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.

Description

[0001]This application claims priority to and benefit of U.S. Provisional Application Ser. No. 60 / 899,173, filed on Feb. 2, 2007, the entire content of which is incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]In certain applications, such as single molecule DNA sequencing or the evaluation of polymerases, it is necessary to wash labeled biomolecules across a surface. This process inevitably results in the nonspecific binding of labeled molecules to the surface and a concominant increased background fluorescence and false-positive features. Many surface attachment chemistries have intrinsic properties designed to enhance specific molecule binding but do little to directly inhibit or suppress the effects of the nonspecific binding of fluorescently labeled molecules. The corresponding increase in background fluorescence as labeled molecules are washed across the surface, combined with the limited fluorescent intensity and lifetime of any single fluorophore, imposes re...

AI Technical Summary

Benefits of technology

[0007]The precise spacing of binding elements depends upon the label used. Diffraction limits of various optically-detectable labels are well known and can be selected at the convenience of the user. In one embodiment, a low-autofluorescence glass (e.g., a coverslip) is coated with a thin metal film and a specific surface coupling chemistry is applied for the attachment of labeled molecules. The metal film may be any appropriate metal (examples are provided below) at the convenience of the user and is applied such that total internal reflection illumination can be conducted on the surface. An evanescent field is generated by total internal reflection and is enhanced by the production of surface plasmons from the thin metal film, which increases the intensity of fluorescently labeled molecules within approximately 150 mn of the surface. This is explained by the fact that surface plasmons tend to stay longer along the surface than the evanescent field, and the electromagnetic field produced by the surface plasmons is intensified near the metal surface. The presence of the thin metal film quenches excited fluorophores near the surface (within tens of mn) by a mechanism of fluorescent energy transfer into the surface plasmon modes of the metal.

[0008]The invention also provides surface deposition methods that are useful independent of the surface chemistry being used. These deposition strategies allow for the fabrication of fully-resolved single molecule arrays at a feature density that surpasses that of any previously-described methods. Methods and devices of the invention improve the total resolvable molecule limit by selectively spacing deposited molecules at least a diffraction limit apart from every other molecule.

Problems solved by technology

This process inevitably results in the nonspecific binding of labeled molecules to the surface and a concominant increased background fluorescence and false-positive features.

The corresponding increase in background fluorescence as labeled molecules are washed across the surface, combined with the limited fluorescent intensity and lifetime of any single fluorophore, imposes restrictions on the overall imaging capabilities of any single molecule surface chemistry.

As a result of these optical limitations, current methods for the surface deposition and visualization of fluorescently-labeled single molecules suffer from a number of fundamental limitations.

Given the sensitivity and capture rate limitations of current CCD technology, it would likely not be practical to use these methods to completely resolve a highly dense surface array of single molecules.

Many of these surface attachment chemistries have intrinsic properties designed to enhance specific molecule binding but some of them do little to inhibit the effects of nonspecific binding.

However, the corresponding increase in background fluorescence as successive fluorescently labeled molecules are washed across the surface limits the overall imaging capabilities of any surface chemistry.

Images