U.S. patent application number 13/930358 was filed with the patent office on 2013-11-07 for selectively functionalized arrays.
The applicant listed for this patent is Pacific Biosciences of California, Inc.. Invention is credited to Ronald Cicero, Stephen Dudek, Jeremy Gray, Gregory Kearns, Natasha Popovich, Robert Sebra.
Application Number | 20130296195 13/930358 |
Document ID | / |
Family ID | 48876322 |
Filed Date | 2013-11-07 |
United States Patent
Application |
20130296195 |
Kind Code |
A1 |
Gray; Jeremy ; et
al. |
November 7, 2013 |
Selectively Functionalized Arrays
Abstract
Methods, substrates, and devices related to arrays of optical
confinements having surfaces with high levels of bias. Substrates
having transparent or silica based portions and opaque or
reflective portions are treated with 1) a selective passivating
agent, that selectively coats the opaque or reflective regions, 2)
a functionalizing agent such as a coupling agent, and 3) a
selective removal agent, which selectively removes functionalizing
agent from the passivated opaque or reflective surfaces.
Inventors: |
Gray; Jeremy; (San
Francisco, CA) ; Cicero; Ronald; (Palo Alto, CA)
; Kearns; Gregory; (San Mateo, CA) ; Dudek;
Stephen; (San Francisco, CA) ; Popovich; Natasha;
(Menlo Park, CA) ; Sebra; Robert; (San Francisco,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pacific Biosciences of California, Inc. |
Menlo Park |
CA |
US |
|
|
Family ID: |
48876322 |
Appl. No.: |
13/930358 |
Filed: |
June 28, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12797260 |
Jun 9, 2010 |
8501406 |
|
|
13930358 |
|
|
|
|
61225507 |
Jul 14, 2009 |
|
|
|
Current U.S.
Class: |
506/18 |
Current CPC
Class: |
B01J 2219/00443
20130101; B01J 2219/00626 20130101; B01J 2219/00317 20130101; B01J
19/0046 20130101; B01J 2219/00637 20130101; B01J 2219/00612
20130101; C12Q 1/6869 20130101; B01J 2219/00617 20130101; B01J
2219/00621 20130101; B01J 2219/00722 20130101; B01J 2219/00596
20130101 |
Class at
Publication: |
506/18 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. An apparatus for obtaining sequence information from a template
nucleic acid comprising: a) a substrate comprising an array of
zero-mode waveguides wherein the zero-mode waveguides comprise
apertures through an upper metal or metal oxide layer disposed on
top of a lower silica-based layer, wherein each zero-mode waveguide
is capable of holding a single molecule of a polymerase enzyme, and
wherein a surface of the substrate is selectively functionalized
by: i) exposing the surface of the substrate to an agent that
preferentially binds to the metal or metal oxide portions of the
surface to produce passivated metal or metal oxide portions of the
surface; ii) exposing the surface of the substrate to a silica
functionalizing agent that binds to both the silica-based portions
and the passivated metal or metal oxide portions of the surface,
then rinsing the surface of the substrate; and iii) after step ii),
exposing the silica-based portions and the passivated metal or
metal oxide portions of the surface of the substrate to a selective
removal compound that preferentially removes the silica
functionalizing agent from the passivated metal or metal oxide
portions of the surface; b) a receptacle for keeping sequencing
reagents in contact with the substrate; c) an optical system
optically coupled to the substrate for delivering light to the
zero-mode waveguides and for measuring light from the zero-mode
waveguides over a period of time, wherein the measured light from
the zero-mode waveguides over a period of time can be used to
obtain sequence information about the template DNA; and d) a
computational system connected to the optical system for
determining sequence information using the measured light over a
period of time.
2. The apparatus of claim 1 wherein the agent that preferentially
binds to the metal or metal oxide portions comprises phosphate or
phosphonate functionality.
3. The apparatus of claim 1 wherein the silica functionalizing
agent comprises a silane coupling agent.
4. The apparatus of claim 3 wherein a single molecule of a
polymerase enzyme is attached to the silane coupling agent in each
zero-mode waveguide.
5. The apparatus of claim 1 wherein the selective removal compound
comprises an acidic compound.
6. The apparatus of claim 1 wherein the selective removal compound
comprises a compound having a pK.sub.a of less than 6.
7. The apparatus of claim 1 wherein the selective removal compound
comprises a polymer.
8. The apparatus of claim 7 wherein the polymer comprises
carboxylate, sulfonate, sulfate, phosphonate or phosphate
functionality.
9. The apparatus of claim 7 wherein the polymer comprises a
homopolymer or copolymer of one or more of the monomers
vinyl(acrylic acid), vinyl(sulfonic acid), vinyl(phosphonic acid),
vinyl(styrenesulfonic acid), maleic acid, or salts thereof.
10. The apparatus of claim 7 wherein the polymer comprises
poly(vinylsulfonic acid) or ##STR00016## where m is about 24 and n
is about 16.
11. A substrate comprising an array of optical confinements,
wherein the substrate has both silica-based portions and metal or
metal oxide portions, and wherein the substrate is selectively
functionalized by: i) exposing a surface of the substrate to an
agent that preferentially binds to the metal or metal oxide
portions to produce passivated metal or metal oxide portions of the
surface; ii) exposing the surface of the substrate to a silica
functionalizing agent that binds to both the silica-based portions
and the passivated metal or metal oxide portions of the surface,
then rinsing the surface of the substrate; and iii) after step ii),
exposing the silica-based portions and the passivated metal or
metal oxide portions of the surface of the substrate to a selective
removal compound that preferentially removes the silica
functionalizing agent from the passivated metal or metal oxide
portions of the surface.
12. The substrate of claim 11 wherein the array of optical
confinements comprises an array of zero-mode waveguides, and
wherein the silica-based portions of the substrate comprise bases
of apertures though a metal or metal oxide layer on a transparent
silica-based substrate.
13. The substrate of claim 11 wherein the agent that preferentially
binds to the metal or metal oxide portions comprises phosphate or
phosphonate functionality.
14. The substrate of claim 11 wherein the silica functionalizing
agent comprises a silane coupling agent.
15. The substrate of claim 11 wherein the selective removal
compound comprises an acidic compound.
16. The substrate of claim 11 wherein the selective removal
compound comprises a compound having a pK.sub.a of less than 6.
17. The substrate of claim 11 wherein the selective removal
compound comprises a polymer.
18. The substrate of claim 17 wherein the polymer comprises
carboxylate, sulfonate, sulfate, phosphonate or phosphate
functionality.
19. The substrate of claim 17 wherein the polymer comprises a
homopolymer or copolymer of one or more of the monomers
vinyl(acrylic acid), vinyl(sulfonic acid), vinyl(phosphonic acid),
vinyl(styrenesulfonic acid), maleic acid, or salts thereof.
20. The substrate of claim 17 wherein the polymer comprises
poly(vinylsulfonic acid) or ##STR00017## where m is about 24 and n
is about 16.
21. An apparatus for obtaining sequence information from a template
nucleic acid comprising: a) a substrate comprising an array of
zero-mode waveguides wherein the zero-mode waveguides comprise
apertures through an upper metal or metal oxide layer disposed on
top of a lower silica-based layer, wherein each zero-mode waveguide
is capable of holding a single molecule of a polymerase enzyme, and
wherein a surface of the substrate is selectively functionalized
by: i) treating the surface of the substrate with a compound
comprising phosphate or phosphonate groups to produce passivated
metal or metal oxide portions of the surface; ii) treating the
surface of the substrate with a silica functionalizing agent that
binds to both the silica-based portions and the passivated metal or
metal oxide portions of the surface, wherein the silica
functionalizing agent comprises a silane coupling agent; and iii)
treating the surface of the substrate with an acidic compound
having a pK.sub.a of less than 6 to remove silica functionalizing
agent from the passivated metal or metal oxide portions of the
surface; b) a receptacle for keeping sequencing reagents in contact
with the substrate; c) an optical system optically coupled to the
substrate for delivering light to the zero-mode waveguides and for
measuring light from the zero-mode waveguides over a period of
time, wherein the measured light from the zero-mode waveguides over
a period of time can be used to obtain sequence information about
the template DNA; and d) a computational system connected to the
optical system for determining sequence information using the
measured light over a period of time.
22. A substrate comprising an array of optical confinements,
wherein the substrate has both silica-based portions and metal or
metal oxide portions, and wherein the substrate is selectively
functionalized by: i) treating the surface of the substrate with a
compound comprising phosphate or phosphonate groups to produce
passivated metal or metal oxide portions of the surface; ii)
treating the surface of the substrate with a silica functionalizing
agent that binds to both the silica-based portions and the
passivated metal or metal oxide portions of the surface, wherein
the silica functionalizing agent comprises a silane coupling agent;
and iii) treating the surface of the substrate with an acidic
compound having a pK.sub.a of less than 6 to remove silica
functionalizing agent from the passivated metal or metal oxide
portions of the surface.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/797,260, filed Jun. 9, 2010, entitled
"SELECTIVELY FUNCTIONALIZED ARRAYS" by Jeremy Gray et al., which
claims the benefit of U.S. Provisional Application No. 61/225,507,
filed Jul. 14, 2009. The full disclosure of each of these
applications is incorporated herein by reference in its entirety
for all purposes.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] Not Applicable.
BACKGROUND OF THE INVENTION
[0003] There are a wide range of analytical operations that may
benefit from the ability to analyze the reaction of individual
molecules or a relatively small numbers of molecules. 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 copy number approaches to obtaining sequence
information in conjunction with the template dependent synthesis of
nucleic acids by the action of polymerase enzymes.
[0004] 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
levels of reactants. In other applications, certain reagents are
immobilized onto bead or planar surfaces such that they may be
monitored without interference from other reaction components in
solution. In still another approach, optical confinement techniques
have been used to ascertain signal information only from a
relatively small number of reactions, e.g., a single molecule,
within an optically confined area.
[0005] For arrays of optical confinements it can be desirable to
have different characteristic properties on various different
portions of the surfaces of the optical confinement structures. For
example, different surface properties for portions of the surfaces
within the observation regions and outside of the observation
regions of the optical confinements. Notwithstanding the
availability of the above-described techniques, there are instances
where greater 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] One aspect of the invention is method for selectively
functionalizing a surface of a substrate having both silica-based
portions and metal or metal oxide portions comprising; a) exposing
a surface of a substrate having both silica-based portions and
metal or metal oxide portions to an agent that preferentially binds
to the metal or metal oxide portions to produce a passivated metal
or metal oxide portions of the surface; b) exposing the surface of
the substrate to a silica functionalizing agent that binds to both
the silica-based portions and the metal or metal oxide portions of
the surface; and c) exposing the surface of the substrate to a
selective removal compound that preferentially removes the silica
functionalizing agent from the passivated metal or metal oxide
portions of the surface. In some embodiments the steps are carried
out in the order a), b), c). In some embodiments the steps are
carried out in the order b), c), a).
[0007] In some embodiments the agent that preferentially binds to
the metal or metal oxide portions comprises phosphate or
phosphonate functionality. In some embodiments the agent that
preferentially binds to the metal or metal oxide portions comprises
a polymer. In some embodiments the agent that preferentially binds
to the metal or metal oxide portions comprises a polymer having
poly(acrylate), poly(sulfonate), or both poly(acrylate) and
poly(sulfonate) portions. In some embodiments the agent that
preferentially binds to the metal or metal oxide portions comprises
polyvinyl phosphonic acid (PVPA) or phosphorous containing
polymeric materials ALBRITECT CP-30, ALBRITECT CP-10, ALBRITECT
CP-90, AQUARITE ESL, or AQUARITE EC4020.
[0008] In some embodiments the silica functionalizing agent
comprises a silane coupling agent. In some embodiments the silane
functionalizing agent comprises an aminosilane. In some embodiments
the silane functionalizing agent comprises a silane comprising a
biotin group. In some embodiments the silane functionalizing agent
comprises a compound having the structure silane-polyethylene
glycol-biotin. In some embodiments the silica functionalizing agent
used for exposing the surface in step (b) comprises a mixture of
silane functionalizing agents.
[0009] In some embodiments the selective removal compound comprises
an acidic compound. In some embodiments the selective removal
compound comprises a compound having a pKa of less than about 6. In
some embodiments the selective removal compound comprises a
polymer. In some embodiments the polymer comprises carboxylate,
sulfonate, sulfate, phosphonate or phosphate functionality. In some
embodiments the polymer comprises a homopolymer or copolymer of one
or more of the monomers vinyl(acrylic acid), vinyl(sulfonic acid),
vinyl(phosphonic acid), vinyl(styrenesulfonic acid), maleic acid,
or salts thereof. In some embodiments the polymer comprises
AQUARITE ESL (an acidic phosphonate containing polymer) or
poly(vinylsulfonic acid) (PVSA).
[0010] In some embodiments the silica-based portions comprise
optical confinement regions.
[0011] In some embodiments the substrate comprises an array of
optical confinement regions wherein the silica-based portions
comprise bases of apertures though a metal or metal oxide layer on
a transparent silica-based substrate.
[0012] An aspect of the invention is method for selectively
functionalizing a surface of a substrate having both silica-based
portions and metal or metal oxide portions comprising; a) treating
a surface of a substrate having both silica-based portions and
metal or metal oxide portions with a compound comprising phosphate
or phosphonate groups; b) treating the surface of the substrate
with a silica functionalizing agent that binds to both the
silica-based and metal or metal oxide portions of the surface; and
c) treating the surface of the substrate with an acidic compound to
remove silica functionalizing agent from the metal or metal oxide
portions of the surface.
[0013] One aspect of the invention is a method for attaching a
desired molecule to a silica-based portion of a surface of a
substrate having both silica-based portions and metal or metal
oxide portions comprising; a) exposing a surface of a substrate
having both silica-based portions and metal or metal oxide portions
to an agent that preferentially binds to the metal or metal oxide
portions to produce passivated metal or metal oxide portions of the
surface; b) exposing the surface of the substrate to a coupling
agent that binds to both the silica-based portions and the metal or
metal oxide portions of the surface; c) exposing the surface of the
substrate to a selective removal compound that preferentially
removes the coupling agent from the passivated metal or metal oxide
portions of the surface; and d) attaching the desired molecule to
the coupling agent bound to the silica-based portions of the
surface.
[0014] In some embodiments step d) of attaching the desired
molecule is carried out prior to step c) of exposing the surface of
the substrate to a selective removal compound.
[0015] In some embodiments the coupling agent comprises a silane
coupling agent. In some embodiments the silane coupling agent
comprises an amino silane or a thiol-silane. In some embodiments
the silane coupling agent comprises biotin. In some embodiments the
silane coupling agent comprises a molecule having the structure
silane-polyethylene glycol-biotin.
[0016] In some embodiments the attaching of the desired molecule to
the to the coupling agent of step (d) comprises process wherein the
coupling agent is reacted with an attaching agent, the attaching
agent having a group which reacts with the coupling agent and a
group for attaching the desired molecule. In some embodiments the
attaching agent comprises biotin.
[0017] In some embodiments the coupling agent exposed to the
surface in step (b) is mixed with a compound that reacts with the
silica-based portions of the surface, but does not have coupling
functionality.
[0018] In some embodiments the desired molecule comprises biotin
and is attached to the silica portions of the surface using a
protein having a high binding affinity for biotin. In some
embodiments the desired molecule comprises an enzyme. In some
embodiments the desired molecule comprises a polymerase enzyme.
[0019] In some embodiments the silica-based portions comprise
optical confinement regions. In some embodiments the substrate
comprises an array of optical confinement regions wherein the
silica-based portions comprise bases of apertures though a metal or
metal oxide layer on a transparent silica-based substrate. In some
embodiments the metal or metal oxide portions comprise
aluminum.
