Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND REAGENTS FOR SURFACE FUNCTIONALIZATION
CROSS REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. provisional application number
60/403,971, entitled, "Biochip Surface Configurations and Methods and
Compositions for Making the Same," filed August 16, 2002, in the name of David
Quincy, which is incorporated herein by reference.
FIELD
This disclosure concerns a class of reagents and a method for surface
functionalization for creating arrays of bioactive compounds on a substrate.
BACKGROUND
Comprehensive understanding of disease states requires the identification
and characterization of all proteins involved in a particular disease pathway.
This
study of proteins and their interactions is termed "proteomics." Due to
advances in
molecular biology, proteins of interest can be produced more rapidly than
current
techniques can characterize the proteins. For example, conventional methods
for
protein identification in proteomics applications rely upon two-dimensional,
polyacrylamide gel electrophoresis (2D-PAGE) technology to isolate proteins,
followed by subsequent identification by mass spectrometry. Typically, 2D-PAGE
can separate as many as 5000 different proteins. However, identification of
each
protein is a tedious, labor-intensive process that requires cutting each of
the
individual separated proteins out of the polyacrylamide gel. Moreover, the
method
has a relatively low sensitivity. For example, when using silver staining, the
detection limit is about 1 nanogram of protein.
Additional drawbacks of 2D-PAGE include a lack of reproducibility, low
throughput, low resolution and protein-dependent sensitivity. For example, 2D-
PAGE generally does not resolve all proteins present in a mixture and systems
are
limited to processing a handful of gels over a two-day period. In addition,
high-
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molecular-weight, low-molecular-weight and membrane-bound proteins are
underrepresented.
Attempts have been made to use protein arrays for the high throughput
characterization of proteins. For example, U.S. Patent No. 6,406,921 to Wagner
et
al. discloses a method of making protein-coated substrates, and U.S. Patent
No.
5,620,850 to Bamdad et al. discloses a method for making a surface including a
plurality of chelating agents, which can be used to bind metal ions. The bound
metal
ions are then reportedly used to capture a biological molecule that also
includes a
chelating agent.
A number of hurdles must be overcome to provide protein arrays of high
quality which produce accurate and reproducible screening results. Typically,
proteins must remain hydrated, be kept at ambient temperatures, and are very
sensitive to the physical and chemical properties of the support materials.
Thus,
maintaining protein activity at the liquid-solid interface requires new
strategies for
assembling arrays that address the sensitivity of the proteins to the
environment.
SUMMARY
Disclosed herein are reagents and a method for using such reagents to
prepare substrates derivatized with affinity agents, such as small molecules,
peptides, proteins, nucleic acids and other bioactive molecules. As disclosed
herein,
densely packed affinity agent arrays exhibiting minimal, non-specific binding
can be
prepared.
In one aspect of the method, a substrate is functionalized with a reagent
having at least two reactive functional groups. The first reactive functional
group
serves to couple the reagent to the substrate and the second reactive
functional group
is an affinity agent or provides a site for operatively associating an
affinity agent.
In a second aspect of the method, non-specific protein adhesion to the arrays
is diminished by the incorporation of a protein resistant component, which
diminishes such non-specific protein adhesion. Thus, in one aspect, the
disclosure
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provides reagents and techniques for assembling arrays of affinity agents that
exhibit
reduced non-specific binding.
BRIEF DESCRIPTION OF THE DRA WINGS
FIG. 1 illustrates one embodiment of the disclosed method for
functionalizing a surface.
FIG. 2A is a schematic representing one embodiment of the disclosed array.
FIG 2B provides examples of monolayer components used to prepare such
arrays.
FIG. 3A illustrates a method for using an asymmetric disulfide reagent to
prepare a monolayer.
FIG. 3B depicts examples of monolayer components prepared via Staudinger
ligation.
FIG. 4 illustrates one example of a process for preparing a novel array.
FIG. 5 is a schematic of a fluorescence detection unit which may be used to
monitor interaction of the proteins of the array with an analyte.
FIG. 6 is a schematic of an ellipsometric detection unit which may be used to
monitor interactions between analytes and the affinity tags of the array.
FIG. 7 illustrates the synthesis of an asymmetric disulfide having N
hydroxysuccinimide and polyethylene glycol groups.
DETAILED DESCRIPTION
The following explanations of terms and methods are provided to better
describe the present compounds, compositions and methods and to guide those of
ordinary skill in the art in the practice of the present disclosure. The
terminology
used in the disclosure is for the purpose of describing particular embodiments
and
examples only and is not intended to be limiting.
A. Definitions
The term "substrate" as used herein refers to a bulk; underlying material used
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in the arrays and devices disclosed herein.
An "array" refers to a two-dimensional distribution or pattern.
The terms "polypeptide" and "protein" are used interchangeably to refer to an
amino acid polymer.
The term "antibody" means an immunoglobulin, whether natural or wholly
or partially synthetically produced. All derivatives thereof which maintain
specific
binding ability are also included in the term. The term also covers any
protein
having a binding domain which is homologous or largely homologous to an
immunoglobulin binding domain. These proteins may be derived from natural
sources, or partly or wholly synthetically produced. An antibody may be
monoclonal or polyclonal. In an exemplary embodiment, the antibody is a
glycosylated antibody. The antibody may be a member of any immunoglobulin
class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. In an
exemplary embodiment, the antibody is of the IgG class.
The term "antibody fragment" refers to any derivative of an antibody which
is less than full-length. In an exemplary embodiment, the antibody fragment
retains
at least a significant portion of the full-length antibody's specific binding
ability.
Examples of antibody fragments include, but are not limited to, Fab, Fab',
F(ab')2,
scFv, Fv, dsFv diabody, and Fd fragments. The antibody fragment may be
produced
by any means. For instance, the antibody fragment may be enzymatically or
chemically produced by fragmentation of an intact antibody or it may be
recombinantly produced from a gene encoding the partial antibody sequence.
Alternatively, the antibody fragment may be wholly or partially synthetically
produced. The antibody fragment may optionally be a single chain antibody
fragment. Alternatively, the fragment may comprise multiple chains which are
linked together, for instance, by disulfide linkages. The fragment may also
optionally be a multimolecular complex. A functional antibody fragment will
typically comprise at least about 50 amino acids and more typically will
comprise at
least about 200 amino acids.
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Single-chain Fvs (scFvs) are recombinant antibody fragments consisting of only
the
variable light chain (VL) and variable heavy chain (VH) covalently connected
to one
another by a polypeptide linker. Either VL or VH may be the NH2-terminal
domain.
The polypeptide linker may be of variable length and composition so long as
the two
variable domains are bridged without serious steric interference. Typically,
the
linkers are comprised primarily of stretches of glycine and serine residues
with some
glutamic acid or lysine residues interspersed for solubility.
A Fv fragment is an antibody fragment which consists of one VH and one VL
domain held together by noncovalent interactions. The term "dsFv" is used
herein
to refer to an Fv with an engineered intermolecular disulfide bond to
stabilize the
VH-VL pair.
A F(ab')Z fragment is an antibody fragment essentially equivalent to that
obtained from immunoglobulins (typically IgG) by digestion with an enzyme
pepsin
at pH 4.0-4.5. The fragment may be recombinantly produced.
A Fab' fragment is an antibody fragment essentially equivalent to that
obtained by reduction of the disulfide bridge or bridges joining the two heavy
chain
pieces in the F(ab')Z fragment. The Fab' fragment may be recombinantly
produced.
A Fab fragment is an antibody fragment essentially equivalent to that
obtained by digestion of immunoglobulins (typically IgG) with the enzyme
papain.
The Fab fragment may be recombinantly produced.
"Amino acid" refers to both naturally occurring and "unnatural" amino acids.
Residues of amino acids also are encompassed by the term amino acid.
"Organic thinfilin" refers to a thin layer of organic molecules formed
directly
on a substrate or on a coating on the substrate. Typically, the organic
thinfilms
disclosed herein are from about 0.5 nm to about 50 nm thick, and more
typically
from about 0.5 nm to about 10 nm thick. The organic thinfilm can be assembled
prior to deposition on the substrate; however, typically the organic thinfilm
is
assembled as the component molecules are attached to the substrate. The
organic
thinfilm optionally includes associated inorganic ions and chelated metals
bound to
the thinfilm. The organic thinfilm can be homogeneous or heterogeneous and can
be
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composed of one or more monolayers. An example of a thinfilm composed of
plural
monolayers is a lipid bilayer. However, typically the organic thinfilm is a
monolayer. Optionally, the organic thinfilm can include more than one type of
organic thinfilm.
Typically, the organic thinfilms disclosed herein include an affinity agent or
a functional group suitable for covalently or noncovalently associating an
affinity
agent to the thinfilm. The organic thinfilm also optionally can bear
functional
groups that reduce the association of molecules with the thinfilm. Typically,
such
functional groups are hydrophilic groups, such as, for example, polyalkylene
oxides,
including polyethylene glycol (PEG) and polypropylene glycol (PPG), which
generally are not bound tightly by proteins. PEG and PPG include oligomers of
ethylene glycol and propylene glycol, respectively. As such, PEG and PPG as
used
herein refer to polymers having as few as two glycol subunits. In working
embodiments, PEG was used as a protein-resistant component of organic
thinfilms
to reduce the nonspecific binding of proteins to the organic thinfilm. In
several
working examples, PEG components that were used were not monodisperse, and
therefore the specific numbers of ethylene glycol units referred to can also
refer to
an average number where a polydisperse mixture of PEG units are used. Other
functional groups of the organic thinfilm serve to tether the thinfilm to the
surface of
the substrate or a coating on the substrate.
The term "monolayer" refers to a layer having a single-molecule thiclmess,
which can be an organic thinfilm or portion thereof. A monolayer can be
ordered or
disordered. Typically the monolayer is ordered and densely packed. The
monolayer
can be homogeneous or heterogeneous, however one face of the monolayer will
include functional groups that can be chemisorbed or physisorbed onto the
surface
of the substrate or a coating on the substrate.
