Note: Descriptions are shown in the official language in which they were submitted.
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FUNCTIONALIZED MATERIALS AND LIBRARIES THEREOF
FIELD OF THE INVENTION
The present invention relates to functionalized materials and libraries
thereof, for the
separation, purification, concentration, immobilization and synthesis of
compounds, as well
as applications for using the same.
BACKGROUND OF THE INVENTION
Isolation and purification of a target molecule is a prerequisite to its study
and use, for
example, the ability to isolate and identify disease causing microorganisms
allows for
accurate diagnosis and treatment of disease states, or isolation of a nucleic
acid is the first
step in the sequencing of the polynucleotide or the polypeptide sequence
encoded by a
nucleic acid, or the determination of the crystal structure of a protein.
There are many
methods for isolating, purifying, and concentrating molecules, but the
compositions for
performing such methods do not have broad application, and are usually
applicable to the
purification of specific molecules. There remains a need in the art for
improved compositions
and methods of isolating and concentrating molecules.
SUMMARY OF THE INVENTION
In general, the invention is based on the discovery that certain materials can
be
fabricated into compositions that have side chains or polymeric molecular
"brushes" which
have particular properties, for example, length, thickness, morphology and
density. The
materials are highly effective for separating, purifying, concentrating and/or
immobilizing
compounds in a three dimensional conformation, and for synthesizing or
otherwise modifying
compounds immobilized thereto. The compositions of the present invention are
useful in
applications that require a high convective flow rate across the material, or
are subjected to
harsh chemicals, or extreme temperature variations.
In one aspect, the invention includes a substrate material having polymer
brushes on
at least a first and a second surface, wherein the polymer brushes on the
first surface further
comprise a first set of functional groups having a charge, and the polymer
brushes on the
second surface f~uther comprise a second set of functional groups having an
opposite charge
to the first set of functional groups. In one embodiment, the polymer brushes
are formed by
radical induced polymerization of the substrate material and the degree of
grafting of the
polymer brushes is greater than 15%. In another embodiment, the degree of
grafting of the
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polymer brushes is greater than 85%. In yet another embodiment, the material
is bipolar and
is capable of dissociating water into H+ and OH- when a voltage is applied.
In another aspect, the invention includes a method of making a bipolar device
by
obtaining a material, forming polymer brushes on the material by graft induced
polymerization on at least a first and second surface, immobilizing a first
functional group to
the polymer brushes on the first surface, wherein the first functional group
has a charge, and
immobilizing a second set of functional groups to the polymer brushes on the
second surface,
wherein the second functional group has a charge opposite to that of the first
functional
group. In one embodiment, the polymer brushes have a degree of grafting
greater than 15%.
In another embodiment, the polymer brushes have a degree of grafting greater
than 85%. In
another aspect, the invention includes a method of using the bipolar device,
to produce a
compound, for example, salicylic acid. In one embodiment, the invention
includes a method
of using the bipolar material to produce acid or alkali from a salt solution.
In another
embodiment, the material is used for an electrodialysis reaction.
In another aspect, the invention includes a device having a plurality of
functionalized
materials, further including a substrate material having at least one surface
having polymer
brushes formed thereon, the polymer brushes presented in a plurality of
domains, and one or
more functional groups, the functional groups immobilized to the polymer
brushes at one or
more domains. In one embodiment, the polymer brushes at a plurality of domains
are of
different morphologies or lengths. In another embodiment, the polymer brushes
at a plurality
of domains are formed from one or more types of reactive monomers. In yet
another
embodiment, the polymer brushes at a plurality of domains have a degree of
grafting from
about 10% to about 500%. In still another embodiment, at least one functional
group is
immobilized at each domain. In even another embodiment, at least two
functional groups are
immobilized at each domain. In one embodiment, the functional groups
immobilized to the
polymer brushes bind one or more targets, including polynucleotide targets,
polypeptide
targets, polysaccharide targets, lipid targets, organelles, cellular membranes
and cell targets,
including animal cells, mammalian cells, human cells, fungal cells, viral
cells and bacterial
cells such as pathogenic bacterial strains e.g., Staphylococcus, Clostridium,
Bacillus, ahd the
like. In one embodiment, the functional groups include immunoglobulins or
antigen binding
fragments thereof. In another embodiment, the functional groups immobilize one
or more
targets to at least one domain. In yet another embodiment, the functional
groups catalyze a
reaction involving a target. In one embodiment, the functional groups are
charged. In still
another embodiment, the functional groups are enzymes, such as restriction
enzymes,
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proteases, kinases or phosphatases. In another embodiment, the functional
groups include
microdelivery functional groups having a compound. In this embodiment, a
target cell is
contacted to microdelivery functional groups at one or more domains, thereby
causing the
target cell to uptake compounds contained in the microdelivery functional
groups.
Compounds that can be delivered to a cell target through microdelivery groups
include, for
example but not limited to, short interfering RNA (siRNA), antisense nucleic
acids,
transposable elements and nucleic acids with similar integration sequences,
vectors, proteins
and drugs, among others.
In still another aspect, the invention includes a method of making a library
of
functionalized materials wherein a substrate material is obtained, polymer
brushes are formed
on the material in a plurality of domains, and at least one functional group
is immobilized to
the polymer brushes at one or more domains. In one embodiment, the polymer
brushes at a
plurality of domains are of different morphologies or lengths. In another
embodiment, the
polymer brushes at a plurality of domains are formed from one or more types of
reactive
monomers. In yet another embodiment, the polymer brushes at a plurality of
domains have a
degree of grafting from about 10% to about 500%. In still another embodiment,
at least one
functional group is immobilized at each domain. In even another embodiment, at
least two
functional groups are immobilized at each domain. In one embodiment, the
functional
groups immobilized to the polymer brushes bind one or more targets, including
polynucleotide targets, polypeptide targets, polysaccharide targets, lipid
targets, organelle
targets, cellular membrane targets and cell targets, including animal cells,
mammalian cells,
human cells, fungal cells, viral cells and bacterial cells such as pathogenic
bacterial strains
e.g., Staphylococcus, Clostridium, Bacillus, and the like. In one embodiment,
the functional
groups include immunoglobulins or antigen binding fragments thereof. In
another
embodiment, the functional groups immobilize one or more targets to at least
one domain. In
yet another embodiment, the functional groups catalyze a reaction involving a
target. In one
embodiment, the functional groups are charged. In still another embodiment,
the functional
groups are enzymes, such as restriction enzymes, proteases, kinases or
phosphatases. In
another embodiment, the functional groups include microdelivery functional
groups having a
compound. In this embodiment, a target cell is contacted to microdelivery
functional groups
at one or more domains, thereby causing the target cell to uptake compounds
contained in the
microdelivery functional groups.
In one aspect, the invention includes methods of using the devices described
to
introduce a compound to a target. In one embodiment, the functional groups
comprise
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microdelivery groups containing a compound. In another embodiment, the
compound is a
nucleic acid, and a cell target is contacted at one or more domains, thereby
delivering the
nucleic acid to the cell. In yet another embodiment, the compound is a
polypeptide, and a
cell target is contacted at one or more domains, thereby delivering the
polypeptide to the cell.
In still another embodiment, the compound is a drug, and a cell target is
contacted at one or
more domains, thereby delivering the drug to the cell. In one embodiment, the
polymer
brushes at a plurality of domains are of different morphologies or lengths,
and have a degree
of grafting from about 10% to about 500%. In another embodiment, the invention
includes
contacting the domains of the library with a solution comprising a target, and
detecting an
interaction with the target at one or more of the domains. In one embodiment,
the interaction
between the taxget and a domain is detected by measuring radioactive emissions
at the
domain. In another embodiment, the interaction between the target and a domain
is detected
by measuring luminescence at the domain. In still another embodiment, the
interaction
between the target and a domain is detected by measuring fluorescence at the
domain. In
even another embodiment, the fluorescence measured is generated by
fluorescence resonance
energy transfer pairs.
In even another aspect, the invention includes a system having a processor in
communication with one or more memory devices, a material library including a
substrate
material having at least one surface having polymer brushes formed thereon,
the polymer
brushes presented in a plurality of domains; and one or more functional
groups, the functional
groups immobilized to the polymer brushes at one or more of the domains, a
reading device
capable of detecting labels at library domain addresses, the reading device in
communication
with the processor, an instruction set stored in at least one memory device,
the instruction set
capable of interacting with the processor, a user controlled input device
capable of entering
information into the memory device, and an output device in communication with
the
processor or memory. In one embodiment, the functional groups are polypeptide
sequences.
In another embodiment, the functional groups are polynucleotide sequences. In
still another
embodiment, the functional groups are immunoglobulins or antigen binding
fragments
thereof. In yet another embodiment, the immunoglobulin concentration varies at
each
domain from about 0.1 fg/mm2 antibody immobilized per domain surface area to
about 100
mg/mm2 antibody immobilized per domain surface area. In even another
embodiment, the
system includes functional groups of human cells, viral cells, or bacterial
cells. In one
embodiment, the system includes a microfluidic device, wherein the library is
contained
within a reaction chamber of the microfluidic device. In another embodiment,
the processor
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is in communication with one or more microfluidic ports on the microfluidic
device. In one
aspect, the system includes a laboratory information management program.
In still another aspect, the invention includes a material library having a
substrate
material further including a plurality of domains having polymer brushes
formed thereon, and
a plurality of polypeptide functional groups immobilized to the polymer
brushes at one or
more of the domains. In another aspect, the invention includes a material
library having a
substrate material further including a plurality of domains having polymer
brushes formed
thereon, and a plurality of cells immobilized to the polymer brushes at one or
more of the
domains. In one embodiment, the cells are human cells. In another embodiment,
the cells are
human cancer cells. In yet another embodiment, the cells are virally infected
cells. In even
another embodiment, the cells are viral cells. In still another embodiment,
the cells are
bacterial cells, for example, pathogenic bacterial strains. In another aspect,
the invention
includes a material library having a substrate material further including a
plurality of domains
having polymer brushes formed thereon, and one or more types of immunoglobulin
molecules immobilized to the polymer brushes at one or more of the domains. In
another
aspect, the invention is a material library having a substrate material
further including a
plurality of domains having polymer brushes formed thereon, and polypeptide
functional
groups immobilized to the polymer brushes at one or more of the domains, the
polypeptide
functional groups capable of interacting with one or more targets. In one
embodiment, the
targets are protein targets, for example from cellular lysates. In yet another
embodiment, the
cellular lysate includes the soluble fraction derived from pulse labeled
cells. In still another
embodiment, the cells are human cells. In another embodiment, the polypeptide
functional
groups include random peptides from about 5 mer to about 50 mer in length. In
one
embodiment, the interaction of targets with functional groups at one or more
domains is
detected. In yet another embodiment, detection of interactions includes
detection of one or
more markers for human disease.
Other features and advantages of the invention will be apparent from following
detailed description, and from the claims.
3O BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a schematic for preparing materials having cation and anion
exchange functional groups.
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FIG. 2 shows an apparatus for introducing cation and anion exchange functional
groups to opposing surfaces of an irradiated high density polyethylene (HDPE)
film to create
graft-polymerized bipolar materials.
FIG. 3 illustrates electrodialysis using graft-polymerized bipolar materials
(BP),
anion exchange materials (A) and cation exchange materials (C).
FIG. 4 illustrates the relationship between the degree of co-grafting as
measured by
changes in material thickness as a function of co-grafting time for (a)
SSS/HEMA (sodium
styrene sulfonate/hydroxyethyl methacrylate) cation exchange materials and (b)
VBTAC/HEMA (vinyl benzyl trimethyl ammonium chloride/hydroxyethyl
methacrylate)
anion exchange materials.
FIG. 5 shows the increase of (a) sulfonic acid group and hydroxyl group
densities in
the cation-exchange material as well as the increase of (b) quaternary
ammonium salt group
and hydroxyl group densities as a function of degree of co-grafting.
FIG. 6 illustrates the distribution profiles of sulfonic acid functional
groups and the
quaternary ammonium functional groups as a measure of the sulfur and the
chlorine,
respectively, across the thickness of both materials as determined by X-ray
microanalysis
(XMA).
FIG. 7 illustrates the relationship between functional group density as
measured by
titration of the salt-splitting capacity and the surface area ratio of X-ray
intensity distribution
of the (a) sulfur and (b) chlorine functional groups, as measured by XMA.
FIG. 8 shows the increase of the degree of co-grafting and the thickness of
the
prepared bipolar material as a function of reaction time (a) and the increase
in functional
group density also as a function of DG (degree of grafting) (b).
FIG. 9 illustrates the functional group density profiles across the thickness
of the
membranes where DG =16% and DG =86%.
FIG. 10 illustrates the voltage-current characteristic of the co-grafted-type
bipolar
material.
FIG. 11 shows the time course for electrodialysis using the co-grafted-type
bipolar
materials having DG =16% (a) and DG =86% (b) as measured by the concentration
changes
of NaCI (salt chamber), HCl (acid chamber) and NaOH (alkali chamber).
FIG. 12 illustrates the decrease in the voltage and the current during the
electrodialysis as a function of operation time using the co-grafted-type
bipolar materials
having DG=16% and DG=86%.
FIG. 13 shows the electrodialysis efficiency for the co-grafted-type bipolar
material.
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FIG. 14 illustrates the chemical structure of the grafted-type GMA-DEA-BC
fiber
having a material of polyethylene (PE) polymer brushes comprising glycidyl
methacrylate
(GMA) polymers, and functional groups of diethylamine (DEA) quaternized with
benzyl
chloride (BC).
FIG. 15 shows the conversion of quaternization of the grafted-type GMA-DEA-BC
material as a function of reaction time.
FIG. 16 illustrates XMA profiles of chloride ion adsorbed on the grafted-type
GMA-
DEA-BC materials as a function of conversion by BC.
FIG. 17 shows adsorption of Staphylococcus au~eus cells using three grafted-
type
GMA-DEA-BC materials with different degrees of quaternization.
FIG. 18 shows the relationship between the adsorption rate constant (k)
describing
the binding of Staphylococcus aureus cells and the functional-group-density of
the grafted-
type GMA-DEA-BC material.
FIG. 19 illustrates the changes in CFU/mL (colony forming units) and pH of the
flow
through solution following contact of the Staphylococcus au~eus cells with the
grafted-type
GMA-DEA-BC material, as a function of contact time.
FIG. 20 illustrates a material library comprising functionalized domains
fabricated in
an array or matrix format, wherein the domains shown vary in terms of polymer
brush length
and functional group density.
FIG. 21 illustrates the material library of FIG. 20, wherein a target compound
is
introduced to one or more domains on the library by a microfluidic device
capable of varying
the composition of an input solution containing the target, and where binding
of a target to
one or more domains indicates the optimum binding conditions, i.e., the brush
length and
functional group density, for isolation of the compound contained in the
particular input
solution.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
Unless otherwise defined, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art of which
this invention
belongs. However, the following terms have the meanings specified below.
As used herein, the term "material" refers to a substrate providing one or
more
surfaces, where at least one surface is capable of forming grafted polymer
brushes, or to
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which polymer brushes can be otherwise affixed. Thus a material may include
two or more
different materials. There is also a wide variety of shapes, into which the
material may be
fabricated or otherwise formed, depending on the particular application for
which the
material will be used. For example, the material may be substantially rigid.
This is
appropriate in applications, for example, where the material is formed into
e.g., a vial, a pipet
tip, a cell culture or ELISA dish, slide or other type of substrate for
forming an array or
matrix of materials. The material may be substantially flexible along one or
more planes, for
example formed into a fiber or membrane. The material may be in the form of a
powder, a
particle preparation or a microparticle suspension. The material may be
substantially
elongated and flexible, and may define a lumen, for example, fabricated into
tubing or pipet
tips. A wide variety of materials are appropriate for the materials and
methods of making the
same which are disclosed herein, and are also described in U.S. Patents
No.:6,009,739,
5,783,608, 5,743,940, 5,738,775, 5,648,400, 5,641,482, 5,506,188, 5,425,866,
5,364,638,
5,344,560, 5,308,467, 5,075,342, 5,071,880, 5,064,866, 4,980,335, 4,897,433,
4,622,366,
4,539,277, 4,407,846, 4,379,200, 4,376,794, 4,288,467, 4,287,272, 4,283,442,
4,273,840,
4,137,137 and 4,129,617, each incorporated herein by reference.
As used herein, the term "brush" or "polymer brush" refers to a polymeric side
chain
that is formed from a polymerization substrate having a radical-polymerizable
terminal
group, wherein the polymerizable substrate is the material itself, or another
polymerizable
material that can be engrafted to, or otherwise affixed to the material. The
side chain can be
any reactive monomer (or polymer), but an easily functionalizable reactive
polyvinyl polymer
is currently preferred, for example such as polyglycidyl methacrylate (GMA) or
polyhydroxyethyl methacrylate (HEMA), which has one reactive epoxide group per
repeat.
Polymer brushes are formed by radical polymerization as described below. A
brush has an
elongated shape of a particular size in one direction related to the degree of
polymerization in
a first direction, its "length", and a cross sectional diameter or thickness
related to the degree
of polymerization in a second direction perpendicular to the first direction,
its "width". The
brushes can assume a coiled or compacted morphology or an extended morphology.
The
width of a brush can vary along its length. In addition, the polymerization
reaction can be
controlled to create branch-like polymer brush structures, as well as
increasing or decreasing
brush density, i. e., number of brushers per surface area or per weight of
material, as described
below. The length, width, branching, and overall morphology of the polymer
brushes in the
present invention can be varied according to the desired end use or purpose as
described
herein and by methods known in the art.
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As used herein the term "reactive monomer" refers to a compound that is
capable of
participating in a radical induced grafting reaction. The reactive monomer can
be any
material capable of forming polymers as described above and herein, for
example but not
limited to glycidyl methacrylate (GMA), or ethylene. The material and reactive
monomer
may be of the same compound, for example, a polyethylene material may utilize
ethelyene
monomers or polymers in the grafting reaction. A wide variety of reactive
monomers are
appropriate for the membrane compositions and methods disclosed herein, and
are described
below and in U.S. Patents No.:6,009,739, 5,783,608, 5,743,940, 5,738,775,
5,648,400,
5,641,482, 5,506,188, 5,425,866, 5,364,638, 5,344,560, 5,308,467, 5,075,342,
5,071,880,
5,064,866, 4,980,335, 4,897,433, 4,622,366, 4,539,277, 4,407,846, 4,379,200,
4,376,794,
4,288,467, 4,287,272, 4,283,442, 4,273,840, 4,137,137 and 4,129,617, each
incorporated
herein by reference.