[0020] One aspect of the invention is a method for attaching a
desired molecule to a silica-based portion of a surface of a
substrate having both silica-based portions and metal or metal
oxide portions comprising; a) treating a surface of a substrate
having both silica-based portions and metal or metal oxide portions
with a compound comprising phosphate of phosphonate groups; b)
treating the surface of the substrate with a coupling agent that
binds to both the silica-based and metal or metal oxide portions of
the surface; c) treating the surface of the substrate with an
acidic compound to remove coupling agent from the metal or metal
oxide portions of the surface; and d) attaching the desired
molecule to the coupling agent bound to the silica-based portions
of the surface.
[0021] One aspect of the invention is an array of optical
confinements having a surface with both a silica-based and a metal
or metal-oxide portion that is prepared by the selective
functionalization methods of the invention.
[0022] One aspect of the invention is an array of optical
confinements having a surface with both a silica-based and a metal
or metal-oxide portion, wherein the array has been treated with a
biotin containing coupling agent and a passivating agent, wherein
when a coupon having regions with the same type of silica as the
silica-based portions and the same type of metal or metal-oxide as
the metal or metal oxide portions of the array is treated in the
same manner as the array, the coupon exhibits a fluorescence
intensity bias of greater than 10 in a labeled neutravidin bead
bias assay.
[0023] One aspect of the invention is an array of optical
confinements having a surface with both a silica-based and a metal
or metal-oxide portion, wherein the array has been treated with a
biotin containing coupling agent and a passivating agent, wherein
when a coupon having regions with the same type of silica as the
silica-based portions and the same type of metal or metal-oxide as
the metal or metal oxide portions of the array is treated in the
same manner as the array, the coupon exhibits a fluorescence
intensity bias of greater than 10 in a labeled template/polymerase
complex bias assay.
[0024] One aspect of the invention is an apparatus for obtaining
sequence information from a template nucleic acid comprising: a) a
substrate comprising an array of zero-mode-waveguides wherein the
zero-mode-waveguides comprise apertures through an upper metal
layer disposed on top of a lower silica-based layer wherein the
bias each zero-mode-waveguide capable of holding a single molecule
of a polymerase enzyme, wherein the substrate is treated by a
selective functionalization methods of the invention; b) a
receptacle for keeping sequencing reagents in contact with the
substrate; c) an optical system optically coupled to the substrate
for delivering light to the zero-mode-waveguides and for measuring
light from the zero mode waveguides over a period of time, wherein
the measured light from the zero-mode-waveguide over a period of
time can be used to obtain sequence information about the template
DNA; and d) a computational system connected to the optical system
for determining sequence information using the measured light over
a period of time.
[0025] One aspect of the invention is an apparatus for obtaining
sequence information from a template nucleic acid comprising: a) an
array of zero-mode-waveguides wherein the zero-mode-waveguides
comprise apertures through an upper metal layer disposed on top of
a lower silica-based layer wherein when a coupon having regions
with the same type of silica as the lower silica-based layer and
the same type of metal as the upper metal layer is treated in the
same manner as the array, the coupon exhibits a fluorescence
intensity bias of greater than 10 in a labeled neutravidin bead
bias assay; b) a receptacle for keeping sequencing reagents in
contact with the substrate; c) an optical system optically coupled
to the substrate for delivering light to the zero-mode-waveguides
and for measuring light from the zero mode waveguides over a period
of time, wherein the measured light from the zero-mode-waveguide
over a period of time can be used to obtain sequence information
about the template DNA; and d) a computational system connected to
the optical system for determining sequence information using the
measured light over a period of time.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 shows a schematic illustration of a
zero-mode-waveguide optical confinement.
[0027] FIG. 2 shows an embodiment of a substrate of the invention
having high bias.
[0028] FIG. 3 shows an alternate embodiment of a substrate of the
invention having high bias.
[0029] FIG. 4 illustrates an embodiment of a method of the
invention for producing a substrate having a functionalized
transparent or silica-based surface with high bias.
[0030] FIG. 5 illustrates an embodiment of a method of the
invention for producing a substrate having coupling agent
selectively bound to a transparent silica surface, and coupling of
a molecule of interest to such surface.
[0031] FIG. 6 illustrates an embodiment of a method of the
invention for producing a substrate having a mixture of
functionalizing agent including coupling agent selectively bound to
a transparent silica surface, and coupling of a molecule of
interest to such surface.
[0032] FIG. 7 shows a schematic illustration of an optical
confinement of the invention having a lipid bilayer coating on a
transparent or silica-based region. FIG. 7(a) illustrates a
molecule of interest tethered to the bilayer. FIG. 7(b) illustrates
a molecule of interest associated directly with the bilayer.
[0033] FIG. 8 shows an embodiment of a system of the invention.
[0034] FIG. 9(A) shows a fluorescence scanner images a coupon
having silica-based and metal or metal oxide regions prior to the
selective removal step and a schematic illustration of the coupon
at that point. FIG. 9(B) shows a fluorescence scanner image of the
coupon after the selective removal step and a schematic
illustration of the coupon. FIG. 9(C) shows a fluorescence scanner
image of a coupon having a bias of greater than 100 as determined
in a fluorescent bead-based assay.
[0035] FIG. 10 shows data for ellipsometric thickness,
demonstrating high bias.
[0036] FIG. 11 shows a data trace for a four-color single-molecule
sequencing reaction performed on a ZMW array produced as described
herein.
DETAILED DESCRIPTION OF THE INVENTION
I. General
[0037] 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).
[0038] The invention provides for a preferentially functionalized
substrates in which, for example, a reflective or opaque region of
a substrate surface is treated to have one set of surface
characteristics, and a transparent portion of the surface is
treated to have a different set of surface characteristics. In some
aspects, an opaque or reflective region of the substrate is coated
with a passivating compound, and a transparent region of the
substrate surface has bound to it coupling agent to which a desired
molecule may be preferentially bound. The methods of the invention
provide for surfaces having either no coupling agent or very low
levels of coupling agent bound to the reflective or opaque regions,
while having sufficient coupling agent on the transparent regions
for the substrates to be used, for example, for molecular analysis.
In some cases, the methods of the invention can also be employed to
functionalize the opaque or reflective portions, while passivating
the transparent or silica based portions of the substrate.
[0039] We have found that a substrate having useful properties can
be prepared a process including the steps of: (1) treating the
substrate with a passivating compound that preferentially binds to
the reflective or opaque surface regions, (2) treating the
substrate with a coupling agent that reacts with the transparent
surface regions, and (3) treating the substrate with a compound
that preferentially removes the coupling agent from the reflective
or opaque regions. We have found that by performing all three of
these steps, surfaces having very little to no coupling agent on
the reflective or opaque surface regions can be produced. The
methods of the invention can be applied, for example, to a
substrate comprising a lower layer made of a silica-based material
such as fused silica and an upper metal cladding layer comprising a
metal such as aluminum. The chemical differences of these surfaces
can be utilized, for example, by incorporating a phosphorous
containing selective passivating agent which reacts preferentially
with the metal, e.g. aluminum, surface. The preferential removal of
coupling agent from a metal, e.g. aluminum, surface can be
accomplished using an acidic compound, such as a polymer comprising
one or more of carboxylic acid, sulfonic acid, or phosphonic acid
moieties. We have found that these compounds can selectively remove
a coupling agent, such as a silane coupling agent from a passivated
metal, e.g. aluminum, surface.
[0040] 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 portion of the 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 about 10 nm to about 200 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. Pat. Nos. 7,170,050, 7,056,661, and 6,917,726, each of
which is incorporated herein by reference in its entirety for all
purposes.
[0041] 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.
[0042] 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 the reactions of a single
molecule 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.
[0043] 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 some cases, it is also desirable to prevent
molecules of interest from binding to the portions of the cladding
that may within the illumination region. 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, in some cases, 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.
[0044] 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.
[0045] 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 Published U.S. Patent Application
No. 2007/0036511, 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.
[0046] 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.
[0047] In at least one aspect, the present invention is directed to
providing enhanced isolation of discrete reaction and/or
observation regions. This is, to provide chemical and/or
environmental isolation for such regions. In some cases, this is
accomplished by providing a barrier or zone between reaction and/or
observation regions that substantially prevents the diffusion of
one or more 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. The creation of such a
demilitarized zone is described, for example, in U.S. patent
application Ser. No. 11/731,748, which is included herein in its
entirety by reference.
[0048] In some aspects, the invention can be utilized to
selectively functionalizing within optical confinement regions on a
substrate. One such optical confinement is the zero-mode-waveguide
(ZMW). 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 layer 104. An aperture or core 106 is disposed through
the cladding layer to expose the transparent layer 104 below. The
aperture 106 has a base 120 that comprises the top surface of the
transparent layer 104. As shown in FIG. 1, the base 120 of the
aperture 106 is at the same level as the planar surface of the
transparent layer 104. In some cases, the base 120 of the aperture
106 is not at the same level, and can be above or below the planar
surface of the transparent layer 104 outside of the aperture. For
example, in some cases, the base of the aperture can be below the
level of the surface of the transparent 104, extending into the
transparent layer 104. The core is dimensioned to provide optical
confinement by attenuating or 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. It will be understood by
those in the art the intensity will fall off in the core with a
certain function, e.g. exponentially, and that line 108 does not
necessarily represent a line above which no light penetrates, but
can represent, for example, a line at which the light falls to a
certain absolute or relative intensity level.
[0049] FIG. 2 shows a ZMW having a surface modified as described in
some aspects of the invention. In FIG. 2, the cladding 102 of the
ZMW is selectively coated with a compound 202, for example a
phosphate containing passivating compound. The exposed surface of
the transparent layer 104 is functionalized, for example, with a
silane coupling agent to produce a functionalized region 204 which
comprises the base of the ZMW. The functionalized base of the ZMW
will tend to be within the observation region of the ZMW, as
illustrated by dashed line 108. The functionalized region can thus
be accessible, for example, to illumination by light for the
excitation of fluorescence within the sample. Where the
functionalized region 204 comprises a coupling agent, the coupling
agent can be used to attach a desired molecule (molecule of
interest) 110 to the top surface 120 of the transparent layer 104.
Methods of the invention allow for obtaining a ZMW having very
little to no functionalizing groups, for example silane coupling
agent, on the surfaces of the cladding, while efficiently
functionalizing the base of the ZMW. These methods ensure that the
desired molecules 110 are attached within the observation regions,
and that desired molecules are not attached outside of the
observation regions. In addition, the methods of the invention can
also result in having very little to none of the passivating
compounds on the transparent layer.
[0050] FIG. 3 shows a ZMW for which the aperture extends into the
transparent layer having a surface modified as described in some
aspects of the invention. In FIG. 3, the cladding 102 is
selectively coated with a compound 202, for example a phosphate
passivating agent. The exposed surface of the transparent layer 104
is functionalized, for example, with a silane coupling agent, to
form a functionalized region 204. In this embodiment, the
functionalized region 204 includes both the base, or floor, of the
ZMW aperture, and also the sidewalls of the aperture where the
aperture extends into the transparent region. The substrate is
treated with a selective removal compound to selectively remove
functionalized groups from the pas sivated surface of the cladding
202 to produce highly selectively coated surfaces. Where the
transparent layer is functionalized with a coupling agent, a
molecule of interest, such as an enzyme, can be attached. Here,
since both the base of the aperture and the sidewalls of the
aperture that extend into the transparent layer are functionalized,
a molecule of interest could attach to either the base or that
portion of the sidewall. Where a desired molecule 110 is attached
to the wall of the aperture that extends into the transparent layer
104, the desired molecule will also be within the observation
volume illustrated by dashed line 108. While illustrated for a ZMW
array, the methods of the present invention can be used to
selectively functionalize other substrates that have transparent or
silica portions and reflective or metal portions such as the
coupons described herein.
II. Methods
[0051] The methods of the invention are generally directed to the
selective functionalization of substrates having both transparent
and reflective or opaque regions. Such substrates can comprise, for
example, an array of optical confinements or zero-mode-waveguide
structures. In such arrays, it is often desirable to have specific
functionality in different regions of the substrate. In some cases,
it is desired that the level of specific functionality be high. One
approach to obtaining high specific functionality is to utilize the
different surface properties of the different types of materials
that make up portions of the substrate surface. We have found that
the methods described herein can provide for higher levels of
specificity than other previously described methods.
[0052] For example, we have found that a surface having high
specific functionality can be obtained with a method that comprises
the three steps of: (a) exposing a surface of a substrate having
both transparent, e.g. silica-based, portions and metal or metal
oxide portions to an agent that preferentially binds to the metal
or metal oxide portions to produce a passivated metal or metal
oxide portions of the surface; (b) exposing the surface of the
substrate to a transparent layer, e.g. silica, functionalizing
agent that binds to both the transparent, e.g. silica-based,
portions and the passivated metal or metal oxide portions of the
surface; and (c) exposing the surface of the substrate to a
selective removal compound that preferentially removes the
transparent layer, e.g. silica, functionalizing agent from the
passivated metal or metal oxide portions of the surface.
[0053] In some cases, the steps are carried out in the order
described in which the selective removal agent is added after
passivation and functionalization. The steps can also be carried
out in the order b), c), then a) in which the passivation step is
carried out after the selective removal step. We have found that
the passivation step can be quite selective on its own, but that
the step of functionalizing the silica or transparent portions of
the surface typically results in some contamination of the metal or
metal oxide portions of the surface. In some cases, the passivation
and selective removal steps (steps a) and c)) can be combined and
carried out at substantially the same time.
[0054] One preferred aspect of the invention provides a method for
attaching a desired molecule selectively to a portion of a
substrate. In one aspect the invention comprises a method for
attaching a desired molecule to a silica-based portion of a surface
of a substrate having both silica-based portions and metal or metal
oxide portions comprising; a) exposing a surface of a substrate
having both silica-based portions and metal or metal oxide portions
to an agent that preferentially binds to the metal or metal oxide
portions to produce passivated metal or metal oxide portions of the
surface; b) exposing the surface of the substrate to a coupling
agent that binds to both the silica-based portions and the
passivated metal or metal oxide portions of the surface; c)
exposing the surface of the substrate to a selective removal
compound that preferentially removes the coupling agent from the
passivated metal or metal oxide portions of the surface; and d)
attaching the desired molecule to the coupling agent bound to the
silica-based portions of the surface.
[0055] In some cases, we have found that it is desirable to carry
out step d) prior to carrying out step c). A reason for carrying
out step d) prior to step c) is provided were step c) of selective
removal results in undesirable reactions with the coupling agent
which lowers the yield of attaching step d). For example, we have
found that where a thiol coupling agent is used, an acidic polymer
selective removal compound can react with the thiol coupling agent
and lower the yield of attaching the desired molecule, for example
by maleimide coupling chemistry. In these cases, carrying out the
attaching step d) first, can lead to a better yield of attachment
of the molecule of interest.
[0056] While the treatment of surfaces made up of different
materials to agents which react selectively with the different
materials has been described, we have found that the use of a
single selective treatment alone generally will not provide the
specificity desired for applications such as producing arrays of
optical confinements, for example, for observing the behavior of
individual molecules. In particular, even where the second step
described above of treating the transparent, e.g. silica-based,
portions of the surface utilizes a highly selective functionalizing
agent, and even where the metal or metal oxide portions are well
passivated, there is generally still some amount of functionalizing
agent bound to the passivated metal or metal oxide surface. We have
found that the desired level of selective functionalization can be
obtained by employing a selective removal compound that selectively
removes functionalizing agent from the passivated metal or metal
oxide surface, while removing little or no functionalizing agent
from the transparent, e.g. silica portions.
