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Patent 2622872 Summary

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(12) Patent Application: (11) CA 2622872
(54) English Title: REACTIVE SURFACES, SUBSTRATES AND METHODS FOR PRODUCING AND USING SAME
(54) French Title: SURFACES REACTIVES, SUBSTRATS ET PROCEDES DE PRODUCTION ET D'UTILISATION CORRESPONDANTS
Status: Dead
Bibliographic Data
(51) International Patent Classification (IPC):
  • G01N 33/53 (2006.01)
(72) Inventors :
  • ROITMAN, DANIEL BERNARDO (United States of America)
  • PELUSO, PAUL (United States of America)
  • FOQUET, MATHIEU (United States of America)
(73) Owners :
  • PACIFIC BIOSCIENCES OF CALIFORNIA, INC. (United States of America)
(71) Applicants :
  • PACIFIC BIOSCIENCES OF CALIFORNIA, INC. (United States of America)
(74) Agent: FETHERSTONHAUGH & CO.
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 2006-09-29
(87) Open to Public Inspection: 2007-04-12
Examination requested: 2011-09-28
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US2006/038243
(87) International Publication Number: WO2007/041394
(85) National Entry: 2008-03-17

(30) Application Priority Data:
Application No. Country/Territory Date
11/240,622 United States of America 2005-09-30

Abstracts

English Abstract




Reactive surfaces, substrates and methods of producing and using such
substrates and surfaces are provided. The substrates and surfaces provide low
density reactive groups preferably on an otherwise non-reactive surface for
use in different applications including single molecule analyses.


French Abstract

L'invention porte sur des surfaces réactives, sur des substrats et sur des procédés de production et d'utilisation de ces substrats et de ces surfaces. Les substrats et les surfaces forment des groupes réactifs de basse densité, de préférence sur une autre surface non réactive destinée à être utilisée dans différentes applications telles que des analyses de molécules simples.

Claims

Note: Claims are shown in the official language in which they were submitted.




What is claimed is:



1. A substrate comprising:
a surface comprising a first reactive moiety coupled thereto; and
wherein the reactive moiety is coupled to the surface at a density of between
about 1
reactive moiety per 50,000 nm2 and 1 reactive moiety per 100 nm2.

2. The substrate of claim 1, wherein the reactive moiety is coupled to the
surface at a
density of between about 1 reactive moiety per 8000 nm2 and 1 reactive moiety
per 300 nm2
3. The substrate of claim 1, wherein the reactive moiety is coupled to the
surface at a
density of between about 1 reactive moiety per 8000 nm2 and 1 reactive moiety
per 1000 nm2
4. The substrate of claim 1, wherein a remainder of the surface other than the
reactive
moieties is a substantially non-reactive surface.

5. The substrate of claim 1, wherein the reactive moiety is coupled to the
surface through a
linker group.

6. The substrate of claim 5, wherein the linker group comprises a polymer.

7. The substrate of claim 5, wherein the linker group comprises polyethylene
glycol.

8. The substrate of claim 4, wherein the reactive moiety is coupled to the
surface through a
linker group, and the substantially non-reactive surface comprises the linker
group without the
reactive moiety.

9. The substrate of claim 1, wherein the first reactive moiety comprises a
binding moiety.
10. The substrate of claim 9, wherein the binding moiety comprises a
nonspecific binding
moiety.

11. The substrate of claim 9, wherein the binding moiety comprises a specific
binding
moiety.



29



12. The substrate of claim 11, wherein the binding moiety comprises one member
of a
specific binding pair.

13. The substrate of claim 9, wherein the binding moiety is selected from the
group of
consisting of an antigen, an antibody, an binding fragment of an antibody, a
polynucleotide, a
binding peptide, biotin, avidin and streptavidin.

14. The substrate of claim 1, wherein the first reactive moiety comprises a
catalytic moiety.
15. The substrate of claim 14, wherein the catalytic moiety comprises a
catalytic metal.

16. The substrate of claim 14, wherein the catalytic moiety comprises an
enzyme.
17. The substrate of claim 16, wherein the enzyme is selected from a nucleic
acid
polymerase, a ligase, a nuclease, a protease, a kinase and a phosphatase.

18. The substrate of claim 16, wherein the enzyme comprises a DNA polymerase.
19. The substrate of claim 1, wherein the surface comprises an observation
area.

20. The substrate of claim 1, wherein the surface comprises an observation
surface of an
optical confinement.

21. The substrate of claim 19, wherein the observation area comprises the
observation
surface of a zero mode waveguide.

22. The substrate of claim 1, wherein the surface of the substrate comprises
silica.

23. The substrate of claim 1, wherein the surface of the substrate is selected
from glass,
quartz, fused silica, and silicon.

24. A device, comprising:
a substrate having at least a first observation area provided therein, the
observation area
having an area of from about 100 nm2 to 50000 nm2; and






from 1 to 3 reactive moieties coupled to the surface within the observation
area.

25. The device of claim 24, wherein the observation area is bounded by an
optically opaque
cladding.

26. The device of claim 24, wherein the reactive moieties comprise an enzyme.
27. The device of claim 26, wherein the enzyme comprises a DNA polymerase.
28. A method of preparing a modified surface, comprising:
providing a substrate having a first reactive surface;
providing a mixture of first and second surface modifying agents, wherein the
first and
second surface modifying agents are each capable of coupling to the reactive
surface, and are
present in the mixture at a first ratio selected so that the first and second
surface modifying
agents couple to the reactive surface at a second ratio;
contacting the reactive surface with the mixture to produce the modified
surface having first and
second modifying agents coupled thereto at the second ratio.

29. A method of preparing a modified surface, comprising:
providing a surface to be modified;
contacting the surface to be modified with a surface modifying composition,
wherein the
surface modifying composition comprises:
a first surface modifying agent coupled to a desired reactive moiety, and a
second surface modifying agent not coupled to the desired reactive moiety; and
the first surface modifying agent and second surface modifying agent are
present
in the surface modifying composition at a ratio that produces the modified
surface,
wherein the reactive moieties are present on the modified surface at a density
of between
about 1 reactive moiety per 50,000 nm2 and 1 reactive moiety per 100 nm2

30. An apparatus, comprising a surface having a reactive moiety coupled
thereto, wherein
the reactive moiety is coupled to the surface at a density of less than 1
reactive moiety per 100
nm2.

31. The apparatus of claim 30, wherein the reactive moiety is coupled to the
surface at a
density of less than 1 reactive moiety per 1000 nm2



31



32. The apparatus of claim 30, wherein the reactive moiety is coupled to the
surface at a
density of less than 1 reactive moiety per 10000 nm2

33. The apparatus of claim 30, wherein the reactive moiety comprises a
molecular binding
group.

34. The apparatus of claim 33, wherein the molecular binding group comprises a
specific
molecular binding group.

35. The apparatus of claim 34, wherein the specific molecular binding group is
selected from
an antibody, an antigen, avidin, streptavidin, biotin, a polynucleotide, a
polynucleotide analog, a
binding peptide.

36. A method of configuring a surface to provide a desired density of reactive
moieties
thereon, comprising treating the surface with a composition that substantially
uniformly couples
to the surface, the composition comprising a first component that does not
contain the reactive
moiety and a second component that comprises the reactive moiety, the second
component being
present in the composition at a concentration relative to the first component
so as to provide the
reactive moieties on the surface at the desired density.



32

Description

Note: Descriptions are shown in the official language in which they were submitted.



CA 02622872 2008-03-17
WO 2007/041394 PCT/US2006/038243
REACTIVE SURFACES, SUBSTRATES AND METHODS OF PRODUCING AND
USING SAME

CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and benefit of USSN 11/240,662
REACTIVE
SURFACES, SUBSTRATES AND METHODS OF PRODUCING AND USING SAME BY
Roitman et al., filed September 30, 2005. This prior application is
incorporated by reference for
all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] Not Applicable

BACKGROUND OF THE INVENTION
"God made the solid state. He left the surface to the Devil." -Enrico Fermi
[0003] This sentiment is not new to materials scientists. The understanding,
or lack
thereof, as to the characteristics of a surface and its interactions with its
environment has been at
the center of monumental discoveries, and monumental failures. This issue
permeates virtually
every technological endeavor, whether it is in the field of engineering,
chemistry or biology,
whether it is focused on nanomaterials technology, extraterrestrial
exploration, semiconductor
technology, biotechnology manufacturing or pharmaceutical administration and
delivery. While
understanding the bulk properties of a material presents one problem, the
point at which that
material ceases, and one must understand and/or deal with the properties of
the surface of that
material and how that surface will interact with its environment, is something
altogether
different.
[0004] The present invention is directed at materials and/or their surfaces
that are
selected and/or configured to meet a variety of different needs, including,
inter alia, a capacity
and ability of selective binding to desired molecules while preventing
excessive binding of
undesired molecules, and other advantageous characteristics that will be
apparent upon reading
the following disclosure.

