U.S. patent application number 17/504115 was filed with the patent office on 2022-03-31 for articles having localized molecules disposed thereon and methods of producing same.
The applicant listed for this patent is Pacific Biosciences of California, Inc.. Invention is credited to Ron L. Cicero, Jonas Korlach, John Lyle, Geoff Otto, Paul Peluso, David R. Rank, Daniel Roitman, Stephen Turner, Jeffery Wegener, Yue Xu.
Application Number | 20220098660 17/504115 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-31 |
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United States Patent
Application |
20220098660 |
Kind Code |
A1 |
Rank; David R. ; et
al. |
March 31, 2022 |
ARTICLES HAVING LOCALIZED MOLECULES DISPOSED THEREON AND METHODS OF
PRODUCING SAME
Abstract
Methods of producing substrates having selected active chemical
regions by employing elements of the substrates in assisting the
localization of active chemical groups in desired regions of the
substrate. The methods may include optical, chemical and/or
mechanical processes for the deposition, removal, activation and/or
deactivation of chemical groups in selected regions of the
substrate to provide selective active regions of the substrate.
Inventors: |
Rank; David R.; (Pacific
Grove, CA) ; Wegener; Jeffery; (Cupertino, CA)
; Korlach; Jonas; (Camas, WA) ; Roitman;
Daniel; (Menlo Park, CA) ; Xu; Yue; (Fremont,
CA) ; Lyle; John; (Fremont, CA) ; Turner;
Stephen; (Eugene, OR) ; Peluso; Paul; (Newark,
CA) ; Otto; Geoff; (Brookline, MA) ; Cicero;
Ron L.; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pacific Biosciences of California, Inc. |
Menlo Park |
CA |
US |
|
|
Appl. No.: |
17/504115 |
Filed: |
October 18, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16848620 |
Apr 14, 2020 |
11186871 |
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17504115 |
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14318324 |
Jun 27, 2014 |
10655172 |
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16848620 |
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11731748 |
Mar 29, 2007 |
8802600 |
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14318324 |
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11394352 |
Mar 30, 2006 |
8975216 |
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11731748 |
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International
Class: |
C12Q 1/6874 20060101
C12Q001/6874; B01J 19/00 20060101 B01J019/00; B82Y 20/00 20060101
B82Y020/00; B82Y 30/00 20060101 B82Y030/00; C12Q 1/6837 20060101
C12Q001/6837 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under NHGRI
Grant No. R01-HG003710-01 awarded by the National Human Genome
Research Institute (NHGRI) of the National Institutes of Health
(NIH). The government has certain rights in the invention. The
preceding statement is included in accordance with 37 C.F.R.
401.14(0(4) because one or more inventions described herein were
made or developed with government grant support. This statement
should not be construed as necessarily covering all inventions
described herein.
Claims
1-13. (canceled)
14. A method of preferentially localizing desired molecules within
optical confinements disposed upon a substrate, comprising:
depositing the desired molecules over the surface of the substrate;
and selectively removing the desired molecules from the surface of
the substrate that is not within the optical confinements.
15. The method of claim 14, wherein the substrate comprises an
opaque layer on a transparent layer, and wherein the optical
confinements comprise nanoscale wells disposed through the opaque
layer and exposing part of the transparent layer.
16. The method of claim 15, wherein the transparent layer comprises
SiO.sub.2.
17. The method of claim 15, wherein the opaque layer comprises a
metal or metal oxide.
18. The method of claim 15, wherein the opaque layer comprises
aluminum and the transparent layer comprises SiO.sub.2.
19. The method of claim 14, wherein the optical confinements
comprise zero mode waveguides.
20. The method of claim 14, wherein selectively removing the
desired molecules from the surface of the substrate that is not
within the optical confinements comprises contacting the substrate
with a deactivation component coupled to an exclusionary component,
which exclusionary component is at least partially excluded from
entering into the optical confinements.
21. The method of claim 20, wherein the deactivation component
comprises an enzyme.
22. The method of claim 21, wherein the enzyme is selected from the
group consisting of a protease, a nuclease, and a carbohydrase.
23. The method of claim 21, wherein the enzyme is a site-specific
protease.
24. The method of claim 20, wherein the exclusionary component is a
large particle or a macromolecule.
25. The method of claim 24, wherein the exclusionary component
comprises a bead.
26. The method of claim 24, wherein the exclusionary component
comprises a rigid or semi-rigid elongated polymer.
27. The method of claim 24, wherein the exclusionary component
comprises a double-stranded nucleic acid molecule.
28. The method of claim 20, wherein the deactivation component is a
protease and the exclusionary component is a double-stranded DNA
molecule.
29. The method of claim 20, wherein the deactivation component is a
protease and the exclusionary component is a bead.
30. The method of claim 14, wherein the desired molecules are
enzymes.
31. The method of claim 14, wherein the desired molecules are
polymerases.
32. The method of claim 14, wherein the desired molecules comprise
binding moieties.
33. The method of claim 32, wherein the binding moieties are
biotins.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application U.S. Ser.
No. 16/848,620, filed Apr. 14, 2020, which is a continuation of
application U.S. Ser. No. 14/318,324, filed Jun. 27, 2014, now U.S.
Pat. No. 10,655,172, which is a continuation of application U.S.
Ser. No. 11/731,748, filed Mar. 29, 2007, now U.S. Pat. No.
8,802,600, which is a continuation-in-part of application U.S. Ser.
No. 11/394,352, filed Mar. 30, 2006, now U.S. Pat. No. 8,975,216.
Each of these applications is incorporated herein by reference in
its entirety for all purposes.
FIELD OF THE INVENTION
[0003] The present invention relates to methods of producing
substrates having selected active chemical regions by employing
elements of the substrates in assisting the localization of active
chemical groups in desired regions of the substrate. Methods that
include optical, chemical and/or mechanical processes for the
deposition, removal, activation and/or deactivation of chemical
groups in selected regions of the substrate to provide selective
active regions of the substrate are described.
BACKGROUND OF THE INVENTION
[0004] There are a wide range of analytical operations that may
benefit from the ability to analyze the reaction of individual
molecules, relatively small numbers of molecules, or molecules at
relatively low concentrations. A number of approaches have been
described for providing these sparsely populated reaction mixtures.
For example, in the field of nucleic acid sequence determination, a
number of researchers have proposed single molecule or low
concentration approaches to obtaining sequence information in
conjunction with the template dependent synthesis of nucleic acids
by the action of polymerase enzymes.
[0005] The various different approaches to these sequencing
technologies offer different methods of monitoring only one or a
few synthesis reactions at a time. For example, in some cases, the
reaction mixture is apportioned into droplets that include low
concentrations of reactants. In other applications, certain
reagents are immobilized onto surfaces such that they may be
monitored without interference from other reaction components in
solution. In still another approach, optical confinement techniques
are used to ascertain signal information only from a relatively
small number of reactions, e.g., a single molecule, within an
optically confined area. Notwithstanding the availability of the
above-described techniques, there are instances where further
selectivity of reaction components for analysis would be desirable.
The present invention meets these and a variety of needs.
BRIEF SUMMARY OF THE INVENTION
[0006] The present invention generally provides methods of
providing functional surface modifications, e.g., active chemical
groups, in selected regions of substrates, as well as the resultant
substrates produced thereby, and the use of such substrates.
[0007] In a first aspect, the present invention provides methods of
producing a substrate having selectively active regions thereon.
The methods comprise providing a substrate having a plurality of
nanostructures defined thereon, wherein each of the nanostructures
limits an ability of a first activation controlling agent to
provide active chemical groups on selected portions of a surface of
the substrate. The substrate is then exposed to at least the first
activation controlling agent to selectively provide active chemical
groups on the selected portions of a surface of the substrate.
Active chemical groups include, for example, chemical functional
groups, active molecules (e.g., enzymes), and coupling or binding
groups.
[0008] In another, related aspect, the invention provides methods
of preparing an analytical substrate having selected active regions
thereon. The method provides a substrate having an optical analysis
structure disposed thereon, the optical analysis structure
providing enhanced optical access to selected regions of the
substrate. Surface functional groups are then provided that are
capable of being activated or deactivated by a first
electromagnetic radiation, which is directed at the substrate
whereby the optical analysis structure directs the electromagnetic
radiation to selectively activate or deactivate the surface
functional groups in the selected regions of the substrate to
provide selectively active regions of the substrate.
[0009] Another aspect of the invention features a method of
providing a zero mode waveguides having an active chemical surface
substantially at a bottom portion of the waveguide. The method
comprises providing a zero mode waveguide disposed in a substrate,
providing functional chemical groups on surfaces of the zero mode
waveguide, and exposing first portions, but not second portions, of
the surfaces of the zero mode waveguide to an activation
controlling agent that selectively activates or deactivates the
functional chemical groups on the first portions to provide an
active chemical surface substantially at a bottom portion of the
zero mode waveguide.
[0010] The invention also provides substrates and devices made
using such methods, including zero mode waveguide arrays that
comprise a plurality of zero mode waveguide cores disposed in a
cladding layer, each core having a bottom surface, and a chemically
active surface substantially only within the cores.
[0011] The invention further provides methods of providing a
substrate having selected active regions disposed thereon that
comprise providing a substrate having an optical enhancement
structure defined thereon, the optical enhancement structure being
capable of directing electromagnetic radiation to provide an
enhanced electromagnetic field proximal to a selected region of the
substrate surface sufficient to create a trapping force proximal to
the selected region. Electromagnetic radiation is directed at the
substrate to provide the enhanced electromagnetic field at the
selected region sufficient to create a trapping force upon an
active molecule proximal to the selected region. The active
molecule is then coupled to the selected region.
[0012] The invention additionally provides methods of producing a
substrate having selected active regions disposed thereon that
comprise providing a substrate having a surface comprised of
chemical functional groups, and a plurality of discrete nanoscale
reaction regions defined thereon, and patterning one or more of a
chemical functional group or an activation controlling agent onto
the surface in selected regions to provide active chemical
functional regions substantially only in the discrete nanoscale
reaction regions.
[0013] Another aspect of the invention provides methods of
identifying a sequence of a nucleic acid molecules. The method
comprises providing a plurality of nucleic acid
polymerase/template/primer complexes within discrete observation
regions on a substrate, and detecting sequential addition of
nucleotides or nucleotide analogs in a template dependent manner to
identify a sequence of incorporation of the nucleotides or
nucleotide analogs in the plurality of observation regions. In such
methods, the substrate has been prepared to substantially reduce
one or more of polymerase activity, polymerase presence, template
presence, and primer presence in regions outside of the observation
regions.
[0014] Relatedly, the invention provides a method of identifying a
sequence of a nucleic acid molecule, comprising providing a
plurality of nucleic acid polymerase/template/primer complexes
within discrete observation regions on a substrate surface, and
detecting sequential addition of nucleotides or nucleotide analogs
in a template dependent manner to identify a sequence of
incorporation of the nucleotides or nucleotide analogs in the
plurality of observation regions. In such methods, an
intra-observation region barrier is provided upon the substrate
surface between at least first and second discrete observation
regions to substantially prevent intra-observation region diffusion
of one or more reactants or products.
[0015] The invention also provides methods of preferentially
localizing desired molecules within an optical confinement disposed
upon a substrate. The methods comprise depositing the desired
molecules over the surface of the substrate, and selectively
removing the desired molecules from the surface of the substrate
that is not within the optical confinement. In one aspect, the
substrate comprises an opaque (non-transparent) layer and a
transparent layer, and the optical confinement comprises a zero
mode waveguide disposed through the opaque layer to the transparent
layer.
[0016] Selectively removing the desired molecules from the surface
of the substrate that is not within the optical confinement
optionally involves contacting the substrate with a deactivation
component coupled to an exclusionary component that is at least
partially excluded from entering into the optical confinement. The
deactivation component removes the molecules it can access from the
surface (e.g., from the upper surface and upper walls of the core
of a zero mode waveguide); it is prevented from accessing the
optical confinement, or the entirety of the optical confinement, by
the exclusionary component.
[0017] In one class of embodiments, the deactivation component
comprises an enzyme, for example, a protease (e.g., a nonspecific
or a site-specific protease), a nuclease, or a carbohydrase. The
exclusionary component is optionally a large particle such as a
bead, a macromolecule, or a rigid or semi-rigid elongated polymer.
In one class of embodiments, the exclusionary component comprises a
double-stranded nucleic acid molecule, e.g., a double-stranded DNA
molecule coupled to a protease deactivation component.
[0018] The desired molecules that are selectively immobilized can
be essentially any molecules, for example, active molecules such as
enzymes (e.g., nucleic acid polymerases) or molecules comprising
binding or coupling moieties (e.g., biotin molecules) which can be
employed in turn to immobilize other molecules.
[0019] Further provided are methods of localizing a molecule within
an optical confinement disposed on a substrate that comprise
providing a light activated coupling group on the surface of the
substrate, including within the optical confinement. Activating
radiation is then directed at the substrate, wherein the optical
confinement permits activating radiation to be incident only within
the optical confinement. The molecule is then coupled to the light
activated coupling group.
[0020] Another aspect of the invention provides methods of
selectively immobilizing a molecule of interest on a substrate. The
methods comprise providing a substrate having a first surface
component and a second surface component where the first and second
surface component have different surface characteristics, and
selectively coupling the molecule of interest to the first surface
component, based upon a difference between the surface
characteristics of the first surface component and the surface
characteristics of the second surface component.
[0021] In one class of embodiments, the differing surface
characteristics comprise surface charge or electrostatic
interactions at the surface. For example, the first surface
component can have or acquire a negative surface charge and the
second surface component a positive surface charge (or vice versa).
As another example, the surface components can have different
surface chemisorption characteristics; for example, the second
surface component can have strong chemical affinity for a
particular group (e.g., phosphonate or phosphate groups), while the
first surface component does not have strong affinity for the
group.
[0022] The methods can be employed to selectively immobilize
molecules of interest, e.g., in a ZMW or other hybrid substrate.
Thus, for example, the substrate optionally comprises a layer of
the second surface component on a layer of the first surface
component, with a zero mode waveguide disposed through the second
surface component layer to the first surface component layer. In
such embodiments (among others), the first surface component can
comprise SiO.sub.2 and/or the second surface component can comprise
a metal or metal oxide (e.g., aluminum or aluminum oxide). In
embodiments in which the hybrid substrate is in contact with a
solution, the substrate can comprise a first surface component that
is a material with a point of zero charge below the pH of the
solution (e.g. SiO.sub.2 at pH>2) and/or a second surface
component that comprises a metal oxide with a point of zero charge
above the pH of the solution (e.g. aluminum oxide at pH<8; other
metal oxides that are positively charged at neutral pH include, but
are not limited to, thallium oxide, iron oxide, yttrium oxide, zinc
oxide, lanthanum oxide, and magnesium oxide).
[0023] In certain embodiments, the molecule of interest associates
preferentially to the first surface component. For example,
positively charged polymerase molecules preferentially associate
with negatively charged silicate over positively charged metal
surfaces. In other embodiments, the substrate is contacted with a
first composition that selectively associates with the first
surface component based upon the difference between the surface
characteristics of the first and second surface components. The
first composition can serve a blocking function, or it can
associate, directly or indirectly, with the molecule of interest.
Thus, for example, in one class of embodiments, the first
composition comprises a first coupling group, and selectively
coupling the molecule of interest to the first surface component
comprises coupling the molecule of interest to the first coupling
group (e.g., a functional chemical group, a binding group such as
biotin, or the like).
[0024] In one class of embodiments, the first composition comprises
a silane, e.g., for selective association with a silicate first
surface component. Exemplary silanes include, but are not limited
to, biotin-PEG-silanes. In another class of embodiments, the first
composition comprises a phospholipid. In yet another class of
embodiments, the first composition comprises polylysine-PEG or
polylysine-PEG-biotin.
[0025] The methods optionally include contacting the substrate with
a second composition that selectively associates with the second
surface component based upon the difference between the surface
characteristics of the first and second surface components.
Treatment with the second composition is optionally performed
before or after coupling of the molecule of interest to the first
surface component, including before or after treatment with any
first composition. In some embodiments, the second composition
comprises a second coupling group, typically different from any
first coupling group. Exemplary second compositions include, e.g.,
polyelectrolytes and polyelectrolyte-PEG copolymers. The method
optionally includes depositing a polyelectrolyte multilayer on the
second surface component.
