U.S. patent application number 11/068936 was filed with the patent office on 2006-01-26 for self-assembling peptide surfaces for cell patterning and interactions.
This patent application is currently assigned to Massachusetts Institute of Technology. Invention is credited to Alexander Rich, George Whitesides, Lin Yan, Shuguang Zhang.
Application Number | 20060019309 11/068936 |
Document ID | / |
Family ID | 25380530 |
Filed Date | 2006-01-26 |
United States Patent
Application |
20060019309 |
Kind Code |
A1 |
Zhang; Shuguang ; et
al. |
January 26, 2006 |
Self-assembling peptide surfaces for cell patterning and
interactions
Abstract
This invention describes self assembled monolayers (SAMs)
manufactured by imprinting reactive peptides onto solid supports.
The invention further relates to methods of preparing and using
these improved SAMs.
Inventors: |
Zhang; Shuguang; (Lexington,
MA) ; Rich; Alexander; (Cambridge, MA) ; Yan;
Lin; (Somerville, MA) ; Whitesides; George;
(Newton, MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
President and Fellows of Harvard College
|
Family ID: |
25380530 |
Appl. No.: |
11/068936 |
Filed: |
February 28, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10317838 |
Dec 11, 2002 |
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11068936 |
Feb 28, 2005 |
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10071500 |
Feb 8, 2002 |
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10317838 |
Dec 11, 2002 |
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08882415 |
Jun 25, 1997 |
6368877 |
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10071500 |
Feb 8, 2002 |
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Current U.S.
Class: |
435/7.1 ;
435/287.2 |
Current CPC
Class: |
B01J 2219/00612
20130101; B01J 2219/00743 20130101; B01J 19/0046 20130101; C07K
2/00 20130101; G01N 33/54353 20130101; B82Y 40/00 20130101; C07K
17/14 20130101; B01J 2219/00605 20130101; C40B 40/10 20130101; B01J
2219/00626 20130101; B01J 2219/0063 20130101; B01J 2219/00635
20130101; B82Y 30/00 20130101; C12N 5/0068 20130101; B82Y 5/00
20130101; G01N 2610/00 20130101; G03F 7/165 20130101; B01J
2219/00382 20130101; C12N 2535/10 20130101; B01J 2219/00617
20130101; B82Y 10/00 20130101; B01J 2219/00637 20130101; B01J
2219/00619 20130101; B01J 2219/00659 20130101; C40B 60/14 20130101;
C07K 7/08 20130101; B01J 2219/00725 20130101; G03F 7/0002
20130101 |
Class at
Publication: |
435/007.1 ;
435/287.2 |
International
Class: |
C40B 20/02 20060101
C40B020/02; C12M 1/34 20060101 C12M001/34 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was supported, in whole or in part, by a grant
(Grant No. DAAH04-94-G-0407) from the Army Research Office. The
Government has certain rights in the invention.
Claims
1. A device comprising: a solid support; and an array of isolated
regions on the support, the array comprising a layer of peptides,
wherein the peptides are bound to the support by a bond between the
support and a terminal amino acid in a preselected, reproducible
pattern.
2. The device of claim 1 further comprising a background region
surrounding the isolated regions, wherein the peptides are not
bound to the background region.
3. The device of claim 2 wherein the background region comprises an
inert compound.
4. A device comprising: a solid support; and an isolated region
comprising a layer of peptides, wherein the peptides are bound to
the support by a bond between the support and a terminal reactive
group in a preselected, reproducible pattern.
5. The device of claim 4 comprising a plurality of isolated regions
of a self-assembled monolayer, the plurality of regions defining an
ordered array on the support.
6. A device comprising: a solid support; an isolated region
comprising a self-assembled monolayer of peptides, wherein the
peptides are bound to the support by a bond between the support and
a terminal reactive group in a preselected, reproducible pattern;
and a background region surrounding the isolated region and
comprising a compound which can react with the support.
7. A device for immobilizing at least one biological material in a
specific and predetermined pattern comprising: a surface, an array
of immobilization islands in a specific and predetermined pattern
over the surface isolated from each other by at least one
background region, the array of immobilization islands comprising a
first self-assembled monolayer comprising at least one first
functional group wherein the at least one first functional group is
selected to biophilic, and wherein the first self-assembled
monolayer comprises a monolayer of linear peptides, the at least
one background region comprising a second self-assembled monolayer
having a second functional group wherein the second functional
group is selected to be biophobic.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/317,838, filed Dec. 11, 2002, which is a continuation of
U.S. application Ser. No. 10/071,500, filed Feb. 8, 2002, which is
a continuation of U.S. application Ser. No. 08/882,415, filed Jun.