SUIVIlVIARY OF THE INVENTION
[0005] The present invention is generally directed to substrates bearing
modified
surfaces that are useful in a variety of different, useful applications, as
well as methods of
producing such substrates and uses and applications of these substrates. In
particular, the
substrates of the invention possess surfaces with a selected density of
reactive groups disposed
on that surface, and preferably, a selected low density of such reactive
groups.
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[0006] In a first aspect, the present invention provides a substrate that
comprises a
surface comprising a first reactive moiety coupled thereto. In accordance with
this aspect of the
invention, the reactive moiety may be coupled to the surface at a density of
between about 1
reactive moiety per 50,000 nm2 and 1 reactive moiety per 100 nm2. Similarly,
the invention
also provides an apparatus, comprising a surface having a reactive moiety
coupled thereto,
wherein the reactive moiety is coupled to the surface at a density of less
than 1 reactive moiety
per 100 nm2.
[0007] Relatedly, the invention also provides devices that comprise a
substrate having at
least a first observation area provided therein, the observation area having
an area of from about
100 nm2 to 50000 nm2. In accordance with these aspects of the invention, the
substrates include
from 1 to 3 reactive moieties coupled to the surface within the observation
area.
[0008] The invention also provides methods of producing the susbstrates and
devices of
the invention by providing a substrate having a first reactive surface, and
then providing a
mixture of first and second surface modifying agents, wherein the first and
second surface
modifying agents are each capable of coupling to the reactive surface, and are
present in the
mixture at a first ratio selected so that the first and second surface
modifying agents couple to
the reactive surface at a second ratio. The reactive surface is then contacted
with the mixture to
produce the modified surface having first and second modifying agents coupled
thereto at the
second ratio.
[0009] Relatedly, also provided is a method of preparing a modified surface,
comprising
providing a surface to be modified, and contacting the surface to be modified
with a surface
modifying composition. In this aspect of the invention, the surface modifying
composition
comprises a first surface modifying agent coupled to a desired reactive
moiety, and a second
surface modifying agent not coupled to the desired reactive moiety. The first
surface modifying
agent and second surface modifying agent are present in the surface modifying
composition at a
ratio that produces the modified surface, wherein the reactive moieties are
present on the
modified surface at a density of between about 1 reactive moiety per 50,000
nm2 and 1 reactive
moiety per 100 nm2.
[0010] In still another aspect, the present invention provides method of
configuring a
surface to provide a desired density of reactive moieties thereon, comprising
treating the surface
with a composition that substantially uniformly couples to the surface, the
composition
comprising a first component that does not contain the reactive moiety and a
second component
that comprises the reactive moiety, the second component being present in the
composition at a
concentration relative to the first component so as to provide the reactive
moieties on the surface
at the desired density.

2


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WO 2007/041394 PCT/US2006/038243
DESCRIPTION OF THE FIGURES
[0011] Figure 1 is a schematic illustration of a surface having a low density
of reactive
groups thereon.
[0012] Figure 2 is a schematic illustration of a substrate having a layered,
or multitiered
functionalization to provide a relatively low density of reactive groups
thereon.
[0013] Figure 3 schematically illustrates one exemplary process for producing
the
surfaces of the invention.
[0014] Figure 4 schematically illustrates another exemplary process for
producing the
low density reactive surfaces of the invention.
[0015] Figure 5 schematically illustrates an example of a steric hindrance
based process
for producing surfaces of the invention to provide a selectively localized
reactive group.
[0016] Figure 6 is a schematic illustration of a surface having two
differently
functionalized derivatization groups disposed thereon, to yield a relatively
low density reactive
surface.
[0017] Figure 7 is a plot of water contact angle on surfaces of varying
concentrations of
reactive groups among a surface of otherwise unreactive groups.

DETAILED DESCRIPTION OF THE INVENTION
[0018] The present invention is generally directed to materials and their
surfaces,
generally referred to hereafter as substrates, where the surfaces have been
selected and or
configured to have desirable properties for a variety of applications. The
invention is also
directed to methods and processes for producing such surfaces, as well as
methods and processes
for using such surfaces in a number of different applications.
[0019] Of particular interest with respect to the present invention are
substrates and
surfaces that possess selective molecular binding or coupling characteristics,
e.g., through the
selective inclusion of molecular binding moieties thereon, and the use of such
surfaces to
selectively bind desired molecules to the surfaces in a selective fashion. Of
still greater interest
is the use of such surfaces when they are selectively coupled to chemically
and/or biologically
active molecules for use in chemical and/or biochemical processes, such as in
preparative
operations and/or analytical operations.
[0020] Although the invention has broad applicability, as will be apparent
from the
ensuing disclosure, in one particularly preferred example, surfaces having low
density reactive
groups include a single reactive group, in preferred cases, an enzyme, such as
a nucleic acid
polymerase, within an area that is being observed and/or monitored, giving the
observer a real-

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CA 02622872 2008-03-17
WO 2007/041394 PCT/US2006/038243
time understanding of the reactions catalyzed by that single enzyme, e.g., DNA
synthesis. Such
systems are particularly useful in template dependent analysis, or sequencing,
of nucleic acids.
1. Substrates and Surfaces
A. Generally
[0021] As alluded to above, the ability and/or propensity of surfaces to
interact on a
molecular level with their surroundings is of particular interest in the
chemical and biological
sciences and industries exploiting those sciences. For example, past efforts
at manipulation of
the reactive groups present on surfaces have focused primarily on one extreme
or another. In
particular, a number of applications benefit from maximizing the density of
molecules bound to
a particular surface by maximizing the number of reactive groups on that
surface, e.g., high
density binding. In other applications, the desired goal has been to exclude
virtually all binding
or other coupling interactions, including adsorbtion, between a surface and
materials exposed to
those surfaces, to create an inert surface for the given application, by
capping or otherwise
masking reactive groups on the surface.
[0022] In the case of biologically reactive surfaces for example, DNA array
technology,
for example, has focused upon binding as many active polynucleotide probes
within a given
area, so as to maximize the signal generated from hybridization reactions with
such probes.
Likewise, affinity surfaces employing, e.g., antibodies, have similarly
focused upon increasing
the density of binding groups on a surface to improve sensitivity.
Alternatively, in a number of
other applications, past efforts have been directed at effectively
neutralizing the binding effects
of surfaces to minimize or eliminate the surface's interaction with the
chemical or biochemical
environment. For example, the field of microfluidics, and particularly
including the capillary
electrophoresis art, is replete with examples of researchers identifying
coating materials or other
surface treatments that are intended to mask any functional groups of fused
silica capillaries to
avoid any molecular associations with those surfaces.
[0023] The present invention, however is directed at surfaces that are neither
intended to
maximize nor completely eliminate reactive chemical groups on a given surface.
Instead, the
present invention is directed at providing a surface with a selected
relatively low density of
reactive groups on a surface, and the use of such surfaces in a number of
valuable applications.
As will be appreciated, the nature of reactive groups does not imply or
require a group capable
of covalent linkage with another group, but includes groups that give rise to
other forms of
interaction, including hydrophobic/hydrophilic interactions, Van der Waals
interactions, and the
like. As such, surface reactivity, as generally described herein, includes,
inter alia, association
by covalent attachment and non-covalent attachment, e.g., adsorption.

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[0024] Although, for ease of discussion, the substrates and surfaces are
generally
described herein in terms of planar solid substrates, it will be appreciated
that the methods,
processes, surfaces, etc. of the invention are applicable to a variety of
different substrate types
where the properties of reactive surfaces of the invention may be useful. In
particular, such
surfaces may comprise planar solid surfaces, including inorganic materials
such as silica based
substrates (i.e., glass, quartz, fused silica, silicon, or the like), other
semiconductor materials
(i.e., Group III-V Group II-VI or Group IV semiconductors), as well as organic
materials such as
polymer materials (i.e., polymethylmethacrylate, polyethylene, polypropylene,
polystyrene,
cellulose, agarose, or any of a variety of organic substrate materials
conventionally used as
supports for reactive media). In addition to the variety of materials useful
as substratres, it will
be appreciated that such materials may be provided in a variety of physical
configurations, such
as microparticles, i.e., beads, nanoparticles, i.e., nanocrystals, fibers,
microfibers, nanofibers,
nanowires, nanotubes, mats, planar sheets, planar wafers or slides, multiwell
plates, optical
slides including additional structures, capillaries, microfluidic channels,
and the like.
[0025] In operation, this selective and limited reactivity of the surfaces of
the invention
is aimed at providing, in a limited fashion, a particular desired molecule or
type of molecule of
interest, typically a selected reactive molecule of interest, on a surface,
e.g., a particular enzyme,
nucleic acid, or the like, while preventing binding of the molecule of
interest and/or other
potentially interfering molecules elsewhere on the surface. For preferred
applications, the
desired result is a surface that includes a relatively low density of the
selected reactive molecule
surrounded by an otherwise non-reactive surface. Although discussed in terms
of a molecule or
type of molecule of interest, it will be appreciated that mixed functionality
surfaces are also
encompassed within the scope of the invention, including, e.g., two, three,
four, or more
different molecules or types of molecules of interest.
[0026] Thus, as used herein, the terms "reactive" and "non-reactive" when
referring to
different groups on the substrate surfaces of the invention refers to (1) the
relative reactivity or
association of such surface components with a given molecule of interest, and
preferably also
refers to (2) the relative reactivity or association of such surface
components with other reagents
in a given application of such surfaces, where such reagents may interfere
with such
applications, such as labeled reactants and or products that might interfere
with detection, as
well as inhibitors or other agents that would interfere with the progress of a
reaction of interest
at the reactive portion of the surface or elsewhere.
[0027] In terms of the first aspect of such reactivity, the reactive portions
or groups on
the surfaces will typically have 10 times greater affinity for the molecule of
interest, preferably
more than 100 times greater affinity and more preferably at least 1000 times
greater affinity for