[0026] Additional exemplary second compositions include compounds
comprising one or more phosphonic acid or one or more phosphate
groups. For example, the second composition can comprise
polyvinylphosphonic acid; 2-carboxyethyl phosphonic acid; amino tri
(methylene phosphonic acid); 1-hydroxyethylidene-1,1,-diphosphonic
acid; hexamethylenediaminetetra (methylenephosphonic acid);
diethylenetriamine penta(methylene phosphonic acid);
ethylenediamine tetra(methylene phosphonic acid); bis(hexamethylene
triamine penta(methylenephosphonic acid));
2-phosphonobutane-1,2,4-tricarboxylic acid; or monoethanloamine
diphosphonate. As additional examples, the second composition can
comprise an alkyl phosphate or an alkyl phosphonate, such as octyl
phosphonic acid, decyl phosphonic acid, dodecyl phosphonic acid,
hexadecyl phosphonic acid, octadecyl phosphonic acid, docosyl
phosphonic acid, hydroxy-dodecyl phosphonic acid,
hydroxy-undecenyl-phosphonic acid, decanediylbis(phosphonic acid),
dodecylphosphate, or hydroxy-dodecylphosphate.
[0027] The two surface components can be differentially modified by
different compositions. Thus, in one class of embodiments, the
methods include contacting the substrate with a first composition
that selectively associates with the first surface component and
coupling the molecule of interest to the first composition to
selectively couple the molecule of interest to the first surface
component, and contacting the substrate with a second composition
that selectively associates with the second surface component
(before or after coupling of the molecule of interest or deposition
of the first composition).
[0028] In an alternative aspect, the invention provides methods of
selectively depositing a molecule of interest on selected regions
of a substrate, comprising providing a substrate having first and
second components, the first component comprising a conductive
material and the second component comprising an insulator, and
applying an electrical potential to the first component so as to
increase or decrease association of a molecule of interest with a
surface of the first component.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 shows a schematic illustration of a Zero Mode
Waveguide (ZMW) in application.
[0030] FIG. 2 provides a schematic illustration of a light directed
surface activation process of the invention.
[0031] FIG. 3 provides a schematic illustration of a process for
providing active surfaces in optically relevant portions of optical
confinements like ZMWs.
[0032] FIG. 4 provides a simulated plot of surface activation level
as a function of the distance from the bottom surface of a ZMW over
two separate activation stages.
[0033] FIG. 5 provides a schematic illustration of an alternate
light activation strategy using a two activation step process.
[0034] FIG. 6 provides a schematic illustration of a diffusion
limited process for providing active surfaces within confined
structures.
[0035] FIG. 7 provides an illustration of process for providing a
printed masking layer on non-relevant surfaces of substrates.
[0036] FIG. 8 schematically illustrates a photocleaving process for
removing active groups from non-relevant portions of substrate
surfaces.
[0037] FIG. 9 illustrates a size excluded particle based process
for removing molecules of interest from non-relevant portions of
substrate surfaces.
[0038] FIG. 10 illustrates selective immobilization of molecules of
interest using an electrically driven system.
[0039] FIG. 11 schematically illustrates a process for removal of
molecules from non-relevant surfaces of substrates using an
entraining matrix followed by a lift-off technique.
[0040] FIG. 12 illustrates the effects of selective immobilization
processes of the invention and particularly using a size excluded
particle process.
[0041] FIG. 13 schematically illustrates a process for selective
localization of molecules using an alternate exclusionary
process.
[0042] FIGS. 14A and 14B schematically illustrate an exemplary
process for selective localization of molecules using an
exclusionary process in which a site-specific deactivation
component removes the molecule of interest from the substrate.
[0043] FIGS. 15A, 15B and 15C schematically illustrate an exemplary
process for selective localization of molecules using an
exclusionary process in which a site-specific deactivation
component removes a coupling moiety from the substrate.
[0044] FIG. 16 schematically illustrates selective immobilization
of molecules of interest by exploiting differing surface
characteristics of different materials in hybrid substrates like
ZMWs and passivation with a PE-PEG copolymer.
[0045] FIG. 17 schematically illustrates formation of a
polyelectrolyte multilayer.
[0046] FIG. 18 schematically illustrates selective immobilization
of molecules of interest by exploiting differing surface
characteristics of different materials in hybrid substrates like
ZMWs and passivation with a polyelectrolyte multilayer.
[0047] FIG. 19 illustrates binding of nucleotide analogs to a
polyelectrolyte multilayer-treated versus a plasma-PDMS treated
(non-biased treated) surface.
[0048] FIGS. 20A and 20B illustrate binding of polymerase to a
polyelectrolyte multilayer-treated versus an untreated aluminum
surface.
[0049] FIG. 21 illustrates the effects of selective immobilization
processes of the invention and particularly using a selective
silanization and polyelectrolyte multilayer passivation
process.
[0050] FIG. 22 illustrates the effects of selective immobilization
processes of the invention and particularly using a selective
silanization and polyelectrolyte multilayer passivation
process.
[0051] FIG. 23 illustrates binding of neutravidin-coated
fluorescent beads to a phosphonate-treated ZMW versus an untreated
ZMW.
[0052] FIG. 24 illustrates binding of nucleotide analogs to a
phosphonate-treated ZMW versus an untreated ZMW.
[0053] Schematic figures are not necessarily to scale.
DETAILED DESCRIPTION OF THE INVENTION
I. General Description of Invention
[0054] The present invention is generally directed to methods and
processes for providing desired molecules in preselected locations
or areas on a substrate or within a set volume, and articles made
from such methods or processes, and particularly, in desired low
concentrations or as individual molecules, within an optical
confinement. In particularly preferred aspects, the invention is
directed to methods for localizing individual molecules within a
particular space or volume, such that the spatial individuality of
the molecule may be exploited, e.g., chemically, optically,
electrically, or the like. The invention also provides the
substrates, devices, receptacles and the like, e.g., the optical
confinements, produced by these processes. While the processes of
the invention may be broadly practical in providing individual
molecules within any of a variety of given desired space or volume
types, in particularly preferred aspects, the processes are used to
selectively deposit or immobilize a desired molecule, such as an
enzyme, within the optically accessible portion of an optical
confinement, and particularly, a zero mode waveguide (ZMW).
[0055] In general, optical confinements are used to provide
electromagnetic radiation to or derive such radiation from only
very small spaces or volumes. Such optical confinements may
comprise structural confinements, e.g., wells, recesses, conduits,
or the like, or they may comprise optical processes in conjunction
with other components, to provide illumination to or derive emitted
radiation from only very small volumes. Examples of such optical
confinements include systems that utilize, e.g., total internal
reflection (TIR) based optical systems whereby light is directed
through a transparent substrate at an angle that yields total
internal reflection within the substrate. Notwithstanding the TIR,
some small fraction of the light will penetrate beyond the outer
surface of the substrate and decay rapidly as a function of
distance from the substrate surface, resulting in illumination of
very small volumes at the surface. Similarly, ZMW structures may be
employed that utilize a narrow core, e.g., from 10 to 100 nm,
disposed through a cladding layer where the core is dimensioned
such that the desired electromagnetic radiation is prevented from
propagating through the core. As a result, the radiation will
permeate the core only a very short distance from the opening of
the core, and consequently illuminate only a very small volume
within the core. A variety of other optical confinement techniques,
including, e.g., field enhancement by sharp metal tips, nanotube
confinement, thin slit confinement, near-field resonant energy
transfer confinement, near field aperture confinement, diffraction
limited optical confinement, and stimulated emission depletion
confinement, are contemplated, as well as all other confinements
described in pending U.S. Ser. Nos. 10/944,106 and 09/572,530 and
U.S. Pat. No. 6,917,726, each of which is incorporated herein by
reference in its entirety for all purposes.
[0056] Zero mode waveguides (ZMWs) are generally characterized by
the existence of a core surrounded by a cladding, where the core is
dimensioned such that it precludes a substantial amount of
electromagnetic radiation that is above a cut-off frequency from
propagating through the core. As a result, when illuminated with
light of a frequency below the cutoff frequency, the light will
only penetrate a short distance into the core, effectively
illuminating only a small fraction of the core's volume. In
accordance with the present invention, the core comprises an empty
or preferably fluid filled cavity surrounded by the cladding layer.
This core then provides a zone or volume in which a chemical,
biochemical, and/or biological reaction may take place that is
characterized by having an extremely small volume, and in some
cases sufficient to include only a single molecule or set of
reacting molecules. ZMWs, their fabrication, structure, and use in
analytical operations are described in detail in U.S. Pat. No.
6,917,726 and Levene, et al., Science 299(5607):609-764 (2003), the
full disclosures of which are hereby incorporated herein by
reference in their entirety for all purposes.
[0057] In the context of chemical or biochemical analyses within
ZMWs as well as other optical confinements, it is clearly desirable
to ensure that the reactions of interest are taking place within
the optically interrogated portions of the confinement, at a
minimum, and preferably such that only a single reaction is
occurring within an interrogated portion of an individual
confinement. A number of methods may generally be used to provide
individual molecules within the observation volume. A variety of
these are described in co-pending U.S. patent application Ser. No.
11/240,662, filed Sep. 30, 2005, incorporated herein by reference
in its entirety for all purposes, which describes, inter alia,
modified surfaces that are designed to immobilize individual
molecules to the surface at a desired density, such that
approximately one, two, three or some other select number of
molecules would be expected to fall within a given observation
volume. Typically, such methods utilize dilution techniques to
provide relatively low densities of coupling groups on a surface,
either through dilution of such groups on the surface or dilution
of intermediate or final coupling groups that interact with the
molecules of interest, or combinations of these.
[0058] In some cases, it may be further desirable that reactions of
interest be reduced or even eliminated from other regions outside
of the observation volume, e.g., on the overall substrate housing
ZMWs, the cladding layer, etc., both inside and outside of the
observation volume. In particular, reactions that are outside of
the range of interrogation may, nonetheless, impact the reaction of
interest or the monitoring of that reaction, by affecting reaction
kinetics through depletion of reagents, increasing concentration of
products, contributing to signal background noise levels, e.g.,
through the generation of products or consumption of reactants,
that may interfere with the interrogated reaction or that provide
excessive detectable background product levels that diffuse into
and out of the interrogation volume of the waveguide. Accordingly,
selective and preferential deposition and/or immobilization of the
reaction components within the observation volume are particular
advantages of the invention. These are generally practicable both
as an alternative to and, preferably, in addition to the low
density deposition methods referenced above. In the context of the
foregoing, molecules of interest may be described as being
preferentially located in a particular region, or localized
substantially in a given region. It will be appreciated that use of
the term preferentially is meant to indicate that the molecule is
localized in a given location at a concentration or surface density
that exceeds that of other locations in which it is not
preferentially localized. Thus preferential immobilization of a
given molecule in a first region will mean that the molecule is
present in such region at a higher density or concentration than in
other regions. Density in such regions may be as much as 20%
greater, 30% greater, 50% greater, 100% greater, or upwards of
200%, up to 1000% or more of the concentration or density in other
regions, and in some cases 100 times greater, 1000 times greater or
more. Similar meaning is generally applicable to indications that a
given molecule is substantially only located in a given region.
[0059] In the case of, for example, ZMWs used for single molecule
enzymatic analysis, it may be desirable to provide a single enzyme
molecule within the illumination volume of a waveguide, and
preferably upon the bottom or base surface of the waveguide. As
noted above, it may therefore be further desirable to ensure that
additional enzyme molecules are not present upon surfaces other
than the bottom surface, e.g., the walls of the core and/or the
surfaces of the cladding layer that are not part of the core, and
the like.
[0060] A particularly valuable application of the substrates
produced by the process of the invention is in processes termed
"single molecule sequencing applications." By way of example, a
complex of a template nucleic acid, a primer sequence and a
polymerase enzyme may be monitored, on a single molecule basis, to
observe incorporation of each additional nucleotide during template
dependent synthesis of the nascent strand. By identifying each
added base, one can identify the complementary base in the
template, and thus read off the sequence information for that
template. In the context of ZMWs, an individual
polymerase/template/primer complex may be provided within the
observation volume of the ZMW. As each of four labeled (e.g.,
fluorescent) nucleotides or nucleotide analogs is incorporated into
the synthesizing strand, the prolonged presence of the label on
such nucleotide or nucleotide analogs will be observable by an
associated optical detection system. Such sequencing processes and
detection systems are described in, e.g., Published U.S. Patent
Application No. 2003/0044781 and pending U.S. patent application
Ser. No. 11/201,768, filed Aug. 11, 2005, the full disclosures of
which are incorporated herein by reference in their entirety for
all purposes. Such single molecule sequencing applications are
envisioned as being benefited by the methods described herein,
through the selected immobilization of polymerases, templates or
primers or complexes of any or all of these, preferentially within
selected regions on a substrate, and/or substantially not on other
portions of the substrate.
[0061] In general, selective provision of a molecule of interest in
a given location, e.g., in the illumination volume within a ZMW,
may be accomplished using either additive or subtractive processes.
By additive process, is generally meant that the individual
molecule is placed or deposited in the desired location and not
elsewhere. By contrast, subtractive processes denote the deposition
of the molecule of interest more ubiquitously and non-selectively,
e.g., over an entire substrate surface, followed by the selected
removal of the molecule of interest from the non-desired locations.
While these descriptions provide convenience in describing various
processes, it will be appreciated that the result of one process
may be indistinguishable from the result of the other process. It
will also be appreciated that many processes may include steps that
may be described as either additive, subtractive, or both. Although
generally discussed in terms of localization of enzymes or other
macromolecular groups, for purposes of the present invention, the
molecule of interest may be any of a variety of different
functional molecules for which one desires to provide spatial
individuality or enhanced localization. Such groups include active
molecules, such as catalytic molecules like enzymes, but also
include molecules with more passive functionality, e.g., non
catalytic groups, such as binding or coupling groups, hydrophobic
or hydrophilic groups, structural enhancement groups, e.g., for
adhesion promotion, activatable or deactivatable groups, or the
like. Binding or coupling groups may include small molecule
coupling groups or they may include macromolecular coupling groups,
e.g., antibodies, antibody fragments, specific binding pairs, such
as avidin/biotin, binding peptides, lectins, complementary nucleic
acids, or any of a variety of other binding groups. Catalytically
active molecules will typically include any catalytically active
molecule for which one desires spatial individuality, e.g., to
exploit in single molecule analyses, or the like.
[0062] In at least one aspect, the present invention is directed to
providing enhanced isolation of discrete reaction and/or
observation regions. This is not simply to provide optical
isolation between such regions, but also to provide chemical and/or
environmental isolation for such regions. In a general sense, this
is accomplished by providing a barrier or zone between reaction
and/or observation regions that substantially prevents the
diffusion of reactants and/or products from outside a particular
reaction zone from entering and potentially interfering with the
reaction taking place therein, or the observation of that reaction.
In providing the requisite isolation, one may focus on one or both
of: (1) providing sufficient separation/isolation between
neighboring reaction/observation regions; and (2) eliminating any
potentially interfering components from the spaces between such
neighboring regions, e.g., clearing any reactants, products and/or
enzymes from such spaces, and creating a type of "demilitarized
zone" between observation regions.
[0063] Providing enhanced isolation generally relates to providing
a barrier of some sort between observation regions. In general,
such barriers may simply include sufficient distance in a fluidic
system such that reactants and products may not diffuse from one
reaction into a particular observation region, whether the reaction
is in a neighboring observation region or is located somewhere
else. One may provide such distance across a planar substrate or
one may increase the effective diffusion distance by providing a
structured or contoured surface on the substrate. For example, in
particularly preferred aspects, one may provide discrete
reaction/observation regions within nanoscale wells to effectively
increase the distance between such regions, as well as treat or
otherwise produce such substrates, to reduce or eliminate any
reactants and/or products from existing or being generated in the
space or regions between the selected regions, e.g., surfaces other
than those at or toward the bottom surface of the nanoscale
wells.
II. Additive Processes
[0064] As noted above, in at least one aspect, an additive process
is employed to provide the desired immobilized molecules of the
invention. The additive processes typically rely upon the selective
provision of binding or coupling groups at the desired location,
followed by the deposition of the molecules of interest. This
deposition may, again, be the result of additive or subtractive
processes.