[0057] Generally, the substrates comprise an opaque or reflective
surface portion and a transparent surface portion, which may be
silica based. It is not always required that the substrate have a
transparent portion. For example, the substrate may have a
silica-based portion that is not transparent, such as, for example,
silicon, and a reflective surface, for example, a metal
surface.
[0058] An embodiment of a method of the invention is shown
schematically in FIG. 4. An optical confinement comprising an
aperture 406 through a metal cladding layer 402, such as aluminum,
to a transparent layer 404 is treated in step (I) with a selective
passivating agent such as a phosphonate containing polymer. The
selective passivating agent produces a passivation coating 420
selectively over the metal cladding layer. Very little to no
selective passivating agent coats onto the transparent layer. In
step (II) the substrate is treated with a transparent layer surface
functionalizing agent such as a silane coupling agent. The
functionalizing agent forms a coating 440 on the exposed
transparent layer surface. While the functionalizing agent may be
somewhat selective, unwanted functionalizing agent 460 can become
deposited onto the metal cladding layer. In step (III), a selective
removal agent is used to selectively remove functionalizing agent
from the metal cladding layer, while leaving the functionalizing
agent on the transparent layer. This process produces a
functionalized transparent layer surface having high bias.
[0059] Another embodiment of a method of the invention is shown
schematically in FIG. 5. An optical confinement comprising an
aperture 406 through a metal cladding layer 402, such as aluminum,
to a transparent layer 404 is treated in step (I) with a selective
passivating agent such as a phosphonate containing polymer. The
selective passivating agent produces a passivation coating 420
selectively over the metal cladding layer. Very little to no
selective passivating agent coats onto the transparent layer. In
step (II) the substrate is treated with a coupling agent that binds
to the exposed transparent layer surface. The coupling agent forms
a coating 540 on the exposed transparent layer surface. While the
coupling agent may be somewhat selective, unwanted coupling agent
560 can become deposited onto the metal cladding layer. In step
(III), a selective removal agent is used to selectively remove
coupling agent from the metal cladding layer, while leaving the
coupling agent on the transparent layer. In step (IV), a molecule
of interest having functionality that can bind to the coupling
agent is added such that the molecule of interest becomes attached
to the coupling agent selectively bound to the transparent layer
surface. Since little or no coupling agent is present on the metal
cladding layer due to the treatment with the selective removal
agent, the molecule of interest is bound to the transparent layer
surface with high bias. In some cases, the molecules of interest
are added at a concentration such that a significant portion of the
optical confinements have only one molecule of interest
present.
[0060] Yet another embodiment of a method of the invention is shown
schematically in FIG. 6. An optical confinement comprising an
aperture 406 through a metal cladding layer 402, such as aluminum,
to a transparent layer 404 is treated in step (I) with a selective
passivating agent such as a phosphonate containing polymer. The
selective passivating agent produces a passivation coating 420
selectively over the metal cladding layer. Very little to no
selective passivating agent coats onto the transparent layer. In
step (II) the substrate is treated with a mixture of a coupling
agent and a non-coupling functionalizing agent that binds to the
exposed transparent layer surface. The coupling/functionalizing
agent mix forms a coating on the exposed transparent layer surface
that has both coupling agent 640 and non-coupling functionalizing
agent 650. The coupling agent can be provided at low concentration
in the mixed functionalizing agent such that a low density of
coupling agents is disposed on the transparent layer surface. While
the functionalizing agent mixture may be somewhat selective,
unwanted coupling agent 660 can become deposited onto the metal
cladding layer. In step (III), a selective removal agent is used to
selectively remove functionalizing agent from the metal cladding
layer, while leaving the functionalizing agents on the transparent
layer. In step (IV), a molecule of interest having functionality
that can bind to the coupling agent is added such that the molecule
of interest becomes attached to the coupling agent selectively
bound to the transparent layer surface. Since little or no coupling
agent is present on the metal cladding layer due to the treatment
with the selective removal agent, the molecule of interest is bound
to the transparent layer surface with high bias. In some cases, the
molecules of interest are added at a concentration such that a
significant portion of the optical confinements have only one
molecule of interest present.
[0061] In some cases, the processes of the invention up to the
functionalization of the transparent or silane portions of the
substrate will be carried out, and the substrate will be stored
prior to carrying out the final treatment with the selective
removal agent. For example, the sample can be stored for 2, 3, 4,
5, 6, or 7 days, or 2, 3, 4, 6, 8, or more weeks prior to treatment
with the selective removal agent. Holding the sample before
performing treating with the selective removal agent can be useful
in cases, which we have observed wherein the surface is more stable
prior to treatment with the selective removal agent than it is
after such treatment.
[0062] Another method for obtaining a selectively functionalized
surface, which is sometimes referred to as an MPP process comprises
first functionalizing the transparent or silica based surface with
a functionalizing agent such as a thiol functionalized silane,
second, conjugating a coupling group or selective binding group
such as biotin to the surface, for example using a
Biotin-PEG-maleimide group, (where the Biotin-PEG-maleimide group
is optionally diluted with a non-coupling surface reactive group or
surface passivating group such as PEG-maleimide at a ratio of from
about 10:1 to about 1:1,000,000, about 1:100 to about 1:100,000, or
about 1:10,000 to about 1:1,000,000, third, treating the surface
with a phosphonate passivating group such as PVPA, and fourth,
treating the surface with an acidic phosphonate containing polymer
such as AQUARITE ESL. In some embodiments the third and fourth
steps are carried out simultaneously.
[0063] While in some cases the invention can involve masking
specific portions of the surface to produce selectively
functionalized regions, it is one aspect of the invention that the
surface comprising the transparent or silica based portions and
reflective or opaque portions are simultaneously treated with the
reagents described herein, thus providing selective
functionalization of the different regions of the surface without
the use of masking the different portions.
A. Substrate
[0064] The substrate of the invention has a surface comprising two
or more regions comprising different materials. The surface of the
substrate may comprise, for example, regions of a transparent
material and regions of an opaque or reflective material. In some
cases, the surface comprises a silica-based material and a metal or
metal oxide based material. The substrate can comprise a
transparent layer which has disposed upon its surfaces regions of
opaque or reflective materials. In addition, the substrate can
comprise a silica-based material which has disposed upon its
surface metal or metal oxide regions. The substrate can comprise an
array of optical confinements or zero-mode-waveguides as described
in U.S. Pat. No. 7,170,050 or 7,302,146 which are incorporated by
reference herein in their entirety. The substrates generally
comprise at least one surface on which a pattern of optical
confinements is present, where the surface may be smooth or
substantially planar, or have irregularities, such as depressions
or elevations. The surface on which the pattern of targets is
presented may be modified with one or more different layers of
compounds that serve to modulate the properties of the surface in a
desirable manner.
[0065] The substrates of the invention are generally rigid, and
often planar, but need not be either. Where the substrate comprises
an array of optical confinements, the substrate will generally be
of a size and shape that can interface with optical instrumentation
to allow for the illumination and for the measurement of light from
the optical confinements. Typically, the substrate will also be
configured to be held in contact with liquid media, for instance
containing reagents and substrates and/or labeled components for
optical measurements.
[0066] Where the substrates comprise arrays of optical
confinements, the arrays may comprise a single row or a plurality
of rows of optical confinement on the surface of a substrate, where
when a plurality of lanes are present, the number of lanes will
usually be at least 2, more commonly more than 10, and more
commonly more than 100. The subject array of optical confinements
may align horizontally or diagonally long the x-axis or the y-axis
of the substrate. The individual confinements can be arrayed in any
format across or over the surface of the substrate, such as in rows
and columns so as to form a grid, or to form a circular,
elliptical, oval, conical, rectangular, triangular, or polyhedral
pattern. To minimize the nearest-neighbor distance between adjacent
optical confinements, a hexagonal array is sometimes preferred.
[0067] The array of optical confinements may be incorporated into a
structure that provides for ease of analysis, high throughput, or
other advantages, such as in a microtiter plate and the like. Such
setup is also referred to herein as an "array of arrays." For
example, the subject arrays can be incorporated into another array
such as microtiter plate wherein each micro well of the plate
contains a subject array of optical confinements.
[0068] In accordance with the invention, arrays of confinements,
e.g., zero mode waveguides, are provided in arrays of more than
100, more than 1000, more than 10,000, more that 100,000, or more
than 1,000,000 separate waveguides on a single substrate. In
addition, the waveguide arrays typically comprise a relatively high
density of waveguides on the surface of the substrate. Such high
density typically includes waveguides present at a density of
greater than 10 zero mode waveguides per mm.sup.2, preferably,
greater than 100 waveguides per mm.sup.2 of substrate surface area,
and more preferably, greater than 500 or even 1000 waveguides per
mm.sup.2 and in many cases up to or greater than 100,000 waveguides
per mm mm.sup.2. Although in many cases, the waveguides in the
array are spaced in a regular pattern, e.g., in 2, 5, 10, 25, 50 or
100 or more rows and/or columns of regularly spaced waveguides in a
given array, in certain preferred cases, there are advantages to
providing the organization of waveguides in an array deviating from
a standard row and/or column format. In preferred aspects, the
substrates include zero mode waveguides as the optical confinements
to define the discrete reaction regions on the substrate.
[0069] The optical confinements can be zero-mode-waveguides. Zero
mode waveguides have been described in, e.g., U.S. Pat. No.
6,917,726, the full disclosure of which is incorporated herein by
reference in its entirety for all purposes. Generally, such
waveguides comprise a core disposed through a cladding layer, which
in the case of applications to reactions, comprises an aperture
disposed through the cladding layer that can receive the reactants
to be monitored. Typically, the aperture has at least one
cross-sectional dimension, e.g., diameter, which is sufficiently
small that light entering the waveguide is prevented in some
measure from propagating through the core, effectively resulting in
a very small portion of the core and its contents being
illuminated, and/or emitting optical signals that exit the core. In
the case of optical signals (and excitation radiation), the
waveguide cores will typically be between about 1 nm and about 300
nm, between about 10 and about 200 nm, or between about 50 and
about 150 nm in diameter where light in the visible range is
used.
[0070] The overall size of the array can generally range from a few
nanometers to a few millimeters in thickness, and from a few
millimeters to 50 centimeters in width and/or length. Arrays may
have an overall size of about few hundred microns to a few
millimeters in thickness and may have any width or length depending
on the number of optical confinements desired.
[0071] The spacing between the individual confinements can be
adjusted to support the particular application in which the subject
array is to be employed. For instance, if the intended application
requires a dark-field illumination of the array without or with a
low level of diffractive scattering of incident wavelength from the
optical confinements, then the individual confinements may be
placed close to each other relative to the incident wavelength.
[0072] The individual confinement in the array can provide an
effective observation volume less than about 1000 zeptoliters, less
than about 900, less than about 200, less than about 80, less than
about 10 zeptoliters. Where desired, an effective observation
volume less than 1 zeptoliter can be provided. In a preferred
aspect, the individual confinement yields an effective observation
volume that permits resolution of individual molecules, such as
enzymes, present at or near a physiologically relevant
concentration. The physiologically relevant concentrations for many
biochemical reactions range from micro-molar to millimolar because
most of the enzymes have their Michaelis constants in these ranges.
Accordingly, preferred array of optical confinements has an
effective observation volume for detecting individual molecules
present at a concentration higher than about 1 micromolar (.mu.M),
or more preferably higher than 50 .mu.M, or even higher than 100
.mu.M.
[0073] As zero-mode-waveguide can provide an optical guide in which
the majority of incident radiation is attenuated, preferably more
than 80%, more preferably more than 90%, even more preferably more
than 99% of the incident radiation is attenuated. As such high
level of attenuation, no significant propagating modes of
electromagnetic radiation exist in the guide. Consequently, the
rapid decay of incident electromagnetic radiation at the entrance
of such guide provides an extremely small observation volume
effective to detect single-molecules, even when they are present at
a concentration as high as in the micromolar range.
[0074] The zero-mode-waveguide of the present invention typically
comprises a cladding surrounding a core (i.e., partially or fully),
wherein the cladding is configured to preclude propagation of
electromagnetic energy of a wavelength higher than the cutoff
wavelength longitudinally through the core of the zero-mode
waveguide. The cladding is typically made of materials that prevent
any significant penetration of the electric and the magnetic fields
of an electromagnetic radiation that is opaque and/or reflective
materials. Suitable materials for fabricating the cladding include
but are not limited to metals, metal oxides, alloys, and
semi-conducting materials, and any combination thereof.
[0075] The internal cavity (i.e., the core) surrounded by the
cladding may adopt a convenient size, shape or volume so long as
propagating modes of electromagnetic radiation in the guide is
effectively prevented. The core typically has a lateral dimension
less than the cutoff wavelength (.lamda..sub.c). For a circular
guide of diameter d and having a clad of perfect conductor,
.lamda..sub.c is approximately 1.7 times d. The cross sectional
area of the core may be circular, elliptical, oval, conical,
rectangular, triangular, polyhedral, or in any other shape.
Although uniform cross sectional area is generally preferred, the
cross sectional area may vary at any given depth of the guide if
desired.
[0076] In some embodiments, the core is non-cylindrical. In one
aspect of this embodiment, a non-cylindrical core comprises an
opening on the upper surface and a base at the bottom surface that
is entirely enclosed by the cladding, wherein the opening is
narrower in lateral dimension than the base. This configuration
significantly restricts the diffusion of reactants, and hence
increases the average residence time in the observation volume.
Such configuration can be useful, for example, for measuring the
association rate constant (on-rate) of a chemical reaction. In
another aspect, the core comprises an opening that is wider in
lateral dimension than the base. Such configuration allows easier
access to large molecules that impose a steric or entropic
hindrance to entering the structure if the open end of the zero
mode waveguide was as small as the base needed to be for optical
performance reasons. Examples include the accessibility for long
strand polyelectrolytes such as DNA molecules that are subject to
entropic forces opposing entry into small openings.
[0077] The surfaces of the substrate are modified according to the
methods described herein. The surface modifications are generally
carried out by laboratory and manufacturing techniques that would
be known to those of skill in the art. In some cases, the compounds
used for surface modification and surface functionalization are
brought into contact with the surface in liquid form. For example,
the surfaces are treated with or exposed to a solution of the
surface modification compound. In other cases, the compounds are
brought into contact with the surface in gaseous form. In general,
the whole surface of the substrate is exposed to or treated with
the compounds or functionalizing agents. In other cases, regions of
the surface can be masked from exposure to one or more of the
compounds or functionalizing agents.
B. Silica-Based Layer/Transparent Layer
[0078] The substrate generally comprises a silica-based and/or a
transparent layer. The term "transparent", as used herein, refers
to a layer will at least partially transmit electromagnetic energy
or light of the wavelength appropriate for the use of the
substrate. Where the substrate comprises an array of optical
confinements, the wavelength is generally from the infrared to the
ultraviolet. In many cases, for example in using fluorescent dyes,
it is desirable that the substrate transmit visible light, for
example, between 400 nm and 800 nm. The optically transparent layer
may generally comprise any of a number of transparent solid
materials, depending upon other components of the substrate. Such
materials include inorganic materials, such as glass, quartz, fused
silica, and the like. In some preferred embodiments, fused silica
comprises the transparent layer. The transparent substrate can
comprise an oxide material such as indium-tin oxide (ITO). In some
cases, a transparent material which can be processed using
semiconductor processing techniques, such as fused silica is
preferred. Alternatively, such materials may include organic
materials, such as polymeric substrates such as polystyrene,
polypropylene, polyethylene, polymethylmethacrylate (PMMA), and the
like. Polymeric materials having low levels of autofluorescence,
such PMMA can be particularly useful in fluorescent or fluorogenic
reactions.
[0079] In some embodiments, the substrate comprises a silica-based
material. The silica-based material can be transparent or opaque.