A first monolayer also can be converted into a second monolayer. For
example, the component molecules in a first monolayer having a first end
chemisorbed or physisorbed to a substrate can be functionalized by covalently
bonding one or more second molecules to plural second ends of the first
monolayer
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component molecules, thereby converting the first monolayer into a new, second
monolayer.
B. Arrays of Affinity Agents
The present disclosure is directed to arrays of affinity agents and methods
for
making and using such arrays. Typically the arrays form a two-dimensional
display
of an affinity agent, which can be used to characterize the interaction of the
affinity
agent with a soluble molecule of interest.
Thus, in one embodiment, the disclosed functionalized substrates are further
functionalized with an affinity agent. The affinity agent can be any agent
that can be
bound to the functionalized substrate and that interacts covalently or
noncovalently
with a molecule of interest. Typically the affinity agent is a small molecule,
oligonucleotide, peptide or protein that binds to or interacts with a soluble
molecule
of interest. Similarly, the soluble molecule of interest or analyte can be a
small
molecule, oligonucleotide, peptide or protein. Interactions between the
affinity
agent and the analyte can be detected by any suitable method, and worl~ing
embodiments used methods such as optical detection methods including
ultraviolet
and visible absorption, chemoluminescence, and fluorescence (including
lifetime,
polarization, fluorescence correlation spectroscopy (FCS), and fluorescence-
resonance energy transfer (FRET)).
In one embodiment, an array of affinity agents includes a substrate, an
organic thinfilm formed on at least a portion of the surface of the substrate,
and
including plural copies of at least one affinity agent operatively associated
with, e.g.,
covalently or noncovalently associated with, the underlying organic thinfilm.
Generally, when plural types of affinity agents are used in the same array,
the
' different types of affinity agents are grouped in "patches," so that
affinity agents
localized in one patch of the array differ from affinity agents localized to
another
patch. In one embodiment, the present invention provides an array of
glycoproteins
containing a substrate, at least one organic thinfilm on some or all of the
substrate
surface, and a plurality of patches arranged in discrete, known regions on
portions of
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the substrate surface covered by organic thinfilm, wherein each of said
patches
comprises a protein immobilized on the underlying organic thinfilm. The array
optionally contains an interlayer between the substrate and coating.
In most cases, the array will comprise at least about ten patches. In an
exemplary embodiment, the array comprises at least about 50 patches. In
another
exemplary embodiment the array comprises at least about 100 patches. In
alternative exemplary embodiments, the array of affinity agents can comprise
more
than 103,104 or 105 patches.
In an exemplary embodiment, the surface area of the substrate covered by
each of the patches is no more than about 0.25 mmz. In another exemplary
embodiment, the area of the substrate surface covered by each of the patches
is
between about 1 ~,mz and about 10,000 ~mz. In another exemplary embodiment,
each patch covers an area of the substrate surface from about 100 ~,m2 to
about
2,500 ~m2. In an alternative embodiment, a patch on the array may cover an
area of
the substrate surface as small as about 2,500 nm2, although patches of such
small
size are generally not necessary for the array to be useful.
The patches of the array may be of any geometric shape. For instance, the
patches may be rectangular or circular. The patches of the array may also be
irregularly shaped.
The distance separating the patches of the array can vary. For example, the
patches of the array are separated from neighboring patches by about 1 ~,m to
about
500 ~.m. Typically, the distance separating the patches is roughly
proportional to the
diameter or side length of the patches on the array if the patches have
dimensions
greater than about 10 Vim. If the patch size is smaller, then the distance
separating
the patches typically will be larger than the dimensions of the patch.
In an exemplary embodiment of the array, the patches of the array are all
contained within an area of about 1 cm2 or less on the surface of the
substrate. In
one exemplary embodiment of the array, therefore, the array comprises 100 or
more
patches within a total area of about 1 cm2 or less on the surface of the
substrate.
Alternatively, an exemplary array comprises 103 or more patches within a total
area
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of about 1 cm2 or less. An exemplary array may even optionally comprise 104 or
105
or more patches within an area of about 1 cm2 or less on the surface of the
substrate.
In other embodiments of the invention, all of the patches of the array are
contained
within an area of about 1 cm2 or less on the surface of the substrate.
In one embodiment, only one type of affinity agent is immobilized on each
patch of the array. In an exemplary embodiment of the array, the affinity
agent
immobilized on one patch differs from the affinity agent immobilized on a
second
patch of the same array. In such an embodiment, a plurality of different
affinity
agents can be present on separate patches of the array. In another aspect, a
single
patch comprises two or more affinity agents that bind to the same analyte.
Such
affinity agents typically bind to different epitopes of the analyte. One class
of
affinity agents that can be used in this aspect is polyclonal antibodies.
Typically the array comprises at least about ten different affinity agents. In
an exemplary embodiment, the array comprises at least about 50 different
affinity
agents. In another exemplary embodiment, the array comprises at least about
100
different affinity agents. Alternative exemplary arrays comprise more than
about 103
different affinity agents or more than about 104 different affinity agents.
The array
may even optionally comprise more than about 105 different affinity agents.
In one embodiment of the array, each of the patches of the array contains a
different affinity agent. For instance, an array comprising about 100 patches
could
comprise about 100 different affinity agents. Likewise, an array of about
10,000
patches could comprise about 10,000 different affinity agents. In an
alternative
embodiment, however, each different affinity agent is immobilized on more than
one
separate patch on the array. For instance, each different affinity agent can
optionally
be present on two to six different patches. Therefore an array can comprise
about
three-thousand affinity agent patches, but only comprise about one thousand
different affinity agents, since each different agent is present on three
different
patches.
In another embodiment, although the affinity agent of one patch is different
from that of another, the affinity agents are related. In an exemplary
embodiment,
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the two different affinity agents are members of the same protein family. The
different proteins on the array can be either functionally related or thought
to be
functionally related. In another embodiment of the invention array, however,
the
function of the immobilized proteins may be unknown. In this case, the
different
glycoproteins on the different patches of the array typically share a
similarity in
structure or sequence or are thought to sharing a similarity in structure or
sequence.
Alternatively, the immobilized proteins can be fragments of different members
of a
protein family.
Any affinity agent that can be operatively associated with an organic thinfilm
on the substrate can be employed in the disclosed arrays. Classes of different
affinity agents include, without limitation, small molecules, peptides,
proteins and
nucleic acids, including DNA and RNA. Optionally, an array can include
different
affinity agents from different classes.
When proteins are selected as affinity agents, the proteins can be members of
a protein family, such as a receptor family, examples of which include growth
factor
receptors, catecholamine receptors, amino acid derivative receptors, cytokine
receptors, and lectins; a ligand family, examples of which include cytokines
and
serpins; an enzyme family, examples of which include proteases, kinases,
phosphatases, ras-like GTPases, and hydrolases; and transcription factors,
examples
of which include steroid hormone receptors, heat-shock transcription factors,
zinc-
finger proteins, leucine-zipper proteins and homeodomain proteins. In one
embodiment, the different immobilized proteins are all HIV proteases or
hepatitis C
virus (HCV) proteases. In another embodiment the associated proteins on the
array
axe all hormone receptors, neurotransmitter receptors, extracellular matrix
receptors,
antibodies, DNA-binding proteins, intracellular signal transduction modulators
and
effectors, apoptosis-related factors, DNA synthesis factors, DNA repair
factors,
DNA recombination factors, or cell-surface antigens.
Antibodies and antibody fragments are particularly useful affinity agents for
use with the disclosed arrays. The antibodies optionally can be polyclonal or
monoclonal antibodies. The production and isolation of antibodies that bind to
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specific targets, including protein targets, using standard hybridoma
technology is
known to those of ordinary skill in the art. Moreover, numerous antibodies are
available commercially. Alternatively, antibodies or antibody fragments can be
expressed in bacteriophage. Such antibody phage display technologies,
including
methods for bacteriophage selection, are well known to those of ordinary skill
in the
art.
C. Reagents and Techniques for Prepariyag Arrays of Affinity Agents
In one aspect, the disclosure provides reagents and techniques for assembling
arrays of affinity agents. According to one embodiment of the method a first
heterobifunctional reagent is covalently or non-covalently associated with a
substrate, thereby forming a monolayer. A second heterobifunctional reagent is
coupled to the monolayer to provide an array of reactive functional groups and
a
third reagent including a protein resistant component is coupled to the
monolayer,
such that the three reagents form an organic thinfilm including reactive
functional
groups. The reactive functional groups are selected such that an affinity
agent can
be coupled to the organic thinfilm, thereby forming an affinity agent array
having
affinity agents operatively coupled to the organic thinfilm. Coupling involves
covalently or non-covalently associating the affinity agent. Non-covalent
associate
may exploit, without limitation, one or more of Coulombic interactions,
hydrogen
bonds, Van der Waals interactions and hydrophobic interactions.
In one embodiment, an affinity agent is coupled to an organic thinfilm by a
chemoselective ligation reaction. Chemoselective ligation reactions generally
refer
to reactions between functional groups that have orthogonal reactivity to
other
functional groups present, particularly those functional groups found in many
biomolecules. Thus, chemoselective ligation reactions are particularly useful
when
the affinity agent is a biomolecule. Typically, chemoselective ligation
reactions
used with biomolecules employ one or more non-native functional groups to
ensure
that the reaction is orthogonal to native functional groups. One example of a
chemoselective ligation reaction is a Staudinger ligation. Other examples
include
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reactions of ketones and aldehydes, such as the condensation of a hydrazide or
aminooxy compound with a ketone or aldehyde to yield the corresponding
hydrazone or oxime. Another example is the reaction of a thiocarboxylate with
an
a-halo carbonyl compound to give a thioester. Versions of these reactions also
can
be used to attach compounds other than biomolecules with the organic thinfilm.