[0065] In at least a first aspect, the additive processes of the
invention typically include the deposition of a coupling group upon
the substrate surface that selectively binds the molecule of
interest only within the desired region on the surface, e.g.,
within the observation area of an optical confinement such as a
ZMW. Coupling of functional groups, including activatable
functional groups, to surfaces may generally be carried out by any
of a variety of methods known in the art. For example, in the
context of silica based substrates, e.g., glass, quartz, fused
silica, silicon, or the like, well characterized silane chemistries
may be used to couple other groups to the surface. Such other
groups may include functional groups, activatable groups, and/or
linker molecules to either of the foregoing, or the actual
molecules of interest that are intended for use in the end
application of the surface. In the context of other substrate
types, e.g., polymeric materials, metals or the like, other
processes may be employed, e.g., using hybrid polymer surfaces
having functional groups coupled thereto or extending from the
polymer surface using, e.g., copolymers with functional groups
coupled thereto, metal associative groups, i.e., chelators, thiols,
or the like.
[0066] In at least a first aspect of the invention, providing
coupling of a molecule of interest only within a desired area or
region is typically carried out by providing an activatable
coupling group coupled to the surface of the overall substrate that
is selectively activated only within the desired region, or by
using a selectively de-activatable coupling group and selectively
deactivating it in all but the desired region. The selective
provision of active coupling groups only where desired allows
selective deposition and coupling of the molecule of interest
substantially only in the desired regions. For ease of discussion,
the portion of a surface or substrate in which one wishes to
selectively provide molecules of interest for a given application
are referred to herein as the "desired regions" while regions
outside of these regions are referred to as the non-desired
regions. Such desired and non-desired regions may include planar
surfaces or may comprise three dimensional structures such as
wells, recesses, surface irregularities, posts, pillars, trenches,
troughs, channels, capillaries, porous materials, or the like.
[0067] A variety of different activatable coupling groups may be
used in conjunction with this aspect of the invention. Typically,
such groups include coupling groups that are capped or blocked with
a selectively removable group. These include groups that are
thermally altered, e.g., thermolabile protecting groups, chemically
altered groups, e.g., acid or base labile protecting groups, and
photo alterable groups, e.g., photo-cleavable or removable
protecting groups.
[0068] Deactivation of coupling groups, e.g., in non-desired
regions, may comprise the use of groups that may be directly
selectively deactivated, e.g., through the use of thermal, chemical
or photo-induced chemistries that cap or result in the removal of
functional groups, i.e., through photo-induced cross-linking,
photocleavage, or the like. Alternatively, and in certain preferred
aspects, such deactivation methods utilize selective activation of
the coupling group in the non-desired regions, followed by blocking
or capping of the resulting active coupling group with a neutral or
inert blocking group, e.g., a group that is substantially incapable
of coupling to the molecule of interest, or an intermediate linking
molecule, under coupling conditions subsequently applied to couple
such groups to the desired regions. This subsequently added
blocking group may be irreversible or reversible. However,
reversibility of such capping, if any, will typically involve a
mechanism other than that of the underlying activatable coupling
group, to avoid re-activating capped groups in the non-desired
regions while activating those underlying activatable groups in the
desired regions. For example, where one is employing a
photoactivation strategy to selectively activate groups in the
desired regions, capping groups applied to non-desired regions will
typically not be photoactivatable or otherwise activated by any
conditions to which the surface will be exposed in application.
[0069] Following the capping of coupling groups in the non-desired
regions, the coupling groups within the desired regions, or area of
interest, may be selectively activated and coupled with the
molecule of interest. For ease of discussion, whether
photoactivation involves photocleavage of a blocking group, or
photoactivation through alteration of a chemical structure without
removal of a larger blocking group, per se, e.g., results in
modified groups or addition of other groups, it will generally be
referred to herein as activation, e.g., photoactivation.
[0070] In at least one particularly preferred aspect,
photoactivatable coupling groups are used to selectively deposit
molecules of interest in desired regions, e.g., using chemically
active coupling groups that are capped with a photo-labile
protecting groups. Such photoactivatable coupling mechanisms are
particularly useful for systems that employ optical confinements
such that light for both observation of an ultimate reaction of
interest and for activation of the coupling group is only capable
of illuminating the desired region, e.g., those regions of a ZMW
closest to the core opening from which the core is illuminated. In
particular, because activating light directed at a ZMW will only
illuminate a restricted volume, e.g., the illumination volume,
molecules of interest will be selectively coupled substantially
only within the illumination volume. Restated, the same optical
confinement effect used to only monitor reactions within the small
confined volume of the illumination volume (which typically
substantially defines the observation volume in the applicable
analytical operations to which the ZMW will be put), likewise only
permits activation (and consequent coupling) within that same
confined volume or portion of the ZMW. As will be appreciated, by
modulating the activation radiation, one can further control the
illumination volume during activation to be a smaller volume than
the illumination volume during application. In particular, by
applying a lower power illumination, using a longer wavelength of
activation light than illumination/interrogation light, one can
illuminate, activate and thus couple molecules of interest only to
a subset of the surface that will ultimately be within the
illumination volume in the ultimate application.
[0071] For a number of the specific aspects of the invention, it is
generally preferred to utilize a substrate that provides for the
selective direction of electromagnetic radiation to desired
regions, both in terms of the ultimate application of such
substrates, e.g., in interrogating chemical, biochemical and/or
biological reactions on those substrates, and in providing
selectively activated surfaces for selectively immobilizing
molecules of interest in those regions for exploitation during such
analyses. In sum, one takes a basic function of the substrate that
is used in its ultimate application, and exploits that function to
improve the fabrication and processing of that substrate for that
application. In the context of directing radiation, a substrate
that is used to focus radiation into desired regions for
interrogation of reactions within such regions is processed using
the same radiation directing properties to selectively
functionalize those desired regions.
[0072] A variety of different coupling groups may be used in this
context, depending upon the nature of the molecule of interest to
be subsequently deposited upon and coupled to the substrate. For
example, the coupling groups may include functional chemical
moieties, such as amine groups, carboxyl groups, hydroxyl groups,
sulfhydryl groups, metals, chelators, and the like. Alternatively
or additionally, they may include specific binding elements, such
as biotin, avidin, streptavidin, neutravidin, lectins,
SNAP-Tags.TM. or substrates therefore (Covalys Biosciences AG; the
SNAP-Tag.TM. is a polypeptide based on mammalian
06-alkylguanine-DNA-alkyltransferase, and SNAP-tag substrates are
derivates of benzyl purines and pyrimidines), associative or
binding peptides or proteins, antibodies or antibody fragments,
nucleic acids or nucleic acid analogs, or the like. Additionally,
or alternatively, the coupling group may be used to couple an
additional group that is used to couple or bind with the molecule
of interest, which may, in some cases include both chemical
functional groups and specific binding elements. By way of example,
a photoactivatable coupling group, e.g., photoactivatable biotin,
may be deposited upon a substrate surface and selectively activated
in a given area. An intermediate binding agent, e.g., streptavidin,
may then be coupled to the first coupling group. The molecule of
interest, which in this particular example would be biotinylated,
is then coupled to the streptavidin.
[0073] Photo-labile protecting groups employed in this aspect of
the invention may include a variety of known photo-cleavable
protecting groups, including, for example, nitroveratryl,
1-pyrenylmethyl, 6-nitroveratryloxycarbonyl,
dimethyldimethoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,
methyl, methyl-6-nitropiperonyloxycarbonyl, 2-oxymethylene
anthraquinone, dimethoxybenzyloxy carbonyl,
5-bromo-7-nitroindolinyl, o-hydroxy-alpha-methyl cinnamoyl, and
mixtures thereof, as described in U.S. Pat. Nos. 5,412,087 and
5,143,854, each of which is incorporated herein by reference in its
entirety for all purposes.
[0074] Coupling of the photoactivatable coupling groups to the
surfaces of interest may be accomplished by a number of methods
known in the art. For example, photoprotected or activatable groups
may include a carboxyl group that is coupled through hydroxyl
groups on the surface or attached to the surface through a linker
group, e.g., a PEG molecule. Alternatively, amine groups on the
photoactivatable groups may be coupled to surface bound epoxy
groups. Alternatively, activatable groups precoupled to linker
molecules, e.g., PEG groups, may be silanated and attached directly
to surfaces through known processes.
[0075] Examples of the compounds used in the foregoing coupling
strategies, e.g., using MeNPOC protected biotin, are illustrated
below:
##STR00001##
[0076] Additional light sensitive protecting groups include groups
useful for coupling amines, such as trimethylphenyloxycarbonyl
(TMPOC), groups useful for coupling acids, such as phenacyl ester
(313 nm cleavage), .alpha.-phenacyl ester, Desyl ester (350 nm),
Bis(o-nitrophenyl)methyl ester (320 nm), 1-pyrenylmethylester(340
nm), N-8-nitro-1,2,3,4-tetrahydroquinolylamide (350 nm), as well as
esters of the following compounds:
##STR00002##
[0077] For those aspects of the invention that use longer
wavelengths for activation or deprotection, appropriate longer
wavelength labile groups would be used, such as brominated
7-hydroxyxoumarin-4yl-methyls, which are photolabile at around 740
nm. Other such groups are known to those of skill in the art.
[0078] Also useful are such photolabile groups for coupling to
alcohols, including, e.g., some of the groups described above, as
well as p-nitrobenzyloxymethyl ether, p-methoxybenzylether,
p-nitrobenzylether, mono, di or trimethoxytrityls,
diphenylmethylsilyl ether, sisyl ether,
3',5'-dimethoxybenzoincarbonate, methanesulfate, tosylate, and the
like. These and a variety of other photocleavable groups may be
employed in conjunction with this aspect of the invention, and are
described in, e.g., the CRC Handbook of Organic Photochemistry and
Photobiology, Second Edition, and Protective Groups in Organic
Synthesis (T. W. Greene and P. G. Wuts, 3.sup.rd Ed. John Wiley
& Sons, 1999), each of which is incorporated herein by
reference in its entirety for all purposes.
[0079] In addition to, or as an alternative to, the use of the
previously described, relatively large, photo-removable protecting
groups, the invention also includes the use of photoactivatable
groups, e.g., groups that are chemically altered, other than
through the removal of such blocking groups. For example, vinyl or
allyl groups may be coupled to surfaces and simultaneously
illuminated and coupled with appropriate groups to be coupled that
bear, e.g., sulfhydryl groups, such as biotin having a sulfhydryl
group coupled to it either directly or through a linker molecule,
which react with the activated vinyl or allyl group to couple to
the surface. Alternatively, other groups, like nitroarylazides may
be employed as the activatable coupling groups. A wide variety of
other photoactivatable compounds may likewise be used, including,
e.g., nitrospiropyran groups (See, Blonder et al., J. Am. Chem.
Soc. 1997, 119:10467-10478, and Blonder et al., J. Am. Chem. Soc.
1997, 119:11747-11757.
[0080] In one aspect, a photoinitiator, e.g., a long wavelength
photoinitiator, is employed, such as Irgacure 784 (bis-(eta
5-2,4-cyclopentadien-1-yl)bis[2,6-difluoro-3-(1H-pyrrol-1-yl)phenyl]titan-
ium; Ciba Specialty Chemicals) that can initiate free radical
reactions at wavelengths as long as 530 nm. Such long wavelength
photoinitiators have a variety of applications. For example, a
surface (e.g., a metal oxide surface) can be coated with
vinyl-alkyl-phosphonate. Exposure of a desired region of the
surface to a 530 nm laser in the presence of Irgacure 784 and
biotin-PEG-SH results in formation of biotin-PEG-alkyl-phosphonate
in that region. The biotin can subsequently be employed to
immobilize a molecule of interest to the desired region.
[0081] In related aspects, the photoactivatable component may be
provided in solution and activated proximal to the surface region
where localization is desired. For example, one may graft an
activatable binding component or other molecule of interest onto an
active silane surface. One example of such a system includes
photoactivatable psoralen-biotin compounds (available from, e.g.,
Ambion, Inc.), that are activatable under UV light for coupling
with a silanated surface, e.g., a trimethoxysilane modified
surface.
[0082] Those aspects of the invention that include an additive
process using a selective surface activation generally encompass a
number of different strategies for selective activation in the
desired locations. Such strategies may include a single activation
step, a multiple activation step process, a multiple step process
that includes both activation and deactivation steps or processes,
or the like. For ease of discussion, such multiple step processes
are described with reference to photoactivation and/or
photodeactivation processes, although it will be appreciated that
other non-photo driven processes may be similarly employed.
[0083] In at least a first, relatively simple aspect, the selective
activation of photo-activatable coupling groups in the desired
region involves a single step of directing activating radiation at
the desired region and coupling the molecule of interest to the
activated coupling groups that are disposed thereon. As noted, in
the case of optical confinements where it is desirable to localize
the molecule of interest, e.g., an enzyme, within the illumination
volume, the single step photo-driven activation should result in
coupling substantially only within the illumination volume.
Further, as noted previously, by modulating the activation
radiation, one can further focus the activation, and thus coupling
of groups of interest, in a subset of the illumination volume that
is interrogated during the ultimate application, e.g., in nucleic
acid sequence determination using an immobilized polymerase
enzyme.
[0084] The basic functional structure of a ZMW structure is
schematically illustrated in FIG. 1. As shown, a ZMW structure 100
is provided that includes a cladding layer 102 deposited upon a
transparent substrate layer 104. A core 106 is disposed through the
cladding layer to expose the transparent layer 104 below. The core
is dimensioned to provide optical confinement by preventing
propagation of electromagnetic radiation that falls below a cut-off
frequency through the core. Instead, the light only penetrates a
short distance into the core, illuminating a relatively small
volume, indicated as bounded by the dashed line 108. By providing
reactants of interest within the observation volume, e.g., enzyme
110 and substrate 112, one can selectively observe their operation
without interference from reactants, e.g., substrates 114 outside
the observation volume, e.g., above line 108.
[0085] As noted previously, it is generally desirable that in
performing molecular analyses, e.g., enzyme analyses, that the
molecule of interest be provided preferentially within the
illumination or observation volume. Accordingly, a simple
activation strategy, as applied to ZMWs, is schematically
illustrated in FIG. 2, with reference to FIG. 1. As shown, the ZMW
structure 100 may be first treated to provide an activatable
surface, e.g., shown as solid line 202. As shown, the treatment
step is not selective, in that it provides such an activatable
surface over the entire surface of the structure, including
cladding layer 102. The activatable groups that are within the
illumination volume, e.g., as bounded by dashed line 108, are then
subjected to activation (as indicated by dashed line 204). In the
context of a ZMW structure, this typically involves exposing the
activatable groups to activating radiation through the transparent
substrate 104, as indicated by waved arrows 206. As will be
appreciated, the activation radiation decays sufficiently beyond
the illumination volume, and as such, substantially activates only
the groups therein, e.g., those below dashed line 108. Molecules of
interest, e.g., enzymes, or enzyme specific coupling groups, are
then coupled to the activated groups within the observation volume,
and nowhere else on the surface. It will be appreciated that the
reference to the illumination volume as having a well defined
border is simplified for ease of discussion, and that decay of
illumination through the ZMW core is not so abrupt. As a result,
both the illumination and, as a result, the level of surface
photo-activation from such illumination would be expected to
decrease in a related fashion with increasing distance from the
illuminated end of the waveguide core. The rate of radiation decay
and the activation levels may decrease at different rates,
depending upon the nature of the activation processes, e.g.,
whether there is saturation at any point, as well as whether the
activation processes are single or multiple photon processes.
[0086] In an alternative process, an additional activation step may
be employed to further select the region to which molecules of
interest may be coupled. In particular, in a given activation step
within an optical confinement, e.g., a ZMW, illumination as shown
in FIGS. 1 and 2 will generally result in a spectrum of activation
within the confinement, with more activated groups being present
where illumination is greatest, e.g., at the bottom surface of the
waveguide. As the illumination decreases with further penetration
into the waveguide, the activation level or efficiency of
activation will decrease depending upon the characteristics of the
activatable group the intensity of the illumination and the amount
of time exposed. This will result in a decreasing probability of
activation of groups in the portions of the illumination region
where light penetration decreases and thus, illumination is less.
By then capping these activated groups with a second photoremovable
group and repeating the activation step, the probability of the
groups present being activated away from high illumination is
similarly limited, but now is applied to a smaller number of
groups. This is further illustrated with the following example: if
one has a uniform distribution of photoactivatable groups in a ZMW
structure that are activatable with a first wavelength of light, at
a particular distance from the bottom of the waveguide, one half of
all activatable groups present are activated. If the active groups
are then capped with a second photoactivatable group that is
activated at a different wavelength, activation of those groups
will again activate only half of the activatable groups present at
the particular distance, or one fourth of the originally
activatable groups. The result when applied over the spectrum of
activation is a more narrowly focused activation/coupling area
approaching the bottom of the waveguide structure.