Suitable silica-based materials include glass, semiconductors
(e.g., silicate, silicon, silicates, silicon nitride, silicon
dioxide), quartz, fused silica,
[0080] In some embodiments, the substrate comprises a semiconductor
material that is not generally transparent. Suitable semiconductor
materials include doped silicon, germanium, or gallium
arsenide.
[0081] In some embodiments the transparent and/or silica based
material comprises a structural component of the substrate. For
example, where the substrate comprises an array of optical
confinements, the array can comprise a transparent layer on which
an opaque or reflective cladding layer is deposited. In such cases,
it is generally desired that the transparent layer be relatively
rigid, and of a thickness which will allow for handling without
significant distortion or breakage. The thickness of the
transparent layer will generally be 10 microns to a millimeter in
thickness.
C. Opaque/Reflective Layer
[0082] The substrates of the invention in some embodiments comprise
an opaque and/or a reflective layer. The opaque or reflective layer
can be disposed upon the upper surface of a transparent lower
layer. By patterning the opaque or reflective layer on the
transparent lower layer, a plurality of optical confinements can be
produced. In some cases the substrate comprises a single opaque or
reflective layer. In some cases, the substrate comprises multiple
opaque or reflective layers, which can comprise the same or
different materials.
[0083] Where the substrate comprises an array of optical
confinements, the opaque and/or reflective layer can also be
referred to as the cladding. The cladding is typically made of
materials that prevent any significant penetration of the electric
and the magnetic fields of an electromagnetic radiation in the
wavelength range of interest, e.g. opaque and/or reflective
materials. Suitable materials for fabricating the opaque and/or
reflective materials include but are not limited to metals, metal
oxides, alloys, and semi-conducting materials, and any combination
thereof. As used herein, the term metal includes alloys comprising
a metal. Alloys generally include any of the numerous substances
having metallic properties but comprising two or more elements of
which at least one is a metal. Alloys may vary in the content or
the amount of the respective elements-whether metallic or non
metallic. Preferred alloys generally improve some desirable
characteristics of the material over a pure elemental material.
Characteristics that can be improved through the use of mixtures of
materials include, chemical resistance, thermal conductivity,
electrical conductivity, reflectivity, grain size, coefficient of
thermal expansion, brittleness, temperature tolerance,
conductivity, and/or reduce grain size of the cladding.
[0084] In some embodiments aluminum or an aluminum alloy comprises
the metal in the zero-mode-waveguide. Non-limiting examples of
materials suitable to alloy with aluminum are antimony, arsenic,
beryllium, bismuth, boron, cadmium, calcium, carbon, cerium,
chromium, cobalt, copper, gallium, hydrogen, indium, iron, lead,
lithium, magnesium, manganese, mercury, molybdenum, nickel,
niobium, phosphorous, silicon, vanadium, zinc, titanium, or
combinations thereof. By way of example of how the introduction of
another element could beneficially impact the ZMW performance, the
introduction of boron to aluminum is known in the art of metallurgy
to increase the conductivity of aluminum. Some embodiments include
an alloy of aluminum that is more than 0.0001% of a dopant. Some
embodiments include an alloy of aluminum that is more than 0.005%
of a dopant. Some embodiments embodiment includes an alloy of
aluminum that is more than 0.1% of a dopant.
[0085] Semi-conducting materials suitable for the opaque and/or
reflective layer are generally opaque, and they include doped or
undoped silicon, germanium, silicates, silicon nitride, silicon
dioxide, gallium phosphide, gallium arsenide, metal oxides or any
combinations thereof.
[0086] In many cases, the surfaces of metals will comprise
metal-oxides. For example, when aluminum is exposed to air, the
exposed surfaces will generally have a surface layer of aluminum
oxide. Some aspects of the invention involve performing reactions
on the surfaces of metals. When describing such reactions herein,
it is to be understood that where the reaction with a metal at the
surface of a metal is described, this reaction may be a reaction
with, for example, a surface metal oxide or surface metal
hydroxide. Alternatively, where a reaction may be described as
occurring with a metal oxide such as an aluminum oxide, it is to be
understood that in the context of the invention such a reaction can
be carried out on the surface of a metal layer.
D. Selective Coating or Selective Passivation of the Opaque or
Reflective Layer
[0087] Some aspects of the invention include the selective
passivation of an opaque or reflective layer. Passivation of a
surface generally means treating a surface to change the properties
of the surface. In some cases, passivation can involve preventing
the surface from participating in a chemical reaction that the
surface would have if not passivated. For example, passivation can
involve minimizing the surfaces' interactions with the environment
(e.g., minimizing or eliminating nonspecific binding to the
surfaces). In particular, passivation can involve minimizing the
binding of biomolecules or other reagents used in carrying out a
reaction with biomolecules, such as proteins, nucleic acids, or
nucleotides. In some cases, passivation can be minimizing a
surface's tendency to undergo corrosion. In the context of the
arrays of optical confinement described herein, it can be desirable
that the opaque or passivation layer passivated with respect to the
adsorption or binding of compounds in a solution which is observed
with the optical confinement. For example, it can be undesirable
for the silica or transparent layer functionalization agents or the
molecules of interest to be bound to the surface of the opaque or
reflective layer.
[0088] Passivation can, in some cases, be accomplished by coating
the opaque or reflective surface with a passivating compound. In
some cases, the passivating compound can be bound covalently to the
opaque or reflective surface. In some cases, the passivating
compound can be deposited onto the opaque or reflective surface
without the formation of a covalent bond, being held in place, for
example by van der Waals, hydrogen bonding, or dipolar forces In
one aspect of the invention, the surface is treated with a
passivating compound that selectively reacts with, and deposits
onto the opaque or reflective surface, while having little or no
deposition onto silica-based, or transparent surfaces.
[0089] Thus, selective coating of a given compound to an opaque or
reflective layer as opposed to a silica-based or transparent layer
means that the compound is present on the surface of an opaque or
reflective layer such as a metal or metal oxide layer 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 than the
concentration or density on the surface of a silica-based or
transparent layer, and in some cases 100 times greater, 1000 times
greater or more.
[0090] In some cases, the opaque or reflective layer comprises a
metal or metal oxide surface such as aluminum or aluminum oxide
surfaces, which tend to be positively charged in aqueous solution,
and which can be passivated using a negatively charged passivating
agent that binds to the metal or metal oxide surface.
[0091] Preferred passivation or coating compounds of the invention
contain phosphorous. These compounds will generally comprise
P.dbd.O and/or P--OH functionality. In particular, compounds
comprising phosphate or phosphonate groups can be used. Such
phosphate or phosphonate compounds can selectively react with metal
or metal oxide surfaces such as aluminum or aluminum oxide, while
having low reactivity to other surfaces, for example silica
surfaces. In addition, in some cases, these compounds will form
strong bonds to a metal or metal oxide surface such as the surface
of aluminum to provide robust passivation to the metal surface.
Preferred passivation or coating compounds include phosphorous
containing polymeric materials. Suitable phosphorous containing
polymeric materials include homopolymers and copolymers of
poly(vinylphosphonic acid), ALBRITECT CP-30, ALBRITECT CP-10,
ALBRITECT CP-90, AQUARITE ESL, and AQUARITE EC4020. ALBRITECT and
AQUARITE compounds are commercially available from Rhodia, Inc.
Phosphate or phosphonic acid moieties can 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 generally 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 layers having aluminum
oxide surfaces of a ZMW or similar hybrid substrate.
[0092] For example, a metal oxide surface can be modified with an
alkyl phosphate or an alkyl phosphonate. The terms phosphonic acid
and phosphonate are alternatively used to refer to the compounds
described herein. It is understood that a phosphonic acid will
generally have hydrogens associated with two of the phosphonic acid
oxygens, and that a phosphonate will generally have other
counterions associated with these oxygens. In aqueous solution,
hydrogen ions and counterions can exchange rapidly. Thus generally
either phosphonic acid and phosphonate compounds can be useful in
the invention.
[0093] 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), melamine, 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.
[0094] 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.
[0095] 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.
[0096] Other suitable phosphonates include high molecular weight
polymeric phosphonates such as polyvinylphosphonic acid (PVPA):
##STR00001##
[0097] wherein n can be from about 1 to about 1000 or from about 10
to about 100.
[0098] Phosphonate end-capped polymers of polymers having acidic
functional groups such as carboxylic acids, sulfonic acids and
mixtures thereof can also be used. These can include phosphonate
end-capped poly(acrylates), poly(sulfonates), and copolymers
thereof. Exemplary phosphonate end-capped compounds include:
##STR00002##
[0099] where n can be from about 1 to 1000, can be between about 10
and about 100, and can be about 20 (available from Rhodia, Inc. as
AQUARITE EC4020), or
##STR00003##
[0100] where n and m can be from about 1 to about 1000, from about
10 to about 100, or can each be about 50, in some cases, m is about
24 and n is about 16 (available from Rhodia, Inc. as AQUARITE ESL).
Exemplary copolymers copolymer include the copolymers:
##STR00004##
[0101] such as vinyl phosphonic acid-acrylic acid copolymers
(commercially available from Rhodia as ALBRITECT CP30). The values
for n and m can range from about 1 to about 1000. In some cases, m
is between about 10 and about 100, and n is between about 100 and
300. In some cases, m is between about 50 and about 70, and n is
between about 80 and 120. In some cases, m is about 60 and n is
about 200.
[0102] 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 PM2) and the compounds listed in Table 1 (commercially
available as DEQUEST compounds from Solutia, Inc., St. Louis Mo.).
Phosphonate compounds can be supplied as salts (e.g., sodium,
potassium, lithium, or ammonium salts) or as free acids.
TABLE-US-00001 TABLE 1 Exemplary phosphonic acid compounds.
Chemical Name Structure Amino tri (methylene phosphonic acid)
##STR00005## 1-Hydroxyethylidene-1,1,- diphosphonic acid
##STR00006## Hexamethylenediaminetetra (methylenephosphonic acid)
##STR00007## Diethylenetriamine penta(methylene phosphonic acid)
##STR00008## ethylenediamine tetra(methylene phosphonic acid)
##STR00009## bis(hexamethylene triamine penta(methylenephosphonic
acid)) Amino methylene phosphonic acid 2-Phosphonobutane-1,2,4-
tricarboxylic acid ##STR00010## Monoethanloamine diphosphonate
[0103] Suitable polymers for use as passivating agents include
polymers produced from the following monomers. Particularly useful
polymers comprise polymers with these monomers and also comprising
one or more phosphate or phosphonate groups, for example copolymers
comprising vinyl(phosphonic acid) (VPA) and at least one other of
the monomers listed below.
##STR00011##
[0104] For PEG-MA and PEG-MA-ME, n is generally chosen such that
the molecular weight is between about 100 and 10,000 or about 200,
400, or 1000. For example n can be from about 1 to about 1000 or
from about 10 to about 100
[0105] Examples of other suitable negatively charged 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] Optionally, 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. 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.
[0107] 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.
[0108] 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.
[0109] Selective passivation can be accomplished with multiple
layers of phosphorous containing polymers such polymers comprising
polyphosphonates. An effective approach to such multilayers uses
multivalent cations to assemble the polyphosphonates containing
polymers. Particularly useful multivalent cations are those of
transition metals, and in particular those of group IV transition
metals, titanium (Ti), zirconium (Zr), or hafnium (Hf). A
multilayer can be constructed with alternating layers. The
multilayers can be produced by alternately treating the surface
with a transition metal compound such as HfCl.sub.2, ZrOCl.sub.2,
or ZrCl.sub.4, and then with the phosphonate-containing polymer
such as PVPA or Cp30. An advantage of a multilayer process is that
while the process requires extra steps, it can result in the
filling in of defect sites that would be present with a single
passivation step.
[0110] In some cases, the transition metal compound and conditions
can be chosen to have little or substantially no reactivity with
SiO.sub.2. In other cases, a transition metal compound, e.g.
HfOCl.sub.2 can be used under conditions where it also reacts with
SiO.sub.2. This approach can be used, for example where the silica
based surface has been functionalized prior to a passivation step.
In this case, the transition metal compound may react with any
unfunctionalized portions of the silica surface in order to
accomplish passivation.
[0111] The transition metal compounds can be introduced to the
surface in solution, for example in water, methanol or a mixture of
water and methanol. The number of layers can be 2, 3, 4, 5, 6, 7,
8, 9, 10 or more than 10 layers. In some embodiments 3 to 6 layers
are used.
[0112] While polymeric phosphorous containing polymers are
generally preferred, in some cases, monomeric compounds such as
bisphosphonic acids, alkyl phosphonic acids, or other phosphonic
acids can be used. In some cases, the phosphonic acid can
incorporate another passivating group such as, for example,
ethylene glycol, polyethylene glycol, or carboxylate
functionality.
[0113] Selective passivation is generally carried out by exposing
the substrate surface to a solution comprising the selective
passivation compounds mixed with a solvent. The solvent used is
typically one that substantially dissolves the passivating
compound. The solvents will generally comprise polar solvents. In
some cases, an aqueous solvent are preferred. While not being bound
by theory, it is believed that the use of an aqueous solvent
assists in providing specificity of the passivation to the metal or
metal oxide regions. In some cases, the aqueous solvent comprises a
mixture of water and an alcohol, for example, water/ethanol or
water/methanol. The pH of the solution can affect the level of
specificity which is obtained. A pH of between about 0 and 5 is
generally used for the passivation reaction. In some cases a pH
between about 1 and 4 used. In some cases a pH between about 3 and
4 is used.
[0114] The passivation agent, such as PVPA in aqueous solution, can
be provided in an aqueous solution at any suitable concentration.
The solution can have from about 5% of the passivation agent, to
about 95% (w/w or w/v) of passivation agent. The selective
passivation reaction can be carried out at a temperature and for a
time that will allow for passivation reaction to occur. In some
cases, temperatures from 20.degree. C. to 100.degree. C. are used.
In some cases, temperatures from 60.degree. C. to 90.degree. C. are
used. The reaction times generally range from minutes to hours.
[0115] In some cases, the addition of salts can improve the
passivation. In some cases, salts having sodium (Na.sup.+) are
present. In some cases, the addition of bivalent salts such as
Ca.sup.++ can be beneficial with respect to passivation.
[0116] It will be understood that the passivation step will often
be preceded by one or more washing steps to remove contaminants
from the surfaces. Pretreatment steps can also be used, for example
to put the metal into the state desired for subsequent reaction. In
some cases, a reduction step will be used to produce, for example,
a pure metal surface having little or no oxide. In other cases, the
surface will intentionally be exposed to an oxidizing environment
such as exposure to air in order, for example, to provide a metal
having oxide to which a passivating compound can bind.
[0117] The level of selective passivation can be evaluated by a
variety of techniques that are known in the art of surface
characterization. For example, techniques such as X-ray
photoelectron spectroscopy (XPS), contact angle, or ellipsometry
can be used to characterize the level of selectivity of the
coating.
E. Functionalizing the Silica-Based or Transparent Layer
[0118] The methods of the invention generally include a step in
which the silica-based or transparent layer is functionalized. This
functionalization is carried out using a functionalizing agent or
coupling agent which reacts with the silica-based or transparent
surface. As used herein, a coupling agent is generally a compound
that binds to the silica or transparent surface, and also comprises
a coupling group that can react with another compound, for example,
to bind a molecule of interest such as an enzyme to the surface. A
functionalizing agent is a compound that binds to the silica-based
or transparent surface that does not necessarily have a separate
coupling group for subsequent binding of another compound. The
functionalizing agent will generally provide a characteristic or
functionality to the silica or transparent region. Such
characteristic or functionality could be a chemical or physical
characteristic. For example, the functionalizing agent could
comprise optically detectable agents that are sensitive to the
medium into which the surface is disposed. The terms coupling agent
and functionalizing agent are not mutually exclusive. In some cases
a functionalizing agent could comprise a reactive group and thus
could be used as a coupling agent.