CA 02622872 2008-03-17
WO 2007/041394 PCT/US2006/038243
the molecule of interest than the non-reactive surface. As such, it will be
appreciated that the
level of association between the molecule of interest and the reactive surface
will be
substantially greater than with the non-reactive surface under uniform
conditions, e.g., more than
times greater, more than 100 times greater and preferably more than 1000 times
greater.
Such greater association includes greater frequency and/or greater duration of
individual
associations.
[0028] In terms of the second aspect of surface reactivity or non-reactivity,
in many
cases, such reactivity is coincident with the first aspect. In particular,
where an enzyme
constitutes the reactive portion of the surface, it will generally have a high
affinity for its
substrate, and thus associate with such substrate at a much greater level than
the non-reactive
portion, e.g., as described above. However, in some cases, the "reactive"
portion of the surface
may not include an ability to associate with certain potential interfering
molecules. In such
cases, the terms adsorptive and non-adsorptive also may be used. Nonetheless,
it is desirable to
prevent such interfering molecules from associating with the remainder of the
surface. As such,
the non-reactive surface may be defined in terms of its reactivity with such
interfering
components.
[0029] Because the primary source of undesirable interference for many
applications lies
in the non-specific interaction of reagents with the non-reactive portions of
the surface, rather
than at the desired reactive portion, the non-reactive surface in such cases,
may generally be
characterized by an association equilibrium constant between the non-reactive
group and a
particular interfering molecule that is preferably 10 fold lower than the
association equilibrium
constant of the reactive surface(s) with the reactive molecule(s), and
preferably 100 fold (or
more) lower. The association reaction for the non-reactive surface is also
characterized by a low
activation barrier, such that the kinetics of the corresponding dissociation
reaction are expected
to be fast, with average binding time preferably at least 10 fold lower than
the significant
timescales of the measurement process of the application, and preferably 100
fold lower or
more.
[0030] As will be appreciated, the characteristics of such non-reactive and
reactive
surfaces will typically depend upon the specific application to which the
surface is to be put,
including environmental characteristics, e.g., pH, salt concentration, and the
like. In particularly
preferred aspects, envirtonmental conditions will typically include those of
biochemical systems,
e.g., pH between about 2 and about 9, and salt levels at biochemically
relevant ionic strength,
e.g., between about 0 mM and 100 mM.
[0031] Figure 1 provides a simplified schematic illustration of the surfaces
of the
invention, in block diagram form. As shown, a substrate 100 includes a surface
102. As noted
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elsewhere herein, the surface 102 is optionally derivatized to provide an
overall active surface
104. As noted below, optionally, the substrate may inherently possess an
overall reactive
surface. The reactive surface 104 is then treated to provide a surface that
includes reactive
groups 106 coupled to the reactive surface 104 at relatively low densities. As
noted below, these
reactive moieties are preferably disposed upon or among an otherwise neutral
or non-reactive
surface 108. In particularly preferred aspects, the reactive groups 106 may
include, or be further
treated to include additional reactive groups, e.g., catalytic components,
such as enzymes 110, or
the lilce, as also shown in Figure 1.
[0032] One important advantage of the surfaces of the invention is the
provision of
relatively isolated reactive groups. Isolation of reactive groups provides the
ability to perform
and/or monitor a particular reactivity without interference from adjacent
reactive groups. This is
of particular value in performing single molecule reaction based analyses,
where detection
resolution necessitates the isolation, e.g., to be able to optically
distinguish between reactive
molecules (optical isolation), electrochemically distinguish between reactions
at different
reactive molecules (electrochemical isolation) or where chemical contamination
from one
reaction at one location may impact reaction at an adjacent location (chemical
isolation).
[0033] An additional advantage of the surfaces of the invention, is the
ability of the
remainder of the surface to be inert to coupling with potentially interfering
molecules, e.g.,
fluorescent analytes or products. In particular, while binding of a few
selected molecules is
desirable for a set of applications, uncontrolled or nonspecific binding the
remainder of the
surface is often highly undesirable. By providing the desired reactive groups
only at a selected,
relatively low density, which themselves comprise a moiety having a desired
reactivity, or which
in some cases, are reacted with another molecule having the desired
reactivity, one can
selectively treat the remainder of the surface as necessary to render it
effectively neutral to
unwanted binding, thus substantially reducing or eliminating such unwanted
binding elsewhere
on the surface. In accordance with preferred aspects of the invention, both
the provision of
selected reactive groups and the provision of non-reactive groups over the
remainder of the
surface to reduce such unwanted surface interactions, are accomplished in the
same process step
or steps.
B. Density
[0034] In accordance with the invention, the low density of the selected
desired reactive
moieties or chemical groups 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. Typically, this means
that any reactive
groups otherwise present upon the remainder of the surface area in question
are capped, masked,

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or otherwise rendered non-reactive. As such, low density reactive groups are
typically present
on a substrate surface at a density of reactive groups of greater than 1/1X106
nm2 of surface area,
but less than about 1/100 nm2. In more preferred aspects, the density of
reactive groups on the
surface will be greater than 1/100,000 nm2, 1/50,000 nm2, 1/20,000 nm2 and
1/10,000 nm2, and
will be less than about 1/100 nm2, 1/1000 nm2, and 1/10,000 nm2. For certain
preferred
applications, the density will often fall between about 1/2500 nm2 and about
1/300 nm2, and in
some cases up to about 1/150 nm2.
C. Observation Areas
[0035] In particularly preferred aspects, the invention provides reactive
groups on a
surface at a density such that one, two, three or a few reactive groups are
present within an area
that is subject to monitoring or observation (an "observation area"). By
providing individual or
few reactive groups within an observation area, one can specifically monitor
reactions with or
catalyzed by the specific individual reactive group. Such observation areas
may be determined
by the detection system that is doing the monitoring, e.g., a laser spot size
directed upon a
substrate surface to interrogate reactions, e.g., that produce, consume or
bind to fluorescent,
fluorogenic, luminescent, chromogenic or chromophoric reactants, or fiber tip
area of an optical
fiber for optical monitoring systems, a gate region of a chemical field effect
transistor
(ChemFET) sensor, or the like, or they may be separately defined, e.g.,
through the use of
structural or optical confinements that further define and delineate an
observation area.
[0036] One example of a particularly preferred observation area includes an
optical
confinement, such as a zero mode waveguide (ZMW). Zero mode waveguides, as
well as their
use in single molecule analyses, are described in substantial detail in U.S.
Patent No. 6,917,726,
which is incorporated herein by reference in its entirety for all purposes.
Such ZMWs have been
exploited for use in single molecule analyses, because they can provide
observation volumes
that are extremely small, e.g., on the order of zeptoliters. In such cases,
the observation area will
generally include the cross sectional area of the observation volume, and
particularly that portion
of the observation volume that intersects the surface in question.
[0037] In preferred aspects, the invention provides one or only a few reactive
groups on
the bottom surface of the waveguide. In such cases, the density is measured by
the number of
reactive groups divided by the surface area of the bottom surface of the
waveguide. Thus,
purely for purposes of exemplification, where a circular waveguide has a
radius of 10 nm, and
includes a single reactive molecule immobilized on its bottom surface, the
density of reactive
groups would be approximately 1/314 nm2. Thus, in terms of zero mode
waveguides or other
observation areas, and for purposes of example, it will be appreciated that
reactive molecules
present at a density of one, two, three or up to 10 reactive molecules in an
area having a radius

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of between about 10 and about 100 nm, or areas from 314 nm2 to about 31,416
nma, respectively
(i.e., larger numbers of molecules in larger areas), are encompassed by the
densities herein
described. In preferred aspects, one, two or three molecules per observation
area is generally
preferred.
[0038] In many cases, ZMWs are provided in arrays of 10, 100, 1000, 10,000 or
more
waveguides. As such, immobilization of a single reactive group, e.g., an
enzyme, within each
and every ZMW would be difficult. However, dilution based protocols, when
combined with
the surfaces of the invention while producing some ZMWs that are not occupied
by an enzyme,
will generally result in the majority of occupied ZMWs (those having at least
one enzyme
molecule immobilized therein) having only one or the otherwise desired number,
of enzymes
located therein. In particular, in the case of ZMWs having reactive molecules
like enzymes
located therein, typically, more than 50% of the occupied ZMWs will have a
single or the
desired number of reactive molecules located therein, e.g., a particular type
of enzyme molecule,
preferably, greater than 75%, and more preferably greater than about 90% and
even greater than
95% of the occupied ZMWs will have the desired number of reactive molecules
located therein,
which in particularly preferred aspects may be one, two, three or up to ten
reactive molecules of
a given type. As noted elsewhere, in some circumstances different reactive
molecules may also
be provided at a desired density to provide a mixed functionality surface. In
accordance with the
present invention, depending upon the types of reactive groups being
referenced, e.g., catalytic
or binding, it will be appreciated that the determination of density may be
applied on a single
occupied ZMW, or upon multiple ZMWs in an array.
D. Specific Reactive Groups
[0039] The reactive groups or moieties present on the surfaces of the
invention include a
wide range of different types of reactive groups having chemical and/or
biological activity,
which are coupled to a surface of a material or substrate, either by exogenous
addition or which
inherently are present on such surface. These reactive groups include groups
on a surface that
possess binding activity for other chemical groups, e.g., the ability to bind
another chemical
moiety through specific or non-specific interactions, through covalent
attachment, Van der
Waals forces, hydrophobic interaction, or the like. Provision of a wide range
of reactive groups
on surfaces is readily understood in the art, and includes, for example, ionic
functional groups,
polyionic groups, epoxides, amides, thiols, hydrophobic groups, e.g.,
aliphatic groups, mono or
polycyclic groups, and the like, e.g., as generally used in reverse phase
and/or hydrophobic
interaction chromatography (HIC), staudinger ligation groups (see, e.g., Lin
et al., J. Am. Chem.
Soc. (2005), 127:2686-95), Click chemistry coupling using chemoselective azide-
acetylene
linkages (See, Deveraj et al., JACS 2005, 127:8600-8601; Lummerstorfer et al.,
J. Phys. Chem.