25, 1997. The entire teachings of the above applications are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Organic surfaces have been employed in numerous methods and
systems, including as substrates for ELISA, cell and tissue
culture. Self-assembled monolayers (SAMs) are a class of organic
surfaces manufactured by imprinting a monolayer of organic
compounds with reactive moieties onto a solid support under
conditions wherein the compounds react with and bind to the solid
support in a single ordered and patterned layer. See, Lopez, et
al., "Convenient Methods for Patterning the Adhesion of Mammalian
Cells to Surfaces Using Self-Assembled Monolayers of
Alkanethiolates on Gold," J. Am. Chem. Soc., 115(13):5877-5878
(1993) and Mrksich and Whitesides, "Using Self-Assembled Monolayers
to Understand the Interactions of Man-Made Surfaces with Proteins
and Cells", Annu. Rev. Biophys. Biomol. Struct., 25:55-78 (1996).
Molecular self-assembly is the spontaneous association of molecules
under equilibrium conditions into stable, structurally well-defined
order joined by non-covalent bonds. SAMs manufactured to date have
linked chemical moieties to solid surfaces through long chain alkyl
linkages. Examples of organic compounds which have been patterned
on a solid support include alkanethiolates and alkylsiloxanes. The
SAMs are manufactured employing a process termed "microcontact
printing."
[0004] It has been suggested that SAMs can be used to pattern cells
on a surface by presenting chemical moieties which bind to the
cells on the solid surface. Mrksich and Whitesides, above. However,
these molecules, and the resulting SAMs, can be difficult and/or
expensive to manufacture. Thus, improvements and cost reductions in
the manufacture of SAMs are desirable and are necessary.
SUMMARY OF THE INVENTION
[0005] This invention is based upon the discovery that improved
SAMs can be manufactured by imprinting reactive self assembling
peptides onto solid supports. The SAMs are characterized by ease of
manufacture and purification. They are versatile in their ability
to readily provide a large variety of chemical reactive moieties,
or "presenting groups", to selected targets. For example, the SAM's
of the present invention can be readily designed to present ligands
to cellular receptors, cell adhesion motifs, antibodies or
antigen-binding fragments thereof to cell surface proteins. This
preferred class of SAMs can be used to bind a target, e.g. a
selected cell or cells, to a predetermined locus on the solid
support.
[0006] Thus, the invention relates to a composition of matter
comprising a solid support and a self-assembled monolayer of linear
peptides wherein said peptides bound directly to said solid support
through a terminal amino acid in a predetermined pattern.
Preferably, the peptides comprise a terminal reactive group, a
central linker and a presenting group. The invention also relates
to the uses and applications of the SAMs described herein, as will
be described in more detail below.
[0007] The invention further relates to a method for manufacturing
an SAM, or a composition of matter comprising a solid support and a
self-assembled monolayer of linear peptides wherein said peptides
bound directly to said solid support through a terminal amino acid
in a predetermined pattern, comprising microcontact printing the
reactive peptides onto the solid support and maintaining the
peptides under conditions suitable for binding.
[0008] The foregoing and other objects, features and advantages of
the invention will be apparent from the following more particular
description of preferred embodiments of the invention, as
illustrated in the accompanying drawings. The drawings are not
necessarily to scale, emphasis instead being placed upon
illustrating the principals of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIGS. 1 and 2 illustrate methods of microcontact printing
reactive peptides to a solid support in a predetermined
pattern.
[0010] FIG. 3 illustrates patterns which may be selected, for
example, in SAMs for immobilizing cells.
DETAILED DESCRIPTION OF THE INVENTION
[0011] As set forth above, the invention relates to improved SAMs
comprising a predetermined pattern of peptides on a solid support.
Preferred peptides of the invention can be characterized by three
regions bound to each other through an amino acid or via peptide
binding, the "terminal reactive group", the "central linker" and
the "presenting group."
[0012] Upon binding the peptides to the solid support, the peptides
are preferably highly ordered and preferably possess a consistent
linear and parallel configuration with each other. Generally, the
peptides, or the central linker thereof, are fully extended beta
strands in configuration under the conditions of use.
[0013] Although in some embodiments, it may be desirable to present
a ligand or other molecule which possesses a tertiary structure,
generally, the peptides are linear (e.g., free or substantially
free of branching or tertiary structure). "Substantially free" of
branching or tertiary structure is intended to include minor
amounts of branching and peptide interactions which do not
significantly interfere with the free movement or function of the
presenting group. The actual degree of branching and peptide
interactions which can be tolerated without deleteriously effecting
the quality of the product will be function of the overall length
of the peptide, the branched peptides, the nature of the amino
acids in each and their ability or tendency to interact with each
other can generally be determined by routine screening or computer
modeling. For example, peptides "substantially free" of branching
may include a peptide composition wherein less than about 5% of the
peptides are characterized by one or more branches.
[0014] While the length of the peptide is not critical to the
invention, the peptide is preferably small to moderate in length.
Thus, the central linker of the peptide can preferably be between
about 2 to about 50 naturally occurring or non-naturally occurring
amino acids in length are preferred, more preferably between about
8 to about 35 amino acids in length. Certain peptides in excess of
50 may present undesirable interactions of the peptides, such as a
possible tendency of the peptide to fold. Peptide interactions can
be predicted by, for example, computer modeling and structural
information available at protein data banks at, for example,
Brookhaven National Laboratories, N.Y.