[0087] A schematic illustration of a double activation method is
provided in FIG. 3. In accordance with the double activation
method, a waveguide structure 300, for example, is provided with a
surface coating of photoactivatable groups uniformly applied over
the surface (shown in panel I, as black diamonds 302). A first
activation step (panel II) is used to activate the activatable
groups within a waveguide (shown as open diamonds 304) by, e.g.,
directing an activation light through the bottom surface 306 of the
waveguide 300. Instead of coupling the molecule of interest to
those activated groups, a second activatable group (shown as black
circles 308 in Panel III), that is activated by a different
wavelength of light can be used to cap the activated groups 304. A
subsequent activation step (Panel IV) then activates a subset of
the newly capped groups (shown as open circles 310), and the
molecule of interest (not shown) is then coupled to these newly
activated groups. FIG. 4 provides an exemplary simulated plot of
surface activation (concentration of activated surface groups) vs.
distance from the bottom surface of a ZMW, for both a first and
second activation step. As shown, a first activation step would be
expected to yield an activation profile that falls off in
conjunction with a rate of decay of activation light away from the
bottom surface of a ZMW. After capping with a second
photo-removable group, and reactivation at a different wavelength,
one would expect a similar decay profile, but based upon only the
previously activated groups. As a result, the activated groups
would be more focused at the bottom surface of the waveguide than
with just a single activation step. While described in terms of two
steps, it will be appreciated that more steps could be performed to
further focus the activated region on the surface.
[0088] As used herein, unless indicated otherwise from the specific
context, capping generally refers to coupling an additional group
to an otherwise reactive group such that the resulting compound is
not active to further applied coupling or other reactions of
interest. Such capping molecules typically comprise groups that
will couple to the exposed coupling group but which are otherwise
natural to the desired reaction, and will vary depending upon the
nature of the groups to be capped. They may include neutral silane
groups for capping silanol surface groups, or they may include
other non-reactive materials, e.g., non-reactive organic materials,
e.g., alcohols, alkyl groups, alkenyl groups, or the like. Such
capping groups may be small molecules or may include larger
polymeric or macromolecular structures, such as polyethylene
glycols (PEGs), or the like. Capping chemistries are widely
practiced in surface modification, derivatization and passivation
processes that are discussed in, e.g., Immobilized Biomolecules in
Analysis: A Practical Approach (Cass and Ligler Eds.) Oxford
University Press, 1998, and Hermansonn et al., Immobilized Affinity
Ligand Techniques, Academic Press, Inc. 1992, the full disclosures
of which are incorporated herein by reference in their entirety for
all purposes.
[0089] In another multi-step approach, iterative steps of
activation and deactivation may be employed to focus the coupling
of the molecule of interest. As noted previously, photoactivatable
groups may be employed in accordance with the deactivation schemes
described above, e.g., where areas other than the desired area are
activated and capped or blocked, followed by activation within the
area of interest and coupling of the molecule of interest. This
method may prove more useful for applications based upon ZMWs. In
particular, through an illumination from the open end of the
waveguide, one will typically activate, and subsequently cap
activatable groups not only on the upper surface of the cladding
layer, but also, some portion of the activatable groups on the
walls of the waveguide core. Subsequent activation from the bottom
or closed end of the core will then only be able to activate those
activatable groups that have not yet been capped. To the extent
activation radiation penetrates greater than half the length of the
core; this will result in a greater selection of activation for
deposition at or toward the bottom of the ZMW. Such a method is
schematically illustrated in FIG. 5.
[0090] In particular, on a substrate having optical confinements,
such as ZMW 500, disposed upon it, one can provide a uniform
surface that includes photo-activatable coupling groups (filled
diamonds 502) over the entire surface, e.g., inside and outside of
the confinement (Step I). In a subsequent step (step II), the
surface is exposed to activation radiation from a top side, e.g.,
the side away from the area where one wishes to immobilize the
molecules of interest. The activated groups (open diamonds 504) are
then inactivated (Step III) by capping them with another protecting
group (filled circles 506), e.g., a non-removable protecting group.
Subsequently, the ZMWs are illuminated from the bottom, so that the
illumination volume includes the desired regions and coupling
groups in that region are activated (Step IV, open diamonds 508).
The molecules of interest are then coupled to these activated
groups. By controlling the initial activation illumination, one can
effectively control the amount of activatable groups that are
capped prior to the later activation step. In particular, by using
activation radiation, or a waveguide geometry or other exposure
conditions, that permit activation radiation to effectively
propagate more than half way through the core of the waveguide, in
the first activation step, one may effectively cap more than half
of the activatable groups in the first activation and capping step.
By then directing activation radiation from the bottom side,
substantially all of the remaining activatable groups, which are
primarily substantially disposed toward the bottom of the core
which would not have been activated and capped in the first steps,
may then be activated and made available for coupling to the
molecules of interest. As will be appreciated, the various
approaches described above may be combined to further enhance
selectivity.
[0091] In an alternative process schematically similar to the
photoactivation methods described above, deep UV etching processes
may be employed in generating an active surface in desired regions,
e.g., at the bottom surface of a ZMW. In particular, deep UV
exposure, e.g., illumination at below 200 nm, i.e., using deep UV
lasers, deep UV lamps, e.g., Xeradex excimer lamp, under vacuum has
been used to selectively degrade surface bound organic or inorganic
materials, as such UV exposure is capable of breaking chemical
bonds directly, e.g., without assistance from oxygen radicals which
may be formed during the process, which may contribute to excessive
etching. By performing such exposure under vacuum or other
restrictions on the ability of oxygen radicals to contact and etch
other surfaces, one can irradiate and consequently controllably
remove organic and inorganic materials from selected substrate
regions.
[0092] In the context of the surfaces of the invention, for
example, a ZMW substrate may be provided with a first blocking
layer that is substantially inert to additional coupling groups,
e.g., it is non-reactive with the coupling strategy to be employed
in eventually joining the molecules of interest to the surface. As
a result, the functional groups on the original surface are
effectively blocked by this blocking layer. Examples of blocking
layers include organosilanes, such as PEG-silane, deep UV resists,
or other long chain organosilanes. Exposure of the waveguides from
the bottom or substrate side to deep UV radiation then degrades the
blocking layer within the waveguides and preferentially at the
bottom surface of the waveguide.
[0093] During the exposure or etching process, it may be desirable
to limit the ability for oxygen radicals to contact other portions
of the surface, e.g., outside of the ZMW or outside the observation
region toward the bottom of the ZMW. In such cases, the system may
be operated under vacuum, or alternatively or additionally, a
sealing layer may be provided over the ZMW. Such sealing layer may
comprise a rigid layer, e.g., a glass or silicon wafer or a more
flexible material, such as a polymer sheet, e.g., PDMS, PTFE,
polypropylene, polyethylene, polystyrene, or any of a variety of
polymeric materials that are capable of sealing the waveguide
structures, preferably without excessive off-gassing or otherwise
contributing undesired chemical residues to the waveguides.
[0094] Following exposure, the substrate is contacted with a
material that includes the functional groups used to couple the
molecule of interest, which binds preferentially to the unblocked
region, e.g., the exposed silanol groups uncovered by the `etching`
process. This additional material may include only functionalized
groups or it may include a mixture of functionalized and inert
groups in order to control density of functional groups, and
consequently, molecules of interest within the waveguide structure.
Such functionalized groups may be reactive chemical species and/or
specific binding moieties, such as avidin, biotin, or the like.
[0095] Once the appropriate density of coupling groups is deposited
in the desired regions, e.g., at the bottom surface of the
waveguide structure, the molecule of interest may be coupled to the
coupling groups, e.g., through the reactive group or through an
appended biotin or avidin group or other specific binding partner
to the coupling group or that is linked to the coupling group.
[0096] In another process similar to the photoactivation methods
described above, tethered or grafted photoinitiators are employed.
Of particular interest are photo-iniferters such as
dithiocarbamates (DTC) which initiate and control the radical
polymerization of acrylates, alkenes or the terminal radical
addition of a capping reagent with a ligand for specific
immobilization of the molecule of interest. The desired region (or
regions) of a surface coated with the photoinitiator is illuminated
to initiate the reaction only in that region. For example, a
hydroxylated silicon substrate can be treated with a photoiniferter
such as N,N-(diethylamino)dithiocarbamoylbenzyl(trimethoxy)-silane
(SBDC), which forms a self-assembled monolayer on the surface of
the substrate. A methyl-methacrylate solution is then supplied, and
UV irradiation of the desired region of the surface initiates
polymerization to form a surface-tethered polymer brush of PMMA
(e.g., including a coupling group) only in that region.
[0097] Another method of selectively immobilizing molecules of
interest in desired regions on substrate surfaces involves the
selective patterning of materials with different characteristics in
different regions and relying upon the differing characteristics of
the surfaces in the selective immobilization process. In the
exemplary ZMW substrates described herein (as well as in other
hybrid substrate types, e.g., metal or semiconductor based sensors
that rely on surface associated molecules of interest, e.g.,
ChemFETS), such patterned hybrid surfaces already exist. In
particular, ZMW substrates typically comprise a metal cladding
layer, e.g., aluminum typically including an aluminum oxide surface
layer, deposited over a silica based layer, e.g., SiO.sub.2, with
an aperture disposed through the cladding layer atop the SiO.sub.2
layer. The resulting structure of the waveguides includes metal or
metal oxide walls, e.g., Al.sub.2O.sub.3 with a SiO.sub.2 base. The
aluminum oxide surface is typically relatively highly positively
charged in aqueous solutions while the SiO.sub.2 surface carries a
substantial negative charge. Such charge differentials may be
readily employed to selectively localize and immobilize molecules
of interest upon one surface relative to the other.
[0098] By way of example, DNA polymerase enzymes typically possess
a relatively high level of positively charged surface residues. As
a result, a polymerase will generally be repelled by the positively
charged metal cladding layer while being attracted and adsorbing to
the negatively charged glass surface at the base of a waveguide
structure. Coupling groups can be similarly deposited, and then
polymerase (or another molecule of interest) coupled to the
coupling groups. One may readily modify the relative
attraction/repulsion of the different surfaces by adjusting the
nature of the environment to alter the charge of the enzyme, e.g.,
ionic strength, pH, additives, etc., by modifying each surface to
enhance or reduce the charge component on the surface or by
electrically (dis)charging the metal, or by modifying the enzyme,
coupling reagent, or other molecule of interest to adjust its level
of surface charge, e.g., through mutation of the enzyme or through
coupling to charged groups, e.g., polyions like polylysine,
polyarginine, or the like. In one aspect, after deposition of the
polymerase (or other group or molecule of interest) on the
negatively charged surface, the positively charged surface is
passivated by coating it with an agent such as bovine serum albumin
(e.g., acetylated BSA), polyglutamate, a polyelectrolyte, a
polyelectrolyte multilayer, a polyelectrolyte-PEG copolymer, a
phosphonate, or a phosphate, as discussed in greater detail below.
Such passivation can, for example, prevent nonspecific binding of
nucleotide analogs to the positively charged metal walls of a ZMW
core during single molecule nucleic acid sequencing applications.
In a related aspect, passivation is accomplished prior to
deposition of the polymerase (or other group or molecule of
interest), and optionally facilitates selective deposition, e.g.,
by blocking polymerase binding to the walls.
[0099] As noted above, the surface charge of a material can, in
some embodiments, be an active, tunable characteristic which can be
addressed, e.g., by pH tuning and/or by external polarization of
the surface. For example, tin oxide (a transparent material) can be
doped to make it conductive, and its surface charge (polarization)
can be modulated to a desired value.
[0100] Other surface selective chemistries may likewise be
employed. For example, different phospholipid compositions have
shown the ability, in the presence and absence of calcium, to form
different levels of supported phospholipid bilayers on metal oxide
surfaces and silicon dioxide based surfaces. By selecting the lipid
composition and the presence or absence of calcium, one can target
deposition of molecules, either as blocking or coupling groups,
onto the different surface types. For example, one can select a
phospholipid that has high binding selectivity for metal oxide
surfaces and use it to block the metal portion of the surface.
Alternatively, one can utilize a phospholipid with an appropriate
coupling group that has high binding selectivity for the underlying
glass substrate, and thus selectively couple additional groups to
the transparent substrate. Examples of these selective phospholipid
compositions are described in, e.g., Rossetti, et al., Langmuir.
2005; 21(14):6443-50, which is incorporated herein by reference in
its entirety for all purposes. Briefly, phospholipid vesicles
containing between 50% and 20% DOPS (dioleoyl phosphatidyl serine)
in DOPC (dioleoyl phosphatidyl choline), added to a hybrid
TiO.sub.2/SiO.sub.2 surface exhibit selective formation of the
lipid bilayer on the SiO.sub.2 surface in the absence of calcium,
whereas calcium presence permits bilayer formation upon the
TiO.sub.2 surface as well.
[0101] As will be appreciated, one may employ the glass selective
phospholipid bilayer (or other surface-selective composition) as
the coupling groups or may use it as a masking layer for a
subsequent blocking layer deposition upon the metallic layer. This
would then be followed by removal of the lipid bilayer from the
glass substrate followed by coupling of the molecules of
interest.
[0102] Alternatively, physical/chemical differences between the
different surfaces may be subjected to differential binding based
upon specifically selective chemistries. For example, specific
groups that associate with particular metal groups may be employed
to selectively localize molecules to one surface relative to the
other, e.g., gold/thiol chemistries, etc.
[0103] As another example, silanes (e.g., methoxy-silane reagents)
form stable bonds with silica surfaces via Si--O--Si bond
formation, but do not significantly modify aluminum or aluminum
oxide surfaces under appropriately selected reaction conditions
(e.g., vapor phase favors modification of silica surfaces, as do
certain conditions in solution). Silanes, for example, silanes
modified with coupling groups for attachment of enzymes or other
molecules of interest (e.g., biotin-PEG-silanes such as those
described in U.S. patent application Ser. No. 11/240,662), can thus
be used to selectively pattern hybrid substrates such as ZMWs that
contain silica surfaces. Ellipsometry and contact angle
measurements on Si surfaces previously modified with
Al.sub.2O.sub.3 show undetectable levels of silane reagent
deposition. In addition, fluorescently labeled neutravidin does not
bind specifically to Al.sub.2O.sub.3-modified fused silica slides
after biotin-PEG-silane deposition on the slides, while, in
contrast, biotin-PEG-silane modification of fused silica slides
(not modified with Al.sub.2O.sub.3) results in very high
specificity of neutravidin binding via the biotin ligand. Such
results demonstrate the feasibility of modifying only the fused
silica bottom of a ZMW or similar device with little or no
modification of the aluminum walls or top surface of the device,
using methoxysilane reagents.
[0104] As another example, negatively charged surfaces can be
selectively modified by adsorption of copolymers containing
positive polyelectrolyte blocks and PEG-ylated (or similar
anti-fouling) blocks. The polycationic blocks bind to regions of
the device that are electronegative, and the PEG components provide
a nonreactive surface to preclude nonspecific binding. Exemplary
polyelectrolyte-PEG copolymers include PLL-PEG
(poly(L-lysine)-poly(ethylene glycol)). The PEG groups, or a subset
thereof, can include a coupling group such as biotin or the other
groups described herein (see, e.g., U.S. patent application
publication 2002/0128234 "Multifunctional Polymeric Surface
Coatings in Analytic and Sensor Devices" by Hubbell et al., Huang
et al. (2002) "Biotin-Derivatized Poly(L-lysine))-g-Poly(ethylene
glycol): A Novel Polymeric Interface for Bioaffinity Sensing"
Langmuir 18(1): 220-230). Thus, for example, the SiO.sub.2 surfaces
of a ZMW can be coated with PLL-PEG-biotin, and biotinylated
polymerase can then be coupled to the bottom of the ZMW via avidin
or streptavidin binding to the PLL-PEG-biotin.
