U.S. patent application number 10/113964 was filed with the patent office on 2002-08-15 for arrays of proteins and methods of use thereof.
Invention is credited to Ault-Riche, Dana, Itin, Christian, Nock, Steffen, Wagner, Peter.
Application Number | 20020110933 10/113964 |
Document ID | / |
Family ID | 22361520 |
Filed Date | 2002-08-15 |
United States Patent
Application |
20020110933 |
Kind Code |
A1 |
Wagner, Peter ; et
al. |
August 15, 2002 |
Arrays of proteins and methods of use thereof
Abstract
Protein arrays for the parallel, in vitro screening of
biomolecular activity are provided. Methods of using the protein
arrays are also disclosed. On the arrays, a plurality of different
proteins, such as different members of a single protein family, are
immobilized on one or more organic thinfims on the substrate
surface. The protein arrays are particularly useful in drug
development, proteomics, and clinical diagnostics.
Inventors: |
Wagner, Peter; (Belmont,
CA) ; Ault-Riche, Dana; (Palo Alto, CA) ;
Nock, Steffen; (Redwood City, CA) ; Itin,
Christian; (Menlo Park, CA) |
Correspondence
Address: |
Zyomyx
26101 Research Road
Hayward
CA
94545
US
|
Family ID: |
22361520 |
Appl. No.: |
10/113964 |
Filed: |
March 29, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10113964 |
Mar 29, 2002 |
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09353215 |
Jul 14, 1999 |
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09353215 |
Jul 14, 1999 |
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09115455 |
Jul 14, 1998 |
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6406921 |
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Current U.S.
Class: |
436/518 ;
435/287.2 |
Current CPC
Class: |
B01J 2219/00621
20130101; B82Y 5/00 20130101; B01J 2219/00702 20130101; B01J
2219/0063 20130101; G01N 33/6845 20130101; B01J 2219/00612
20130101; B82Y 30/00 20130101; B01J 2219/00637 20130101; B01J
2219/00617 20130101; B01J 2219/00619 20130101; B01J 2219/0061
20130101; B01J 2219/00605 20130101; B01J 2219/00635 20130101; G01N
33/551 20130101; G01N 33/54393 20130101; B01J 2219/00641 20130101;
B01J 2219/00626 20130101; B01J 2219/00725 20130101; B01J 2219/00659
20130101; C07K 2319/20 20130101; C40B 40/10 20130101 |
Class at
Publication: |
436/518 ;
435/287.2 |
International
Class: |
G01N 033/543; C12M
001/34 |
Claims
What is claimed is:
1. An array of proteins, comprising: (a) a substrate; (b) at least
one organic thinfilm on some or all of the substrate surface; and
(c) a plurality of patches arranged in discrete, known regions on
portions of the substrate surface covered by organic thinfilm,
wherein each of said patches comprises a protein immobilized on the
underlying organic thinfilm.
2. The array of claim 1 which comprises at least about 10 of said
patches.
3. The array of claim 2 which comprises at least about 100 of said
patches.
4. The array of claim 3 which comprises at least about 10.sup.3 of
said patches.
5. The array of claim 1 which comprises at least about 10 different
immobilized proteins.
6. The array of claim 5 which comprises at least about 100
different immobilized proteins.
7. The array of claim 6 which comprises at least about 1000
different immobilized proteins.
8. The array of claim 1, wherein the area of the substrate surface
covered by each of the patches is no more than about 0.25
mm.sup.2.
9. The array of claim 8, wherein the area of the substrate surface
covered by each of the patches is between about 1 .mu.m.sup.2 and
about 10,000 .mu.m.sup.2.
10. The array of claim 1, wherein the patches are all contained
within an area of about 1 cm.sup.2 or less on the surface of the
substrate.
11. The array of claim 1, wherein all of the proteins immobilized
on the array are functionally related.
12. The array of claim 1, wherein all of the proteins immobilized
on the array are structurally related.
13. The array of claim 1, wherein all of the proteins immobilized
on the array are members of the same family.
14. An array of claim 13, wherein said family is selected from the
group consisting of growth factor receptors, hormone receptors,
neurotransmitter receptors, catecholamine receptors, amino acid
derivative receptors, cytokine receptors, extracellular matrix
receptors, antibodies, lectins, cytokines, serpins, proteases,
kinases, phosphatases, ras-like GTPases, 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, hepatitis C virus (HCV) proteases and HIV
proteases.
15. The array of claim 1, wherein the proteins are antibodies or
antibody fragments.
16. The array of claim 1, wherein the proteins are protein-capture
agents.
17. The array of claim 1, wherein the organic thinfilm on the array
is less than about 20 nm thick.
18. The array of claim 1, wherein the organic thinfilm on the array
comprises a monolayer.
19. The array of claim 18, wherein the monolayer comprises a
self-assembled monolayer comprising molecules of the formula
(X).sub.aR(Y).sub.b wherein R is a spacer, X is a functional group
that binds R to the surface, Y is a functional group for binding
the protein onto the monolayer, and a and b are, independently,
integers.
20. The array of claim 19, wherein both a and b are 1.
21. The array of claim 19, wherein: said substrate is selected from
the group consisting of silicon, silicon dioxide, indium tin oxide,
alumina, glass, and titania; and X, prior to incorporation into
said monolayer, is selected from the group consisting of a
monohalosilane, dihalosilane, trihalosilane, trichlorosilane,
trialkoxysilane, dialkoxysilane, monoalkoxysilane, carboxylic
acids, and phosphates.
22. The array of claim 19, wherein the substrate comprises silicon
and X is an olefin.
23. The array of claim 1, wherein the substrate comprises a
polymer.
24. The array of claim 19, further comprising at least one coating
between said substrate and said monolayer, wherein said coating is
formed on the substrate or applied to the substrate.
25. The array of claim 24, wherein: the coating comprises a noble
metal film; and X prior to incorporation into said monolayer, is a
functional group selected from the group consisting of an
asymmetrical or symmetrical disulfide, sulfide, diselenide,
selenide, thiol, isonitrile, selenol, trivalent phosphorus
compounds, isothiocyanate, isocyanate, xanthanate, thiocarbamate,
phosphines, amines, thio acid and dithio acid.
26. The array of claim 24, wherein the coating comprises titania or
tantalum oxide and X is a phosphate group.
27. The array of claim 1, wherein each protein is immobilized on
the organic thinfilm by an affinity tag.
28. A biosensor comprising an array of proteins of claim 1.
29. A micromachined device comprising an array of proteins of claim
1.
30. A diagnostic device comprising an array of proteins of claim
1.
31. A method for screening a plurality of proteins for their
ability to interact with a component of a sample, comprising: (a)
delivering the sample to the array of claim 1 comprising the
proteins to be screened; and (b) detecting, either directly or
indirectly, for the interaction of said component with the
immobilized protein of each patch.
32. The method of claim 31, wherein the component is a protein.
33. A method for screening a plurality of proteins for their
ability to bind a particular component of a sample, comprising: (a)
delivering said sample to the array of claim 1 comprising the
proteins to be screened; and (b) detecting, either directly or
indirectly, for the presence or amount of said particular component
retained at each patch.
34. The method of claim 33, wherein said particular component is a
protein.
35. The method of claim 33, further comprising the step: (d)
further characterizing said particular component retained on at
least one patch.
36. A method of assaying for protein-protein binding interactions,
comprising: (a) delivering a sample comprising at least one protein
to be assayed for binding to the array of claim 1; and (b)
detecting, either directly or indirectly, for the presence or
amount of the protein from the sample which is retained at each
patch.
37. A method of assaying in parallel for a plurality of analytes in
a sample, comprising: (a) delivering the sample to the array of
claim 1, wherein at least one of the immobilized proteins of said
array can react with each of said analytes; and (b) detecting for
the interaction of the analytes with the immobilized protein at
each patch.
38. A method of assaying in parallel for a plurality of analytes in
a sample, comprising: (a) delivering the fluid sample to the array
of claim 1, wherein at least one of the immobilized proteins of
said array can bind each of said analytes; and (b) detecting,
either directly or indirectly, for the presence or amount of
analyte retained at each patch.
39. The method of claim 38, further comprising the step: (d)
further characterizing the analyte retained on at least one patch.
Description
[0001] This application is a continuation of co-pending application
Ser. No. 09/353,215, filed Jul. 14, 1999, which is a
continuation-in-part of co-pending application Ser. No. 09/115,455,
filed Jul. 14, 1998, both of which are incorporated herein by
reference in their entirety for all purposes and the specific
purposes disclosed throughout this application.
BACKGROUND OF THE INVENTION
[0002] a) Field of the Invention
[0003] The present invention relates generally to arrays of
proteins and methods for the parallel in vitro screening of a
plurality of protein-analyte interactions. More specifically, the
present invention relates to uses of the arrays for drug
development, proteomics, and clinical diagnostics.
[0004] b) Description of Related Art
[0005] A vast number of new drug targets are now being identified
using a combination of genomics, bioinformatics, genetics, and
high-throughput biochemistry. Genomics provides information on the
genetic composition and the activity of an organism's genes.
Bioinformatics uses computer algorithms to recognize and predict
structural patterns in DNA and proteins, defining families of
related genes and proteins. The information gained from the
combination of these approaches is expected to greatly boost the
number of drug targets (usually, proteins).
[0006] The number of chemical compounds available for screening as
potential drugs is also growing dramatically due to recent advances
in combinatorial chemistry, the production of large numbers of
organic compounds through rapid parallel and automated synthesis.
The compounds produced in the combinatorial libraries being
generated will far outnumber those compounds being prepared by
traditional, manual means, natural product extracts, or those in
the historical compound files of large pharmaceutical
companies.
[0007] Both the rapid increase of new drug targets and the
availability of vast libraries of chemical compounds creates an
enormous demand for new technologies which improve the screening
process. Current technological approaches which attempt to address
this need include multiwell-plate based screening systems,
cell-based screening systems, microfluidics-based screening
systems, and screening of soluble targets against solid-phase
synthesized drug components.
[0008] Automated multiwell formats are the best developed
high-throughput screening systems. Automated 96-well plate-based
screening systems are the most widely used. The current trend in
plate based screening systems is to reduce the volume of the
reaction wells further, thereby increasing the density of the wells
per plate (96-well to 384- and 1536-well per plate). The reduction
in reaction volumes results in increased throughput, dramatically
decreased bioreagent costs, and a decrease in the number of plates
which need to be managed by automation.
[0009] However, although increases in well numbers per plate are
desirable for high throughput efficiency, the use of volumes
smaller than 1 microliter in the well format generates significant
problems with evaporation, dispensing times, protein inactivation,
and assay adaptation. Proteins are very sensitive to the physical
and chemical properties of the reaction chamber surfaces. Proteins
are prone to denaturation at the liquid/solid and liquid/air
interfaces. Miniaturization of assays to volumes smaller than 1
microliter increases the surface to volume ratio substantially.
(Changing volumes from 1 microliter to 10 nanoliter increases the
surface ratio by 460%, leading to increased protein inactivation.)
Furthermore, solutions of submicroliter volumes evaporate rapidly,
within seconds to a few minutes, when in contact with air.
Maintaining microscopic volumes in open systems is therefore very
difficult.
[0010] Other types of high-throughput assays, such as miniaturized
cell-based assays are also being developed. Miniaturized cell-based
assays have the potential to generate screening data of superior
quality and accuracy, due to their in vivo nature. However, the
interaction of drug compounds with proteins other than the desired
targets is a serious problem related to this approach which leads
to a high rate of false positive results.
[0011] Microfluidics-based screening systems that measure in vitro
reactions in solution make use of ten to several-hundred micrometer
wide channels. Micropumps, electroosmotic flow, integrated valves
and mixing devices control liquid movement through the channel
network. Microfluidic networks prevent evaporation but, due to the
large surface to volume ratio, result in significant protein
inactivation. The successful use of microfluidic networks in
biomolecule screening remains to be shown.
[0012] Drug screening of soluble targets against solid-phase
synthesized drug components is intrinsically limited. The surfaces
required for solid state organic synthesis are chemically diverse
and often cause the inactivation or non-specific binding of
proteins, leading to a high rate of false-positive results.
Furthermore, the chemical diversity of drug compounds is limited by
the combinatorial synthesis approach that is used to generate the
compounds at the interface. Another major disadvantage of this
approach stems from the limited accessibility of the binding site
of the soluble target protein to the immobilized drug
candidates.
[0013] Miniaturized DNA chip technologies have been developed (for
example, see U.S. Pat. Nos. 5,412,087, 5,445,934 and 5,744,305) and
are currently being exploited for nucleic acid hybridization
assays. However, DNA biochip technology is not transferable to
protein arrays because the chemistries and materials used for DNA
biochips are not readily transferable to use with proteins. Nucleic
acids withstand temperatures up to 100.degree. C., can be dried and
re-hydrated without loss of activity, and can be bound directly to
organic adhesion layers supported by materials such as glass while
maintaining their activity. In contrast, proteins must remain
hydrated, kept at ambient temperatures, and are very sensitive to
the physical and chemical properties of the support materials.
Therefore, maintaining protein activity at the liquid-solid
interface requires entirely different immobilization strategies
than those used for nucleic acids. Additionally, the proper
orientation of the protein at the interface is desirable to ensure
accessibility of their active sites with interacting molecules.
With miniaturization of the chip and decreased feature sizes the
ratio of accessible to non-accessible antibodies becomes
increasingly relevant and important.
[0014] In addition to the goal of achieving high-throughput
screening of compounds against targets to identify potential drug
leads, researchers also need to be able to identify highly specific
lead compounds early in the drug discovery process. Analyzing a
multitude of members of a protein family or forms of a polymorphic
protein in parallel (multitarget screening) enables quick
identification of highly specific lead compounds. Proteins within a
structural family share similar binding sites and catalytic
mechanisms. Often, a compound that effectively interferes with the
activity of one family member also interferes with other members of
the same family. Using standard technology to discover such
additional interactions requires a tremendous effort in time and
costs and as a consequence is simply not done.
[0015] However, cross-reactivity of a drug with related proteins
can be the cause of low efficacy or even side effects in patients.
For instance, AZT, a major treatment for AIDS, blocks not only
viral polymerases, but also human polymerases, causing deleterious
side effects. Cross-reactivity with closely related proteins is
also a problem with nonsteroidal anti-inflammatory drugs (NSAIDs)
and aspirin. These drugs inhibit cyclooxygenase-2, an enzyme which
promotes pain and inflammation. However, the same drugs also
strongly inhibit a related enzyme, cyclooxygenase-1, that is
responsible for keeping the stomach lining and kidneys healthy,
leading to common side-effects including stomach irritation.
[0016] For the foregoing reasons, there is a need for miniaturized
protein arrays and for methods for the parallel, in vitro,
screening of the interactions between a plurality of proteins and
one or more analytes in a manner that minimizes reagent volumes and
protein inactivation problems.
SUMMARY OF THE INVENTION
[0017] The present invention is directed to miniaturized protein
arrays and methods of use thereof that satisfy the need for
parallel, in vitro, screening of the interactions between a
plurality of proteins and one or more analytes in a manner that
minimizes reagent Volumes and protein inactivation problems.