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B (2004) 108:3963-3966, and Collman et al., Langmuir (2004) 20:1051-1053, each
of which is
incorporated herein by reference in its entirety for all purposes) and other
groups that associate
or are capable of being coupled with otlier groups in a non-specific fashion.
Additionally, use
of specific binding groups on surfaces, e.g., groups that specifically
recognize a complementary
binding partner has been described, including, e.g., complementary nucleic
acid pairs, antibody-
epitope pairs, binding peptides that recognize specific macromolecular
structures, e.g.,
recognition sequences in proteins, peptides or nucleic acids, lectins,
chelators, biotin-avidin
linkages, and the like.
[0040] Identification of the number and/or density of reactive groups may
generally be
ascertained through the use of a reporter molecule, which in many cases, may
be the reactive
group itself. In particular, and by way of example, one can ascertain the
number of enzyme
molecules coupled to a surface area by assaying for the activity of that
enzyme. Likewise, other
reactive groups may be quantified through other methods, e.g., titration,
coupling of labeling
groups, or the like.
[0041] As used herein, both reactive groups and non-reactive groups envision
an
environment in which the surfaces are to be applied, and in which the
reactivity, or non-
reactivity is evident. As will be appreciated, different groups may be
reactive in certain
environments and non-reactive in others, and the invention, as broadly
practiced, envisions
applicability in a wide range of different environments. For ease of
discussion, and in preferred
aspects, the surfaces of the invention are most often to be applied in
biological or biochemical
reactions, and as such are subjected to appropriate environments. Such
environments typically
include aqueous systems having biochemically relevant ionic strength, that
range in pH between
about 2 and about 9, and preferably between about 5 and about 8, but may vary
depending upon
the reactions being carried out.
[0042] In certain preferred aspects, the reactive chemical groups also include
groups
having catalytic activity, e.g., the ability to interact with another moiety
to alter that moiety other
than through binding, i.e., enzymatic activity, catalytic charge transfer
activity, or the like. In
particularly preferred aspects, the active chemical groups of the invention
include chemical
binding groups, and optionally and additionally, catalytic groups, where the
binding group is
used to couple the catalytic group to a given surface in accordance with the
invention. For
example, an enzyme or other catalytic group may be coupled to a surface via an
intermediate
binding or linker group that is, in turn, coupled directly to a reactive group
that is disposed upon
the surface material at a desired density.
[0043] A number of different reactive groups may be employed in accordance
with the
invention, and may to some extent, depend upon the surface being used, and
whether the



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reactive group is intended to provide a low-density general or non-specific
binding or
associative function, a low-density specific binding function, or a low
density catalytic function.
[0044] For example, for silica based surfaces, e.g., glass, quartz, fused
silica, silicon or
the like, reactive groups may be provided by silane treatment of the surface,
e.g., using
epoxysilane, aminosilane, activated carboxylic acid silane, isocyanatosilane,
aldehyde silane,
mercaptosilane, vinyl silane, hydroxyterminated silanes, acrylate silane and
the lilce. Such
treatments may yield the reactive groups, e.g., in terms of low density, non-
specific associative
groups, or they may result in or be further treated, to provide a specific
binding group or
catalytic group, as the ultimate reactive group. Alternatively or
additionally, other inorganic or
organic reactive groups may be provided upon a surface. In the case of
inorganic surfaces like
silica based substrates, such additional materials may be coupled to the
surface via an
intermediate chemical coupling, e.g., using silane chemistry, i.e., as
described above. These
additional materials may include small molecules, e.g., ionic groups, metal
ions, small organic
groups, as well as larger or polymeric/oligomeric molecules, e.g., organic
polymers. For ease of
discussion, polymer and oligomer are used interchangeably herein to refer to
molecules that
include multiple subunits of similar chemical structure.
[0045] In particularly preferred aspects, a longer linker molecule, and
preferably an
organic linker molecule may be used to link the reactive group to the surface
to provide further
flexibility to the overall linkage, e.g., by providing greater spacing between
the surface and
reactive group. In particular, polymeric or oligomeric chains that bear the
desired reactive
group at one end, may be linked at the other end to the surface, e.g., via
silane linkage in the
case of a glass surface. By selecting different types and lengths of polymer
linkers, one can
further adjust the properties of the surface, e.g., relative hydrophobicity of
different
groups/areas, relative distance to the surface, overall or local surface
charge, and the like.
Examples of useful polymer linkers include, e.g., cellulosic polymers (such as
hydroxyethyl-
cellulose, hydroxypropyl-cellulose, etc.), alkane or akenyl linkers,
polyalcohols (such as
polyethyleneglycols (PEGs), polyvinylalcohols (PVA)), acrylic polymers (such
as
polyacrylamides, polyacrylates, and the like), polyethylene polymers (such as
polyethyleneoxides), biopolymers (such as polyamino acids like polylysine,
polyarginine,
polyhistidine, etc.), other carbohydrate polymers (such as xanthan, alginate,
dextrans), synthetic
polyanions or polycations (such as polyacrylic acid, carboxyl terminated
dendrimers,
polyethyleneimine, etc.) and the like. Again, depending upon the type of
linker used, the linker
may further include a desired reactive group coupled to it.
[0046] While described generally in terms of application of a reactive group
to the
surface, it will be appreciated that the active group may be applied to the
surface as an inactive
11


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or less reactive precursor to the desired reactive group, and subsequently
activated to yield the
desired reactive group. In particular, the reactive groups may be provided as
photo, thermally
or chemically activatable precursor groups, e.g., bearing a photolytic capping
group, a
temperature sensitive capping group or an acid or base labile capping group,
blocking the
reactive moiety of interest. The group may then be selectively activated,
e.g., through the use of
photo, thermal or chemical treatment to yield the desired surface. A variety
of such groups are
known in the art and are described in, e.g., Guillier, et al., Linkers and
Cleavage Strategies in
Solid Phase Organic Synthesis and Combinatorial Chemistry, Chem. Rev. 100:2091-
2157
(2000).
[0047] As noted above, the reactive groups on a surface may be comprised of
the
aforementioned specific or non-specific binding moieties, or may include
catalytic groups that
are coupled to the surface, either directly to the surface, through the above
mentioned specific or
non specific binding or associative groups, that are, in turn, coupled
directly or indirectly to the
surface, or through additional specific or non-specific binding groups coupled
to the surface.
Catalytic groups may include catalytic chemicals, e.g., catalytic metals or
metal containing
compounds, such as nickel, zinc, titanium, titanium dioxide, platinum, gold,
or the like. In
preferred aspects, however, the catalytic groups present at a desired low
density on the surfaces
of the invention comprise bioactive molecules including, e.g., nucleic acids,
nucleic acid
analogs, biological binding compounds, e.g., peptides or proteins, biotin,
avidin, streptavidin,
etc., and enzymes. In the case of nucleic acids or nucleic acid analogs, such
surfaces find use in
a variety of specific binding assays, e.g., to interrogate mixtures of nucleic
acids for a nucleic
acid segment of interest (See, e.g., U.S. Patent Nos. 5,153,854, 5,405,783,
and 6,261,776).
Likewise, binding proteins and peptides are often useful in interrogating
biological samples for
the presence or absence of a given molecule of interest. Typically such
proteins or peptides are
embodied in antibodies or their binding fragments or binding epitopes of such
antibodies. In
particularly preferred aspects, the surfaces of the invention bearing the
catalytic groups comprise
an enzyme of interest and are used to monitor the activity of that enzyme. A
wide variety of
enzymes are regularly monitored and detected in biological, biochemical and
pharmaceutical
research and diagnostics. Examples of preferred enzymes include those
monitored in genetic
analyses like DNA sequencing applications, such as polymerases, e.g., DNA and
RNA
polymerases, nucleases (endo and exonucleases), ligases, and those involved in
a variety of other
pharmaceutically and diagnostically relevant reactions, such as kinases,
phosphatases, proteases,
lipases, and the like.
[0048] With respect to immobilization of enzymes on surfaces in accordance
with the
invention, yet a further advantage of the surfaces of the invention stems from
the combined
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advantages set forth elsewhere herein. In particular, in selectively
immobilizing biomolecules,
lilce enzymes, through specific linkages, and rejecting their adsorption
elsewhere on the surface,
the activity of the biomolecules present on the surface can be more
selectively preserved, where
mere adsorption may have yielded a significant population of inactive or less
active molecules.
Thus, the resulting surface, the biomolecules present, while present at low
density, will
nonetheless be present at a relatively high specific activity, e.g., number of
active biomolecules
of interest vs. total number of biomolecules present).
[0049] In the case of certain catalytic reactive groups, e.g., enzymes, the
density of such
reactive groups further envisions the density of active molecules, as opposed
to immobilized
inactive molecules. For example, in the case of enzymes immobilized on a
surface at a
relatively low density, such density will typically include an allocation for
the specific activity
of the immobilized enzyme, e.g., the efficacy of the immobilization process.
Thus, where the
immobilization process yields only 50% viable or active enzymes, the overall
density of enzyme
molecules active and otherwise, will generally be 2X the density of active
molecules.
Accordingly, in ascertaining the desired density of such reactive groups, it
is often desirable to
assess the relative efficacy of the immobilization process in depositing
active molecules. As
noted elsewhere herein, certain aspects of the methods of the invention are
particularly useful at
retaining very high specific activity of enzymes immobilized on the surfaces
(See Examples),
and preferably will provide specific activities (fraction of immobilized
enzyme having activity)
of greater than 20%, greater than 30%, more preferably, greater than 50% and
in still more
preferred aspects, greater than 75%, and in some cases greater than 90%.
[0050] In contrast to the low density of desired reactive groups on the
substrates of the
invention, it is also typically preferred that the remainder of the surfaces
in question be non-
reactive. As noted previously, such non-reactivity includes a substantially
lower affinity for a
molecule of interest as compared to the reactive groups, but additionally,
preferably includes a
lack of excessive binding or association with molecules that would potentially
interfere with the
end-application of the surface. For example, where additional catalytic groups
are to be coupled
to a desired low density population of desired reactive groups on a surface,
it is generally desired
that such catalytic groups not associate substantially with the remainder of
the surface, either
specifically or non-specifically. Likewise, where in applications, additional
chemical groups
will be exposed to the surfaces of the invention, it will generally be desired
that the remainder or
non-reactive surface not catalyze reactions with such materials or bind or
otherwise associate
with the materials that might provide adverse or noisy signals that do not
correspond to the
reactions of the reactive groups of interest.