Exemplary methods disclosed herein employ a Staudinger ligation reaction
to associate a reagent or affinity agent with an organic thinfilm. Staudinger
ligation
functions as an amide bond forming reaction and typically involves two
reactive
components, the first typically having the formula Y-Z-PRZR3 where Z is an
aryl
group substituted with Rl, wherein Rl is preferably in the ortho position on
the aryl
ring relative to the PR2 R3; and wherein Rl is an electrophilic group to trap
(e.g.,
stabilize) an aza-ylide group, including, but not necessarily limited to, a
carboxylic
acid, an ester (e.g., alkyl ester (e.g., lower alkyl ester, benzyl ester),
aryl ester,
substituted aryl ester), aldehyde, amide, e.g., alkyl amide (e.g., lower alkyl
amide),
aryl amide, alkyl halide (e.g., lower alkyl halide), thioester, sulfonyl
ester, alkyl
ketone (e.g., lower alkyl ketone), aryl ketone, substituted aryl ketone,
halosulfonyl,
nitrile, nitro and the like; R2 and R3 are generally aryl groups, including
substituted
aryl groups, or cycloalkyl groups (e.g., cyclohexyl groups) where RZ and R3
may be
the same or different, preferably the same; and Y is H, a protein resistant
group, a
reactive group that facilitates covalent attachment of an affinity agent or a
molecule
of interest, wherein Y can be at any position on the aryl group (e.g., para,
meta,
ortho); where exemplary reactive groups include, but are not necessarily
limited to,
carboxyl, amine, (e.g., alkyl amine (e.g., lower alkyl amine), aryl amine),
ester (e.g.,
alkyl ester (e.g., lower alkyl ester, benzyl ester), aryl ester, substituted
aryl ester),
thioester, sulfonyl halide, alcohol, thiol, succinimidyl ester,
isothiocyanate,
iodoacetamide, maleimide, hydrazine, and the like. Exemplary affinity agents
further include dyes (e.g., fluorescein or modified fluorescein, and the
like),
antibodies, toxins (including cytotoxins), linkers, peptides, and the like. An
exemplary and preferred engineered phosphine reactant is 2-diphenylphosphanyl-
benzoic acid methyl ester.
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The second reagent comprises an azide. Molecules comprising an azide and
suitable for use in the present invention, as well as methods for producing
azide-
comprising molecules suitable for use in the present invention, are well known
to
those having ordinary skill in the art.
In a working embodiment a monolayer comprising a 2-diphenylphosphanyl-
benzoic acid methyl ester derivative was prepared on a gold substrate.
According to
this embodiment, a first reagent, 11-thio-undecanionic-N hydroxysuccinimide
ester
was linked to the substrate via the sulfhydryl group. The resulting monolayer
was
derivatized with compound 25, thereby forming a second monolayer from the
first,
where the second monolayer is suitable for coupling of an affinity agent
containing
an azide moiety. A fluorescently labeled, azidoalanine-containing peptide was
then
coupled to the second monolayer via Staudinger ligation.
H
N!~ NHS
O
15 In another aspect of the method, a native functional group of a biomolecule
is used to attach it to an organic thinfilm. For example, proteins that have
one or
more cysteine residues can be selectively alkylated with reagents, such as a-
halo
carbonyl compounds. When an a-halo carbonyl compound is associated with a
thinfilm, this can be a useful reaction for associating certain proteins to
the thinfilm.
20 In a parallel method, a natural or non-natural amino acid can be
incorporated
into a peptide or protein to provide a functional group for associating the
protein
with an organic thinfilm. For example, as discussed above, in a working
embodiment an azide-containing amino acid was incorporated into a peptide,
which
is then attached to an organic thinfilm via a Staudinger ligation protocol.
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In one embodiment, the substrate is fwctionalized with a chelator, such as an
N nitrilotriacetic acid (NTA) derivative or an imidodiacetic acid (IDA)
derivative,
which chelate metals, such as nickel, cobalt, iron and copper. Using such
derivatives, a histidine-tagged protein can be noncovalently bound to the
substrate
via, for example, mutual nickel chelation by both the substrate-associated
chelator,
such as an NTA or IDA derivative, and the histidine tag. Proteins having
histidine
tags are known to those of ordinary skill in the art. See, Hochuli, et al.,
Biotechnology, 1988, 6, 1321. In another aspect of this method for attaching
an
affinity agent, the initial, non-covalent association can be followed by
covalent bond
fornzation between the affinity agent and the substrate associated organic
thinfilm.
This can be accomplished by using, for example a photoactivatable group, such
as
an aryl azide, particularly haloaryl azides, for example a pentafluorophenyl
aryl
azide, benzophenones, diazocompounds, particularly diazopyruvates. Additional
suitable photoactivatable groups are taught by Hermanson, G.T. Bioconjugate
Techniques, Academic Press: San Diego, 1996, which is incorporated herein by
reference.
In a second example, the first affinity agent is a multivalent protein, and is
associated with a substrate-associated organic thinfilm. The first affinity
agent can
then serve as the site of attachment for a second affinity agent having a
portion for
binding to the first affinity agent and a portion for interacting with a
molecule of
interest. In a specific example, the first affinity agent is streptavidin,
which is bound
to the substrate via a biotin derivative displayed on the substrate-associated
organic
thinfilm, and the second affinity agent is a biotin-conjugated molecule.
Because
streptavidin has plural biotin binding sites, the biotin conjugated molecule
is then
associated with the organic thinfilm via the streptavidin.
One aspect of the method includes one or more optional "quenching" steps.
Typically a quenching step is perfornled to deactivate reactive functional
groups
associated with an organic thinfilm. In working embodiments cysteine was used
to
quench thiol-reactive functional groups associated with a thinfilin. In other
working
embodiments glycine was used to quench amino-reactive functional groups
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associated with a thinfilm. Suitable quenching agents and procedures can be
selected as is known to those of ordinary skill in the art, however, typically
the
quenching reagent can be selected so that it has a functional group having a
similar
reactivity as a functional group used to associate a reagent or affinity agent
to an
organic thinfilm.
In one embodiment of an array of affinity agents, an array of specifically
oriented immobilized antibodies is provided. The antibodies are immobilized by
conjugating a biotin molecule to the antibody. In one example the antibody is
conjugated to the biotin molecule by first oxidizing a vicinal diol
functionality on an
antibody glycosyl moiety to form the corresponding aldehydes. The glycosyl
moiety aldehydes are reacted with a biotin molecule functionalized with an
aminooxy group. The reaction yields an antibody-biotin compound covalently
bound through an oxime bond. Finally, the antibody-biotin compound is
immobilized to an organic thinfilm comprising a streptavidin compound.
Additional reagents and functional groups useful for assembling monolayers
and conjugating affinity reagents to organic thinfilms can be selected from
those
disclosed by Hermanson, G.T. Bioconjugate Techniques, Academic Press: San
Diego, 1996, which is incorporated herein by reference.
One disclosed embodiment for derivatizing substrates is illustrated in FIG. 1.
With reference to FIG. 1, substrate 2 is functionalized with reagent 4 having
at least
two functional groups X and Y.
Classes of materials useful as substrate 2 for forming arrays as disclosed
herein include inorganic materials, metals, and organic polymers. Examples of
inorganic materials include silicon, silica, quartz, glass, controlled pore
glass,
carbon, alumina, titanium oxide, tantalum oxide, indium tin oxide, germanium,
silicon nitride, gallimn arsenide, zeolites, mica and combinations thereof.
Useful
metals include aluminum, copper, gold, platinum, titanium, alloys thereof, and
combinations thereof. Examples of useful polymers include, without limitation,
polyethylene, polyethyleneimine, polyvinylethylene, polystyrene,
poly(tetra)fluoroethylene, polycarbonate, polymethylmethacrylate,
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polydimethylsiloxane, polyvinylphenol, polyoxymethylene, polymethacrylamide,
polyimide and copolymers of such materials. Optionally, the substrate coated
with
one or more different materials, which typically are selected from those
listed above.
For example, the coating optionally may be a metal film. Possible metal films
include aluminum, Chrolnlunl, titanium, tantalum, nickel, stainless steel,
zinc, lead,
iron, copper, magnesium, manganese, cadmium, tungsten, cobalt, and alloys or
oxides thereof. In an exemplary embodiment, the metal film is a noble metal
film.
Noble metals that may be used for a coating include, but are not limited to,
gold,
platinum, silver, and copper. Electron-beam or ion beam evaporation may be
used
to provide a thin coating of gold on the surface of the substrate. In another
exemplary embodiment, the coating comprises an alloy of a noble metal. The
metal
film may have a thickness ranging from about 50 nm to about 1,000 nm, more
particularly from about 100 nm to about 500 nm in thickness. In alternative
embodiments, the coating can include silicon, silicon oxide, silicon nitride,
titanium
oxide, tantalum oxide, silicon nitride, silicon hydride, indium tin oxide,
magnesium
oxide, alumina, glass, hydroxylated surfaces, and polymers.
X can be any group that yields chemisorption or physisorption of reagent 4
on substrate 2, thereby forming a monolayer. X typically is a group that
reacts
chemically with substrate 2, and thus X is selected to be compatible with the
substrate material. For example, when the substrate is an oxide, such as
silicon
oxide, indium tin oxide, magnesium oxide, alumina, quartz, glass, or is a
material
such as silicon or aluminum having an oxidized surface layer, silanes and
siloxanes
are useful groups for functionalizing the substrate. For example, halosilanes,
including mono-, di- and tri-halosilanes can be used to attach a reagent to
such
substrates. Useful siloxanes that can be used to attach functional groups to
the
substrate include mono-, di-, and tri-, alkoxysilanes. In working embodiments
X
was a siloxane, such as a triethoxysiloxane, when substrate 2 was silicon
oxide.
