two or more spatially distinct regions, each region comprising a plurality of immobilized nucleic acids.  
     
     
         15 . The method of  claim 14 , wherein the solid support further comprises a polymer layer.  
     
     
         16 . The method of  claim 15 , wherein the solid support comprises a microarray.  
     
     
         17 . The product formed by the method of  claim 10 .  
     
     
         18 . A method of immobilizing an affinity ligand on microarray comprising the steps of: 
 providing a solid support comprising immobilized latent thiol groups;    activating the latent thiol groups; and    reacting the activated thiol groups with an affinity ligand having at least one α,β unsaturated carbonyl functional group, thereby immobilizing an affinity ligand on a solid support.    
     
     
         19 . The method of  claim 18 , wherein the ligand is selected from the group consisting of a nucleic acid, a modified nucleic acid and a nucleic acid analog.  
     
     
         20 . The method of  claim 19 , wherein the steps of activating the latent thiol groups and reacting the activated thiol groups occur essentially simultaneously.  
     
     
         21 . The method of  claim 10 , further comprising the steps of: 
 contacting a glass solid support with a silane compound represented by the following structural formula:                          wherein:    X is a halogen; and    RI, R, and R, are each, independently, a halogen, an alkyl group. an alkenyl group or a group having at least one α,β-unsaturated carbonyl, provided that at least one of RI, R, or R, is an alkenyl group or a group having at least one α,β-unsaturated carbonyl, thereby forming a solid support having an unsaturated aliphatic surface; and    contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing free radical initiator, disulfide bisacrylamide, and optionally an acrylamide, wherein the disulfide bisacrylamide is represented by the following structural formula:                          wherein n and m are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups.    
     
     
         22 . The method of  claim 21 , wherein the latent thiol groups are activated by contacting the solid support with a disulfide reducing agent.  
     
     
         23 . The method of  claim 21 , wherein the polymerization solution further includes alkylene bisacrylamide.  
     
     
         24 . The method of  claim 21 , wherein the free radical initiator is added to the polymerization solution after the solution is in contact with the unsaturated aliphatic surface of the solid support.  
     
     
         25 . The method of  claim 21 , further comprising the step of derivatizing a solid support with a latent thiol group, thereby forming a solid support having immobilized latent thiol groups.  
     
     
         26 . The method of  claim 21 , wherein the solid support has an amine functional group and the solid support is derivatized by contacting the solid support with a compound represented by the following structural formula:  
       
         
           
           
               
               
           
         
         wherein: 
 Y is a leaving group;  
 L is a linking group; and  
 R 4  is a thiol protecting group, thereby forming a solid support having immobilized latent thiol groups.  
 
       
     
     
         27 . The method of  claim 26  wherein Y consists of a group selected from the following groups:  
       
         
           
           
               
               
           
         
         wherein R 6  and R 7  are aliphatic groups.  
       
     
     
         28 . The method of  claim 26 , wherein R, is selected from the group consisting of:  
       
         
           
           
               
               
           
         
         wherein R 8  is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted aralkyl, or a substituted or unsubstituted heteroaralkyl group.  
       
     
     
         29 . A method of preparing a solid support having immobilized thiol groups, comprising the steps of: 
 contacting a glass solid support with a silane compound represented by the following structural formula:                          wherein: 
 X is a halogen; and  
 R 1 , R 2 , and R 3 , are each, independently, a halogen, an alkyl group, an alkenyl group or a group having at least one α,β-unsaturated carbonyl, provided that at least one of R 1 , R 2 , or R 3  is an alkenyl group or a group having at least one α,β-unsaturated carbonyl, thereby forming a solid support having an unsaturated aliphatic surface;  
   contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing free radical initiator, disulfide bisacrylamide, and optionally an acrylamide, wherein the disulfide bisacrylamide is represented by the following structural formula:                          wherein:    n and m are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups; and contacting the latent thiol groups with a disulfide reducing agent, thereby forming a solid support having immobilized thiol groups.    
     
     
         30 . The method of  claim 29 , wherein a plurality of nucleic acids are immobilized on the solid support.  
     
