SURFACE CHEMISTRY AND DEPOSITION TECHNIQUES
First Claim
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 Assignments
0 Petitions
Accused Products
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.
16 Citations
46 Claims
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1. A method for preparing a surface for chemical analysis, the method comprising the steps of:
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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. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12)
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2. A surface for single molecule analysis, the surface comprising:
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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. - View Dependent Claims (13, 14, 20, 21)
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3. A surface for visualizing labeled single molecules, the surface comprising:
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a glass surface coated with a metal film; and a binding ligand for attachment of single molecules. - View Dependent Claims (15, 19)
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4. A method for single molecule visualization, the method comprising the steps of:
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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.
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16. A method for sequencing a nucleic acid, the method comprising the steps of:
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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. - View Dependent Claims (17)
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18. The method of claim 18, wherein said detectable labels are fluorescent labels.
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22. A method for preparing a surface, the method comprising the steps of:
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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. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)
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46. A method for sequencing a nucleic acid, the method comprising the steps of:
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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.
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Specification