[0018] In one embodiment, the present invention provides an array
of proteins which comprises a substrate, at least one organic
thinfilm on some or all of the substrate surface, and a plurality
of patches arranged in discrete, known regions on portions of the
substrate surface covered by organic thinfilm, wherein each of said
patches comprises a protein immobilized on the underlying organic
thinflm. Preferably, a plurality of different proteins are present
on separate patches of the array.
[0019] In a second embodiment, the invention provides a method for
screening a plurality of proteins for their ability to interact
with a component of a sample. The method of this embodiment
comprises delivering the sample to the array of proteins of the
invention, and detecting, either directly or indirectly, for the
interaction of the component with the immobilized protein of each
patch.
[0020] In a third embodiment, the invention provides a method for
screening a plurality of proteins for their ability to bind a
particular component of a sample. The method of this embodiment
comprises first delivering the sample to the array of proteins of
the invention. In a final step, the method comprises detecting,
either directly or indirectly, for the presence or amount of the
particular component which is retained at each patch. Optionally,
the method comprises the additional step of further characterizing
the particular component retained at the site of at least one
patch.
[0021] In an alternative embodiment, the invention provides a
method of assaying for protein-protein binding interactions. The
first step of the method of this embodiment comprises delivering a
sample comprising at least one protein to be assayed for binding to
the protein array of the invention. The last step comprises
detecting, either directly or indirectly, for the presence or
amount of the protein from the sample which is retained at each
patch.
[0022] In another embodiment of the invention, a method for
assaying for a plurality of analytes in a sample is provided which
comprises delivering the sample to a protein array of the invention
and detecting for the interaction of the analytes with the
immobilized protein at each patch.
[0023] In still another embodiment of the invention, an alternative
method for assaying for a plurality of analytes in a sample is
provided which comprises delivering the fluid sample to a protein
array of the invention and detecting either directly or indirectly,
for the presence or amount of analyte retained at each patch.
BRIEF DESCRIPTION OF THE FIGURES
[0024] FIG. 1 shows the top view of an array of protein-reactive
patches.
[0025] FIG. 2 shows the cross section of an individual patch of the
array of FIG. 1.
[0026] FIG. 3 shows the cross section of a row of monolayer-covered
patches of the array of FIG. 1.
[0027] FIG. 4 shows a thiolreactive monolayer on a substrate.
[0028] FIG. 5 shows an aminoreactive monolayer on a coated
substrate.
[0029] FIG. 6 shows the immobilization of a protein on a
monolayer-coated substrate via an affinity tag.
[0030] FIG. 7 shows the immobilization of a protein on a
monolayer-coated substrate via an affinity tag and an adaptor.
[0031] FIG. 8 shows a schematic of a fluorescence detection unit
which may be used to monitor interaction of the proteins of the
array with an analyte.
[0032] FIG. 9 shows a schematic of an ellipsometric detection unit
which may be used to monitor interactions between analytes and the
proteins of the array.
DETAILED DESCRIPTION OF THE INVENTION
[0033] A variety of protein arrays, methods, and protein-coated
substrates useful for drug development, proteomics, clinical
diagnostics, and related applications are provided by the present
invention.
[0034] (a) Definitions
[0035] A "protein" means a polymer of amino acid residues linked
together by peptide bonds. The term, as used herein, refers to
proteins, polypeptides, and peptides of any size, structure, or
function. Typically, however, a protein will be at least six amino
acids long. Preferably, if the protein is a short peptide, it will
be at least about 10 amino acid residues long. A protein may be
naturally occurring, recombinant, or synthetic, or any combination
of these. A protein may also be just a fragment of a naturally
occurring protein or peptide. A protein may be a single molecule or
may be a multi-molecular complex. The term protein may also apply
to amino acid polymers in which one or more amino acid residues is
an artificial chemical analogue of a corresponding naturally
occurring amino acid. An amino acid polymer in which one or more
amino acid residues is an "unnatural" amino acid, not corresponding
to any naturally occurring amino acid, is also encompassed by the
use of the term "protein" herein.
[0036] A "fragment of a protein" means a protein which is a portion
of another protein.
[0037] For instance, fragments of a proteins may be polypeptides
obtained by digesting full-length protein isolated from cultured
cells. A fragment of a protein will typically comprise at least six
amino acids. More typically, the fragment will comprise at least
ten amino acids. Preferably, the fragment comprises at least about
16 amino acids.
[0038] The term "antibody" means an immunoglobulin, whether natural
or wholly or partially synthetically produced. All derivatives
thereof which maintain specific binding ability are also included
in the term. The term also covers any protein having a binding
domain which is homologous or largely homologous to an
immunoglobulin binding domain. These proteins may be derived from
natural sources, or partly or wholly synthetically produced. An
antibody may be monoclonal or polyclonal. The antibody may be a
member of any immunoglobulin class, including any of the human
classes: IgG, IgM, IgA, IgD, and IgE. Derivatives of the IgG class,
however, are preferred in the present invention.
[0039] The term "antibody fragment" refers to any derivative of an
antibody which is less than full-length. Preferably, the antibody
fragment retains at least a significant portion of the full-length
antibody's specific binding ability. Examples of antibody fragments
include, but are not limited to, Fab, Fab', F(ab').sub.2, scFv, Fv,
dsFv diabody, and Fd fragments. The antibody fragment may be
produced by any means. For instance, the antibody fragment may be
enzymatically or chemically produced by fragmentation of an intact
antibody or it may be recombinantly produced from a gene encoding
the partial antibody sequence. Alternatively, the antibody fragment
may be wholly or partially synthetically produced. The antibody
fragment may optionally be a single chain antibody fragment.
Alternatively, the fragment may comprise multiple chains which are
linked together, for instance, by disulfide linkages. The fragment
may also optionally be a multimolecular complex. A functional
antibody fragment will typically comprise at least about 50 amino
acids and more typically will comprise at least about 200 amino
acids.
[0040] Single-chain Fvs (scFvs) are recombinant antibody fragments
consisting of only the variable light chain (V.sub.L) and variable
heavy chain (V.sub.H) covalently connected to one another by a
polypeptide linker. Either V.sub.L or V.sub.H may be the
NH.sub.2-terminal domain. The polypeptide linker may be of variable
length and composition so long as the two variable domains are
bridged without serious steric interference. Typically, the linkers
are comprised primarily of stretches of glycine and serine residues
with some glutamic acid or lysine residues interspersed for
solubility.
[0041] "Diabodies" are dimeric scFvs. The components of diabodies
typically have shorter peptide linkers than most scFvs and they
show a preference for associating as dimers.
[0042] An "Fv" fragment is an antibody fragment which consists of
one VH and one VL domain held together by noncovalent interactions.
The term "dsFv" is used herein to refer to an Fv with an engineered
intermolecular disulfide bond to stabilize the V.sub.H-V.sub.L
pair.
[0043] A "F(ab').sub.2" fragment is an antibody fragment
essentially equivalent to that obtained from immunoglobulins
(typically IgG) by digestion with an enzyme pepsin at pH 4.04.5.
The fragment may be recombinantly produced.
[0044] A "Fab'" fragment is an antibody fragment essentially
equivalent to that obtained by reduction of the disulfide bridge or
bridges joining the two heavy chain pieces in the F(ab').sub.2
fragment. The Fab' fragment may be recombinantly produced.
[0045] A "Fab" fragment is an antibody fragment essentially
equivalent to that obtained by digestion of immunoglobulins
(typically IgG) with the enzyme papain. The Fab fragment may be
recombinandy produced. The heavy chain segment of the Fab fragment
is the Fd piece.
[0046] The term "protein-capture agent" means a molecule or a
multi-molecular complex which can bind a protein to itself.
Protein-capture agents preferably bind their binding partners in a
substantially specific manner. Protein-capture agents with a
dissociation constant (K.sub.D) of less than about 10.sup.-6 are
preferred. Antibodies or antibody fragments are highly suitable as
protein-capture agents. Antigens may also serve as protein-capture
agents, since they are capable of binding antibodies. A receptor
which binds a protein ligand is another example of a possible
protein-capture agent. Protein-capture agents are understood not to
be limited to agents which only interact with their binding
partners through noncovalent interactions. Protein-capture agents
may also optionally become covalently attached to the proteins
which they bind. For instance, the protein-capture agent may be
photocrosslinked to its binding partner following binding.
[0047] The term "binding partner" means a protein which is bound by
a particular protein-capture agent, preferably in a substantially
specific manner. In some cases, the binding partner may be the
protein normally bound in vivo by a protein which is a
protein-capture agent. In other embodiments, however, the binding
partner may be the protein or peptide on which the protein-capture
agent was selected (through in vitro or in vivo selection) or
raised (as in the case of antibodies). A binding partner may be
shared by more than one protein-capture agent. For instance, a
binding partner which is bound by a variety of polyclonal
antibodies may bear a number of different epitopes. One
protein-capture agent may also bind to a multitude of binding
partners (for instance, if the binding partners share the. same
epitope),
[0048] "Conditions suitable for protein binding" means those
conditions (in terms of salt concentration, pH, detergent, protein
concentration, temperature, etc.) which allow for binding to occur
between a protein and its binding partner in solution. Preferably,
the conditions are not so lenient that a significant amount of
nonspecific protein binding occurs.
[0049] A "body fluid" may be any liquid substance extracted,
excreted, or secreted from an organism or tissue of an organism.
The body fluid need not necessarily contain cells. Body fluids of
relevance to the present invention include, but are not limited to,
whole blood, serum, urine, plasma, cerebral spinal fluid, tears,
sinovial fluid, and amniotic fluid.
[0050] An "array" is an arrangement of entities in a pattern on a
substrate. Although the pattern is typically a two-dimensional
pattern, the pattern may also be a three-dimensional pattern.
[0051] The term "substrate" refers to the bulk, underlying, and
core material of the arrays of the invention.
[0052] The terms "micromachining" and "microfabrication" both refer
to any number of techniques which are useful in the generation of
microstructures (structures with feature sizes of sub-millimeter
scale). Such technologies include, but are not limited to, laser
ablation, electrodeposition, physical and chemical vapor
deposition, photolithography, and wet chemical and dry etching.
Related technologies such as injection molding and LIGA (x-ray
lithography, electrodeposition, and molding) are also included.
Most of these techniques were originally developed for use in
semiconductors, microelectronics, and Micro-ElectroMechanical
Systems (MEMS) but are applicable to the present invention as
well.
[0053] The term "coating" means a layer that is either naturally or
synthetically formed on or applied to the surface of the substrate.
For instance, exposure of a substrate, such as silicon, to air
results in oxidation of the exposed surface. In the case of a
substrate made of silicon, a silicon oxide coating is formed on the
surface upon exposure to air. In other instances, the coating is
not derived from the substrate and may be placed upon the surface
via mechanical, physical, electrical, or chemical means. An example
of this type of coating would be a metal coating that is applied to
a silicon or polymer substrate or a silicon nitride coating that is
applied to a silicon substrate. Although a coating may be of any
thickness, typically the coating has a thickness smaller than that
of the substrate.
[0054] An "interlayer" is an additional coating or layer that is
positioned between the first coating and the substrate. Multiple
interlayers may optionally be used together. The primary purpose of
a typical interlayer is to aid adhesion between the first coating
and the substrate. One such example is the use of a titanium or
chromium interlayer to help adhere a gold coating to a silicon or
glass surface. However, other possible functions of an interlayer
are also anticipated. For instance, some interlayers may perform a
role in the detection system of the array (such as a semiconductor
or metal layer between a nonconductive substrate and a
nonconductive coating).
[0055] An "organic thinfilm" is a thin layer of organic molecules
which has been applied to a substrate or to a coating on a
substrate if present. Typically, an organic ini is less than about
20 nm thick. Optionally, an organic thinfilm may be less than about
10 nm thick. An organic thinfilm may be disordered or ordered. For
instance, an organic thinfim can be amorphous (such as a
chemisorbed or spin-coated polymer) or highly organized (such as a
Langmuir-Blodgett film or self-assembled monolayer). An organic
thinfilm may be heterogeneous or homogeneous. Organic thinfilms
which are monolayers are preferred. A lipid bilayer or monolayer is
a preferred organic thinfilm. Optionally, the organic thinfilm may
comprise a combination of more than one form of organic thinfilm.
For instance, an organic thinfilm may comprise a lipid bilayer on
top of a self-assembled monolayer. A hydrogel may also compose an
organic thinfilm. The organic thinfilm will typically have
functionalities exposed on its surface which serve to enhance the
surface conditions of a substrate or the coating on a substrate in
any of a number of ways. For instance, exposed functionalities of
the organic thinfilm are typically useful in the binding or
covalent immobilization of the proteins to the patches of the
array. Alternatively, the organic thinflim may bear functional
groups (such as polyethylene glycol (PEG)) which reduce the
non-specific binding of molecules to the surface. Other exposed
functionalities serve to tether the thinfilm to the surface of the
substrate or the coating. Particular functionalities of the organic
thinfilm may also be designed to enable certain detection
techniques to be used with the surface. Alternatively, the organic
thinfilm may serve the purpose of preventing inactivation of a
protein immobilized on a patch of the array or analytes which are
proteins from occurring upon contact with the surface of a
substrate or a coating on the surface of a substrate.
[0056] A "monolayer" is a single-molecule thick organic thinfilm. A
monolayer may be disordered or ordered. A monolayer may optionally
be a polymeric compound, such as a polynonionic polymer, a
polyionic polymer, or a block-copolymer. For instance, the
monolayer may be composed of a poly(amino acid) such as polylysine.
A monolayer which is a self-assembled monolayer, however, is most
preferred. One face of the self-assembled monolayer is typically
composed of chemical functionalities on the termini of the organic
molecules that are chemisorbed or physisorbed onto the surface of
the substrate or, if present, the coating on the substrate.
Examples of suitable functionalities of monolayers include the
positively charged amino groups of poly-L-lysine for use on
negatively charged surfaces and thiols for use on gold surfaces.
Typically, the other face of the self-assembled monolayer is
exposed and may bear any number of chemical functionalities (end
groups). Preferably, the molecules of the self-assembled monolayer
are highly ordered.
[0057] A "self-assembled monolayer" is a monolayer which is created
by the spontaneous assembly of molecules. The self-assembled
monolayer may be ordered, disordered, or exhibit short- to
long-range order.
[0058] An "affinity tag" is a functional moiety capable of directly
or indirectly immobilizing a protein onto an exposed functionality
of the organic thinfilm. Preferably, the affinity tag enables the
site-specific immobilization and thus enhances orientation of the
protein onto the organic thinfilm. In some cases, the affinity tag
may be a simple chemical functional group. Other possibilities
include amino acids, poly(amino acid) tags, or full-length
proteins. Still other possibilities include carbohydrates and
nucleic acids. For instance, the affinity tag may be a
polynucleotide which hybridizes to another polynucleotide serving
as a functional group on the organic thinfilm or another
polynucleotide serving as an adaptor. The affinity tag may also be
a synthetic chemical moiety. If the organic thinfilm of each of the
patches comprises a lipid bilayer or monolayer, then a membrane
anchor is a suitable affinity tag. The affinity tag may be
covalently or noncovalently attached to the protein. For instance,
if the affinity tag is covalently attached to the protein it may be
attached via chemical conjugation or as a fusion protein. The
affinity tag may also be attached to the protein via a cleavable
linkage. Alternatively, the affinity tag may not be directly in
contact with the protein. The affinity tag may instead be separated
from the protein by an adaptor. The affinity tag may immobilize the
protein to the organic thinfilm either through noncovalent
interactions or through a covalent linkage.