     
         31 . The method of  claim 30 , wherein the solid support comprises two or more spatially distinct regions, each region comprising a plurality of immobilized nucleic acids.  
     
     
         32 . The method of  claim 29 , wherein the thiol groups comprise disulfide groups.  
     
     
         33 . The method of  claim 31 , wherein the latent thiol groups in selected regions of the support are activated, thereby providing a support comprising selected regions of reactive thiol groups.  
     
     
         34 . A method of forming an array of nucleic acids immobilized on a solid support comprising: 
 forming an amine-derivatized region on the support;    treating the amine-derivatized region with a thiolating agent such that latent thiol groups immobilized on the support are formed;    activating the latent thiol groups;    contacting the activated thiol groups with a plurality of nucleic acids comprising acrylamide functional groups; and    forming a covalent bond between the two groups,    thereby forming an array of nucleic acids immobilized on the solid support.    
     
     
         35 . The method of  claim 34 , wherein each nucleic acid comprises a nucleotide sequence substantially identical to the nucleotide sequence of the other nucleic acids of the array.  
     
     
         36 . The method of  claim 34 , wherein nucleic acids with a plurality of nucleotide sequences are contained in the array.  
     
     
         37 . The method of  claim 34  comprising a plurality of amine-derivatized regions.  
     
     
         38 . The method of  claim 34  further comprising a step of blocking unbonded reactive thiol groups remaining following the binding of the nucleic acids to the thiol groups.  
     
     
         39 . The microarray prepared by the method of  claim 34 .  
     
     
         40 . A kit for attaching nucleic acids to a solid support comprising a solid support component comprising a plurality of immobilized latent thiol groups and instructions for activating the thiol groups to form covalent bonds with nucleic acids comprising acrylamide functional groups.  
     
     
         41 . The kit of  claim 40  further comprising at least one component selected from the group consisting of an activator component, an acrylamide functional nucleic acids component, a blocking component, a wash buffer and a wash buffer.  
     
     
         42 . A kit for attaching nucleic acids to a solid support comprising a solid support component comprising a plurality of immobilized thiol groups and nucleic acids comprising acrylamide functional groups.  
     
     
         43 . The kit of  claim 42 , wherein the nucleic acids are immobilized on a solid support by a covalent bond between the immobilized thiol groups and the nucleic acids.  
     
     
         44 . The kit of  claim 43  further comprising at least one component selected from the group consisting of an activator component, an acrylamide functional nucleic acids component, a blocking component and a wash buffer.  
     
     
         45 . A method for detecting or separating target nucleic acids from other components contained in a sample comprising: 
 providing a solid support comprising a plurality of immobilized nucleic acids comprising nucleotide sequences complementary to a subsequence of the nucleotide sequence of the target nucleic acid, wherein the nucleic acids are immobilized by a covalent bond formed between a thiol group immobilized on the solid support and an acrylamide functional group contained on the nucleic acid;    contacting the immobilized nucleic acid with the sample; and    hybridizing target nucleic acids to immobilized nucleic acids with complementary subsequences,    thereby detecting or separating target nucleic acids from other components contained in the sample.    
     
     
         46 . The method of  claim 45 , wherein the target nucleic acids are amplified after detection or separation.  
     
     
         47 . The method of  claim 45 , wherein the method is used in an assay selected from the group of assays for detecting a contaminant in a sample, for medical diagnosis of a medical condition, for genetic and physical mapping of genomes, for monitoring gene expression and for DNA sequencing.  
     
     
         48 . A method for detecting or separating target nucleic acids from other components contained in a sample comprising: 
 providing a solid support comprising a plurality of immobilized thiol groups;    contacting the thiol groups with a plurality of nucleic acids comprising nucleotide sequences complementary to a subsequence of the nucleotide sequence of the target nucleic acid and acrylamide functional groups;    forming a covalent bond between the thiol and acrylamide functional groups, thereby immobilizing the nucleic acids on the solid support;    contacting the immobilized nucleic acids with the sample; and    hybridizing target nucleic acids to immobilized nucleic acids with complementary subsequences,    thereby detecting or separating target nucleic acids from other components contained in the sample.    
     