[0119] Coupling of functional groups to the transparent materials
may 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 transparent material types, e.g., polymeric materials, 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.
[0120] Where the transparent material comprises a silica-based
surface, silanes (e.g., methoxy-, or ethoxy-, silane reagents) can
form stable bonds with silica surfaces via Si--O--Si bond
formation, and are less reactive to metal or metal oxide surfaces
such as aluminum or aluminum oxide surfaces under appropriately
selected reaction conditions (e.g., vapor phase, solution-based
treatments). 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 bind desired
molecules to silica surfaces such as those in a ZMW.
[0121] In some cases, the coupling groups are activatable or
deactivatable coupling groups. A variety of different activatable
or deactivatable 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.
Suitable activatable and deactivatable coupling groups are
provided, for example, in U.S. patent application Ser. No.
11/394,352.
[0122] 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 or SNAP-TAGS
and their substrates (Covalys Biosciences AG; the SNAP-TAG is a
polypeptide based on mammalian
O6-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. Click chemistry
including the Azide-Alkyne Huisgen Cycloaddition catalyzed, for
example, by copper can also be used.
[0123] 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. A
preferred set of embodiments utilizes biotin to attach a molecule
of interest to the silica-based or transparent substrate. The
attachment of biotin or other selective binding group to the
surface can be accomplished in a number of ways.
[0124] One exemplary approach involves reacting a silica-based
surface region with a compound having a silane group directly
coupled to the selective binding group, for example, a
silane-polyethylene glycol-biotin compound to produce a surface
having selective binding groups, e.g. biotin bound to the
silica-based region. This method provides a one step process for
obtaining a silica-based surface having selective binding groups
such as biotin attached thereto. In some cases, the silane compound
having the selective binding group is diluted with a silane that
does not contain the selective binding group, e.g.
silane-polyethylene glycol in order to control the density of
selective binding groups on the silica-based surface.
[0125] Another exemplary approach involves first reacting a
silica-based surface with a coupling agent, and reacting the
coupling agent on the surface with an attaching agent that has both
functionality for reacting with the coupling agent, and
functionality for attaching the desired molecule (e.g. a selective
binding agent such as. Biotin). For example, the silica-based
surface is reacted with an aminosilane or thiol-silane under
conditions where the aminosilane or thiol-silane becomes bound to
the substrate. The aminosilane or thiol-silane surface is
subsequently reacted with an attaching agent, for example having an
activated ester coupled to biotin to link the biotin to the
aminosilane surface, or a maleimide group coupled to biotin to link
to the thiol-silane surface. The attaching agent can be diluted as
described herein with molecules that react, for example, with the
aminosilane or thiol-silane, but do not have selective binding
groups. This process incorporating an attaching group results in
the coupling the selective binding agent to the surface in two
steps. While this approach uses two steps rather than the one step
described above, it can have some advantages in development,
processing, and quality control.
[0126] The linking chemistry between the coupling agent and the
compound having the selective binding agent can comprise any
suitable linking chemistry. The linking chemistry can comprise, for
example, thiol-maleimide, anhydride-amine, alkyne-azide,
epoxide-amine, or amine-activated ester. As with the one step
method, the compound having the selective binding agent can be
diluted with a compound with the same reactive functionality, but
not having the selective binding agent to control the density of
selective binding agent on the surface.
[0127] In some cases, the compound comprising the selective binding
agent is not diluted with another agent such as a capping agent
that can bind to the surface, but does not have selective binding
agent. Where there is no dilution, a relatively highly density of
selective binding agent can be achieved. This high level of
selective binding agent allows either for attaching a relatively
high density of molecules of interest to the surface, or can be
used to attach relatively few molecules of interest to the
surface.
[0128] In some cases, the compound comprising the selective binding
agent is diluted with another agent such as a capping agent that
can bind to the surface, but does not have selective binding agent.
In accordance with the invention, the low density of the coupling
agent on a surface is designed to provide a single reactive moiety
within a relatively large area for use in certain applications,
e.g., single molecule analyses, while the remainder of the area is
substantially non-reactive. As such, coupling groups can be diluted
to provide a low density of reactive groups that are typically
present on a substrate surface at a density of reactive groups of
greater than 1/1.times.10.sup.6 nm.sup.2 of surface area, but less
than about 1/100 nm.sup.2. In more preferred aspects, the density
of reactive groups on the surface will be greater than 1/100,000
nm.sup.2, 1/50,000 nm.sup.2, 1/20,000 nm.sup.2 and 1/10,000
nm.sup.2, and will be less than about 1/100 nm.sup.2, 1/1000
nm.sup.2, and 1/10,000 nm.sup.2. For certain preferred
applications, the density will often fall between about 1/2500
nm.sup.2 and about 1/300 nm.sup.2, and in some cases up to about
1/150 nm.sup.2.
[0129] The conditions for the attachment of the molecule of
interest can be controlled such that, for example, only one
molecule of interest or one active molecule of interest is
delivered to one or more optical confinements on a surface. In some
cases, the conditions for the attachment of the molecule of
interest are controlled such that 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90% or more of the optical confinements have only one
molecule of interest or one active molecule of interest. Approaches
for obtaining a high fraction of optical confinements having one
molecule of interest is described in copending U.S. patent
application Ser. No. 12/384,097, which is incorporated herein by
reference for all purposes.
[0130] 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. Many silica-based surfaces can be rendered
negatively charged under the appropriate conditions in order to
facilitate this approach. 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. Other polycationic
blocks can include, for example, PEI (poly(ethylenimine), PDDA
(poly(diallyldimethyl ammonium chloride), and PAH (poly(allylamine
hydrochloride). In some cases, poly(methyl methacrylate) (PMMA) and
copolymers thereof can bee used to functionalize the transparent or
silica-based portions of the substrate.
[0131] Phospholipid chemistries can also be used to functionalize
the surface of the transparent or silica-based portions of the
substrate. Chemistries using 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 surface of the transparent or silica-based portion of the
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.
[0132] According to the methods of the invention, the exposure of
the transparent or silica-based surface is performed in the
presence of passivated opaque or reflective surfaces such as metal
or metal oxide surfaces. The treatment of the transparent or
silica-based surface can be performed in a selective manner, such
that relatively more of the functionalizing agent or coupling agent
is bound to the transparent or silica-based surface than is bound
to the opaque or reflective surface. The treatment can even be
carried out in a highly selective manner, whereby significantly
more functionalizing agent or coupling agent is bound to the
transparent or silica-based surface than is bound to the opaque or
reflective surface. However, we have found that for some
applications, such as many single-molecule detection applications,
even a highly selective treatment will result in more
functionalizing agent or coupling agent bound to the opaque or
reflective area than is desired. Where this is the case, a
subsequent selective removal step can be employed to remove the
unwanted groups from the opaque or reflective regions.
F. Selective Removal of Coupling Agent or Functionalizing Agent
[0133] We have found that surfaces having the desired low level of
functionalizing agent or coupling agent bound to the opaque or
reflective regions can be obtained by following the steps described
above followed by a step involving the selective removal of
unwanted functionalizing agent or coupling agent from the opaque or
reflective regions while removing little to no functionalizing
agent or coupling agent from the transparent or silica-based
regions. The use of a selective removal step can allow for the use
of less-specific chemistry for functionalizing the transparent or
silica surfaces than might otherwise be used without the selective
removal step.
[0134] The selective removal compounds of the invention will
generally remove or deactivate a significant portion of the
functionalizing agent or coupling agent that is bound to, or
otherwise associated with the passivated opaque or reflective
surfaces of the substrate. In some embodiments, the selective
removal agent will remove or deactivate substantially all of the
functionalizing agent or coupling agent bound or associated with
the passivated opaque or reflective surfaces of the substrate. The
deactivation of the functionalizing agent can occur in various
ways. Deactivation of the functionalizing agent can include
chemical reaction with the functionalizing agent, and can include
covering or sequestering the functionalizing agent. In some
embodiments the selective removal agent will remove or deactivate
greater than 99.9%, greater than 99%, greater than 98%, greater
than 95%, greater than 90%, greater than 80%, greater than 75%,
greater than 70% or greater than 60% of the functionalizing agent
or coupling agent that is bound to, or otherwise associated with
the passivated opaque or reflective surfaces of the substrate.
[0135] A suitable selective removal agent will leave enough
functionalizing agent on the transparent or silica-based surface to
allow for the substrate to function, e.g. for molecular analysis.
In some embodiments, the selective removal agent will remove little
or no functionalizing agent or coupling agent from the transparent
or silica-based surfaces. In some embodiments, the selective
removal agent will remove substantially no functionalizing agent or
coupling agent from the transparent or silica-based surfaces of the
substrate. In some embodiments the functionalizing agent will
remove less than 0.1%, less than 1%, less than 2%, less than 5%,
less than 10%, less than 15%, less than 20%, less than 25%, less
than 30%, or less than 50% of the functionalizing agent or coupling
agent from the transparent or silica-based surfaces of the
substrate.
[0136] Suitable selective removal agents include acidic compounds,
for example, compounds having one or more functional groups with a
pKa of less than about 6, less than about 5, less than about 4,
less than about 2, less than about 1 or lower than 1. We have found
that the acidic selective removal agents are particularly useful
where the opaque or reflective layer comprises a metal or metal
oxide, for example aluminum or aluminum oxide.
[0137] The selective removal agents include compounds having, for
example carboxylic acid (--CO.sub.2H), sulfonic acid (-SO.sub.3H),
phosphonate (--PO.sub.3H.sub.2), or phosphate (--OPO.sub.3H.sub.2)
functional groups or combinations thereof.
[0138] Particularly useful selective removal agents comprise
polymers having acidic functionality. These polymers include, for
example, carboxylic acid groups, e.g. acrylates, including
poly(acrylic acid) (PAA), and poly(methacrylic acid), sulfonic acid
groups, e.g. poly(vinylsulfonic acid) (PVSA), phosphonic acid
groups, e.g. poly(vinylphosphonic acid) (PVPA), or phosphoric acid
groups or copolymers having two or more of these groups.
[0139] While not to be bound by theory, it is believed that the
polymeric selective removal agents are beneficial in some cases
because they can effectively remove functionalizing agents or
coupling agents from the passivated opaque or reflective surfaces,
yet because of their higher molecular weight, will not diffuse
through a passivation layer to react with the underlying opaque or
reflective layer, causing unwanted reactions, for example
corrosion, especially in the case of a metal layer. In some cases
it is desirable to minimize or eliminate corrosion, which can
remove the passivation layer on the opaque or reflective layer, and
can result in removal of portions of the layer, causing pitting,
and changing its dimensions, e.g. thinning the layer, and enlarging
apertures.
[0140] The number average molecular weight (Mn) of the polymeric
selective removal agents can be from about 1,000 to about 100,000
or from about 5,000 to about 50,000.
[0141] Polymeric selective removal agents can comprise
poly(vinylsulfonic acid) PVSA, having the structure:
##STR00012##
[0142] wherein n can be about 1 to about 1000, or about 10 to about
100. In some cases, n is selected to provide a suitable molecular
weight for acting as a selective removal agent. In the molecular
weight is from about 1,000 to about 100,000 or from about 1,000 to
about 50,000. In some embodiments, the PVSA has a number average
molecular weight of Mn from about 4,000 to about 9,000. In some
embodiments the PVSA has a polydispersity from about 1.2 to about
1.6.
[0143] Selective removal agents of the invention can also comprise
poly(styrenesulfonic acid) (PSSA), and poly(styrenesulfonic
acid-co-maleic acid) (PSSA-MA) with the structures shown below.
##STR00013##
[0144] wherein n and m are selected to provide a suitable molecular
weight for acting as a selective removal agent. In some cases n and
m are from 1 to about 1000. In some cases, m and n are from about
10 to about 500.
[0145] Suitable copolymers include compounds comprising PAA-PVSA,
PAA-PVPA, PVSA-PVPA. In some cases, the polymer or copolymer
selective removal agents have attached to them polyethylene glycol,
creating PEG-ylated polymers.
[0146] One class of selective removal agents comprise compounds
having the structure below:
##STR00014##
[0147] wherein n and m are selected to provide a suitable molecular
weight for acting as a selective removal agent. In some
embodiments, n is from about 1 to about 1000, and m is from about 1
to about 1000. In some embodiments, n is from about 10 to about
100, and m is from about 10 to about 100. In some embodiments n is
about 50 and m is about 50. One preferred embodiment comprises
AQUARITE ESL, available from Rhodia, Inc.
[0148] Other exemplary copolymers which comprise selective removal
agents of the invention include the copolymers below:
##STR00015##
[0149] wherein n and m are selected to provide a suitable molecular
weight for acting as a selective removal agent. In some
embodiments, n is from about 1 to about 1000, and m is from about 1
to about 1000. In some embodiments, n is from about 10 to about
100, and m is from about 10 to about 100.
[0150] For copolymers described herein having two polymeric
regions, the ratio of the regions can be about 10:90, 20:80, 30:70,
40:60, 50:50, 60:40, 70:30, 80:20, or 90:10 (molar or weight
ratio).
[0151] Suitable materials for use as a selective removal agent
include DEQUEST compounds available from ThermPhos Trading GmbH;
including, for example, DEQUEST P9000 a homopolymer of maleic acid,
DEQUEST P9020, a modified polyacrylic acid, sodium salt, and
DEQUEST P9030, a sulphonated polyacrylic acid copolymer.
[0152] Generally, corrosion of the opaque or reflective layer is
undesirable, and should be minimized or eliminated. However, there
are certain instances, such as those for creating an island of
functionalizing agent or coupling agent as described in U.S. patent
application Ser. No. 12/384,097, filed Mar. 30, 2009, where
corrosion, if controlled, can be beneficial.
[0153] The substrates can be treated with the selective removal
agents using methods described herein and methods known in the art.
The selective removal agents are typically delivered to the
substrates in solution, but other methods of delivery, such as
delivery of the selective removal agents in a gaseous form can be
used. The solutions of the selective removal agent for treatment of
the substrate will generally utilize solvents in which the
selective removal agent is substantially soluble. In some cases
aqueous solutions are used.
[0154] An exemplary method of treating the substrate with selective
removal agents comprises putting a solution containing the
selective removal agent into contact with the substrate and
bringing the solution to a temperature for a period of time. In
some cases agitation or stifling of the solution is carried out
during such time. The solution is then removed, after which the
substrate may be rinsed, for example, with pure solvent, e.g. pure
water, and dried.
[0155] The concentration of the selective removal agent will
typically be between 0.01% and 20% (weight by volume, e.g. g/mL).
In many cases the concentration of the selective removal agent will
be less than 1%, for example between 0.1% and 0.8% or between 0.2%
and 0.6%.
[0156] The treatment with selective removal agent is generally
carried out under acidic conditions, e.g. at a pH of less than 6.
In some cases it is carried out at a pH of less than 5, less than
4, less than 3, less than 2, less than 1, or lower. The treatment
with selective removal agent can be carried out at between pH 6 and
pH 0, between pH 5 and pH 1, between pH 4 and pH 2. We have found
that in some cases, the selective removal reaction tends to proceed
more rapidly at lower pH. In some cases, if the pH is too low, it
is difficult to control the level of corrosion. The reaction can be
carried out at any effective temperatures. For example,
temperatures from 20.degree. C. to 100.degree. C. can be employed.