[0059] An "adaptor", for purposes of this invention, is any entity
that links an affinity tag to the immobilized protein of a patch of
the array. The adaptor may be, but need not necessarily be, a
discrete molecule that is noncovalently attached to both the
affinity tag and the protein. The adaptor can instead be covalently
attached to the affinity tag or the protein or both (via chemical
conjugation or as a fusion protein, for instance). Proteins such as
full-length proteins, polypeptides, or peptides are typical
adaptors. Other possible adaptors include carbohydrates and nucleic
acids.
[0060] The term "fusion protein" refers to a protein composed of
two or more polypeptides that, although typically unjoined in their
native state, are joined by their respective amino and carboxyl
termini through a peptide linkage to form a single continuous
polypeptide. It is understood that the two or more polypeptide
components can either be directly joined or indirectly joined
through a peptide linker/spacer.
[0061] The term "normal physiological condition" means conditions
that are typical inside a living organism or a cell. While it is
recognized that some organs or organisms provide extreme
conditions, the intra-organismal and intra-cellular environment
normally varies around pH 7 (i.e., from pH 6.5 to pH 7.5), contains
water as the predominant solvent, and exists at a temperature above
0.degree. C. and below 50.degree. C. It will be recognized that the
concentration of various salts depends on the organ, organism,
cell, or cellular compartment used as a reference.
[0062] "Proteomics" means the study of or the characterization of
either the proteome or some fraction of the proteome. The
"proteome" is the total collection of the intracellular proteins of
a cell or population of cells and the proteins secreted by the cell
or population of cells. This characterization most typically
includes measurements of the presence, and usually quantity, of the
proteins which have been expressed by a cell. The function,
structural characteristics (such as post translational
modification), and location within the cell of the proteins may
also be studied. "Functional proteomics" refers to the study of the
functional characteristics, activity level, and structural
characteristics of the protein expression products of a cell or
population of cells.
[0063] (b) Arrays of Proteins.
[0064] The present invention is directed to arrays of proteins.
Typically, the protein arrays comprise micrometer-scale,
two-dimensional patterns of patches of proteins immobilized on an
organic thinfilm coating on the surface of the substrate.
[0065] In one embodiment, the present invention provides an array
of proteins which comprises a substrate, at least one organic
thinfilm on some or all of the substrate surface, and a plurality
of patches arranged in discrete, known regions on portions of the
substrate surface covered by organic thinfilm, wherein each of said
patches comprises a protein immobilized on the underlying organic
thinfilm.
[0066] In most cases, the array will comprise at least about ten
patches. In a preferred embodiment, the array comprises at least
about 50 patches. In a particularly preferred embodiment the array
comprises at least about 100 patches. In alternative preferred
Embodiments, the array of proteins may comprise more than 10.sup.3,
10.sup.4 or 10.sup.5 patches.
[0067] The area of surface of the substrate covered by each of the
patches is preferably no more than about 0.25 mm.sup.2. Preferably,
the area of the substrate surface covered by each of the patches is
between about 1 .mu.m.sup.2 and about 10,000 .mu.m.sup.2. In a
particularly preferred embodiment, each patch covers an area of the
substrate surface from about 100 .mu.m.sup.2 to about 2,500
.mu.m.sup.2. In an alternative embodiment, a patch on the array may
cover an area of the substrate surface as small as about 2,500
nm.sup.2, although patches of such small size are generally not
necessary for the use of the array.
[0068] The patches of the array may be of any geometric shape. For
instance, the patches may be rectangular or circular. The patches
of the array may also be irregularly shaped.
[0069] The distance separating the patches of the array can vary.
Preferably, the patches of the array are separated from neighboring
patches by about 1 .mu.m to about 500 .mu.m. Typically, the
distance separating the patches is roughly proportional to the
diameter or side length of the patches on the array if the patches
have dimensions greater than about 10 .mu.m. If the patch size is
smaller, then the distance separating the patches will typically be
larger than the dimensions of the patch.
[0070] In a preferred embodiment of the array, the patches of the
array are all contained within an area of about 1 cm.sup.2 or less
on the surface of the substrate. In one preferred embodiment of the
array, therefore, the array comprises 100 or more patches within a
total area of about 1 cm.sup.2 or less on the surface of the
substrate. Alternatively, a particularly preferred array comprises
10.sup.3 or more patches within a total area of about 1 cm.sup.2 or
less. A preferred array may even optionally comprise 10.sup.4 or
10.sup.5 or more patches within an area of about 1 cm.sup.2 or less
on the surface of the substrate. In other embodiments of the
invention, all of the patches of the array are contained within an
area of about 1 mm.sup.2 or less on the surface of the
substrate.
[0071] Typically, only one type of protein is immobilized on each
patch of the array. In a preferred embodiment of the array, the
protein immobilized on one patch differs from the protein
immobilized on a second patch of the same array. In such an
embodiment, a plurality of different proteins are present on
separate patches of the array. Typically the array comprises at
least about ten different proteins. Preferably, the array comprises
at least about 50 different proteins. More preferably, the array
comprises at least about 100 different proteins. Alternative
preferred arrays comprise more than about 10.sup.3 different
proteins or more than about 10.sup.4 different proteins. The array
may even optionally comprise more than about 10.sup.5 different
proteins.
[0072] In one embodiment of the array, each of the patches of the
array comprises a different protein. For instance, an array
comprising about 100 patches could comprise about 100 different
proteins. Likewise, an array of about 10,000 patches could comprise
about 10,000 different proteins. In an alternative embodiment,
however, each different protein is immobilized on more than one
separate patch on the array. For instance, each different protein
may optionally be present on two to six different patches. An array
of the invention, therefore, may comprise about three-thousand
protein patches, but only comprise about one thousand different
proteins since each different protein is present on three different
patches.
[0073] In another embodiment of the present invention, although the
protein of one patch is different from that of another, the
proteins are related. In a preferred embodiment, the two different
proteins are members of the same protein family. The different
proteins on the invention array may be either functionally related
or just suspected of being functionally related. In another
embodiment of the invention array, however, the function of the
immobilized proteins may be unknown. In this case, the different
proteins on the different patches of the array share a similarity
in structure or sequence or are simply suspected of sharing a
similarity in structure or sequence. Alternatively, the immobilized
proteins may be just fragments of different members of a protein
family.
[0074] The proteins immobilized on the array of the invention may
be members of a protein family such as a receptor family (examples:
growth factor receptors, catecholamine receptors, amino acid
derivative receptors, cytokine receptors, lectins), ligand family
(examples: cytokines, serpins), enzyme family (examples: proteases,
kinases, phosphatases, ras-like GTPases, hydrolases), and
transcription factors (examples: steroid hormone receptors,
heat-shock transcription factors, zinc-finger proteins,
leucinezipper proteins, homeodomain proteins). In one embodiment,
the different immobilized proteins are all HIV proteases or
hepatitis C virus (HCV) proteases. In other embodiments of the
invention, the immobilized proteins on the patches of the array are
all hormone receptors, neurotransmitter receptors, extracellular
matrix receptors, antibodies, DNA-binding proteins, intracellular
signal transduction modulators and effectors, apoptosis-related
factors, DNA synthesis factors, DNA repair factors, DNA
recombination factors, or cell-surface antigens.
[0075] In a preferred embodiment, the protein immobilized on each
patch is an antibody or antibody fragment. The antibodies or
antibody fragments of the array may optionally be single-chain Fvs,
Fab fragments, Fab' fragments, F(ab').sub.2 fragments, Fv
fragments, dsFvs diabodies, Fd fragments, full-length,
antigen-specific polyclonal antibodies, or full-length monoclonal
antibodies. In a preferred embodiment, the immobilized proteins on
the patches of the array are monoclonal antibodies, Fab fragments
or single-chain Fvs.
[0076] In another preferred embodiment of the invention, the
proteins immobilized to each patch of the array are protein-capture
agents.
[0077] In an alternative embodiment of the invention array, the
proteins on different patches are identical.
[0078] Biosensors, micromachined devices, and diagnostic devices
that comprise the protein arrays of the invention are also
contemplated by the present invention.
[0079] (c) Substrates, Coating, and Organic Thinfilms.
[0080] The substrate of the array may be either organic or
inorganic, biological or non-biological, or any combination of
these materials. In one embodiment, the substrate is transparent or
translucent. The portion of the surface of the substrate on which
the patches reside is preferably flat and firm or semi-film.
However, the array of the present invention need not necessarily be
flat or entirely two-dimensional. Significant topological features
may be present on the surface of the substrate surrounding the
patches, between the patches or beneath the patches. For instance,
walls or other barriers may separate the patches of the array.
[0081] Numerous materials are suitable for use as a substrate in
the array embodiment of the invention. For instance, the substrate
of the invention array can comprise a material selected from a
group consisting of silicon, silica, quartz, glass, controlled pore
glass, carbon, alumina, titania, tantalum oxide, germanium, silicon
nitride, zeolites, and gallium arsenide. Many metals such as gold,
platinum, aluminum, copper, titanium, and their alloys are also
options for substrates of the array. In addition, many ceramics and
polymers may also be used as substrates. Polymers which may be used
as substrates include, but are not limited to, the following:
polystyrene; poly(tetra)fluoroethylene (PTFE);
polyvinylidenedifluoride; polycarbonate; polymethylmethacrylate;
polyvinylethylene; polyethyleneimine; poly(etherether)ketone;
polyoxymethylene (POM); polyvinylphenol; polylactides;
polymethacrylimide (PI); polyalkenesulfone (PAS); polypropylene;
polyethylene; polyhydroxyethylmethacrylate (HEMA);
polydimethylsiloxane; polyacrylanmide; polyimide; and
block-copolymers. Preferred substrates for the array include
silicon, silica, glass, and polymers. The substrate on which the
patches reside may also be a combination of any of the
aforementioned substrate materials.
[0082] An array of the present invention may optionally further
comprise a coating between the substrate and organic thinfilm on
the array. This coating may either be formed on the substrate or
applied to the substrate. The substrate can be modified with a
coating by using thin-film technology based, for example, on
physical vapor deposition (PVD), thermal processing, or
plasma-enhanced chemical vapor deposition (PECVD). Alternatively,
plasma exposure can be used to directly activate or alter the
substrate and create a coating. For instance, plasma etch
procedures can be used to oxidize a polymeric surface (i.e.,
polystyrene or polyethylene to expose polar functionalities such as
hydroxyls, carboxylic acids, aldehydes and the like).
[0083] The coating is optionally a metal film. Possible metal films
include aluminum, chromium, titanium, tantalum, nickel, stainless
steel, zinc, lead, iron, copper, magnesium,. manganese, cadmium,
tungsten, cobalt, and alloys or oxides thereof. In a preferred
embodiment, the metal film is a noble metal film. Noble metals that
may be used for a coating include, but are not limited to, gold,
platinum, silver, and copper. In an especially preferred
embodiment, the coating comprises gold or a gold alloy.
Electron-beam evaporation may be used to provide a thin coating of
gold on the surface of the substrate. In a preferred embodiment,
the metal film is from about 50 nm to about 500 nm in thickness. In
an alternative embodiment, the metal film is from about 1 nm to
about 1 .mu.m in thickness.
[0084] In alternative embodiments, the coating comprises a
composition selected from the group consisting of silicon, silicon
oxide, titania, tantalum oxide, silicon nitride, silicon hydride,
indium tin oxide, magnesium oxide, alumina, glass, hydroxylated
surfaces, and polymers.
[0085] In one embodiment of the invention array, the surface of the
coating is atomically flat. In this embodiment, the mean roughness
of the surface of the coating is less than about 5 angstroms for
areas of at least 25 .mu.m.sup.2. In a preferred embodiment, the
mean roughness of the surface of the coating is less than about 3
angstroms for areas of at least 25 .mu.m.sup.2. The ultraflat
coating can optionally be a template-stripped surface as described
in Hegner et al., Surface Science, 1993, 291:3946 and Wagner et
al., Langmuir, 1995, 11:3867-3875, both of which are incorporated
herein by reference.
[0086] It is contemplated that the coatings of many arrays will
require the addition of at least one adhesion layer between said
coating and the substrate. Typically, the adhesion layer will be at
least 6 angstroms thick and may be much thicker. For instance, a
layer of titanium or chromium may be desirable between a silicon
wafer and a gold coating. In an alternative embodiment, an epoxy
glue such as Epo-tek 377.RTM., Epo-tek 301-2.RTM., (Epoxy
Technology Inc., Billerica, Mass.) may be preferred to aid
adherence of the coating to the substrate. Determinations as to
what material should be used for the adhesion layer would be
obvious to one skilled in the art once materials are chosen for
both the substrate and coating. In other embodiments, additional
adhesion mediators or interlayers may be necessary to improve the
optical properties of the array, for instance, in waveguides for
detection purposes.
[0087] Deposition or formation of the coating (if present) on the
substrate is performed prior to the formation of the organic
thinfilm thereon. Several different types of coating may be
combined on the surface. The coating may cover the whole surface of
the substrate or only parts of it. The pattern of the coating may
or may not be identical to the pattern of organic thinfilms used to
immobilize the proteins. In one embodiment of the invention, the
coating covers the substrate surface only at the site of the
patches of the immobilized. Techniques useful for the formation of
coated patches on the surface of the substrate which are organic
thinfilm compatible are well known to those of ordinary skill in
the art. For instance, the patches of coatings on the substrate may
optionally be fabricated by photolithography, micromolding (PCT
Publication WO 96/29629), wet chemical or dry etching, or any
combination of these.
[0088] The organic thinfilm on which each of the patches of
proteins is immobilized forms a layer either on the substrate
itself or on a coating covering the substrate. The organic thinfilm
on which the proteins of the patches are immobilized is preferably
less than about 20 nm thick. In some embodiments of the invention,
the organic thinfilm of each of the patches may be less than about
10 nm thick.
[0089] A variety of different organic thinfilms are suitable for
use in the present invention. Methods for the formation of organic
thinfilms include in situ growth from the surface, deposition by
physisorption, spin-coating, chemisorption, self-assembly, or
plasma-initiated polymeriztion from gas phase. For instance, a
hydrogel composed of a material such as dextran can serve as a
suitable organic thinfilm on the patches of the array. In one
preferred embodiment of the invention, the organic thinfilm is a
lipid bilayer. In another preferred embodiment, the organic
thinfilm of each of the patches of the array is a monolayer. A
monolayer of polyarginine or polylysine adsorbed on a negatively
charged substrate or coating is one option for the organic
thinfilm. Another option is a disordered monolayer of tethered
polymer chains. In a particularly preferred embodiment, the organic
thinfilm is a self-assembled monolayer. A monolayer of polylysine
is one option for the organic thinfilm. The organic thinfilm is
most preferably a self-assembled monolayer which comprises
molecules of the formula X-R-Y, wherein R is a spacer, X is a
functional group that binds R to the surface, and Y is a functional
group for binding proteins onto the monolayer. In an alternative
preferred embodiment, the self-assembled monolayer is comprised of
molecules of the formula (X).sub.aR(Y).sub.b where a and b are,
independently, integers greater than or equal to 1 and X R, and Y
are as previously defined. In an alternative preferred embodiment,
the organic thinfilm comprises a combination of organic thinfilms
such as a combination of a lipid bilayer immobilized on top of a
self-assembled monolayer of molecules of the formula X-R-Y. As
another example, a monolayer of polylysine can also optionally be
combined with a self-assembled monolayer of molecules of the
formula X-R-Y (see U.S. Pat. No. 5,629,213).
[0090] In all cases, the coating, or the substrate itself if no
coating is present, must be compatible with the chemical or
physical adsorption of the organic thinfilm on its surface. For
instance, if the patches comprise a coating between the substrate
and a monolayer of molecules of the formula X-R-Y, then it is
understood that the coating must be composed of a material for
which a suitable functional group X is available. If no such
coating is present, then it is understood that the substrate must
be composed of a material for which a suitable functional group X
is available.
[0091] In a preferred embodiment of the invention, the regions of
the substrate surface, or coating surface, which separate the
patches of proteins are free of organic thinfilm. In an alternative
embodiment, the organic thinfilm extends beyond the area of the
substrate surface, or coating surface if present, covered by the
protein patches. For instance, optionally, the entire surface of
the array may be covered by an organic thinfilm on which the
plurality of spatially distinct patches of proteins reside. An
organic thinfilm which covers the entire surface of the array may
be homogenous or may optionally comprise patches of differing
exposed functionalities useful in the immobilization of patches of
different proteins. In still another alternative embodiment, the
regions of the substrate surface, or coating surface if a coating
is present, between the patches of proteins are covered by an
organic thinfilm, but an organic thinfilm of a different type than
that of the patches of proteins. For instance, the surfaces between
the patches of proteins may be coated with an organic thinfilm
characterized by low non-specific binding properties for proteins
and other analytes.
[0092] A variety of techniques may be used to generate patches of
organic thinfilm on the surface of the substrate or on the surface
of a coating on the substrate. These techniques are well known to
those skilled in the art and will vary depending upon the nature of
the organic thinfilm, the substrate, and the coating if present.
The techniques will also-vary depending on the structure of the
underlying substrate and the pattern of any coating present on the
substrate. For instance, patches of a coating which is highly
reactive with an organic thinfilm may have already been produced on
the substrate surface. Arrays of patches of organic thinfilm can
optionally be created by microfluidics printing, microstamping
(U.S. Pat. Nos. 5,512,131 and 5,731,152), or microcontact printing
(.mu.CP) (PCT Publication WO 96/29629). Subsequent immobilization
of proteins to the reactive monolayer patches results in
two-dimensional arrays of the agents. Inkjet printer heads provide
another option for patterning monolayer X-R-Y molecules, or
components thereof, or other organic thinfilm components to
nanometer or micrometer scale sites on the surface of the substrate
or coating (Lemmo et al., Anal Chem., 1997, 69:543-551; U.S. Pat.
Nos. 5,843,767 and 5,837,860). In some cases, commercially
available arrayers based on capillary dispensing (for instance,
OmniGridT.TM. from Genemachines, inc, San Carlos, Calif., and
High-Throughput Microarrayer from Intelligent Bio-Instruments,
Cambridge, Mass.) may also be of use in directing components of
organic thinfilms to spatially distinct regions of the array.
[0093] Diffusion boundaries between the patches of proteins
immobilized on organic thinfilms such as self-assembled monolayers
may be integrated as topographic patterns (physical barriers) or
surface functionalities with orthogonal wetting behavior (chemical
barriers). For instance, walls of substrate material or photoresist
may be used to separate some of the patches from some of the others
or all of the patches from each other. Alternatively,
non-bioreactive organic thinfilms, such as monolayers, with
different wettability may be used to separate patches from one
another.
[0094] In a preferred embodiment of the invention, each of the
patches of proteins comprises a self-assembled monolayer of
molecules of the formula X-R-Y, as previously defined, and the
patches are separated from each other by surfaces free of the
monolayer.
[0095] FIG. 1 shows the top view of one example of an array of 25
patches reactive with proteins. On the array, a number of patches
15 cover the surface of the substrate 3.
[0096] FIG. 2 shows a detailed cross section of a patch 15 of the
array of FIG. 1. This view illustrates the use of a coating 5 on
the substrate 3. An adhesion interlayer 6 is also included in the
patch. On top of the patch resides a self-assembled monolayer
7.
[0097] FIG. 3 shows a cross section of one row of the patches 15 of
the array of FIG. 1. This figure also shows the use of a cover 2
over the array. Use of the cover 2 creates an inlet port 16 and an
outlet port 17 for solutions to be passed over the array.
[0098] A variety of chemical moieties may function as monolayer
molecules of the formula X-R-Y in the array of the present
invention. However, three major classes of monolayer formation are
preferably used to expose high densities of reactive
omegafinctionalities on the patches of the array: (i) alkylsiloxane
monolayers ("silanes") on hydroxylated and non-hydroxylated
surfaces (as taught in, for example, U.S. Pat. No. 5,405,766, PCT
Publication WO 96/38726, U.S. Pat. No. 5,412,087, and U.S. Pat. No.
5,688,642); (ii) alkyl-thiol/dialkyldisulfide monolayers on noble
metals (preferably Au(111)) (as, for example, described in Allara
et al., U.S. Pat. No. 4,690,715; Bamdad et a., U.S. Pat. No.
5,620,850; Wagner et al., Biophysical Journal, 1996, 70:2052-2066);
and (iii) alkyl monolayer formation on oxide-free passivated
silicon (as taught in, for example, Linford et al., J. Am. Chem.
Soc., 1995, 117:3145-3155, Wagner et al., Journal of Structural
Biology, 1997, 119:189-201, U.S. Pat. No. 5,429,708). One of
ordinary skill in the art, however, will recognize that many
possible moieties may be substituted for X, R, and/or Y, dependent
primarily upon the choice of substrate, coating, and affinity tag.
Many examples of monolayers are described in Ulman, An Introduction
to Ultrathin Organic Films: From Langmuir-Blodgett to Self
Assembly, Academic press (1991).
[0099] In one embodiment, the monolayer comprises molecules of the
formula (X).sub.aR(Y).sub.b wherein a and b are, independently,
equal to an integer between 1 and about 200. In a preferred
embodiment, a and b are, independently, equal to an integer between
1 and about 80. In a more preferred embodiment, a and b are,
independently, equal to 1 or 2. In a most preferred embodiment, a
and b are both equal to 1 (molecules of the formula X-RY).
[0100] If the patches of the invention array comprise a
self-assembled monolayer of molecules of the formula
(X).sub.aR(Y).sub.b, then R may optionally comprise a linear or
branched hydrocarbon chain from about 1 to about 400 carbons long.
The hydrocarbon chain may comprise an alkyl, aryl, alkenyl,
alkynyl, cycloalkyl, alkaryl, aralkyl group, or any combination
thereof. If a and b are both equal to one, then R is typically an
alkyl chain from about 3 to about 30 carbons long. In a preferred
embodiment, if a and b are both equal to one, then R is an alkyl
chain from about 8 to about 22 carbons long and is, optionally, a
straight alkane. However, it is also contemplated that in an
alternative embodiment, R may readily comprise a linear or branched
hydrocarbon chain from about 2 to about 400 carbons long and be
interrupted by at least one hetero atom. The interrupting hetero
groups can include --O--, --CONH--, --CONHCO--, --NH--, --CSNH--,
--CO--, --CS--, --S--, --SO--, --(OCH.sub.2CH.sub.2).sub.n-- (where
n=1-20), --(CF.sub.2).sub.n-- (where n=1-22), and the like.
Alternatively, one or more of the hydrogen moieties of R can be
substituted with deuterium. In alternative, less preferred,
embodiments, R may be more than about 400 carbons long.
[0101] X may be chosen as any group which affords chemisorption or
physisorption of the monolayer onto the surface of the substrate
(or the coating, if present). When the substrate or coating is a
metal or metal alloy, X, at least prior to incorporation into the
monolayer, can in one embodiment be chosen to be an asymmetrical or
symmetrical disulfide, sulfide, diselenide, selenide, thiol,
isonitrile, selenol, a trivalent phosphorus compound,
isothiocyanate, isocyanate, xanthanate, thiocarbamate, a phosphine,
an amine, thio acid or a dithio acid. This embodiment is especially
preferred when a coating or substrate is used that is a noble metal
such as gold, silver, or platinum.
[0102] If the substrate of the array is a material such as silicon,
silicon oxide, indium tin oxide, magnesium oxide, alumina, quartz,
glass, or silica, then the array of one embodiment of the invention
comprises an X that, prior to incorporation into said monolayer, is
a monohalosilane, dihalosilane, trihalosilane, trialkoxysilane,
dialkoxysilane, or a monoalkoxysilane. Among these silanes,
trichlorosilane and trialkoxysilane are particularly preferred.
[0103] In a preferred embodiment of the invention, the substrate is
selected from the group consisting of silicon, silicon dioxide,
indium tin oxide, alumina, glass, and titania; and X, prior to
incorporation into said monolayer, is selected from the group
consisting of a monohalosilane, dihalosilane, trihalosilane,
trichlorosilane, trialkoxysilane, dialkoxysilane, monoalkoxysilane,
carboxylic acids, and phosphates.
[0104] In another preferred embodiment of the invention, the
substrate of the array is silicon and X is an olefin.
[0105] In still another preferred embodiment of the invention, the
coating (or the substrate if no coating is present) is titania or
tantalum oxide and X is a phosphate.
[0106] In other embodiments, the surface of the substrate (or
coating thereon) is composed of a material such as titanium oxide,
tantalum oxide, indium tin oxide, magnesium oxide, or alumina where
X is a carboxylic acid or phosphoric acid. Alternatively, if the
surface of the substrate (or coating thereon) of the array is
copper, then X may optionally be a hydroxamic acid.
[0107] If the substrate used in the invention is a polymer, then in
many cases a coating on the substrate such as a copper coating will
be included in the array. An appropriate functional group X for the
coating would then be chosen for use in the array. In an
alternative embodiment comprising a polymer substrate, the surface
of the polymer may be plasma-modified to expose desirable surface
functionalities for monolayer formation. For instance, EP 780423
describes the use of a monolayer molecule that has an alkene X
functionality on a plasma exposed surface. Still another
possibility for the invention array comprised of a polymer is that
the surface of the polymer on which the monolayer is formed is
functionalized by copolymerization of appropriately functionalized
precursor molecules.
[0108] Another possibility is that prior to incorporation into the
monolayer, X can be a free-radical-producing moiety. This
functional group is especially appropriate when the surface on
which the monolayer is formed is a hydrogenated silicon surface.
Possible free-radical producing moieties include, but are not
limited to, diacylperoxides, peroxides, and azo compounds.
Alternatively, unsaturated moieties such as unsubstituted alkenes,
alkynes, cyano compounds and isonitrile compounds can be used for
X, if the reaction with X is accompanied by ultraviolet, infrared,
visible, or microwave radiation.
[0109] In alternative embodiments, X, prior to incorporation into
the monolayer, may be a hydroxyl, carboxyl, vinyl, sulfonyl,
phosphoryl, silicon hydride, or an amino group.
[0110] The component, Y, of the monolayer is a functional group
responsible for binding a protein onto the monolayer. In a
preferred embodiment of the invention, the Y group is either highly
reactive (activated) towards the protein or is easily converted
into such an activated form. In a preferred embodiment, the
coupling of Y with the protein occurs readily under normal
physiological conditions not detrimental to the activity of the
protein. The functional group Y may either form a covalent linkage
or a noncovalent linkage with the protein (or its affinity tag, if
present). In a preferred embodiment, the functional group Y forms a
covalent linkage with the protein or its affinity tag. It is
understood that following the attachment of the protein (with or
without an affinity tag) to Y, the chemical nature of Y may have
changed. Upon attachment of the protein, Y may even have been
removed from the organic thinfilm.
[0111] In one embodiment of the array of the present invention, Y
is a functional group that is activated in situ. Possibilities for
this type of functional group include, but are not limited to, such
simple moieties such as a hydroxyl, carboxyl, amino, aldehyde,
carbonyl, methyl, methylene, alkene, alkyne, carbonate, aryliodide,
or a vinyl group. Appropriate modes of activation would be obvious
to one skilled in the art. Alternatively, Y can comprise a
functional group that requires photoactivation prior to becoming
activated enough to trap the protein.
[0112] In an especially preferred embodiment of the array of the
present invention, Y is a complex and highly reactive functional
moiety that is compatible with monolayer formation and needs no in
situ activation prior to reaction with the protein and/or affinity
tag. Such possibilities, for Y include, but are not limited to,
maleimide, N-hydroxysuccinimide (Wagner et al., Biophysical
Journal, 1996, 70:2052-2066), nitrilotriacetic acid (U.S. Pat. No.
5,620,850), activated hydroxyl haloacetyl, bromoacetyl, iodoacetyl,
activated carboxyl, hydrazide, epoxy, aziridine, sulfonylchloride,
trifluoromethyldiaziridine, pyridyldisulfide, N-acyl-imidazole,
imidazolecarbamate, vinylsulfone, succinimidylcarbonate, arylazide,
anhydride, diazoacetate, benzophenone, isothiocyanate, isocyanate,
imidoester, fluorobenzene, and biotin.
[0113] FIG. 4 shows one example of a monolayer on a substrate 3 In
this example, substrate 3 comprises glass. The monolayer is
thiolreactive because it bears a maleimidyl functional group Y.
[0114] FIG. 5 shows another example of a monolayer on a substrate 3
which is silicon. In this case, however, a thinfilm gold coating 5
covers the surface of the substrate 3. Also, in this embodiment, a
titanium adhesion interlayer 6 is used to adhere the coating 5 to
the substrate 3. This monolayer is aminoreactive because it bears
an N-hydroxysuccinimidyl functional group Y.
[0115] In an alternative embodiment, the functional group Y of the
array is selected from the group of simple functional moieties.
Possible Y functional groups include, but are not limited to, --OH,
--NH.sub.2, --COOH, --COOR, --RSR, --PO.sub.4.sup.-3,
--OSO.sub.3.sup.-2, --SO.sub.3.sup.-, --COO.sup.-, --SOO.sup.-,
--CONR.sub.2, --CN, --NR.sub.2, and the like.
[0116] The monolayer molecules of the present invention can
optionally be assembled on the surface in. parts. In other words,
the monolayer need not necessarily be constructed by chemisorption
or physisorption of molecules of the formula X-R-Y to the surface
of the substrate (or coating). Instead, in one embodiment, X may be
chemisorbed or physisorbed to the surface of the substrate (or
coating) alone first. Then, R or even just individual components of
R can be attached to X through a suitable chemical reaction. Upon
completion of addition of the spacer R to the X moiety already
immobilized on the surface, Y can be attached to the ends of the
monolayer molecule through a suitable covalent linkage.
[0117] Not all self-assembled monolayer molecules on a given patch
need be identical to one another. Some patches may comprise mixed
monolayers. For instance, the monolayer of an individual patch may
optionally comprise at least two different molecules of the formula
X-R-Y, as previously described. This second X-R-Y molecule may
optionally immobilize the same protein as the first. In addition,
some of the monolayer molecules X-R-Y of a patch may have failed to
attach any protein.
[0118] As another alternative embodiment of the invention, a mixed,
self-assembled monolayer of an individual patch on the array may
comprise both molecules of the formula X-R-Y, as previously
described, and molecules of the formula, X-R-V where R is a spacer,
X is a functional group that binds R to the surface, and V is a
moiety which is biocompatible with proteins and resistant to the
non-specific binding of proteins. For example, V may consist of a
hydroxyl, saccharide, or oligo/polyethylene glycol moiety (EP
Publication 780423).