As used herein the term "degree of polymerization" refers to the extent of
radical
induced polymerization of a polymerizable substrate having a radical-
polymerizable terminal
group, with one or more types of a reactive monomer, wherein the
polymerization reaction
forms a polymer brush. The degree of polymerization is thus determinative of
the overall
brush surface characteristics. The polymeric side chains can, for example, be
a monomer, an
oligomer, or have an average length between about 10 nm and about 2000 nm
corresponding
to anywhere from about several hundred to tens of thousands of monomer units
or longer, for
example about 5000 nm or more. The degree of polymerization depends on, e.g.,
the
crystalinity of the polymerizable substrate, the degree of radicalization, the
length of time the
reaction is allowed to progress, and on the physical properties of the
polymerizable substrate,
i.e., its strength or rigidity (see, Lee, et al., (1999) Chenz, Mater., 11,
3091-3095, incorporated
herein by reference).
As used herein the term "degree of grafting" or "DG" refers to the brush
density, i.e.,
the number of the side chains brushes per unit surface area of material.
Anywhere from
about 1.0 x 108 to about 1.0 x 103° of the side chains brushes can be
present per square meter
of surface area or per weight of material, for example, from about 1.0 x 1016
to about 1.0 x
102° of the side chains brushes represents a degree of grafting between
about 10% and about
500%. The degree of grafting is essentially a ratio describing the initial
weight of a material
and the additional weight of the polymer brush structures (see, Lee, et al.,
(1999).
As used herein a "functional group" refers to a compound having a particular
chemical property, biological activity or affinity for a ligand, or a
particular structure. A
functional group is immobilized, bound, entrapped, cross-linked or otherwise
substantially
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affixed to the polymer brushes grafted to the material. A wide variety of
functional groups
are suitable for the present invention, imparting such ftmctionality to the
brushes.
Combinations of functional groups are preferred where mufti-functionalized
materials are
desirable. A functional group can be any molecule or complex of molecules
which has the
ability to bind a target and immobilize it to the polymer brush. Preferably,
the functional
group binds its target in a substantially specific manner. Hence, the
functional group may
optionally be a target whose natural function in a cell is to specifically
bind another protein,
such as an antibody or a receptor. Alternatively, the functional group may
instead be a
partially or wholly synthetic or recombinant polypeptide which specifically
binds a target.
Alternatively, the ftmctional group may be a polypeptide which has been
selected in vitro
from a mutagenized, randomized, or completely random and synthetic library by
its binding
affinity to a specific target. The selection method used may optionally have
been a display
method such as ribosome display or phage display. Alternatively, the
functional group
obtained via in vitro selection may be a DNA or RNA aptamer which specifically
binds a
protein target (for example: Potyrailo et al., Anal. Chem., 70:3419-25, 1998;
Cohen, et al.,
Proc. Natl. Acad. Sci. USA, 95:14272-7, 1998; Fukuda, et al., Nucleic Acids
Symp. Ser.,
(37):237-8, 1997, each incorporated by reference). Alternatively, the ih vitro
selected
functional group may be a polypeptide (Roberts and Szostak, Proc. Natl. Acad.
Sci. USA,
94:12297-302, 1997, incorporated by reference). In an alternative embodiment,
the
functional group may be a small molecule which has been selected from a
combinatorial
chemistry library or is isolated from an organism. Functional groups may also
be selected on
their ability to bind a target and catalyze a biological reaction. Suitable
functional groups
include, for example and without limitation, anionically dissociating groups
(e.g., primary,
secondary, tertiary, or quaternary amines), cationically dissociating groups
(e.g., acid groups)
with or without coexisting hydrophilic or hydrophobic groups (nonionic groups
such as,
GMA or other hydrophobic reactive groups), polypeptides, polynucleotides,
proteins or
active domains thereof, ions, epitopes and affinity tags, nucleic acids,
ribonucleic acids,
polypeptides, glycopolypeptides, mucopolysaccharides, lipoproteins,
lipopolysaccharides,
carbohydrates, enzymes or co-enzymes, hormones, chemokines, lymphokines,
antibodies,
ribozyrnes, aptamers, siRNA's, IFN-alpha, IFN-gamma, SpA, SpG, immunoglobulins
including monoclonal and polyclonal preparations, TNF-alpha, TNF-beta, v-Ras,
c-Ras,
reverse transcriptase, G-coupled protein receptors (GPCR's), FcRn, FcyR's,
FcER's,
nicotinicoid receptors (nicotinic receptor, GABAA and GABA~ receptors, glycine
receptors,
5-HT3 receptors and some glutamate activated anionic channel receptors), ATP-
gated
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channels (also referred to as the P2X purinoceptors), glutamate activated
cationic channels
(NMDA receptors, AMPA receptors, Kainate receptors, etc.), hemagglutinin (HA),
receptor-tyrosine kinases (RTI~'s) such as EGF, PDGF, NGF and insulin receptor
tyrosine
kinases, SH2-domain proteins, PLC-y, c-Ras-associated GTPase activating
protein
(RasGAP), phosphatidylinositol-3-kinase (PI-3I~) and protein phosphatase 1 C
(PTP 1 C), as
well as intracellular protein tyrosine kinases (PTK's), such as the Src family
of tyrosine
kinases, protein-tyrosine phosphatases, such as receptor tyrosine phosphatase
rho, protein
tyrosine phosphatase receptor J, receptor-type tyrosine phosphatase D30,
protein tyrosine
phosphatase receptor type C polypeptide associated protein, protein tyrosine
phosphatase
receptor-type T, receptor tyrosine phosphatase gamma, leukocyte-associated Ig-
like receptor
1D isoform, LAIR-1D, LAIR-1C, MAP kinases, neuraminidase (NA), proteases,
polymerases, serine/threonine kinases, second messengers, antigenic or
tumorigenic markers,
transcription factors, and other such important metabolic building blocks or
regulators, and
fluorescent labeled polypeptides or fluorescent polypeptides like GFP, growth
factor
receptors, hormone receptors, neurotransmitter receptors, catecholamine
receptors, amino
acid derivative receptors, cytokine receptors, extracellular matrix receptors,
lectins, serpins,
hydrolases, steroid hormone receptors, transcription factors, heat-shock
transcription factors,
DNA-binding proteins, zinc-finger proteins, leucine-zipper proteins,
homeodomain proteins,
intracellular signal transduction modulators and effectors, apoptosis-related
factors, DNA
synthesis factors, DNA repair factors, DNA recombination factors, cell-surface
antigens,
disease markers, hepatitis C virus (HCV) proteases or HIV proteases. Selection
and use of
functional groups will depend on the application desired, as illustrated
herein.
The term "anionically dissociating functional groups" as used herein means
those ion-
exchange groups whose counter ion is an anion. Anionically dissociating groups
have the
ability catalyze chemical reactions and to absorb and/or immobilize target
compounds or
other functional groups and are capable of entering into neutralizing
reactions with acidic
substances such as hydrogen sulfide or mercaptans, allowing for a wide range
of uses with
effective removal of the acidic substances.
The term "cationically dissociating functional groups" as used herein means
those
ion-exchange groups whose counter ion is a cation. A typical cationically
dissociating group
is an acid group. Cationically dissociating groups have the ability to
catalyze chemical
reactions and adsorb and/or immobilize target compounds or other functional
groups and are
capable of releasing a proton (hydrogen ion) to enter into neutralizing
reaction with basic
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substances, say, ammonia or amines. As a result, these groups provide a wide
range of uses
with basic substances.
The term "hydrophilic functional groups" as used herein refers to groups that
have an
affinity for water but do not undergo significant ionic dissociation upon
contact with water.
Hydrophilic groups have the ability to catalyze chemical reactions and adsorb
and/or
immobilize target compounds or other functional groups, by providing a
hydration shell, or
by providing a reactive surface. An example of such group, without limitation,
is a hydroxyl
group.
The term "hydrophobic functional groups" as used herein refers to groups that
do not
have an affinity for water. Hydrophobic groups have the ability to catalyze
chemical
reactions and adsorb andlor immobilize target compounds or other functional
groups, by
excluding water, or by providing a surface for hydrophobic interactions, or by
providing a
reactive surface. An example of such group, without limitation, is a nonionic
group, an ester
group, a succinimide group or an epoxy group.
Detaileel Description of the Invention
The present invention provides for compositions and methods of immobilizing
functional groups to polymer brushes grafted to one or more materials.
Immobilization
methods include entrapment, gelification, physical retention or adsorption,
ionic binding,
covalent binding or cross-linking (see, Biotechnol. Bioeng., 22:735-756, 1980;
Chem. Eng.
Prog., 86:81-89, 1990; J. Am. Chem. Soc., 117:2732-2737, 1995; Enzyme Microb.
Technol.,
14:426-446, 1997; Trends Biotechnol., 13:468-473, 1997; Nat. Biotechnol.,
15:789-793,
1997, each incorporated herein by reference). The immobilization method and
the amount
and kind of the functional groups used both determine the activity of the
composition of the
present invention. The resulting activity of the immobilized functional group
can often
further reduced by mass-transfer effects (see, Methods Enzymol., 44:397-413,
1976; J. Am.
Chern. Soc., 114:7314-7316, 1992; Trends Biotechnol., 14:223-229, 1996; Angew.
Chem.,
109:746-748, 1997, each incorporated herein by reference). The activity
following
immobilization can be further reduced as a result of the diminished
availability of the
functional groups, i.e., due to steric hindrance, entrapment within brushes,
pores or other
structures on the material substrate, or by slow diffusion of the functional
groups. Such
limitations lead to lowered efficiency. It is an objective of the present
invention to provide
materials having a high capacity for functional groups immobilized thereto,
The invention is usable with a wide variety of materials, i. e., all polymeric
plastics,
such as, for example, polyurethanes, polyamides, polyesters, polyethers,
polyether-block
12
CA 02469301 2004-06-08
WO 03/049671 PCT/US02/33942
amides, polystyrene, polyvinyl chloride, polycarbonates, polyorganosiloxanes,
polyolefins,
polysulfones, polyisoprene, polychloroprene, polytetrafluoroethylene (PTFE),
corresponding
copolymers and blends, as well as natural and synthetic rubbers, metal, glass
or wooden
bodies. The compositions have multifiuictional properties and can be used to
separate,
remove, purify, synthesize, concentrate and immobilize compounds, and axe
particularly
suited to the harsh operating environments, i. e., extreme temperatures and
pressures,
chemical concentrations, electrical charges, etc., from commercial processes.
In general, the desired target compound is in a sample solution, which can be
passed
directly through the compositions, as in a filtration membrane, tube, pipet
tip or a
chromatography matrix. Liquids containing cells or other large insoluble
particles may
require pre-treatment to separate the larger particles from the smaller
soluble ones. However,
the polymer brush sizes and brush density provide a degree of physical
filtration, and the
compositions can be woven or otherwise fabricated into filtration devices if
appropriate.
While an aqueous sample solution is often described, one skilled in the art
will realize that
gaseous samples may be employed. Examples of filter elements for adsorbing
gaseous
components of a gas stream are described in, for example, United States Patent
Application
20020002904 A1, to Gentilcore, et al., published January 10, 2002, herein
incorporated by
reference. In addition, a membrane or fiber is often described, but the
compositions of the
invention illustrated below can comprise other forms as described herein. Thus
the following
is illustrative and are not meant to be limiting examples of the present
invention.
Materials Useful in the P~ese~zt Invention
In general, the material of the present invention is not limited to any
particular type,
and any substrate that permits grafting or affixation of the polymer brush is
an appropriate
material. Treatment of a material surface is acceptable if the original
material is not itself
sufficient for the polymerization reaction. In such cases, the surface
treatment according to
the invention can be, for example, a coating formed from a polymeric material.
Materials
useful in the present invention are widely available, for example polyolefins
(low density or
high density) including polyethylene and polypropylene, cellulose (see,
Radiat. Phys. Chem.
1990, 36:581; J. Membr. Sci. 1993, 85:71), poly(isobutylene oxide) (see,
Radiat. Phys.
Chem. 1987, 30:151), ethylene-tetrafluoroethylene copolymer (see, J.
Electrochem. Soc.
1996, 143: 2795) ethylene-propylene-dime terpolymer (see, Radiat. Phys. Chem.
1991, 37:
83) ethylene-propylene rubber (see, Nippon Gensiryoku Gakkaishi, 1977, 19:340)
chlorosulfonated polyethylene (see, Radiat. Phys. Chem. 1991, 37:83)
polytetrafluoroethylene (PTFE) (see, React. Polym. 1993, 21:187; Radiat. Phys.
Chem.
13
CA 02469301 2004-06-08
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1989, 33:539) tetrafluroethylene-hexafluoropropylene copolymer (see, Radiat.
Phys. Chem.
1988, 32:193) polyvinyl chloride) (see, Radiat. Phys. Chem. 1978, 11:327)
silicone rubber
(see, Radiat. Phys. Chem. 1988, 32: 605) polyurethanes (see, Radiat. Phys.
Chem. 1981, 18:
323) polyesters (see, Radiat. Phys. Chem. 1988, 31: 579) butadiene-styrene
copolymer (see,
Radiat. Phys. Chem. 1990, 35: 132) natural and nitrite rubbers (see, Radiat.
Phys. Chem.
1989, 33: 87) cellulose acetate and propionate (see, Radiat. Phys. Chem. 1990,
36: 581)
starch and cotton fabric (see, Zhurn, Vsesoyuz. I~him. Ob-va im. D. I.
Mendeleeva. 1981,
26:401) polyester-cellulose fabric (see, Radiat. Phys. Chem. 1981, 18:253)
natural leather
(see, Radiat. Phys. Chem. 1980, 16:411) and medical gauze (see, Zhurn.
Vsesoyuz. Khim.
Ob-va im. D. I. Mendeleeva. 1981, 26:401) hydrophilic polyurethanes,
polyureas, olefins,
acrylics, as well as other hydrophilic components. Particular materials
include polyethylene
glycol, polyethylene glycol or polypropylene glycol copolymers and other
poloxamers,
heterocyclic monomers (see, Applied Radiation Chemistry: Radiation Processing,
Robert J.
Woods and Alexei K. Pikaev, John Wiley 8c Sons, Inc., 1994 (ISBN 0-471-54452-
3)),
polyethylene glycol) methacrylate or dimethacrylate (see, J. Appl. Polym.
Sci., 1996,
61:2373-2382), polyamine (such as polyethyleneimine), polyethylene oxide), and
styrene.
These coatings preferably are covalently bonded to the surface which is being
treated. Many
methods for forming the coating exist, and include the steps of adsorbing the
polymeric
material to the surface, and then covalently attaching the polymeric material
to the surface by
exposure to UV radiation, RF energy, heat, X-ray radiation, gamma radiation,
electron
beams, chemical initiated polymerization or the like.
A material provides a plurality of surfaces, and may be itself a polymerizable
substrate having a radical-polymerizable terminal group, for example,
celluloses, polyolefins,
polyacrylonitriles, polyesters such as PET and PBT, polyamides such as nylon 6
and nylon
66, as well as combinations of these. An appropriate material may not itself
be
polymerizable, but is suitable for the present invention provided polymer
brushes can be
grafted, affixed, or otherwise adhered to the non-polymerizable material.
A carbohydrate polymer, such as cellulose or lignin, or a similar material,
can be used
as the material. An example of a composition and method of a grafted
carbohydrate polymer
having pendant 3-amino-2-hydroxy propyl groups grafted thereon, for use as a
retention aid
and strengthening additive in paper manufacture is described in United States
Patent
Application 20020026992 A1, to Antal, et al., published March 7, 2002,
incorporated herein
by reference. The method of radiatioxi induced grafting to cellulose is
described in,
Yamagishi et al., (1993) J. Membr. Sci., 85, 71-80, incorporated herein by
reference.
14
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WO 03/049671 PCT/US02/33942
When the carbohydrate polymer is a component of wood pulp the resulting
chemically modified wood pulp may be employed in conjunction with unmodified
wood pulp
to incorporate therein the retention and strengthening characteristics.
Typical sources of the
carbohydrates, specifically celluloses that can be used as the material
include wood celluloses
such as paper pulp and wood chips. In addition to these celluloses, leaf fiber
cellulose, stem
fiber cellulose and seed tomentous or pubescent fiber cellulose can also be
used. Examples
of such celluloses include bast fibers (e.g., hemp, flax, ramie and Manila
hemp) and cotton.
If desired, rice straw, coffee bean husk, spent tea leaves, soy pulp and other
waste can be
recycled for use as cellulose. Such waste is very convenient to use as a
material because it
does not require any special preliminary treatments. One such source for
cellulose for use in
the present invention is paper pulp.
Metallic materials can be grafted with biologically active compounds, for
example
surface-modified medical metallic materials having a gold or silver thin layer
plated onto a
base metal, as described in United States Patent Application 20010037144, Al
to I~im, et al.,
published November 1, 2001 and incorporated herein by reference.
Animal tissues such as fiber, hair, and leather can be used as the material.
One skilled
in the art would be able to determine if an animal product provided the
desired properties for
use as a material. For example, where it is desired that the invention be used
in a mechanical
filtration, fibers, for example, can be woven or otherwise fabricated into
among other forms,
membrane compositions or sheets. Examples of fibers or animal hairs that can
be used as
materials include wool, camel hair, alpaca, cashmere, mohair, goat hair,
rabbit hair, and silk.
Examples of natural leather that can be used as materials include cowskin,
goatskin, and the
skin or hide of reptiles. Examples of synthetic leather that can be used as
materials include
CORFAM~ (DuPont), CLARINO~ (Kuraray), and ECSAINE~ (Toray).
Polyolefins can also be used as materials (see, Applied Radiation Chemistry:
Radiation Processing, Robert J. Woods and Alexei K. Pikaev, John Wiley & Sons,
Inc.,
1994 (ISBN 0-471-54452-3), Introduction to Radiation Chemistry 3rd Edition,
J.W.T. Spinks
and R.J. Woods, John Wiley & Sons, Inc., 1990 (ISBN 0-471-61403-3), Radiation
Chemistry of Polymeric Systems, A. Chapiro, Interscience, New York, 1962,
Atomic
Radiation and Polymers, A Charlesby, Pergamon Press, 1960, Radiat. Phys. Chem.
1991,
37:175-192, and Prog. Polym. Sci. 2000, 25:371-401 (all incorporated herein by
reference in
their entirety). Polyolefins can be fabricated into many shapes and forms.