[0015] Peptides which can be used in the invention can be
characterized by a reactive moiety which can react and bind to the
solid support, the "terminal reactive group". Typically, the
terminal reactive group is an amino acid characterized by a
functional group pendant from the side chain, the amino group or
the carboxy group. Thus, the terminal reactive group which binds to
the solid support can be an amino acid substituted by a hydroxy,
thiol, carboxy, amino, amido, imido or guanidino group. Preferred
terminal amino acids, thus, include serine, cysteine, tyrosine,
asparagine, glutamine, aspartic acid, glutamic acid, lysine,
arginine and histidine. Alternatively, the terminal reactive group
can be a nonnaturally-occurring amino acid characterized by a
functionality which can react with the solid support. Examples
include beta amino acids (amino acids wherein the amino and/or
carboxy group are not substituted on the same alpha carbon, such as
beta-alanine) or amino acids which have been chemically modified,
by electrophilic substitution, nucleophilic substitution,
activation reactions or addition reactions, for example. See March,
"Advanced Chemistry," Third Edition (1985), Chapters 10-16, the
contents of which are incorporated herein by reference.
[0016] It is further desirable that the peptide be of sufficient
length to provide a flexible, spatial separation between the solid
support (upon reaction with the reactive terminal group) and the
opposing reactive terminus of the peptide (e.g., the presenting
group). Thus, the peptides of the invention preferably comprise a
"central linker", which is a peptide bound to the terminal reactive
group and presenting group through peptide or amide bonds. The
amino acids employed in the peptide and/or central linker are
selected to promote or optimize a beta strand configuration at the
conditions for use. It is further preferred that the amino acids in
this portion of the peptide be substantially free of large or bulky
side chains or bonds which will interfere with the configuration
(e.g. proline). The amino acids can further be selected considering
material strength, permeability and degradation rate of the
resulting peptide and SAM. Preferably, the amino acids selected for
the central section of the peptide are glycine, L-alanine and
D-alanine. D-amino acids have advantages in many applications due
to their resistance to L-protease degradation.
[0017] The length of the central linker where present, is also
generally not critical to the invention. Preferably, the central
linker is between about 2 and about 30 amino acids in length, more
preferably between about 2 and about 8 amino acids.
[0018] The peptide can also be characterized by a "presenting
moiety" which will bind to one or more targets. The term
"presenting group" is defined herein to include one or more
chemical atoms, functional groups, amino acids or peptides that
possess an affinity to or resistance for a target entity. For
example, a presenting group which presents a resistance for a
target entity, e.g., a protein or cell, can be poly(ethylene
glycol) or another compound which is inert to the target. A
presenting group which is resistant to water, as a target molecule,
is a hydrophobic group, such as a high chain alkyl or
hydrophobically blocked amino acid (e.g., an alkyl ester of valine,
leucine, isoleucine or phenylalanine).
[0019] Generally, one or more peptides employed in the present
invention possess a presenting group with an affinity for a target,
e.g. a target molecule. In such embodiments, the presenting group
can be specific or non-specific for the target molecule. For
example, where the target is a cell, the target molecule can be a
cell surface protein. The presenting group can be a ligand for that
protein, an antibody or an antigen-binding fragment thereof which
binds specifically to the cell surface protein.
[0020] The presenting group can be a non-peptide or, preferably, a
peptide. As discussed above, the presenting group can be a ligand
for or an antibody or antibody fragment which binds to the target
molecule.
[0021] Particularly suitable presenting groups are oligopeptides
which self assemble to form a beta sheet under conditions for the
desired or selected application. Examples of oligopeptides which
self assemble under these conditions are described in U.S.
application Ser. Nos. 08/346,849 and 08/784,606, which are
incorporated herein by reference in their entireties. Briefly,
these oligopeptides are amphiphilic, have alternating hydrophobic
and hydrophilic amino acids and are complementary. As will be
described in more detail below, particularly preferred
oligopeptides for self assembly are RADX.sub.n and EAXX.sub.n
wherein X is an amino acid and n is an integer between about 2 and
about 8.
[0022] Particularly preferred targets include cells. Examples of
cells which can be targeted include prokaryotic and eukaryotic
cells. The cells can be mammalian, plant, bacterial, and yeast.
Mammalian cells which can be targeted include tumor cells, normal
somatic cells and stem cells. The cells can be fibroblasts,
endothelial cells, neuronal cells, hepatocytes, blood cells, smooth
muscle cells, and progenitors thereof, for example. Bacterial cells
can be gram positive or gram negative bacteria and can include
Escherichia coli, Streptococcus, Staphylococcus, as well as many
others. Bacterial cells which may be desirable to target and, thus
detect and/or culture, can include pathogens and non-pathogens,
e.g., contaminants in a food sample, a mammalian tissue sample or
serum sample or in a plant tissue sample. Similarly, yeast can be
targeted and include, for example, Candida and Saccharomyces.
[0023] Cells can preferably be targeted by selecting a presenting
group which will react with and bind to the cell surface.