[0119] In still another embodiment of the invention, the array
comprises at least one unreactive patch of organic thinfilm on the
substrate or coating surface which is devoid of any protein. For
instance, the unreactive patch may optionally comprise a monolayer
of molecules of the formula X-R-V, where R is a spacer, X is a
functional group that binds R to the surface, and V is a moiety
resistant to the non-specific binding of proteins. The unreactive
patch may serve as a control patch or be useful in background
binding measurements.
[0120] Regardless of the nature of the monolayer molecules, in some
arrays it may be desirable to provide crosslinking between
molecules of an individual patch's monolayer. In general,
crosslinking confers additional stability to the monolayer. Such
methods are familiar to those skilled in the art (for instance, see
Ulman, An Introduction to Ultrathin Organic Films: From
Langmuir-Blodgett to Self-Assembly, Academic Press (1991)).
[0121] After completion of formation of the monolayer on the
patches, the protein may be attached to the monolayer via
interaction with the Y-functional group. Y-functional groups which
fail to react with any proteins are preferably quenched prior to
use of the array.
[0122] (d) Affinity Tags and Immobilization of the proteins.
[0123] In a preferred embodiment, the protein-immobilizing patches
of the array further comprise an affinity tag that enhances
immobilization of the protein onto the organic thinfilm. The use of
an affinity tag on the protein of the array typically provides
several advantages. An affinity tag can confer enhanced binding or
reaction of the protein with the functionalities on the organic
thinfilm, such as Y if the organic thinfilm is a an X-R-Y monolayer
as previously described. This enhancement effect may be either
kinetic or thermodynamic. The affinity tag/thinfilm combination
used in the patches of the array preferably allows for
immobilization of the proteins in a manner which does not require
harsh reaction conditions that are adverse to protein stability or
function. In most embodiments, immobilization to the organic
thinfilm in aqueous, biological buffers is ideal.
[0124] An affinity tag also preferably offers immobilization on the
organic thinfilm that is specific to a designated site or location
on the protein (site-specific immobilization). For this to occur,
attachment of the affinity tag to the protein must be
site-specific. Site-specific immobilization helps ensure that the
active site or binding site of the immobilized protein, such as the
antigen-binding site of the antibody moiety, remains accessible to
ligands in solution. Another advantage of immobilization through
affinity tags is that it allows for a common immobilization
strategy to be used with multiple, different proteins.
[0125] The affinity tag is optionally attached directly, either
covalently or noncovalently, to the protein. In an alternative
embodiment, however, the affinity tag is either covalently or
noncovalently attached to an adaptor which is either covalently or
noncovalendy attached to the protein.
[0126] In a preferred embodiment, the affinity tag comprises at
least one amino acid. The affinity tag may be a polypeptide
comprising at least two amino acids which is reactive with the
functionalities of the organic thinfilm. Alternatively, the
affinity tag may be a single amino acid which is reactive with the
organic thinfilm. Examples of possible amino acids which could be
reactive with an organic thinfilm include cysteine, lysine,
histidine, arginine, tyrosine, aspartic acid, glutamic acid,
typtophan, serine, threonine, and glutamine. A polypeptide or amino
acid affinity tag is preferably expressed as a fusion protein with
the immobilized protein of each patch. Amino acid affinity tags
provide either a single amino acid or a series of amino acids that
can interact with the functionality of the organic thinfilm, such
as the Y-functional group of the self-assembled monolayer
molecules. Amino acid affinity tags can be readily introduced into
recombinant proteins to facilitate oriented immobilization by
covalent binding to the Y-functional group of a monolayer or to a
functional group on an alternative organic thinfilm.
[0127] The affinity tag may optionally comprise a poly(amino acid)
tag. A poly(amino acid) tag is a polypeptide that comprises from
about 2 to about 100 residues of a single amino acid, optionally
interrupted by residues of other amino acids. For instance, the
affinity tag may comprise a poly-cysteine, polylysine,
poly-arginine, or poly-histidine. Amino acid tags are preferably
composed of two to twenty residues of a single amino acid, such as,
for example, histidines, lysines, arginines, cysteines, glutamines,
tyrosines, or any combination of these. According to a preferred
embodiment, an amino acid tag of one to twenty amino acids includes
at least one to ten cysteines for thioether linkage; or one to ten
lysines for amide linkage; or one to ten arginines for coupling to
vicinal dicarbonyl groups. One of ordinary skill in the art can
readily pair suitable affinity tags with a given functionality on
an organic thinfilm.
[0128] The position of the amino acid tag can be at an amino-, or
carboxy-terminus of the protein of a patch of the array, or
anywhere in-between, as long as the active site or binding site of
the protein remains in a position accessible for ligand
interaction. Where compatible with the protein chosen, affinity
tags introduced for protein purification are preferentially located
at the C-terminus of the recombinant protein to ensure that only
full-length proteins are isolated during protein purification. For
instance, if intact antibodies are used on the arrays, then the
attachment point of the affinity tag on the antibody is preferably
located at a C-terminus of the effector (Fc) region of the
antibody. If scFvs are used on the arrays, then the attachment
point of the affinity tag is also preferably located at the
C-terminus of the molecules.
[0129] Affinity tags may also contain one or more unnatural amino
acids. Unnatural amino acids can be introduced using suppressor
tRNAs that recognize stop codons (i.e., amber) (Noren et al.,
Science, 1989, 244:182-188; Ellman et al., Methods Enzym., 1991,
202:301-336; Cload et al., Chem. Biol., 1996, 3:1033-1038). The
tRNAs are chemically amino-acylated to contain chemically altered
("unnatural") amino acids for use with specific coupling
chemistries (i.e., ketone modifications, photoreactive groups).
[0130] In an alternative embodiment the affinity tag can comprise
an intact protein, such as, but not limited to, glutathione
S-transferase, an antibody, avidin, or streptavidin.
[0131] Other protein conjugation and immobilization techniques
known in the art may be adapted for the purpose of attaching
affinity tags to the protein. For instance, in an alternative
embodiment of the array, the affinity tag may be an organic
bioconjugate which is chemically coupled to the protein of
interest. Biotin or antigens may be chemically cross linked to the
protein. Alternatively, a chemical crosslinker may be used that
attaches a simple functional moiety, such as a thiol or an amine to
the surface of a protein to be immobilized on a patch on the array.
Alternatively, protein synthesis or protein ligation techniques
known to those skilled in the art may be used to attach an affinity
tag to a protein. For instance, intein-mediated protein ligation
may optionally be used to attach the affinity tag to the protein
(Mathys, et al., Gene 231:1-13, 1999; Evans, et al., Protein
Science 7:2256-2264, 1998).
[0132] In an alternative embodiment of the invention, the organic
thinfilm of each of the patches comprises, at least in part, a
lipid monolayer or bilayer, and the affinity tag comprises a
membrane anchor. Optionally, the lipid monolayer or bilayer is
immobilized on a self-assembled monolayer.
[0133] FIG. 6 shows a detailed cross section of a patch on one
embodiment of the invention array. In this embodiment, a protein 10
is immobilized on a monolayer 7 on a substrate 3. An affinity tag 8
connects the protein 10 to the monolayer 7. The monolayer 7 is
formed on a coating 5 which is separated from the substrate 3 by an
interlayer 6.
[0134] In an alternative embodiment of the invention, no affinity
tag is used to immobilize the proteins onto the organic thinfilm.
An amino acid or other moiety (such as a carbohydrate moiety)
inherent to the protein itself may instead be used to tether the
protein to the reactive group of the organic thinfilm. In preferred
embodiments, the immobilization is site-specific with respect to
the location of the site of immobilization on the protein. For
instance, the sulfhydryl group on the C-terminal region of the
heavy chain portion of a Fab' fragment generated by pepsin
digestion of an antibody, followed by selective reduction of the
disulfide between monovalent Fab' fragments, may be used as the
affinity tag. Alternatively, a carbohydrate moiety on the Fc
portion of an intact antibody can be oxidized under mild conditions
to an aldehyde group suitable for immobilizing the antibody on a
monolayer via reaction with a hydrazide-activated Y group on the
monolayer. Examples of immobilization of proteins without any
affinity tag can be found in Wagner et al., Biophys. J.,
70:2437-2441, 1996 and the specific examples, Examples 8-10,
below.
[0135] When the proteins of at least some of the different patches
on the array are different from each other, different solutions,
each containing a different, preferably, affinity-tagged protein,
must be delivered to their individual patches. Solutions of
proteins may be transferred to the appropriate patches via arrayers
which are well-known in the art and even commercially available.
For instance, microcapillary-based dispensing systems may be used.
These dispensing systems are preferably automated and
computer-aided. A description of and building instructions for an
example of a microarrayer comprising an automated capillary system
can be found on the internet at
http://cmgm.stanford.edu/pbrown/array.html and
http://cmgm.stanford.edu/pbrown/mguidelindex.html. The use of other
microprinting techniques for transferring solutions containing the
proteins to the protein-reactive patches is also possible. Inkjet
printer heads may also optionally be used for precise delivery of
the proteins to the protein-reactive patches. Representative,
non-limiting disclosures of techniques useful for depositing the
proteins on the patches may be found, for example, in U.S. Pat. No.
5,731,152 (stamping apparatus), U.S. Pat. No. 5,807,522 (capillary
dispensing device), U.S. Pat. No. 5,837,860 (ink-jet printing
technique, Hamilton 2200 robotic pipetting delivery system), and
U.S. Pat. No. 5,843,767 (ink-jet printing technique, Hamilton 2200
robotic pipetting delivery system), all incorporated by reference
herein.
[0136] (e) Adaptors.
[0137] Another embodiment of the arrays of the present invention
comprises an adaptor that links the affinity tag to the immobilized
protein. The additional spacing of the protein from the surface of
the substrate (or coating) that is afforded by the use of an
adaptor is particularly advantageous since proteins are known to be
prone to surface inactivation. The adaptor may optionally afford
some additional advantages as well. For. instance, the adaptor may
help facilitate the attachment of the protein to the affinity tag.
In another embodiment, the adaptor may help facilitate the use of a
particular detection technique with the array. One of ordinary
skill in the art will be able to choose an adaptor which is
appropriate for a given affinity tag. For instance, if the affinity
tag is streptavidin, then the adaptor could be a biotin molecule
that is chemically conjugated to the protein which is to be
immobilized.
[0138] In a preferred embodiment, the adaptor is a protein. In a
preferred embodiment, the affinity tag, adaptor, and protein to be
immobilized together compose a fusion protein. Such a fusion
protein may be readily expressed using standard recombinant DNA
technology. Adaptors which are proteins arm especially useful to
increase the solubility of the protein of interest and to increase
the distance between the surface of the substrate or coating and
the protein of interest. Use of an adaptor which is a protein can
also be very useful in facilitating the preparative steps of
protein purification by affinity binding prior to immobilization on
the array. Examples of possible adaptors which are proteins include
glutathione-S-transferase (GST), maltose-binding protein,
chitin-binding protein, thioredoxin, green-fluorescent protein
(GFP). GFP can alsq be used for quantification of surface binding.
If the protein immobilized on the patches of the array is an
antibody or antibody fragment comprising an Fc region, then the
adaptor may optionally be protein G, protein A, or recombinant
protein A/G (a gene fusion product secreted from a nonpathogenic
form of Bacillus which contains four Fc binding domains from
protein A and two from protein G).
[0139] FIG. 7 shows a cross section of a patch on one particular
embodiment of the invention array. The patch comprises a protein 10
immobilized on a monolayer 7 via both an affinity tag 8 and an
adaptor molecule 9. The monolayer 7 rests on a coating 5. An
interlayer 6 is used between the coating S and the substrate 3.
[0140] (f) Preparation of the Proteins of the Array.
[0141] The proteins immobilized on the array may be produced by any
of the variety of means known to those of ordinary skill in the
art.
[0142] In preparation for immobilization to the arrays of the
present invention, the protein can optionally be expressed from
recombinant DNA either in vivo or in vitro. The cDNA of the protein
to be immobilized on the array is cloned into an expression vector
(many examples of which are commercially available) and introduced
into cells of the appropriate organism for expression. A broad
range of host cells and expression systems may be used to produce
the proteins to be immobilized on the array. For in vivo expression
of the proteins, cDNAs can be cloned into commercial expression
vectors (Qiagen, Novagen, Clontech, for example) and introduced
into an appropriate organism for expression. Expression in vivo may
be done in bacteria (for example, Escherichia coli), plants (for
example, Nicotiana tabacum), lower eukaryotes (for example,
Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris), or
higher eukaryotes (for example, bacculovirus-infected insect cells,
insect cells, mammalian cells). For in vitro expression
PCR-amplified DNA sequences are directly used in coupled in vitro
transcription/translation systems (for instance: Escherichia coli
S30 lysates from T7 RNA polymerase expressing, preferably
protease-deficient strains; wheat germ lysates; reticulocyte
lysates (Promega, Pharmacia, Panvera)). The choice of organism for
optimal expression depends on the extent of post-translational
modifications (i.e., glycosylation, lipid-modifications) desired.
One of ordinary skill in the art will be able to readily choose
which host cell type is most suitable for the protein to be
immobilized and application desired.
[0143] DNA sequences encoding amino acid affinity tags and adaptor
protein sequences are engineered into the expression vectors such
that the genes of interest can be cloned in frame either 5' or 3'
of the DNA sequence encoding the affinity tag and adaptor.
[0144] The expressed proteins are purified by affinity
chromatography using commercially available resins.
[0145] Preferably, production of families of related proteins
involves parallel processing from cloning to protein expression and
protein purification. cDNAs for the protein of interest will be
amplified by PCR using cDNA libraries or EST (expressed sequence
tag) clones as templates. Any of the in vitro or in vivo expression
systems described above can then be used for expression of the
proteins to be immobilized on the array.
[0146] Escherichia coli-based protein expression is generally the
method of choice for soluble proteins that do not require extensive
post-translational modifications for activity. Extracellular or
intracellular domains of membrane proteins will be fused to protein
adaptors for expression and purification.
[0147] The entire approach can be performed using 96-well assay
plates. PCR reactions are carried out under standard conditions.
Oligonucleotide primers contain unique restriction sites for facile
cloning into the expression vectors. Alternatively, the TA cloning
system (Clontech) can be used. Expression vectors contain the
sequences for affinity tags and the protein adaptors. PCR products
are ligated into the expression vectors (under inducible promoters)
and introduced into the appropriate competent Escherichia coli
strain by calcium-dependent transformation (strains include: XL-1
blue, BL21, SG13009(1on-)). Transformed Escherichia coli cells are
plated and individual colonies transferred into 96-array blocks.
Cultures are grown to mid-log phase, induced for expression, and
cells collected by centrifugation. Cells are resuspended contaiinng
lysozyme and the membranes broken by rapid freeze/thaw cycles, or
by sonication. Cell debris is removed by centrifugation and the
supernatants transferred to 96-tube arrays. The appropriate
affinity matrix is added, protein of interest bound and
nonspecifically bound proteins removed by repeated washing steps
using 12-96 pin suction devices and centrifugation. Alternatively,
magnetic affinity beads and filtration devices can be used
(Qiagen). The proteins are eluted and transferred to a new 96-well
array. Protein concentrations are determined and an aliquot of each
protein is spotted onto a nitrocellulose filter and verified by
Western analysis using an antibody directed against the affinity
tag. The purity of each sample is assessed by SDS-PAGE and silver
staining or mass spectrometry. Proteins are snap-frozen and stored
at -80.degree. C.