Examples of suitable X groups for derivatizing metal substrates or a metal
coating on a substrate include those sulfur-bearing compounds, such as thiols,
thioethers (sulfides), isothiocyanates, xanthanates, thioacids, thiocarbamate,
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disulfides (including symmetric and asymmetric disulfides), dithioacids
(including
symmetric and asymmetric dithioacids) and sulfur-containing heterocycles;
selenium
bearing molecules, such as selenols, selenides and diselenides (including
symmetric
and asymmetric diselenides; nitrogen-bearing compounds, such as 1 °,
2° and perhaps
3° amines, aminooxides, pyridines, isocyanates, isonitriles, nitrites,
and hydroxamic
acids; phosphorus-bearing compounds, such as phosphines; and oxygen-bearing
compounds, such as carboxylates, hydroxyl-bearing compounds, such as alcohols,
and mixtures thereof.
Sulfur-containing compounds are particularly useful for functionalizing
silver, gold and platinum surfaces. When the substrate is a metal, such as
silver,
gold or platinum, thiols and disulfides are preferred reagents for
functionalizing the
substrate. In working embodiments, thiols and disulfides were used to attach
various reagents to gold surfaces
In other embodiments, the surface of the substrate (or coating thereon) is
composed of a metal oxide such as titanium oxide, tantalum oxide, indium tin
oxide,
magnesium oxide, or alumina and X is a carboxylic acid. Alternatively, if the
surface of the substrate (or coating thereon) of the device is copper, then X
typically
is a hydroxamic acid.
If the substrate used in the invention is a polymer, then in many cases a
coating on the substrate such as a copper coating will be included in the
device. An
appropriate functional group X for the coating would then be chosen for use in
the
device. In an alternative embodiment comprising a polymer,substrate, the
surface of
the polymer may be plasma-modified to expose desirable surface functionalities
for
monolayer formation. For instance, European Patent Publication No. 70423
describes using a monolayer molecule that has an alkene X functionality on a
plasma
exposed surface. Still another possibility for the invention device comprised
of a
polymer is that the surface of the polymer on which the monolayer is formed is
functionalized due to copolymerization of appropriately functionalized
precursor
molecules.
Another possibility is that prior to incorporation into the monolayer, X can
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be a free-radical-producing or free-radical-activated moiety. Such a
functional
group is especially appropriate when the surface on which the monolayer is
formed
is a hydrogenated silicon surface. Possible free radical producing moieties
include,
but are not limited to, diacylperoxides, peroxides, and azo compounds.
Alternatively, unsaturated moieties, such as unsubstituted alkenes
(particularly a-[3
unsaturated ketones), alkynes, cyano compounds and isonitrile compounds can be
used for X as free radical activated moieties, particularly if the reaction
with X is
accompanied by ultraviolet, infrared, visible, or microwave radiation.
In alternative embodiments, X can be a vinyl, sulfonyl, phosphoryl or silicon
hydride group.
The linker group in reagent 4 between groups X and Y can be any suitable,
chemically compatible spacer group. In one embodiment the linker group is
selected
to promote the self assembly of reagent 4 into monolayer 6, and/or to ensure
that the
monolayer is ordered and densely packed. The factors that contribute to self
assembly are known to those of ordinary skill in the art, as described by
Laibinis, et
al. Science 1989, 245, 845 and Ulman, Ara Introductiofz to Ult~athin Or~gafaic
Filfras:
From Laragmuir-Blodgett to Self Assembly, Academic Press (1991).
Typically, the linker group in reagent 4 between functional groups X and Y
is a hydrocarbon chain, optionally branched and/or including heteroatoms, such
as
oxygen. Where the linker group is a hydrocarbon chain, the group typically
includes
from 2 to about 22 carbon atoms. The term "hydrocarbon chain" as used herein
therefore typically refers to a chain of carbon atoms, typically comprising
from 2 to
about 22 carbon atoms. The chain can comprise aliphatic and aryl groups and
can
comprise straight chain, bra~lched chain and/or cyclic groups. In one working
embodiment the linker group between X and Y was a three carbon chain, and in
another working embodiment the linker group was an unbranched 11 carbon
hydrocarbon chain.
In one aspect, regardless of the nature of the monolayer molecules, it may be
desirable to provide crosslinking between component molecules of a monolayer.
In
this case, the linker group in reagent can include the functional groups to
facilitate
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crosslinking. In general, crosslinking confers additional stability to the
monolayer.
Such methods are familiar to those of ordinary skill in the art (for instance,
see
Ulman, An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to
Self Assembly, Academic Press (1991)).
With continued reference to FIG. 1, functionalization of substrate 12 with
reagent 4 yields monolayer 6 formed on substrate 2, which is further
derivatized
with reagents 8 and 10 to yield monolayer 14. Thus, the component, Y, of
monolayer 6 is a functional group responsible for attaching a second set of
reagents,
e.g. 8 and 10. Y can function to form a covalent bond with reagents 8 and 10
or can
interact with 8 and 10 non-covalently so that 8 and 10 are operatively
associated
with monolayer 6. Optionally, a reversible covalent bond can be formed so that
reagent 8, 10, or both can be dissociated from monolayer 6.
Where 8 and/or 10 are to be covalently attached to monolayer 6, thereby
forming a new monolayer, Y typically is either reactive with functional groups
V
and V' on reagents 8 and 10, respectively, or alternatively is readily
activated for
reaction with V and V'.
Examples of functional groups suitable for use as Y include electrophiles,
such as activated carboxylic acids, including, for example, acyl imidazolides,
acyl
azides, and activated esters, such as pentafluorophenol, p-nitrophenol, N
hydroxysuccinimide and sulfo-N hydroxysuccinimide esters. Additional useful
electrophiles include Michael acceptors, such as a,(3-unsaturated carbonyl
groups
(including acids, amides, esters, ketones and aldehydes), a,(3-unsaturated
sulfoxides,
a,(3,-unsaturated sulfones, and the like. Preferred Michael acceptors include
maleimide and maleimide derivatives. Additional examples of electrophiles
useful
as Y groups include aldehydes and alkylating agents, such as epoxides,
aziridines,
and alkyl halides, such as alkyl iodides, bromides and chlorides, particularly
a-
haloacetyl groups and primary and tertiary alkyl iodides, bromides and
chlorides.
Similarly, alkylating agents such as alkyl sulfonic acid esters also are
useful
alkylating agents, with specific examples including methylenesulfonic acid,
trifluoromethylenesulfonic acid andp-toluenesulfonic acid esters.
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Additional examples of functional groups suitable for use as Y include
nucleophilic groups, such as hydroxyl, peroxide, peroxyacid, sulfhydryl,
thioacid,
selenol, amino, aminooxy, hydrazide and thiosemicarbazide groups.
Another class of reactive Y group that can be used includes functional
groups that engage in cycloaddition reactions. Examples of useful
cycloaddition
reactions include, without limitation 2+2, 3+2, particularly 1,3-dipolar
cycloadditions, and 4+2 cycloaddition reactions. For example, Y can be a
dienophile used in a Diels-Alder reaction. Examples of suitable dienophiles
include
electron poor alkenes or, alternatively, in an inverse-demand Diels-Alder
reaction,
electron-rich alkenes. Conversely, electron-rich dimes or electron-poor dimes
are
used in Diels-Alder and inverse-demand Diels-Alder cycloaddition reactions,
respectively.
Azide-containing compounds can be used in several different types of
cycloaddition reactions. For example, 1,3-dipolar cycloaddition reactions can
be
used to link a compound to monolayer 6 when Y includes an azide, which can be
reacted with a suitable alkene compound to yield, following thermal
elimination of
nitrogen, the corresponding aziridine compound. Conversely, Y can include an
alkene group that can react with an azide-containing compound.
Azides also can be used to react with an in situ-formed ketene in Staudinger-
type cycloadditions, which yields a (3-lactam-containing product. Suitable
precursors and conditions for using cycloaddition reactions to functionalize
monolayer 6 can be selected by those of ordinary skill in the art.
Another class of reaction that can be used to functionalize monolayer 6
covalently includes organometallic reactions. Specific organometallic
reactions
include, without limitation, metathesis reactions, for example, in one
embodiment an
aldehyde- or alkene-functionalized surface can be reacted with reagents 8 and
10
wherein V and V' include an aldehyde or alkene functional group. Examples of
catalysts and conditions for such metathesis reactions are known to those of
ordinary
skill in the art. Certain examples of such catalysts include the metals
titanium,
tungsten, molybdenum or ruthenium. See, for example, Ivin, K.J., Mol, J.C.
Olefifa
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Metatlzesis and Metatlzesis Polynzerizatiorz; Academic Press: London, 1997.
Where Y is a functional group that is activated in situ, possibilities for Y
include, but are not limited to, moieties such as an azide, hydroxyl,
carboxyl, amino,
aldehyde, carbonyl, methyl, methylene, alkene, alkyne, carbonate, aryliodide,
or a
vinyl group. Appropriate modes of activation of such functional groups are
known
to those of ordinary skill in the art. For example, where Y is a carboxyl
group, Y
can be activated in situ for acylation by, for example, converting Y into an
activated
ester, such as a pentafluorophenol, p-nitrophenol, N hydroxysuccinimide or
sulfo-N
hydroxysuccinimide ester. Moreover, such activated esters react readily with a
variety of suitable nucleophiles, such as amines, thiols, alcohols, and the
like. In one
aspect, Y may be a protected functional group that can be removed in situ to
umnask
a reactive functional group. Suitable protecting groups that can be used can
be
selected, installed and removed as is known to those of ordinary skill in the
art, and
include protecting groups disclosed in Greene, T.W.; Wuts P.G. M. Protective
Groups in Organic Syntlzesis, 3rd ed.; Wiley-Interscience, 2002. In a working
embodiment a protected aldehyde was used to produce a masked aldehyde-
functionalized substrate.
In an alternative embodiment, the functional group Y of the monolayer is
selected from the group of charged moieties. Possible charged Y functional
groups
include, but are not limited to, phosphate, sulfate, carboxylate, and the
like. Simple
groups such as these can be used for Y when reagent 8, 10, or both include a
ionic
group, such as poly-lysine, that coats the exposed portion of the monolayer.