     
         49 . The method of  claim 48 , wherein the target nucleic acids are amplified after detection or separation.  
     
     
         50 . The method of  claim 49 , wherein the method is used in an assay selected from the group of assays for detecting a contaminant in a sample, for medical diagnosis of a medical condition, for genetic and physical mapping of genomes, for monitoring gene expression and for DNA sequencing.  
     
     
         51 . The method of  claim 10 , wherein the solid support is doped or undoped silica, alumina, quartz or glass, and wherein the method further comprises the steps of: 
 contacting the solid support with a compound comprising a silane group or a carboxylic acid and a substituted or unsubstituted alkenyl group or a group having at least one (α,β-unsaturated carbonyl, thereby forming a solid support having an unsaturated aliphatic surface; and    contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing free radical initiator, a disulfide bisacrylamide and optionally containing an acrylamide, wherein the disulfide bisacrylamide is represented by the following structural formula:                          wherein: 
 n and m are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups.  
   
     
     
         52 . The method of  claim 51 , wherein the compound is represented by the following structural formula:  
       
         
           
           
               
               
           
         
         wherein: 
 X is a halogen; and  
 RI, R2 and R3 are each, independently, a halogen, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group or a group having at least one a,P-unsaturated carbonyl, provided that at least one of RI, R2 or R3 is a substituted or unsubstituted alkenyl group or a group having at least one a,p-unsaturated carbonyl.  
 
       
     
     
         53 . The method of  claim 51 , wherein the latent thiol groups are activated by contacting the solid support with a disulfide reducing agent.  
     
     
         54 . The method of  claim 51 , wherein the polymerization solution further includes alkylene bisacrylamide.  
     
     
         55 . The method of  claim 51 , wherein the free radical initiator is added to the polymerization solution after the solution is in contact with the unsaturated aliphatic surface of the solid support.  
     
     
         56 . The method of  claim 10 , wherein the solid support is selected from the group consisting of gold, silver, copper, cadmium, zinc, palladium, platinum, mercury, lead, iron, chromium, manganese, tungsten, and alloys thereof, and wherein the method further comprises the steps of: 
 contacting the solid support with a compound comprising a thiol group, sulfide or disulfide group and a substituted or unsubstituted alkenyl group or a group having at least one (x,p-unsaturated carbonyl, thereby forming a solid support having an unsaturated aliphatic surface; and    contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing free radical initiator, a disulfide bisacrylamide and optionally containing a conmonomer, wherein the disulfide bisacrylamide is represented by the following structural formula:                          wherein: 
 n and m are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups.  
   
     
     
         57 . The method of  claim 10 , wherein the solid support is selected from the group consisting of platinum or palladium, and wherein the method further comprises the steps of: 
 contacting the solid support with a compound comprising a nitrile or isonitrile group and a substituted or unsubstituted alkenyl group or a group having at least one a,p-unsaturated carbonyl, thereby forming a solid support having an unsaturated aliphatic surface; and    contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing free radical initiator, a disulfide bisacrylamide and optionally containing an acrylamide, wherein the disulfide bisacrylamide is represented by the following structural formula:                          wherein: 
 n and m in are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups.  
   
     
     
         58 . The method of  claim 10 , wherein the solid support is copper, and wherein the method further comprises the steps of: 
 contacting the solid support with a compound comprising a hydroxamic acid group and a substituted or unsubstituted alkenyl group or a group having at least one α,β-unsaturated carbonyl, thereby forming a solid support having an unsaturated aliphatic surface; and    contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing free radical initiator and disulfide bisacrylamide and optionally containing an acrylamide, wherein the disulfide bisacrylamide is represented by the following structural formula:                          wherein n and mn are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups.    
     