Generally, this is accomplished by binding to a cell surface
molecule, such as a protein, lipid, or sugar at the surface of the
protein. These surface molecules are included herein as "target
molecules." For example, a target molecule can be a cell surface
protein and can be specific to the target or, in this case, cell,
or the target molecule can be non-specific. Where the object of the
application is to detect the presence of a cell in a sample, e.g.,
a tumor cell in a sample which can contain normal cells, it is
desirable that the target molecule be specific to the tumor cell
(e.g., present on tumor cells and absent on the normal cells).
These molecules are generally known in the art as tumor markers.
Where the object of the invention is to detect the presence of
bacteria in a sample, such as in food, tissue sample, blood sample,
or pharmaceutical, it can be desirable to select a target molecule
which is present on many types of bacteria which are potentially
contaminating the sample to be tested. In other instances, e.g.,
where a substantially pure cell culture is being targeted or
transferred to the solid support, the selection of specific or
non-specific target molecule is immaterial.
[0024] Suitable target molecules include tumor markers, cellular
receptors, such as CD4 and, CD8. Neuronal cellular receptors
include N-CAMs, the L1 receptors, NGF receptor, the netrin receptor
and others.
[0025] Targets can include non-cellular products as well, including
viruses (such as retroviruses, influenza viruses, and
herpesviruses, for example), and proteins (such as prostate soluble
antigen (PSA), cytokines, cytokine receptors, growth factors, and
growth factor recpetors. Where the target is a virus, the target
molecule can be a surface protein as well, such as a cellular
receptor implicated in the infection of cells. A particularly
preferred target molecule for HIV is, for example, gp120.
[0026] Examples of presenting groups include cellular adhesion
motifs, ligands or binding fragments of ligands for the target
molecule (e.g., the ligand for gp120 is CD4), antibodies or antigen
binding fragments of antibodies which bind to the target
molecule.
[0027] A ligand is defined here to include molecules which are the
same as or substantially the same as the native molecule which
binds the target molecule. For example, CD4 is a native ligand for
the HIV env protein, gp120. Thus, where the target molecule is
gp120, the term "ligand" and, thus, the presenting groups include
native CD4, a ligand-binding fragment of CD4 (such as, an
extracellular domain), and mutations thereof which bind to
gp120.
[0028] The terminal reactive group, central linker and presenting
group are preferably arranged linearly with the central linker
bonded directly or indirectly to both the reactive group and the
presenting group through, e.g., peptide bonds. Preferably, the
peptide has the formula:
X--(CH.sub.2).sub.n--CH(NH.sub.2)CO(AA).sub.m-L or
X--(CH.sub.2).sub.n--CH(COOH)NH(AA).sub.m-L
[0029] wherein X is an inert group, such as H, alkyl, alkoxy,
alkylthio or dialkylamine, or is a labile or reactive group, such
as a thiol, hydroxy, amino, carboxy, acylhalide, carboxy ester, or
halide;
[0030] AA is, independently, the same or different,
naturally-occurring or non-naturally-occurring amino acid, and is
preferably, glycine, L-alanine or D-alanine;
[0031] L is a group which binds specifically or non-specifically to
a target and is preferably a peptide, such as a ligand, an antibody
or an antibody fragment;
[0032] n is zero or an integer between 1 to about 5;
[0033] m is an integer of at least about 2 and, preferably, between
about 2 and about 50, more preferably between about 2 and about
8.
[0034] The peptides of the invention can be manufactured by known
and industry stadnard peptide synthesis technology. For example,
the peptides can be synthesized chemically or recombinantly (e.g.
by the expression of a recombinant nucleic acid molecule which
encodes the peptide or a precursor thereof). A precursor of the
peptide can be particularly beneficial where one or more of the
amino acids are non-naturally occurring (e.g. a beta amino acid or
an amino acid with a non-naturally occurring side chain). The
manufacture of peptides chemically and recombinantly are generally
practiced in the art and are described in, for example, U.S.
application Ser. Nos. 08/346,849 and 08/784,606 and Ausubel,
Current Protocols in Molecular Biology (1997). The peptides can
preferably be purified prior to use in the manufacture of the SAMs
by standard techniques, including HPLC.
[0035] The peptides employed in the invention are imprinted or
patterned on a solid support. The shape of the solid support is not
critical to the invention and can be selected to optimize ease of
use in the particular application. Thus, the solid support can be
substantially spherical (e.g., a bead) or non-spherical, such as in
a container (e.g., a petri dish or cup), cylinder or cone, or a
substantially flat film, stick, chip or disc, of essentially any
size suitable for the ultimate application. The solid support can
be porous (as in a membrane) or non-porous (as in a petri dish or
container).
[0036] The material employed in the manufacture of the solid
support is not critical as well. Thus, a variety of materials can
be employed in the manufacture of the solid support. For example,
the solid support can be an inorganic material such as a metal,
including as gold, copper, zinc, silver or nickel or a metal alloy.