With continued reference to FIG. 1, reagents 8 and 10 are attached to
monolayer 6, thereby forming new, second monolayer 14, which includes the
components of first monolayer 6. Reagent 8 has first functional group V for
attachment to monolayer 6 and second functional group Z, which can be used,
for
example, to attach an affinity reagent. Reagent 10 includes functional group
V' for
attachment to monolayer 6 and also includes a protein resistant component W.
Functional groups V and V' can be the same or different functional groups.
Optionally, reagents 8 and 10 can be delivered in two different steps. Thus,
reagent
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8 could be deposited either before or after reagent 10. In one embodiment V
and V'
are optionally covalently linked in, for example, a disulfide bond, such that
reagents
8 and 10 having functional groups Z and W, respectively, are delivered in the
same
step. Functional groups V and V' are selected with reference to functional
group Y
displayed on monolayer 6. As noted above, with respect to functional group Y,
V
and/or V' can form a covalent linkage to monolayer 6, or can associate non-
covalently with monolayer 6. Generally, V and V' are selected to be
complementary
to functional group Y, thus, for example, where Y is an electrophilic group, V
and
V' typically are nucleophilic groups, and conversely, where Y is a
nucleophilic
group, V and V' typically are electrophilic groups.
Functional group Z on monolayer 14 is used to attach an affinity agent to
yield monolayer 16 having affinity agent 18 bound thereto.
FIG. 2 includes a schematic representation 20 of arrays disclosed herein and
also depicts several exemplary reactive monolayer functional groups. With
reference to array 20, substrate 22 can be any suitable substrate. X
represents a
functional group for non-covalently or covalently (reversibly or irreversibly)
associating a reagent with substrate 22. Optionally, one or more functional
groups X
depicted in array 20 are linked directly or indirectly to one another. For
example,
groups X can be linked before association with the substrate 22 by a polymer
backbone, such as a poly-L-lysine backbone. Alternatively, X can be a group
(or
derived from a group) such as a siloxane, which can react with one or more
during
or after association with substrate 22.
R represents a linker group, such as a linker group of the type discussed with
respect to reagent 4 above. Typically, R is a substantially unbranched chain,
such as
an unbranched hydrocarbon chain. Where R includes a hydrocarbon chain, the
chain typically has from 2 to about 22 carbon atoms, and more typically R has
from
3 to about 1 ~ carbon atoms. In working embodiments the various hydrocarbon
chains used included those having 3, 4, 10, 11, and 12 carbon atoms.
Y represents a linkage, typically formed as disclosed herein when a second
reagent is associates covalently or noncovalently with a substrate-associated
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monolayer. Where Y represents a covalent linkage, Y is typically formed by the
reaction of two functional groups where one can be described as electrophilic
and
the second as nucleophilic. Useful examples of electrophilic and nucleophilic
groups include those discussed above, with respect to FIG. 1. Other functional
groups that can react to form Y, and that typically are not characterized as
either
nucleophilic or electrophilic, include functional groups that engage in
cycloaddition
and organometallic reactions.
With continued reference to FIG. 2, structures 24, 26, 28, 30 and 32 depict
representative monolayer functional groups used in working embodiments to
incorporate at least a second reagent into a monolayer formed on a glass or
silicon
substrate. For example, the maleimide functional group in structure 24 can be
used
as an electrophile in a Michael reaction, or, alternatively can be used in a
cycloaddition reaction, such as a Diels-Alder reaction.
The azide moiety in structure 26 can be activated to function as a
nucleophile via reduction to the corresponding amine, or can be used in a
Staudinger
ligation reaction, which accomplishes activation, reduction and acylation in a
single
step in situ. Alternatively the azide functionality can be used directly in a
cycloaddition reaction to react with, for example an in situ-formed ketene
reagent.
Structure 28 includes a bromoacetate moiety that is an excellent
electrophile, with the bromine being subj ect to facile nucleophilic
displacement by a
variety of nucleophiles, particularly thiolates. With reference to structure
28, X can
be a nitrogen or an oxygen.
Structure 30 includes an aldehyde, which can be used as an electrophile for
reaction with a various nucleophilic reagents. A variety of nucleophilic
groups
capable of reacting with an aldehyde to produce a covalent bond are useful in
the
current invention. Useful reactive groups include, for example, alkoxides,
enolates,
carbanion equivalents, such as Grignard-type reactive groups, alcohol groups
(yielding the corresponding acetal or hemiacetal), cyanide reactive groups,
amines,
including primary or secondary amines, hydrazines, phosphorus glides,
phosphine
oxide anions, aminooxy reagents, hydrazides thiosemicarbazides, and the like.
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Alternatively, an aldehyde can be used in an organometallic reaction, such as
an
aldehyde-alkene metathesis reaction.
Structure 32 represents an acylating group, such as an activated ester. Thus,
X is a group that is readily displaced, as is known to those of ordinary skill
in the art,
such as a halide, N hydroxysuccinimide, sulfo-N hydroxysuccinimide,
pentafluorophenol, p-nitrophenol, and the like.
FIG. 3 illustrates a method for preparing organic thinfilm arrays by first
derivatizing a substrate 34 using an asymmetric disulfide 33. The disulfide 33
is
asymmetric in the sense that it is composed of two different substituents.
Deposition
according to this method yields a monolayer 35, which includes two different
monolayer components. Optionally, one or more additional symmetric or
asymmetric disulfides can be deposited to provide a monolayer including three
or
more different monolayer components, or simply a different ratio of the two
monolayer components provided by the asymmetric disulfide. For example, where
a
three-to-one ratio of X to Y groups is desired, deposition of asymmetric
disulfide 33
can be accompanied by deposition of an equal amount of a symmetric disulfide
having the formula Y R-S-S-R-X. X and Y in monolayer 35 function as an
affinity agent and a protein resistant component or as reactive functional
groups for
incorporating an affinity agent and protein resistant reagent. Optionally,
where X
and Y are such reactive functional groups, they can be the same or different
functional groups.
FIG. 3 also illustrates representative monolayer components formed by a
Staudinger ligation method as disclosed herein. With reference to structure
37, X is
a functional group suitable for associating a monolayer or organic thinfilm
with
substrate 36. X typically is attached to a hydrocarbon linking group as
illustrated in
structure 37; however, other linking groups also can be used in such
structures.
Typically, the selected hydrocarbon chains have from 2 to about 22 carbon
atoms,
and thus 'n' is from 2 to about 22. More typically n is from 3 to about 18. G
can be
any group useful for covalently or non-covalently associating an affinity
agent or
protein resistant component, or another reactive functional group, which can
be used
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to covalently or non-covalently associate an affinity agent or protein
resistant
reagent.
Refernng again to FIG 3, structures 39 and 41 represent monolayer
components formed on a substrate 36. With reference to structure 39, substrate
36 is
functionalized with a siloxane functional group linked to a hydrocarbon chain.
The
illustrated hydrocarbon chain includes 'n' methylene groups, where n typically
is
from 2 to about 22, and more typically is from 3 to about 1~. In one working
embodiment n was 11. The hydrocarbon chain is functionalized with an aminoacyl
aryl phosphine oxide moiety, which is installed via a Staudinger ligation
protocol.
At the papa position relative to the hydrocarbon amino acyl group is an acyl
amino
group, bonded to a polyethylene glycol chain comprising 'm' ethylene glycol
units.
The ethylene glycol chain can be any length, however typically m is from 2 to
about
100 and more typically from 2 to about 70 ethylene glycol units. Most
typically m
is from about 2 to about 20. One working example included monolayer components
according to structure 39 where m was 6.
With continued reference to structure 39, X is a group that renders the
monolayer component an active acylating agent. Such acylating agents can be
prepared from the corresponding carboxy group as is known to those of ordinary
skill in the art. Typically, 39 is an activated ester group, such that X is a
pentafluorophenol, p-nitrophenol, N hydroxysuccinimide, sulfo-N
hydroxysuccinimide, or the like. Additional suitable X groups include halides,
imidazolides, anhydrides, and the like. Structure 41, like structure 39,
illustrates a
monolayer component formed on a glass or silicon substrate. However, 41
functions
as a protein resistant component. With reference to 41 'p' is from 2 to about
100
ethylene glycol units and more typically from 2 to about 70 ethylene glycol
units.
Most typically, 'p' is from 2 to about 20 ethylene glycol units. R represents
an alkyl
group, hydrogen or a hydroxyl bearing group. Typically, R is either a methyl
group
or a hydrogen.
FIG. 4 illustrates another embodiment for functionalizing a substrate. In this
embodiment, substrate 45 is glass or has a silicon oxide coating, which is
derivatized
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with a 3-maleimidopropyl-1-silane derivative to give the monolayer coated
substrate
shown having a maleimide (MAL) functional group on one face. The maleimide
group can be used to attach a sulfhydryl-containing reagent with high
efficiency. In
the illustrated example a heterobifunctional reagent having a sulfhydryl group
at a
first end and a biotin group at a second end is attached to the substrate via
the
maleimide group. A reagent containing a protein-resistant component is then
attached via remaining maleimide functional groups.
D. Uses for the Disclosed Arrays
The disclosed arrays are useful for characterizing the interaction of soluble
analytes with arrayed affinity agents. In particular, the arrays can be used
to identify
affinity agents that bind to a molecule of interest, particularly a
biomolecule of
interest, such as a protein or nucleic acid. The arrays are especially useful
for high
throughput drug screening, using both small molecule and biomolecule arrays.
A wide range of detection methods is applicable to characterizing the
interactions of soluble analytes with the disclosed arrayed affinity agents.
For
example the arrays can be incorporated into a device that is interfaced with
optical
detection methods such as absorption in the visible range, chemoluminescence,
and
fluorescence (including lifetime, polarization, fluorescence correlation
spectroscopy
(FCS), and fluorescence-resonance energy transfer (FRET)). Furthermore, built-
in
detectors such as optical waveguides as disclosed in PCT Publication WQ
96/26432
and U.S. Pat. No. 5,677,196, incorporated herein by reference, surface plasmon
resonance, and surface charge sensors are compatible with embodiments of the
arrays disclosed herein.