In some cases temperatures between 40.degree. C. and 95.degree. C.
are used. In some cases, temperatures between 80.degree. C. and
90.degree. C. are used. The time for the selective removal reaction
is generally between 1 minute and 1 day. The optimal time may vary
depending, for example, on the pH and temperature employed. Times
between about 10 min. to about 120 min., or between about 20 min.
and about 60 min. can be used.
[0157] In some cases, the addition of salts can improve the
selective removal reaction. In some cases, salts having sodium
(Na.sup.+) are present. In some cases, the addition of bivalent
salts such as Ca.sup.++ can be beneficial with respect to selective
removal.
[0158] In one exemplary embodiment the substrate is a fused silica
lower layer having an aluminum cladding layer disposed upon the
fused silica layer, and having a series of apertures having
diameters between 30 nm and 200 nm extending through the aluminum
cladding layer. The aluminum cladding layer is exposed to air such
that the surface of the aluminum would comprise aluminum oxide. The
substrate is immersed in an aqueous solution comprising a
phosphonate containing polymer such as AQUARITE CP-30 or PVPA,
heated, and with deionized water. This treatment results in the
selective coating of the aluminum portions of the surface of the
substrate. The substrate is then immersed in a solution of silane
coupling agent and treated in a manner such that the silane
coupling agent becomes bound to the fused silica portions of the
surface. While the silica binds preferentially to the fused silica
surface as compared to the passivated aluminum surface, there is a
measurable amount of silane coupling agent bound to the aluminum
regions. The substrate is submersed into an aqueous solution of
selective removal agent, e.g. PVSA, and heated. The substrate is
rinsed with deionized water and dried. The resulting surface may
have a high density of coupling agent on the fused silica regions,
and very low to undetectable amounts of coupling agent on the
aluminum portions.
[0159] While not being bound by theory, it is believed that where
silane functionalizing agents or silane coupling agents are used,
that in some cases, the selective removal agent acts by the
selective scission of P--O--Si, and/or Metal-O--Si (e.g. Al--O--Si)
bonds on metal portions of the substrate while not reacting with,
or reacting slowly with the Si--O--Si bonds connecting the silanes
to the silica surface.
G. Molecules of Interest--Attachment
[0160] The methods described herein can be used, for example, for
the selective attachment of one or molecules of interest to a
transparent or silica-based region of the surface of a substrate.
These molecules of interest can thereby be disposed into reaction
and or observation regions, such as within an optical
confinement.
[0161] The molecules of interest are generally attached to the
coupling agents selectively placed onto the transparent or
silica-based portions of the surface as described above. A variety
of chemistries are available for specifically attaching a molecule
of interest to the coupling agents bound to the surface.
[0162] For example, where biotin is bound to the transparent or
silica-based regions of the surface, this surface can be used to
attach the molecule of interest using a binding agent such as
streptavidin, which has a very high affinity for biotin. In one
approach, the molecule of interest has a biotin tag which can then
be attached to the surface using an intermediate binding agent,
e.g., streptavidin, which acts to bind to both the surface and the
molecule of interest. In another approach, streptavidin is attached
directly to the molecule of interest.
[0163] A variety of analytes can be delivered to
reaction/observation regions using the methods and compositions
herein. These include enzyme substrates, nucleic acid templates,
primers, etc., as well as polypeptides such as enzymes (e.g.,
polymerases).
[0164] A wide variety of nucleic acids can be analytes in the
methods herein. These include cloned nucleic acids (DNA or RNA),
expressed nucleic acids, genomic nucleic acids, amplified nucleic
acids cDNAs, and the like. Details regarding nucleic acids,
including isolation, cloning and amplification can be found, e.g.,
in Berger and Kimmel, Guide to Molecular Cloning Techniques,
Methods in Enzymology volume 152 Academic Press, Inc., San Diego,
Calif. (Berger); Sambrook et al., Molecular Cloning--A Laboratory
Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., 2000 ("Sambrook"); Current Protocols in
Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a
joint venture between Greene Publishing Associates, Inc. and John
Wiley & Sons, Inc; Kaufman et al. (2003) Handbook of Molecular
and Cellular Methods in Biology and Medicine Second Edition Ceske
(ed) CRC Press (Kaufman); and The Nucleic Acid Protocols Handbook
Ralph Rapley (ed) (2000) Cold Spring Harbor, Humana Press Inc
(Rapley).
[0165] Similarly, a wide variety of proteins, e.g., enzymes, can
also be delivered using the methods herein. A variety of protein
isolation and detection methods are known and can be used to
isolate enzymes such as polymerases, e.g., from recombinant
cultures of cells expressing the recombinant polymerases of the
invention. A variety of protein isolation and detection methods are
well known in the art, including, e.g., those set forth in R.
Scopes, Protein Purification, Springer-Verlag, N.Y. (1982) and
Handbook of Bioseparations, Academic Press (2000). Sambrook,
Ausubel, Kaufman, and Rapley supply additional useful details.
[0166] For a description of polymerases and other enzymes that are
active when bound to surfaces, which is useful in single molecule
sequencing reactions in which the enzyme is fixed to a surface
(e.g., to a particle or to a wall of a reaction/observation region,
e.g., in a ZMW), e.g., conducted in a ZMW, see Hanzel et al. ACTIVE
SURFACE COUPLED POLYMERASES, WO 2007/075987 and Hanzel et al.
PROTEIN ENGINEERING STRATEGIES TO OPTIMIZE ACTIVITY OF SURFACE
ATTACHED PROTEINS, WO 2007/075873). For a description of
polymerases that can incorporate appropriate labeled nucleotides,
useful in the context of sequencing, see, e.g., Hanzel et al.
POLYMERASES FOR NUCLEOTIDE ANALOGUE INCORPORATION, WO 2007/076057.
For further descriptions of single molecule sequencing applications
utilizing ZMWs, see Levene et al. (2003) "Zero Mode Waveguides for
single Molecule Analysis at High Concentrations," Science
299:682-686; Eid et al. (2008) "Real-Time DNA Sequencing from
Single Polymerase Molecules" Science DOI:
10.1126/science.322.5905.1263b; Korlach et al. (2008) "Selective
aluminum passivation for targeted immobilization of single DNA
polymerase molecules in zero-mode waveguide nanostructures"
Proceedings of the National Academy of Sciences U.S.A. 105(4):
1176-1181; Foquet et al. (2008) "Improved fabrication of zero-mode
waveguides for single-molecule detection" Journal of Applied
Physics 103, 034301; "Zero-Mode Waveguides for Single-Molecule
Analysis at High Concentrations" U.S. Pat. No. 7,033,764, U.S. Pat.
No. 7,052,847, U.S. Pat. No. 7,056,661, and U.S. Pat. No.
7,056,676, the full disclosures of which are incorporated herein by
reference in their entirety for all purposes. In some cases, the
enzyme can be covalently attached to the substrate through
functional groups on the enzyme such as amine, carboxylate, or
thiol groups, for example with NHS or maleimide linking
chemistry.
[0167] In order to attach an enzyme to the surface, binding
elements can be added to the polymerase (recombinantly or, e.g.,
chemically) including, e.g. biotin, avidin, GST sequences, modified
GST sequences, e.g., that are less likely to form dimers, biotin
ligase recognition (BiTag) sequences, S tags, SNAP-TAGS
(polypeptides based on mammalian
O6-alkylguanine-DNA-alkyltransferase), enterokinase sites, thrombin
sites, antibodies or antibody domains, antibody fragments,
antigens, receptors, receptor domains, receptor fragments, ligands,
dyes, acceptors, quenchers, or combinations thereof.
[0168] Multiple surface binding domains can be added to orient the
polypeptide relative to a surface and/or to increase binding of the
polymerase to the surface. By binding a surface at two or more
sites, through two or more separate tags, the polymerase is held in
a relatively fixed orientation with respect to the surface. Further
details on attaching tags is available in the art. See, e.g., U.S.
Pat. Nos. 5,723,584 and 5,874,239 for additional information on
attaching biotinylation peptides to recombinant proteins.
[0169] In some cases the desired molecule or molecule of interest
can be attached to the surface in a one step process in which a
coupling group, for example biotin, is bound to the transparent or
silica based portion of the surface, and the
[0170] The attached molecule of interest can then be observed using
the optical containments to characterize its behavior.
H. Substrates Having High Levels of Bias
[0171] The methods of the invention can be used to produce
substrate surfaces having high levels of bias. The term bias as
used herein is generally used to refer a measure of a difference in
surface properties of two different surfaces or two different
portions of a surface. The measured bias can represent the
selective attachment of an agent to one portion of the surface over
another portion of the surface. The bias can be represented as a
ratio of a property associated with the agent on one portion of the
surface to that measured on a different portion of the surface. It
will be understood that there may be multiple methods for measuring
bias, and that the bias measured by one method may not provide the
same level of bias as another method. Bias can be measured, for
example, using optical methods (including fluorescence), X-ray
photoelectron spectroscopy (XPS), ellipsometry, or contact
angle.
[0172] The methods of the invention provide for producing
substrates having surfaces with high bias. In particular, the
substrates of the invention can have a transparent or silica-based
surface portion and an opaque or reflective surface portion,
wherein the bias for agents bound to one portion as compared to the
other portion is high. Substrates having these characteristics can
be, for example, arrays of optical confinements. In some cases, the
level of bias can be measured on the array of optical confinements.
In some cases, it is not practical to measure the bias on the array
of optical confinements. In such cases, the bias that is achievable
by a surface preparation method can be determined on a surrogate
surface. For example, where the array of optical confinements
comprises a fused silica layer having a metal cladding layer having
nanometer scale apertures through the cladding, the amount of fused
silica available for the measurement can be small, making the
measurement of bias difficult. In such cases, coupons of fused
silica coupons having regions of metal coated on their top surfaces
can be used to determine the bias that is obtained by a method of
the invention.
[0173] In some cases, the bias represents the relative levels of a
coupling agent on the transparent or silica-based portions of the
substrate as compared to the levels of coupling agent on the opaque
or reflective portions of the substrate. In these cases, bias can
be determined by measuring the amount of attachment of a compound
that reacts with the coupling agent. Examples of chemistries that
can be used for coupling groups and compounds that react with the
coupling groups described herein. Other chemistries are well known
in the art. In some cases, the coupling agent can comprise a
selective binding agent.
[0174] In some cases, the bias represents the relative levels of a
selective binding agent on the transparent or silica-based portions
of the substrate as compared to the levels of selective binding
agent on the opaque or reflective portions of the substrate. For
example, where the selective binding agent is biotin, bias can be
determined by measuring the relative amount of a labeled avidin,
streptavidin, or neutravidin bound to each surface region. In some
cases, for example where fluorescent labels are used, a metal or
metal oxide surface may tend to quench the fluorescence of the
labeled avidin, streptavidin, or neutravidin. In these cases, the
labeled avidin, streptavidin, or neutravidin can be bound to beads,
allowing for fluorescence in the bound state.
[0175] The bias for a substrate treated with a selective binding
agent can also be determined using a molecule of interest, e.g. an
enzyme of interest that is labeled. The enzyme of interest can be
labeled covalently, or the enzyme could be labeled by having the
enzyme bound to a molecule that is labeled. For example, the enzyme
could be bound to a labeled substrate molecule. Where the enzyme of
interest is a polymerase, the polymerase can be bound to a
fluorescently labeled template nucleic acid. For example, a surface
having been selectively functionalized with a biotin containing
selective binding agent as described herein can be reacted first
with neutravidin, and then with an enzyme bound to a fluorescently
bound template where the enzyme comprises a biotin group. The
relative fluorescence measured on the transparent or silica based
portions of the surface relative to that on the opaque or
reflective portions provides a measure of the bias.
[0176] In one aspect, the invention provides an array of optical
confinements having a surface with both a silica-based and a metal
or metal-oxide portion, wherein the array has been treated with a
passivating agent and a coupling agent, wherein when a coupon
having regions with the same type of silica as the silica-based
portions and the same type of metal or metal-oxide as the metal or
metal oxide portions as the array is treated in the same manner as
the array, the coupon exhibits a fluorescent intensity bias of
greater than about 5, greater than about 8, greater than about 10,
greater than about 20, greater than about 30, greater than about
40, greater than about 50, greater than about 60, greater than
about 70, greater than about 80, greater than about 90, or greater
than about 100 in a fluorescent bias assay such as a neutravidin
labeled bead assay. The coupling agent can comprise a selective
binding agent such as biotin.
[0177] In one aspect, the invention provides an array of optical
confinements having a surface with both a silica-based and a metal
or metal-oxide portion, wherein the array has been treated with a
passivating agent and a coupling agent, wherein when a coupon
having regions with the same type of silica as the silica-based
portions and the same type of metal or metal-oxide as the metal or
metal oxide portions as the array is treated in the same manner as
the array, the coupon exhibits a fluorescent intensity bias of
greater than about 5, greater than about 8, greater than about 10,
greater than about 20, greater than about 30, greater than about
40, greater than about 50, greater than about 60, greater than
about 70, greater than about 80, greater than about 90, or greater
than about 100 in a labeled enzyme assay. The coupling agent can
comprise a selective binding agent such as biotin.
[0178] In one aspect, the invention provides an array of optical
confinements having a surface with both a silica-based and a metal
or metal-oxide portion, wherein the array has been treated with a
phosphorous containing passivating agent and a coupling agent;
wherein when a coupon having regions with the same type of silica
as the silica-based portions and the same type of metal or
metal-oxide as the metal or metal oxide portions as the array is
treated in the same manner as the array, the metal or metal oxide
portion of the coupon exhibits an XPS signal for phosphorous of,
greater than about 10, greater than about 20, greater than about
30, greater than about 40, greater than about 50, greater than
about 60, greater than about 70, greater than about 80, greater
than about 90, or greater than about 100 times phosphorous signal
on the transparent, or silica based portion of the coupon.
[0179] In one aspect, the invention provides an array of optical
confinements having a surface with both a silica-based and a metal
or metal-oxide portion, wherein the array has been treated with a
passivating agent and a coupling agent; wherein when a coupon
having regions with the same type of silica as the silica-based
portions and the same type of metal or metal-oxide as the metal or
metal oxide portions as the array is treated in the same manner as
the array, the coupon exhibits an ellipsometric bias of greater
than about 10, greater than about 20, greater than about 30,
greater than about 40, greater than about 50, greater than about
60, greater than about 70, greater than about 80, greater than
about 90, or greater than about 100. The coupling agent can
comprise a selective binding agent such as biotin.
I. Lipid Bilayer Functionalization of Optical Confinements
[0180] An aspect of the invention is the functionalization of the
transparent or silica-based portions of the substrate surface with
a lipid bilayer. The lipid bilayer can then be used to immobilize
the molecule of interest within the optical confinement.
[0181] By choosing the appropriate conditions, the lipid bilayer
can be formed by self-assembly onto the transparent or silica-based
surface. Conditions can be chosen such that the lipid bilayer will
form selectively on the transparent or silica based surfaces and
not on the opaque or reflective surfaces of an array of optical
confinements. For example, an array of optical confinements having
a metal cladding layer on a silica-based surface can be treated by
the methods described herein to selectively passify the metal
cladding layer. The pacified metal cladding layer can be produced
to have different chemical and physical properties that the
silica-based portion, e.g. positive vs. negative, or neutral vs.
negative, such that the lipid bilayer will form only on the, e.g.
negatively charged, silica layer.
[0182] A lipid bilayer is a thin membrane made of two layers of
lipid molecules. Any suitable lipid or mixture of lipids can be
used to functionalize the optical confinement such as a ZMW.
Suitable bilayers include those made mostly of phospholipids, which
have a hydrophilic head and two hydrophobic tails. When
phospholipids are exposed to water, they tend to arrange themselves
into a two-layered sheet (a bilayer) with their tails pointing
toward the center of the sheet. In some cases, a positively charged
head group of the phospholipid can associate with negative groups
on the transparent or silica based surface, thereby anchoring the
lipid bilayer to the surface. The bilayers used in the invention
may also include other molecules which can improve the stability of
the bilayer, such as, for example molecules of cholesterol.
