Method for immobilizing a biomolecule on a solid substrate at a high density by using the substrate having an active carboxyl group on a surface thereof and microarray produced using the method

Abstract

Provided is a method for immobilizing a biomolecule on a solid substrate. The method includes coating the solid substrate with silane anhydride to introduce an anhydride functional group onto a surface of the solid substrate; obtaining a carboxyl group from the anhydride functional group by hydrolysis; reacting the carboxyl group with carbodiimide and succinimide to activate the carboxyl group; and contacting the biomolecule with the solid substrate having the activated carboxyl group on its surface to immobilize the biomolecule on the solid substrate.

1 . A biomolecule microarray produced using a method for immobilizing a biomolecule on a solid substrate comprising 
 coating a solid substrate with silane anhydride such that the silane portion of the silane anhydride is bound to a surface of the solid substrate;    exposing two carboxyl groups from the anhydride functional group by hydrolysis;    reacting each exposed carboxyl group with carbodiimide and succinimide to activate the carboxyl group; and    contacting a biomolecule with the solid substrate having the activated carboxyl groups on its surface such that the biomolecule reacts with an activated carboxyl group derived from the silane anhydride to immobilize the biomolecule on the solid substrate.    
     
     
         2 . A method for immobilizing a biomolecule on a solid substrate, comprising: 
 coating a solid substrate with an aminosilane such that the silane portion of the aminosilane is bound to a surface of the solid substrate;    reacting an amino functional group with a tetracarboxylic dianhydride to introduce an anhydride functional group onto the surface of the solid substrate;    exposing a carboxyl group from the anhydride functional group by hydrolysis;    reacting the carboxyl group with a carbodiimide and a succinimide to activate the carboxyl group; and    contacting a biomolecule with the solid substrate having the activated carboxyl group on its surface such that the biomolecule reacts with the activated carboxyl group to immobilize the biomolecule on the solid substrate.    
     
     
         3 . The method of  claim 2 , wherein the solid substrate is coated with a solution containing 0.01 to 90% by weight of the aminosilane.  
     
     
         4 . The method of  claim 2 , wherein the aminosilane is a primary aminosilane represented by formula V or a secondary aminosilane represented by formula VI:  
         H 2 N—R 8 —Si(OR 9 ) 3   (V),  HN R 8 —Si(OR 9 ) 3 ] 2   (VI),  wherein    R 8  is an alkylene group, an arylene group, an arylenealkylene group, alkylenearylene group, ether, ester, or an imine containing group, and    R 9  is a C 1 -C 20  alkyl group, an aryl group, an arylenealkyl group, or an alkylenearyl group, or a hydrogen atom.    
     
     
         5 . The method of  claim 2 , wherein coating the solid substrate with the aminosilane comprises coating the solid substrate with an aminosilane solution comprising 1,2-bis(triethoxysilyl)ethane.  
     
     
         6 . The method of  claim 2 , wherein coating the solid substrate with the aminosilane is performed using a method selected from the group consisting of self-assembly molecular coating, spin coating, spraying, and chemical vapor deposition.  
     
     
         7 . The method of  claim 2 , wherein the tetracarboxylic dianhydride is represented by formula VII:  
       
         
           
           
               
               
           
         
         wherein  
         R 10  is a quaternary organic group, selected from a quaternary carboncyclic aromatic, heterocyclic, alicyclic, or aliphatic group.  
       
     
     
         8 . The method of  claim 7 , wherein the tetracarboxylic dianhydride is selected from the group consisting of pyromellitic dianhydride, 3,3′,4,4′-biphenyltetracarboxylic dianhydride, 2,2′,3,3′-biphenyltetracarboxylic dianhydride, 2,3,3′,4′-biphenyltetracarboxylic dianhydride, 1,2,4,5-benzenetetracarboxylic dianhydride, 3,3′,4,4′-benzophenonetetracarboxylic dianhydride, 2,2′,3,3′-benzophenonetetracarboxylic dianhydride, 2,3,3′,4′-benzophenonetetracarboxylic dianhydride, bis(3,4-dicarboxylphenyl) ether dianhydride, bis(3,4-dicarboxylphenyl)sulfone dianhydride, 1,4,5,8-naphthalenetetracarboxylic dianhydride, 1,2,5,6-naphthalenetetracarboxylic dianhydride, 2,3,6,7-naphthalenetetracarboxylic dianhydride, 2,2-bis(3,4-dicarboxylphenyl)-hexafluoropropane dianhydride, cyclobutanetetracarboxylic dianhydride, methylcyclobutanetetracarboxylic dianhydride, and 1,2,3,4-tetracarboxybutane dianhydride.  
     
     
         9 . The method of  claim 2 , wherein the tetracarboxylic dianhydride is reacted at 0.02 to 90% by weight.  
     
     
         10 . The method of  claim 2 , wherein the tetracarboxylic dianhydride is dissolved in at least one solvent selected from the group consisting of acetone, methyl ethyl ketone, dimethyl formamide, and N-methyl pyrrolidone.  
     
     
         11 . The method of  claim 2 , wherein the hydrolysis of the anhydride is performed in an aqueous solution at 20 to 100° C.  
     
     
         12 . The method of  claim 2 , wherein the carbodiimide is 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC) or N,N′-dicyclohexyl carbodiimide (DCC).  
     
     
         13 . The method of  claim 2 , wherein the succinimide is N-hydroxysuccinimide or N-hydroxysulfosuccinimide.  
     
     
         14 . The method of  claim 2 , wherein the carbodiimide or the succinimide is reacted at 20 to 200 mM.  
     
     
         15 . The method of  claim 2 , wherein the biomolecule has an amino group.  
     
     
         16 . The method of  claim 15 , wherein the biomolecule is DNA, RNA, PNA, or a protein.  
     
     
         17 . The method of  claim 2 , wherein the solid substrate is selected from the group consisting of glass, silicon wafer, polyethylene, polypropylene, polycarbonate, polyester, polyacrylate, and polyurethane.  
     
     
         18 . A biomolecule microarray produced using the method of  claim 2.