They are capable
of being molded, thermoformed, poured, extruded and otherwise shaped by
processes well
known in the art, such as the formation of fibers or filaments by conventional
melt spinning
CA 02469301 2004-06-08
WO 03/049671 PCT/US02/33942
processes. In addition, polyolefm compounds are useful in among other
industries, the
biotechnology industry, largely because polyolefin products are resistant to
chemical
degradation from common laboratory reagents, are durable and can be reused,
and are
chemically inert, and are inexpensive and often disposable. Polyolefin
compounds are
currently preferred materials as they demonstrate these properties and
additionally provide a
polymerizable substrate having a radical-polymerizable terminal group. Olefin
monomers
and polymers are well suited to the grafting techniques of the invention both
as materials and
additionally as reactive monomers. Examples of polyolefins include, for
example,
polyethylene and polypropylene. If desired, these materials can be modified,
for example by
incorporating halogens into the polymer, such as chlorine, fluorine, or
bromine, for example
the halogenated polyolefm, polytetraflurorethylene. Other modifications such
as
incorporation of hydroxyl groups into the polymer are also appropriate.
Polyolefinic
polymers having weight-averaged molecular weights in the range of from 20,000
to 750,000
daltons are suitable for the present invention. One skilled in the art would
know which
molecular weights are appropriate for the particular purpose. For example, a
polyolefin
having a molecular weight from about 50,000 daltons to about 500,000 daltons
is suitable to
use in the production of fiber or filament, used for example, in a membrane
comprising
polyolefin filaments or fibers (see, above) further comprising brushes having
combinations of
functional groups affixed thereto. When the molecular weight of a polyolefin
is greater than
about 500,000 daltons, the fluidity ofthe resultant polyolefin is low, and it
is difficult to form
the polyolefin into such a filament by conventional melt spinning processes.
However, the
structural rigidity of a polyolefin greater than about 500,000 daltons is
suitable, for example,
in high density applications such as containers, freezing vials for cells, and
the like. By
contrast, when the molecular weight of a polyolefin is lower than about 50,000
daltons, the
strength and rigidity of the polymer is lessened and a filament obtained
therefrom does not
have a sufficient tensile strength. However the structural rigidity of a
polyolefm when the
molecular weight of a polyolefin is lower than about 50,000 daltons is
suitable, for example,
in a powder or microcrystaline composition. An example of a polymerized
grafted and
crosslinkable thermoplastic polyolefin powder composition in the form of a
powder intended
for the production of flexible coatings by its free flow over a hot mold is
described in United
States Patent Application 20020019487 Al, to Valligy, et al., published
February 14, 2002,
hereby incorporated by reference. Another polymerized grafted and
crosslinkable
thermoplastic polyolefin powder composition is described in EP0409992,
incorporated by
reference, is directed to a process for the preparation of particles of
crosslinkable
16
CA 02469301 2004-06-08
WO 03/049671 PCT/US02/33942
thermoplastic polyolefin polymers according to which said particles are
brought into contact,
in the solid state, with the crosslinking agent, in particular by means of a
mineral oil.
The shape of the material is not limited in any particular way, and various
shapes can
be employed as selected from among fibers, films, flakes, powders, sheets,
mats and spheres.
The material of the present invention has the function of serving as a
structural member that
supports the polymer brushes. From the viewpoint of maximizing the area of
adsorption
and/or immobilization and enhancing the efficiency of adsorption and/or
immobilization, the
use of fibrous materials is advantageous. Grafted fibers in porous hollow
fiber membrane
configurations or woven or otherwise fabricated into sheets provide two
examples of a
substantially enhanced brush surface area. Materials with lumenal inner
surfaces can also be
adapted to enhance brush surface area by manufacturing the material such that
the lumenal
surface contains irregularities or projections. An analogous illustration of
this embodiment
may be found in physiology, using the small intestine as an example: the
mucosal surface
contains villiar projections (i.e., the surface irregularities) and upon each
villus are thousands
of microvillar structures (i.e., the polymer brushes).
Woven fiber sizes appropriate for the present invention range from about 10 nm
to
about 100,000 nm. It is particularly advantageous to use woven fibrous
materials having
fiber diameters of from about 1000 nm to about 50,000 nm. One of the reasons
why fibrous
materials are advantageous is that they can be easily worked or woven into a
desired shape,
i.e., a fabric, and assembled in a device. Further, fibrous materials
generally have no
potential to release fine particles or dust into the atmosphere and, hence,
they can be used in
semiconductors and other areas of precision machining. If fibrous materials
are to be used,
they can be staple fibers or filaments. Such fibers can be processed into
woven or nonwoven
fabrics. If the membrane of the present invention employs a fibrous substrate,
it can be used
in admixture with other fibrous materials. Combinations of fibers thereby
comprising
different functional groups can be fabricated, thus providing for
multifunctional properties in
a single membrane composition.
Fibers can also be porous hollow fibers manufactured as nonwoven substrates.
Examples of commercially available porous hollow fibers are those manufactured
by Asahi
Chemical Industry, Corp., described herein. These can have a broad range of
porosity and be
fabricated into, for example, filtration devices. Furthermore, a combination
of porosity and
fiber composition thereby provides physical and molecular immobilization,
filtration or
concentration. If fibrous materials are to be used in a spherical form, their
diameter is
advantageously adjusted to lie between about 2 and 20 mm, simply from the
viewpoint of
17
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WO 03/049671 PCT/US02/33942
ease of handling. The porosity of the material of the present invention has an
average pore
diameter of about 0.1 nm to about 50,000 nm, and preferably about 1 nm to 5000
nm, and
more preferably 10 to 1000 nm from the standpoint of the desired functional
activity and
permeability of the material. ~ne skilled in the art could determine the
optimal composition
and porosity for a given application. When the average pore diameter is too
small, the
permeability of the membrane composition is decreased. When the average pore
diameter is
too large, desired substances would are not well adsorbed on the brush surface
of the porous
material. Instead, the subject sample would pass through the pores of the
porous material
without contacting the brush surface and functional groups, so that the
activity of the desired
functional group cannot be attained. The porosity of the porous material of
the present
invention is preferably in the range of from 20 to 90 %, more preferably SO to
90 %. The
degree of porosity depends e.g., on the physical properties of the material
used.
Measurement of porosity and pore size etc. of a material is generally well
known in the art,
for example, the bubble point method, mercury pressure method, Scanning
Electron
Microscopy (SEM) or Tunneling Electron Microscopy (TEM) or the nitrogen
adsorption
method (see, ASTM F316, 1970; Pharmaceutical Tech., 1978, 2:65-75; Filtration
in the
Pharmaceutical Industry, Marcel Dekker, 1987, incorporated herein by
reference).
Agents for Ge~e~ati~g Radicals
The agent generating radicals which are capable of creating radical sites is
an organic
peroxide or a perester such as, for example, tert-butylperoxy 3,5,5-
trimethylhexanoat- e, 2,5-
dimethyl-2,5-di(benzoylperoxy)hexane, tent-butyl-peroxy 2-ethylhexyl
carbonate, tert-
butylperoxy acetate, tert-amylperoxy benzoate, tent-butylperoxy benzoate, 2,2-
di(tert-
butylperoxy)butane, n-butyl 4,4-di(tert-butyl-peroxy)valerate, ethyl 3,3-
di(tert-butylperoxy)-
butyrate, dicumyl peroxide, tert-butyl cumyl peroxide, di-tert-amyl peroxide,
di(2-tert-
butylperoxyisopropyl)benzene, 2,5-dimethyl-2,6-di(ter- t-butylperoxy)hexane,
di-tert-butyl
peroxide, 2,5-dimethyl-2,5-di-tert-but- ylperoxy-3-hexyne, 3,3,6,6,9,9-
hexamethyl-1,2,4,5-
tetraoxacyclononane, tert-butyl hydroperoxide, 3,4-dimethyl-3,4-
diphenylhexane, 2,3-
dimethyl-2,3-diphenylbutane and tert-butyl perbenzoate and azo compounds, for
example
azobisisobutyronitrile and dimethyl azodiisobutyrate; the said agent is
preferably chosen
within the group consisting of dicumyl peroxide, tert-butyl cumyl peroxide, di-
tert-amyl
peroxide, di-tert-butyl peroxide and 2,5-dimethyl-2,5-di(tertbutylperoxy)- -3-
hexyne.
Radiation Induced Graft Polyme~izatio~
Graft polymerization can be carried out, for example, by polymerization in the
presence of a chemical or inducible polymerization initiator, thermal
polymerization,
18
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WO 03/049671 PCT/US02/33942
irradiation-induced polymerization using ionizing radiation (e.g., alpha rays,
beta rays,
gamma rays, accelerated electron rays. X-rays, or ultraviolet rays).
Polymerization induced
by gamma rays or accelerated electron rays provides a convenient graft
polymerization
method.
Several methods of graft polymerization of a reactive monomer to a material
exist.
The material can be a formed article or can be manufactured into a product or
device at a later
time. Liquid phase polymerization, in which a formed article is directly
reacted with a liquid
reactive monomer, and gaseous or vapor phase polymerization, in which a formed
axticle is
brought into contact with vapor or gas of a reactive monomer, are two
polymerization
methods that are useful in the present invention according to the end use or
purpose. Vapor
phase grafting is described in J. Membr. Sci. 1993, 85:71-80, Chem. Mater.
1991, 3:987-
989, Chem. Mater. 1990, 2:705-708, and AIChE J. 1996, 42:1095-1100, all of
which axe
herein incorporated by reference.
Graft polymerization of the reactive monomer to the material is performed.
Grafting
proceeds in three different ways: (a) pre-irradiation; (b) peroxidation and
(c) mutual
irradiation technique. In the pre-irradiation technique, the first polymer
backbone is
irradiated in vacuum or in the presence of an inert gas to form radicals. The
irradiated
polymer substrate is then treated with the monomer, which is either liquid or
vapor or as a
solution in a suitable solvent. However, in the peroxidation grafting method,
the trunk
polymer is subjected to high-energy radiation in the presence of air or
oxygen. The result is
the formation of hydroperoxides or diperoxides depending on the nature of the
polymeric
backbone and the irradiation conditions. The peroxy products, which are
stable, are then
treated with the monomer at higher temperature, whence the peroxides undergo
decomposition to radicals, which then initiate grafting. The advantage of this
technique is
that the intermediate peroxy products can be stored for long periods before
performing the
grafting step. On the other hand, with the mutual irradiation technique the
polymer and the
monomers are irradiated simultaneously to form the radicals and thus addition
takes place.
Since the monomers are not exposed to radiation in the preirradiation
technique, the obvious
advantage of that method is that it is relatively free from the problem of
homopolymer
formation which occurs with the simultaneous technique. However, the decided
disadvantage of the pre-irradiation technique is the scission of the base
polymer due to its
direct irradiation, which brings forth predominantly the formation of block
copolymers rather
than graft copolymers.
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WO 03/049671 PCT/US02/33942
The material substrate surfaces activated in this way are coated in a solution
including
reactive monomers, for example, tert-butylaminoethyl methacrylate, by known
methods, such
as by dipping, spraying or brushing. Suitable solvents have proved to be water
and
water/ethanol mixtures, although other solvents can also be used if they have
a sufficient
dissolving power for tent-butylaminoethyl methacrylate, and wet the material
substrate
surfaces thoroughly. Solutions having reactive monomer contents of 0.1 % to
10% by weight,
for example about 5% by weight, have proved suitable in practice and in
general give
continuous coatings which cover the substrate surface and have coating
thicknesses which
can be more than 0.1 ~,m in one pass. Two, three, or more different reactive
monomers can
be cografted to the material, see, Chem. Mater. 1999, 11:1986-1989, J. Membr.
Sci. 1993,
81:295-305, J. Electrochem. Soc. 1995, 142:3659-3663, and React. Polym. 1993,
21:187-191,
all incorporated herein by reference.
A reactive monomer is any compound that is capable of participating in a
radical
induced graft polymerization reaction. The reactive monomer thus incorporates
in the side
chain reaction, and forms polymer brushes. The term monomer is used for
simplicity, as side
reactions between reactive monomers can create oligomers before these are in
turn involved
in the polymerization reaction with the material, and oligomers or even
polymers are also
useful reactive species for the present invention. As described above, monomer
side chain
brushes can be obtained, comprising multiple functional groups, i. e., three
functional groups
on a single monomeric brush.
The material and reactive monomer may be the same compound, for example, a
polyethylene material may utilize ethylene monomers or polymers in the
grafting reaction.
Reactive monomers that can be used in the present invention include, for
example, vinyl
monomers and heterocyclic monomers. Other specific examples of suitable
reactive
monomers include vinyl monomers containing a glycidyl group, e.g., glycidyl
methacrylate,
glycidyl acrylate, glycidyl methylitaconate, ethyl glycidyl maleate, and
glycidyl vinyl
sulfonate; and vinyl monomers containing a cyano group, e.g., acrylonitrile,
vinylidene
cyanide, crotononitrile, methacrylonitrile, chloroacrylonitrile, 2-cyanoethyl
methacrylate, and
2-cyanoethyl acrylate. These have epoxide groups for immobilization of
functional groups
and vinyl groups, which provide reactive polymerization sites and are thereby
useful as
reactive monomers. Ring-opening, i. e., the conversion of the epoxy groups
into diol groups
of the poly-GMA brushes is described in J. Membr. Sci. 1996, 117:33-38
(incorporated by
reference).
CA 02469301 2004-06-08
WO 03/049671 PCT/US02/33942
The reactive monomers are covalently bonded to the material through the
polymerization reaction, or are separately formed and affixed or adhered to
the material. The
reactive monomers form polymer brushes that are thereby grafted to the
material. The degree
of grafting is determined by the choice of material and reactive monomer, the
polymerization
method, and the desired length and width of the brushes. In certain cases, the
resultant
polymer brushes of the invention have bioactive properties themselves, for
example, tert-
butylaminoethyl methacrylate on a surface of an article or apparatus displays
antimicrobial
activity.
Measurement of modified or grafted materials can be determined by, for example
degree of grafting, assaying thickness or weight, water content, IR method
(FTIR-ATR, etc),
titration for ion-exchange groups, zeta-potential, Donnan method, atomic force
microscopy
(AFM), scanning electron microscopy (SEM), determination of contact angle, XPS
(X-ray
photoelectron spectroscopy), XMA (x-ray microanalysis), and SIMS (secondary
ion mass
spectrometry).
The grafting copolymerization of the reactive monomer applied to the activated
surfaces is also effected by radical induced polymerization initiated by, for
example, short
wavelength radiation in the visible range or in the long wavelength segment of
the UV range
of electromagnetic radiation. The radiation of a UV-Excimer of wavelengths 250
to 500 nm,
preferably 290 to 320 nm, for example, is particularly suitable. Mercury vapor
lamps are also
suitable here if they emit considerable amounts of radiation in the ranges
mentioned. The
exposure times generally range from 10 seconds to 30 minutes, preferably 2 to
15 minutes. A
suitable source of radiation is, for example, a UV-Excimer apparatus HERAEUS
Noblelight,
Hanau, Germany. However, mercury vapor lamps are also suitable for activation
of the
substrate if they emit considerable proportions of radiation in the ranges
mentioned. The
exposure time generally ranges from 0.1 second to 20 minutes, preferably 1
second to 10
minutes.
The activation of the reactive monomers and materials with UV radiation can
furthermore be carried out with an additional photosensitizer. Suitable such
photosensitizers
include, for example, benzophenone, as such are applied to the surface of the
substrate and
irradiated. In this context, irradiation can be conducted with a mercury vapor
lamp using
exposure times of 0.1 second to 20 minutes, preferably 1 second to 10 minutes.
According to the invention, the activation can also be achieved by a high
frequency or
microwave plasma (Hexagon, Technics Plasma, X5551 Kirchheim, Germany) in air
or a
nitrogen or argon atmosphere. The exposure times generally range from 30
seconds to 30
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WO 03/049671 PCT/US02/33942
minutes, preferably 2 to 10 minutes. The energy output of laboratory apparatus
is between
100 and 500 W, preferably between 200 and 300 W.
For example, a Corona apparatus (SOFTAL, Hamburg, Germany) can furthermore be
used
for the polymer activation. In this case, the exposure times are, as a rule, 1
to 10 minutes,
preferably 1 to 60 seconds.
The flaming of surfaces likewise leads to activation of the reactive monomers
and
materials. Suitable apparatus, in particular those having a barrier flame
front, can be
constructed in a simple manner or obtained, for example, from ARCOTEC, 71297
Monsheim, Germany. The apparatus can employ hydrocarbons or hydrogen as the
combustible gas. In all cases, harmful overheating of the materials must be
avoided, which is
easily achieved by intimate contact with a cooled metal surface on the
substrate surface
facing away from the flaming side. Activation by flaming is accordingly
limited to relatively
thin, flat materials. The exposure times generally range from 0.1 second to 1
minute,
preferably 0.5 to 2 seconds. The flames without exception are nonluminous and
the distances
between the substrate surfaces and the outer flame front ranges from 0.2 to 5
cm, preferably
O.Sto2cm.
In the case of ionizing radiation initiated polymerization, in addition to the
ultraviolet
radiation discussed above, electron beams, X-rays, alpha rays, beta rays,
gamma rays, etc.,
can be used. Graft polymerization condition changes with such variables, as
the crystalline
and amorphous structure of the material polymer, the influence of solvent or
gasses,
temperature, pH, the hydrophobicity/hydrophilicity of the material, reactive
monomers,
irradiation dose and intervals of exposure, aid the type of radicals generated
by irradiation.
One skilled in the art would recognize such variables and adjust experimental
conditions
accordingly, for example activation by electron beams or gamma-rays, from a
cobalt-60
source allow short exposure times which generally range from about 0.1 to
about 60 seconds
and employ dose ranges of about 1 to about 500 kGy. These high energy
radiation sources
are appropriate for applications where it is desirable to initiate a radical
induced
polymerization reaction on one or more intraluminal surfaces of a material.
Multiple grafting steps can also be used to create the polymer brushes.
Radicals are
generated in the material, for example a polymer material is irradiated at an
ambient
temperature under nitrogen atmosphere to create radicals for polymer grafting.