An exemplary type of fluorescence detection unit that may be used to
monitor interaction of immobilized affinity agents of an array with an analyte
is
described in U.S. Patent No. 6,406,921 to Wagner et al. (Wagner), which is
incorporated herein by reference in its entirety. FIG. 5 is a schematic
diagram of
this type of fluorescence detection unit, which may be used to monitor
interaction of
immobilized affinity agents of an array with an analyte. In the illustrated
detection
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unit, the array 54 is positioned on a base plate 52. Light from a 100W mercury
arc
lamp 62 is directed through an excitation filter 60 and onto a beam splitter
58. The
light is then directed through a lens 56, such as a Micro Nikkor 55 mm 1:2:8
lens,
and onto the array 54. Fluorescence emission from the array returns through
the lens
56 and the beam splitter 58. After next passing through an emission filter 64,
the
emission is received by a cooled CCD camera 66, such as the Slowscan TE/CCD-
1024SF&SB (Princeton Instruments). The camera is operably connected to a CPU
68, which is in turn operably connected to a VCR/monitor 70.
FIG. 6 is a schematic diagram of an alternative detection method based on
ellipsometry. Ellipsometry allows information about the sample to be
determined
from the observed change in the polarization state of a reflected light wave.
Interaction of an analyte with a layer of immobilized affinity agents on a
substrate
results in a thickness change and alters the polarization status of a plane-
polarized
light beam reflected off the surface. This process can be monitored in situ
from
aqueous phase and, if desired, in imaging mode. With reference to FIG. 6, in a
typical setup 80, monochromatic light (e.g. from a He--Ne laser, 84) is plane
polarized (polarizer 86) and directed onto the surface of the sample on
substrate 82
and detected by a detector 92. A compensator 88 changes the elliptically
polarized
reflected beam to plane-polarized. The corresponding angle is determined by an
analyzer 90 and then translated into the ellipsometric parameters Psi and
Delta
which change upon binding of analyte with th.e immobilized proteins.
Additional
information can be found in Azzam, et al., Ellipsometry and Polarized Light,
North-
Holland Publishing Company: Amsterdam, 1977.
E. Examples
The foregoing disclosure is further explained by the following non-limiting
examples. Efforts have been made to ensure accuracy with respect to numbers
(e.g.,
amounts, temperature, etc.) but some errors and deviations should be accounted
for.
Unless indicated otherwise, parts are parts by weight, temperature is in
°C or is at
ambient temperature and pressure is at or near atmospheric.
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Gefze~al Methods
Glass and silicon substrate materials were cleaned prior to derivatization
according to the following procedure: 200 mL of 30% H2O2 (Aldrich, Milwaukee,
Wisconsin) and 200 mL concentrated NH40H (Aldrich) were added to a 65
°C bath
containing 1000 mL of nanopure water (18 MS2 resistance). A Teflon rack
holding
glass microscope slides (1" by 3", Fisher Scientific) or 1 cm x 2 cm silicon
wafers
was immersed in the cleaning bath. After about 15 minutes, the slide rack was
removed from the bath and placed into a 300 mL bath of nanopure water for 10
minutes with gentle agitation, followed by immersion in a second 300 mL bath
of
nanopure water for 10 minutes with gentle agitation. The slides were stored
under
nanopure water and dried immediately before use via spin rinse drying, using a
cycle
of 3 minutes washing at 600 rpm, 10 seconds nitrogen purging at 1500 rpm,
followed by two drying cycles of 1.5 and 20 minutes at 2500 and 600 rpm,
respectively.
Example 1
This example describes the formation of an aminoreactive organic thin film
on a gold substrate using disulfide thin film precursors. A freshly prepared
Au(111)
chip was immersed in a 1 mM solution of asymmetric disulfide compound 120 (the
synthetic route to compound 120 is illustrated in FIG. 7 and described in
detail
below) at room temperature for 1 hour. The chip was then rinsed with ethanol
(3 x
10 mL). After rinsing, the coated chip was dried under a nitrogen stream and
used
immediately for reaction with an amine-containing reagent.
O
~O~O~ O~ N
S O
O~ O
S O~ CHs O
3
10
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Example 2
This example describes functionalizing an aminoreactive organic thin film to
form a biotin-functionalized chip. A chip having an aminoreactive coating
prepared
according to example 1 was incubated with a 5 mM solution of either (+)-
biotinyl-
3,6-dioxaoctanediamine or (+)-biotinyl-3,6,9,11-tetraoxatridecanediamine
(commercially available from Pierce, Rockford, Illinois and Molecular
BioSciences,
Boulder, Colorado, respectively) in 50 mM phosphate buffer, pH 7.5 containing
0.05% Tween-20 (polyoxyethylene(20) sorbitan monolaurate) commercially
available from Sigma-Aldrich, Milwaukee, Wisconsin) for 10 minutes at room
temperature. The chip was washed with 500 mM ionic strength PBS buffer, pH 7.5
(total wash volume 30 mL). The chip was then immersed in a 37° C 1M
glycine
solution to deactivate any remaining aminoreactive NHS groups.
Example 3
This example describes the general protocol used for silanization of silicon
oxide substrates. Reagents deposited by this method included 11-azidoundecyl-1-
triethoxysilane and 3-maleimidopropyl-1-triethoxysilane, which was prepared
according to the protocol of Shaltout et al. Mat Res Soc. Synap. Proc. 1999,
576, 15-
20, which is incorporated herein by reference. The Shaltout procedure also was
applied to the synthesis of other maleimide derivatives. 3-Maleimidopropyl-1-
triethoxysilane reagent was deposited as follows: To a clean, dry 120 mL PTFE
vial
(Savillex, Minnetonka, Minnesota) was added 50 mL dry toluene (99.x%
anhydrous,
Aldrich catalog number, 24,451-l, Milwaukee, Wisconsin) and 1 mL hexanoic acid
(Aldrich catalog number 15374-5), followed by 0.5 mL of neat silane. The vial
was
swirled and dry, clean silicon oxide wafers were added to the silane solution.
The
silanization was allowed to proceed for 24 hours at room temperature on a
shaker
(ca. 100-150 rpm). The wafers were rinsed with dry toluene and absolute
ethanol
and then sonicated under absolute ethanol for 10 minutes. The sonicated wafers
were dried under nitrogen and stored in a fluoroware container in a dessicator
under
argon. The silanized chips can be used for about 2 to 3 weeks.
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Exafnple 4
This example describes the general Staudinger ligation protocol used to
couple aminoreactive and protein resistant components to the 11-azidoundecyl-1-
triethoxysilane functionalized substrate prepared according to the protocol of
example 3. The protein resistant Staudinger reagent 20 and aminoreactive
Staudinger reagent 30 are depicted below. Staudinger reagents 20 and 30 were
prepared according to the method disclosed by Saxon and Bertozzi in U.S.
Patent
No. 6,570,040, which is incorporated herein by reference in its entirety.
CH3
w
M
O
H O
N~0 O O,N
3 0
10 30
Staudinger reagent was dissolved in 4 mL of oxygen purged (via nitrogen
sparging)
3:1 tetrahydrofuxan:nanopure (deionized, 18 MS2) water to give a concentration
of
20 mM. The 11-azidoundecyl-1-triethoxysilane chip was placed in a teflon
container and was immersed in the Staudinger reagent solution. The container
was
15 shaken on a shaker for 24 hours at room temperature, and then the chip was
removed
and rinsed with 30 mL each of 3:1 tetrahydrofuran:nanopure water, nanopure
water,
and absolute ethanol. Each chip was dried under nitrogen and stored until
further
v r
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use. The progress of the Staudinger ligation can be monitored by IR
spectroscopy,
and the yield can be determined by quantitative IR spectroscopy.
Example 5
This example describes the functionalization of a 3-maleimidopropyl-1-
triethoxysilane substrate with a biotin derivative 40 and a protein resistant
component 50. A maleimide functionalized chip prepared according the protocol
of
example 3 was coated with 250 ~,L of an 80 ~,M solution of compound 40 in
buffer
(20 mM citrate, 150 mM sodium, 5 mM EDTA, 0.05% Tween 20 buffer at pH 6.5).
The chip was incubated at room temperature for 30 minutes with slight
agitation on
a shaker. The chip was washed with buffer (0.5 M sodium in 1X PBS, 0.05%
Tween 20, pH 7.5), and then incubated with 150 ~,L an 80 ~M solution of
compound
50 in buffer (20 mM citrate, 150 mM sodium, 5 mM EDTA, 0.05% Tween 20 buffer
at pH 6.5) at room temperature for 30 minutes with slight agitation. The chip
was
washed with buffer (0.5 M sodium in 1X PBS, 0.05% Tween 20, pH 7.5), and then
immersed in a cysteine quenching solution (O.1M cysteine, 20 mM citrate, 150
mM
sodium, 5 mM EDTA, 1 mM tris-(2-carboxyethyl)phosphine hydrochloride (TCEP),
0.05% Tween 20, pH 6.5) for 30 minutes at 37° C. The quenched chip was
washed
with 30 mL of buffer (0.5 M sodium in 1X PBS, 0.05% Tween 20, pH 7.5) and 30
mL of nanopure water. The chip was spin dried and used immediately. The biotin
functionalized chips prepared according to this protocol can be stored under
argon at
4° C for up to about 3 weeks.
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0
H
HS
O
HS p p~pMe
5
The biotin functionalized surfaces prepared as described above in example 5
can be used to immobilize different biotin-derivatized reagents on the surface
via
mutual streptavidin binding. The following examples describe four different
5 antibody immobilization strategies. The four types are: randomly
biotinylated IgG;
oriented IgG (biotinylated on carbohydrate on Fc domain); oriented Fab'
fragments
(biotinylated in hinge region) and randomly biotinylated Fab' fragments.