     
         59 . The method of  claim 10 , wherein the solid support is a polymer comprising a reactive functional group, and wherein the method further comprises the steps of: 
 contacting the solid support with a compound comprising a functional group which can react to form a bond with the reactive functional group and a substituted or unsubstituted alkenyl group or a group having at least one α,β-unsaturated carbonyl, thereby forming a solid support having immobilized unsaturated aliphatic groups; and    contacting the unsaturated aliphatic groups of the solid support with a polymerization solution containing free radical initiator, a disulfide bisacrylamide and optionally containing an acrylamide, wherein the disulfide bisacrylamide is represented by the following structural formula:                          wherein: 
 n and m are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups.  
   
     
     
         60 . The method of  claim 59 , wherein the polymeric solid support is polystyrene.  
     
     
         61 . The method of  claim 59 , wherein hod of  claim 59 , wherein the reactive functional group of the polymeric solid support is an amine group or a hydroxyl group and the compound is represented by the following structural formula:  
       
         
           
           
               
               
           
         
         wherein: 
 Y is a leaving group;  
 L is a linking group; and  
 R 10  is a substituted or unsubstituted alkenyl group or a group having at least one α,β-unsaturated carbonyl.  
 
       
     
     
         62 . The compound of  claim 61 , wherein Y is selected from the group consisting of:  
       
         
           
           
               
               
           
         
         wherein R 6  and R 7  are each, independently, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted aralkyl, or a substituted or unsubstituted heteroaralkyl group.  
       
     
     
         63 . The method of  claim 10 , further comprising the step of derivatizing a solid support with a latent thiol group, thereby forming a solid support having immobilized latent thiol groups.  
     
     
         64 . The method of  claim 63 , wherein the solid support is selected from the group consisting of doped or undoped silica, alumina, quartz or glass, and the solid support is derivatized by contacting it with a compound comprising a silane group or a carboxylic acid group and a latent thiol group.  
     
     
         65 . The method of  claim 63 , wherein the solid support is selected from the group consisting of platinum or palladium, and the solid support is derivatized by contacting it with a compound comprising a nitrile or isonitrile group and a latent thiol group.  
     
     
         66 . The method of  claim 63 , wherein the solid support is a polymer comprising reactive functional groups, and the solid support is derivatized by contacting it with a compound comprising a functional group which can react to form a bond with the reactive functional group and a latent thiol group.  
     
     
         67 . The method of  claim 63 , wherein the polymeric solid support is polystyrene.  
     
     
         68 . The method of  claim 66 , wherein the reactive functional group of the polymeric solid support is an amine or a hydroxyl group and the solid support is derivatized by contacting the solid support with a compound represented by the following structural formula:  
       
         
           
           
               
               
           
         
         wherein: 
 Y is a leaving group;  
 L is a linking group; and  
 R 4  is a thiol protecting group, thereby forming a solid support having immobilized latent thiol groups.  
 
       
     
     
         69 . The method of  claim 68 , wherein Y is selected form the group consisting of:  
       
         
           
           
               
               
           
         
         wherein R 6  and R 7  are each, independently, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted aralkyl, or a substituted or unsubstituted heteroaralkyl group.  
       
     
     
         70 . The method of  claim 68 , wherein is selected from the group consisting of:  
       
         
           
           
               
               
           
         
         wherein R 6 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted aralkyl, or a substituted or unsubstituted heteroaralkyl group.  
       
     
     
         71 . A method of making a solid support having immobilized thiol groups, comprising the steps of: 
 contacting a glass solid support with a silane compound represented by the following structural formula:                        wherein: 
 X is a halogen; and  
 R 1 , R 2  and R 3  are each, independently, a halogen, an alkyl group, an alkenyl group or a group having at least one α,β-unsaturated carbonyl, provided that at least one of RI, R, or R, is an alkenyl group or group having at least one α,β-unsaturated carbonyl, thereby forming a solid support having an unsaturated aliphatic surface;  
     contacting the unsaturated aliphatic surface of the solid support with a polymerization solution containing free radical initiator, a disulfide bisacrylamide and optionally containing an acrylamide, wherein the disulfide bisacrylamide is represented by the following structural formula:                        wherein: 
 n and m are each, independently, a positive integer, thereby forming a solid support comprising immobilized latent thiol groups; and  
 contacting the latent thiol groups with a disulfide reducing agent, thereby forming a solid support having immobilized thiol groups.