In the
currently preferred embodiment, irradiation is performed by using an electron
beam
accelerator. Graft polymerization of reactive monomers (for example, liquid
phase grafting)
is performed on the material to allow the formation of polymer brushes. As
such, grafted
22
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WO 03/049671 PCT/US02/33942
polymer #1 is obtained. The above processes are repeated to obtain grafted
polymer #2,
grafted polymer #3 and so on. Moreover, the grafting process can be stopped at
any step
depending on the desired complexity of the brush structure. Different reactive
monomers can
be used at each grafting step, providing a plurality of brush compositions for
immobilizing
numerous types of functional groups or bioactive molecules thereto. The
process can include
immobilization of functional groups followed by additional grafting reactions.
Functionalized Polymer Brushes
The present invention provides materials having grafted polymer brushes, and
methods of making the same as. While these polymer brush structures are
designed to have
specific physical properties themselves, due to, for example, their size,
brush density and
brush morphology, the invention provides that these polymer brushes may be
functionalized,
i.e., the brushes have one or more types of functional groups immobilized
thereto. Methods
of immobilizing functional groups to particular substrates are known in the
art, and axe
applicable for immobilizing the same to the present materials using the
teachings provided
herein. One or more types of functional groups can be immobilized to the
materials, i. e., one,
two, three, four, or five or more different types of functional groups may be
used, depending
on the desired functionality.
Agents for Immobilizing Functiov~al Groups to the Brushes
The polymer brush structures of the present invention include reactive groups
on the
brush surface, thus permitting the immobilization of functional groups thereto
resulting in
materials having functional or multifunctional properties. Different methods
for
immobilization of functional groups to the polymer brushes include, for
example, physical
adsorption (non-covalent bridges such as ionic and hydrogen bonds, hydrophobic
interactions
and van der Waals forces), immobilization via reactive groups,
aminopropyltriethoxysilane
, bridges, glutaraldehyde, or bis(sulfosuccinimidyl) suberate activation, or
via aldehydye
groups, phosphoramidite groups, peptide groups, binding through biotin or
avidin, protein A
or G, attachment via metal-carrying media, such as chelate-forming
.iminodiacetate groups,
copper ions, nickel ions, ferric or ferrous ions, zinc ions, magnesium ions,
manganese ions,
cobalt ions or similar charged species including complexes of the same,
covalent attachment
of oxidized groups, for example to oxidize the carbohydrate moieties in an
antibody's Fc
region with periodate to form aldehyde groups, which are then chemically bound
to
hydrazide-activated solid supports such as agarose, silica, acrylic-based
copolymers, and
cellulose. Methods for immobilization of nucleic acids include, for example,
adsorption: (i)
electrochemical adsorption: electrostatic attraction between the positively
charged solid
23
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WO 03/049671 PCT/US02/33942
support and the negatively charged oligonucleotides. (ii) hybridization
between
electrochemically adsorbed oligonucleotides and its complementary target for
sequence
specific hybridization, avidin-biotin complexation, covalent attachment: (i)
through
deoxyguaosine group using carbodiimide method (in other words, carboxylic
group (-
COOH)), (ii) amino groups (-NH2), phosphoric acid groups. Organic synthesis
(or peptide
synthesis) can be performed directly on the polymer brushes or on functional
groups
immobilized thereto (see, U.S. patent number 6,306,975, incorporated by
reference). Other
coupling chemistries are well known in the art, and by using graft
polymerization, one can
prepare solid supports having a plurality of functional groups (see, J.
Biochem. Biophys.
Methods 2001, 49:467-480, Radiat. Phys. Chem. 1987, 30:263-270, Biosens.
Bioelectron.
2000, 15:291-303, Analytica Chimica Acta 1997, 346:259-275, Chem. Rev. 2000,
100:
2091-2157, Tetrahedron 1998, 54: 15383-15443, Radiat. Phys. Chem. 1986, 27:265-
273, and
Solid-Phase Synthesis and Combinatorial Technologies by Pierfausto Seneci,
John Wiley &
Sons, Inc:, 2000, all incorporated herein by reference).
Another method of immobilizing a molecule to the brush surface includes,
without
limitation, silanes of the formula SiX3-R, wherein X is a methyl group or a
halogen atom
such as chlorine and R is a functional group which can be a coating material
as described
herein or a group which is reactive with a coating material. Particular silane-
terminated
compounds include vinyl silanes, silane-terminated acrylics, silane-terminated
polyethylene
glycols (PEGs), silane-terminated isocyanates and silane-terminated alcohols.
The silanes
can be reacted with the surface by various means known to those skilled in the
art. For
example, dichloro methyl vinyl silane can be reacted with the surface in
aqueous ethanol.
This strongly binds to the surface via --O--Si bonds or directly with the
silicon atom. The
vinyl group of the silane can then be reacted with polymeric materials as
described herein
using appropriate conventional chemistries. For example, a methacrylate-
terminated PEG
can be reacted with the vinyl group of the silane, resulting in a PEG that is
covalently bonded
to the surface of the present device.
In addition, spacer molecules may be inserted between the functional group and
the
polymer brush, as is known in the art, to facilitate binding or improve the
activity of the
functional group or bioactive molecule. The extended morphology of the brushes
can
function as spacers, or additional chemical spacers can be used. Branch-like
grafted brush
structures often provide optimal steric positioning of laxger functional
groups, or ones having
larger cognate targets.
24
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WO 03/049671 PCT/US02/33942
These functional groups impart to the compositions of the invention particular
properties. For example, the functional groups can change the effective or
active surface area
and thereby change the adsorptive capacity. In certain embodiments, they
provide for
particular brush shapes. In other embodiments they impart a particular
strength, chemical
resistance, enzymatic property, affinity for a target, or provide other
effective functionality to
the materials, such as the ability to catalyze a reaction or deliver a
compound to cells.
Functional groups that are appropriate for immobilization by the brushes in
the
compositions of the present invention include, for example, ion exchange
functional groups,
i.e., anionically dissociating groups and cationically dissociating groups,
hydrophilic
functional groups, affinity functional groups, enzymatic functional groups,
and other such
functional groups that have the ability to adsorb and/or immobilize other
molecules, or cause
a catalytic reaction, alone or in combination with mechanical or physical
properties of the
polymer brush structures, i. e., adjusting the density and morphology of the
brushes to
optimize the surface area of a charged group, or to increase the activity of
catalytic sites, or
sterically optimize the binding sites of the functional groups immobilized
thereto, thereby
optimizing their association with a particular ligand or target.
One or more kinds of anionically dissociating substances can be immobilized by
the
polymer brushes. Examples of suitable anionically dissociating groups include
quaternary
ammonium salts and primary, secondary, and tertiary amino or amido groups.
Specific
examples include an amino group, a methylamino group, a dirnethylamino group,
and a
diethylamino group. Preferred anionically dissociating groups include the
amino group and
quaternary ammonium salts. Reactive monomers that have such anionically
dissociating
groups and that are useful in the present invention include, for example,
vinylbenzyltrimethyl
ammonium salt, diethylaminoethyl methacrylate, dimethylaminoethyl acrylate,
dimethylaminoethyl methacrylate, diethylaminoethyl acrylate,
diethylaminomethyl
methacrylate, tertiary-butylaminoethyl acrylate, tertiary-butylaminoethyl
methacrylate and
dimethylaminopropylacrylamide. Also useful in the present invention are
reactive monomers
that have epoxide groups capable of conversion to anionically dissociating
groups. An
example of such a reactive monomer is glycidyl methacrylate. An example of an
amine
capable of converting the epoxide group to an anionically dissociating group
is diethylamine.
One or more kinds of cationically dissociating groups can be immobilized by
the
polymer brushes. Examples of such cationically dissociating groups include,
for example, a
carboxyl group, a sulfone group, a phosphate group, a sulfoethyl group, a
phosphomethyl
group, a carbomethyl group. Preferred cationically dissociating groups include
a sulfone
CA 02469301 2004-06-08
WO 03/049671 PCT/US02/33942
group and a carboxyl group. Reactive monomers that have such cationically
dissociating
groups and that are useful include, for example, acrylic acid, methacrylic
acid,
styrenesulfonic acid and salts thereof, and 2-acrylamido-2-
methylpropanesulfoni.c acid.
One or more kinds of hydrophilic substances can be immobilized by the polymer
brushes. Such hydrophilic groups are capable of trapping the water molecules
present in air,
forming a layer of adsorbed water on the surface of the membrane of the
present invention.
Such hydrophilic groups will function in water in the same manner as in air.
Examples of
suitable hydrophilic groups include, for example, a hydroxyl group, a
hydroxyalkyl group
(where the alkyl group is preferably a lower alkyl group), an amino group and
a pyrrolidonyl
group. Preferred hydrophilic groups include a hydroxyl group, a hydroxyalkyl
group and a
pyrrolidonyl group. One or more kinds of hydrophilic groups can be immobilized
onto the
polymer brush. Reactive monomers that have such hydrophilic groups and that
are useful in
the present invention include, for example, ethanolamine, hydroxyethyl
methacrylate,
hydroxypropyl acrylate, vinylpyrrolidone, dimethylacrylamide, ethylene glycol
monomethacrylate, ethylene glycol monoacrylate, ethylene glycol
dimethacrylate, ethylene
glycol diacrylate, triethylene glycol diacrylate and triethylene glycol
methacrylate. Thus a
polymer brush may itself comprise a functional group, or one may be
immobilized to the
brush.
One or more kinds of functional groups can be immobilized on the polymer
brushes.
Such groups can be combined or immobilized in discrete multi-layers to impart
an additional
degree of functionality to the composition. For example, the present invention
provides
materials having enzymatic activity such as the ability to phosphorylate or
dephosphorylate a
target polypeptide substrate, the ability to digest, i. e., a nucleic acid at
a restriction site, or
hydrolyze a polypeptide, the ability to radiolabel a polynucleotide or
polypeptide, i. e., using
llas~ hay Psa~ Sss~ Crsi ~d other radionuclides, or the ability to catalyze a
biological or
chemical reaction. Examples of enzyme functional groups that can be bound to
or isolated
using the polymer brushes, and potential uses for those enzymes, include, but
are not limited
to ascorbic acid oxidase (e.g., for avoidance of interference of ascorbic acid
on diagnostic
assays of blood, urine, or other samples), aspartase (e.g., for conversion of
fiunaric acid to L-
aspartic acid), aminoacylase (e.g., for conversion of acetyl-D,L-amino acids
to L-amino
acids), tyrosinase (e.g., for synthesis of tyrosine from phenol, pyruvate and
ammonia), lipase
(e.g., for hydrolysis of a cyano-ester to ibuprofen or hydrolysis of a
diltiazem precursor),
penicillin amidase (e.g., for production of ampicillin and amoxycillin),
hydantoinase and
carbamylase (e.g., for hydrolysis of 5-p-HP-hydantoine to d-p-HP-glycine),
DNase (e.g., for
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CA 02469301 2004-06-08
WO 03/049671 PCT/US02/33942
hydrolysis of DNA to oligonucleotides), bovine liver catalase (e.g., for
hydrolysis of
hydrogen peroxide), trypsin and chymotrypsin (e.g., for hydrolysis of whey
proteins),
arginase and aspaxaginase (e.g., for hydrolysis of arginine and asparagine),
proteases (e.g., to
remove organic stains from fabrics), lipases (e.g., to remove greasy stains
from fabrics),
amylase (e.g., to remove residues of starchy foods from fabrics), cellulase
(e.g., to restore a
smooth surface to fibers of fabrics and restore fabrics to their original
colors), proteases and
lipases (e.g., to intensify flavor and accelerate the aging process of foods),
lactase (e.g., to
produce low-lactose milk and related products for special dietary
requirements), beta-
glucanase (e.g., to help the clarification process of wines), cellulase (e.g.,
to aid the
breakdown of cell walls in winemaking), pectinase (e.g., to improve fruit
juice extraction and
reduce juice viscosity), cellulase (e.g., to improve juice yield and color of
fruit juice), lipase
(e.g., for hydrolysis of fats and oils or the production of fatty acids,
glycerine, fatty acids
(e.g., used to produce pharmaceuticals, flavors, fragrances and cosmetics),
alpha-amylase
(e.g., for liquefaction of starch or fragmentation of gelatinized starch),
aminoglucosidase
(e.g., for saccharification or complete degradation of starch and dextrins
into glucose), alpha-
amylase (e.g., for conversion of starch to fructose), glucoamylase and
pullulanase (e.g., for
saccharification), glucose isomerase (e.g., for isomerization of glucose),
beta-glucanase (e.g.,
for reduction of beta-glucans), beta-glucanase (e.g., for reduction of beta-
glucans and
pentosans), lipase, amidase and nitrilase (e.g., for manufacture of
enantiomeric intermediates
for drugs and agrochemicals), lipase (e.g., to remove fats in the de-greasing
process in the
leather industry), amylase and cellulase (e.g., to produce fibers from less
valuable raw
materials in the textiles industry), xylanase (e.g., as a bleaching catalyst
during pretreatment
for the manufacture of bleached pulp for paper), beta-galactosidase (e.g., for
hydrolysis of
lactose to glucose), trypsin and chymotrypsin (e.g., for hydrolysis of high-
molecular-weight
protein in milk), alpha-galactosidase and invertase (e.g., for hydrolysis of
raffinose), alpha-
amylase, beta-amylase, and pullulanase (e.g., for hydrolysis of starch to
maltose), pectinase
(e.g., for hydrolysis of pectins), endopeptidase (e.g., for hydrolysis of k-
casein), protease and
papain (e.g., for hydrolysis of collagen and muscle proteins), glucose oxidase
and catalase
(e.g., for conversion of glucose to gluconic acid), lipase (e.g., for
hydrolysis of triglycerides
to fatty acids and glycerol, hydrolysis of olive oil triglycerides, hydrolysis
of soybean oil,
butter oil glycerides and milk fat), cellulase and beta-glucosidase (e.g., for
hydrolysis of
cellulose to cellobiose and glucose), and fumarase (e.g., for hydrolysis of
fiunaric acid to 1-
malic acid). Alternatively, microorganisms or fragments thereof can be
functional groups,
for example, such as Pseudomonas dacunhae (e.g., for conversion of L-aspartic
acid to L-
27
CA 02469301 2004-06-08
WO 03/049671 PCT/US02/33942
alanine), Curvularia lunata/Candida simplex (e.g., for conversion of
cortexolone to
hydrocortisone and prednisolone), or Saccharomyces and other yeasts (e.g., for
fermentation
of sugars and anaerobic fermentation); all can be immobilized on the polymer
brushes.
The functional groups can include all hydrophilic groups, anionically
dissociating
groups and/or cationically dissociating groups, and enzymes. Stated more
specifically, the
polymer brush can include multiple functional groups (e.g., anionically
dissociating groups
and hydrophilic groups, or alternatively cationically dissociating groups and
hydrophilic
groups) or three kinds of functional groups (e.g., hydrophilic groups,
anionically dissociating
groups, and cationically dissociating groups), or more (e.g., hydrophilic
groups, anionically
dissociating groups, cationically dissociating groups, enzymes, SpA and one or
more
immunoglobulins). Combinations of functional groups that are appropriate in
the present
invention include, for example, an ionic group and a non-ionic group, i. e.,
an amine group
with a coexisting hydrophilic group. A preferred embodiment additionally
comprises a
second functional group in combination with the first functional groups
described above. In a
currently preferred embodiment, the first, second, third, and fourth
functional groups are
immobilized on the polymer brushes in multilayers. Thus, one of the major
features of the
present invention is that different kinds of molecules having hydrophilic
domains (non-ions)
present in a sample solution with molecules having ionic domains (anions
and/or cations), or
molecules having a phosphorylation state, or a binding site or nucleotide or
polypeptide
sequence can be recovered, purified, concentrated and isolated, modified,
synthesized, or
otherwise utilized with the compositions of the invention. The functional
group may be
altered to change the binding of a substrate bioactive molecule, to thereby
tailor the
dissociation rate ih vivo, and provide controlled release of the substrate
bioactive molecule
bound thereto. Such alteration or chemical modification may be effectuated on
the
compositions of the present invention, or the modifications may be effectuated
before
immobilization to the polymer brush surface.
The functional groups can include antibodies or domains or fragments thereof.
Hydroxysuccinimide esters, for example, provide one method for immobilizing
one or more
antibodies to the polymer brushes via lysine residues, thereby producing
functionalized
materials with affinities for antigenic targets. The carbohydrate moieties,
described above,
provide yet another source for immobilization to the polymer brushes or to
fractional groups.
The basic antibody structural unit is known to comprise a tetramer. Each
tetramer is
composed of two identical pairs of polypeptide chains, each pair having one
"light" (about 25
kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of
each chain
28
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WO 03/049671 PCT/US02/33942
includes a variable region of about 100 to 110 or more amino acids primarily
responsible for
antigen recognition. The carboxy-terminal portion of each chain defines a
constant region
primarily responsible for effector function. Human light chains are classified
as kappa and
lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha,
or epsilon, and
define the antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Within
light and
heavy chains, the variable and constant regions are joined by a "J" region of
about 12 or more
amino acids, with the heavy chain also including a "D" region of about 10 more
amino acids.
See generally, Fu~cdamehtal Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven
Press, N.Y.
(1989)) (incorporated by reference in its entirety for all purposes). The
variable regions of
each light/heavy chain pair form the antibody binding site. Thus, an intact
antibody has two
binding sites. Except in bifunctional or bispecific antibodies, the two
binding sites are the
same. The chains all exhibit the same general structure of relatively
conserved framework
regions (FR) joined by three hyper variable regions, also called
complementarity determining
regions or CDRs. The CDRs from the two chains of each pair are aligned by the
framework
regions, enabling binding to a specific epitope. From N-terminal to C-
terminal, both light
and heavy chains comprise the domains FRl, CDRl, FR2, CDR2, FR3, CDR3 and FR4.
The
assignment of amino acids to each domain is in accordance with the definitions
of Rabat
Sequences of Proteins of Immu~ological Interest (National Institutes of
Health, Bethesda,
Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987);
Chothia et al.
Nature 342:878-883 (1989). All such domains or fragments or sequences
therefrom may be
immobilized on polymer brushes by the methods described herein. In one aspect,
immobilization of an immunoglobulin to the polymer brushes through the CH2 or
CH3
domains is preferred. Without being restricted to theory, it is believed such
immobilization
decreases steric hindrance and increases the surface area available for
antigen binding.
A bispecific or bifunctional antibody is an artificial hybrid antibody having
two
different heavy/light chain pairs and two different binding sites. These
provide for increased
functionality of the materials, as two targets can be bound by one
immunoglobulin molecule,
useful for example, where it is desirable to crosslink two targets, such as in
an enzymatic
reaction. Bispecific antibodies can be produced by a variety of methods
including fusion of
hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann
Clin. Exp.
