Microfabrication methods for the optimal patterning of substrates

Abstract

The invention is directed to a method of fabricating a microarray. The method includes: (a) providing a substrate having at least two layers of different chemical reactivity, wherein a well in an outer layer exposes an inner layer; (b) contacting the substrate with a first reagent specifically reactive with the outer layer to produce a first modified layer; (c) contacting the substrate with a second reagent specifically reactive with the inner layer of the substrate to produce a modified inner layer, wherein the modified inner layer has a higher affinity for a biopolymer than the modified outer layer, and (d) depositing the biopolymer onto the modified inner layer within the well, wherein the higher affinity of the modified inner layer facilitates localization of the biopolymer onto the well. Methods of fabricating a microarray which include polishing a substrate or functionalizing a plurality of features with a reactive reagent also are provided. A method of fabricating a microarray which includes loading a plurality of discrete nanochannels is additionally provided.

1 . A method of fabricating a microarray, comprising:
 (a) providing a substrate having at least two layers of different chemical reactivity, wherein a well in an outer layer exposes an inner layer;   (b) contacting said substrate with a first reagent specifically reactive with said outer layer to produce a first modified layer;   (c) contacting said substrate with a second reagent specifically reactive with said inner layer of said substrate to produce a modified inner layer, wherein said modified inner layer has a higher affinity for a biopolymer than said modified outer layer, and   (d) depositing said biopolymer onto said modified inner layer within said well, wherein said higher affinity of said modified inner layer facilitates localization of said biopolymer onto said well.   
     
     
         2 . The method of  claim 1 , wherein said inner layer comprises silicon and said outer layer comprises silicon oxide. 
     
     
         3 . The method of  claim 2 , wherein said first reagent comprises a chlorosilane having a moiety that repels or is inert to said biopolymer. 
     
     
         4 . The method of  claim 3 , wherein said silicon is converted to silicon oxide after step (b) and prior to step (c). 
     
     
         5 . The method of  claim 4 , wherein said second reagent comprises a chlorosilane having a moiety with affinity for said biopolymer. 
     
     
         6 . The method of  claim 1 , wherein said deposited biopolymer is covalently attached to said modified inner layer. 
     
     
         7 . The method of  claim 1 , wherein said biopolymer comprises a nucleic acid clonal ball or a nucleic acid attached to a microsphere. 
     
     
         8 . A method of fabricating a microarray, comprising:
 (a) contacting a substrate having wells with a reagent reactive with said substrate to produce a surface modification within said wells and a surface modification surrounding said wells;   (b) polishing said substrate to produce a polished surface surrounding said wells , whereby said surface modification surrounding said wells is removed and said surface modification within said wells is retained, and   (c) depositing a biopolymer onto said substrate, wherein higher affinity of said surface modification within said wells compared to said polished surface facilitates localization of said biopolymer within said wells.   
     
     
         9 . The method of  claim 8 , wherein said polishing comprises removing a first portion of a layer to expose a second portion of the layer. 
     
     
         10 . The method of  claim 8 , wherein said substrate comprises two or more layers. 
     
     
         11 . The method of  claim 10 , wherein a first layer comprises silicon. 
     
     
         12 . The method of  claim 10 , wherein a second layer comprises silicon oxide. 
     
     
         13 . The method of  claim 11 , wherein said first layer comprises an inner layer of said substrate corresponding to a bottom of a well. 
     
     
         14 . The method of  claim 12 , wherein said second layer comprises an outer layer of said substrate corresponding to at least a portion of the sides of a well. 
     
     
         15 . The method of  claim 8 , wherein said deposited biopolymer is covalently attached to said modified substrate within said wells. 
     
     
         16 . A method of fabricating a microarray, comprising:
 (a) functionalizing a plurality of features on a substrate to create discrete single biopolymer anchor sites, said functionalization comprising:
 (1) contacting said substrate with a reagent reactive with said substrate to produce a modified substrate; 
 (2) applying a protecting reagent to discrete sites on said modified substrate, said discrete sites having an area of between about 5-40 nm 2 ; 
 (3) modifying said reagent located in unprotected regions of said modified substrate surrounding said discrete sites, thereby rendering said regions unreactive to a target biopolymer, and 
 (4) removing said protecting reagent to produce a substrate having a plurality of discrete functionalized features, and 
   (b) attaching a single target biopolymer to one or more of said discrete functionalized features.   
     
     
         17 . The method of  claim 16 , wherein a target biopolymer having a different target nucleotide sequence is attached at each of said discrete functionalized features. 
     
     
         18 . The method of  claim 17 , wherein each different single target biopolymer further comprises a common nucleotide priming site. 
     
     
         19 . The method of  claims 17 , further comprising, (c) attaching a plurality of secondary biopolymers to said regions surrounding said discrete sites, wherein said secondary biopolymers comprise a common nucleotide primer sequence complementary to said common nucleotide priming sequence. 
     
     
         20 . The method of  claim 19 , further comprising amplifying each of said target biopolymers, thereby forming a plurality of biopolymers comprising said target nucleotide sequence at said region surrounding each of said discrete functionalized features. 
     
     
         21 . A method of fabricating a microarray, comprising:
 (a) contacting a substrate having a plurality of discrete nanochannels with a plurality of biopolymers, said nanochannels having a length and diameter sufficient for entry of only a single biopolymer molecule;   (b) applying an electric potential to said substrate sufficient to translocate said single biopolymer molecules into said nanochannels to produce a substrate containing a plurality of single biopolymer molecules each in said plurality of discrete nanochannels, and   (c) transferring said plurality of single biopolymer molecules contained in said plurality of discrete nanochannels to a solid support.   
     
     
         22 . The method of  claim 21 , wherein said transferring step comprises reversing the polarity of said electric potential. 
     
     
         23 . The method of  claim 21 , wherein said transferring step comprises subjecting said substrate containing a plurality of single biopolymers each in said plurality of discrete nanochannels to centrifugal force.