Alternatively, the solid support can be glass, silica, or silicon
oxide. In yet another embodiment, the solid support can be an
organic material, such as a polymer or resin, including nylon,
poly(ethylene glycol), and polyfluoropolymers. It can be desirable
in some embodiments to employ a transparent solid support. In this
embodiment, the detection of the binding of an opaque target (e.g.,
a cell) can be determined readily and accurately visually or
electronically and/or robotically employing, for example, a laser
under the control of a computer.
[0037] The solid support is selected with a view towards its
ability to react with the terminal reactive group of the peptide.
For example, the thiol group (e.g., X) can react with gold under
standard methods, as described, for example in Mrkisch and
Whitesides, above. Likewise, the hydroxy group (e.g., X) can react
with siloxane under relatively mild conditions. Xia, et al.
"Microcontact Printing of Octadecylsiloxane on the Surface of
Silicon Dioxide and Its Application in Microfabrication," J. Am.
Chem. Soc. 117:9576-9577 (1995).
[0038] Solid supports which are inert to the peptide can be
derivatized to render them reactive. For example, the solid support
can be coated with a reactive material, chemically treated (e.g.,
by electrophilic or nucleophilic substitution reaction, addition
reactions, etc.) to introduce reactive groups.
[0039] The peptides are printed on the solid support, as will be
described below. The terms "printed", "patterned" or "predetermined
pattern" are defined herein to mean that the solid support has
ordered areas where the peptides are bonded and not bonded to the
solid support. That is, a printed or patterned solid support is
expressly not intended to include a support with random or
substantially homogeneous distribution of the peptide over its
entire surface(s). Furthermore, the peptides are printed on the
solid support in a single layer in a substantially consistent
configuration. Thus, the terms are further not intended to include
solid supports wherein peptides are bonded to the solid support via
distinct and different functional groups across the same molecule
(e.g., distinct cysteine residues in a protein containing multiple
cysteines along its sequence).
[0040] The patterns which can be selected in this invention are not
particularly critical. Preferred patterns for SAMs useful as
research tools in the study of cell/cell interactions are linear
tracks of alternating peptides which can adhere to the cells and
inert tracks of solid support or an inert compound bound to the
solid support. Depending upon the thickness of the tracks, the
orientation of the cell can further be manipulated. That is a thin
track can result in the orientation of the cells linearly. FIG. 3
exemplifies suitable patterns.
[0041] As stated above, methods for the manufacture of SAMs are
generally known in the art. U.S. Pat. Nos. 5,620,850 and 5,512,131
and PCT Published Application Nos.: WO97/07429 and WO96/29629
decribed suitable methods for manufacture. Additional examples
include Deng, Li, Milan Mrksich and George M. Whitesides,
"Self-Assembled Monolayers of Alkanethiolates Presenting
Tri(propylene sulfoxide) Groups Resist the Adsorption of Protein,"
J. Am. Chem. Soc., 118(21):5136-5137 (1996); Chen, Christopher S.,
Milan Mrksich, Sui Huang, George M. Whitesides, Donald E. Ingber,
"Geometric Control of Cell Life and Death," Science, 276:1425-1428
(1997); Lopez, Gabriel P., Mark W. Albers, Stuart L. Schreiber,
Reed Carroll, Ernest Peralta, and George M. Whitesides, "Convenient
Methods for Patterning the Adhesion of Mammalian Cells to Surfaces
Using Self-Assembled Monolayers of Alkanethiolates on Gold," J. Am.
Chem. Soc., 115(13):5877-5878 (1993); Kumar, Amit, Nicholas L.
Abbott, Enoch Kim, Hans A. Biebuyck, and George M. Whitesides,
"Patterned Self-Assembled Monolayers and Meso-Scale Phenomena,"
Acc. Chem. Res., 28(5):219-226 (1995); DiMilla, Paul A., John P.
Folkers, Hans A. Biebuyck, Ralph Harter, Gabriel P. Lopez, and
George M. Whitesides, "Wetting and Protein Adsorption of
Self-Assembled Monolayers of Alkanethiolates Supported on
Transparent Films of Gold," J. Am. Chem. Soc., 116(5):2225-2226
(1994); Singhvi, Rahul, Amit Kumar, Gabriel P. Lopez, Gregory N.
Stephanopoulos, Daniel I. C. Wang, George M. Whitesides, Donald E.
Ingber, "Engineering Cell Shape and Function," Science, 264:696-698
(1994); Mrksich, Milan and George M. Whitesides, "Using
Self-Assembled Monolayers to Understand the Interactions of
Man-Made Surfaces with Proteins and Cells," Annu. Rev. Biophys.
Biomol. Struct., 25:55-78 (1996); Wilbur, James L., Amit Kumar,
Enoch Kim, George M. Whitesides, "Microfabrication by Microcontact
Printing of Self-Assembled Monolayers," Adv. Mater. 6(7/8):600-604
(1994); Xia, Younan, Enoch Kim, Milan Mrksich and George M.