Immu~ol. 79: 315-321 (1990), I~ostelny et al. J. Immunol. 148:1547-1553
(1992).
Production of bispecific antibodies can be a relatively labor intensive
process compared with
production of conventional antibodies and yields and degree of purity are
generally lower for
bispecific antibodies. Bispecific antibodies do not exist in the form of
fragments having a
29
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WO 03/049671 PCT/US02/33942
single binding site (e.g., Fab, Fab', and Fv) but a bispecific antibody can be
immobilized as
described, and provides an additional functional property for the polymer
brushes, i.e., an
additional specificity for a ligand. Multiple isotypes, species, and epitope
recognition
properties can be imported to the polymer brushes by the methods described
herein.
Humanized, or chimeric antibodies are also appropriate immunoglobulins for
developing functionalized materials according to the present invention. Such
approaches for
generating these are further discussed and delineated in U.S. Patent
Application Serial Nos.
07/466,008, filed January 12, 1990, 07/610,515, filed November 8, 1990,
07/919,297, filed
July 24, 1992, 07/922,649, filed July 30, 1992, filed 08/031,801, filed March
15,1993,
08/112,848, filed August 27, 1993, 08/234,145, filed April 28, 1994,
08/376,279, filed
January 20, 1995, 08/430, 938, April 27, 1995, 08/464,584, filed June 5, 1995,
08/464,582,
filed June 5, 1995, 08/463,191, filed June 5, 1995, 08/462,837, filed June 5,
1995,
08/486,853, filed June 5, 1995, 08/486,857, filed June 5, 1995, 08/486,859,
filed June 5,
1995, 08/462,513, filed June 5, 1995, 08/724,752, filed October 2, 1996, and
081759,620,
filed December 3, 1996 and U.S. Patent Nos. 6,162,963, 6,150,584, 6,114,598,
6,075,181,
and 5,939,598 and Japanese Patent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068
507 B2.
See also Mendez et al. Nature Genetics 15:146-156 (1997) and Green and
Jakobovits J.
Exp. Med. 188:483-495 (1998). See also European Patent No., EP 0 463 151 Bl,
grant
published June 12, 1996, International Patent Application No., WO 94/02602,
published
February 3, 1994, International Patent Application No., WO 96/34096, published
October 31,
1996, WO 98/24893, published June 11, 1998, WO 00/76310, published December
21, 2000.
The disclosures of each of the above-cited patents, applications, and
references are hereby
incorporated by reference in their entirety. Humanized, or chimeric antibodies
or domains or
fragments thereof can be immobilized to the polymer brushes as described.
Liposomes, microsponges and microspheres act as functional microdelivery
groups,
and may be immobilized to the materials described herein. These are useful for
delivery of a
compound contained within the microdelivery device to a target cell, where the
cell is
contacted with the functionalized materials having the liposomes, microsponges
and
microspheres containing the compound, immobilized thereto. Liposomes are lipid
molecules
formed into a typically spherically shaped arrangement defining aqueous and
membranal
inner compartments. Liposomes can be used to encapsulate compounds within the
inner
compartments, and deliver such compounds to desired sites within a cell. The
compounds
contained by the liposome may be released by the liposome and incorporated
into a cell, as
for example, by virtue of the similarity of the liposome to the lipid bilayer
that makes up the
CA 02469301 2004-06-08
WO 03/049671 PCT/US02/33942
cell membrane. A variety of suitable liposomes may be used, including those
available from
NeXstar Pharmaceuticals or Liposome, Inc. Liposomes may be immobilized to the
polymer
brushes by several methods, for example through interactions with the
hydrophobic polymer
brushes, or by a functional group, for example, a fatty acid functional group.
Uses include
transfection of cell targets introduced to the functionalized materials having
immobilized
liposomes with nucleic acids contained within the liposomes.
Microsponges are high surface area polymeric spheres having a network of
cavities
which may contain compounds. The microsponges are typically synthesized by
aqueous
suspension polymerization using vinyl and acrylic monomers. The monomers may
be mono
or difunctional, so that the polymerized spheres may be cross-linked, thus
providing shape
stability. Process conditions and monomer selection can be varied to tailor
properties such as
pore volume and solvent swellability, and the microsponges may be synthesized
in a
controlled range of mean diameters, including small diameters of about 2
micrometers or
less. A standard bead composition would be a copolymer of styrene and di-vinyl
benzene
(DVB). The compounds contained by the polymeric microsponges may be gradually
released therefrom due to mechanical or thermal stress or sonication. A
variety of suitable
microsponges may be used, if functionalized as by the procedures described
herein, including
those commercially available from Advanced Polymer Systems. Since these are
themselves
polymers, they can be grafted to the polymer brushes or otherwise immobilized
by standard
chemical techniques known in the art in view of the teachings described
herein. Uses of such
functionalized materials having microsponges include introduction of bioactive
molecules or
compounds contained within the microsponges into cell targets introduced to
the
functionalized materials.
Bipolar Functionalized Materials
The techniques disclosed herein are applicable for the development of
materials that
have bipolar properties. These bipolar functionalized materials include
materials having, for
example, charged functional groups on one region or surface and oppositely
charged
functional groups on another region or surface. Example One describes the
preparation and
use of a bipolar membrane, but other embodiments are possible, and the
morphology of the
devices according to the invention is not limited to preparation of a
functionalized membrane.
Other materials may be utilized, and selective grafting and immobilization of
oppositely
charged functional groups and including other functional groups of the present
invention can
be developed by stepwise grafting and masking techniques.
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The bipolar membrane of Example One is substantially planar, having cation-
exchange functional groups immobilized on one surface and anion-exchange
functional
groups immobilized on the opposing surface. The characteristic of this bipolar
membrane is
that when a voltage is applied, both functional groups at the center phase of
the membrane
will dissociate water into H+ and OH-. Without being restricted to theory,
there are two
assumptions that may explain the mechanism of water dissociation. First, the
voltage
generated in the center phase of the membrane electrically dissociates water
into H+ and OH-
according to the second Wien effect. Second, the reaction between immobilized
charged
functional groups with water molecule leads to the dissociation of water into
H+ and OH-
according to a chemical reaction (see, R. Simons, J. Membr. Sci., 78, 13
(1993) and H.
Strathmann et al., J. Membr. Sci., 125, 123 (1997) each incorporated herein by
reference).
Applications for using such a bipolar functionalized material are numerous.
For
example, a substrate including graft polymerized brushes with affinity
purification functional
groups and charged functional groups can be used for high-throughput isolation
and
subsequent isoelectric separation of polypeptides. In another aspect, a
substrate is used for
the production of compounds for the pharmaceutical and biotechnology
industries, such as
salicylic acid. Other applications include but are not limited to,
electrodialysis, recycling of
salts from waste treatment, production of acid and alkali from concentrated
seawater.
Methods of producing bipolar functionalized materials include pasting, casting
and
plasma graft polymerization methods (see, Iwamoto et al., Nihon Kagaku Kashi,
No.6, 425
(1997) in Japanese, G. S. Trivedi et al., React. Funct. Polym., 28, 243
(I996), Y.
Yokoyama et al., J. Membr. Sci., 43, 165 (1989) each incorporated herein by
reference).
Each have their advantages as would be known to one of skill in the art. The
present device
illustrated at Example One demonstrates electrolytic performance comparable to
casted
electroseparation devices, but is believed to be easier and more cost
effective to manufacture
and operate.
Immobilization of Cells and Microbes to the Functionalized Materials
The materials of the present invention are capable of interaction with a wide
range of
biological and chemical targets, including nucleic acids, peptides, organic
and inorganic
molecules and compounds, enzymes and mufti-subunit polypeptides and the like.
In one
aspect, the functionalized materials interact with these targets a and
catalyze a reaction. In
another aspect, the materials bind the targets. In addition to these small
compounds, the
functionalized materials are useful for with larger target structures such as
polypeptide
complexes like mufti-subunit enzymes, organelles, membrane receptors and
membranes. In
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another aspect, the materials and devices of the present invention are capable
of interacting
with targets including intact cells, for example but not limited to, human and
animal cells,
bacteria, viruses, and fungi. These targets can, in turn, be immobilized to
the polymer
brushes for use as subsequent functional groups, thereby providing additional
levels of
functionality to the materials.
Immobilization of larger structures including multi-subunit polypeptides,
organelles,
cells and microorganisms can be effectuated by many of the numerous types of
functional
groups disclosed herein. Charged functional groups, for example, tertiary
amino functional
groups, can be used as highly effective filters to capture viruses and virus
particles from
liquids with minimal removal of proteins (see, U. S. Patent Application
No.:20010034055 to
Lee et al., incorporated herein by reference). Example Two describes the use
of quaternary
amino functional groups for the immobilization of Staphylococcus aureus
bacteria from
liquid culture preparations. This bacteria can, in turn, be used to immobilize
immunoglobulin
molecules through its surface Protein A, or used for other purposes.
Applications for
isolation of bacteria from solutions are numerous, and include purification
techniques.
Immobilization of a bacterium on a device of the present invention also
includes applications
in the field of cloning and expression of recombinant genes and polypeptides.
For example,
device comprising a membrane capable of immobilizing bacteria and nucleic
acids further
comprises bipolar functional groups, and can be used for single step
electroporation and
transformation of the bacteria with the nucleic acids. Another application is
the
immobilization and transfection of mammalian cells using, for example
transfections to
create cell lines, hybridomas, and transgenic organisms, where the device
comprises polymer
brushes having functional groups for the immobilization of cells and nucleic
acids, and
chemical transfection methods are used to cause uptake of the polynucleotides
into the cells.
Many charged molecules on the cellular surfaces axe targets for immobilization
via
charged functional groups. Acidic sugars including those incorporated into
many
polysaccharides, glycoproteins and glycolipids are potential targets for anion
exchange
functional groups, such as but not limited to, N-acetylneuraminic acid (sialic
acid) or N-
acetylmuramic acid. Sialic acid, for example, is found on many human cells
including
erythrocytes, and can be targeted for immobilizing the same on the materials
of the present
invention. Cation exchange functional groups are useful in the isolation of
cells or
microorganisms having surface polysaccharide amino sugars, such as but not
limited to, N-
acetylglucosamine, N-acetylgalactosamine, galactosamine, and mannosamine.
Other
compounds such as peptidoglycans, glycosaminoglycans (e.g., chondroitin,
keratin, and
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hyaluronic acid among others), and proteoglycans all display negative charges
and can serve
as targets for anion exchange functional groups, permitting the binding of
cells to the
materials of the present invention. Further targets for immobilization of
bacteria include
lipopolysaccharide (LPS), for example O antigens and H antigens a major
component of the
membrane of gram negative pathogenic bacteria like E. coli and Salmonella
typhinaurium.
Other pathogenic bacteria, such as Staphylococcus, Streptomyces, Clostf~idium,
Ye~sinia, and
Bacillus can be similarly isolated by the materials of the present invention
and evaluated for
pathogenicity as described.
Immobilization of cells and microbes need not be based solely on the binding
of
charged targets to charged functional groups on the polymer brushes. For
example,
polysaccharides have numerous hydroxyl groups which may serve as targets based
on their
ability to form hydrogen bonds. In addition, lipids and fatty acids are the
major components
of biological membranes, and can be immobilized by non-polar functional
groups, as well as
functional groups comprising lipid anchors, e.g., glycophosphoinosital,
phosphatidylserine,
sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine, and
phosphatidylinosital, among others. In addition, specific surface receptors,
integral
membrane proteins, peripheral proteins, annexins, and ion channels can provide
targets for
affinity purification functional groups that can immobilize cells. In a
preferred aspect, these
affinity purification functional groups include immunoglobulins or fragments
thereof, capable
of specific binding to targets on the cells. For example, hemagglutinin (HA)
and
neuraminidase on orthomyxovirus or paramyxovirus virions are targets for anti-
HA and anti-
neuraminidase antibodies, VP1 is a target for immobilization of
picornanviruses, and gp120
is a target for the immobilization of HIV virions using an anti-gp120
antibody. Enveloped
viruses in particular, are amenable to the targeted separation based on the
techniques
disclosed herein. In particular, polypeptides in the lipoprotein membrane
surrounding
poxvirus virions, e.g, variola and vaccinia, provide targets for
immobilization of virions by
affinity based methods, and are also markers for identification and phenotypic
determination.
A comprehensive listing of surface antigens for bacteria, for viruses, and for
fungi can be
found respectively in e.g., Bergy's Manual of Systematic Bacteriology, Field's
hirology, and
Atlas ~f Cliv~ical Fuhgi G. S. De Hoog, et al, each incorporated by reference
in their entirety.
Other surface antigens on mammalian and in particular human cells, are well
know in the
medical and biotechnology arts (see, Altered Glycosylation in Tumor Cells
(UCLA Symposia
on Molecular an Cellular Biology, Vol 79) Christopher L. Reading (Editor),
Immune
Complexes and Human Cancer (Contemporary Topics in Immunobiology, Vol 15)
Fernando
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A. Salinas, Michael G., Jr. Hanna (Editor), Tumor Markers: Biology and
Clinical
Applications (Cancer Research Monographs, Vol 4) Nasser Javadpour (Editor),
and the
American Type Culture Collection catalogue found online at
http://www.atcc.org/, each
incorporated herein by reference. These surface antigens are useful for
targets for
immobilization according to the present invention, i.e., based on the
affinities of antibodies to
the surface antigen, or based on weak covalent interactions, or other physical
properties. The
selection of particular functional groups for immobilization of cells is thus
believed to be
routine for one of ordinary skill in the art in view of the targets expressed
on the particular
cell or organism desired, in view of the teachings provided herein. For
example, Bacillus
anthraces spore biomarkers can be used as targets to isolate the bacillus from
a sample
mixture. Samples are obtained from water supplies or from clothing, or tissues
of organisms.
The sample is introduced to the materials, which have functional groups with
affinity for the
spore biomarkers, and the isolated organisms are evaluated for pathogenicity,
for example, by
determination of expression levels of the anthrax toxin components PA
(protective antigen),
LF (lethal factor) and EF (edema factor), from isolated organisms, see,
Elhanany E., et al.,
Rapid Commun Mass Spectrom 2001;15(22):2110-6, incorporated by reference,
which
details isolation of the organism and detection by matrix-assisted laser
desorption/ionization
time-of flight mass spectrometry (MALDI-TOFMS). In this aspect, B. anthraces
spores are
immobilized to the materials, which further include sinapinic acid or alpha-
cyano-4-
hydroxycinnamic acid immobilized thereto as the matrix, followed by linear
mode analysis of
the materials.
Polypeptide or oligosaccharide markers are cellular targets and functional
groups
having affinity thereto are used to immobilize cells to the materials. In one
aspect, the
materials are used to isolate stem cells based on the presence of surface
antigenic
determinants, indicating cells committed to a particular lineage, or
differentiated immature
cells e.g., hematapoetic stem cells from cord blood or bone marrow having one
or more of the
markers CD10+, CD19+, CD34, +PAXS, E2A, EBF, ATM, PDGFRA, SIAH1, PIM2,
C/EBPB, WNT16, and TCLl, each expressed at intervals during B-cell maturation,
see,
Muschen et al., Proc Natl Acad Sci LT S A 2002 Jul 23;99(15):10014-9,
incorporated by
reference. These are useful for the creation of cell lines, for characterizing
cellular
development, and such other applications requiring isolated cells. In another
aspect, the
materials have an affinity for a target such as a marker for a disease state.
For example,
transcription of CBF 1 responsive genes lead to the overexpression of CD23, a
marker of B-
cell chronic lymphocytic leukemia. In this embodiment, the isolated cells are
used in
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subsequent diagnosis or to monitor for therapeutic endpoints in the treatment
of a disease
characterized by aberrant expression levels of the markers. For example, the
cells are
immobilized and the presence or absence of the markers is determined and
correlated to the
presence or absence of a disease state. Many such markers for disease states
are known in
the medical arts. In a preferred aspect, human cells are immobilized to the
materials of the
present invention by appropriate functional groups having affinity for such
marker taxgets.
Using such isolated cells in routine assays, the phenotypes and genotypes of
these isolated
cells can be determined and correlated with information about disease states.
The materials of the present invention are inert to a wide variety of reagents
and
conditions. For example, polypropylene and polyethylene are resistant to acids
and alkalis, as
well as numerous solvents and denaturants, such as urea, chloroform,
formaldehyde and
dimethylsulfoxide (DMSO). Advantages of the present materials include
applications where
carbohydrate, lipid or protein fractions are extracted from the immobilized
cells. In addition,
the materials can withstand temperature extremes of heating and in particular,
freezing
temperatures. Thus, the materials are particularly advantageous where it is
desirable to
immobilize cells and subsequently freeze them, without necessitating an
elution step. For
example, E. coli cells are immobilized and treated with media including 10%
DMSO, then
stored at -80 degrees C, directly on the materials in the appropriate
cryogenic nutrient
solution. Without being restricted to theory, it is believed that viability of
the immobilized
cell is enhanced as the polymer brushes immobilize the cell in multiple
dimensions over its
surface, thereby without substantially deforming the cellular membranes.
Functionalized Material Libraries
Current graft polymerized materials have been produced using a directed
process
whereby each material is custom-made and evaluated for a specific
biotechnology
application. Although successful, this process is laborious and time
consuming, and thus
limited in its ability to produce new products. To accelerate the discovery
process, a library
of functionalized materials is needed, having varying properties, for example
a plurality of
polymer brush domains, each provided as small surfaces (e.g., about 1
micrometer2 to about 2
cm2) that vary over a wide range of physical and functional properties in both
the (x) and (y)
dimensions. (see, FIG. 20). A "library" or "material library" as used herein,
refers to a
substrate including one or more surfaces having a plurality of functionalized
materials
presented thereon. A "domain" as used herein, refers.to an independent region
of a particular
functionalized material. The same functionalized material may be used at more
than one
domain, thereby providing redundancy in the library. A domain may be in direct
proximity
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to another domain, and may contact it, or the domains may not be in physical
contact but may
be in fluid communication, or the domains may be isolated. An "address" or
"domain
address" as used herein, refers to the region or location of a domain within
the library, where
the location can be described by e.g., Cartesian coordinates or other means of
providing a
spatial description. Thus, a library comprises a plurality of domains, each
having an address.
Similar in concept to the libraries produced by combinatorial chemistry
companies for
identifying promising drug candidates, the library can be rapidly produced and
rapidly
screened for optimizing the development of new materials or optimization of
existing ones.