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[0051] In the case of fluorescent single molecule assays, one particular
desire is to avoid
excessive (e.g., in duration and/or frequency), nonspecific binding or
association or "sticking" of
unreacted fluorescent reagents or fluorescent products, with the surface other
than with the
reactive groups of interest, e.g., an enzyme, as such associations can lead to
erroneous signal
production, background signal noise and signal noise build-up over time. In
general, it will be
desired that non-specific association of compounds with the non-reactive
portion of the surface
will be comparable to the rate of diffusion of such compounds in solution.
Rephrased in terms
of labeled compounds being observed in observation areas or optical
confinements, signal
resulting from the non-specific association of compounds with the non-reactive
surface will
typically be on the same or similar order, e.g., less than 100 times such
diffusion based signals
and preferably less than 10 times suchg diffuson based signals (in either or
both of duration and
frequency), as signal resulting from random diffusion of such compounds into
and out of the
observation area or volume of fluid for a given analysis. In terms of
fluorescent compounds or
other signal generating compounds that might potentially interfere with the
desired application,
it will generally be desirable that any signal resulting from association of
such compounds with
the non-reactive surface (referred to herein as "non-specific signal
generation"), will be at least
fold lower than signal generated by the reactive groups, preferably more than
100 fold less,
and still more preferably, more than 1000 fold less than signal resulting from
action of the
reactive molecules ("specific signal generation"), e.g., desired enzyme
activity. Such reductions
in non-specific signal generation includes reductions in either or both of
frequency or duration,
e.g., reductions in the number of signal events or a reduction in the
aggregate amount of signal
emanating from such non-specific signal generation.
[0052] A variety of non-reactive groups may be employed upon the remainder of
the
surface that will, again, depend upon the environment to which the surface
will be subjected. In
general, however, terminal hydroxyl groups, methyl groups, ethyl groups,
cyclic alkyl groups,
methoxy groups, hydroxyl groups, e.g., in non-reactive alcohols and polyols,
inactivated
carboxylate groups, ethylene oxides, sulfolene groups, hydrophilic
acrylamides, and the like.
E. Layered Surfaces/Thickness
[0053] As repeatedly described above, the reactive groups, as set forth above,
may be
coupled directly to the surfaces of the substrates or coupled through one or
more intermediate
linking groups that provide one or more intermediate molecular layers between
the desired
reactive group and the inherent or native surface of the substrate material.
Restated, each
component of the surface, reactive or non-reactive, may result from one or
more layers of
components to provide the desired resulting surface component.

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[0054] For example, in its simplest form, both reactive and non-reactive
groups may be
coupled directly to a substrate's native surface to yield the low-density
reactive surfaces of the
invention. Alternatively, one or more layers of linking groups may be added to
the surface to
yield a layered surface, to which the reactive and non-reactive groups are
then coupled to yield
the desired surface. In either of these cases, the process for apportioning
reactive and non-
reactive groups on the surface occurs in the deposition of the final layer.
[0055] In still more complex configurations, apportionment of the reactive and
non-
reactive groups on the final surface layer may occur in the selection and
deposition of earlier
layers on the surface. In other words, a first low-density reactive layer may
be used to dictate
the deposition of a subsequent or desired low-density reactive layer. By way
of example, a first
layer that includes a low density of non-specific binding groups may be used
as a template for
the deposition of a subsequent layer with a low density of catalytic groups,
e.g., where the
catalytic groups couple to the binding groups. In still further aspects, such
apportionment may
take place over multiple layers, to more finely tune the deposition process.
For example, a first
apportioned layer, e.g., including a mixture of binding groups and nonbinding
groups, may
underlie an additional layer that includes a further apportionment. Such
complex layers are also
particularly useful in depositing surfaces according to the invention that
include a number of
different types of reactive groups on an otherwise non-reactive surface, e.g.,
different enzymes,
different nucleic acids, different antibodies, and the like.
[0056] In accordance with the foregoing, in some cases, a surface's inherent
properties
may permit coupling of reactive or intermediate groups thereto, in many cases,
the surfaces must
first be derivatized to provide reactive groups, either for use as such, or
for further coupling to
intermediate linking groups. In many cases, the derivatization process may be
concurrent with
the coupling of reactive groups by providing the desired reactive group as a
constituent of the
derivatizing chemical. In such cases, the derivatizing agent bearing the
reactive group of interest
is coupled to the surface at a relatively low density. Typically, and as set
forth in greater detail
below, this is accomplished by providing the derivatizing agent bearing the
reactive group of
interest in an appropriate ratio with derivatizing agent that, other than its
ability to modify the
surface, is substantially non-reactive.
[0057] In alternative configurations, the entire surface may be derivatized
using any of
the aforementioned reactive groups to provide a reactive surface to which an
intermediate
linking group may be coupled. In such cases, the intermediate linking group,
which is provided
in a ratio of linking group bearing a reactive group of interest and a non-
reactive linking group is
then contacted with the reactive surface to provide the desired density of
reactive groups of
interest on the ultimate surface. As will be appreciated, an intermediate
reactive or coupling



CA 02622872 2008-03-17
WO 2007/041394 PCT/US2006/038243
group may be provided at a higher density than the density at which the
desired, final reactive
group is provided, depending upon the level of coupling of that final group to
the intermediate
group. For example, if it is anticipated (or even planned) that the final
reactive group will
couple to the intermediate coupling group at a rate of 1 linkage for every ten
intermediate
groups, then such intermediate reactive groups may be present at a level 10
times higher.
Typically, when employing such intermediate reactive groups, their density
will be between
about 1 and about 1000 times greater than the final reactive group, often
between about 1 and
about 100 times, and in some cases from 1 to about 10 times greater than the
density of the final
reactive group, e.g., an enzyme.
[0058] One example of such a"layered" surface is schematically illustrated in
Figure 2,
which provides a substrate 200 having an initial or inherent surface 202 that
is treated or
derivatized to provide an overall reactive surface 204 to which additional
chemicals may be
coupled. Reactive surface 204 has coupled thereto a linker/spacer layer 206
which in turn bears
the terminal layer 208. Terminal layer 208 includes at relatively low density,
reactive groups
210 interspersed among the remainder of the terminal layer 208. The number,
type, and order of
layers may be varied in accordance with the various aspects of the invention
described herein
and to achieve the desired final surface.

II. Methods of Preuaring Substrates and Surfaces
[0059] A number of methods may be used to prepare the surfaces of the
invention. In at
least a first approach, the methods of the invention that are used, apply a
ratio or dilution based
treatment to a surface to yield the desired density. In particular, a simple
surface modification
protocol would employ a mixture of at least two different surface modifying
agents where the
first agent included, in addition to the moieties for coupling to the surface,
the reactive group of
interest, such that coupling of the first agent to the surface would also
provide the reactive group
in a configuration that preserved its reactivity. The second or diluent agent
would, other than its
ability to couple to the surface, be otherwise unreactive to the environment
of the application to
which the surface was to be put.
[0060] A schematic illustration of the process is illustrated in Figure 3,
similar to the
diagram shown in Figures 1 and 2. As shown, a surface, i.e., glass or silica
based surface 304 on
substrate 302 is initially derivatized, e.g., using silane chemistry to
provide an overall reactive
surface 304. The reactive surface 304 is then treated with a mixture of
surface modifying agents
306 and 308, that are capable of coupling to the reactive surface 304. The
mixture of agents
includes a concentration of agents bearing a reactive group of interest 306,
and a concentration
of agents that provide non-reactive groups 308. The two agents are present in
a ratio that

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provides for binding of the agents to the reactive surface 304 at the desired
density of reactive
groups 306 on that surface. For example, where binding kinetics of coupling of
both modifying
agents to the reactive surface are equal, a 1:10 ratio of reactive agent 306
to non-reactive agent
308 would effectively yield a density ratio of 1 reactive group 306 coupled to
the reactive
surface 304 for every ten non-reactive groups 308 on that surface. Following
contact and
coupling to the reactive surface 304, a ultimate substrate surface 310 bears
the desired reactive
groups (provided by agents 306) at a desired relative density. For ease of
discussion, the surface
modifying agents and the groups that result on the surface from their use are
illustrated using the
same diagrammatical and numerical representations, although it will be
appreciated that the
agents and the resulting groups may have different chemical structures.
[0061] In a related, but alternative exemplary method, the initial
derivatization process
yields a substrate surface having low density reactivity, using a mixture of
different surface
derivatizing agents. For example, a silica based surface may be initially
derivatized using a
mixture of silane reagents including a first silane reagent that includes a
reactive group when
coupled to the surface, e.g., an aminosilane, with a silane reagent that is
capped or otherwise
nonreactive, e.g., a hydroxylsilane, methylsilane, fluoroalkylsilane, or the
like. With reference
to Figure 3, for example, the initial derivatization process might employ a
mixture of silanes that
bear no additional reactive moiety, e.g., they are capped, blocked or
otherwise non-reactive, and
silanes that include the reactive group of interest. Derivatization with this
mixture would
produce substrate surface that included a low density reactive layer, e.g.,
similar to layer 310, in
place of overall reactive layer 304.
[0062] As noted previously, in any of the cases described above, the reactive
group of
interest that is first coupled to the surface at a relatively low density, may
be the ultimate desired
reactive group for the desired end-application, or it may be an intermediate
linking group that is
capable of linking to the ultimate reactive group. By way of example, the low
density reactive
surface 310 shown in Figure 3, may be further treated with the particular
reactive group of
interest, e.g., enzyme 312, to couple enzyme 312 to reactive groups 306. The
enzyme may
additionally be treated to render it more amenable to coupling to the reactive
groups, e.g.,
through the incorporation of linker moieties, specific binding partners to
reactive groups 306, or
the like. By way of example, the reactive groups may include one member of a
specific binding
pair, e.g., avidin, while the enzyme 312 includes the complementary member of
the binding pair,
e.g., biotin. Coupling of the enzyme 312 (the ultimately desired reactive
group) to the existing
or intermediate reactive group 306 then involves contacting the enzyme with
the surface under
conditions conducive to the affinity interaction between the complementary
binding pair
members.