[0148] Saccharomyces cerevisiae allows for core glycosylation and
lipid modifications of proteins. The approach described above for
Escherichia coli can be used with slight modifications for
transformation and cell lysis. Transformation of Saccharomyces
cerevisiae is by lithium-acetate and cell lysis is either by
lyticase digestion of the cell walls followed by freeze-thaw,
sonication or glass-bead extraction. Variations of
post-translational modifications can be obtained by different yeast
strains (i.e. Saccharomyces pombe, Pichia pastoris).
[0149] The advantage of the bacculovirus system or mammalian cells
are the wealth of post-translational modifications that can be
obtained. The bacculo-system requires cloning of viruses, obtaining
high titer stocks and infection of liquid insect cell suspensions
(cells are SF9, SF21). Mammalian cell-based expression requires
transfection and cloning of cell lines. Soluble proteins are
collected from the medium while intracellular or membrane bound
proteins require cell lysis (either detergent solubilization,
freeze-thaw). Proteins can then be purified analogous to the
procedure described for Escherichia coli.
[0150] For in vitro translation the system of choice is Escherichia
coli lysates obtained from protease-deficient and T7 RNA polymerase
overexpressing strains. Escherichia coli lysates provide efficient
protein expression (30-50 .mu.g/ml lysate). The entire process is
carried out in 96-well arrays. Genes of interest are amplified by
PCR using oligonucleotides that contain the gene-specific sequences
containing a T7 RNA polymerase promoter and binding site and a
sequence encoding the affinity tag. Alternatively, an adaptor
protein can be fused to the gene of interest by PCR Amplified DNAs
can be directly transcribed and translated in the Escherichia coli
lysates without prior cloning for fast analysis. The proteins are
then isolated by binding to an affinity matrix and processed as
described above.
[0151] Alternative systems which may be used include wheat germ
extracts and reticulocyte extracts. In vitro synthesis of membrane
proteins and or post-translationally modified proteins will require
reticulocyte lysates in combination with microsomes.
[0152] In one preferred embodiment of the invention, the proteins
immobilized on the patches of the array are antibodies. Optionally,
the immobilized proteins may be monoclonal antibodies. The
production of monoclonal antibodies against specific protein
targets is routine using standard hybridoma technology. In fact,
numerous monoclonal antibodies are available commercially.
[0153] As an alternative to obtaining antibodies or antibody
fragments which have been produced by cell fusion or from
continuous cell lines, the antibody moieties may be expressed in
bacteriophage. Such antibody phage display technologies are well
known to those skilled in the art. The bacteriophage expression
systems allow for the random recombination of heavy- and
light-chain sequences, thereby creating a library of antibody
sequences which can be selected against the desired antigen. The
expression system can be based on bacteriophage .lambda. or, more
preferably, on filamentous phage. The bacteriophage expression
system can be used to express Fab fragments, Fv's with an
engineered intermolecular disulfide bond to stabilize the
V.sub.H-V.sub.L pair (dsFv's), scFvs, or diabody fragments.
[0154] The antibody genes of the phage display libraries may be
from pre-immunized donors. For instance, the phage display library
could be a display library prepared from the spleens of mice
previously immunized with a mixture of proteins (such as a lysate
of human T-cells). Immunization can optionally be used to bias the
library to contain a greater number of recombinant antibodies
reactive towards a specific set of proteins (such as proteins found
in human T-cells). Alternatively, the library antibodies may be
derived from naive or synthetic libraries. The naive libraries have
been constructed from spleens of mice which have not been contacted
by external antigen. In a synthetic library, portions of the
antibody sequence, typically those regions corresponding to the
complementarity determining regions (CDR) loops, have been
mutagenized or randomized.
[0155] The phage display method involves batch-cloning the antibody
gene library into a phage genome as a fusion to the gene encoding
one of the phage coat proteins (pIII, pVI, or pVIII). The pIII
phage protein gene is preferred. When the fusion product is
expressed it is incorporated into the mature phage coat. As a
result, the antibody is displayed as a fusion on the surface of the
phage and is available for binding and hence, selection, on a
target protein. Once a phage particle is selected as bearing an
antibody-coat protein fusion with the desired affinity towards the
target protein, the genetic material within the phage particle
which corresponds to the displayed antibody can be amplified and
sequenced or otherwise analyzed.
[0156] In a preferred embodiment, a phagemid is used as the
expression vector in the phage display procedures. A phagemid is a
small plasmid vector that carries gene III with appropriate cloning
sites and a phage packaging signal and contains both host and phage
origins of replication. The phagemid is unable to produce a
complete phage as the gene III fusion is the only phage gene
encoded on the phagemid. A viable phage can be produced by
infecting cells containing the phagemid with a helper phage
containing a defective replication origin. A hybrid phage emerges
which contains all of the helper phage proteins as well as the gene
III-rAb fusion. The emergent phage contains the phagemid DNA
only.
[0157] In a preferred embodiment of the invention, the recombinant
antibodies used in phage display methods of preparing antibody
fragments for the arrays of the invention are expressed as genetic
fusions to the bacteriophage gene III protein on a phagemid vector.
For instance, the antibody variable regions encoding a single-chain
Fv fragment can be fused to the amino terminus of the gene III
protein on a phagemid. Alternatively, the antibody fragment
sequence could be fused to the amino terminus of a truncated pIII
sequence lacking the first two N-terminal domains. The phagemid DNA
encoding the antibody-pIII fusion is preferably packaged into phage
particles using a helper phage such as M13KO7 or VCS-M13, which
supplies all structural phage proteins.
[0158] To display Fab fragments on phage, either the light or heavy
(Fd) chain is fused via its C-terminus to pIII. The partner chain
is expressed without any fusion to pIII so that both chains can
associate to form an intact Fab fragment. Any method of selection
may be used which separates those phage particles which do bind the
target protein from those which do not. The selection method must
also allow for the recovery of the selected phages. Most typically,
the phage particles are selected on an immobilized target protein.
Some phage selection strategies known to those skilled in the art
include the following: panning on an immobilized antigen; panning
on an immobilized antigen using specific elution; using
biotinylated antigen and then selecting on a streptavidin resin or
streptavidin-coated magnetic beads; affinity purification;
selection on Western blots (especially useful for unknown antigens
or antigens difficult to purify); in vivo selection; and pathfinder
selection. If the selected phage particles are amplified between
selection rounds, multiple iterative rounds of selection may
optionally be performed.
[0159] Elution techniques will vary depending upon the selection
process chosen, but typical elution techniques include washing with
one of the following solutions: HCl or glycine buffers; basic
solutions such as triethylamine; chaotropic agents; solutions of
increased ionic strength; or DTT when biotin is linked to the
antigen by a disulfide bridge. Other typical methods of elution
include enzymatically cleaving a protease site engineered between
the antibody and gene III, or by competing for binding with excess
antigen or excess antibodies to the antigen.
[0160] A method for producing an array of antibody fragments
therefore comprises first selecting recombinant bacteriophage which
express antibody fragments from a phage display library. The
recombinant bacteriophage are selected by affinity binding to the
desired antigen. (Iterative rounds of selection are possible, but
optional.) Next at least one purified sample of an antibody
fragment from a bacteriophage which was selected in the first step
is produced. This antibody production step typically entails
infecting E. coli cells with the selected bacteriophage. In the
absence of helper phage, the selected bacteriophage then replicate
as expressive plasmids without producing phage progeny.
Alternatively, the antibody fragment gene of the selected
recombinant bacteriophage is isolated, amplified, and then
expressed in a suitable expression system. In either case,
following. amplification, the expressed antibody fragment of the
selected and amplified recombinant bacteriophage is isolated and
purified. In a third step of the method, the earlier steps of phage
display selection and purified antibody fragment production are
repeated using affinity binding to antigens from before until the
desired plurality of purified samples of different antibody
fragments with different binding partners are produced. In a final
step of the method, the antibody fragment of each different
purified sample is immobilized onto organic thinfilm on a separate
patch on the surface of a substrate to form a plurality of patches
of antibody fragments on discrete, known regions of the substrate
surface covered by organic thinfilm.
[0161] For instance, to generate an antibody array with antibody
fragments against known antigens, open reading frames of the known
protein targets identified in DNA databases are amplified by
polymerase chain reaction and transcribed and translated in vitro
to produce proteins on which a recombinant bacteriophage expressing
single-chain antibody. fragments are selected. Once selected, the
antibody fragment sequence of the selected bacteriophage is
amplified (typically using the polymerase chain method) and
recloned into a desirable expression system. The expressed antibody
fragments are purified and then printed onto organic thinfilms on
substrates to form the high density arrays.
[0162] In the preparation of the arrays of the invention, phage
display methods analogous to those used for antibody fragments may
be used for other proteins which are to be immobilized on an array
of the invention as long as the protein is of suitable size to be
incorporated into the phagemid or alternative vector and expressed
as a fusion with a bacteriophage coat protein. Phage display
techniques using non-antibody libraries typically make use of some
type of protein host scaffold structure which supports the variable
regions. For instance, .beta.-sheet proteins, a-helical handle
proteins, and other highly constrained protein structures have been
used as host scaffolds.
[0163] Alternative display vectors may also be used to produce the
proteins which are printed on the arrays of the invention.
Polysomes, stable protein-ribosome-mRNA complexes, can be used to
replace live bacteriophage as the display vehicle for recombinant
antibody fragments or other proteins (Hanes and Pluckthun, Proc.
Natl. Acad. Sci USA, 94:4937-4942, 1997). The polysomes are formed
by preventing release of newly synthesized and correctly folded
protein from the ribosome. Selection of the polysome library is
based on binding of the antibody fragments or other proteins which
are displayed on the polysomes to the target protein. mRNA which
encodes the displayed protein or antibody having the desired
affinity for the target is then isolated. Larger libraries may be
used with polysome display than with phage display.
[0164] (g) Uses of the Arrays.
[0165] The present invention also provides for methods of using the
invention array. The arrays of the present invention are
particularly suited for the use in drug development. Other uses
include medical diagnostics, proteomics and biosensors.
[0166] Use of one of the protein arrays of the present invention
may optionally involve placing the two-dimensional protein array in
a flowchamber with approximately 1-10 microliters of fluid volume
per 25 overall surface area. The cover over the array in the
flowchamber is preferably transparent or translucent. In one
embodiment, the cover may comprise Pyrex or quartz glass. In other
embodiments, the cover may be part of a detection system that
monitors interaction between biological moieties immobilized on the
array and an analyte. The flowchambers should remain filled with
appropriate aqueous solutions to preserve protein activity. Salt,
temperature, and other conditions are preferably kept similar to
those of normal physiological conditions. Analytes and potential
drug compounds may be flushed into the flow chamber as desired and
their interaction with the immobilized proteins determined.
Sufficient time must be given to allow for binding between the
immobilized proteins and an analyte to occur. No specialized
microfluidic pumps, valves, or mixing techniques are required for
fluid delivery to the array.
[0167] Alternatively, fluid can be delivered to each of the patches
of the array individually. For instance, in one embodiment, the
regions of the substrate surface may be microfabricated in such a
way as to allow integration of the array with a number of fluid
delivery channels oriented perpendicular to the array surface, each
one of the delivery channels terminating at the site of an
individual protein-coated patch.
[0168] The sample which is delivered to the array is typically a
fluid.
[0169] In general, delivery of solutions containing proteins to be
bound by the proteins of the array may optionally be preceded,
followed, or accompanied by delivery of a blocking solution. A
blocking solution contains protein or another moiety which will
adhere to sites of non-specific binding on the array. For instance,
solutions of bovine serum albumin or milk may be used as blocking
solutions.
[0170] A wide range of detection methods is applicable to the
methods of the invention. As desired, detection may be either
quantitative or qualitative. The invention array can be interfaced
with optical detection methods such as absorption in the visible or
infrared range, chemoluminescence, and fluorescence (including
lifetime, polarization, fluorescence correlation spectroscopy
(FCS), and fluorescence-resonance energy transfer (FRET)).
Furthermore, other modes of detection such as those based on
optical waveguides (PCT Publication WO 96/26432 and U.S. Pat. No.
5,677,196), surface plasmon resonance, surface charge sensors, and
surface force sensors are compatible with many embodiments of the
invention. Alternatively, technologies such as those based on
Brewster angle microscopy (Schaaf et al., Langmuir, 3:1131-1135
(1987)) and ellipsometry (U.S. Pat. Nos. 5,141,311 and 5,116,121;
Kim, Macromolecules, 22:2682-2685 (1984)) can be used in
conjunction with the arrays of the invention. Quartz crystal
microbalances and desorption processes (see for example, U.S. Pat.
No. 5,719,060) provide still other alternative detection means
suitable for at least some embodiments of the invention array. An
example of an optical biosensor system compatible both with some
arrays of the present invention and a variety of non-label
detection principles including surface plasmon resonance, total
internal reflection fluorescence (TIRF), Brewster Angle microscopy,
optical waveguide lightmode spectroscopy (OWLS), surface charge
measurements, and ellipsometry can be found in U.S. Pat. No.
5,313,264.
[0171] Although non-label detection methods are generally
preferred, some of the types of detection methods commonly used for
traditional immunoassays which require the use of labels may be
applied to use with at least some of the arrays of the present
invention, especially those arrays which are arrays of
protein-capture agents. These techniques include noncompetitive
immunoassays, competitive immunoassays, and dual label, ratiometric
immunoassays. These particular techniques are primarily suitable
for use with the arrays of proteins when the number of different
proteins with different specificity is small (less than about 100).
In the competitive method, binding-site occupancy is determined
indirectly. In this method, the proteins of the array are exposed
to a labeled developing agent, which is typically a labeled version
of the analyte or an analyte analog. The developing agent competes
for the binding sites on the protein with the analyte. The
fractional occupancy of the proteins on different patches can be
determined by the binding of the developing agent to the proteins
of the individual patches. In the noncompetitive method, binding
site occupancy is determined directly. In this method, the patches
of the array are exposed to a labeled developing agent capable of
binding to either the bound analyte or the occupied binding sites
on the protein. For instance, the developing agent may be a labeled
antibody directed against occupied sites (i. e., a "sandwich
assay"). Alternatively, a dual label, ratiometric, approach may be
taken where the immobilized protein is labeled with one label and
the second, developing agent is labeled with a second label (Ekins,
et al., Clinica Chimica Acta., 194:91-114, 1990). Many different
labeling methods may be used in the aforementioned techniques,
including radioisotopic, enzymatic, chemiluminescent, and
fluorescent methods. Fluorescent methods are preferred.
[0172] FIG. 8 shows a schematic diagram of one type of fluorescence
detection unit which may be used to monitor interaction of
immobilized proteins of an array with an analyte. In the
illustrated detection unit, the protein array 21 is positioned on a
base plate 20. Light from a 100W mercury arc lamp 25 is directed
through an excitation filter 24 and onto a beam splitter 23. The
light is then directed through a lens 22, such as a Micro Nikkor 55
mm 1:2:8 lens, and onto the array 21. Fluorescence emission from
the array returns through the lens 22 and the beam splitter 23.