Among the parameters to be varied include the chemical composition of base
polymers, type
of monomers used for forming brushes, brush densities, brash lengths, and type
and degree of
chemical functionalization of polymer brushes. To illustrate the potential
complexity of the
library, there are well over 1000 different reactive monomers available off
the-shelf that are
suitable for forming polymer brush structures using the grafting techniques
disclosed herein.
Moreover, these monomers can be used in various blends, and can be
functionalized using
different immobilization chemistries and functional groups to generate an
enormous library
of unique materials spanning a wide range of functionalities. Furthermore, the
process of
producing and screening material libraries according to the present invention
is suitable for
miniaturization and automation.
These libraries may further be housed in a microfluidic device that will
facilitate the
rapid screening of the library using high-throughput methods to identify
domains of candidate
materials that interact with target molecules (FIG. 21 ). For example, a
membrane for
purification of a therapeutic protein is desired. Candidate materials are
first selected based
on high-throughput screens of a Library, for domains of functionalized
materials that bind this
specific target protein. This screen will identify the optimal composition of
material (i.e.,
material, brush composition, brush length/density and functional group
density), based upon
binding of the target to one or more domains in the library. In addition, this
screen will also
determine the appropriate environmental conditions (e.g., salt concentration,
temperature,
cofactor requirements, etc.) needed for the optimal binding and elution of the
target protein.
Thus, by performing a high-throughput screen, the library not only identifies
a unique
material that binds a commercially important protein or biological target, it
has also identified
the conditions for the optimal use and performance of this material.
To create the library, a plurality of domains of functionalized materials,
each varying
from the others in terms of its physical or functional properties, is
presented or formed on at
least one surface of a substrate. One or more of the materials disclosed
herein are suitable for
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use as a substrate, for example, an organic thin film coating on a glass
surface provides an
appropriate substrate, to which polymer brushes can be grafted and
subsequently
functionalized. Alternatively, functionalized materials may be prepared and
later affixed to
the substrate.
The ftmctionalized materials or polymer brushes that are grafted or otherwise
affixed
to the substrate are presented as independent domains on a surface of the
substrate. Through
the techniques described herein, brush densities as well as brush length and
morphology can
be varied. Further, standard masking techniques can be applied, allowing the
grafting process
to be controlled with respect to selected domains. A material library of the
present
invention includes at least two domains, and more preferably 10, 50, 100,
1000, or 10,000
domains. In a currently' preferred embodiment, the array comprises about 105
domains. Each
domain covers a mean surface area of the substrate less than about 0.25 mma.
Preferably, the
area of the substrate surface covered by each of the domains is between about
1 ~,m2 to about
10,000 ~.ma. In a particularly preferred embodiment, each domain covers an
area of the
substrate surface from about 100 ~.ma to about 2,500 ~,m2. The domains of the
array may be
of any geometric shape. For instance, the domains may be square, rectangular
or circular.
The domains of the array may also be irregularly shaped. The domains are
optionally
elevated from the median plane of the underlying substrate. The distance
separating the
domains of the array can vary. Preferably, the domains of the array are
separated from
neighboring domains by about 1 ~ma to about 5000 ~,ma. Typically, the distance
separating
the domains is roughly proportional to the diameter or side length of the
domains on the array
if the domains have dimensions greater than about 10 ~,m2. If the domain size
is smaller, then
the distance separating the domains will typically be larger than the
dimensions of the
domain. Each domain has an independent location or domain address, on the
substrate. In a
currently preferred embodiment, the domains are presented in a two-dimensional
matrix
format and the address of each domain is described by a set of Cartesian
coordinates having
the values (x) and (y). Each domain includes one or more functional groups
immobilized on
the polymer brushes. Functional groups are preferably covalently immobilized
on the
polymer brushes, either directly or indirectly, at concentrations varying from
about 0.001
fg/mma material surface area to about 100 g/mm2 material surface area. The
number of
different functional groups immobilized at a domain address will vary
depending on the
application desired. It is possible to create multifunctional domains by using
different
functional groups, for example, renaturation of a polypeptide and binding to a
particular
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functional group that recognizes the native polypeptide may be accomplished by
incorporating into a particular domain, groups capable of hydrolyzing urea in
addition to
groups that bind the folded polypeptide. Additionally, a series of domain
locations with
different urease activity levels may be created, and the degree of
renaturation required for
polypeptide binding may be determined by detecting protein binding at each
domain address.
Similar arrangements of functionalized materials can evaluate a target's
ability to bind or
perform a biological function in terms of in other variables, for example but
not limited to
over a range of pH values or over a range of salt concentrations, or over a
range of
concentrations of cofactors such as metal ions, phosphates, or antioxidants,
or over a
concentration range of antagonists or agonists. For example, an antibody to
HIV protein
gp120 is used as a universal functional group and can capture the virus,
immobilizing it to the
polymer brushes at each domain. Two candidate binding site antagonists axe
introduced to
the library, varying the concentrations at each domain. Fluorescently labeled
CD4 is
introduced to the library. Quantitation of CD4 binding to the virus with
respect to the
combinations of receptor antagonists is evaluated by detection of fluoresence
at each domain
address. This is related to the degree of antagonist activity, which in turn,
is a function of the
antagonist concentrations, known for each domain address. Therefore, the
optimal antagonist
concentrations are determined.
Many combinations of functionalized materials are possible to create a
material
library. For example, in a single material library each of the domains may be
a distinct
functionalized material in that each may comprise different functional groups,
i. e., a material
library having 100 domains could comprise 100 different functionalized
materials each with a
unique functional group. Likewise, a material library of 10,000 different
functionalized
materials could comprise 10,000 domains, each having three distinct functional
groups not
used at any other domain address, leading to a library with an overall
complexity of 30,000
different functional groups. In contrast, in a single material library each of
the domains may
be a distinct functionalized material but may share in part, a common
functionality with other
domains in that each domain may comprise combinations of similar and different
functional
groups, i.e., a material library having 100 domains could comprise 100
different
functionalized materials, each having a unique first functional group, but
half of the domains
may share a common second functional group and the remaining half may share a
common
third functional group. In alternative embodiments, a plurality of domains
have common
functional groups, i. e., a library has three-thousand domains, but only
comprises one
thousand different functionalized materials, as each different functional
group is immobilized
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at three common domains. In a currently preferred embodiment, a plurality of
domains have
common functional groups but each of these domains is still a distinct
functionalized material
as each varies from the others in terms of polymer brush density, brush length
and brush
morphology.
The type of functionalized materials and number of domains used to create a
material
library will depend on the library's prospective use. For instance, if the
library is to be used
as a diagnostic tool in evaluating the status of a tumor or other diseased
tissue in a patient, a
library may comprise about 10 different functionalized materials, 5 domains
having
functional groups with specificity for the target marker proteins whose
expression is known
to be indicative of the disease condition and 5 domains having functional
groups with
specificity for target marker proteins whose expression is known to exclude
the disease
condition. However, if the library is to be used to measure a significant
portion of the soluble
protein content of a cell, then the library preferably comprises at least
about 10,000 different
functionalized materials. Alternatively, a more limited proteomics study, such
as a study of
the abundances of various human transcription factors, for instance, might
only require a
library of about 100 different functional materials.
The material libraries are useful for a wide range of applications, depending
on the
types of functionalized materials. They are used as capture agents for binding
of particular
targets, such as polypeptides, polynucleotides, membranes, organelles,
microorganisms,
viruses, prions or cells, particularly human cells. To accomplish this,
functionalized
materials are selected having groups that bind nucleic acids, polypeptides,
complexes of
polypeptides, receptors, receptor ligands, oligosaccharides and
polysaccharides, lipids,
prions, bacterial cells, viruses, fungi, and cells. The libraries are also
useful for evaluating
the chemical or physical properties of targets, or for performing reactions
involving targets.
Functional materials are selected having groups that are catalytic agents for
reactions
involving particular targets, for example, restriction digestion of target
nucleic acids,
proteolytic cleavage of target polypeptides, and other enzymatic reactions
involving target
lipids or polysaccharides. In one embodiment, the targets are proteins which
are all
expression products, or fragments thereof, of a cell or population of cells of
a single
organism. The expression products may be proteins, including peptides, of any
size or
function. They may be intracellular proteins or extracellular proteins. The
expression
products may be from a one-celled or multicellular organisms. The organisms
may be plants
or animals. In a preferred embodiment of the invention, the binding partners
are human
expression products, or fragments thereof. In one embodiment, the targets
include randomly
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chosen subsets of all the proteins, including peptides, which are expressed by
a cell or
population of cells in a given organism or a subset of all the fragments of
those proteins. The
targets of some or all of the functional materials on the library need not
necessarily be
known. For instance, the different functional materials of the library may
together bind a
wide range of cellular proteins from a single cell type, many of which are of
unknown
identity and/or function. In another embodiment the targets of the functional
materials are
related proteins. The different proteins bound by the functional materials may
optionally be
members of the same protein family. The different targets of the functional
materials may be
either functionally related or just suspected of being functionally related.
The different
proteins bound by the functional materials may also be proteins which share a
similarity in
structure or sequence or are simply suspected of sharing a similarity in
structure or sequence.
Likewise, intact cells may be targets of specific functional materials, for
example but not
limited to, cells expressing a desired surface antigen may be isolated e.g.,
CD4+ cells, or cells
expressing cancer markers. This can provide for detection of particular
phenotypes of cells
isolated from patient tissue samples, i.e., evaluation of an immunocompromised
patient's T-
cell count, or identification of malignant cells in a sample of tumor tissue.
In one aspect, the material library is used in a microfluidics device. The
substrate
having the library presented thereon is placed in a reaction chamber. The
reaction chamber
includes a device that houses the substrate and regulates the environment.
Methods for
regulating the supply (and removal) of reagents to the reaction chamber, as
well as the
environment of the reaction chamber (e.g., the temperature, and oxidative
environment) are
incorporated into the reaction chamber using techniques common in the art.
Examples of this
technology are outlined in: I~ricka, Clinical Chem. 44: 2008-2014 (1998); see
also U.S.
Patent No. 5,846,727, both incorporated by reference. For example, the
substrate is fixed
into a reaction chamber and reagents and buffers as well as solutions of
targets are pumped
into and out-of the reaction chamber through microfluidic ports on either side
of the chamber.
In one embodiment, the substrate has etched channels in communication with a
plurality of
domains, thereby permitting the microfluidic ports to be in fluid
communication with the
desired functionalized materials on the substrate. Complete exchanges of
volume can take
place rapidly, i.e., within about 1 second and is mediated by electronically
controlled valves
and pumps that control the flow of solutions through the microfluidic ports.
Control of the
same is effectuated by an operator directed system, preferably an automated
system. Robotic
introduction of fluids onto microtiter plates, gene and proteomics chips or
arrays is
commonly performed to speed mixing of reagents and to enhance experimental
throughput,
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and the present invention is compatible with such robotic systems. More
recently, microscale
devices for lugh throughput mixing and assaying of small fluid volumes have
been
developed, (for example, U.S. Pat. No. 6,046,056 to Parce et al. incorporated
by reference).
The microfluidics devices are generally suitable for assays relating to the
interaction of
biological and chemical species, including enzymes and their substrates,
ligands and ligand
binders, receptors and ligands, antibodies and antibody ligands, as well as
many other assays.
Because the devices provide the ability to mix fluidic reagents and assay
mixing results in a
single continuous process, and because minute amounts of reagents can be
assayed, these
microscale devices represent a fundamental advance for laboratory science.
According to the present invention, the substrate is positioned in an
integrated
microfluidic system including a microfluidic device. The device has at least a
first reaction
channel and at least a first reagent introduction channel, typically etched,
machined, printed,
or otherwise manufactured in or on a surface of the device that will contain
the substrate
having the material library. Optionally, the device can have a second reaction
channel and/or
reagent introduction channel, a third reaction channel and/or reagent
introduction channel or
the like, up to and including hundreds or even thousands of reaction and/or
reagent
introduction channels. The reaction channel and reagent introduction channels
are in fluid
communication, i.e., fluid can flow between the channels under selected
conditions. The
device has a material transport system for controllably transporting a
material through and
among the reagent introduction channel and reaction channel. For example, the
material
transport system can include electrokinetic, electroosmotic, electrophoretic
or other fluid
manipulation aspects (micro-pumps and microvalves, fluid switches, fluid
gates, etc.) which
permit controlled movement and mixing of fluids. The device also has a fluidic
interface in
fluid communication with the reagent introduction channel. Such fluidic
interfaces optionally
include capillaries, channels, pins, pipettors, electropipettors, or the like,
for moving fluids,
and optionally further include microscopic, spectroscopic, fluid separatory or
other aspects.
The fluidic interface samples a plurality of reagents or mixtures of reagents
from a plurality
of sources of reagents or mixtures of reagents and introduces the reagents or
mixtures of
reagents into the reagent introduction channel. The fluid is thus directed to
domains on the
substrate, for example by one or more channels in the substrate in
communication with one
or more domains. Essentially any number of reagents or reagent mixtures can be
thus
introduced to the substrate by the fluidic interface, depending on the desired
application.
Because microfluidic manipulations axe performed in a partially or fully
sealed environment,
contamination and fluidic evaporation in the systems axe minimized.
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A first reagent from the plurality of sources of reagent or mixtures of
reagents is
selected. A first reagent or mixture of reagents (for example, comprising a
target compound
and a buffer solution) is introduced into the first reaction channel, and is
then introduced to a
first domain of the library, whereupon the first reagent or mixture of
reagents react with the
functional groups immobilized to the polymer brushes of the material at the
first domain.
This reaction can take a variety of different forms depending on the nature of
the reagents.
For example, where the reagents bind to one another, such as where the
reagents axe an
antibody or cell receptor and a ligand, or an amino acid and a binding ligand,
the reaction
results in a bound component such as a bound ligand. Where the reagents are
sequencing
reagents, a primer extension product results from the reaction. Where the
reagents include
enzymes and enzyme targets (substrates), a modified form of the enzyme target
typically
results. Where two reacting chemical reagents are introduced to the
functionalized materials,
a third product chemical typically results at the applicable domains. In
another aspect, a
reaction product from a reaction at one or more domains is analyzed. This
analysis can take
any of a variety of forms, depending on the application. For example, where
the product is a
primer extension product, the analysis can take the form of separating
reactants by size,
detecting the sized reactants and translating the resulting information to
give the sequence of
a template nucleic acid. Similarly, because microscale fluidic devices of the
invention are
optionally suitable for heating and cooling a reaction, a PCR reaction
utilizing PCR reagents
(thermostable polyrnerase, nucleotides, templates, primers, buffers and the
like) can be
performed and the amplicons detected. Amplified or transcribed nucleic acids
obtained from
a first domain may be transferred to a second domain for subsequent
processing, for example,
restriction digestion to determine the presence or absence of a single
nucleotide
polymorphism, or radiolabeling, or hybridization. Where the reaction results
in the formation
of a new product, such as an enzyme-substrate product, a chemical species, or
an
immunological component such as a bound ligand, the product is typically
detected by any of
a variety of detection techniques, including autoradiography,
chemiluminescence
microscopy, spectroscopy, or the like.
Based upon the reaction product, a second reagent or mixture of reagents is
selected
and introduced to the first domain or one or more second domains, as described
above. The
second reaction product is similarly assessed. For example, where the product
is a DNA
sequence, a sequencing primer and/or template for extension of available
sequence
information is selected. Where the product is a new product such as those
above, an
appropriate second domain may include components such as an enzyme, ligand,
antibody,
43
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receptor molecule, chemical, or the like, selected to further test the binding
or reactive
characteristics of the first or second reaction product. The second reagent or
mixture of
reagents is introduced to the appropriate domain via the first reaction
channel, or optionally
the second (or third or fourth . . . or nth) reaction channel in the
microfluidic device. The
results of the analysis of any reaction product can serve as the basis for the
selection and
analysis of additional reactants and domains for subsequent introduction to
the same or
different functionalized materials. For example, a single type of DNA template
is optionally
sequenced in several serial reactions. Alternatively, completing a first
sequencing reaction,
as outlined above, serves as the basis for selecting additional templates
(e.g., overlapping
clones, PCR amplicons, or the like, see, U.S. Patent No.:6,403,338
incorporated herein by
reference).
Analysis of Target Interactions with the Library
Detection of taxget interactions with functional groups at particular domains
within
the material library can be accomplished by numerous technologies known and
described in
the art, including detection of binding or detection of enzymatic or
functional activity.
Hybridization of nucleic acid targets to immobilized nucleic acid or
polypeptide functional
groups, or binding of a polypeptide target to a functional group at one or
more domains can
be detected using labeled targets, labeled functional groups, reagents
introduced to the same
having detectable labels, or combinations of these approaches. A detectable
label may
include but is not limited to a luminescent compound such as fluorescein, a
chromophore, a
fluorescent compound, a radioactive isotope or group containing same, or a
nonisotopic label,
such as an enzyme or dye, a catalyst, a polynucleotide coding for a catalyst
or a promoter,
horseradish peroxidase (HRP), alkaline phosphatase, a chemiluminescer such as
luminol, a
coenzyme, an enzyme substrate, a radioactive group, a small organic molecule,
an
amplifiable polynucleotide sequence, a particle such as latex or carbon
particle, metal,
crystallite, liposome, cell, etc., which may or may not be further labeled
with a dye, catalyst
or other detectable group, a terbium chelator such as N-(hydroxyethyl)
ethylenediaminetriacetic acid that is capable of detection by delayed
fluorescence, and the
like. The detectable label may be directly linked to a polypeptide or
polynucleotide target or
indirectly linked, e.g., by its presence on a partner molecule that interacts
with or binds to a
target, for example an HRP conjugated antibody. Such labeling is well known in
the art. In
general, any label that is detectable can be used. Detectable labels thus
include, for example
but not limited to (i) labels that can be detected directly by virtue of
generating a signal, (ii)
specific binding pair members that may be detected indirectly by subsequent
binding of a
44
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target to a functional group, where either the functional group or target or
both contain one or
more detectable labels.
In one aspect, the detectable labels are fluorescent, for example but not
limited to,
fluorescence resonance energy transfer pairs. These refer to a pair of
fluorophores
comprising a donor fluorophore and acceptor fluorophore, wherein the donor
fluorophore is
capable of transferring resonance energy to the acceptor fluorophore. In other
words the
emission spectrum of the donor fluorophore overlaps the absorption spectrum of
the acceptor
fluorophore. In preferred fluorescence resonance energy transfer pairs, the
absorption
spectrum of the donor fluorophore does not substantially overlap the
absorption spectrum of
the acceptor fluorophore. Suitable visible light fluorophores (excitation
maximum of the
donor above about 350 nm) include fluorescein, Lucifer Yellow, acridine
Qrange, rhodamine
and its derivatives, for example tetramethylrhodamine and Texas Red, and
fluorescent
chelates or cryptates of Europium. A preferred fluorophore is fluorescein.
