Surface chemistry and deposition techniques

Abstract

Surface chemistries for the visualization of labeled single molecules (analytes) with improved signal-to-noise properties are provided. To be observed, analyte molecules are bound to surface attachment features that are spaced apart on the surface such that when the analytes are labeled adjacent analytes are optically resolvable from each other. One way to express this concept is that binding elements should be spaced apart such that the Guassian point spread functions of adjacent labels do not overlap. Another way of expressing this concept is that the surface binding elements should be spaced apart by a distance equal to at least the diffraction limit for an optical label attached to the bound analytes.

1 . A method for preparing a surface for chemical analysis, the method comprising the steps of:
 providing a surface comprising a coating for binding an optically-labeled analyte; and   depositing said analyte on said surface such that individual analyte molecules are spaced apart by a distance equal to at least the diffraction limit for said optical label.   
     
     
         2 . A surface for single molecule analysis, the surface comprising:
 a coating;   a plurality of surface attachment features, each capable of binding to a detectably-labeled analyte; wherein said surface attachment features are spaced on said surface such that Guassian Gaussian point spread functions of said surface attachment features do not overlap when analyte is bound.   
     
     
         3 . A surface for visualizing labeled single molecules, the surface comprising:
 a glass surface coated with a metal film; and   a binding ligand for attachment of single molecules.   
     
     
         4 . A method for single molecule visualization, the method comprising the steps of:
 providing a glass surface comprising a metal film;   binding a plurality of single molecules to a binding member on said metal film;   producing an evanescent electromagnetic field on said surface; and   visualizing said single molecules using an optical microscope.   
     
     
         5 . The method of  claim 1 , wherein said analyte is a nucleic acid. 
     
     
         6 . The method of  claim 5 , wherein said optically-detectable label is a fluorescent label. 
     
     
         7 . The method of  claim 1 , wherein said surface is selected from a polyelectrolyte multilayer and an epoxide. 
     
     
         8 . The method of  claim 1 , wherein said nucleic acid is DNA. 
     
     
         9 . The method of  claim 5 , further comprising obtaining a sequence of said nucleic acid. 
     
     
         10 . The method of  claim 9 , wherein said obtaining step comprises conducting a template-dependent sequencing-by-synthesis reaction. 
     
     
         11 . The method of  claim 5 , further comprising the step of detecting said label by optical microscopy. 
     
     
         12 . The method of  claim 11 , wherein said optical microscopy comprises a total internal reflection objective. 
     
     
         13 . The surface of  claim 2 , wherein said attachment feature is selected from an antibody, a ligand, a gold particle, a bead, a well, a surface dimple, an amine, and an epoxide. 
     
     
         14 . The method of  claim 2 , wherein said detectably-labeled analyte is a fluorescently-labeled analyte. 
     
     
         15 . The surface of  claim 3 , wherein said binding ligand is selected from an antibody, a ligand, a gold particle, a bead, a well, a surface dimple, an amine, and an epoxide. 
     
     
         16 . A method for sequencing a nucleic acid, the method comprising the steps of:
 attaching a plurality of nucleic acid primers to a surface comprising binding elements spaced apart such that primers, when attached to said binding elements, are individually optically resolvable;   exposing said primers to one or more template nucleic acids that are capable of hybridizing thereto;   introducing a detectably-labeled nucleotide and a polymerase under conditions that permit template-dependent incorporation of said nucleotide into said primer;   rinsing said surface to remove unincorporated nucleotides;   detecting said incorporated nucleotides;   neutralizing detectable labels associated with said incorporated nucleotides; and   repeating said introducing, rinsing, detecting, and neutralizing steps at least once, thereby to determine a sequence of said template.   
     
     
         17 . The method of  claim 16 , wherein said binding elements are spaced apart such that when adjacent primers have incorporated a detectably-labeled nucleotide, the Gaussian point spread function of adjacent detectable labels do not substantially overlap. 
     
     
         18 . The method of  claim 18 , wherein said detectable labels are fluorescent labels. 
     
     
         19 . A method for sequencing a nucleic acid, the method comprising the step of conducting a template-dependent sequencing-by-synthesis reaction on a surface of  claim 3 . 
     
     
         20 . A surface according to  claim 2 , wherein said surface attachment features are bins spaced apart such that the Gaussian point spread function for excited fluorophores in adjacent bins do not overlap. 
     
     
         21 . The surface according to  claim 2 , wherein said surface attachment feature is a virus or a viral capsid. 
     