After next passing through an emission filter 26, the emission is
received by a cooled CCD camera 27, such as the Slowscan
TE/CCD-1024SF&SB (Princeton Instruments). The camera is
operably connected to a CPU 28 which is in turn operably connected
to a VCR 29 and a monitor 30.
[0173] FIG. 9 shows a schematic diagram of an alternative detection
method based on ellipsometry. Ellipsometry allows for information
about the sample to be determined from the observed change in the
polarization state of a reflected light wave. Interaction of an
analyte with a layer of immobilized proteins on a patch results in
a thickness change and alters the polarization status of a
plane-polarized light beam reflected off the surface. This process
can be monitored in situ from aqueous phase and, if desired, in
imaging mode. In a typical setup, monochromatic light (e.g. from a
He--Ne laser, 30) is plane polarized (polarizer 31) and directed
onto the surface of the sample and detected by a detector 35. A
compensator 32 changes the elliptically polarized reflected beam to
plane-polarized. The corresponding angle is determined by an
analyzer 33 and then translated into the ellipsometric parameters
Psi and Delta which change upon binding of analyte with the
immobilized proteins. Additional information can be found in Azzam,
et al., Ellipsometry and Polarized Light, North-Holland Publishing
Company: Amsterdam, 1977.
[0174] In one embodiment, the invention provides a method for
screening a plurality of proteins for their ability to interact
with a component of a sample comprising the steps of delivering the
sample to a protein array of the invention comprising the proteins
to be screened and detecting for the interaction of the component
with the immobilized protein of each patch. Optionally, the
component may be a protein.
[0175] Possible interactions towards which the present invention
may be directed include, but are not limited to, antibody/antigen,
antibody/hapten, enzyme/substrate, carrier protein/substrate,
lectin/carbohydrate, receptor/hormone, receptor/effector,
protein/DNA, protein/RNA, repressor/inducer, or the like. The
interaction may involve binding and/or catalysis. The array of he
invention is even suitable for assaying translocation by a membrane
through a lipid bilayer. In preferred embodiments of use of the
array, the assayed interaction is a binding interaction. The
assayed interaction may be between a potential drug candidate and a
plurality of potential drug targets. For instance, a synthesized
organic compound may be tested for its ability to act as an
inhibitor to a family of immobilized receptors.
[0176] Another aspect of the invention provides for a method for
screening a plurality of proteins for their ability to bind a
particular component of a sample. This method comprises delivering
the sample to a protein array of the invention comprising the
proteins to be screened and detecting, either directly or
indirectly, for the presence or amount of the particular component
retained at each patch. In a preferred embodiment, the method
further comprises the intermediate step of washing the array to
remove any unbound or nonspecifically bound components of the
sample from the array before the detection step. In another
embodiment, the method further comprises the additional step of
further characterizing the particular component retained on at
least one patch. The particular component may optionally be a
protein.
[0177] The optional step of further characterizing the particular
component retained on a patch of the array is typically designed to
identify the nature of the component bound to the protein of a
particular patch. In some cases, the entire identity of the
component may not be known and the purpose of the further
characterization may be the initial identification of the mass,
sequence, structure and/or activity (if any) of the bound
component. In other cases, the basic identity of the component may
be known, but some information about the component may not be
known. For instance it may be known that the component is a
particular protein, but the post-translational modification,
activation state, or some other feature of the protein may not be
known. In one embodiment, the step of further characterizing
components which are proteins involves measuring the activity of
the proteins. Although in some cases it may be preferable to remove
the component from the patch before the step of further
characterizing the protein is carried out, in other cases the
component can be further characterized while still bound to the
patch. In still further embodiments, the proteins of the patch
which binds a component can be used to isolate and/or purify the
component on a larger scale, such as by purifying a component which
is a protein from cells. The purified sample of the component can
then be characterized through traditional means such as
microsequencing, mass spectrometry, and the like.
[0178] In another embodiment of the invention, a method of assaying
for protein-protein binding interactions is provided which
comprises the following steps: first, delivering a sample
comprising at least one protein to be assayed for binding to the
protein array of the invention; and then detecting, either
directly, or indirectly, for the presence or amount of the protein
from the sample which is retained at each patch. In a preferred
embodiment, the method further comprises an additional step prior
to the detection step which comprises washing the array to remove
unbound or nonspecifically bound components of the sample from the
array. Typically, the protein being assayed for binding will be
from the same organism as the proteins immobilized on the
array.
[0179] Another embodiment of the invention provides a method of
assaying in parallel for the presence of a plurality of analytes in
a sample which can react with one or more of the immobilized
proteins on the protein array. This method comprises delivering the
sample to the invention array and detecting for the interaction of
the analyte with the immobilized protein at each patch.
[0180] In still another embodiment of the invention, a method of
assaying in parallel for the presence of a plurality of analytes in
a sample which can bind one or more of the immobilized proteins on
the protein array comprises delivering the fluid sample to the
invention array and detecting, either directly or indirectly, for
the presence or amount of analyte retained at each patch. In a
preferred embodiment, the method further comprises the step of
washing the array tot remove any unbound or non-specifically bound
components of the sample from the array.
[0181] The array may be used in a diagnostic manner when the
plurality of analytes being assayed are indicative of a disease
condition or the presence of a pathogen in an organism. In such
embodiments, the sample which is delivered to the array will then
typically be derived from a body fluid or a cellular extract from
the organism.
[0182] The array may be used for drug screening when a potential
drug candidate is screened directly for its ability to bind or
otherwise interact with a plurality of proteins on. the invention
array. Alternatively, a plurality of potential drug candidates may
be screened in parallel for their ability to bind or otherwise
interact with one or more immobilized proteins on the array. The
drug screening process may optionally involve assaying for the
interaction, such as binding, of at least one analyte or component
of a sample with one or more immobilized proteins on an invention
array, both in the presence and absence of the potential drug
candidate. This allows for the potential drug candidate to be
tested for its ability to act as an inhibitor of the interaction or
interactions originally being assayed.
[0183] (h) EXAMPLES
[0184] The following specific examples are intended to illustrate
the invention and should not be construed as limiting the scope of
the claims:
Example 1
Fabrication of a Two-Dimensional Array by Photolithography
[0185] In a preferred embodiment of the invention, two-dimensional
arrays are fabricated onto the substrate material via standard
photolithography and/or thin film deposition. Alternative
techniques include microcontact printing. Usually, a computer-aided
design pattern is transferred to a photomask using standard
techniques, which is then used to transfer the pattern onto a
silicon wafer coated with photoresist.
[0186] In a typical example, the array ("chip") with lateral
dimensions of 10.times.10 mm comprises squared patches of a
bioreactive layer (here: gold as the coating on a silicon
substrate) each 0.1.times.0.1 mm in size and separated by
hydrophobic surface areas with a 0.2 mm spacing. 4" diameter
Si(100) wafers (Virginia Semiconductor) are used as bulk materials.
Si(100) wafers are first cleaned in a 3:1 mixture of
H.sub.2SO.sub.4, conc.: 30% H.sub.2O.sub.2 (90.degree. C., 10 min),
rinsed with deionized water (18 M.OMEGA.m), finally passivated in
1% aqueous HF, and singed at 150.degree. C. for 30 min to become
hydrophobic. The wafer is then spincoated with photoresist (Shipley
1813), prebaked for 25 minutes at 90.degree. C., exposed using a
Karl Suss contact printer and developed according to standard
protocols. The wafer is then dried and postbaked at 110.degree. C.
for 25 min. In the next step, the wafer is primed with a titanium
layer of 20 nm thickness, followed by a 200 nm thick gold layer.
Both layers were deposited using electron-beam evaporation (5
.ANG./s). After resist stripping and a short plasma treatment, the
gold patches can be further chemically modified to achieve the
desired bioreactive and biocompatible properties (see Example 3,
below).
Example 2
Fabrication of a Two-Dimensional Array by Deposition Through a Hole
Mask
[0187] In another preferred embodiment the array of gold patches is
fabricated by thin film deposition through a hole mask which is in
direct contact with the substrate. In a typical example, Si(100)
wafers are first cleaned in a 3:1 mixture of H.sub.2SO.sub.4,
conc.: 30% H.sub.2O.sub.2 (90.degree. C., 10 min), rinsed with
deionized water (18 M.OMEGA.cm), finally passivated in 1% aqueous
HF and singed at 150.degree. C. for 30 min to become hydrophobic.
The wafer is then brought into contact with a hole mask exhibiting
the positive pattern of the desired patch array. In the next step,
the wafer is primed with a titanium layer of 20 nm thickness,
followed by a 200 nm thick gold layer. Both layers were deposited
using electron-beam evaporation (5 .ANG./s). After removal of the
mask, the gold patches can be further chemically modified to
achieve the desired bioreactive and biocompatible properties (see
Example 3, below).
Example 3
Synthesis of an Aminoreactive Monolayer Molecule (Following the
Procedure Outlined in Wagner et al., Biophys. J., 1996,
70:2052-2066)
[0188] General.
[0189] .sup.1H- and 13C-NMR spectra are recorded on Bruker
instruments (100 to 400 MHz). Chemical shifts (.delta.) are
reported in ppm relative to internal standard ((CH.sub.3).sub.4Si,
.delta.=0.00 (.sup.1H- and .sup.13C-NMR)). FAB-mass spectra are
recorded on a VG-SABSEQ instrument (Cs.sup.+, 20 keV). Transmission
infrared spectra are obtained as dispersions in KBr on an FTIR
Perkin-Elmer 1600 Series instrument. Thin-layer chromatography
(TLC) is performed on precoated silica gel 60 F254 plates (MERCK,
Darmstadt, FRG), and detection was done using Cl.sub.2/toluidine,
PdCl.sub.2 and UV-detection under NH3-vapor. Medium pressure liquid
chromatography (MPLC) is performed on a Labomatic MD-80 (LABOMATIC
INSTR. AG, Allschwil, Switzerland) using a Buechi column
(460.times.36 mm; BUECHK FlawiL Switzerland), filled with silica
gel 60 (particle size 15-40 .mu.m) from Merck.
[0190] Synthesis of 11,11'-dithiobis(succinimidylundecanoate)
(DSU).
[0191] Sodium thiosulfate (55.3 g, 350 mmol) is added to a
suspension of 11-bromo-undecanoic acid (92.8 g, 350 mmol) in 50%
aqueous 1,4-dioxane (1000 ml). The mixture is heated at reflux (90
.degree. C.) for 2 h until the reaction to the intermediate Bunte
salt was complete (clear solution). The oxidation to the
corresponding disulfide is carried out in situ by adding iodine in
portions until the solution retained with a yellow to brown colour.
The surplus of iodine is retitrated with 15% sodium pyrosulfite in
water. After removal of 1,4-dioxane by rotary evaporation the
creamy suspension is filtered to yield product
11,11'-dithiobis(undecanoic acid). Recrystallization from ethyl
acetate/THF provides a white solid (73.4 g, 96.5%): mp 94.degree.
C.; .sup.1H NMR (400 MHz, CDC.sub.3/CD.sub.3OD 95:5): .delta.2.69
(t, 2H, J=7.3 Hz), 2.29 (t, 2H, J=7.5 Hz), 1.76-1.57 (m, 4H), and
1.40-1.29 (m, 12H); FAB-MS (Cs.sup.+, 20 keV): m/z (relative
intensity) 434 (100, M.sup.+). Anal. Calcd. for
C.sub.22H.sub.42O.sub.4S.sub.2: C, 60.79; H, 9.74; S, 14.75. Found:
C, 60.95; H, 9.82; S, 14.74. To a solution of
11,11'-dithiobis(undecanoic acid). (1.0 g, 2.3 mmol) in THF (50 ml)
is added N-hydroxysuccinimide (0.575 g, 5 mmol) followed by DCC
(1.03 g, 5 mmol) at 0.degree. C. After the reaction mixture is
allowed to warm to 23.degree. C. and is stirred for 36 h at room
temperature, the dicyclohexylurea (DCU) is filtered. Removal of the
solvent under reduced pressure and recrystallization from
acetone/hexane provides 11,11'-dithiobis(succinimidylundecanoate)
as a white solid. Final purification is achieved by medium pressure
liquid chromatography (9 bar) using silica gel and a 2:1 mixture of
ethyl acetate and hexane. The organic phase is concentrated and
dried in vacuum to afford 11,11'-dithiobis(succinimidylundecanoate)
(1.12 g, 78%): mp 95.degree. C.; IH NMR (400 MHz, CDCl.sub.3):
.delta. 2.83 (s, 4H), 2.68 (t, 2H, J=7.3 Hz), 2.60 (t, 2H, J=7.5
Hz), 1.78-1.63 (m, 4H), and 1.43-1.29 (m, 12H); FAB-MS (Cs.sup.+,
20 keV): m/z (relative intensity) 514 (100), 628 (86, M.sup.+).
Anal. Calcd. for C.sub.30H.sub.48N.sub.2O.sub.8S.sub.2: C, 57.30;
H, 7.69; N, 4.45; S, 10.20. Found: C, 57.32; H, 7.60; N, 4.39; S,
10.25.
Example 4
Formation of an Aminoreactive Monolayer on Gold (Following the
Procedure of Wagner et al., Biophys. J., 1996, 70:2052-2066)
[0192] Monolayers based on
11,11'-dithiobis(succinimidylundecanoate) (DSU) can be deposited on
Au(111) surfaces of microarrays described under Examples 1 and 2 by
immersing them into a 1 mM solution of DSU in chloroform at room
temperature for 1 hour. After rinsing with 10 volumes of solvent,
the N-hydroxysuccinimidyl-terminated monolayer is dried under a
stream of nitrogen and immediately used for protein
immobilization.
Example 5
Expression and Purification of Human Caspase Fusion Proteins
[0193] Caspases are cysteine proteases of the papain superfamily,
with a different active site and catalytic mechanism than observed
for papain, Wilson, K. P. et al., Nature, 1994 370:270-275.
Caspases are important enzymes in the promotion of the cell death
pathways and inflammation, Villa, et al, TIBS, 1997, 22:288-392.
Identification of selective caspase inhibitors is essential to
prevent cross-inhibition of other caspase-dependent pathways.
Caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, Villa, et al., TIBS, 1997,
22:288-392 and new caspase homologs identified by the human genome
project are PCR amplified and cloned into an E. coli expression
vector containing an N-terminal histidine tag, Hochuli, et al.,
Biotechnology, 1988 6:1321, a factor Xa cleavage site, a lysine tag
and a tri-glycine linker. Fusion proteins are expressed, purified
by nickel-nitrilotriacetic acid (NTA) agarose chromatography, the
histidine tag removed by factor Xa cleavage, followed by gel
filtration. Caspases are snap-frozen and stored in 20 mM PIPES, pH
7.2, 150 mM NaCl, 0.1% CHAPS, 10% sucrose at -80.degree. C.
Example 6
Immobilization of Fusion Proteins on a 2D-Protein Array
[0194] Caspase-fusion proteins can be immobilized to the
aminoreactive monolayer surface of the bioreactive patches of the
two-dimensional array (see Examples 1, 2, and 4 above). Caspase
fusion proteins can be diluted to concentrations of 1 .mu.g/ml in
20 mM PIPES, pH 7.2, 150 mM NaCl, 0.1% CHAPS, 10% sucrose and
applied onto the bioreactive patches using a computer-aided,
capillary-based dispensing system. After an immobilization period
of 30 min, the 2D array was rinsed and subjected to analysis.