[0105] In one aspect, selective immobilization of the molecule of
interest on one type of material in a hybrid substrate (e.g., a
ZMW) is complemented or facilitated by modification of the other
type of material. For example, for a ZMW that is to be used in an
application such as single-molecule nucleic acid sequencing, it is
desirable to selectively immobilize the polymerase to the bottom
silica surface of the ZMW, and it is also desirable to passivate
the metal walls and top surface of the device (before or after
immobilization of the polymerase). Unmodified aluminum or aluminum
oxide ZMW surfaces, which as noted above tend to be positively
charged in aqueous solution, can demonstrate undesirable
nonspecific binding of proteins (such as neutravidin or
streptavidin and polymerase), nucleotide analogs (e.g., through the
analog's phosphate groups), and dyes (e.g., dyes with sulfonic or
carboxylic acid groups). As noted above, such undesirable
electrostatic interactions can be minimized by binding of
passivating agents to the surface; additional examples of suitable
passivating agents include, but are not limited to, anionic
polyelectrolytes such as poly(styrenesulfonate) and poly(acrylic
acid) and macromolecules such as heparin and alginine.
[0106] In some instances, however, the adsorption of anionic
polyelectrolytes to a positively charged surface may result in
overcompensation of the net charge of the surface, where adsorption
of the polyanion results in a change in the net surface charge from
positive to negative. This change in principle minimizes the
nonspecific adsorption of nucleotide analogs or other negatively
charged compounds to the surface, but has the disadvantage that
many proteins (e.g., polymerases) have affinity for electronegative
surfaces. Thus, an electronegative surface produced by such
overcompensation may result in undesirably high levels of
polymerase nonspecific binding. This problem can be addressed by
using high salt immobilization conditions; however, the high salt
regime can cause swelling of the polyelectrolyte layer as well as
partial loss of polyelectrolytes. In addition, coating of surfaces
with polyelectrolytes is a dynamic process, and it is possible that
the polymerase may eventually form stable activity-blocking
complexes with the polyelectrolytes.
[0107] Optionally, instead of passivating the positively charged
surface by adsorption of anionic polyelectrolytes, positively
charged surfaces can be passivated by binding of copolymer
structures containing polyelectrolyte blocks (negative) and
PEG-ylated blocks. The polyelectrolyte blocks of the copolymer
adsorb or anchor the macromolecules to regions of the device that
are electropositive (e.g., the aluminum or aluminum oxide areas of
a ZMW), and the PEG components provide a non-ionic cushion that
precludes the surface attachment or the complexation of the
polymerase with the polyelectrolyte blocks. The
polyelectrolyte(PE)-PEG copolymers can, for example, be diblock
(PEG-PE) or multi-block copolymers (e.g., PE-PEG-PE or PEG-PE-PEG),
as well as branched polymers, comb polymers, or dendron-like
polymers. A few exemplary linear and branched copolymers are
schematically illustrated in FIG. 16 Panel VI. It will be evident
that, while the exemplary copolymers described herein employ PEG,
any anti-fouling backbone is applicable, for example,
polypyrrolidone, polyvinyl alcohol, dextrans, and polyacrylamides.
See, e.g., U.S. patent application publication 2002/0128234, Voros
et al. (2003) "Polymer Cushions to Analyze Genes and Proteins"
BioWorld 2:16-17, Huang et al. (2002) Langmuir 18(1): 220-230, and
Zoulalian et al. (2006) J. Phys. Chem. B 110(51):25603-25605.
[0108] Orthogonal modification of a hybrid substrate with two
compositions with different selectivities for different surface
characteristics is schematically illustrated in FIG. 16. As shown
in Panel I, ZMW 1600 includes core 1602 disposed through aluminum
cladding layer 1604 to transparent silica substrate 1606. The
aluminum core has a thin aluminum oxide layer 1605 on its surface.
As shown in Panel II, the bottom surface of the ZMW is selectively
modified with a mixture of biotin-PEG-silane 1620 and PEG-silane
1622 (e.g., at a ratio selected to provide a desired density of
biotin coupling groups, and thus ultimately of molecules of
interest, on the surface, optionally, one per core). As illustrated
in Panel III, the walls and top surfaces of the device are then
selectively modified with polyanion-PEG copolymer 1630. As shown in
the expanded view in Panel V, copolymer 1630 includes polyanion (A)
blocks 1631 and PEG (B) blocks 1632. (It is worth noting that
modification of the aluminum surface is optionally performed
before, rather than after, modification of the silica surface.)
Biotinylated polymerase 1608 is then bound via neutravidin 1609 to
the biotin coupling group on biotin-PEG-silane 1620, as shown in
Panel IV.
[0109] In one aspect, the compositions used to passivate the
surface to which the molecule of interest is not attached (e.g.,
the aluminum surface) can also have a selected density of moieties
that add functionality to the surface. For example, in the PE-PEG
copolymers described above, fluorescence quenching moieties 1640
can be attached to the functional ends of the PEG blocks (FIG. 16
Panel V). As another example, orthogonal ligand schemes can be used
to attach proteins to work in tandem with polymerases or other
molecules of interest; e.g., in embodiments in which biotin is used
to immobilize polymerase 1608, functional group 1640 can be a SNAP,
HA, GST, or similar non-biotin coupling group, to bind a suitably
modified second protein. These second proteins can be used to break
up newly synthesized DNA chains, assist in removing reaction
products from solution, assist in bringing reactants to the region
of reaction, assist in regeneration of triplet quenchers, or the
like.
[0110] As another example, the surface of the hybrid substrate to
which the molecule of interest is not immobilized can be passivated
using a polyelectrolyte multilayer. Polyelectrolyte multilayers are
conveniently formed through successive deposition of alternating
layers of polyelectrolytes of opposite charge. See, e.g., Decher
(1997) Science 277:1232. Formation of a polyelectrolyte multilayer
is schematically illustrated in FIG. 17. As shown in Panels I and
II, in step 1 positively charged substrate 1705 is contacted with
polyanion 1732, which adsorbs to the surface of the substrate.
Excess polyanion is washed away in step 2. In step 3, a layer of
polycation 1734 is deposited over the layer of polyanion 1732
formed in step 1; excess polycation is washed away in step 4. Steps
1-2 and/or 3-4 are repeated as desired, to deposit alternating
layers of oppositely charged polyelectrolytes and form multilayers
of essentially any desired thickness and resulting surface charge
(negative when the polyanion is deposited last, or positive when
the polycation is deposited last). Panel III illustrates exemplary
polycation poly(ethyleneimine) and exemplary polyanion poly(acrylic
acid), which are optionally employed to form polyelectrolyte
multilayers.
[0111] Optionally, the final layer in a polyelectrolyte multilayer
comprises a polyelectrolyte-PEG copolymer, for example, a copolymer
such as those described above containing polyelectrolyte blocks
(either positive or negative, depending on the charge of the
preceding layer in the multilayer) and PEG-ylated blocks. As just
one example, a poly(acrylic acid) layer in a polyelectrolyte
multilayer can be followed by a layer of PLL-PEG or
polyglutamate-PEG, to provide a PEG finish. It will be evident
that, while the exemplary copolymers described herein employ PEG,
any anti-fouling backbone is applicable, for example,
polypyrrolidone, polyvinyl alcohol, dextrans, and
polyacrylamides.
[0112] Differential surface derivatization of a hybrid substrate
with two compositions having different selectivities for different
surface characteristics and formation of a polyelectrolyte
multilayer is schematically illustrated in FIG. 18. As shown in
Panel I, ZMW 1800 includes core 1802 disposed through aluminum
cladding layer 1804 to transparent fused silica layer 1806. The
aluminum walls have a thin aluminum oxide layer 1805 on their
surface. As shown in Panel II, the bottom surface of the ZMW is
selectively modified with a mixture of biotin-PEG-silane 1820 and
PEG-silane 1822. As illustrated in Panel III, polyelectrolyte
multilayer 1830 is then deposited on the walls and top surfaces of
the device. The polyelectrolyte multilayer can be deposited as
illustrated in FIG. 17, for example; a layer of polyanion (e.g.,
poly(acrylic acid)) is deposited on the positively charged aluminum
oxide layer 1805, followed by a layer of polycation (e.g.,
poly(ethyleneimine)), then another layer of polyanion, etc. For
single molecule sequencing or similar applications, the final layer
of the polyelectrolyte multilayer is typically a polyanionic layer,
such that the surface of the polyelectrolyte multilayer is
negatively charged to repel nucleotide analogs (or optionally a
polyelectrolyte-PEG copolymer or similar, again to provide a
surface with low binding to the analogs). As shown in panel IV,
biotinylated polymerase 1808 is then bound via neutravidin 1809 to
the biotin coupling group on biotin-PEG-silane 1820. Such
exploitation of the differences in surface properties of the
materials constituting a ZMW, e.g., silanization specific to the
glass bottom and passivation of the sides and top aluminum oxide
surfaces with polyelectrolyte multilayers to prevent nonspecific
binding, can limit polymerase occupancy to the ZMW bottom, avoiding
polymerase occupancy on side wall and top surfaces while limiting
nonspecific binding of nucleotide analogs or the like.
[0113] As yet another example of ways in which the different
materials in a hybrid substrate can be differentially modified
based on their different surface characteristics, phosphate and
phosphonic acid compounds can be employed (as can other compounds
that exhibit surface specific chemisorption and/or self-assembled
monolayer formation). Phosphate or phosphonic acid moieties bind
strongly to metal oxides (e.g., aluminum oxide, titanium oxide,
zirconium oxide, tantalum oxide, niobium oxide, iron oxide, and tin
oxide) but do not bind strongly to silicon oxide. Thus, compounds
that comprise at least one phosphate group (--OP(O)(OH).sub.2,
whether protonated, partially or completely deprotonated, and/or
partially or completely neutralized) or phosphonic acid group
(--P(O)(OH).sub.2, whether protonated, partially or completely
deprotonated, and/or partially or completely neutralized) can be
used to selectively modify the aluminum oxide surfaces of a ZMW or
similar hybrid substrate.
[0114] For example, a metal oxide surface can be modified with an
alkyl phosphate or an alkyl phosphonate. (The terms phosphonic acid
and phosphonate are used interchangeably herein.) Exemplary alkyl
phosphates and alkyl phosphonates include, but are not limited to,
an alkyl phosphate or alkyl phosphonate in which the alkyl group is
a straight chain unsubstituted alkyl group (e.g., a straight chain
alkyl group having from 1 to 26 carbons, e.g., from 8 to 20
carbons, e.g., from 12 to 18 carbons). Additional exemplary alkyl
phosphates and alkyl phosphonates include functionalized or
substituted alkyl phosphonates and alkyl phosphates, for example,
functionalized X-alkyl-phosphonates and X-alkyl-phosphates where X
is a terminal group comprising or consisting of a vinyl (CH.sub.2),
methyl (CH.sub.3), amine (NH.sub.2), alcohol (CH.sub.2OH), epoxide,
acrylate, methacrylate, thiol, carboxylate, active ester
(NHS-ester), maleimide, halide, phosphonate, or phosphate group, or
an ethylene glycol (EG) oligomer (EG4, EG6, EG8) or polyethylene
glycol (PEG), photo-initiator (e.g., photo-iniferters such as
dithiocarbamates (DTC)), photocaged group, or photoreactive group
(e.g., psoralen). The alkyl chain spacer in the X-alkyl-phosphonate
or X-alkyl-phosphate molecule is a hydrophobic tether that
optionally has 1 to 26 methylene (CH.sub.2) repeat units,
preferably from 8 to 20, and more preferably from 12 to 18. The
alkyl chain may contain one or more (up to all) fluorinated groups
and/or can instead be a hydrocarbon chain with one or more double
or triple bonds along the chain. The X-alkyl-phosphate or
X-alkyl-phosphonate layer can furthermore be used as a substrate to
anchor other ligands or components of the surface stack, such as a
polyelectrolyte multilayer or chemisorbed multilayer. The alkyl
phosphates/phosphonates can form a stable, solvent resistant
self-assembled monolayer that can protect the underlying material
(e.g., aluminum) from corrosion etc.; the role of the alkyl tether
in the above structures is to enhance the lateral stability of the
chemisorbed monolayer in aqueous environments. In embodiments in
which the phosphonate or phosphate compound includes an unsaturated
hydrocarbon chain, the double or triple bond(s) can serve as
lateral crosslinking moieties to stabilize a self-assembled
monolayer comprising the compound.
[0115] Specific exemplary alkyl phosphates and alkyl phosphonates
include, but are not limited to, octyl phosphonic acid, decyl
phosphonic acid, dodecyl phosphonic acid, hexadecyl phosphonic
acid, octadecyl phosphonic acid, docosyl phosphonic acid (i.e., C22
phosphonic acid), hydroxy-dodecyl phosphonic acid
(HO(CH.sub.2).sub.12P(O)(OH).sub.2), hydroxy-undecenyl-phosphonic
acid, decanediylbis(phosphonic acid), dodecylphosphate, and
hydroxy-dodecylphosphate. Ellipsometry and/or contact angle
measurements show that octyl phosphonic acid, octadecyl phosphonic
acid, hydroxy-dodecyl phosphonic acid, and dodecyl phosphonic acid
exhibit specificity toward aluminum/aluminum oxide surfaces
relative to Si/SiO.sub.2 surfaces. Modification of metal oxides
with such phosphates and phosphonates has been described, e.g., in
Langmuir (2001) 17:3428, Chem. Mater. (2004) 16:5670; J. Phys.
Chem. B (2005) 109:1441, Langmuir (2006) 22:6469, Langmuir (2006)
22:9254, Langmuir (2006) 22:3988, J. Phys. Chem. B (2003)
107:11726, J. Phys. Chem. B (2003) 107:5877, Langmuir (2001)
17:462, J. Phys. Chem. B (2006) 110:25603, Langmuir (2002) 18:3957,
Langmuir (2002) 18:3537, and Langmuir (2001) 17:4014.
[0116] Metal oxide surfaces can similarly be modified with
polyphosphates or polyphosphonates. Chemisorption, e.g., of
polyphosphonates differs from the previous description of
polyelectrolyte adsorption in that the ligands (phosphonic acid
moieties) form a chemical complex with the substrate (e.g.,
alumina, zirconia, or titania). Such interaction is stronger and
less reversible to salt exchange than are simple electrostatic
interactions. Examples include, but are not limited to,
PEG-phosphonates such as those described in Zoulalian et al. (2006)
"Functionalization of titanium oxide surfaces by means of
poly(alkyl-phosphonates)" J. Phys. Chem. B 110(51):25603-25605 or
PEG-polyvinyl(phosphonate) copolymers. (In general, copolymers
including chemisorbing moieties plus PEG or other anti-fouling
moieties are contemplated herein.)
[0117] Other suitable phosphonates include high molecular weight
polymeric phosphonates such as polyvinylphosphonic acid (PVPA)
##STR00003##
end-capped phosphonates such as
##STR00004##
(commercially available from Rhodia as Aquarite.RTM. EC4020 and
Aquarite.RTM. ESL, respectively); and copolymers such as vinyl
phosphonic acid-acrylic acid copolymer
##STR00005##
(commercially available from Rhodia as Albritect.TM. CP30).
[0118] Suitable phosphonates also include low molecular weight
phosphonates such as 2-carboxyethyl phosphonic acid (also known as
3-phosphonopropionic acid; commercially available from Rhodia as
Albritect.TM. PM2) and the compounds listed in Table 1
(commercially available as Dequest.RTM. compounds from Solutia,
Inc., St. Louis Mo.). Phosphonate compounds can be supplied as
salts (e.g., sodium, potassium, lithium, or ammonium salts) or,
preferably, as free acids.
TABLE-US-00001 TABLE 1 Exemplary phosphonic acid compounds.
Chemical Name Structure Amino tri(methylene phosphonic acid)
##STR00006## 1-Hydroxyethylidene-1,1,- diphosphonic acid
##STR00007## Hexamethylenediaminetetra (methylenephosphonic acid)
##STR00008## Diethylenetriamine penta(methylene phosphonic acid)
##STR00009## ethylenediamine tetra(methylene phosphonic acid)
##STR00010## bis(hexamethylene triamine penta(methylene- phosphonic
acid)) Amino methylene phosphonic acid 2-Phosphonobutane-1,2,4-
tricarboxylic acid ##STR00011## Monoethanloamine diphosphonate
[0119] A few exemplary uses of phosphonates and phosphates follow,
with respect to treatment of a ZMW where a molecule of interest
such as a polymerase is to be immobilized selectively on the
bottom, silica surface of the ZMW waveguide cores. It will be
evident that similar considerations apply to treatment of other
hybrid substrates. As one example, a ZMW chip can be treated with a
phosphonate to passivate the aluminum oxide surface of the ZMW, and
a positively charged polymerase can then be immobilized by
selective binding to the negatively charged silica surface.