Suitable energy
transfer pairs for detectable labels may be found in, for example,
Applications of
Fluorescence in hnmunoassays (I. A. Hemmila, Wiley Interscience, 1991 and
"Molecular
Probes: Handbook of Fluorescent Probes and Research Chemicals"--Richard P.
Haughland--
Molecular Probes Inc.) or may be devised by a person of ordinary skill in
accordance with
energy transfer principles (for example as outlined by J. R. Lakowicz,
Principles of
Fluorescence Spectroscopy, Plenum, 1983), each incorporated by reference. When
used as
detectable labels, fluorophores are generally attached to polypeptides via a
side chain of an
amino acid in the polypeptide chain, or at the N-terminus or C-terminus.
Convenient
reagents for labeling amino groups include donor/acceptors derivatized with
isothiocyanates,
active esters, such as succinimidyl esters, of carboxylic acids or sulphonyl
halides. These are
generally reacted at moderately alkaline pH in an excess over the compound to
be labeled
(target or functional groups or the like). Convenient reagents for labeling at
thiol groups
include donor/acceptors derivatized with maleimido or haloacetyl groups. Two
derivatives of
GFP are useful for FRET standards, based on their spectral properties. The
mutant BFP11,
constructed by Lossau et al. (Chew. Physics. 213: 1-16, 1996) using
combinatorial
mutagenesis, contains the mutations F64M/Y66H. BFP11 has blue-shifted
excitation and
emission maxima relative to wild-type GFP. The mutant RSGFP4 (Delagrave et
al.,
Bio/Technology, 13:151-154, 1995), generated by combinatorial mutagenesis,
contains the
mutations F64M/S65G/Q69L. RSGFP4 has spectral properties similar to the S65T
mutant
reported by Heim et al. (Proc. Natl. Acad. Sci. LJSA 91:12501-12504, 1994). As
the
acceptor in a FRET pair, both S65T and RSGFP4 are superior to other red-
shifted excitation
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mutants such as RSGFP8 (F64L+S65T), because the latter mutant has significant
excitation
in the violet.
After introduction of targets to one or more domains and optional washing to
remove
unreacted or nonspecifically bound targets, the properties of the target
following its
interaction with specific domains are determined for the whole library or at
specified domain
address. The detection means will depend on the detectable label used. For
example, with
radioactively labeled targets, the activity at a domain address is measured
through detection
of radioactive emissions at that address, and may be further measured against
reference
standards at other domain addresses to provide quantitative information about
target
interactions with the materials library. For fluorescent labeled targets,
detection at a domain
address includes quantitation of color spectra and intensity, determined, for
example, using a
scanning confocal microscope in photon counting mode. Appropriate scanning
devices for
confocal microscopy are described by e.g., Trulson et al., U.S. Pat. No.
5,578,832 and Stern
et al., U.S. Pat. No. 5,631,734, each incorporated herein by reference.
Further examples of
particular labels and their detection can be found in U.S. Pat. No. 5,508,178,
the relevant
disclosure of which is incorporated herein by reference.
Many systems may be used to detect labels and evaluate interactions between
targets
and functional groups at one or more domains, for example but not limited to,
by detection of
electromagnetic radiation, x-ray or particle emission, or by optical
examination. Detection
systems that may be employed with the present libraries include those
described in U.S. Pat.
No. 5,508,178, the relevant disclosure of which is incorporated herein by
reference. A
currently preferred method of detecting labels and quantitating reactions at
domains is based
on epifluorescence microscopy using FRET as detailed in U.S. Patent 6,456,734
to Youvan et
al., hereby incorporated by reference in its entirety, which discloses imaging
hardware,
software, calibrants, and methods to visualize and quantitate the amount of
fluorescence
resonance energy transfer (FRET) occurring between donor and acceptor labels.
Such a
detection system is applicable to the libraries of the present invention when
the donor and
acceptor labels are affixed to targets and to functional groups or reagents as
described above.
Where colormetric or photon based detectable labels are used, an optical
reader or
other such imaging device is used to read the library. A suitable optical
imaging device
provides a means for acquiring spatially co-registered electronic images, thus
determining
detection values for labels at each domain address. Alternatively, the optical
imaging device
provides a means for reading an impression of the library, e.g., an
autoradiograph or
chemiluminescent image of the domains of the library. For example, one optical
imaging
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device includes a microscope and digital camera. The microscope can be a
steady-state,
wavelength-scanning fluorescence microscope (i.e., not a time-resolved system
or not an
interferometer-based system). The optical imaging device can also provide for
background
subtraction, spectral overlap corrections, and transformation of data from
three channels set
into a color space defined by the primary colors of red, green, and blue.
Labels first detected
at domain addresses and rendered as images with such an imaging device may be
further
processed to produce an enhanced image, for example, an image in which FRET,
acceptor,
and donor pixels are more clearly differentiated and pseudocolored. An optical
imaging
device may include a light source, to provide illumination of the library or
for excitation of
detectable labels, for example, a 75 watt quartz tungsten halogen (QTH) light
source can be
coupled directly to the fluorescence microscope, although other light sources
may be used if
desired. The method of the invention is not limited to an epifluorescence
microscope.
Macroscopic lens-based systems could be used in place of the microscope's
objectives to
achieve detection and quantitation over a macroscopic field of view, where the
library is large
or where the individual domains are easily visualized.
Systems for Evaluating Material Libraries
To control the application of targets and reagents to the material library;
and to detect
and quantify interactions between the targets and the functionalized materials
and relate such
interactions to domain addresses, a computer system is utilized. Further, to
interpret the
information obtained from using the material libraries, and to control and to
guide the use and
evolution of the libraries, the computer system includes one or more programs
in informatics.
In addition to cataloging the composition of, and physical and functional
properties of each
material used at particular domains in the libraries, the informatics program
will track the
target detection data obtained from all uses involving a particular material.
This information
is used to direct future assays involving related target molecules to or
domains of similar
function or composition, thus accelerating the discovery process. Moreover,
these data sets
will also guide new directions for the expansion of the library, i.e., the
evolution of materials
with specific properties. For example, a high-throughput screen might
determine that the
optimal binding/elution of a particular polyclonal antibody target occurs with
materials
containing a narrow range of brush densities or lengths and that this occurs
at domains where
there are specific blends of monomers that contain certain concentrations of
ionic functional
groups and immunospecific (antigenic) functional groups. The library thus can
be used to
indicate the optimal materials for isolating immunoglobulins having specific
affinity ranges
for the antigen. Lastly, the informatics program is used to develop
sophisticated molecular
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models of the polymer brushes that will simulate brush structure/function by
taking into
account critical factors such as brush composition, density, length and
flexibility. When
combined with the data sets obtained from the target screens, these molecular
models will
help predict binding properties of the polymer brushes, thus speeding the
discovery process
and guiding continued expansion of new functionalized materials for the
library.
In one aspect, the systems of the present invention provides an integrated
computer
program that compares digital profiles of images of library domains and causes
the system to
select one or more addresses, and generates instructions that direct a robotic
device to isolate
reaction products from the domains. In one embodiment, the system directs the
robotic
device to introduce these reaction products to secondary domains and directs
the microfluidic
device to introduce reagents to the secondary domains. In yet a further
embodiment, the
system includes a library information management system that tracks materials
used in the
libraries and tracks data associated with targets, such as clinical data,
operations performed
on the targets, and data generated by analysis of the reactions of the targets
and materials.
According to the present invention, targets are introduced to a library
comprising
functionalized materials presented as domains having addresses, as described
above. The
interactions of targets with functional groups is detected and evaluated by a
reading means,
such as the optical imaging device described. The reading means thus provides
both
qualitative and quantitative information about the interaction of targets to
functional groups
through detection of labels at domain addresses on the library, which is
communicated to
computer system. The system includes an instruction set having modules for
data
management, e. g. , a data input means, a data storage means, a data retrieval
means, a
relational database, and a data output means, as well as an instruction set
comprising data
analysis algorithms e.g., detection and quantitation of labels. The
instruction set may further
include control modules, e.g., for control of robotic or microfluidic devices.
Processors
appropriate for executing the instruction set of the system include any
processors capable of
recognizing an instruction set written in an appropriate language, for example
but not limited
to PowerPC based Apple~ computers, Pentium~ or similar PC type computers, SUN~
or
Silicon Graphics~ workstations, or systems running LINUX or UNIX. The
instruction set
includes a computer readable algorithm for data analysis, which is stored in
computer
readable media as part of a program written in a suitable computer readable
language, for
example C, C++, iJNIX, FORTRAN, BASIC, PASCAL, or the like. The program
provides
the processor with instructions for performing calculations on the input data,
as well as other
functional elements contained in one or more modules or subroutines (e.g.,
relational
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database capabilities, search features, and other user defined functions). The
program
includes input modules for entering data into the system in computer readable
format and a
selection module instructing the system to select and read data entered. In
one aspect, the
system includes an input module. Users of the system enter data into the
system in computer
readable format, which can be stored in RAM or ROM, or a more permanent
storage medium
such as a disk or tape drive. The information entered through the input module
is thus
accessible to the system processor. The system further comprises an output
module. The
output of the computer system can be represented on a display or monitor, as a
word
processing text file, formatted in commercially-available software such as
WordPerfect~ and
Microsoft Word~, or represented in the form of an ASCII file, stored in a
database
application, such as DB2, Sybase, Oracle, or the like. A skilled artisan can
readily adapt any
number of data processor structuring formats (e.g. text file or database) in
order to obtain
computer readable medium having recorded thereon the expression information of
the present
invention.
Computer assisted data analysis minimally includes detection of the labels and
determination of signal intensity for each domain address, rendered as an
image of the
domain address, and where the address is further given as a set of two-
dimensional Cartesian
coordinates. Where multiple detectable labels are used, i.e., for FRET, a set
of three such
images from each channel (donor, acceptor, and FRET) can then be processed as
three
spatially coregistered images or treated as a single image in which each pixel
has three color
space coordinates corresponding to the monochrome wavelengths. In one
embodiment, the
quantitative method of the invention performs quantitative FRET measurements
and
analyses. FRET image data is obtained with standard filter sets using a
fluorescence
microscope and processed as described above. Equations for quantitative FRET
useful in
designing specific data analysis algorithms for the program are provided in
Gordon, et al.,
Quantitative Fluorescence Resonance Energy Transfer Measurements Using
Fluorescence
Microscopy, Biophysical Journal, Vol. 74, May 199 2702:2713, which is hereby
incorporated by reference. In a preferred embodiment, the data analysis
further includes
information about the materials and the reactions occurring and detected at
each domain
location, i.e., number and type of functional groups, polymer brush density
and morphology,
and reaction conditions such as flow rate, target concentrations, salt
concentrations, buffer
composition, ionic strength, temperature, pH, and time, binding and affinity
or catalytic
activity. In a more preferred embodiment, the data analysis includes
information obtained
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from or stored within a database, that contains information about the targets,
such as their
source and method of isolation, and correlation with a disease state.
The invention thus provides a computer-generated digital profile representing
the
identity and relative abundance of a plurality of target biomolecules detected
in view of a
plurality of reaction conditions, thereby permitting computer-mediated
comparison of profiles
from multiple target samples for multiple materials. This automatable
technology for
screening biological targets and comparing their profiles permits rapid and
efficient
identification of individual targets whose presence, absence or altered
expression is
associated with a disease or condition of interest. Such targets are useful in
the design and
evaluation of their potential as therapeutic agents, as targets for
therapeutic intervention, and
as markers for diagnosis, prognosis, and evaluating response to treatment.
This technology
also permits rapid and efficient identification of sets of targets whose
pattern of expression is
associated with a disease or condition of interest; such sets of targets
provide constellations of
markers for diagnosis, prognosis, and evaluating response to treatment.
As those skilled in the art will recognize, the invention described herein can
be
modified to accommodate and/or comply with any one or more computer based
technologies
and standards. In addition, variations, modifications, and other
implementations of what is
described herein can occur to those of ordinary skill in the art without
departing from the
spirit and the scope of the invention as claimed. Further, virtually any
aspect of the
embodiments of the invention described herein can be implemented using
software,
hardware, or in a combination of hardware and software.
Example One--Method and Process for Producing Bipolar Membranes
This following example describes a new method for preparing bipolar
functionalized
materials, in this case, fabricated in the form of a membrane. Using this
method, polymer
brushes that contain cation-exchange and anion-exchange functional groups are
introduced
separately on opposing surfaces of a membrane in a single step. Electron-beam
treated high
density polyethylene film (HHOPE) was used as the material for grafting, and
was sandwiched
between a monomer solution containing sulfonic acid groups and a monomer
solution
containing quaternary ammonium salt groups. X-ray microanalysis (XMA), was
used to
measure the distribution of sulfur and chlorine across the membrane thickness,
i. e., the
distribution of cation-exchange and anion-exchange groups, respectively. The
bipolar
membrane was used for electrodialysis to regenerate HCl and NaOH from a NaCI
solution.
This bipolar membrane demonstrates similar electricolytic potential as
compared to bipolar
membranes prepared by conventional methods.
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Preparation of a bipolar membrane ahd its characters
In order to determine the conditions to produce a bipolar membrane, ion-
exchange
membranes were prepared as shown in FIG. 1. A nonporous high density
polyethylene film
(HDPE) with a thickness of 50 micrometers was used as the polymeric base
membrane for
grafting. The base membrane was irradiated with electron beams at a total dose
of 200 kGy.
As shown in FIG. 2, the electron-beam treated HDPE film was interposed between
two
identical cylindrical cells having an inner diameter about 4 cm.
Sodium styrene sulfonate (CHa=CHC6H4SO3Na; SSS) and 2-hydroxylethyl
methacrylate (CH2=CCH3COOCHaCH20H; HEMA) were co-grafted onto the electron-
beam-
treated base membrane to form a cation-exchange membrane (see, S. Tsuneda et
al., J.
Electrochem. Soc., 142, 3659 (1995) incorporated herein by reference).
Similarly, vinyl
benzyl trimethyl ammonium chloride (CH2=CHC6H4CHaN(CH3)3C1; VBTAC) and HEMA
were co-grafted to form an anion-exchange membrane (see, W. Lee et al., J.
Membr. Sci.,
81, 295 (1993) incorporated herein by reference). All reaction conditions are
listed in Table
l, below. The degree of co-grafting (dg) is defined as:
Degree of co-grafting (d~ _
(weight of grafted polymer brushes) l (weight of base membrane) x
100°fo
Table 1. Pre aration Conditions for Bi olar Ion-Exchan a Membranes
Cation-exchan a Anion-exchange
Base olymer HDPE film
Total irradiation200 kGy
dose
Monomer SSS/HEMA VBTAC/HEMA
Solvent Water DMF/MeOH=1/1 vol.
ratio
Reaction time 1 ~9 h
Reaction tem erature323 I~
The sulfonic acid (-S03H) group density and the quaternary ammonium salt
(-N(CH3)3+Cl-) group density of the prepared membrane were determined by
methods known
in the art for salt-splitting capacity, and the hydroxyl group density was
calculated by
subtracting the ion-exchange group density from the total functional group
density. The
sulfur (S) profile of the sulfonic acid group and the chlorine (Cl) profile of
the quaternary
anunonium salt group across the thickness of the prepared membranes were
determined by an
electron-probe X-ray microanalysis (WA). In order to measure the electrical
resistance of
the bipolar membrane, the membrane was sandwiched between two identical
cylindrical cells
having an effective membrane area of approximately 7 cma with the quaternary
ammonium
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salt group side facing the anode and the sulfonic acid group side facing the
cathode. The
electrical current was measured across a 0.1 M NaCI solution at 298 K, using
an applied
voltage of 15 V.
Elect~odialysis usihg a bipolay~ membrane
As shown in FIG 3, with the bipolar membrane placed in the center,
conventional
cation-exchange and anion-exchange membranes (2 membranes each) were placed in
between the electrodes in a electrodialysis chamber. The quaternary ammonium
salt group
side of the bipolar membranes faced the anode. A voltage of 5.1 V was applied.
The
operation conditions for the electrodialysis axe summarized in Table 2, below.
The effective
membrane area of all membranes was 10 cm2. The NaCI concentration in the salt
chambers
(2 and 5) was determined by measuring the conductivity. The HCl concentration
in the acid
chamber (3) and the NaOH concentration in the alkali chamber (4) were measured
by titration
of acid and base. The concentrations of sodium, chlorine and sulfonic acid
ions before and
after the electrodialysis were determined by ion chromatography. The electric
power unit
was determined using the equation:
electric p~wer unit (Whlk~ = j V i dtlw
where V, I, t and w are the voltage (V), current (A), time (h) and weight of
removed NaCI
(kg), respectively.
Table 2. Measurement Conditions of lysis
Electrodia
Applied voltage 5.1 V
Duration 5 h
Salt chamber 1.0 M NaCI
Acid chamber 1.0 M HCl
Alkali chamber 1.0 M NaOH
Electrode chamber0.5 M NaaSO4
Circulation rate 50 mL/h
Temperature 298 K
-~-
The degrees of co-grafting of SSSIHEMA and VBTAC/HEMA onto the electron-
beam-treated HDPE base membranes and their changes in membrane thickness as a
function
of co-grafting time are shown in FIG. 4. The degree of co-grafting increased
with the
reaction time. The thickness of the prepared cation-exchange membrane (Na-
type) and the
anion-exchange membrane (CI-type) increased to a maximum of 1.8-fold and 1.4-
fold,
respectively. FIG. 5 shows the increase of sulfonic acid group and hydroxyl
group densities
in the cation-exchange membrane as well as the increase of quaternary ammonium
salt group
and hydroxyl group densities as a function of degree of co-grafting (dg). At a
reaction time
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of 9 hours, the sulfonic acid group and the quaternary ammonium group
densities reached 2.3
and 0.81 mmol/g-product, respectively. By XMA, the profiles of sulfonic acid
group
(measuring the sulfur) and the quaternary ammonium salt group (measuring the
chlorine)
across the thickness of both membranes were measured (FIG. 6). The surface
area ratio of X-
ray intensity distribution was compared to the functional group density ratio
from salt-
splitting capacity measurement (FIG. 7). Both ratios matched each other to a
maximum ratio.