[0183] Suitable lipids include phospholipids including
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
and phosphatidylglycerol. The lipids can comprise a zwitterionic
headgroup, such as that in phosphatidylcholine, which a negative
charge on the phosphate group and a positive charge on the amine
but, because these local charges balance, having no net charge. In
other cases, lipids having a net positive or net negative charge
can be used. Mixtures of lipids having different net charges can
also be used.
[0184] Any suitable lipids that can form a lipid bilayer, either
alone or in combination with other lipids can be used. Suitable
lipids include fatty acids, glycerolipids, glycerophospholipids,
sphingolipids, sterol lipids, prenol lipids, saccharolipids, and
polyketides. Suitable lipids include monoglycerides, diglycerides,
and triglycerides.
[0185] The molecule of interest can be attached to or associated
with the lipid bilayer. For example, the molecule of interest, for
instance an enzyme or other protein, can be attached to one or more
lipid components which will serve to bind or associate the molecule
of interest with the lipid bilayer. The tether can be constructed
such that the molecule of interest is held within the observation
region of the optical confinement. FIG. 7(a) shows an optical
confinement having a silica base layer and an aluminum cladding
with a lipid bilayer disposed upon the silica surface. The molecule
of interest, e.g. an enzyme such as a polymerase, is tethered to a
lipid forming component, thus attaching the enzyme to the bilayer
and to the base of the optical confinement, for example ZMW.
[0186] The tether can either be short, having just a few atoms in
the linking group, or can be long, such as a polymeric tether. The
tether will generally comprise water soluble functional groups. The
linker can comprise, for example polyethylene glycol. The tether
can be made of other water soluble functionality, for example,
polymers with ether, hydroxyl, carboxyl, amine, sugar, or sulfonate
functionality. The tether will generally be from 1 nm to about 50
nm in length.
[0187] The structure having the molecule of interest tethered to
the lipid bilayer can be formed by a variety of methods. In one
method, the lipid membrane is disposed upon the base of the optical
confinement, and the lipid membrane has a functionalized tether
attached to it. Subsequently, the molecule of interest, having a
group that is reactive to the functionalized tether is added.
Chemistries for attaching the molecule of interest to the
functionalized tether include the chemistries described herein for
coupling agents (e.g. maleimide, thiol, amine) and selective
binding agents (e.g. biotin). Alternatively, the membrane can first
be formed on the optical confinement, and the molecule of interest
attached via a tether to a lipid associating component (e.g. a
lipid or membrane protein) can be subsequently added.
[0188] In some cases, the molecule of interest can be associated
directly with the lipid bilayer. FIG. 7(b) shows an optical
confinement wherein the molecule of interest is disposed within the
lipid bilayer. The lipid bilayer is generally hydrophobic in its
interior. Some proteins, e.g. membrane proteins, have hydrophobic
portions which result in their association with membranes. In some
cases, the molecule of interest will comprise a membrane protein,
which will thermodynamically associate with the membrane. In other
cases, the molecule of interest can be modified, for example by
mutation or with the attachment of hydrophobic moieties such that
it will associate with the enzyme. The molecule of interest can
alternatively be attached to a membrane protein, for example in the
form of a fusion protein.
[0189] In some cases the attachment of the molecule of interest can
be carried out such that some or all of the optical confinements on
the optical confinement array have one molecule of interest per
optical confinement.
III. Apparatus
[0190] In certain preferred embodiments, the substrates of the
present invention comprise arrays of optical confinements that are
monitored using an optical system capable of detecting and/or
monitoring interactions between reactants at the single-molecule
level. Such an optical system achieves these functions by first
generating and transmitting an incident wavelength to the
reactants, followed by collecting and analyzing the optical signals
from the reactants. Such systems typically employ an optical train
that directs signals from the reactions to a detector, and in
certain embodiments in which a plurality of reactions is disposed
on a solid surface, such systems typically direct signals from the
solid surface (e.g., array of confinements) onto different
locations of an array-based detector to simultaneously detect
multiple different optical signals from each of multiple different
reactions. In particular, the optical trains typically include
optical gratings or wedge prisms to simultaneously direct and
separate signals having differing spectral characteristics from
each confinement in an array to different locations on an array
based detector, e.g., a CCD, and may also comprise additional
optical transmission elements and optical reflection elements.
[0191] An optical system applicable for use with the present
invention preferably comprises at least an excitation source and a
photon detector. The excitation source generates and transmits
incident light used to optically excite the reactants in the
reaction. Depending on the intended application, the source of the
incident light can be a laser, laser diode, a light-emitting diode
(LED), a ultra-violet light bulb, and/or a white light source.
Further, the excitation light may be evanescent light, e.g., as in
total internal reflection microscopy, certain types of waveguides
that carry light to a reaction site (see, e.g., U.S. Application
Pub. Nos. 20080128627, 20080152281, and 200801552280), or zero mode
waveguides, described below. Where desired, more than one source
can be employed simultaneously. The use of multiple sources is
particularly desirable in applications that employ multiple
different reagent compounds having differing excitation spectra,
consequently allowing detection of more than one fluorescent signal
to track the interactions of more than one or one type of molecules
simultaneously. A wide variety of photon detectors or detector
arrays are available in the art. Representative detectors include
but are not limited to optical reader, high-efficiency photon
detection system, photodiode (e.g. avalanche photo diodes (APD)),
camera, charge couple device (CCD), electron-multiplying
charge-coupled device (EMCCD), intensified charge coupled device
(ICCD), and confocal microscope equipped with any of the foregoing
detectors. For example, in some embodiments an optical train
includes a fluorescence microscope capable of resolving fluorescent
signals from individual sequencing complexes. Where desired, the
subject arrays of optical confinements contain various alignment
aides or keys to facilitate a proper spatial placement of the
optical confinement and the excitation sources, the photon
detectors, or the optical train as described below.
[0192] The subject optical system may also include an optical train
whose function can be manifold and may comprise one or more optical
transmission or reflection elements. Such optical trains preferably
encompass a variety of optical devices that channel light from one
location to another in either an altered or unaltered state. First,
the optical train collects and/or directs the incident wavelength
to the reaction site (e.g., optical confinement). Second, it
transmits and/or directs the optical signals emitted from the
reactants to the photon detector. Third, it may select and/or
modify the optical properties of the incident wavelengths or the
emitted wavelengths from the reactants. In certain embodiments, the
optical train controls an on/off cycle of the illumination source
to provide illuminated and non-illuminated periods to one or more
illuminated reaction sites. Illustrative examples of such optical
transmission or reflection elements are diffraction gratings,
arrayed waveguide gratings (AWG), optic fibers, optical switches,
mirrors (including dichroic mirrors), lenses (including
microlenses, nanolenses, objective lenses, imaging lenses, and the
like), collimators, optical attenuators, filters (e.g.,
polarization or dichroic filters), prisms, wavelength filters
(low-pass, band-pass, or high-pass), planar waveguides,
wave-plates, delay lines, and any other devices that guide the
transmission of light through proper refractive indices and
geometries. One example of a particularly preferred optical train
is described in U.S. Patent Pub. No. 20070036511, filed Aug. 11,
2005, and incorporated by reference herein in its entirety for all
purposes.
[0193] In a preferred embodiment, a reaction site (e.g., optical
confinement) containing a reaction of interest is operatively
coupled to a photon detector. The reaction site and the respective
detector can be spatially aligned (e.g., 1:1 mapping) to permit an
efficient collection of optical signals from the reactants. In
certain preferred embodiments, a reaction substrate is disposed
upon a translation stage, which is typically coupled to appropriate
robotics to provide lateral translation of the substrate in two
dimensions over a fixed optical train. Alternative embodiments
could couple the translation system to the optical train to move
that aspect of the system relative to the substrate. For example, a
translation stage provide a means of removing a reaction substrate
(or a portion thereof) out of the path of illumination to create a
non-illuminated period for the reaction substrate (or a portion
thereof), and returning the substrate at a later time to initiate a
subsequent illuminated period. An exemplary embodiment is provided
in U.S. Patent Pub. No. 20070161017, filed Dec. 1, 2006.
[0194] In particularly preferred aspects, such systems include
arrays of reaction regions, e.g., zero mode waveguide arrays, that
are illuminated by the system, in order to detect signals (e.g.,
fluorescent signals) therefrom, that are in conjunction with
analytical reactions being carried out within each reaction region.
Each individual reaction region can be operatively coupled to a
respective microlens or a nanolens, preferably spatially aligned to
optimize the signal collection efficiency. Alternatively, a
combination of an objective lens, a spectral filter set or prism
for resolving signals of different wavelengths, and an imaging lens
can be used in an optical train, to direct optical signals from
each confinement to an array detector, e.g., a CCD, and
concurrently separate signals from each different confinement into
multiple constituent signal elements, e.g., different wavelength
spectra, that correspond to different reaction events occurring
within each confinement.
[0195] The systems of the invention also typically include
information processors or computers operably coupled to the
detection portions of the systems, in order to store the signal
data obtained from the detector(s) on a computer readable medium,
e.g., hard disk, CD, DVD or other optical medium, flash memory
device, or the like. For purposes of this aspect of the invention,
such operable connection provide for the electronic transfer of
data from the detection system to the processor for subsequent
analysis and conversion. Operable connections may be accomplished
through any of a variety of well known computer networking or
connecting methods, e.g., Firewire.RTM., USB connections, wireless
connections, WAN or LAN connections, or other connections that
preferably include high data transfer rates. The computers also
typically include software that analyzes the raw signal data,
identifies signal pulses that are likely associated with
incorporation events, and identifies bases incorporated during the
sequencing reaction, in order to convert or transform the raw
signal data into user interpretable sequence data (See, e.g.,
Published U.S. Patent Application No. 2009-0024331, the full
disclosure of which is incorporated herein by reference in its
entirety for all purposes).
[0196] Exemplary systems are described in detail in, e.g., U.S.
patent application Ser. No. 11/901,273, filed Sep. 14, 2007 and
U.S. patent application Ser. No. 12/134,186, filed Jun. 5, 2008,
the full disclosures of which are incorporated herein by reference
in their entirety for all purposes.
[0197] Further, as noted above, the invention provides data
processing systems for transforming sequence read data into
consensus sequence data. In certain embodiments, the data
processing systems include machines for generating sequence read
data by interrogating a template nucleic acid molecule. In certain
preferred embodiments, the machine generates the sequence read data
using a sequencing-by-synthesis technology, as described elsewhere
herein, but the machine may generate the sequence read data using
other sequencing technologies known to those of ordinary skill in
the art, e.g., pyrosequencing, ligation-mediated sequencing, Sanger
sequencing, capillary electrophoretic sequencing, etc. Such
machines and methods for using them are available to the ordinary
practioner.
[0198] In another aspect, the invention provides data processing
systems for transforming sequence read data from one or more
sequencing reactions into consensus sequence data representative of
an actual sequence of one or more template nucleic acids analyzed
in the one or more sequencing reactions. Such data processing
systems typically comprise a computer processor for processing the
sequence read data according to the steps and methods described
herein, and computer usable medium for storage of the initial
sequence read data and/or the results of one or more steps of the
transformation (e.g., the consensus sequence data), such as a
computer-readable medium.
[0199] As shown in FIG. 8, the system 800 includes a substrate 802
that includes a plurality of discrete sources of chromophore
emission signals, e.g., an array of zero mode waveguides 804. An
excitation illumination source, e.g., laser 806, is provided in the
system and is positioned to direct excitation radiation at the
various signal sources. This is typically done by directing
excitation radiation at or through appropriate optical components,
e.g., dichroic element 808 and objective lens 810, that direct the
excitation radiation at the substrate 802, and particularly the
array of zero mode waveguides 804. Emitted signals from the array
of zero mode waveguides 804 are then collected by the optical
components, e.g., objective 810, and passed through additional
optical elements, e.g., dichroic element 808, prism 812 and lens
814, until they are directed to and impinge upon an optical
detection system, e.g., detector array 816. The signals are then
detected by detector array 816, and the data from that detection is
transmitted to an appropriate data processing system, e.g.,
computer 818, where the data is subjected to interpretation,
analysis, and ultimately presented in a user ready format, e.g., on
display 820, or printout 822, from printer 824. As will be
appreciated, a variety of modifications may be made to such
systems, including, for example, the use of multiplexing components
to direct multiple discrete beams at different locations on the
substrate, the use of spatial filter components, such as confocal
masks, to filter out-of focus components, beam shaping elements to
modify the spot configuration incident upon the substrates, and the
like (See, e.g., Published U.S. Patent Application Nos.
2007/0036511 and 2007/095119, and U.S. patent application Ser. No.
11/901,273, all of which are incorporated herein by reference in
their entireties for all purposes.).
IV. Uses
[0200] In certain aspects, the subject invention provides
substrates and methods for performing single-molecule observation.
The optical arrays of the invention can provide information on
individual molecules whose properties are hidden in the statistical
mean that is recorded by ordinary ensemble measurement techniques.
In addition, because of multiplexing, the arrays are conducive to
high-throughput implementation, requiring small amounts of
reagent(s), and taking advantage of the high bandwidth of modern
avalanche photodiodes for extremely rapid data collection.
Moreover, because single-molecule counting automatically generates
a degree of immunity to illumination and light collection
fluctuations, single-molecule analysis can provide greater accuracy
in measuring quantities of material than bulk fluorescence or
light-scattering techniques. As such, the subject substrates and
devices may be used in a wide variety of circumstances including
sequencing individual human genomes as part of preventive medicine,
rapid hypothesis testing for genotype-phenotype associations, in
vitro and in situ gene-expression profiling at all stages in the
development of a multi-cellular organism, determining comprehensive
mutation sets for individual clones and profiling in various
diseases or disease stages. Other applications involve profiling of
cell receptor diversity, identifying known and new pathogens,
exploring diversity towards agricultural, environmental and
therapeutic goals.
[0201] In preferred embodiments, the instant invention is directed
to observing nucleic acid sequencing reactions, e.g.,
sequencing-by-incorporation reactions. In preferred embodiments,
such an illuminated reaction analyzes a single molecule to generate
nucleotide sequence data pertaining to that single molecule. For
example, a single nucleic acid template may be subjected to a
sequencing-by-incorporation reaction to generate one or more
sequence reads corresponding to the nucleotide sequence of the
nucleic acid template. For a detailed discussion of such single
molecule sequencing, see, e.g., U.S. Pat. Nos. 6,056,661,
6,917,726, 7,033,764, 7,052,847, 7,056,676, 7,170,050, 7,361,466,
7,416,844; Published U.S. Patent Application Nos. 2007-0134128 and
2003/0044781; and M. J. Levene, J. Korlach, S. W. Turner, M.
Foquet, H. G. Craighead, W. W. Webb, SCIENCE 299:682-686, January
2003 Zero-Mode Waveguides for Single-Molecule Analysis at High
Concentrations, all of which are incorporated herein by reference
in their entireties for all purposes.
V. Examples
Example 1
Preparing Substrates Having High Bias Using: a) Selective
Passivation, b) Functionalization of the Transparent/Silica-Based
Surface, and c) Selective Removal
[0202] The process described herein is applied to various types of
samples including a 1) ZMW array comprising a layer of aluminum on
fused silica having about 3000 apertures with diameters between
about 60 nm and 130 nm; 2) mixed surface coupons having aluminum
and fused silica portions, and 3) separate aluminum substrates and
silicon-oxide substrates, each treated identically.
[0203] The mixed surface coupons can be, for example, fused silica
having deposited onto its surface a 100 nm thick Aluminum.
Substrates are patterned at the wafer level utilizing standard
photolithographic techniques. The finished pattern consists of 1 mm
squares evenly spaced on a fused silica substrate. Prior to
aluminum deposition, the wafers are cleaned in a sulfuric and
hydrogen peroxide mixture (Piranha solution) to remove organic
residues from the surface.
[0204] Where the assay is done on two separate substrates treated
identically, the substrates can be, for example 1) a silicon
substrate with 20 nm thermally grown silicon oxide and 2) a fused
silica wafer having a layer of aluminum. For the silicon substrate
with thermally grown silicon oxide, silicon substrates are
commercially available and are typically 1-12'' diameter wafers
that are approximately 500 microns thick. A 20 nm oxide is grown on
the surface by high temperature oxidation in a tube furnace. In
some instances, the wafer is scribed with a diamond tipped scribing
tool and is broken into small pieces to enable multiple tests per
wafer. To prepare the aluminum coated fused silica substrates, a
100 nm thick Aluminum film is deposited (evaporated or sputtered)
on high purity fused silica was used for passivation testing. The
fused silica wafers are commercially available and are typically
1-12'' that are approximately 500 microns thick. Prior to aluminum
deposition, the wafers are cleaned in a sulfuric and hydrogen
peroxide mixture (Piranha solution) to remove organic residues from
the surface.
[0205] Just prior to surface deposition, samples are plasma treated
in medical grade air for about 2 minutes at a backfill pressure of
1900 mTorr. The plasma treatment removes organic residues from the
surface and provides enhanced bonding for subsequent deposited
molecules. Immediately following plasma treatment, the samples are
placed in a pre-heated (90 C) container containing 0.5% w/v of the
vinyl phosphonic acid-acrylic acid copolymer ALBRITECT CP30
(Rhodia) in water. After 2 minutes, the samples are removed from
the CP30 vessel, rinsed in a stream of water, and then placed in a
90 C oven. After 10 minutes in the oven, the samples are then
placed in a vessel containing biotin-peg-silane dissolved in
ethanol at a concentration of 150 micromolar. After 2 hours, the
samples are removed from the biotin-peg-silane vessel, rinsed in a
stream of methanol, and then placed in a 90 C oven. After 10
minutes in the oven, the samples are placed in a pre-heated (90 C)
vessel containing 0.5% w/v of the acidic phosphonate containing
polymer AQUARITE ESL (Rhodia). After 10 minutes, the samples are
removed from the AQUARITE ESL vessel, rinsed in a stream of water,
and then placed in a 90 C oven for 10 minutes. The samples are then
removed from the oven and either tested immediately or stored in a
dessicated vacuum container for later use.
Example 2
Bead Assay for Determination of Bias by Fluorescence
[0206] Labeled latex beads (488 Oregon Green (OG)(Invitrogen))
coated with neutravidin are used as a bias probe for determining
adsorption bias on a mixed surface, where one of the components (in
this case, the fused silica (fusi)) is functionalized with biotin,
and the other component (e.g. metal) is coated with a protein
rejection agent. Neutravidin, a 53 kD tetrameric protein, forms a
strong bond (Kd .about.10-15 mol/L) with biotin. The bead is
utilized in order to visualize fluorescence on the metallic
surface, which may otherwise be undetectable due to quenching.
[0207] The labeled neutravidin beads are diluted in a pH 7.5 buffer
prior to sample exposure. A small droplet is then pipetted onto a
two-component coupon (consisting of equal parts fused silica and
metal), where the surface of the coupon has been treated to produce
a bias. The droplet and coupon are placed in a humidity chamber to
mitigate evaporation. After 15 minutes, the coupon is washed in
Millipore or DI water and dried in a nitrogen stream. A Typhoon
fluorescence scanner with a 488 laser and 520 notch filter is used
to visualize amount of the labeled beads that adsorbed on the
surfaces. Commercially available software packages (Image J,
ImageQuant) are then used for quantification of the subsequent
fluorescence on each surface component. The bias is defined as the
fluorescence intensity of the fusi surface divided by the intensity
at the metal surface. Bias numbers in excess of 100:1 have been
regularly observed.
[0208] Substrates treated as described in Example 1 were assayed
for bias using the fluorescent neutravidin bead assay. After
thorough washing, the coupon was placed on a typhoon scanner that
employed a 488 laser excitation and 520 nm notch filter. FIGS. 9
(A) and (B) show Typhoon fluorescence scanner image of 488
nm-fluorescently labeled latex beads coated with neutravidin
immobilized to the surface of a mixed substrate consisting of three
Al squares deposited on a fused silica substrate (regions that are
fluorescent appear dark in the figure). FIG. 9A (above) shows the
fluorescent scanner image protein adsorption following the first
two steps of the process where the surface consists of both metal
passivating agent and fused silica functionalizing agent. FIG. 9A
(below) shows a schematic illustration of the surface after the
treatment of the substrate with silane-PEG-Biotin and before the
selective removal step. FIG. 9(B) (above) shows the fluorescent
scanner image after the selective removal step, demonstrating the
selective removal of the functionalizing agent from the metal as
evidenced by the low fluorescence (white appearance) on the Al,
while the fused silica substrate retains the functionalizing
reagent in order to immobilize protein. FIG. 9(B) (below) shows a
schematic illustration of the substrate after the selective removal
step. FIG. 9(C) shows a Typhoon fluorescence scanner image of a
single square of Al deposited on the fused silica substrate
(regions that are fluorescent appear dark in the figure) after the
selective removal process. The bias of the coupon, defined as the
ratio of fluorescence intensity on the fused silica surface to that
of the Al surface, in the figure is 135:1.
Example 3
Labeled Template Assay for Determination of Bias
[0209] An alexa-488 labeled IDT-32 primer annealed to a 72base
mini-circle DNA template and subsequently immobilized to phi29 DNA
polymerases is used as a bias probe. The metal-oxide, atomic layer
deposition (ALD) alumina is generally used in this case to mitigate
metal fluorescence quenching.
[0210] The labeled primer/template complex is immobilized to DNA
polymerase in a DNA low-bind tube in a suitable buffer for 15
minutes prior to sample exposure. A small droplet is then pipetted
onto a two-component coupon (consisting of equal parts fusi and
metal-oxide), where the surface of the coupon has been treated. The
droplet and coupon are placed in a humidity chamber to mitigate
evaporation. After 15 minutes, the coupon is washed in Millipore or
DI water and dried in a nitrogen stream. A Typhoon fluorescence
scanner with a 488 laser and 520 notch filter is used to visualize
amount of protein that adsorbed on the surfaces. Commercially
available software packages (Image J, ImageQuant) are used for
quantification of the subsequent fluorescence on each surface
component. The bias is defined as the fluorescence intensity of the
fusi surface divided by the intensity at the metal surface. Bias
numbers in excess of 10:1 and in some cases, 100:1 have been
observed for surfaces prepared as described herein.
Example 4
Assay for the Specificity of Biotin-Neutravidin Binding
[0211] 488 OG (Invitrogen) labeled neutravidin is used as a
specificity probe. To quantify the specificity of immobilization to
surfaces functionalized with biotin, such as the transparent or
silica-based surface, blocked neutravidin can be used. Blocked
neutravidin is produced by mixing neutravidin in a concentrated
biotin buffer solution (1 uM biotin, pH7.5). As a result, the
blocked neutravidin binding sites are filled prior to exposure to
the biotinylated surface. The specificity of immobilization is
determined by measuring the ratio fluorescent signal from portions
of the surface treated with unblocked and biotin-blocked
neutravidin.
[0212] Equal volumes of unblocked and biotin blocked neutravidin
are diluted to 35 nM in pH7.5 buffer. One small droplet of each
solution (blocked/unblocked) is then pipetted in close proximity
onto a fused silica coupon, where the surface of the coupon has
been treated. The droplets and coupon are placed in a humidity
chamber to mitigate evaporation. After 15 minutes, the coupon is
washed in Millipore or DI water and dried in a nitrogen stream. A
Typhoon fluorescence scanner with a 488 laser and 520 notch filter
is used to visualize amount of protein that adsorbed on the
surfaces. Commercially available software packages (Image J,
ImageQuant) are used for quantification of the subsequent
fluorescence on each surface component. The specificity is defined
as the fluorescence intensity of the unblocked portion of the
neutravidin surface divided by the intensity of the biotin-blocked
portion of the neutravidin surface. Specificity numbers in excess
of 100:1 have been measured for the surfaces prepared as described
herein.
Example 5
Assay for Bias by Ellipsometry
[0213] Immobilization bias is confirmed by measurements of the
ellipsometric thickness of the adsorbed protein layers on the two
different surfaces, each treated identically. Because the
ellipsometry measurement requires a relatively large area, in some
cases, separate silica-based and metal or metal oxide based samples
are treated identically and used. A variable angle spectroscopic
ellipsometer (VASE) (J. Wollam) is used to assess changes in
thickness due to protein immobilization (e.g. neutravidin or
streptavidin) on a silicon dioxide surface and aluminum surface.
The ellipsometric bias is defined as the change in thickness on the
silicon oxide surface divided by the change in thickness on the
aluminum oxide surface (.DELTA.d.sub.Si/.DELTA.d.sub.Al).
[0214] Commercially available neutravidin (MW.about.60 kD) is used
as the protein assay probe due to its high specificity to biotin.
Stock concentrations of neutravidin are diluted to 30 nM in pH7.5
MOPS buffer and vortexed thoroughly. An 8 uL aliquot of the dilute
neutravidin reagent is pipetted onto a surface prepared in the
methods described above. The samples are then placed in a humidity
chamber to mitigate evaporation. After 15 minutes the sample is
removed and rinsed in a stream of nanopure water and then dried in
a nitrogen stream. The samples were then mounted on the VASE and
psi and delta values were acquired across the visible wavelength
range (400 to 700 nm). A four layer model is employed to correlate
psi and delta to film thicknesses which included the ambient (air),
protein layer, oxide, and metal. Dielectric constants are input
from literature values (Palik). Example ellipsometric bias results
are shown in FIG. 10.
[0215] In FIG. 10, the gray columns represent the measured
ellipsometric thickness on the Al and SiO2 surface regions before
passivation, indicating a significant amount of binding of the
neutravidin to the aluminum surface. The dark columns in FIG. 10
represent the measured ellipsometric thickness of the Al and SiO2
surfaces after treatment by a method of the invention. It can be
seen that after the treatment, very little of the neutravidin was
on the Al surface, while the amount bound to the SiO2 surface is
very similar to the amount bound prior to treatment. The
ellipsometric bias can be quantified, for example, by taking the
ratio of the dark columns. For the experiment shown in FIG. 10,
ellipsometric bias was determined to be about 60.
Example 6
Bias by XPS
[0216] Coupons of fused silica having regions of aluminum deposited
thereon were treated as described in Example 1. Phosphonate
deposition on aluminum was confirmed by X-ray photoelectron
spectroscopy by the presence of peaks attributable to phosphorous.
In this method, the passivation bias (defined here as the ratio of
the atom % of individual components to a metal passivating molecule
(e.g. atom % of P from CP30) from the metal and glass surfaces,
respectively (% P.sub.Al:% P.sub.fusi)), can be qualitatively
assessed. Measurements showed that the % P on Al was 10% and was
<0.02% on fusi (below detection limit)
Example 8
Observation of DNA Polymerase Activity--Single Molecule
Sequencing
[0217] A zero-mode waveguide array having about 3000 apertures
through a 100 nm layer of Al on fused silica is treated as
described in Example 1. Prior to sequencing, a single DNA
polymerase molecule having a biotin label is complexed with a
double stranded and primed DNA to form a polymerase-template-primer
complex. This complex is immobilized on the biotin functionalized
fused silica substrate of the zero-mode waveguide using
streptavidin or neutravidin. A sequencing reaction was carried in
the manner described in Eid et al. Science, 323, 134-138 (2009).
The zero-mode waveguide array was exposed to a solution comprising
labeled nucleotide bases. Each base was labeled with a unique
fluorescent marker (organic dye) that served as signatures for
detection, 555-T, 568-G, 647-A and Cy5.5-C. Following
immobilization of the DNA polymerase/template complex, the four
bases were added in equal concentrations in buffer were added to
the system along with manganese to catalyze the reaction. The
fluorophores were excited by 532 nm and 641 nm lasers. Fluorescence
emission was monitored using a cooled CCD camera and the time
averaged spectra were converted to trace data acquired prior to
data acquisition. FIG. 11 shows a portion of a data trace from the
single molecule real-time four-color sequencing reaction in a
zero-mode waveguide. The data has been analyzed using base calling
software in order to correlate the observed peaks with the labeled
bases that comprise DNA, (A) adenine, (C) cytosine, (G) guanine and
(T) thymine.
[0218] Several aspects of the sequencing system were markedly
improved by employing surfaces prepared by the methods described
above as compared to surfaces prepared in other ways, including
lower surface stickiness (lower background (non-sequencing related)
fluorescent signal), higher sequencing yield of statistically
significant sequencing and higher overall sequencing accuracy. An
experiment was performed in which single molecule sequencing
reactions were carried out using a zero-mode-waveguide treated by a
method of the invention, and the results were compared to
sequencing reactions identically performed on a surface treated in
another manner, referred to herein as a PDMS treated surface. PDMS
surfaces were produced through repetitive steps of deposition poly
dimethyl siloxane source and subsequent oxygen plasma treatment.
The result of the PDMS process is a ZMW array that is passivated
with a thin layer of SiOx. As compared to the PDMS treated ZMW,
fluorophore stickiness decreased by 7.times. as compared with PDMS
passivated surfaces. The decrease in stickiness may be due to the
specific attachment of the DNA polymerase do the bottom of the ZMWs
in conjunction with the passivation of the aluminum oxide
surface.
[0219] In addition, yields of statistically significant sequencing
increased by 12.times. when directly compared with PDMS passivated
surfaces. In some cases a 40.times. increase in yield has been
demonstrated. The yield improvements are believed to be due to
several factors: (1) The biased surface enables a higher percentage
of ZMWs to be loaded in the detection volume of the zero-mode
waveguide (models predict a limit of 13% for PDMS and 37% for the
surface described above having high bias). (2) The biased surface
enables sequencing polymerases to be located at the fused-silica
ZMW bottom where excitation and emission electric fields are
optimal, thus yielding higher average signal-to-noise traces. (3)
The decrease in stickiness enables higher accuracy and thus higher
yields of significant sequencing. (4) The specific attachment via a
flexible linker enables a native conformation of DNA
polymerase.
[0220] Also, sequencing readlengths were improved .about.2.times.
when directly compared with PDMS passivated surfaces. In some cases
a 25.times. improvement has been demonstrated. The readlength
improvements can be attributed part to: (1) the use of lower laser
powers which is afforded by the biased surface described herein,
and (2) the specific attachment at the bottom of the ZMWs reduces
the instances of desorbed polymerases present when there is
non-specific attachment.
[0221] It is to be understood that the above description is
intended to be illustrative and not restrictive. It readily should
be apparent to one skilled in the art that various embodiments
and-modifications may be made to the invention disclosed in this
application without departing from the scope and spirit of the
invention. The scope of the invention should, therefore, be
determined not with reference to the above description, but should
instead be determined with reference to the appended claims, along
with the full scope of equivalents to which such claims are
entitled. All publications mentioned herein are cited for the
purpose of describing and disclosing reagents, methodologies and
concepts that may be used in connection with the present invention.
Nothing herein is to be construed as an admission that these
references are prior art in relation to the inventions described
herein. Throughout the disclosure various patents, patent
applications and publications are referenced. Unless otherwise
indicated, each is incorporated by reference in its entirety for
all purposes.
* * * * *