Enzyme immobilization for electroosmotic flow

Abstract

Disclosed herein is a method and apparatus of immobilizing a biocatalyst on a microfluidic biochip for conducting reactions in the presence of electroosmotic flow. The biochip includes a polymer on its microfluidic flow surfaces, wherein the polymer includes a first substituent selected from ionic groups of the same polarity or precursors thereof, a second substituent that is a hydrophobic group, and a third substituent comprising an immobilized biocatalyst-or precursor thereof. The biochip can be used to conduct multiple sequential biocatalyzed reactions in the presence of electroosmotic flow.

1 . A microfluidics biochip, comprising: 
 a) a microfluidic component comprising a flow surface;    b) a plurality of electrodes, whereby an electroosmotic flow can be generated at the flow surface;    c) an immobilizing polymer coated on the flow surface, wherein the polymer is substantially adhered to the surface, wherein the polymer comprises 
 a first substituent selected from ionic groups of the same polarity and covalent precursors of the ionic groups;  
 a second substituent that is a hydrophobic group; and  
 an immobilized biomolecule.  
   
   
   
       2 . The biochip of  claim 1 , further comprising a microfluidic channel, wherein a cross-sectional area of the channel is less than about 0.1 mm 2 .  
   
   
       3 . The biochip of  claim 2 , wherein the hydrophobic group is an optionally substituted C8-C30 alkyl, polycyclic alkyl, polycyclic aryl, or polycyclic heteroaryl, or a C8-C30 alkyl optionally interrupted by a cyclic or polycyclic alkyl, aryl, or heteroaryl.  
   
   
       4 . The biochip of  claim 3 , wherein the polymer is substantially adhered to the flow surface by hydrophobic interactions, electrostatic interactions, or by a covalent bond.  
   
   
       5 . The biochip of  claim 4 , wherein the polymer is substantially adhered to the flow surface by covalent attachment.  
   
   
       6 . The biochip of  claim 5 , wherein the biomolecule is a biocatalyst comprising 
 a catalytically functional portion of an antibody, an enzyme, a peptide, or an oligonucleotide; and    optionally, a cofactor.    
   
   
       7 . The biochip of  claim 6 , wherein the biocatalyst comprises 
 a catalytically functional portion of an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, or a ligase; and    optionally, a cofactor.    
   
   
       8 . The biochip of  claim 7 , wherein the biocatalyst is immobilized to the polymer through an amide bond.  
   
   
       9 . The biochip of  claim 8 , wherein the flow surface comprises surface —Si—O— groups.  
   
   
       10 . The biochip of  claim 9 , wherein the covalent attachment of the polymer to the flow surface is represented by structural formula B:  
     
       
         
         
             
             
         
       
     
     wherein 
 R s  represents one of the surface —Si—O— groups;  
 R a  is a C1-C8 alkylene chain;  
 each R b  is independently a C1-C4 alkyl group; and  
 D represents an amide bond to the polymer.  
 
   
   
       11 . The biochip of  claim 10 , further comprising at least two catalytically distinct immobilized biocatalysts.  
   
   
       12 . The biochip of  claim 11 , further comprising 
 at least two spatially distinct flow surfaces, wherein 
 the flow surfaces are in microfluidic communication;  
 each flow surface is coated with the polymer;  
 at least two said catalytically distinct biocatalysts are respectively immobilized to the polymer at the spatially distinct flow surfaces; and  
   whereby the biocatalysts are catalytically and spatially distinct.    
   
   
       13 . The biochip of  claim 12  wherein the polymer is represented by a structure derived from a copolymer of ethylenically unsaturated monomers, wherein the monomers are substituted with groups selected from the ionic groups or precursors thereof, the hydrophobic groups, the biomolecules, and the linker.  
   
   
       14 . The biochip of  claim 13 , wherein 
 the ionic groups are selected from carboxylate, carbamate, sulfate, thiosulfate, sulfonate, phosphate, phosphonate, and hydroxyl;    the precursors are selected from optionally substituted alkyl esters, alkyl anhydrides, cyclic anhydrides, alkyl ethers, aryl esters, aryl anhydrides, and aryl ethers of the ionic groups.    
   
   
       15 . The biochip of  claim 13 , wherein 
 the ionic groups are selected from ammonium, phosphonium, and sulfonium groups substituted with one or more groups selected from C1-C8 alkyl and aryl.    
   
   
       16 . The biochip of  claim 14 , wherein the polymer is derived from a maleic anhydride-α-olefin polymer, wherein 
 the ionic groups consist of anhydride or carboxyl derived from a maleic anydride monomer;    the hydrophobic substituent is a C12-C28 alkyl chain;    the biocatalysts are each immobilized through an amide bond to a carboxyl derived from a maleic anhydride monomer;    the amide bond represented by D comprises a carboxyl derived from a maleic anhydride monomer.