Whitesides, "Microcontact Printing of Alkanethiols on Copper and
Its Application in Microfabrication," Chem. Mater. 8(3):601-603
(1996); Mrksich, Milan, Jocelyn R. Grunwell and George M.
Whitesides, "Biospecific Adsorption of Carbonic Anhydrase to
Self-Assembled Monolayers of Alkanethiolates that Present
Benzenesulfonamide Groups on Gold," J. Am. Chem. Soc.,
117(48):12009-12010 (1995); Jeon, Noo Li, Ralph G. Nuzzo, Younan
Xia, Milan Mrksich, and George M. Whitesides, "Patterned
Self-Assembled Monolayers Formed by Microcontact Printing Direct
Selective Metalization by Chemical Vapor Deposition on Planar and
Nonplanar Substrates," Langmuir, 11(8):3024-3026 (1995); Xia,
Younan, Milan Mrksich, Enoch Kim and George M. Whitesides,
"Microcontact Printing of Octadecylsiloxane on the Surface of
Silicon Dioxide and Its Application in Microfabrication," J. Am.
Chem. Soc., 117(37):9576-9577 (1995). The contents of these
articles are incorporated herein by reference. The method is
illustrated in FIGS. 1 and 2.
[0042] Referring specifically to FIG. 1, a polymeric or elastomeric
stamp 1 (e.g. a polydimethylsiloxane stamp) is contacted or "inked"
with a solution 2 containing the peptide in a suitable solvent and
then the inked stamp is pressed against the solid support 3,
thereby transferring the peptide solution in a controlled fashion
to the solid support 3. The peptide is then maintained in contact
with the solid support 3 under conditions suitable for binding,
resulting in a SAM 4.
[0043] Upon binding of the peptide to the solid support, the
solvent is generally removed, for example, by washing (e.g.,
extraction), evaporation or lyophilization.
[0044] The patterned SAM thus formed can then be used directly or
can be further derivatized, e.g., by subjecting the SAM to a second
printing step to ink a different chemical compound thereon. The
second chemical compound can preferably be a peptide of the claimed
invention or can be different, such as an alkanethiol or
poly(ethylene glycol), as described in Mrksich and Whitesides,
above.
[0045] In yet another alternative, the SAM can be subjected to
additional steps which can modify the peptide on the SAM. This
embodiment may be desirable where the presenting group (e.g., L) or
the chemical bond to the central linker ((AA).sub.m) is labile
under the conditions for binding the peptide to the solid support.
Thus, the presenting group can be chemically reacted with a peptide
precursor bonded directly to the solid support, thereby obtaining a
SAM of the present invention.
[0046] In many instances, it can be desirable to modify the exposed
areas of the solid support, for example, by exposing the SAM to
ultraviolet light or oxidize the SAM. This can be done to improve
the reactivity or eliminate reactivity of the material of the solid
support with one or more materials encountered in storage or in use
of the SAM.
[0047] Referring to FIG. 2, the solid support 3 is stamped with a
solution containing a first compound 5 (such as a
poly(alkoxyglycothiol)) which can react with the solid support and
presents an imprint or pattern of the solid support, as described
above. The printed solid support 6 is then contacted with a
solution containing the peptide 2 under conditions suitable for
reacting the peptide with the exposed solid support. The thus
formed SAM 7 possess a pattern of the peptide in the relief of the
imprint of the first compound. The SAM can then be washed and
dried,. as above. The printed solid support 6 can be immersed into
a solution of the peptide or the peptide can be poured or sprayed
onto the surface of the SAM, as is convenient.
[0048] Solvents which can be used to ink the peptide onto the stamp
and, then, onto the solid support include solvents which can
disperse or, preferably, solubilize the peptide. The solvent is
preferably readily removed, for example, by evaporation,
lyophilization or extraction, from the solid support. Examples of
preferred solvents include alcohols, such as ethanol, acetone,
acetonitrile, DMSO and DMF and miscible combinations thereof. The
peptide solution concentration is selected such that the desired
amount of peptide is delivered to the solid support. That is, if it
is desired to print the peptide upon the solid support at a high
concentration or density, then the peptide solution can be at or
near the saturation level of a good solvent. If it is desired to
imprint a low concentration of the peptide sparsely upon the solid
support, the solution can be characterized by a low concentration
such as employing a dilute solution.
[0049] The solution comprising the peptide can also include
additional components. For example, a dispersant or solubilizer can
be added to the solution to solubilize or disperse, for example,
the peptide. It can be desirable in some instances to include a
colorant in the solution, particularly where the solution is
colorless or is difficult to observe on the solid support or stamp,
so that the area of the solid support which has been inked can be
visually observed. It is generally desirable where additional
components are added to the solution that they can be readily
removed from, e.g. washed free of, the solid support.
[0050] It is clear that, in the method for manufacturing the SAMs,
either the stamp, solid support or both can be mobile, relative to
the other. That is, the stamp can be fixed and the solid support
pressed firmly against it or vice versa. Alternatively, both the
stamp and support can be mobilized. This process can be readily
achieved employing robotics, which ensures a high degree of
consistency and accuracy in the printing step.