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[0063] As will be appreciated, the methods of providing the low density
reactive surface
may be approached from a number of different directions, while still yielding
similar results.
For example, with reference to and as shown in Figure 3, above, the surface
modifying reactive
groups 306 which are used to couple enzyme 312 to the substrate surface 302
are first diluted
with surface modifying non-reactive groups 308, to yield a low density
template to which the
enzyme (or other reactive group of interest) is coupled.
[0064] Alternatively, a subset of'surface modifying reactive groups 306 may be
pre-
coupled to the enzyme 312 and then diluted with reactive surface binding
agents 306 (or
optionally, non-reactive surface modifying agents 308) in an appropriate
dilution to yield the
desired density when coupled to the reactive surface 304.
[0065] An example of this process is schematically illustrated in Figure 4. As
shown, a
substrate 400 having a derivatized surface 404 is substantially uniformly
biotinylated to provide
a substantially uniformly biotinylated surface 406. An enzyme 410, bearing a
coupled biotin
moiety 412 is then coupled to the biotinylated surface 406 using an
intermediate
avidin/streptavidin linkage 414 (hereafter referred to as avidin, for
simplicity of description). In
one aspect, the biotinylated surface 406 may be uniformly treated with avidin
414 to provide a
uniform avidin surface. In order to then provide the enzyme 410 at a
relatively low density, the
biotinylated enzyme 410/412 is optionally mixed with biotin 416 that does not
include the
enzyme, at a ratio intended to yield the desired density of enzyme when the
mixture is coupled
to the overall biotinylated surface 406. Alternatively, instead of treating
the biotinylated surface
406 with avidin 414, and applying a mixture of biotinylated enzyme 410/412 and
free biotin
416, one could mix biotinylated enzyme 410/412 with an excess of avidin 414 at
a desired ratio
and apply it to biotinylated surface 406 to yield the same low density of
enzyme coupled to
biotinylated surface 406 via the avidin/biotin linkage. In this case, assuming
purely for the sake
of example that the rate of coupling to a biotinylated surface is equivalent
for avidin coupled to a
biotinylated enzyme and uncomplexed avidin, a mixture of such species at a
ratio of 1:10 would
yield approximately a density of enzyme to uncomplexed avidin on the surface
of 1:10. As will
be appreciated, this assumption is simplified for ease of example, and actual
binding rates would
likely vary significantly, but would generally be readily calibrated through
routine
experimentation.
[0066] Again, although described in terms of enzymes linked via
biotin/avidin/biotin
linkages to a derivatized surface, it will be appreciated that a wide range of
different linkages,
different reactive groups, different surfaces, and different orders of
mixture/dilution and the like
may be used to accomplish the surfaces of the invention.

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[0067] In another, alternative aspect, the invention provides the relative low
density of
reactive moieties on a surface by steric exclusion of such reactive groups
from intervening
spaces. Such methods utilize other molecules or molecular components to create
excluded zones
upon a surface where the reactive moiety cannot be coupled. Such molecules or
components
may be a component of the reactive moiety or a linking molecule for the
reactive moiety, or they
may comprise separate molecular components. By way of example, relatively
large molecules,
e.g., disordered polymers, proteins, polyamino acids, large organic species,
e.g., dendrons, and
the like, that bear a single or a few reactive moieties may be used as spatial
separators between
reactive moieties that they bear (See, e.g., Hong et al., (2003) Langmuir 2357-
2365.
[0068] Alternatively, one class or species of surface associating molecule may
be
provided on a portion of a surface to exclude binding of the surface
associating molecule that
bears the reactive moiety within that portion. By way of example, in the case
of a zero mode
waveguide, or other structural confinement, one group of surface associating
molecules may be
selectively provided upon a wall portion of the confinement, but not on the
observation area
surface of the confinement. The presence of the surface associating molecules
on the walls
effectively reduces the cross sectional dimension of the confinement to
localize any molecules
coupled to the observation area nearer the center of that area. A schematic
illustration of this is
shown in Figure 5. As shown, a zero mode waveguide 502 includes a base
substrate 504 having
a cladding layer 506 disposed thereon. The cladding layer 506 includes the
wave guide core
508, constituting an aperture, disposed through it. Although such cores may
vary in cross-
sectional dimension, in preferred aspects, they are between about 20 nm and
200 nm in diameter.
By coupling exclusion molecules 510 to the wall surfaces, one effectively
reduces the radius of
the substrate surface within waveguide core available for binding of reactive
groups, e.g.,
enzymes (as indicated by dimension 514), by the effective exclusionary length
of the molecules
510 on the walls (as indicated by dimensions 512). Also, because the materials
used to fabricate
cladding layers, e.g., layer 506, are typically a different material from the
underlying substrate,
e.g., metals like aluminum, gold or chrome, or silicon as opposed to silicon
dioxide, one can
take advantage of their different surface properties to selectively couple the
exclusion molecules
510 to the wall surfaces. In the case of metal cladding materials, e.g.,
aluminum, chrome or
gold, surface binding groups are selected that preferentially bind to the
cladding surface as
opposed to the underlying glass or silica based surface. For example,
exclusion molecules
comprising metal or metal chelating groups may be associated with the metal
cladding layer, but
not the underlying glass or silica based substrate. Examples of such groups
include thiol groups,
e.g., mercaptoundecanoic acids, in associating with thin gold layers on the
cladding layer, or
nitriloacetic acids, in associating with nickel layers on the cladding
material, to which are

19


CA 02622872 2008-03-17
WO 2007/041394 PCT/US2006/038243
coupled large molecules, e.g., polymers, disordered polymers, polyamino acids,
proteins, and the
like. These large molecules then shield the active groups present on the
overall surface to
provide a relatively low density of accessible reactive groups, or provide
only sufficient space
for a single catalytic reactive group, e.g., an enzyme, to localize within a
given area on the
observation area surface.
[0069] In yet a further alternative aspect, a low density of reactive groups
of interest may
be accomplished through interactions of layers within a surface structure. In
particular, in some
cases, the interactions of a first reactive layer coupled to the surface, with
a subsequent layer
may result in some subset of the subsequent layer being rendered unreactive,
in the context of
the reactive group of interest. In such cases, that interaction effectively
operates in the same
fashion as a dilution step and may be used to accomplish the goals of the
invention. By way of
example, a first reactive layer may include a field of biotin groups coupled
to the surface
through, e.g., a PEG linker. It has been observed that deposition of an avidin
or streptavidin
layer over the biotin layer appears to result in some level of saturation of a
subset of the avidin
layer. Without being bound to a particular theory of operation, it is believed
that the underlying
biotin linkers may be causing some measure of saturation of the avidin layer,
yielding a subset
of unreactive avidins, e.g., the PEG/avidin linkage may be capable of
sufficient conformational
flexibility to permit binding by the surface bound biotin/linker groups to
multiple recognition
sites on individual avidin molecules, while other avidin molecules remain
unsaturated by the
underlying layer. Effectively, such surfaces have resulted in a functionally
diluted avidin
surface, where a subset of the surface is reactive (e.g., unsaturated avidin)
while the remainder
of the surface is blocked (e.g., saturated avidin).
[0070] A variety of interactive sublayers may be prepared by utilizing
conformationally
flexible linkers, or linkers of varying lengths, e.g., a mixture of shorter
and longer linkers, to
engineer a sublayer that partially blocks the overlaying layer, to yield the
desired density of
reactive groups on the final surface.
[0071] In addition to the foregoing, it will be appreciated that while a
number of
methods are described that refer to the mixture of different components to
create the surfaces of
the invention, it many cases, it will be desirable to synthesize the desired
material as a mixture,
to ensure the proper ratio of each component as used. Synthesis of the ratio
mixture as such,
prevents surface binding variability in the constituent elements used in
producing the surface,
resulting from different synthesis reactions (both in terms of synthetic
scheme variations, and in
terms of lot to lot variability). By way of example, synthesis of mixed
reactive and nonreactive
groups, e.g., mixed silanes, by combining two or more precursor backbones,
e.g., bearing a



CA 02622872 2008-03-17
WO 2007/041394 PCT/US2006/038243
reactive or a non-reactive end, that are reactive with a single silane
precursor, one secures
uniform reactivity of the resulting silane mixture toward the surface.

III. Exemulary Applications of Substratesand Surfaces
[0072] The selectively reactive surfaces of the invention have a variety of
different
applications where it may be desirable to isolate individual molecules or
their reactions from
each other. For example, bead substrates bearing single or few reactive
molecules may be
readily interrogated using FACS or other bead sorting methods, to ascertain a
desired reactive
group in, e.g., a combinatorial chemistry library, directed evolution library,
or phage display
library. The surface modification techniques of the invention are applicable
to such systems.
[0073] Alternatively, single molecule analyses may be performed on a given
enzyme
system to monitor a single reaction and effectors of that reaction. Such
analyses include enzyme
assays that may be diagnostically or therapeutically important, such as kinase
enzymes,
phosphatase enzymes, protease enzymes, nuclease enzymes, polymerase enzymes,
and the like.
[0074] In preferred aspects, the surfaces are used to couple DNA polymerase
enzymes at
low densities in optically isolated locations on a substrate so as to analyze
sequencing reactions
in real-time, and monitor and identify the sequence of the synthesis reactions
as they occur.
Examples of a particularly preferred application of the surfaces of the
invention are described in
published U.S. Patent Application No. 2003/004478 1, which is incorporated
herein by reference
in its entirety for all purposes, and particularly, the application of such
methods in zero mode
waveguide structures as described in U.S. Patent No. 6,917,726, previously
incorporated herein
by reference in its entirety for all purposes. In particular, sequencing data
from the above
described sequencing methods is more easily analyzed when data from individual
reactions, i.e.,
individual polymerase enzymes, can be isolated from data from other enzymes.
By providing
such enzymes on a surface at a low density, one provides physical isolation,
and thus the ability
to optically isolate one enzyme from another. In its most preferred aspect, a
single enzyme
molecule would be provided upon the observation surface of each zero mode
waveguide, to
permit each waveguide to provide data for a reaction of a single enzyme
molecule. Because it
may be difficult to assure that every wave guide or other observation area
possesses a single
enzyme, a density is selected whereby many waveguides will include a single
enzyme, while
some will include 2 or 3 or more enzymes.
[0075] As will be appreciated, the highly defined surfaces of the invention
may have
application across a wide spectrum of applications, technologies and
industries. For example, in
other applications, the surfaces of the invention may be used in any of a
variety of applications
where it is desirable to precisely control the level of functionality of a
surface to control the