This also means that XMA can be used for quantification. With the increase of
the reaction
time, i.e., the increase of degree of co-grafting (dg), the brushes that
contain sulfonic acid
group or quaternary ammonium salt group invaded the membrane from both sides
and
reached the center.
FIG. 8a shows the increase of the degree of co-grafting and the membrane
thickness
of the prepared bipolar membrane as a function of reaction time. The
functional group
density also increased with the increase of dg (FIG. 8b). The sulfonic acid
group density was
about 2-fold of the quaternary ammonium salt group density within the range of
dg from 16%
to 86%. For example, at dg=86%, the sulfonic acid group density and the
quaternary
ammonium salt group density were 1.0 and 0.57 mmol/g-product, respectively.
The
functional group density profiles across the thickness of the dg=16% and
dg=86%
membranes were compared (FIG. 9). Both membranes have shown the invasion of
sulfonic
acid groups from the left and the invasion of quaternary ammonium salt groups
from the right
of the membrane. At dg=16%, both functional groups did not reach the center of
the
membrane. However, from an observation by scanning electron microscopy (SEM),
results
have shown that the poly-HEMA brushes have invaded the center of the membrane.
In
contrast, at dg=86%, both functional groups intersected each other forming a
neutral area in
the center of the bipolar membrane. The voltage-current characteristic of the
prepared
bipolar membrane is shown in FIG. 10. At dg=16%, no flow of current was
observed even
with the increase of voltage. This corresponds to the XMA result that showed
no intersection
of the functional groups.
Regeneration of Acid and Alkali from a Salt Solution
The bipolar membranes with dg's of 16% and 86% were used for electrodialysis.
FIG. 11 shows the time course for the concentration changes of NaCI (salt
chamber), HCl
(acid chamber) and NaOH (alkali chamber). In 5 hours of electrodialysis, M
molar of HCl
and M molar of NaOH were regenerated from 1M of NaCI solution. Table 3, below
summarizes the mass balance of all ions before and after the electrodialysis
of the prepared
bipolar membrane (dg=86%). The molar numbers of Na and 5042- agreed with each
other
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before and after the electrodialysis. However, the molar number of Cl- before
the
electrodialysis did not agree with the one after. Without being restricted to
theory, this is
believed to be due to the electrode reaction. Calculations using Faraday's law
indicate that
3.40 x 10-3 mol of Cl- was needed for the electrode reaction. This value
agrees with the
shortage of 3.25 x 10-3 mol of Cl' seen before and after electrodialysis.
Table 3. Mass Balance of Ions Before and After Electrodialysis of the Co-
f'raftPrl_tvnP Rinnlar Memhrane ldu=R6°/nl
Before After
electrodialysis electrodialysis
(0 (300
min) min)
H+ Nab Cl- S04 H+ Na+ S04
OH' ' OH' -
Cl-
(10-Lmol) (10-
mol)
Salt 1.000 1.000 0.424 0.424 0.024
chamber
Acid 1.000 1.000 1.262 0.016 1.231
chamber
Alkali 1.000 1.000 1.220 1.2350.010
chamber
Electrode 3.000 1.500 3.320 0.010 1.470
chamber
Total 1.000 5.000 1.0002.000 1.5001.262 4.980 1.2351.675 1.494
The voltage and current of electrodialysis as a function of operation time
showed that
the current decreased from the initial 0.07 A to 0.02 A (FIG. 12). The
electrical power unit
calculated in FIG. 11 and 12 using the above equation is shown in FIG. 13. The
electrical
power unit was within the range of 2.22.6 kWh/kg. The electrical power unit
obtained by
radiation induced graft polymerization (RGIP) was compared to those of
previous studies
involving pasting or grafting, summarized in Table 4, below, (see, J.
I~assotis et al., J.
Electrochem. Sci., 131, 2810 (1984), S. I~. Adhikary et al., React. Polym., 1,
197 (1983), F.
Alvarez et al., J. Membr. Sci., 123, 61 (1997) and G. S. Trivedi et al.,
React. Funct.
Polym., 32, 209 (1997), each incorporated herein by reference). The electrical
power unit of
the prepared bipolar membrane exhibited similar order of unit (1.62.6 kWh/kg)
to the
membrane prepared by a casting method.
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Table 4. Previous Studies of Bipolar Membranes
Prep. Functional (V) Salt Acid regen.Alkali Electric
method group rate x regen. power
density 10-a rate unit
(mmol/g- (M/min) x 10-2 (kWh/kg-
roduct)
S03Na N(CH3)3C1 (M/min)salt
grou ou removed)
Pasting 3.8 CH3COONa 0.025 0.097
Pasting 50 Na2SOd 0.037 0.071 7.1
Pasting 25 C6H4(OH)COONa4.00 g/1-min
Casting1.33 1.00 25 NaZS04 0.440 0.650 2.6
Casting1.56 1.41 20 NaZS04 0.570 1.300 1.6
Casting1.32 1.25 25 CH3COONa 0.930 1.000 1.7
RIGP 1.00 0.57 5.1 NaCl 0.087 0.070 2.22.6
Example Two-Immobilization of Microbes Using Graft Polymerized Materials
Preparation of the GMA-1~EA-BC Fiber
This example describes the immobilization of microbes, in particular the
bacteria
Staphylococcus aureus, to a material comprising a polyethylene hollow-fiber
membrane. The
inner and outer diameters of the membrane were 1.9 and 3.2 mm, respectively,
with a
porosity of 70°/~ and a pore size of 0.34 micrometers. FIG. 14 shows
the preparation scheme.
A vinyl monomer, glycidyl methacrylate (GMA, CHa=CCH3COOCH2CHOCH2) was grafted
onto the PE membrane by the electron-beam-induced grafting method. The PE
membrane
was irradiated with an electron beam at a total dose of 200 kGy at room
temperature. The
irradiated membrane was then immersed in a GMA solution (10% vol/vol in
methanol) and
reacted at 313 K. The amount of GMA grafted onto the backbone membrane was
calculated
as the degree of GMA grafting, dg, using the equation provided below.
The GMA-grafted membrane was reacted with diethylamine (DEA, HN(C2H5)2), and
then quaternized with benzyl chloride (BC, C6HSCH2C1). The resulting strong
base anion-
exchange membranes are referred to as GMA-DEA-BC (dg/Xt/Xq), where dg, Xt and
Xq in
parentheses designate degree of grafting, conversions of introduction of the
tertiary amino
group and subsequent quaternization, respectively. The values of dg, Xt and X9
were
calculated from the weight change as follows:
dg =100 ((Wl - Wo) l Wo) ~~~
X~ =100 (((1'~a - Wi) l 73) l ((WI - Wo) l 142)) ~~~
X9 =100 (((W3 - W~) l 127) l (WZ - WI ) l 73)) ~/,
where Wo, Wl, W2 and W3 are the weights of the starting film, GMA-grafted, DEA-
introduced and quaternized membranes, respectively. The FIG.s 142, 73 and 127
correspond
to the molecular weights of GMA, DEA and BC, respectively.
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Culture of Microorganisms
Staphylococcus aureus strain IFO 12732 was obtained from the Institute for
Fermentation, Osaka, Japan, and was used as a model microorganism for
microbial-cell-
immobilization studies. One loopful of the bacteria was inoculated into 10 mL
of rehydration
fluid (polypeptone 1.0%, yeast extract 0.2%, MgS04~7HaO 0.1%, pH 7.0) and
cultured at
305 K for 18 to 24 h in a test-tube shaker at 100 strokes/min. After the cells
in the cultured
cell suspension were collected by centrifugation at 5600xg for 15 min in a
refrigerated
centrifuge at a temperature below 277 K to arrest cell division and hold the
cells in the
stationary phase, and then washed twice with 10 mL of distilled, deionized and
sterilized
water. The cells were then resuspended in fresh sterilized water to a final
volume of 10 mL.
The cells were serially diluted in sterilized water to the desired cell
concentration (about 105
to about 106 cells/mL) before contact with the prepared grafted membranes.
Microbial Cell Adsorption onto the Grafted Type GMA-DEA-BC Membranes
Forty milliliters of the prepared cell suspension was added into a 100 mL
flask. Then,
prepared GMA-DEA and GMA-DEA-BC membranes were brought into contact with the
cells by shaking the flask at 130 rpm at 298 K. GMA-DEA and GMA-DEA-BC
membranes
of 10-cm length (cut into 1-cm sections and subsequently sliced vertically in
half) were used.
One-tenth milliliter of the contact suspension was pipetted from the flask at
specific time
intervals, inoculated into 9.9 mL of sterilized water, and serially diluted
with sterilized water.
One-tenth milliliter of the diluted suspension was spread on an agar plate
containing growth
media. The plate was incubated at 310 K for 18 to 20 h, and the number of
viable cells in the
contact suspension was calculated from the number of colonies formed on the
plate.
The adsorption rate of microbial cells by both the GMA-DEA and GMA-DEA-BC
membranes was examined in terms of the decrease in the number of free viable
cells. Values
of the adsorption rate constant, taking into consideration of the contact
surface area for the
microbial-cell-capturing action, were determined from the slopes of the
logarithm of viable
cell number (CFU/mL; colony forming units per milliliter) versus contact time
plots. The
adsorption rate constant, k, of the various DEA-EO membranes and DEA beads was
defined
as:
h(dCldt) _ -kAC
where k can be derived as follows under an initial condition of C = Co at t =
0, and the
adsorption rate constant is defined by:
k = - (hlA) (1/t) In (ClCo) ~mlsJ
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where V, A, t, C and Co are the volume of viable cell suspension, the contact
surface area, the
contact time, the viable cell number at contact time t and the initial viable
cell number,
respectively.
Since the pore size of the membrane is about 0.3 micrometer, S. aureus, which
has an
average diameter of about 1 micrometer, cannot enter the pores of the
membrane. As a
result, the contact surface area of the fiber was calculated from the outside
surface area only
excluding the surface area of the pore. In this experiment excess surface area
of membrane
was provided (maximum cell coverage of 1 %) in order to calculate k. FIG. 15
shows the
conversion of quaternization of the grafted-type GMA-DEA-BC fiber. FIG. 16
shows the X-
ray microanalysis (XMA) profiles of chloride ion adsorbed on the grafted-type
GMA-DEA-
BC fibers as a function of conversion of BC. The fibers were converted into Cl-
form before
the performance of XMA. FIG. 17 shows the adsorption experiments for the
grafted-type
GMA-DEA-BC fibers against Staphylococcus aureus cells. FIG. 18 shows the
relationship
between the adsorption rate constant and functional-group-density of the
grafted-type GMA-
DEA-BC fiber. FIG. 19 shows the changes of CFU/mL and pH when the grafted-type
GMA-
DEA-BC fiber was brought contact with Staphylococcus aureus cells a function
of contact
time.
The materials and methods disclosed above are also applicable for the creation
of
bacterial and viral libraries where, for example, variant strains are
immobilized at different
domains. Such libraries are useful for detection and characterization of
microbial samples,
e.g., identification of a pathological strain. In addition, the libraries can
be used in the
production of antibiotics, for example, to determine the effectiveness of an
antibiotic
contacted to the bacteria at particular domains at varying dilutions.
Example Three-Library of Cells Used for Diagnostic Assays
Libraries of cells from one or more different tissue sources or types are
created.
These are used, for example, to determine the levels of disease markers in a
sample tissue, or
for toxicological assays such as for detecting targets in the environment.
This example
describes the manufacture and use of a human ovarian cell library, for
diagnosis of ovarian
cancer.
Preparation of the Library
A glass slide having a coating of high density polyethylene film (HDPE) of
about 0.5
micrometers in thickness was used as the base material for grafting. The
substrate was
irradiated with electron beams from a cascade-type accelerator in a nitrogen
atmosphere at a
total dose of 200 kGy. Glycidyl methacrylate (GMA) was grafted onto the HDPE
film by
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immersion of the substrate in a 10 vol/vol% GMA/1-butanol solution with the
grafting
reaction temperature at 313 I~, to form polymer brushes. After 10 minutes, the
substrate was
removed and washed to remove any residual GMA and poly-GMA homopolymers.
Degree
of grafting was calculated as described, and determined to be about 200%.
Masking of the
substrate prior to grafting permitted the formation of 100 domains of polymer
brushes, each
having dimensions of 2 mm2, presented in a 5 x 20 matrix pattern.
MUC 1 is expressed on the surface of ovarian cancer cells. Nine different
splice
variants of MUCl have been described. Obermair, et al., (Int J Cancer 2002 Jul
10;100(2):166-71, incorporated by reference) compares patterns of expression
of MUC 1
splice variants of malignant and benign ovarian tumors. In this study, ovarian
tissue samples
were taken from patients with benign ovarian tumors (n = 34) and from patients
who had
surgery for primary (n = 47) or recurrent (n = 8) ovarian cancer. RT-PCR for
MUC1 splice
variants A, B, C, D, X, Y, Z, REP and SEC was performed and their expression
compared to
clinical and histopathologic parameters. Variants A, D, X, Y and Z were more
frequently
expressed in malignant than in benign tumors. All primary ovarian cancer cases
were
positive for variant REP but negative for variant SEC. Expression of MUCl
splice variants
A, D, N, Y, Z and REP is associated with the presence of malignancy, whereas
expression of
MUC1/SEC is associated with the absence of malignancy.
Goodheart et al, (Gynecol Oncol 2002 Ju1;86(1):85-90, incorporated by
reference)
showed that an ovarian cancer p53 mutation is associated with tumor
microvessel density, as
measured by CD31 staining, and other histopathologic factors. In this study,
histopathologic
and mutational data were related to CD31 staining utilizing the Mantel
correlation statistic.
The microvessel density was scored by averaging counts from three high-power
(200x)
fields. The mean microvessel density counts based on CD31 staining
(vessels/HPF) for each
FIGO stage and mutation type are reported as: Stage I (10.2), Stage II (10.7),
Stage III (13.8),
Stage IV (22.0), wild-type p53 (9.3), missense p53 mutation (14.4), and null
p53 mutation
(23.1) with a significant correlation between microvessel density count and
FIGO stage (P =
0.026), grade (P = 0.04), and p53 mutation type (P = 0.02).
Lindgren et al., (Int J Oncol 2002 Sep;21(3):583-9, incorporated by reference)
showed
that the pattern of estradiol and progesterone differs in serum and tissues of
benign and
malignant ovarian tumors. Production of both estradiol and progesterone by
ovarian cancers
has been demonstrated and can be detected. In this study, ovarian tissue,
ovarian tumor cyst
fluid, ovarian vein samples and peripheral serum concentrations of estradiol
and progesterone
in pre- and post-menopausal women, subdivided into groups with normal ovaries,
benign,
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borderline and malignant ovarian tumors, were quantitatively assessed. Both
ovarian tissue
concentrations of estradiol and progesterone were more than 100-fold higher
than in serum.
Lower concentrations of estradiol, but not progesterone, were found in ovarian
cancer tissue,
ovarian cyst fluid and peripheral serum in patients with FIGO stages 3 and 4
than in stages 1
and 2. Finding a large ovarian tissue to serum difference of both estradiol
and progesterone
indicates an important role of ovarian tissue concentrations in tumor biology
and can
influence anti-hormonal therapy in women with ovarian cancer.
Ovarian cells obtained from tissue biopsies are immobilized to each domain at
constant cell numbers. Reference tissues are obtained from ATCC, selected as
control cells
in view of the detectable markers disclosed. The cells from the patient and
reference tissues
are immobilized to the brushes at each domain by lipid rich functional groups.
Immunoglobulins specific to MUC-1 isoforms A,D, X, Y, Z, REP, and SEC were
developed
using the methods of Takeuchi et al., (Immunol Methods 2002 Dec 15;270(2):199
,
incorporated by reference). Monoclonal preparations of anti-CD31, anti-bcl-2,
anti-estradiol,
and anti-progesterone immunoglobulins were obtained from commercial sources.
The
immunoglobulins are fluorescently labeled and applied to the cells at
different domains and
over concentrations ranging from about 0.001 mg protein/ml to about 10 mg
protein/ml.
Detection of labeled targets at each domain is performed by confocal
microscopy.
Anti-estradiol, and anti-progesterone immunoglobulins were each prepared in
serial
dilutions to concentrations of antibodies ranging from 15 mg/ml to 1.5 ng/ml
in 0.025 M
borate buffer (pH about 10). Ten microliters of each of the dilutions were
placed on
independent domains of the library (each having a mean surface area of about 1
mm2) and
reacted with the epoxy groups of the GMA grafted brushes for 24 hours at room
temperature,
after which the unbound antibodies were removed by washing. Ovarian cells
obtained from
patient and reference tissues were pulse labeled with 35S and cell lysates
were contacted to
the domains of the library. The domains were washed, and activity at each
domain was
assayed by counting the beta emissions at each domain address. The data was
interpreted
using standard ELISA computer algorithms. The data obtained using both
libraries are
compared to reference standards and predictive values for these markers known
in the
medical literature, and a diagnosis is made in view of such information.
Example Four-Library of Randomized Peptides
A library is developed as described, having approximately 10,000 domains. The
library is manufactured on a substrate adapted for a microfluidic chamber.
Glycine is
immobilized to the polymer brushes at each domain, and the unbound glycine
removed by
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WO 03/049671 PCT/US02/33942
washing. The substrate is placed in an automated microfluidic device, and
random 6-mer
polypeptides are synthesized at each domain, with the peptide sequence at each
domain
address monitored by the controller computer system. The resultant peptide
library is used
for screening pulse labeled cell lysates as described. Interactions are
detected by counting
beta emissions at each domain address. Bound targets at domain addresses are
recovered and
identified by MALDI-TOF spectroscopy. Data pertaining to the interactions and
identification of targets is tracked by a laboratory information management
system (LIMS).
EQUIVALENTS
From the foregoing detailed description of the specific embodiments of the
invention,
it should be apparent that a unique compositions comprising graft polymerized
materials
having functional groups immobilized thereto in multiple layers, as well as
methods of
making and using such compositions, has been described. Although particular
embodiments
have been disclosed herein in detail, this has been done by way of example for
purposes of
illustration only, and is not intended to be limiting with respect to the
scope of the appended
claims that follow. In particular, it is contemplated by the inventor that
substitutions,
alterations, and modifications may be made to the invention without departing
from the spirit
and scope of the invention as defined by the claims. For instance, the number
and kind of
functional group combinations, or the use of such compositions in particular
devices is
believed to be matter of routine for a person of ordinary skill in the art
with knowledge of the
embodiments described herein.