[0051] The peptide can be bound to the solid support via covalent
bonding, ionic bonding or other chemical interactions. It is
preferred that the bonding be of a high affinity and be essentially
irreversible under the conditions for use. The conditions suitable
for bonding the peptide to the solid support can be dependent upon
the nature of the chemical reaction relied upon and can generally
be determined by the person of skill employing no more than routine
skill.
[0052] Clearly, other methods for the manufacture of the SAMs of
the present invention will be apparent to the person of skill in
the art and are intended to be included within the scope of the
present invention.
[0053] The SAMs of the invention can be employed in a variety of
processes in biology, biotechnology, medicine, material science,
biomedical engineering and computer-related inventions. A preferred
example of an application includes the use of the SAMs as
substrates for ELISA.
SAMs to Screen for the Presence of a Target in a Sample
[0054] The SAMs of the present invention can be used to screen for
the presence of a target in a sample. As set forth above, the SAMs
of the invention can be designed to possess a presenting group
which binds specifically or non-specifically to a target or target
molecule. Where the presence of a cell is to be detected and
distinguished from other cells in the sample (e.g.,the presence of
a tumor cell in a tissue sample which can further comprise normal
diploid cells), the presenting group is "specific" to the target,
i.e. does not bind substantially to other materials or cells which
can be present. Where the presence of many different cells in a
sample (e.g., the presence of bacterial contaminants in a
pharmaceutical process stream), the presenting group is
non-specific to a particular target but can bind to a large number
of targets.
[0055] The method of screening for the presence of a target can
comprise the steps of contacting an SAM, as described above, with a
sample under conditions suitable for the target or target molecule
to bind to the presenting group on the SAM and detecting the
presence of the target or target molecule. The target or target
molecule can be a cell, such as a mammalian cell (e.g.,tumor cell,
normal diploid somatic cell, or stem cell), a bacterium or yeast
(e.g., a causative agent for disease or contaminant).
Alternatively, the target or target molecule can be a virus (e.g.,
a causative agent for disease or contaminant), toxin or protein,
etc.
[0056] The sample can be obtained from an animal or patient, such
as a tissue sample or biopsy, body fluid, e.g., serum, milk, saliva
or urine or fecal matter. Alternatively, the sample can be obtained
from manufacturing process, such as a pharmaceutical process or
food process. Thus, the method can be used to screen for
contaminants or sterile conditions in manufacturing or it can be
used to screen for or diagnose disease in a patient.
[0057] It is generally desirable that the sample be contacted with
the SAM as a liquid, e.g. a dispersion or solution. Thus, the
sample can be mixed with a diluent or buffer. Examples of diluents
include water, such as sterile water, polar and non-polar solvents,
e.g. alcohols, dimethylformamide, acetonitrile, alkanes, benzene,
toluene, etc. Buffers include physiological buffers, such as
phosphate buffered solution, culture media, etc.
[0058] The person of skill in the art can determine empirically the
conditions for contacting the SAM and the sample such that the
target or target molecule can react with each other and bind. Such
conditions are well known in the art. Generally, where the method
is a diagnostic tool and the sample is a tissue sample or other
biological sample, the conditions will physiologic. That is,
physiological pH is generally employed. Room temperature can also
be employed in many instances. Where the method is detecting the
presence of contaminants in a sample, neutral pH can be generally
employed, as well as room temperature.
[0059] The SAM can be contacted with the sample in a number of
ways. For example, the SAM can be immersed into the sample, as in
dipping a stick. Alternatively, the sample can be poured over or
through the SAM. Optionally, the SAM can be rinsed after the
contacting step, such as with sterile water.
[0060] After the SAM has been contacted with the sample, the
presence of the target or target molecule is detected. This can
also be performed in a number of ways. In one embodiment, the SAM
can be contacted with a second solution which possesses a labeled
compound which can react with the target molecule, as in an ELISA
method. The label (e.g., a colorimetric label or radiolabel) can
then be detected. In many embodiments, the target can be detected
visually, with the naked eye, under a microscope or robotically.
This can be advantageous, for example, where the target is a cell.
In many embodiments, it may be desirable to permit any cells bound
to the SAM to colonize prior to detection. The method of the
invention can accurately determine the presence of an individual
cell or determine a precise cell count in a sample.
[0061] In a particularly preferred method, the solid support for
the SAM is transparent. In such an embodiment, the presence of an
opaque target, such as a cell, can be determined by scanning the
SAM with a laser and determining the number of targets or cells
present thereon, which accurately correlates to the number of
interruptions in scanning. This method can be performed in an
automated system (e.g. robotically), thereby improving efficiency
and avoiding inaccurate results due to human error.
SAMs in Cell Culture
[0062] The SAMs of the invention can be used as a solid support in
culturing cells. Cells can be attached to the SAMs by contacting
the cells to be attached with the SAM and maintaining the cells
under conditions suitable for growth. As above, it is generally
desirable to contact the cells with the SAM as a liquid, e.g., in
the presence of a diluent or solvent. The cells can be attached to
the solid support in a predetermined fashion, order and
orientation.