Similarly, the ZMW chip can be treated with a phosphonate, a
capture reagent that can be used for subsequent immobilization of
the polymerase (e.g., neutravidin) can be immobilized by binding to
the silica surface, and then the polymerase can be immobilized by
binding to the capture agent. In these examples, the phosphonate
passivates the aluminum oxide surface, providing bias (e.g., by
blocking the aluminum oxide) and providing a surface with low
nonspecific binding of nucleotide analogs, etc. In related
examples, after immobilization of the polymerase or the capture
agent, a polyelectrolyte multilayer is formed on the aluminum oxide
surfaces to passivate them. Phosphonates and phosphates can also be
employed in combination with compounds that selectively modify the
silica surfaces of the ZMW. Thus, for example, the aluminum oxide
surface can be passivated and/or blocked with a phosphonate, and
silane reagent(s) can then be employed to modify the silica surface
(or vice versa, with modification of the silica surface preceding
phosphonate deposition).
[0120] In one class of embodiments, the phosphate or phosphonate
compound serves as the first layer on which a polyelectrolyte
multilayer is built on the surface, e.g., by successive deposition
of oppositely charged polyelectrolytes as described above. In a
related class of embodiments, a chemisorbed multilayer is formed on
the surface. The chemisorbed multilayer can include, e.g.,
alternating layers of a multi-phosphonate-containing reagent (for
example, a diphosphonate, such as 1,n-alkyl diphosphonic acid, or a
polyphosphonate, such as polyvinylphosphonate) and zirconium (IV)
ligands. The zirconium (IV) ligand for the phosphonate can be
provided by providing a precursor such as zirconium t-butoxide,
zirconium acetylacetonate, or zirconium ethoxide, from which the
phosphonate displaces the ligand around the zirconium. The
multilayers can be formed using methods well known in the art, for
example, by alternately dipping the substrate or surface in a
solution of the phosphonate and in a solution of the zirconium
precursor (with an intermediate heat annealing step), or by
alternating dipping in a solution of the phosphonate with
organometallic chemical vapor deposition (MOCVD) or rapid thermal
chemical vapor deposition (RT-CVD) of the zirconium (with an
annealing step if necessary). Such chemisorbed multilayers are
robust, and are similar to polyelectrolyte multilayers but with the
advantage of having the equivalent of chemisorbed "cross-links"
between adjacent layers rather than physical electrostatic ones as
in polyelectrolyte multilayers.
[0121] In another aspect, thermodynamic or diffusion limited
processes may be employed in the selective activation and/or
deactivation of desired or non-desired regions. In particular,
active coupling groups may be disposed over a substrate surface,
including within ZMW structures, and may be provided in active
form. They are then subsequently and selectively deactivated by
exposing the substrate to capping or blocking groups that will
prevent any additional coupling to those groups. Because the
coupling groups that are present on the desired regions reside
within the ZMW, e.g., at the bottom surface, diffusion of the
capping or blocking agents to these groups is somewhat limited. As
a result, those coupling groups will be less prone to being blocked
(will likely be the last groups to be blocked), and may be used to
couple the molecules of interest toward the bottom surface of the
ZMW. In particular, by controlling the time of exposure of the
substrate as a whole to the blocking agent, the concentration of
the blocking agent, and other conditions of the capping reaction,
e.g., temperature, etc., one can control the degree to which the
coupling groups within the waveguide become blocked or capped. In
this aspect of the invention, it will be appreciated that the
blocking component need not specifically bind to particular
coupling groups to prevent coupling of the molecule of interest. In
some cases, such blocking or capping groups may prevent such
binding through its presence within the waveguide or other portions
of the surface. This may include hydrophobic or hydrophilic coating
materials that may form a thin or monolayer over the surface and
thus block binding of the molecule of interest, or which provide a
spatial or steric barrier to binding at a given coupling group
without actually binding to the active coupling component of the
coupling groups.
[0122] The foregoing aspects of the invention are schematically
illustrated in FIG. 6. As shown, a waveguide structure 600 is
provided with a uniform coating of coupling groups 602 disposed
upon it (shown as open diamonds). Contacting the overall structure
with capping groups 604 (shown as closed circles) results in
diffusion limited capping within the waveguide structure, and as a
result, leaves more active (uncapped) coupling groups 602 toward
the bottom surface of the waveguide structure for coupling
molecules of interest in a subsequent contacting step.
[0123] As will be appreciated, the initial step of providing active
coupling groups over an entire surface may be avoided where one
simply wishes to couple groups directly to the underlying surface,
e.g., silanol groups on glass substrates, or the like. In
particular, by initially blocking any active coupling groups on the
surface for a relatively short period, those groups that are most
accessible, e.g., not within the bottom regions of a ZMW, will be
blocked first. A subsequent, longer exposure of the partially
blocked or capped surface groups to coupling groups that are
capable of binding to such surface groups will yield such coupling
groups immobilized upon the bottom regions of the waveguide
structures. The amount of time, concentration, temperature, and
other conditions of each step may be varied to provide optimal
conditions for each of the blocking steps and coupling steps, and
may be determined based upon readily identifiable characteristics
and simple experimentation.
[0124] An alternative approach to additively providing molecules of
interest in a desired location is through the optical trapping of
the molecule in the desired location, e.g., using optical "tweezer"
techniques. In particular, using the strongly enhanced electric
field created by focused laser energy within an optical
confinement, such as a ZMW, one may enhance the concentration of
particles such as molecules of interest, or enrich for their
presence within the focal region of a ZMW and subsequently couple
it to a binding group located within that region. The molecule of
interest may be provided coupled to additional groups, e.g.,
avidin, streptavidin, neutravidin, biotin, or particles, such as
beads, e.g., heparin sparse beads, or the like, etc., in order to
provide a sufficiently large particle for trapping. The use of such
optical trapping/enhancement techniques has been described in
detail for exerting trapping forces on particles as small as 10 nm.
See, e.g., Novotny, et al., Phys. Rev. Letts. 79(4):645-648 (July
1997), which is incorporated herein by reference in its entirety
for all purposes.
[0125] As an alternative or additional process to the selective
activation/deactivation processes discussed above, or below, the
present invention optionally or additionally may include an initial
patterning step to provide neutral or inert groups upon areas where
it is not desired to couple the molecules of interest. Such
patterning typically provides a coarse selectivity to the
localization, in that it is not specifically intended to yield the
final selective surface. For example, in the context of micro or
nanowells, or other structures provided in an otherwise planar
surface, inert groups may be printed, applied or otherwise
patterned upon the upper planar surface of the substrate without
depositing such materials into the nanostructures, e.g., ZMWs. By
first blocking the non-relevant surfaces with inert groups, one can
then deposit and couple active groups within the relevant areas.
Again, in the context of a ZMW array, depending upon the density of
the array, e.g., the percentage of overall substrate occupied by
waveguide structures, a substantial amount of non-relevant surface
can be blocked and thus prevented from harboring molecules of
interest that might otherwise interfere with the ultimate
application of the device, e.g., through substrate depletion,
excessive product formation, etc.
[0126] Such patterning may include simple stamping of the inert
molecules onto a surface whereby the inert groups will not
penetrate the depressions on that surface, or it may involve more
complex printing patterns using either nanolithographically
produced stamps to provide selective deposition, ink jet printing,
or the like, to selectively deposit inert groups upon the overall
substrate surface. An example of the process of the invention is
schematically illustrated in FIG. 7.
[0127] As shown, a substrate 700 that includes an array of ZMWs 702
disposed in its surface 704 (in panel I), is contacted with a
separate substrate 706 bearing a printable material 708 thereon
that prevents coupling of active functional groups to the substrate
surface 704 (Panel II). By contacting surface 704 with the
printable material 708, the material is transferred to the surface
704 while not penetrating ZMWs 702 (Panels III and IV). As a
result, subsequent coupling of molecules of interest to the upper
surface 704 of substrate 700 is blocked. The printable material may
include any of a variety of different materials, including, e.g.,
inert surface associating groups that simply cap any active groups
on the surface. Alternatively, such material may include coating
materials that prevent association with the molecules of interest,
e.g., hydrophobic or hydrophilic materials, highly charged
materials that repel any binding or other association, or materials
that provide an impenetrable barrier to such materials, e.g.,
polymer coatings, resists, or the like.
[0128] As will be appreciated, any of the foregoing processes may
be practiced in conjunction with other processes described herein
to further enhance surface selectivity and/or localization.
III. Subtractive Processes
[0129] As noted previously, in alternative aspects, subtractive
processes are employed to provide the molecule(s) of interest in
the desired regions of a substrate and at the desired concentration
and/or density of molecules. As noted, subtractive processes are
generally characterized and differentiated from the additive
processes described above, in that they deposit the molecule of
interest more ubiquitously, e.g., over an entire substrate surface
including in the desired regions. Subsequently, the excess
molecules of interest, e.g., that are located in non-desired
regions, are removed. A variety of different processes may be
employed in such subtractive processes.
[0130] In one example, a process may be employed that is roughly
the inverse of the photoactivatable processes described above. In
particular, coupling of the molecule of interest may be
accomplished over the substrate surface using a selectively
cleavable linker or coupling group. A variety of photocleavable
linker chemistries are known in the art and include 2-nitrobenzyl
linkers (See, e.g., Rodebaugh, R.; Fraser-Reid, B.; Geysen, H. M.
Tetrahedron Lett. 1997, 38, 7653-7656), as well as a number of
other known photocleavable linker types, see e.g., Org. Lett., 2
(15), 2315-2317, 2000.
[0131] In the context of the present invention, a coupling group
may be broadly applied to a substrate surface using a
photocleavable linker group. The molecule of interest is then
coupled to the coupling groups substantially non-selectively.
Selective illumination of areas that are outside the desired
regions then releases the molecules of interest from these areas,
leaving such molecules substantially only coupled within the
desired regions. Washing of the substrate then removes the
molecules from any potential interference with the desired
application.
[0132] This aspect of the invention is schematically illustrated in
FIG. 8. In particular, coupling groups 802 (shown as open diamonds)
are provided in a uniform coating over the surface of the waveguide
structure, but are attached to that surface through photocleavable
linker groups 804 (shown as filled circles). The surface that is
outside of the area of interest, e.g., not at the bottom surface
806 of ZMW core, is then exposed to light (shown as wavy arrows
808) to cleave the linker groups in the non-desired regions, where
coupling is not ultimately desired, leaving those coupling groups
in the desired regions for subsequent coupling, e.g., at bottom
surface 806, available for coupling.
[0133] Another subtractive approach to the selective immobilization
of molecules of interest, particularly within nanostructured wells
or other constrained spaces, e.g., optical confinements like ZMWs,
utilizes deactivation components, e.g., that deactivate either the
molecule of interest or the component linking that molecule to the
surface, or otherwise cause the digestion, deactivation, release or
removal of such molecules from the surface. For ease of discussion,
such components are referred to herein as "deactivation components"
regardless of whether such components degrade and/or digest the
molecules of interest, inactivate such molecules, e.g., through
nonreversible binding to active sites or other modification of such
molecules of interest, or the like, or merely release them from the
surface, e.g., through the cleavage of a linking group or
otherwise.
[0134] Such approaches may rely upon thermodynamics to selectively
avoid deactivation or removal within a ZMW, as diffusion of larger
deactivation components, e.g., enzymes, i.e., proteases or other
larger macromolecular compounds, or the like, will diffuse into a
waveguide more slowly, similar to the diffusion limited capping of
coupling groups shown in FIG. 6.
[0135] Alternatively, the method may rely upon the use of
additional components to prevent the deactivation components from
accessing the molecules of interest within the constrained space,
e.g., a ZMW. One particularly preferred aspect of such prevention
involves the coupling of the deactivation component to a large
component, such as a bead or other particle, or a large polymeric
molecule or aggregation of molecules, that are at least partially
incapable of entering into the ZMW. Such larger components are
generally referred to as exclusionary components as they are sized
or shaped to be at least partially excluded from recesses such as
ZMWs on substrates. Because the deactivation component is coupled
to the exclusionary component, it is only capable or more capable
of accessing molecules of interest that are exposed upon or
proximal to the upper surface of the substrate incorporating the
ZMW(s), and are thus accessible to the deactivation component, and
not those molecules that are well within the structures.
[0136] In accordance with this aspect of the invention, the
deactivation component might include digestive molecules, e.g.,
proteases, such as serine proteases, i.e. proteinase K, subtilisin,
and the like, threonine proteases, aspartic acid proteases,
cysteine proteases, metalloproteases, and glutamic acid proteases,
e.g., for digestion, cleaving or release of protein or peptide
based molecules of interest or linking components in either
non-specific or specific fashion, e.g., using a target protease to
cleave a particular linking molecule, e.g., a biotin.
Alternatively, such deactivation components might include
carbohydrate digesting enzymes (also termed carbohydrases), such as
cellulases and amylases, or nucleases, such as exo- or
endonucleases, etc., for the digestion or cleaving of carbohydrate
or nucleic acid based linking molecules or the molecules of
interest. This aspect of the invention is schematically illustrated
in FIG. 9.
[0137] As shown, an array 900 of confining structures, e.g., ZMWs
902, is provided with molecules of interest 904 randomly deposited
over its entire surface, e.g., including the surface of cladding
layer 908 and substrate layer 915 (Step I). Large particles, such
as beads 912, having deactivation components immobilized upon their
surface (or components that otherwise deactivate, cleave or release
the molecules of interest), are then contacted with the array 900.
Because beads 912 are larger than the openings to the waveguides
902, the deactivation components immobilized on the beads are only
capable of accessing and inactivating, digesting, cleaving or
releasing molecules of interest that are deposited on surfaces
outside the structures 902 or that are sufficiently proximal to the
opening of such structures as to be accessible by the immobilized
components on the beads 912. As a result, molecules upon or near
the surface outside of the ZMW structures are removed or otherwise
deactivated, leaving only those molecules that are well within the
constrained or exclusionary space of the waveguide (Step III). This
aspect of the invention is also further illustrated, below.
[0138] In related aspects, the beads may be provided with a binding
or crosslinking component that binds or crosslinks with or to the
molecule of interest. Subsequently, the bead may be mechanically
removed from the surface taking at least a portion of the molecules
of interest with it.
[0139] A variety of different types of beads may be used, including
beads generally used in chemical and biochemical analyses, i.e.,
agarose, acrylic, silica, or polyacrylamide beads or the like, or
other chromatographic or enzyme immobilization media/matrices, such
as F7m or G3m matrices, available from MoBiTec, GmbH (Gottingen,
Germany), magnetic beads or other metallic beads. Similarly,
methods for linking the deactivation component to the beads may be
varied to achieve desired results. For example, linker groups
having varied lengths may be used to permit penetration of the
deactivation component partially into a ZMW or other constrained
space. Likewise, linker stiffness may be adjusted through the
chemical structure and/or crosslinking of the linkers to provide
greater or lesser ability for the deactivation component to enter
into a confined space such as a ZMW.
[0140] In an alternative approach to the use of beads, other
scaffold materials may be used to support the deactivation
component and provide that component with accessibility to the
upper surface of the overall substrate, and in some cases, a subset
of the surfaces within recesses on that surface, e.g., a waveguide
core. In particular, the scaffold component would result in the
deactivation component being not entirely excluded from a given
recess on the substrate surface, e.g., a zero mode waveguide core.
By way of example, the deactivation component may be provided
tethered or coupled to a scaffold or supporting molecule that is
either only partially excluded from the recess or is only excluded
when provided in certain orientations. For example, a rigid or
semi-rigid linear molecule, such as a double stranded nucleic acid
or other rigid or semi-rigid elongated polymer, may be used that
includes the deactivation component, e.g., a protease, coupled to
it at an intermediate position. The supporting molecule is provided
of sufficient length that it can only move into the recess if
oriented appropriately, e.g., lengthwise. As a result of entering
the recess lengthwise or being retained upon the upper surface,
only those molecules on the upper surface or within the recess but
within reach of the deactivation component will be deactivated. By
way of analogy, the supporting molecule and intermediate
deactivation component function as a chimney sweep to remove
molecules of interest from the upper surface of the substrate and a
certain distance within the recesses, as dictated, at least in
part, by the intermediate positioning of the deactivation component
on the supporting molecule.