Methods
for preparing these biotinylated antibodies are described below.
10 Example 6
This example describes the preparation of an aldehyde functionalized organic
thinfilm. Ethylene glycol (28 mL, 31.4 g, 505 mmol, 1.05 equiv.) was added to
a 1
L round bottom flask containing undecylenic aldehyde 60, toluene (500 mL),
followed byp-toluenesulfonic acid hydrate (13.7 g, 72 mmol, 0.15 equiv.) was
15 added and the solution heated to 125 °C. The solution was refluxed
under a Dean-
Stark trap for 3 hours. After approximately 13 mL of water was collected
in.the
trap, the solution was allowed to cool to room temperature, was diluted with
toluene,
and washed with aqueous sodium bicarbonate solution (1N, 2x 300mL) and brine
(ca. 400 mL). The organic solution was dried over NaaS04 and concentrated in
20 vacuo to a brown oil. The crude material was purified by distillation at 85-
88 °C
and 48 mTorr. Compound 70 was isolated as clear liquid (approximately 110 mL).
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1H NMR (400 MHz, CDC13): 8 1.3 (m, 12H), 1.6 (m, 2H), 2.0 (m, 2H), 3.85
(AA'BB', 2H), 3.95 (AA'BB', 2H), 4.85 (t, 1H), 4.9-5.0 (two d, 2H), 5.8 (ddt,
1H).
0
HO OH .~
\ H+ ~ \ H
60 70
O O
+ Et03SiH
\ H Et03Si
80 90
11-triethoxysilylundecyl ethylene glycol acetal 90 was prepared from alkene
acetal
70 (38 mL, 34.2 g, 161 mmol) was added to a 100 mL round bottom flask.
Pt(Ph3P)4 (0.20 g, 0.161.mmol, 0.001 equivalent)was added, followed by Pt
octanal/octanol (2.5 Pt solution, 1.25 mL, 0.161 mmol, 0.001 equivalent).
Triethoxysilane (29.75 mL, 26.5 g, 161 mmol, 1.0 equivalent) was added to the
alkene catalyst solution at ambient temperature, resulting in a slight
exotherm. The
solution was stirred at ambient temperature for 1.5 hours, and then the
reaction was
heated to 105 °C for two hours. Completion of the reaction was
determined by
monitoring the disappearance of the alkene resonances in the 1H NMR spectrum.
Desired product was obtained by distillation at 114-130 °C and 13
mTorr. A clear,
colorless liquid was obtained, yielding 34 mL. 1H NMR (400 MHz, CDC13): 8 0.57
(m, 2H, -CHZSi-), 1.176 (m, 25H, C-CH2-C and C-CH3), 1.35 (m, 2H, Si-C-CHZ-C),
1.6 (m, 2H, glycol AA'BB'), 2.8 (m, 1H, C-CH(OR). 13C NMR (100 MHz, CDCl3):
8 10.4 (Si-C), 18.2 (OC-CH3), 22.8, 24.1, 29.3-29.6 multiple resonances, 33.2,
33.9,
58.3, (O-C-CH3), 64.8 (O-CHz-CH2-), 104 (C-CH(OR)-OR).
Silanization 'vvas carried out as follows. To a glass Copeland jar, prerinsed
with a small amount of dry toluene, was added 50 mL dry toluene and 0.5 mL
aldehyde silane. The j ar was placed on a shaker to mix the solution and five
clean
glass microscope slides were placed into the slots inside. After 24 hours of
gentle
agitation, the solution was decanted and replaced with 50 mL dry toluene and
the jar
was shaken for five minutes; this procedure was repeated twice. After
decanting the
final toluene rinse, 50 mL of ethanol was added and the jar was allowed to
shake for
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minutes. After decanting the first ethanol rinse, an additional 50 mL of
ethanol
was added and the jar sonicated for 10 minutes. Finally, the slides were
removed
from the Copeland jar and dried under nitrogen. The slides were stored under
nitrogen to avoid aldehyde oxidation.
5 Silicon wafers coated with acetal silane 90 was immersed in a 0.1 M HCl
solution and agitated for 0.25, l, 4, 8 and 16 hours. Upon removal from the
acid
bath, the wafer was immersed in 1 M pH 7 NaHC03 for 1 minute, removed,
immersed in water and agitated for 5 minutes and then dried using a stream of
nitrogen gas.
The deprotection reaction can be followed by X-ray photoelectron
spectroscopy (XPS, 45 °C take off angle). The disappearance of the peak
corresponding to the C-O bond at 287 eV can be monitored, and the deprotection
reaction is nearly complete at 1 hour. The resulting aldehyde functionalized
surface
can be derivatized with 3'-aminofunctionalized oligonucleotides via reductive
amination. Oligonucleotide, 5 ~.M, is spotted on the aldehyde functionalized
surface, and the surface is allowed to dry. The spotted surface is rinsed in a
solution
of 0.2% SDS in deionized water for 2 minutes at room temperature with
agitation.
The surface is then immersed in a fresh reducing solution (1.5 g NaBH4, 133 mL
absolute ethanol, and 450 mL PBS at pH 7.2-7.4) and soaked for 5 minutes at
room
temperature.
Exatrtple 7
This example describes the preparation of 11-triethoxysilylundecyl-2-
bromoacetate. 10-Undecenyl alcohol (100 g, 0.59 mole) and a-bromoacetic acid
(90
g, 0.65 mole) were weighed into a 1 L round bottom flask and dissolved in 600
mL
of dichloromethane. Dicyclohexylcarbodiimide (DCC, 133.3 g, 0.65 mole) was
dissolved in 250 mL of dichloromethane and added in portions to the
acid/alcohol
mixture. A white precipitate quickly formed and some heat was evolved. The
mixture was allowed to stir overnight, and then the precipitate (dicyclohexyl
urea,
DCU) was removed via filtration. The filtrate was concentrated to an oil and
taken
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up in 500 mL of hexanes to precipitate more DCU, which was removed via
filtration. The solution was concentrated to give a colorless oil and the
product was
distilled (74 °C at 4.0 mTorr) yielding a colorless liquid (115.77 g,
68% yield). 1H
NMR (CDCl3) ~ 1.24-1.44 (m, 14H)1.62 (m, 2H), 2.05 (dd, 2H), 3.79 (s, 2H),
4,20
(t, 2H), 4.93 (m, 2H) 5.79 (dddd, 1H). 13C NMR (CDC13) 8 25.69, 25.92, 28.36,
28.86, 29.03, 29.11, 29.32, 29.37, 33.74, 66.33, 114.12, 138.97, 167.09.
The undecenyl a-bromo acetate, >97% pure, (20g, 0.07 mole) and 280 mg of
hydrogen hexachloplatinate (1 mole %) were added to a round bottom flask.
Triethoxysilane (12.46 g, 76 mmol) was added by syringe under Ar. The mixture
was placed in a preheated (90 °C) oil bath. Over the course of the
reaction (12
hours), the mixture turns a dark color. The product was purified by
distillation, 150
°C at 6.0 mTorr. The product was isolated as a clear colorless liquid
(13.6 g, 43.5%
yield). 1H NMR (CDC13) 8 0.55 (t, 2H), 1.15-1.45 (m, 17H), 1.60 (m, 2H), 2.05
(m,
2H), 3.79 (m, lOH), 4.15 (t, 2H). Elemental Analysis (Theoretical %C 50.10, %H
8.63, %Si 6.17, %Br 17.54), Found: %C 50.18, %H 9.16, %Si 8.10, %Br 15.31.
Silanation using the bromoacetal silane was performed as with the aldehyde
silane (example 6), except that the deposition time was only 2 hours (instead
of 24)
and that care was taken to keep all jars in the dark. Bromoacetyl silane
coated slides
were stored in a desiccator in the dark.
Materials for Example ~-10
MAB9647 is a mouse IgGI that was raised against the human IL-8; it was
produced by Covance Inc. (Princeton, NJ) from mouse ascites fluid using the
hybrodoma cell line HB-9647 from ATCC (Manassas, VA); it is protein G-
purified.
MAB208 is a mouse IgGI that was raised against human IL-8 by R&D Systems
(Minneapolis, MIA; it is protein G-purified from mouse ascites fluid, clone
number
6217.11 l, catalog number MAB208. MAB602 is a mouse IgGaA that was raised
against human IL-2 by R&D Systems; it is protein G-purified from mouse ascites
fluid, clone number 5355.111, catalog number MAB602. As a detection antibody
for the IL-8 assays, an R-phycoerythrin (PE)-conjugated mouse anti-human IL-8
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antibody was used (BD Pharmingen, cat. No. 20795A). For the IL-2 assays, two
detection antibodies were used: a goat anti-human IL-2 antibody (R&D, cat. No.
AF-202-NA) and an R-PE-conjugated donkey anti-goat IgG F(ab')2 (Jackson
Immuno Research, cat. No. 705-116-147). Unless noted, chemicals were purchased
from Sigma-Aldrich (St. Louis, MO). Pepsin-agarose was purchased from Pierce
(Rockford, IL), product number 20343. PNGase F was obtained from New England
Biolabs (Beverly, MA), product number P0704. Aldehyde-reactive probe was from
Molecular Probes (Eugene, OR). Human interleukin-2 (IL-2) was purchased from
Leinco Technologies (St. Louis, MO), product number O11R455, and was
reconstituted in phosphate buffered saline (PBS; 11.9 mM sodium phosphate, 137
mM NaCI, 2.7 mM KCI, pH 7.4). A DNA sequence encoding the mature version of
human Interleukin-8 (IL-8; AVLPRSAKELRCQCIKT'YSKPFHPKFIKELRVIES
GPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLI~RAENS) SEQ ID 1
with a C-terminal Factor Xa cleavage site and Protein Kinase A site
(GIEGRRRASV) SEQ ID 2 was created by gene assembly of oligonucleotides.