[0063] Conditions for maintaining cells can be those employed
routinely for the cell or cell type to be cultures. For example,
the culture can be maintained under temperatures (e.g. between
about 25.degree. C. to about 60.degree. C.) and pH (e.g. between
about 4 and about 10) appropriate for growth. Nutrients appropriate
for growth can also advantageously be provided to the culture.
[0064] The invention permits very accurate control of cell
population and density. The invention can be utilized to study cell
growth and cellular interactions to external stimuli, including
other cells, growth factors, repellants and inhibitors. Thus, the
invention represents a significant advance in the ability to
conduct research in biology and medicine.
[0065] In yet another embodiment, the method can be employed in
screenings or assays employing cells, such as screening for drugs
which may inhibit the growth of a cell or cells (such as in a
screen for anti-tumor agents, anti-bacterials). Alternatively, the
method can be employed in the screening for drugs which increase or
activate the growth of a cell or cells, including fibroblasts,
endothelial cells, smooth muscle cells, hematopoietic cells and
neuronal cells, etc.
[0066] The method can also be used to maintain cell cultures,
including tissue cultures, in the manufacture of cellular products
(e.g., proteins, hormones, etc.), artificial tissues, etc. Examples
of tissues which can be cultured in this manner include
fibroblasts, endothelial cells, smooth muscle cells and neuronal
cells. Such tissues can be employed as grafts, such as autologous
grafts.
[0067] The understanding of complex neuronal connections is central
to our comprehension of central nervous system function, and
advances in doing so will benefit from combining engineering with
molecular cell biology to analyze neuronal behavior under
well-characterized and controlled conditions. Neurite outgrowth,
guidance and connections can be studied on surfaces patterned with
self-assembling peptides that contain cell-adhesion motifs.
Controlling neurite outgrowth, including distances, angles and
direction, can be important in controlling and studying synapse
formation between neuronal cells guided into proximity. Neuronal
cells attached to the described SAMs can be employed in the study
of neuronal cell culture, synapse formation, neuronal connection
engineering, screening neuropeptides, as well as pharmaceutical
agents that stimulate, inhibit or alter the nature of nerve growth,
and inter-connections. For example, attractants, e.g., growth
factors, neuropeptides, neurotrophins, and drugs can be screened
for their ability to alter the direction or growth behavior of
neurites or their ability to induce, stimulate, suppress or inhibit
neurite growth. These attractants can be placed or randomly
contacted with the neuronal cell-bound SAMs.
[0068] Preferred peptides for the manufacture of the SAMs for this
application include peptides wherein the presenting group is a cell
adhesion motif or peptide which binds to neuronal cells. Examples
of suitable cell adhesion motifs are (RADX).sub.n, (RADS).sub.n,
(EAKX).sub.n, and (EAKS).sub.n, wherein X is an amino acid, such as
S, and n is an integer, preferably between about 2 to about 8.
Oligopeptides of these sequences have been shown to promote neurite
outgrowth in culture (U.S. application Ser. No. 08/784,606, which
is incorporated herein by reference in its entirety).
EXAMPLE 1
Preparation of Patterned SAMs Glass Chip
[0069] A 10:1 (w:w) mixture of Sylgard Silicone Elastomer 184 and
Sylgard Curing Agent 184 (Dow Corning Corp., Midland, Mich.) was
casted over a master, which was generated by photolithography, and
pressure degassed. After sitting at room temperature for 1 hour,
the PDMS was cured at 60.degree. C. for 2 hours. The stamp was
carefully peeled off the master after cooling to room temperature
and rinsed with ethanol. The PDMS stamp was inked by a cotton swab
which has been moistened with a 5 mM solution of
(1-mercaptoundec-11-yl)hexa(ethylene glycol)
(HO(CH.sub.2CH.sub.2O).sub.6(CH.sub.2).sub.11SH) in ethanol. The
resulting stamp was placed on the gold substrate (125 .star-solid.
gold on a titanium-primed 24.times.50-2 microscope cover glass) and
gentle hand pressure was applied to aid in complete contact between
the stamp and the glass chip. After 1 minute, the stamp was peeled
off the glass chip and the resulting substrate was immersed
directly in a 2 mM solution of (RADC).sub.3 AAAC (SEQ ID NO: 1) in
distilled, deionized water. After approximately 2 hours of
immesion, the glass chip was removed from the solution, rinsed
extensively with water and ethanol, and dried with a stream of
filtered nitrogen gas.
[0070] In our preliminary experiments, when the cells (of various
types) are plated on surfaces coated with hexa-ethyleneglycolthiol,
(EG)6-SH, they rarely attach to the surface. In contast, cells
attached very well when plated on the surface coated with the
"RADSC" peptide. In these experiments, after cell attachment, the
plates containing cells wee shacked at 150 rpm for 10 minutes and
the coated cover-slides were washed in new medium and transferred
to new plates in order to eliminate unattached cells.
[0071] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *