Selectively Functionalized Arrays

Abstract

Methods, substrates, and devices related to arrays of optical confinements having surfaces with high levels of bias. Substrates having transparent or silica based portions and opaque or reflective portions are treated with 1) a selective passivating agent, that selectively coats the opaque or reflective regions, 2) a functionalizing agent such as a coupling agent, and 3) a selective removal agent, which selectively removes functionalizing agent from the passivated opaque or reflective surfaces.

What is claimed is: 
     
         1 . An apparatus for obtaining sequence information from a template nucleic acid comprising:
 a) a substrate comprising an array of zero-mode waveguides wherein the zero-mode waveguides comprise apertures through an upper metal or metal oxide layer disposed on top of a lower silica-based layer, wherein each zero-mode waveguide is capable of holding a single molecule of a polymerase enzyme, and wherein a surface of the substrate is selectively functionalized by:
 i) exposing the surface of the substrate to an agent that preferentially binds to the metal or metal oxide portions of the surface to produce passivated metal or metal oxide portions of the surface; 
 ii) exposing the surface of the substrate to a silica functionalizing agent that binds to both the silica-based portions and the passivated metal or metal oxide portions of the surface, then rinsing the surface of the substrate; and 
 iii) after step ii), exposing the silica-based portions and the passivated metal or metal oxide portions of the surface of the substrate to a selective removal compound that preferentially removes the silica functionalizing agent from the passivated metal or metal oxide portions of the surface; 
   b) a receptacle for keeping sequencing reagents in contact with the substrate;   c) an optical system optically coupled to the substrate for delivering light to the zero-mode waveguides and for measuring light from the zero-mode waveguides over a period of time, wherein the measured light from the zero-mode waveguides over a period of time can be used to obtain sequence information about the template DNA; and   d) a computational system connected to the optical system for determining sequence information using the measured light over a period of time.   
     
     
         2 . The apparatus of  claim 1  wherein the agent that preferentially binds to the metal or metal oxide portions comprises phosphate or phosphonate functionality. 
     
     
         3 . The apparatus of  claim 1  wherein the silica functionalizing agent comprises a silane coupling agent. 
     
     
         4 . The apparatus of  claim 3  wherein a single molecule of a polymerase enzyme is attached to the silane coupling agent in each zero-mode waveguide. 
     
     
         5 . The apparatus of  claim 1  wherein the selective removal compound comprises an acidic compound. 
     
     
         6 . The apparatus of  claim 1  wherein the selective removal compound comprises a compound having a pK a  of less than 6. 
     
     
         7 . The apparatus of  claim 1  wherein the selective removal compound comprises a polymer. 
     
     
         8 . The apparatus of  claim 7  wherein the polymer comprises carboxylate, sulfonate, sulfate, phosphonate or phosphate functionality. 
     
     
         9 . The apparatus of  claim 7  wherein the polymer comprises a homopolymer or copolymer of one or more of the monomers vinyl(acrylic acid), vinyl(sulfonic acid), vinyl(phosphonic acid), vinyl(styrenesulfonic acid), maleic acid, or salts thereof. 
     
     
         10 . The apparatus of  claim 7  wherein the polymer comprises poly(vinylsulfonic acid) or 
       
         
           
           
               
               
           
         
         where m is about 24 and n is about 16. 
       
     
     
         11 . A substrate comprising an array of optical confinements, wherein the substrate has both silica-based portions and metal or metal oxide portions, and wherein the substrate is selectively functionalized by:
 i) exposing a surface of the substrate to an agent that preferentially binds to the metal or metal oxide portions to produce passivated metal or metal oxide portions of the surface;   ii) exposing the surface of the substrate to a silica functionalizing agent that binds to both the silica-based portions and the passivated metal or metal oxide portions of the surface, then rinsing the surface of the substrate; and   iii) after step ii), exposing the silica-based portions and the passivated metal or metal oxide portions of the surface of the substrate to a selective removal compound that preferentially removes the silica functionalizing agent from the passivated metal or metal oxide portions of the surface.   
     
     
         12 . The substrate of  claim 11  wherein the array of optical confinements comprises an array of zero-mode waveguides, and wherein the silica-based portions of the substrate comprise bases of apertures though a metal or metal oxide layer on a transparent silica-based substrate. 
     
     
         13 . The substrate of  claim 11  wherein the agent that preferentially binds to the metal or metal oxide portions comprises phosphate or phosphonate functionality. 
     
     
         14 . The substrate of  claim 11  wherein the silica functionalizing agent comprises a silane coupling agent. 
     
     
         15 . The substrate of  claim 11  wherein the selective removal compound comprises an acidic compound. 
     
     
         16 . The substrate of  claim 11  wherein the selective removal compound comprises a compound having a pK a  of less than 6. 
     
     
         17 . The substrate of  claim 11  wherein the selective removal compound comprises a polymer. 
     
     
         18 . The substrate of  claim 17  wherein the polymer comprises carboxylate, sulfonate, sulfate, phosphonate or phosphate functionality. 
     
     
         19 . The substrate of  claim 17  wherein the polymer comprises a homopolymer or copolymer of one or more of the monomers vinyl(acrylic acid), vinyl(sulfonic acid), vinyl(phosphonic acid), vinyl(styrenesulfonic acid), maleic acid, or salts thereof. 
     
     
         20 . The substrate of  claim 17  wherein the polymer comprises poly(vinylsulfonic acid) or 
       
         
           
           
               
               
           
         
         where m is about 24 and n is about 16. 
       
     
     
         21 . An apparatus for obtaining sequence information from a template nucleic acid comprising:
 a) a substrate comprising an array of zero-mode waveguides wherein the zero-mode waveguides comprise apertures through an upper metal or metal oxide layer disposed on top of a lower silica-based layer, wherein each zero-mode waveguide is capable of holding a single molecule of a polymerase enzyme, and wherein a surface of the substrate is selectively functionalized by:
 i) treating the surface of the substrate with a compound comprising phosphate or phosphonate groups to produce passivated metal or metal oxide portions of the surface; 
 ii) treating the surface of the substrate with a silica functionalizing agent that binds to both the silica-based portions and the passivated metal or metal oxide portions of the surface, wherein the silica functionalizing agent comprises a silane coupling agent; and 
 iii) treating the surface of the substrate with an acidic compound having a pK a  of less than 6 to remove silica functionalizing agent from the passivated metal or metal oxide portions of the surface; 
   b) a receptacle for keeping sequencing reagents in contact with the substrate;   c) an optical system optically coupled to the substrate for delivering light to the zero-mode waveguides and for measuring light from the zero-mode waveguides over a period of time, wherein the measured light from the zero-mode waveguides over a period of time can be used to obtain sequence information about the template DNA; and   d) a computational system connected to the optical system for determining sequence information using the measured light over a period of time.   
     
     
         22 . A substrate comprising an array of optical confinements, wherein the substrate has both silica-based portions and metal or metal oxide portions, and wherein the substrate is selectively functionalized by:
 i) treating the surface of the substrate with a compound comprising phosphate or phosphonate groups to produce passivated metal or metal oxide portions of the surface;   ii) treating the surface of the substrate with a silica functionalizing agent that binds to both the silica-based portions and the passivated metal or metal oxide portions of the surface, wherein the silica functionalizing agent comprises a silane coupling agent; and   iii) treating the surface of the substrate with an acidic compound having a pK a  of less than 6 to remove silica functionalizing agent from the passivated metal or metal oxide portions of the surface.