21


CA 02622872 2008-03-17
WO 2007/041394 PCT/US2006/038243
physical properties of such surfaces. For example, in a number of
applications, precise control
of ionic groups on a surface may provide precise control of the impact of such
ionic groups on
the surface's interaction with its environment. By way of example, in systems
used for
electrophoretic and/or electroosmotic transport of materials, e.g., in
microfluidic conduits, e.g.,
channels, capillaries, etc., precise control of the zeta potential of the
surface can have broad
impacts upon the electroosmotic mobility of materials within such conduits,
which can, in turn,
impact the relative effectiveness of the system, e.g., in electrophoretic
applications.
[0076] Further, in application of high surface area conduits, e.g.,
capillaries or channels,
one may be desirous of maintaining a certain low level of functionality at a
surface while
preventing excessive interactions between materials and the surface. For
example, in providing
dynamic coatings for capillary electrophoresis a certain level of interaction
between the coating
material and the surface may be desired, while little of no interaction
between analytes and the
surface is desired.
[0077] In still other applications, the surfaces of the invention may be used
to fine tune
surface modifications on medical implants and grafts, to enhance
biocompatibility of such
devices, by more precisely controlling the level of surface modification
thereon.
[0078] One example of a modified surface according to the invention can be
produced
by coating the substrate surface with a mixed layer of molecules harboring
reactive/attachment
sites and capped or nonreactive attachement sites. In the case of silica based
substrate surfaces,
an exemplary mixed layer composition includes silane-PEG-X, where the X may be
-OH or
CH2, for a nonreactive or capped site, and carboxyl, epoxide, amine, biotin,
glutathione, Ni-
NTA, or other well known binding groups (see, e.g., G.T. Hermanson,
Bioconjugate Techniques
(Academic Press 1996). To achieve the relatively low density of
reactive/attachment sites on the
surface, the molar content of capped/nonreactive molecules and the molar
content of reactive
molecules in the applied mixture is selected to yield the desired ratio or
density on the resulting
surface. This ratio is determined by the binding kinetics of each component to
the surface, as
well as the desired end-ratio of reactive to non-reactive groups on the final
surface. In addition,
where a vapor deposition method is used, the relative concentration in the
deposited vapor is of
import, and as such, the vapor pressure of each component in the deposition
chamber is factored
in.
[0079] A schematic of the modified surface is shown in Figure 6. As shown, a
glass
substrate includes an upper surface upon which silane-PEG-OH and silane-PEG-
epoxide
molecules are coupled, where the nonreactive silane-PEG-hydroxyl molecules far
outnumber the
reactive epoxide bearing groups. In related aspects, the epoxide moiety may be
used to link to
specific binding molecules, e.g., biotin, avidin, streptavidin, binding
peptides, antibodies,

22


CA 02622872 2008-03-17
WO 2007/041394 PCT/US2006/038243
antigens, glutathione, GST, which may in turn be used to couple additional
molecules, e.g.,
catalytic molecules like enzymes, or such catalytic molecules may be directly
coupled via the
epoxide group. As will be appreciated, the nature of function of operation of
zero mode
waveguides in a variety of applications dictates that the reactive groups that
are the subject of
observation be relatively near the bottom surface of the waveguide. As such, a
proper linkage
scheme for zero mode waveguide applications will typically result in a
reactive group of interest
being disposed within the relevant observation volume of the waveguide, which
will depend in
some part upon the wavelength of light that is being used. In certain aspects,
that distance is
preferably between about 0 nm and about 20 nm from the bottom surface. In the
case of
preferred surfaces of the invention, the surface thickness that yields a
desired level of reactive
groups and otherwise is non-reactive wiIl typically range from 0.8 nm to about
20 nm, and more
preferably from about 1 nm to about 10 nm. Such surfaces provide sufficient
rejection of
interfering interactions while presenting the reactive groups within an
observation volume of a
preferred optical confinement.
[0080] As noted previously, the substrates of the invention are, in preferred
aspects, used
in conjunction with optical detection systems to monitor particular reactions
occurring on these
low density surfaces. In particular, these systems typically employ
fluorescence detection
systems that include an excitation source, an optical train for directing
excitation radiation
toward the surface to be interrogated, and focusing emitted light from the
substrate onto a
detector. One example of such a system is set forth in U.S. Patent Application
No. 11/201,768,
filed August 11, 2005, and incorporated herein by reference in its entirety
for all purposes.
IV. Examples
Example 1: Silane-PEG20-Biotin/Avidin Surface
[0081] A trimethoxysilane PEG20-biotin molecule was synthesized by BioLink
Life
Sciences (Cary, North Carolina), where the "20" refers to the number of
repeating ethlylene
glycol units. The precursor reagents are commercially available.
[0082] The PEG20-biotin surface was fabricated by incubating substrates in lug
PEG20-
biotin per g of solvent for four hours. The solvent consisted in Methanol, 1%
water by weight,
and 0.1% Tween 20 non-ionic surfactant. The thickness of the PEG20-biotin coat
was t=1.1 nm
as measured by ellipsometry, and the water contact angle (measured within 5
seconds of drop
deposition) was 0=35 degrees.
[0083] The PEG20-biotin forms a thin coating on fused silica slides and Si
substrates
containing a native oxide (ca 2nm) from dilute methanol solutions (ca 1 mg
PEG20-Biotin in 1 g
of methanol), where the thickness varies depending upon solvent composition,
and incubation

23


CA 02622872 2008-03-17
WO 2007/041394 PCT/US2006/038243
time at room temperature. It was observed that when the thickness of the
coating (measured by
ellipsometry) reached 1 nm or more, the surfaces generally showed very good
specificity
towards directed binding (e.g. biotin-avidin) and high degree of rejection of
protein non-specific
binding. Coatings thicker than 2 nm were found not to present further
improvements, while
films below 0.8 nm showed less specificity towards protein binding than those
1 nm or thicker.
Ellipsometric measurements carried out on Si surfaces (with native oxide)
modified with
PEG20-biotin showed that the thickness of the layer when coupled to Alexa488
labeled
streptavidin (dry) is approximately between 3 nm and 4.3 nm thick.
[0084] A test panel was designed to demonstrate and to quantify these
observations. The
panel consisted of four separate areas on a fused silica slide (ca 25mm by 75
mm). Each square
is 6 rnm X 6 mm and was isolated from the other areas by a
polydimethylsiloxane (pdms) gasket
on a plastic frame window that forms a 96-well format pattern (Fast Frame O
sold by

Schleicher & Schuell Biosicences). Typically, each quadrant or well requires -
70 L of
solution.
[0085] The positive and negative control panels interrogated the PEG20-Biotin
Surface
with fluorescently labeled streptavidin (Alexa-488) or NeutrAvidin , or
labeled streptavidin or
NeutrAvidin preincubated with an excess of biotin to provide a measure of
specific vs. non-
specific interaction. The test panels were then interrogated with-labeled 029
polymerase to
determine the level of nonspecific association of the protein (or the level of
protein "rejection"
provided by the surface), under different ionic strength buffer conditions,
e.g., "low salts" (ca 25
mM) and "high salts (ca 150 mM).
[0086] Streptavidin (or NeutrAvidin ) binding specificity and polymerase
rejection
characteristics of the PEG20-biotin surfaces, were compared to the association
with bare fused
silica slide. All slides were rigorously cleaned with Nanostrip followed by
oxygen plasma
cleaning (medium power, 5 minutes @ 2000 mTorr, Harrick xx). The protein-bound
slides were
scanned with a fluorescent scanning instrument. Typically the photomultiplier
gain was set at
550V, and the pixel resolution was 100 m.
[0087] The incubation conditions were as follows: A488-SA (Molecular
Probes/Invitrogen) was dissolved in buffer. The A488-SA was preincubated with
biotin. <A-29
polymerase was labeled with A488 using a commercial labeling kit. The enzyme
was diluted in
25mM tris, 1 mM and 5mM (3-mercaptoethanol ((3ME), and with added 150 mM KC1.
The
solutions were typically incubated for one hour. Afterwards, each well was
rinsed with buffer,
then water and blown-dry with an air gun.

24


CA 02622872 2008-03-17
WO 2007/041394 PCT/US2006/038243
[0088] Fused Silica and Si substrates modified with PEG20-biotin demonstrated
high
levels of protein "rejection" when challenged with (~--29 polymerase, which
was then stained
with a fluorescently labeled antibody epitope, yielding fluorescent
intensities comparable to the
biotin-bloclced fluorescently labeled streptravidin or NeutrAvidin0 in both
low and high salt
conditions. In particular, the results showed fluorescent intensities that
were greater than 100X
lower than that of the positive control (A488-SA) or protein adsorption on
untreated fused silica.
[0089] A comparison of non-specific binding of 0-29 polymerase on fused silica
and on
PEG20-biotin surfaces showed high polymerase surface coverage (40-60%) on
fused silica
under low ionic strength immobilization conditions, and nearly 200 fold
reduction of non-
specific adsorption on PEG20-biotin (at low ionic strengths) as compared to
fused silica. It also
shows that PEG20-biotin presents a high degree of binding specificity towards
streptavidin. The
polymerase rejection characteristics of the surface decrease as the PEG layer
becomes less than
lnm thick.
[0090] Next, a panel bearing the PEG20-biotin surface was interrogated with
biotinylated A488-Polymerase, mediated by non-labeled Streptavidin. Briefly,
all wells on the
slide bearing the PEG20-biotin surface were incubated first with streptavidin,
and then
interrogated with biotinylated A488-labeled polymerase that was either pre-
incubated or not pre-
incubated with an excess of unlabeled streptavidin, to determine the level of
specific streptavidin
mediated linkage to the surface. The specific interaction between the surface
and the
biotinylated protein showed approximately 50X increase over the non-specific
interaction
(where the biotinylated protein was pre-blocked with streptavidin prior to
contacting it with the
surface).