Ultrapure water with a resistance of 18 M.OMEGA.m is generally
useable for all aqueous buffers (purified by passage through a
Barnstead Nanopure.RTM. system).
Example 7
Assay of Caspase Activity on a Two-Dimensional Array
[0195] Caspase activity can be determined by a binding assay using
three fluorescently labeled peptide aldehyde inhibitors that form a
reversible thiohemiacetal moiety with the active site cysteine,
Thornberry, Methods in Enzymology, 1994, 244:615-631. The peptides
are adapted to caspase 1, 3, 4, 7: Dns (dansyl)-SS-DEVD-CHO,
caspase 1: Dns-SS-VDVAD-CHO, caspase 6: Dns-SS-VQID-CHO, Talanian,
J. Biol. Chem., 1997, 272:9677-9682. The affinity for Ac-DEVD-CHO
to caspase 1 is determined to be in the low nanomolar range,
Thornberry, Methods in Enzymology, 1994, 244:615-631. The assay
buffer is 20 mM PIPES, pH 7.2, 150 mM NaCl, 0.1% CHAPS, 10%
sucrose, Stennicke, and Salvesen, J. Biol. Chem., 1997,
272:25719-25723. Fluorescently labeled peptides are mixed to a
final concentration of 1 to 5 nM each, the potential drug compound
added and flushed onto the 2D array. Peptides are allowed to bind
for 10-60 min., unbound peptide removed by washing with buffer and
the fluorescence intensity measured (excitation at 360 nm, emission
at 470 nm).
Example 8
Formation and Use of an Array of Immobilized Fab' Antibody
Fragments to Detect Concentrations of Soluble Proteins Prepared
from Cultured Mammalian Cells
[0196] Collections of IgG antibodies are purchased from commercial
sources (e.g. Pierce, Rockford, Ill.). The antibodies are first
purified by affinity chromatography based on binding to immobilized
protein A. The antibodies are diluted 1:1 in binding buffer(0.1 M
Tris-HCl, 0.15 M NaCl, pH 7.5). A 2 ml minicolumn containing a gel
with immobilized protein A is prepared. (Hermanson, et. al.,
Immobilized Affinity Ligand Techniques, Academic Press, San Diego,
1992.) The column is equilibrated with 10 ml of binding buffer.
Less than 10 mg of immunoglobulin is applied to each 2 ml
minicolumn and the column is washed with binding buffer until the
absorbance at 280 nm is less than 0.02. The bound immunoglobulins
are eluted with 0.1 M glycine, 0.15 M NaCl, pH 2.8, and immediately
neutralized with 1.0 M Tris-HCl, pH 8.0 to 50 mM final
concentration and then dialyzed against 10 mM sodium phosphate,
0.15 M NaCl, pH 7.2 and stored at 4.degree. C.
[0197] The purified immunoglobulin are digested with immobilized
pepsin. Pepsin is an acidic endopeptidase and hydrolyzes proteins
favorably adjacent to aromatic and dicarboxylic L-amino acid
residues. Digestion of IgG with pepsin generates intact F(ab')
fragments. Immobilized pepsin gel is washed with digestion buffer;
20 mM sodium acetate, pH 4.5. A solution of purified IgG at 10
mg/ml is added to the immobilized pepsin gel and incubated at
37.degree. C. for 2 hours. The reaction is neutralized by the
addition of 10 mM Tris-HCl, pH 7.5 and centrifuged to pellet the
gel. The supernatant liquid is collected and applied to an
immobilized protein A column, as described above, to separate the
F(ab') .sub.2 fragments from the Fc and undigested IgG. The pooled
F(ab').sub.2 is dialyzed against 10 mM sodium phosphate, 0.15 M
NaCl, pH 7.2 and stored at 4.degree. C. The quantity of pooled,
eluted F(ab').sub.2 is measured by peak area absorbance at 280
nm.
[0198] The purified F(ab').sub.2 fragments at a concentration of 10
mg/ml are reduced at 37.degree. C. for 1 hour in a buffer of 10 mM
sodium phosphate, 0.15 M NaCl, 10 mM 2mercaptoethylamine, 5 mM
EDTA, pH 6.0. The Fab' fragments are separated from unsplit
F(ab').sub.2 fragments and concentrated by application to a
Sephadex G-25 column (M.sub.r=46,000-58,000). The pooled Fab'
fragments are dialyzed against 10 mM sodium phosphate, 0.15 M NaCl,
pH 7.2. The reduced Fab' fragments are diluted to 100 .mu.g/ml s
and applied onto the bioreactive patches containing exposed
aminoreactive functional groups using a computer-aided,
capillary-based microdispensing system (for antibody immobilization
procedures, see Dammer et al., Biophys. J, 70:2437-2441, 1996).
After an immobilization period of 30 minutes at 30.degree. C., the
array is rinsed extensively with 10 mM sodium phosphate, 0.15 M
NaCl, 5 mM EDTA, pH 7.0.
[0199] Transformed human cells grown in culture are collected by
low speed centrifugation, briefly washed with ice-cold
phosphate-buffered solution (PBS), and then resuspended in ice-cold
hypotonic buffer containing DNase/RNase (10 .mu.g/ml each, final
concentration) and a mixture of protease inhibitors. Cells are
transferred to a microcentrifuge tube, allowed to swell for 5
minutes, and lysed by rapid freezing in liquid nitrogen and thawing
in ice-cold water. Cell debris and precipitates are removed by
high-speed centrifugation and the supernatant is cleared by passage
through a 0.45 .mu.M filter. The cleared lysate is applied to the
Fab' fragment array described above and allowed to incubate for 2
hours at 30.degree. C. After binding the array is washed
extensively with 10 mM sodium phosphate, 0.15 M NaCl, 5 mM EDTA, pH
7.0. The location and amount of bound proteins are determined by
optical detection.
Example 9
Formation and Use of an Array of Immobilized Antibody Fragments to
Detect Concentrations of Soluble Proteins Prepared from Cultured
Mammalian Cells
[0200] A combinatorial library of filamentous phage expressing scFv
antibody fragments is generated based on the technique of
McCafferty and coworkers; McCafferty, et al., Nature, 1990,
348:552-554; Winter and Milstein, Nature, 1991, 349:293-299.
Briefly, mRNA is purified from mouse spleens and used to construct
a cDNA library. PCR fragments encoding sequences of the variable
heavy and light chain immunoglobulin genes of the mouse are
amplified from the prepared cDNA. The amplified PCR products are
joined by a linker region of DNA encoding the 15 amino acid peptide
(Gly.sub.4SerGly.sub.2CysGlySerGly.sub.4Ser) (SEQ ID NO: 1) and the
resulting fill-length PCR fragment is cloned into an expression
plasmid (PCANTAB 5 E) in which the purification peptide tag (E Tag)
has been replaced by a His.sub.6 peptide (SEQ ID NO: 2).
Electrocompetent TG1 E. coli cells are transformed with the
expression plasmid by electroporation. The pCANTAB-transformed
cells are induced to produced functional filamentous phage
expressing scFv fragments by superinfection with M13KO7 helper
phage. Cells are grown on glucose-deficient medium containing the
antibiotics ampicillin (to select for cells with the phagemid) and
kanamycin (to select for cells infected with M13KO7). In the
absence of glucose, the lac promoter present on the phagemid is no
longer repressed, and synthesis of the scFv-gene 3 fusion
begins.
[0201] Proteins from a cell lysate are adsorbed to the wells of a
96-well plate. Transformed human cells grown in culture are
collected by low speed centrifugation and the cells are briefly
washed with ice-cold PBS. The washed cells are then resuspended in
ice-cold hypotonic buffer containing DNase/RNase (10 .mu.g/ml each,
final concentration) and a mixture of protease inhibitors, allowed
to swell for 5 minutes, and lysed by rapid freezing in liquid
nitrogen and thawing in ice-cold water. Cell debris and
precipitates are removed by high-speed centrifugation and the
supernatant is cleared by passage through a 0.45 .mu.m filter. The
cleared lysate is diluted to 10 .mu.g/ml in dilution buffer; 20 mM
PIPES, 0.15 M NaCl, 0.1% CHAPS, 10%, 5 mM EDTA, 5 mM
2-mercaptoethanol, 2 mM DTT, pH 7.2 and applied to the 96-plate
wells. After immobilization for 1 hour at 30.degree. C., the well
is washed with the dilution buffer and then incubated with dilution
buffer containing 10% nonfat dry milk to block unreacted sites.
After the blocking step, the well is washed extensively with the
dilution buffer.
[0202] Phage expressing displayed antibodies are separated from E.
coli cells by centrifugation and then precipitated from the
supernatant by the addition of 15% w/v PEG 8000, 2.5 M NaCl
followed by centrifugation. The purified phage are resuspended in
the dilution buffer containing 3% nonfat dry milk and applied to
the well containing the immobilized proteins described above, and
allowed to bind for 2 hours at 37.degree. C., followed by extensive
washing with the binding buffer. Phage are eluted from the well
with an elution buffer; 20 mM PIPES, 1 M NaCl, 0.1% CHAPS, 10%, 5
mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT, pH 7.2. The well is then
extensively washed with purge buffer; 20 mM PIPES, 2.5 M NaCl, 0.1%
CHAPS, 10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT, pH 7.2.
The well is then extensively washed with dilution buffer; 20 mM
PIPES, 0.15 M NaCl, 0.1% CHAPS, 10%, 5 mM EDTA, 5 mM
2-mercaptoethanol, 2 mM DTT, pH 7.2. The eluted phage solution is
then re-applied to a new well containing adsorbed antigen and the
panning enrichment is repeated 4 times. Finally, the phage are
eluted from the well with 2M of NaCl in 20 mM PIPES, 0.1% CHAPS,
10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT, pH 7.2. Eluates
are collected and mixed with log-phase TG1 cells, and grown at
37.degree. C. for 1 hour and then plated onto SOB medium containing
ampicillin and glucose and allowed to grow for 12-24 hours.
[0203] Individual colonies are picked and arrayed into 96-well 2ml
blocks containing SOB medium and M13KO7 helper phage and grown for
8 hours with shaking at 37.degree. C. The phage are separated from
cells by centrifugation and precipitated with PEG/NaCl as described
above. Concentrated phage are used to infect HB2151 E. coli. E.
coli TG1 produces a suppressor tRNA which allows readthrough
(suppression) of an amber stop codon located between the scFv and
phage gene 3 sequences of the pCANTAB 5 E plasmid. Infected HB2151
cells are selected on medium containing ampicillin, glucose, and
nalidixic acid. Cells are grown to mid-log and then centrifiged and
resuspended in medium lacking glucose and growth continued Soluble
scFv fragments will accumulate in the cell periplasm. A periplasmic
extract is prepared from pelleted cells by mild osmotic shock. The
soluble scFv released into the supernatant is purified by affinity
binding to Ni-NTA activated agarose and eluted with 10 mM EDTA.
[0204] The purified scFv antibody fragments are diluted to 100
.mu.g/ml and applied onto the bioreactive patches with exposed
aminoreactive groups using a computer-aided, capillary-based
microdispensing system. After an immobilization period of 30
minutes at 30.degree. C., the array is rinsed extensively with 10
mM sodium phosphate, 0.15 M NaCl, 5 mM EDTA, pH 7.0.
[0205] Transformed human cells grown in culture are collected by
low speed centrifugation, briefly washed with ice-cold PBS, and
then resuspended in ice-cold hypotonic buffer containing
DNase/RNase (10 .mu.g/ml each, final concentration) and mixture of
protease inhibitors. Cells are transferred to a microcentrifige
tube, allowed to swell for 5 minutes, and lysed by rapid freezing
in liquid nitrogen and thawing in ice-cold water. Cell debris and
precipitates are removed by high-speed centrifugation and the
supernatant is cleared by passage through a 0.45 .mu.m filter. The
cleared lysate is applied to the scFv fragment array described
above and allowed to incubate for 2 hours at 30.degree. C. After
binding, the array is washed extensively with 0.1 M sodium
phosphate, 0.15 M NaCl, 5 mM EDTA pH 7.0. The location and amount
of bound proteins are determined by optical detection.
[0206] Patterns of binding are established empirically by testing
dilutions of a control ,cell extract. Extracts from experimental
cells are diluted to a series of concentrations and then tested
against the array. Patterns of protein expression in the
experimental cell lysates are compared to protein expression
patterns in the control samples to identify proteins with unique
expression profiles.
Example 10
Formation and Use of an Array of Immobilized Monoclonal Antibodies
to Detect Concentrations of Soluble Proteins Prepared from Cultured
Mammalian Cells
[0207] Collections of monoclonal antibodies are purchased from
commercial suppliers as either raw ascities fluid or purified by
chromotography over protein A, protein G, or protein L. If from raw
ascites fluid, the antibodies are purified using a HiTrap Protein G
or HiTrap Protein A column (Pharmacia) as appropriate for the
immunoglobulin subclass and species. Prior to chromotography the
ascites are diluted with an equal volume of 10 mM sodium phosphate,
0.9% NaCl, pH 7.4 (PBS) and clarified by passage through a 0.22
.mu.m filter. The filtrate is loaded onto the column in PBS and the
column is washed with two column volumes of PBS. The antibody is
eluted with 100 mM Glycine-HCl, pH 2.7 (for protein G) or 100 mM
citric acid, pH 3.0 (for protein A). The eluate is collected, into
{fraction (1/10)} volume 1 M Tris-HCl. pH 8.0. The final pH is 7.5.
Fractions containing the antibodies are confirmed by SDS-PAGE and
then pooled and dialyzed against PBS.
[0208] The different samples of purified antibodies are each
diluted to 100 .mu.g/ml. Each different antibody sample is applied
to a separate patch of an array of aminoreactive monolayer patches
(see Example 4, above) using a computer-aided, capillary-based
microdispensing system. After an immobilization period of 30
minutes at 30.degree. C., the array is rinsed extensively with 10
mM sodium phosphate, 0.15 M NaCl, 5 mM EDTA, pH 7.0.
[0209] Transformed human cells grown in culture are collected by
low speed centrifugation, briefly washed with ice-cold PBS, and
resuspended in ice-cold hypotonic buffer containing Dnase/Rnase (10
.mu.g/ml each, final concentration) and a mixture of protease
inhibitors. Cells are transferred to a microcentrifuge tube,
allowed to swell for 5 minutes, and lysed by rapid freezing in
liquid nitrogen and thawing in ice-cold water. Cell debris and
precipitates are removed by high-speed centrifugation and the
supernatant is cleared by passage through a 0.45 .mu.m filter. The
cleared lysate is applied to the monoclonal antibody array
described above and allowed to incubate for 2 hours at 30.degree.
C. After binding the array is washed extensively as in Example 9,
above. The location and amount of bound proteins are determined by
optical detection.
[0210] All documents cited in the above specification are herein
incorporated by reference. In addition, the co-pending U.S. patent
application "Arrays of Protein-Capture Agents and Methods of Use
Thereof", filed on Jul. 14, 1999, with the identifier 24406-0006,
for the inventors Peter Wagner, Steffen Nock, Dana Ault-Riche, and
Christian Itin, is herein incorporated by reference in its
entirety. Various modifications and variations of the present
invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in the art are
intended to be within the scope of the following claims.
* * * * *
References