This construct was inserted into the NdeI and XhoI sites of pET24a (Novagen,
Madison, WI), resulting in the further addition of a His(6) tag at the extreme
C-
terminus (LEHHHHHH SEQ ID 3; where the LE codons comprise the XhoI site).
This plasmid was then used to direct expression of IL-8 in BL21 DE3 cells
(Novagen) grown in EZMix modified Ternfic Broth (Sigma, St. Louis, MO; CAT
No. T-9179) by the addition of 1mM IPTG at an OD of 0.6 and growth for four
hours in a BioF1o3000 fermentor (New Brunswick Scientific, Edison, New Jersey)
at 30°C. The cells were collected by centrifugation, resuspended in Sml
buffer/g
with 300mM NaCI, SOmM sodium phosphate, SmM beta-mercaptoethanol, 5 mM
imidazole, pH 8.0 including one Complete Protease Inhibitor Cocktail tablet
(Roche
Applied Science; Cat No. 1697498) per SOmI and lysed using a microfluidizer.
The
soluble fraction of IL-8 was purified by metal affinity chromatography using
TALON Superflow beads (Clontech, Palo Alto, CA; Cat No. 8908-2) followed by
gel filtration in PBS using a Superdex 75 prep grade column (Amersham
Biosciences). The IL-8 containing fractions were concentrated to 0.4 - 0.5
mg/ml
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using a model 8400 stirred ultrafiltration cell (Millipore, Bedford, MA; CAT
No
5124) with a 3K MWCO membrane and then dialyzed into PBS with 10% glycerol
for storage. Identity and Correct secondary structure was confirmed by mass
spectrometry, ELISA and circular dichroism (Spectrapolarimeter J-810, Jasco,
Easton MD, www.jascoinc.com).
Example 8
' This example describes the preparation of biotinylated Fab' fragments. The
conserved N-linked glycosylation of IgG's were removed from the antibodies
under
the following reaction conditions: 1-4 mg/ml antibody in 50 mM sodium
phosphate,
pH 7.5, 10-20 U/p l PNGase F (from New England Biolabs, using their unit
definition), 24 48h at 37°C. After deglycosylation, antibodies were
buffer-
exchanged (using ultrafiltration or dialysis) into 20 mM sodium acetate, pH
4.5.
Conditions for pepsinolysis were as follows: 30% (by volume) pepsin agarose
(settled bed volume, beads washed in 20 mM NaOAc, pH 4.5), 0.5 - 2 mg/ml IgG,
mM NaOAc, 260 mM KC1, 0.1% Triton X-100, pH 4.5. Reactions were
incubated at 37°C with agitation for an amount of time that had
previously been
optimized (MAB9647, 12 h; MAB208, 4.5 h;MAB602, 3.5 h). After pepsin-
treatment, the fragments were recovered from the pepsin agarose by washing the
20 resin with O.1M NaOAc, pH 4.5. The products of the pepsin-cleavage were
then
concentrated and exchanged into 0.1 M Na2HPO4, 5 mM EDTA, pH 6.0, and then
treated with 20 mM 2-mercaptoethylamine (MEA) in the same buffer for 90 min at
37°C. The MEA was then removed by dialyzing for 6 hours at 4°C
against 0.1 M
Na~P04, 5 mM EDTA, using a 10 kD cutoff membrane, and then residual MEA was
removed by running sample over a desalting column (PD-10, Amersham-Pharmacia,
Piscataway, NJ). Immediately after this step, the reduced F(ab)' was treated
with 20
mM N ethylmaleimide or maleimide-activated biotin (Pierce product number
21901)
for 2 h at room temperature, and the unincorprated NEM or biotin-maleimide was
then removed by dialysis. The samples were concentrated and the Fab' fragments
were purified from other fragments by FPLC using a Superdex-75 gel filtration
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column (Amersham-Pharmacia). In the case of the NEM-treated Fab' fragments,
random biotinylation was as described below. Samples of the FPLC-purified Fab'
fragments were diluted 1:1 with non-reducing protein loading buffer (62.5 mM
TrisHCl, 25% glycerol, 2% SDS, 0.01% Bromophenol Blue) and loaded onto a 4-
20% gradient SDS polyacrylamide gel (Product number 161-1123, Biorad,
Hercules,
CA). SDS-PAGE was performed according to Laemmli (1970). In each case the
~SOkD band corresponding to the Fab' was observed, and the only observable
contaminants correspond to the sizes of the light and cleaved heavy chains.
Because
these contaminants co-migrated with the Fab' in a high resolution gel
filtration
colmnn, they presumably correspond to a non-covalent but otherwise
structurally
native complex between the light and cleaved heavy chains, in which the
disulfide
bond that normally links them has been reduced and alkylated. These fragments
are
likely to be functional since the disulfide bond between the heavy and light
chain is
not in the antigen-binding site. They correspond to about 20% of the purified
protein, and some of these Fab' fragments are >80% active.
The biotinylation of all capture agents used in this study was verified by
Western blot analysis using an HRP-conjugated streptavidin (SA) probe (data
not
shown). In addition, the extent of biotinylation was estimated by using a SA
resin
pull-down assay (data not shown). Biotinylation was 60% or greater in each of
these
reactions. No attempt was made to remove non biotinylated protein prior to
surface
immobilization.
Example 9
This example describes the carbobiotinylation protocol. IgG (3-5 mg/ml)
were dialyzed into coupling buffer (0.1 M NaO.Ac pH 5.5) and then incubated
with
20 mM sodium meta periodate, NaI04 in the dark for 1 h at 0 °C to
oxidize the
vicinal diols in the carbohydrate to aldehydes. The reaction was then quenched
by
the addition of 30 mM glycerol for 10 minutes, filtered to remove insoluble
salts,
and then dialyzed against coupling buffer for 6 hours. The aldehyde reactive
probe
(ARP, N-(aminooxyacetyl)-N-(D biotinyl) hydrazine, trifluoroacetic acid salt,
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Molecular Probes, Eugene, OR) is added at 1 mg/ml and incubated at room
temperature for 2 h, after which the sample is extensively dialyzed against
PBS.
Example 10
This example describes the random biotinylation of IgG and Fab 'fragment
molecules. IgG and NEM-treated Fab' fragments were modified with the amine
reactive probe, EZ LinkT"" Sulfo-NHS-Biotin (Pierce). Reactions were performed
with a 20-fold molar excess of biotinylation reagent over protein in 1X PBS at
room
temperature for 2 hours. The biotinylation reagent was then quenched by adding
Tris, pH 7.4, to a final concentration of l OmM. The samples were then
dialyzed
against a 1000-fold excess of PBS 5 times in order to remove free biotin
probe.
After dialysis, the biotinylated proteins were analyzed on a gel and tested
for extent
of biotinylation on UltraLinkT"" Plus Immobilized Streptavidin Gel (Pierce).
Typically 60% - 100% of the protein would be biotinylated (data not shown).
Example 11
This example describes using surface plasmon resonance (SPR) to measure
surface coverage and activity of affinity agents linked to a thinfilm formed
on a gold
substrate. All SPR assays were performed in a BIAcore 3000 sing a biotinylated
self assembled monolayer formed on a gold-coated glass surface. The surface
was
prepared using iuisymmetrical alkanedisulfide compound 10 according to the
protocol of example 1. The prepared surface comprised N hydroxysuccinimide and
methoxy groups on its exposed face. This monolayer was then reacted with (+)-
biotinyl-3,6-dioxaoctanediamine (tri-ethyleneglycol amino biotin) reagent to
give a
biotinylated surface. The biotin groups on the surface allow for the binding
of
streptavidin (SA). All assays were performed at 25° C in PBS with 0.05%
Tween-
20. SA was loaded onto the surface at a flow rate of 20 ~.1/min at 0.1 mg/ml.
Typically 320 ~,1 was loaded to achieve a saturated surface of SA whereby a
surface
coverage of 3.7-4.0 pmol/cma was obtained, as calculated according to Jung et
al.,
Langnauif~ 14:5636-5648 (1998). After SA deposition, the various capture
agents
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were loaded at 20-100 nM at a flow rate of 20 ~1/min until saturation was
observed.
For analyte binding, flow rates of 80 ~,1/min were used unless otherwise
noted.
Various analyte concentrations over a broad range were assayed in order to
determine the upper limit of analyte binding. For comparison, non-specific
binding
of the analytes to SA was tested independently at all analyte concentrations
studied.
Exaynple 12
This example describes the synthesis of asymmetric NHS disulfide 120.
With reference to FIG. 7, alkenyl bromide compound 100 is used as the starting
material for both "arms" of the asymmetric disulfide. To produce the protein
resistant arm, 100 is reacted with alkoxide triethylene glycol monomethyl
ether
(produced by deprotonation with NaH) to afford compound 102 in 60% yield.
Compound 102 is subjected to radical addition to give the terminal thioacetyl
compound 104 in 88% yield. The acetyl group is removed under acidic conditions
(4N HCl/dioxane at 75 °C for 4 hours) to afford thiol 106 (95%), which
is treated
with 2',2'-dipyridyl disulfide to furnish the corresponding 2'-pyridyl mixed
disulfide
108 in 98% yield.
The NHS "arm" of the mixed .disulfide 120 is prepared from 100 via reflux
with tetraethylene glycol in aqueous NaOH for 24 hours to yield compound 110
in
42% yield. Terminal alcohol 110 is treated with NaH and alkylated with t
butylbromoacetate to afford terminal ester compound 112 in 42% yield. Compound
112 is converted to thioacetyl compound 114 in 87% yield via free radical
addition
of thioacetic acid. Removal of the acetyl and t-butyl blocking groups by
treatment
with 2N aqueous HCl for 48 to 60 hours at 75 °C produced thiol 116 in
80% yield.
Compounds 108 and 116 were then combined in the presence of Amberlite
CG-50 (pH ca. 3-4) for about 12 to about 16 hours at room temperature to yield
asymmetric disulfide 118, which was converted to desired compound 120 via DCC-
catalyzed condensation with N hydroxysuccinimide.