[0141] In the case of a relatively typical zero mode waveguide
structure of approximately 100 nm in depth and 70 nm in diameter,
for example, a double-stranded DNA oligonucleotide 150 nm in length
could be used with the deactivation component, e.g., a protease or
the like, affixed to it. Positioning and coupling are accomplished
through covalent coupling chemistry to a nucleotide analog that has
been inserted in the oligonucleotide sequence at a selected
position a given distance from one or both ends. Because
double-stranded DNA is mechanically rigid, the center portion of
the oligonucleotide to which the deactivation component is affixed
is away from the end of the supporting molecule. Upon entry into a
waveguide core, only the end of the supporting double stranded DNA
molecule will be able to reach the bottom of the core, and thus the
deactivation component will be geometrically constrained away from
the bottom of the core, or other confined space. Thus, molecules of
interest that are on the top surface or on the side walls of (for
example) a ZMW would be removed, while a molecule of interest on or
near the floor of the ZMW, e.g., within the illumination volume,
would remain. Positional coupling of deactivation components to
double stranded nucleic acids may be carried out by a variety of
methods. For example, in the case of coupling proteins, such as
proteases or other enzymes, to nucleic acid supporting molecules, a
protease or other enzyme can be maleimide activated by conjugation
with a bifunctional crosslinker such as GMBS (available from
PIERCE). This maleimide-activated protein can be directly coupled
to a single strand or double strand of DNA possessing an internal
thiol modification (e.g., a THSS internally labeled molecule
available from, e.g., Operon, Inc.). The thiol modification is
capped via a disulfide which is removed during the conjugation by
TCEP (also available from PIERCE). Similarly, a nucleic acid with
an internal thiol can be conjugated with a heterobifunctional
crosslinker (e.g., MAL-NHS, maleimide-N-hydroxysuccinimide) and
then conjugated to a protease via an amine-NHS reaction. Similar
reactions can be employed to conjugate amino-modified DNA to a
protease with thiols available on or near its surface.
[0142] The foregoing process is schematically illustrated in FIG.
13. As shown, a ZMW device 1300 includes a core 1302 disposed
within a cladding layer 1304, again extending to an underlying
transparent substrate 1306. As shown in panel I, a number of active
molecules of interest, e.g., polymerase molecules 1308, are
adsorbed or otherwise coupled to the surface of the overall
substrate, including both within a desired illumination region (as
indicated by dashed line 1310), on upper surface 1312 and at the
upper wall surfaces of the core 1302. In the context of the
invention, and as shown in panel II, a deactivation component, such
as protease molecule 1314, is coupled at an intermediate position
to a rigid, linear or elongated supporting molecule, such as dsDNA
molecule 1316. Because of its size and structural rigidity, the
supporting molecule 1316 with associated deactivation component
1314 only penetrates the core 1302 of the waveguide structure 1300
in an end-on orientation, or it lays across the upper surface 1312
of the overall structure. As a result of this, only polymerases
that are disposed upon the upper surface or within reach of the
deactivation component that penetrates a partial distance into the
waveguide core will be potentially affected by the deactivation
component. As such, polymerase molecules that are disposed at or
near the bottom surface of the waveguide core, e.g., within the
illumination region, will be spared deactivation (Panel III). As
will be appreciated, the positioning of the deactivation component
and/or the rigidity of the supporting molecule may generally be
chosen to adjust the depth within a core structure at which
deactivation can occur.
[0143] As noted above, the deactivation component is optionally a
protease such as Proteinase K that nonspecifically digests the
active molecule or a coupling group etc., thereby removing it from
the surface of the substrate. In other embodiments, the
deactivation component is a site-specific protease (e.g.,
enterokinase, thrombin, TEV protease, or any of the variety of
other site-specific proteases available in the art). Use of a
site-specific protease can avoid autoproteolytic cleavage of the
protease from the exclusionary component, which would release
soluble active protease able to undesirably access the optimal
confined illumination volume of the structures.
[0144] An exemplary embodiment employing a site-specific protease
is schematically illustrated in FIGS. 14A-14B. As shown, a ZMW
device 1400 includes a core 1402 disposed within a cladding layer
1404, again extending to an underlying transparent layer 1406. In
this example, polymerase molecule 1408 is covalently linked to
biotin 1420 through peptide linker 1421, which includes a cleavage
recognition site for site-specific protease 1415. Binding of biotin
1420 to streptavidin 1409, which is in turn bound to biotin 1422
that is adsorbed or otherwise coupled to the surface of the
substrate, couples polymerase 1408 to the surface. As shown in FIG.
14A, a number of active molecules of interest, e.g., polymerase
molecules 1408, are coupled to the surface of the overall
substrate, including both within a desired illumination region (as
indicated by dashed line 1410) and at the upper wall surfaces of
the core 1402 (and optionally also on upper surface 1412). As shown
in FIG. 14B, cleavage of linker 1421 by protease 1415 releases
polymerase 1408 from the surface. The site-specific protease
molecule 1415 is coupled at an intermediate position to a rigid,
linear or elongated supporting molecule, such as dsDNA molecule
1416. As for the embodiments described above, because of its size
and structural rigidity, the exclusionary component 1416 with
associated protease 1415 only penetrates the core 1402 of the
waveguide structure 1400 in an end-on orientation, or it lies
across the upper surface 1412 of the overall structure. As a result
of this, only polymerases that are disposed upon the upper surface
or within reach of the deactivation component that penetrates a
partial distance into the waveguide core are potentially affected
by the deactivation component. As such, polymerase molecules that
are disposed at or near the bottom surface of the waveguide core,
e.g., within the illumination region, will remain attached to the
surface since their linkers are inaccessible to the protease and
are not cleaved.
[0145] Another exemplary embodiment employing a site-specific
protease is schematically illustrated in FIGS. 15A-15C. As shown,
ZMW device 1500 includes core 1502 disposed within cladding layer
1504 that extends to underlying transparent layer 1506. In this
example, as illustrated in FIG. 15A, biotin coupling group 1522 is
coupled to the surface of the overall substrate via peptide linker
1521, which includes a cleavage recognition site for site-specific
protease 1515. Cleavage of linker 1521 by protease 1515 releases
biotin 1522 from the surface. Since protease 1515 is coupled to
exclusionary component double-stranded DNA 1516, as shown in FIG.
15B the protease removes biotin 1522 from the surface everywhere
except the lowest portion of core 1502. As shown in FIG. 15C,
streptavidin 1509 (or neutravidin etc.) and polymerase 1508 coupled
to biotin 1520 are then deposited on the substrate and are retained
by binding to biotin 1522 only in optimal confined illumination
volume 1510.
[0146] Another alternative subtractive method for the selective
localization of molecules of interest involves the use of that
molecule's own activity against it within the undesired regions.
For example, in the case of immobilized nucleic acid polymerase
enzymes, it has been determined that such enzymes, when
incorporating fluorescently labeled nucleotides under excitation
illumination, can suffer from substantial inactivation as a result
of photodamage. In accordance with the subtractive aspects of the
present invention, by subjecting enzymes at the upper surface of a
waveguide substrate to prolonged illumination during nucleic acid
synthesis in the presence of fluorescently labeled nucleotides or
nucleotide analogs, one can effectively inactivate those molecules.
As with the activation/inactivation based additive approaches
described above, it will be appreciated that damaging illumination
would not penetrate to the bottom surface, or area of interest, of
the ZMW, and thus, such enzymes present at these locations would
remain active. Fluorophore mediated inactivation of polymerases is
discussed at length in commonly assigned U.S. patent application
Ser. No. 11/293,040, filed Dec. 2, 2005, and incorporated herein in
its entirety for all purposes. Other enzyme/fluorescent substrate
pairs would be expected to yield similar characteristics, e.g., ATP
binding proteins/fluorescently labeled ATP. Additionally, other
components may be employed that generate radicals upon irradiation,
that will damage those molecules that are within diffusive contact.
By illuminating the upper surface of a waveguide structure in the
presence of such compounds, for example, one could generate oxygen
or other free radicals, that will deactivate molecules of interest
within diffusive reach of such compounds. A variety of such
compounds are known in the art and include, e.g., methylene blue,
hypocrellin A, hypocrellin B, hypericin, Rose Bengal Diacetate,
Merocyanine 540, and other dyes available from, e.g.,
Invitrogen/Molecular Probes (Eugene, Oreg.).
[0147] In another aspect of the invention, the structural
characteristics of a substrate may be actively employed in
subtractively selecting molecules of interest. In particular,
substrates including optical confinements, such as ZMWs, typically
include a metal layer deposited upon a transparent layer, e.g.,
glass or quartz, through which the waveguides are disposed,
exposing the transparent substrate at the bottom surface of the
waveguide. In accordance with the invention, an overall substrate
that includes molecules of interest both coupled to the metal layer
and the glass layer may be selectively partitioned, e.g., removing
molecules of interest from the metal surfaces, by applying an
electrical potential between the metal layer and the solution
deposited over it, e.g., through the use of an electrode in contact
with such fluid. Because the underlying substrate is not
electrically conductive, the field between the surface of the
substrate and the fluid will be substantially less than that
between the metal layer and the fluid. The electrical potential may
then be employed to selectively drive the molecules of interest
from the metal surface and into solution (see FIG. 10). This
driving force may be selected and/or controlled to result in
electrophoretic forces, e.g., driving charged molecules of interest
away from the surface in the non-desired surface regions or driving
capping groups toward such surfaces, or alternatively or
additionally, changes in the local environment at the metal
surface, e.g., pH changes resulting from the generation of protons
at the metal surface, that result in release from the surface,
e.g., through the use of acid labile linkers, charge based
linkages, e.g., hydrogen bonding, hydrolytic degradation of
molecules of interest on the metal surfaces through the generation
of locally harsh environments, or the like.
[0148] In another aspect, electrochemically releasable linker
compounds may be employed to release molecules of interest from
electrically active surfaces. By way of example, linking molecules
that include electrochemically controllable coupling may be
patterned upon the overall surface of a hybrid (metal/insulator)
substrate. Applying a current through the metal portion of the
surface results in release of the coupled molecule. Examples of
such electrically switchable linkers include self assembled
monolayers of biotin linked to quinone propionic ester bearing
linker compounds, i.e., alkanethiolates on gold surfaces.
Application of a potential to the underlying metal substrate
results in reduction of the quinine to hydroquinone that rapidly
undergoes lactonization with the release of the tethered molecule,
e.g., biotin (See, e.g., Hodneland, et al., J. Am. Chem. Soc. 2000,
122:4235-4236).
[0149] In addition to the use of such methods in optical
confinements, it will be appreciated that such electrophoretic
and/or electrochemical selection and immobilization processes may
be similarly applied to other hybrid analytical substrate types,
including, e.g., metal or semiconductor based sensors that rely on
surface associated molecules of interest, e.g., ChemFETS (chemical
field effect transistors), and the like. In particular, the metal
or semiconductor sensor element may be employed as one electrode in
the repulsion or attraction of different groups from or to the
surface of the sensor to enhance coupling.
[0150] Other subtractive processes may employ lift-off methods
where an otherwise active surface is coated with a lift-off layer
that entrains the molecules of interest on the upper surface of the
substrate, and in some cases penetrating a certain distance into a
ZMW. Lifting off of the layer brings the entrained molecules of
interest with it, allowing those not entrained, e.g., those at the
bottom surface of the ZMW, to remain.
[0151] This technique is schematically illustrated in FIG. 11. As
shown, a uniform or random distribution of molecules of interest
1104 is deposited over a substrate 1100 that includes selected
regions where such molecules are desired (Step I). In the case of
FIG. 11, such areas include optical confinements like ZMWs 1102. A
coating layer 1106 is then deposited over the surface as a viscous
liquid, e.g., having a viscosity of 1 or greater (Step II). Because
of its relative viscosity and the relatively small dimensions of
the waveguides 1102, and/or the material's relatively slow
diffusion in a liquid material present in the waveguide core, the
coating layer 1106 will typically not flow completely into the
waveguide structure. The coating layer is then typically allowed to
cure, e.g., through air drying, heating or exposure to UV
radiation, chemical crosslinking, entraining molecules of interest
within the coating layer, e.g., molecules of interest 1108. Upon
removal, any molecules of interest entrained in the coating layer
are removed as well, leaving only those molecules of interest that
were well within the waveguide structure, e.g., molecules 1110
(Step III). Although the above described method relies upon the
limited ability of the coating layer to penetrate the waveguide
structure to leave molecules of interest within such structures, it
will be appreciated that such methods may be applied in the absence
of such constrained structures. For example, the coating layer may
be selectively patterned upon the surface, e.g., through screening
or ink jet printing methods, to entrain and remove molecules of
interest from selected regions.
[0152] Another subtractive, selective immobilization process relies
generally upon masking strategies to ensure localization of the
molecule of interest where desired. In particular, such masking
strategies typically utilize a masking layer that may be either
removed to eliminate molecules of interest from undesired
locations, or which is deposited over a uniformly distributed
population of the molecules of interest to render those in
undesired locations inaccessible to the desired operation.
[0153] Other simpler brute force techniques are also within certain
aspects of the invention, particularly related to subtractive
processes. For example, one may use simple ablative processes to
remove coupling groups from exposed surfaces, e.g., surfaces upon
or near the upper surfaces of waveguide array substrates. Removal
of such groups would be expected to reduce the amount of molecules
of interest that are bound to surfaces outside of the waveguide
structure. Such ablative processes include, e.g., laser ablation
techniques, high sheer fluid ablation techniques, mechanical
abrasion techniques, and the like that will remove materials upon
contact or exposure. By directing such ablative processes at the
upper surfaces, it is expected that little or none of the ablative
forces will propagate into waveguide structures. Additional
adjustments may be made to further enhance the selectivity of the
process. For example, using laser ablation techniques, one could
direct the beam at an oblique angle to the upper surface of the
substrate, thereby penetrating only a minimal distance into high
aspect ratio recesses, e.g., ZMWs. Likewise, ablation energy could
be modulated to focus on regions that did not include the regions
where eventual coupling of molecules of interest is desired, e.g.,
focused upon substrate surface regions or spaces between ZMWs in an
array.
[0154] Once the coupling groups have been provided upon the surface
of the substrate, e.g., in the desired regions such as at the
bottom surface of a ZMW, the molecules of interest are then coupled
to those active groups. As noted elsewhere herein, coupling may be
via functional chemical groups, e.g., hydroxyl groups, amino
groups, epoxy groups or the like. Alternatively, coupling may occur
through specific binding partners, e.g., where one member of a
specific binding pair is the coupling group attached to the surface
(or is attached to a coupling group that is attached to the
surface), and the other member of the binding pair is attached to
or is integral with the molecule of interest. In particularly
preferred aspects, such specific binding pairs are used to couple
the molecule of interest to the surface, including, e.g., the use
of avidin, streptavidin or neutravidin as one member of the binding
pair, and biotin as the other member. Additionally, sandwich
binding strategies may be employed, e.g., coupling biotin to the
surface in the area of interest, followed by linkage to avidin,
which is in turn, linked to a biotin molecule coupled to the
molecule of interest. Typically, a linker silane group is used as
the initial functional group. This group may be provided directly
upon the surface or, as alluded to previously, diluted with similar
linker silanes that are inert to additional coupling. In
particularly preferred aspects, a linker silane bearing, e.g., a
biotin group is immobilized in the initial step, followed by
coupling of a molecule of interest, e g., a polymerase enzyme,
through a bridging avidin group coupled with an enzyme linked
biotin group. As will be appreciated any of a variety of different
configurations may be practiced within the context of the
invention.
[0155] In the case of molecules of interest that are enzymes or
otherwise active proteins, the orientation of immobilization may be
an important characteristic to optimizing activity of the enzyme.
For example, in the case of DNA polymerases, random adsorption of
polymerases to a surface can yield substantially less than 100%
activity at least partially as a result of some molecules being
oriented in a way so as to prevent them from exhibiting optimal
activity. As such, it may be desirable to provide for a specific
orientation of the molecule by providing an anchoring group or
groups on the molecule to increase the probability of correct
orientation. Such methods have been previously described in
commonly owned U.S. Patent Application No. 60/753,446, filed Dec.
22, 2005, and incorporated herein by reference in its entirety for
all purposes. Alternatively, one may provide the enzyme with a
substrate molecule or substrate proxy that can prevent surface
adsorption in a manner that blocks the active site of the enzyme.