     
         22 . A method for preparing a surface, the method comprising the steps of:
 a) providing a surface having a plurality of binding elements thereon;   b) binding a plurality of spacer objects to the binding elements so that the spacer objects occupy a subset of the binding elements;   c) optionally, blocking or inactivating unoccupied binding elements;   d) removing the spacer objects, thereby exposing attachment sites, each site containing no more than a single attachment element; and   e) attaching one or more analytes directly or indirectly to the attachment elements,   wherein individual analyte molecules attached to adjacent attachment sites are optically resolvable.   
     
     
         23 . The method of  claim 22 , wherein step c) is required. 
     
     
         24 . The method of  claim 22 , wherein the analyte molecules are optically labeled. 
     
     
         25 . The method of  claim 24 , wherein the analyte molecules are fluorescently labeled. 
     
     
         26 . The method of  claim 22 , wherein the analyte molecules are spaced apart such that the Gaussian point spread functions for excited fluorophores at adjacent attachment sites do not substantially overlap. 
     
     
         27 . The method of  claim 22 , wherein the analyte molecules are labeled nucleic acids or labeled nucleotides. 
     
     
         28 . The method of  claim 22 , wherein the size of the spacer objects is equal to or greater than the optical diffraction limit for optical resolution of individual analytes. 
     
     
         29 . The method of  claim 22 , wherein the spacer objects are chosen from beads, gold particles and viral capsids. 
     
     
         30 . The method of  claim 22 , wherein the spacer objects are beads with a diameter from 50 nm to 1 micron. 
     
     
         31 . The method of  claim 22 , wherein the binding elements comprise epoxy and/or amine reactive groups. 
     
     
         32 . The method of  claim 22 , wherein the binding elements are one member of a binding pair selected from biotin/streptavidin, receptor/ligand, hapten/antibody, and a nucleic acid duplex. 
     
     
         33 . The method of  claim 22 , wherein the surface is coated with a polyelectrolyte multilayer. 
     
     
         34 . The method of  claim 22 , wherein the surface is coated with a metal film. 
     
     
         35 . The method of  claim 22 , wherein the spacer objects are bound to the surface via a latent attachment element which is exposed following the removal of the spacer objects. 
     
     
         36 . The method of  claim 22 , wherein the spacer objects are removed using enzymatic cleavage, light absorption, acid or alkaline hydrolysis, or reducing conditions. 
     
     
         37 . The method of  claim 22 , wherein the spacer objects are bound to the binding elements via a nucleic acid. 
     
     
         38 . The method of  claim 37 , wherein the nucleic acid contains a restriction site. 
     
     
         39 . A surface prepared using the method of  claim 22 . 
     
     
         40 . The surface of  claim 34 , wherein the surface is a glass surface coated with a metal film and at least one member of a binding pair for attachment of analytes. 
     
     
         41 . A method for single-molecule analysis, the method comprising:
 a) preparing a surface using the method of  claim 22 ;   b) attaching a nucleic acid template to attachment elements;   c) conducting a chemical reaction thereby incorporating optically labeled nucleotides into individual analyte molecules; and   d) detecting the optically labeled analyte molecules by optical microscopy.   
     
     
         42 . A method of  claim 41 , wherein the method further comprises determining a sequence of the nucleic acid template based on the detection of incorporated nucleotides. 
     
     
         43 . The method of  claim 41 , wherein the chemical reaction is chosen from sequencing by synthesis and sequencing by ligation. 
     
     
         44 . The method of  claim 41 , wherein the detecting step is performed by optical microscopy. 
     
     
         45 . The method of  claim 44 , wherein the detection is performed using a Total Internal Reflection Fluorescence (TIRF) microscope. 
     
     
         46 . A method for sequencing a nucleic acid, the method comprising the steps of:
 a) providing a surface having a plurality of binding elements thereon;   b) binding a plurality of spacer objects to the binding element so that the spacer objects occupy a subset of the binding elements;   c) blocking or inactivating unoccupied binding elements;   d) removing the spacer objects, thereby exposing attachment sites, each site containing no more than a single attachment element;   e) directly or indirectly attaching a nucleic acid template to attachment elements;   f) introducing one or more optically labeled nucleotides and a polymerase under conditions that permit template-dependent incorporation of said nucleotide into said primer;   g) rinsing the surface to remove unincorporated nucleotides;   h) optically detecting nucleotides incorporated into the individual nucleic acid molecules associated with respective attachment sites;   i) removing or neutralizing labels associated with said incorporated nucleotides; and   j) repeating said introducing, rinsing, detecting, and neutralizing steps, thereby to determine a sequence of said template.