Exam~le 2: Silane-PEG24-Biotin/Avidin Surface
[0091] a-biotinyl-co-trimethoxysilyl terminated poly(ethylene glycol) (24
units) (PEG24-
biotin) was synthesized by Polymer Source (Dorval, Montreal Canada).
[0092] A solution was prepared by dissolving PEG24-biotin in Ethanol at a
concentration of 0.6 mg PEG24-Biotin per gram of solvent. A small amount of
methanol (ca
0.4% by weight) was added to the mix to adjust the rate of deposition of the
silane reagent to a
Si chip (with a native oxide of ca 2nm) between 1nm and 1.5nm (by
ellipsometry) in three to
five hours.
[0093] Fused Silica and Si substrates modified with PEG24-biotin again
demonstrated
low levels of non-specific interaction when challenged with fluorescently-
labeled 0-29
polymerase as well as biotin-blocked Neutravidin, while at the same time
showing high levels of



CA 02622872 2008-03-17
WO 2007/041394 PCT/US2006/038243
(specific) binding towards Neutravidin and streptavidin. Restated, the
surfaces of the invention
have demonstrated a high level of specific interaction with desired groups,
e.g., biotinylated
protein, while showing low levels of non-specific binding, boith of proteins,
e.g., excess
enzymes, as well as potentially interfering label molecules, e.g., fluorescent
avidin (or by
analogy, in the case of nucleic acid analyses, labeled nucleotides or
nucleotide analogs, which if
allowed to non-specifically adsorb within an observation area, might otherwise
interfere with
detection of enzymatic reactions). In this experiment, the fluorescently
labeled streptavidin and
polymerase were premixed with unlabeled proteins in the ratio of 1/10 in order
to maintain a
linear relationship between fluorescence intensity and fluorophore surface
density.

Example 3: Adjusting Density of Reactive Groups
[0094] A system was prepared to test the ability to adjust the surface density
of reactive
functional groups using a dilution protocol. In particular, PEG24-biotin
surfaces were prepared
using varying dilutions of the PEG24-Biotin in a solution of N-(3-
triethoxysilylpropyl)gluconamide (TES-G) is a commercial reagent (Gelest Inc,
Morrisville, PA
product #SIT8189-0).
[0095] A TES-G stock solution was prepared by dilution of 2 mg of reagent per
g of a
solvent mix of ethanol and 0.4% methanol. A PEG24-biotin stock solution was
prepared at the
concentration of 1.5mg per gram of solvent (ethanol/methanol mix). Five
aliquots of 10 mL
each were prepared, and increasing amounts of PEG24-biotin stock were added to
each aliquot:
0, 40 L (0.3%), 80 L (0.6%), 200 L (1.4%), 400 L (2.7%) and 800 L (5.4%).
Fused silica
blanks and Si wafers were incubated simultaneously in the five aliquots for
15h. Afterwards the
substrates were removed from the reaction solutions, washed three times with
methanol,
annealed in air at 80C for 10 minutes, washed in water for 10minutes with
ultrasonic agitation to
remove weakly bound molecules, and dried with nitrogen gas gun. A measure of
the amount of
PEG24-Biotin on the surface was provided by measuring the water contact angle
on the surface,
where a pure PEG24 surface would have a contact angle of approximately 35 .
[0096] Figure 7 shows a plot of water contact angle vs. the concentration of
PEG24-
biotin in TES-G, and illustrates that surface density of the active groups is
readily controllable
using a dilution approach.

Example 4: Mixed Functionality PEG24 Surfaces
[0097] In order to provide uniformity of surfaces, structurally similar groups
were used
to provide the binding or reactive group, as well as the non-reactive
component of the surface.
In particular, a PEG24-Biotin was diluted with a PEG24-methoxy group, similar
to the PEG24
26


CA 02622872 2008-03-17
WO 2007/041394 PCT/US2006/038243
groups described in Example 2 (supra), in order to provide a surface having
uniform
characteristics, other than in its ability to interact with other molecules,
e.g., thickness. PEG24-
Methoxy was custom synthesized by Polymer Source Inc. (Dorval, Montreal,
Canada) and was
used as received. PEG24-methoxy was deposited on fused silica slides and Si
wafers by
incubating the substrates at room temperature in a solution containing 0.6 mg
of reagent in
ethanol with 0.4% methanol. Si substrates were removed from the solution at
different time
intervals to evaluate contact angle and film thickness. The rate of deposition
and film thiclcness
were comparable to PEG24-biotin films deposited under similar solvent and
concentration
conditions.
[0098] Silane films deposited from mixed solutions of PEG24-Methoxy and PEG24-
biotin were investigated using four-quadrant panels. The PEG24-Methoxy
concentration was
0.83 mg/g of ethanol/methanol solvent (0.4% methanol) and four aliquots were
prepared by
mixing increasing amounts of a PEG24-biotin stock solution containing 0.67
mg/g of solvent.
The final solution concentrations in terms of mole percent of biotin relative
to end methoxy
group were 0%, 0.2%, 0.7%, and 2%. Si wafers and fused silica slides were
incubated in each
aliquot for 3.5 hours at room temperature. Afterwards, the substrates were
rinsed three times in
methanol, dried in air, annealed at 80 degrees C for 10 minutes, and washed in
water with
ultrasonic mixing to remove weakly-bound PEG molecules. The thickness of the
films were
close to t=1.3 nm for all samples, and the corresponding contact angles were
between 42 (no
PEG24-biotin) to 38 (2% biotin). Binding of labeled streptavidin to the mixed
surface showed
good correlation with increasing molar percent of PEG24-biotin in the mixed
surface, while
binding of pre-blocked streptavidin was largely unchanged.

Example 5: Deposition of Low Density Reactive Polymerases on Surfaces Through
Dilution of
Linkage Mediating L"er
[0099] The methods of the invention were used in the deposition of relatively
low
density of 029 DNA polymerase enzyme in a zero-mode waveguide array, by
providing a
PEG24-biotin surface within each waveguide, and depositing a mixture of
streptavidin and
biotinylated polymerase. The ratio of Neutravidin to biotinylated polymerase
was adjusted to
yield a desired level of Neutravidin mediated linkage between the polymerase
and the PEG-24-
biotin surface, while blocking the remaining surface with excess Neutravidin.
[00100] Initially, statistical analysis was performed to determine the
relative dilution of
polymerase in streptavidin to yield an acceptable probability that each
occupied waveguide
would include no more than one polymerase enzyme. In particular, the
probability of
occupation (P(,c,) of a Poisson statistics of average density is given as P
cc = 1 - P (0) = 1- e(-

27


CA 02622872 2008-03-17
WO 2007/041394 PCT/US2006/038243
). For small occupation numbers Pocc = - 2/2 + 3/6 +O( 4). So using
Probability of
occupancy as a means of measuring creates an error of order 2. Therefore,
one can use
Probability of occupancy PoCC as a measure of average occupation number
whenever is
small. For a probability of occupancy of 0.3, one can calculate the actual
value of to be
approximately 0.3567. That is, in this case. the use of the probability of
occupancy as an
estimate of average occupation yields an error of 16%. It is worth noting that
for an average
occupation number of 0.3567, there is a 70% chance that any waveguide will be
empty, a 25%
chance that the waveguide will contain one and exactly one polymerase and a 5%
chance the
waveguide will contain one or more polymerases. This illustrates that for
sufficiently low
occupation numbers, the use of probability of occupancy is an acceptable
estimate of average
occupation number.
[00101] This was then achieved by providing a dilution of biotinylated enzyme
in a 50-
fold excess of Neutravidin. Following dilution and deposition, the resulting
waveguide arrays
were tested and found to have roughly a 30% occupancy, correlating well with
the probability.
Further, the specific activity of the enzyme within these waveguides
correlated very well to
specific activity data generated on planar surfaces (which showed very high
specific activity of
immobilized enzyme), further validating the statistical approach.
[00102] Although described in some detail for purposes of illustration, it
will be readily
appreciated that a number of variations known or appreciated by those of skill
in the art may be
practiced within the scope of present invention. Unless otherwise clear from
the context or
expressly stated, any concentration values provided herein are generally given
in terms of
admixture values or percentages without regard to any conversion that occurs
upon or following
addition of the particular component of the mixture. To the extent not already
expressly
incorporated herein, all published references and patent documents referred to
in this disclosure
are incorporated herein by reference in their entirety for all purposes.

28

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Administrative Status

Title Date
Forecasted Issue Date Unavailable
(86) PCT Filing Date 2006-09-29
(87) PCT Publication Date 2007-04-12
(85) National Entry 2008-03-17
Examination Requested 2011-09-28
Dead Application 2013-10-01

Abandonment History

Abandonment Date Reason Reinstatement Date
2012-10-01 FAILURE TO PAY APPLICATION MAINTENANCE FEE

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Registration of a document - section 124 $100.00 2008-03-17
Application Fee $400.00 2008-03-17
Maintenance Fee - Application - New Act 2 2008-09-29 $100.00 2008-08-22
Maintenance Fee - Application - New Act 3 2009-09-29 $100.00 2009-09-14
Maintenance Fee - Application - New Act 4 2010-09-29 $100.00 2010-09-13
Maintenance Fee - Application - New Act 5 2011-09-29 $200.00 2011-09-06
Request for Examination $800.00 2011-09-28
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
PACIFIC BIOSCIENCES OF CALIFORNIA, INC.
Past Owners on Record
FOQUET, MATHIEU
PELUSO, PAUL
ROITMAN, DANIEL BERNARDO
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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