By way of example, it has been determined that immobilization of
nucleic acid polymerase enzymes, such as DNA polymerases, in the
presence of template nucleic acid molecules yields substantially
higher activity of surface immobilized polymerases. Without being
bound to a particular theory of operation, it is believed that the
presence of the template molecule within the active site of the
polymerase prevents immobilization of the polymerase in a manner
that interferes with the active site, due to steric or other
interference from the associated template. While template nucleic
acid molecules can be used, other template-like molecules may also
be used, including, e.g., LNA polymer strands, PNA polymers, or
other nucleic acid analogs.
IV. Examples
Example 1: Photoactivatable Groups for Selective Immobilization of
DNA Polymerases
[0156] A substrate may be used that includes a glass substrate
layer with an aluminum cladding layer deposited over the glass
layer. An array of ZMW cores is fabricated into the cladding layer
to provide apertures through the cladding layer to the glass
substrate. The overall substrate is optionally further treated to
provide a thin insulating layer over the cladding layer and cores,
e.g., to provide a substantially uniform surface. Such layers
typically include SiO.sub.2 coatings applied by vapor deposition
techniques, including, e.g., CVD and MVD methods, as well as other
methods such as fluid deposition or in situ formation using, e.g.,
spin on glass systems. The substrate surface is derivatized to
first provide a relatively uniform population of amino terminated
groups coupled to the surface. For example, for glass surfaces,
such derivatization typically employs standard aminosilane
chemistries known in the art. Alternatively, amine groups may be
provided upon a linker molecule that is coupled to the surface
through existing hydroxyl groups or surfaces otherwise derivatized.
Such coupling groups may be provided at limited densities in order
to further control the density of the molecules of interest that
will ultimately be bound to the surface (see, e.g., commonly
assigned U.S. patent application Ser. No. 11/240,662, filed Sep.
30, 2005, incorporated herein by reference in its entirety for all
purposes).
[0157] Biotin molecules capped with an appropriate photolabile
protecting group, such as MeNPOC, are then coupled to the
derivatized surface using known chemistries, e.g., through an
included epoxy group on the biotin molecule.
[0158] Following washing of the surface, appropriate illumination
radiation is directed at the substrate through the transparent
glass substrate layer, illuminating and deprotecting only the
biotin groups at or near the bottom surface of the ZMW. DNA
polymerase enzyme linked to avidin, streptavidin or neutravidin is
then contacted with the substrate and selectively binds with the
exposed biotin at the bottom of the waveguides.
[0159] In a second exemplary process, a photoactivatable acid
group, e.g., surface coupled .alpha.-methylphenacyl ester, is
coupled to the surface in the same fashion provided above.
Illumination, e.g., at 313 nm, through the ZMW yields the acid
groups at the bottom surface of the waveguides, which are then
contacted with amino biotin groups followed by coupling to avidin
linked polymerase enzymes, to yield enzyme groups only at or near
the bottom surface of the waveguide.
Example 2: Selective Digestion of DNA Polymerase Enzymes Using Bead
Bound Proteases
[0160] ZMWs that had previously been plasma treated in the presence
of a PDMS gasket (to provide a priming layer), were provided with
.PHI.29.sup.N62D DNA Polymerase (complexed with a circular template
nucleic acid) substantially uniformly surface adsorbed over the
entire surface of the array, including upon the upper surface of
the cladding layer.
[0161] The array was then contacted with beads bearing immobilized
Proteinase-K (Sigma Chemical Co., P0803 or P9290) for 5 minutes at
room temperature in 25 mM Tris-HCl, pH 7.5, 10 mM
.beta.-mercaptoethanol, 1 mM EDTA. The bead diameter far exceeded
the nominal diameter of the waveguide cores on the array,
preventing entry to the bead or its associated protease molecules
into the cores to any substantial degree.
[0162] Polymerization reaction mixture including four dNTPs was
then contacted with the array under conditions suitable for DNA
synthesis (50 mM Tris-HCl, pH 7.5, 75 mM KCl, 20 mM
(NH.sub.4).sub.2SO.sub.4, 10 mM .beta.-mercaptoethanol, 0.7 mM
MnCl.sub.2), and synthesis was allowed to proceed for 30 minutes at
30.degree. C.
[0163] Following synthesis any synthesized DNA on the array was
stained with SybrGold stain. The array was then imaged using a
standard fluorescence microscope. The array images, as well as
images of the negative control experiment, are shown in FIG. 12. As
shown in the negative control (Row I), bottom side illumination
(Column A) shows the presence of a significant amount of DNA within
the waveguide structures, while top side illumination and
observation (Column B) shows a uniform layer of DNA produced over
the entire surface of the array. In the proteinase treated array
(Row II), both the bottom side (Column A) and top side (Column B)
show a similar pattern of DNA presence within specific waveguides.
Further, as can be seen, there is little DNA present upon the upper
surface other than within waveguides in the array, showing a
substantial reduction from the high level of DNA synthesis present
in the control experiment. Also of note is that the waveguides
showing DNA presence from the upper surface track to the same
waveguides showing DNA presence from the lower surface, indicating
that DNA synthesis is occurring within the waveguide structure, and
not outside the waveguide core. This also indicates that DNA being
synthesized within the waveguide structure is of substantial
length, e.g., greater than 500 bases, potentially up to 1000 or
more bases in length, as it spans the illumination regions at the
top and bottom portions of a waveguide structure having a core
region of approximately 70 nm in diameter and 100 nm deep.
[0164] DNA synthesis experiments were also carried out in the
presence of labeled nucleoside polyphosphate analogs, labeled at
the terminal phosphate group (see, e.g., Published U.S. Patent
Application No. 2003-0044781 and Levene, et al., Science (2003)
299:609-764, the full disclosures of which are incorporated herein
in their entirety for all purposes). These assays indicated
substantially better signal to noise ratios than waveguide arrays
that were not proteinase treated, showing markedly less
interference from other noise sources, e.g., labeled by products of
the polymerase reaction. As a result, it appears clear that
provision of molecules of interest such as polymerase enzymes only
within a desired region of an analytical substrate, i.e., an
observation region, can have profoundly beneficial results on the
application to which the substrate is to be put.
Example 3: Selective Immobilization of DNA Polymerases by
Differential Modification of Surfaces
[0165] The following sets forth a series of experiments that
demonstrate selective immobilization of a DNA polymerase on the
bottom surface of ZMWs and passivation of the remaining ZMW
surfaces with a polyelectrolyte multilayer. The process, which
exploits the differential reactivity of silanes with glass and
aluminum oxide, is schematically illustrated in FIG. 18. PEG-biotin
silanization is specific to glass under the conditions employed,
thereby resulting in chemical derivatization of only the ZMW bottom
surface. The aluminum layer is then passivated using a
polyelectrolyte multilayer, in this example, a 2.5.times.
multilayer of PAA/PEI/PAA/PEI/PAA (where PAA is poly(acrylic acid)
and PEI is poly(ethyleneimine)). Biotin tagged polymerase is
rejected by the polyelectrolyte multilayer but binds to the
biotinylated PEG surface via avidin chemistry, thereby resulting in
biased immobilization of the polymerase to the bottom surface of
the ZMW. In addition, the polyelectrolyte multilayer limits
nonspecific binding of nucleotide analogs to the aluminum
layer.
[0166] Biased immobilization of polymerase on the bottom surface of
ZMWs was accomplished as follows. ZMW chips are cleaned in an
oxygen plasma for 2 minutes at 2 torr (medium power setting). The
PEG-Biotin silanization is carried out for 3 hours at 4.degree. C.
using a mixture of PEG methoxy silane and Biotin-PEG silane
(Polymer Source Inc.) in 270:1 (w/w) ethanol:methanol solvent. The
samples are rinsed with methanol, sonicated for 3 minutes in hot
(70.degree. C.) water, and washed with cold water. The
polyelectrolyte procedure consists of consecutive immersion of the
chips for 5 minutes at room temperature in 20 mg/ml Polyacrylic
acid and Polyethylenimine (Sigma-Aldrich, pH 7.5 adjusted with
HCl), each step followed by 3.times. rinsing with water, in the
order: PAA/PEI/PAA/PEI/PAA. The last wash is with 5 volume
equivalents of water.
[0167] Nonspecific binding of four nucleotide analogs to the biased
immobilization surface (ZMW chip treated with the mixture of
PEG-silanes followed by polyelectrolyte multilayer formation) and
to a control surface (a plasma-PDMS treated chip) was compared
(FIG. 19). The plasma-PDMS treatment used on the control chip
removes bias because it coats the entire structure with a uniform
layer; see International Application Number PCT/US 2006/045,429
filed Nov. 27, 2006. Chips were incubated with a mixture of
fluorescently labeled nucleotide analogs (A488-dA4P, FAM-A532-dG4P,
FAM-A594-dT4P, A633-dC4P, 5 .mu.M each; see, e.g., U.S. patent
application Ser. No. 11/645,223 for analog nomenclature), and
subjected to laser illumination. Movies were acquired for 1 minute
at 100 fps camera speed. Fluorescence traces were analyzed by a
custom-build analysis software, using a threshold algorithm to
determine the number of non-specific adsorption events shown in the
graph for each, spectrally separated analog. As shown in FIG. 19,
the biased immobilization surface is as good at preventing
nonspecific analog binding as is the plasma-PDMS surface (which
exhibits good nonspecific binding characteristics). Analog binding
to an untreated surface was not quantified, since the analogs bind
the untreated surface to such an extent that single pulses cannot
be identified.
[0168] Polyelectrolyte multilayer deposition on an aluminum surface
inhibits nonspecific binding of polymerase, as illustrated in FIGS.
20A-20B. Essentially no DNA synthesis is observed on the aluminum
surface treated with a 2.5.times. PAA/PEI/PAA/PEI/PAA
polyelectrolyte multilayer (FIG. 20A), while DNA is produced over
the entire surface of a control surface not treated with the
polyelectrolyte multilayer (FIG. 20B). Polymerization reactions
were carried out as follows: 100 nM polymerase was bound to
Neutravidin (present in excess at 150 nM) in a BF-300 buffer
containing 25 mM Tris-acetate, pH 7.5, 300 mM potassium acetate,
0.05% Tween 20 and 5 mM dithiothreitol for 30 minutes at 4.degree.
C. The solution was diluted to an effective potassium acetate
concentration of 150 mM by 2-fold dilution with the same buffer as
above but lacking potassium acetate (BF-0). The
polymerase/Neutravidin mixture was incubated for 30 minutes at
4.degree. C. on the ZMW chip, and washed 3.times. with BF-150
buffer (the same buffer as BF-300 but including 150 mM potassium
acetate). Template at 100 nM was added for 20 minutes at 4.degree.
C., in reaction buffer (50 mM Tris acetate, pH 7.5, 75 mM potassium
acetate, 20 mM ammonium sulfate, 0.05% Tween 20 and 5 mM
dithiothreitol) supplemented with 4 mM EDTA. Template solution was
removed and the extension reaction mixture was added, containing
0.7 mM MnCl.sub.2, 10 .mu.M of each dATP, Alexa Fluor ChromaTide
488-dCTP (Invitrogen), dGTP and dTTP in reaction buffer. DNA
synthesis proceeded for 10 minutes at room temperature, followed by
5.times. washing with BF-150 supplemented with 1 mM EDTA.
ChromaTide nucleotide incorporation into DNA was visualized on a
wide-field fluorescence microscope (Olympus), using a
60.times.0.9NA physiology objective lens to image the top
(solution) side of the ZMW chips, and a 60.times.1.2NA objective
lens for the bottom side.
[0169] The biased immobilization procedure (treatment of the ZMW
chip with the mixture of PEG-silanes followed by polyelectrolyte
multilayer formation) results in selective immobilization of the
polymerase within the waveguides. Polymerization reactions were
carried out as described in the preceding paragraph on a biased
immobilization ZMW chip and on a control ZMW chip (uniformly
coated, with a plasma-PDMS layer underneath followed by
PEG-methoxy/Biotin-PEG silane derivatization). Images of the biased
immobilization ZMW array, as well as images of the control array,
are shown in FIG. 21. As shown in the control (Column II), bottom
side illumination (Row B) shows the presence of a significant
amount of DNA within the waveguide structures, while top side
illumination (Row A) shows a uniform layer of DNA produced over the
entire surface of the array. For the biased immobilization ZMW
array (Column I), in contrast, both the bottom side (Row B) and top
side (Row A) show a similar pattern of DNA presence within specific
waveguides. Further, as can be seen, there is little DNA present
upon the upper surface other than within waveguides in the array,
showing a substantial reduction from the high level of DNA
synthesis present in the control experiment. Also of note is that
the waveguides showing DNA presence from the upper surface track to
the same waveguides showing DNA presence from the lower surface,
indicating that DNA synthesis is occurring within the waveguide
structure, and not outside the waveguide core; see FIG. 22, in
which an image of the top surface of a biased immobilization ZMW
array (Panel I) and an image at the bottom surface of the same
array (Panel II) are overlaid (Panel III).
[0170] These results indicate that the polyelectrolyte multilayer
is relatively non-sticky to nucleotide analogs and that the
polyelectrolyte multilayer passivates well against polymerase
binding to aluminum surfaces. Differential PEG-biotin-silane
chemistry, followed by polyelectrolyte multilayer passivation,
yields biased immobilization of the polymerase with high
contrast.
Example 4: Selective Immobilization and Passivation Using a
Phosphonic Acid
[0171] Deposition of polyvinylphosphonic acid (PVPA) onto untreated
ZMWs results in a ZMW that is passivated from nonspecific protein
(e.g., neutravidin and polymerase) and nucleotide analog binding to
the aluminum surface. PVPA is specific to aluminum and does not
affect the SiO.sub.2 bottom surface of the ZMW, which can be used
for nonspecific capture agent or polymerization immobilization or
subsequent derivatization (e.g., by silanization or binding of
compounds such as PLL-PEG) for specific polymerase deposition.
[0172] Treatment of a mixed material substrate (e.g., 100 nm
aluminum film on glass) with PVPA results in immobilization of a
neutravidin capture agent preferentially on the SiO.sub.2, rather
than the aluminum, portion of the substrate, as illustrated in FIG.
23. On a substrate not treated with PVPA, more neutravidin is
deposited on the aluminum portion of the substrate (Panel I Row A)
than on the SiO.sub.2 portion (Panel I Row B). On a PVPA-treated
substrate, in contrast, neutravidin is immobilized preferentially
on the SiO.sub.2 portion of the substrate (Panel II Row B), while
little neutravidin sticks to the aluminum portion of the substrate
(Panel II Row A).
[0173] To assess neutravidin binding, chips are cleaned from a
protective photoresist layer by first rinsing them in acetone,
followed by rinsing in isopropanol and drying with a stream of
nitrogen. They are cleaned in a plasma cleaner (Harrick) for 2
minutes at 2 torr (medium power setting). PVPA treatment proceeds
on a heat block set to 90.degree. C., the chips are put on the heat
block, and 90.degree. C. PVPA solution (molecular weight 24,000,
from Polysciences Inc. (Warrington, Pa.), 25% stock diluted to 2%
working solution concentration in water) is put on the chip for 2
minutes, followed by rinsing with water. Excess water is blown away
by a stream of nitrogen, followed by heat treatment for 10 minutes
at 80.degree. C. in a dry oven. 40 nm A488-Neutravidin latex beads
(Invitrogen) are diluted to 0.01% in buffer (50 mM MOPS-acetate, pH
7.5, 75 mM potassium acetate, 5 mM DTT) and incubated with the
chips for 15 minutes at room temperature. The chips are rinsed with
water and imaged on a wide-field fluorescence microscope, using a
60.times.0.9 NA physiology objective lens (Olympus).
[0174] PVPA treatment reduces nucleotide analog binding, as
illustrated in FIG. 24. ZMW chips were treated with PVPA, and
nonspecific binding of nucleotide analogs to the chips was analyzed
as described above in Example 3. As shown in FIG. 24, the analogs
exhibit considerable nonspecific binding to an untreated ZMW (Panel
II), while little analog binding to a PVPA-treated ZMW is observed
(Panel I).
[0175] Although described in some detail for purposes of
illustration, it will be readily appreciated that a number of
variations known or appreciated by those of skill in the art may be
practiced within the scope of present invention. Unless otherwise
clear from the context or expressly stated, any concentration
values provided herein are generally given in terms of admixture
values or percentages without regard to any conversion that occurs
upon or following addition of the particular component of the
mixture. To the extent not already expressly incorporated herein,
all published references and patent documents referred to in this
disclosure are incorporated herein by reference in their entirety
for all purposes.
* * * * *