U.S. patent application number 12/261508 was filed with the patent office on 2010-06-03 for sensors employing combinatorial artificial receptors.
This patent application is currently assigned to RECEPTORS, LLC. Invention is credited to Robert E. Carlson.
Application Number | 20100133102 12/261508 |
Document ID | / |
Family ID | 34557964 |
Filed Date | 2010-06-03 |
United States Patent
Application |
20100133102 |
Kind Code |
A1 |
Carlson; Robert E. |
June 3, 2010 |
SENSORS EMPLOYING COMBINATORIAL ARTIFICIAL RECEPTORS
Abstract
The present invention relates to sensors and sensor systems that
utilize combinational artificial receptors. Embodiments of the
present invention employ combinational artificial receptors in
electromagnetic (e.g. optical) and electrochemical sensors.
Inventors: |
Carlson; Robert E.;
(Minnetonka, MN) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
RECEPTORS, LLC
Chasaka
MN
|
Family ID: |
34557964 |
Appl. No.: |
12/261508 |
Filed: |
October 30, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10934193 |
Sep 3, 2004 |
7469076 |
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12261508 |
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60499752 |
Sep 3, 2003 |
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60500081 |
Sep 3, 2003 |
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60499776 |
Sep 3, 2003 |
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60499867 |
Sep 3, 2003 |
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60499965 |
Sep 3, 2003 |
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60499975 |
Sep 3, 2003 |
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60526511 |
Dec 2, 2003 |
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60526699 |
Dec 2, 2003 |
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60526703 |
Dec 2, 2003 |
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60526708 |
Dec 2, 2003 |
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60527190 |
Dec 2, 2003 |
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Current U.S.
Class: |
204/403.15 ;
257/253; 257/E29.166 |
Current CPC
Class: |
G01N 21/763 20130101;
G01N 21/6445 20130101; G01N 21/6428 20130101; G01N 2021/772
20130101; G01N 2021/7786 20130101; G01N 2021/6484 20130101; C12Q
1/6804 20130101; G01N 21/645 20130101; G01N 21/553 20130101; C12Q
2565/631 20130101; G01N 2021/7776 20130101; G01N 21/6408 20130101;
C12Q 2565/629 20130101; C12Q 1/6804 20130101; G01N 2021/6432
20130101; G01N 21/76 20130101; G01N 21/7703 20130101 |
Class at
Publication: |
204/403.15 ;
257/253; 257/E29.166 |
International
Class: |
G01N 33/00 20060101
G01N033/00; H01L 29/66 20060101 H01L029/66 |
Claims
1. An electrochemical sensing system comprising: a working
electrode; a reference electrode; and a working artificial receptor
that is coupled to the working electrode, wherein the sensing
system is configured to generate a sensing signal.
2. The electrochemical sensing system of claim 1 further comprising
a membrane between the working electrode and the reference
electrode, the working artificial receptor being coupled to the
membrane.
3. The electrochemical sensing system of claim 1 wherein the system
is configured to detect a chemical reaction by detecting a flow of
electrons between the working electrode and the reference
electrode.
4. The electrochemical sensing system of claim 1 wherein the system
is configured to detect a chemical reaction by detecting a
potential difference between the reference electrode and the
working electrode.
5. The electrochemical sensing system of claim 1 comprising an
array of working electrodes.
6. The electrochemical sensing system of claim 1 wherein the
working receptors are coupled to a carbon paste that is
electrically coupled to the working electrode.
7. The sensing system of claim 1, wherein the system is configured
to detect at least one of: pathogenic microorganism, cancerous
cell, pollutant in water, airborne pollutant, explosive-related
vapor, protein, polynucleotide.
8. An electrochemical sensing system comprising: a field effect
transistor; a working artificial receptor that is coupled to the
field effect transistor, wherein a signal can be generated when a
test ligand binds to a working artificial receptor.
9. The electrochemical sensing system of claim 8 wherein the field
effect transistor is one of: a ChemFET, an ISFET, a SAFET, an
SGFET.
10. The electrochemical sensing system of claim 8 wherein the
working artificial receptor is coupled to a gate.
11. The electrochemical sensing system of claim 8 wherein the
working artificial receptor is coupled to an insulating layer.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/934,193, filed Sep. 3, 2004, which claims priority to
U.S. Provisional Patent Application Nos. 60/499,752, 60/500,081,
60/499,776, 60/499,867, 60/499,965, and 60/499,975 each filed Sep.
3, 2003; and 60/526,511 60/526,699, 60/526,703, 60/526,708, and
60/527,190 each filed Dec. 2, 2003. Each of these patent
applications is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to sensors employing
artificial receptors, such as combinatorial artificial receptor
arrays. The present receptors include heterogeneous and immobilized
combinations of building block molecules. In certain embodiments,
combinations of 2, 3, 4, or 5 distinct building block molecules
immobilized near one another on a support provide molecular
structures that can be employed in sensor systems. Sensors
employing the present artificial receptors can detect the
receptor's ligand.
BACKGROUND
[0003] The preparation of artificial receptors that bind ligands
like proteins, peptides, carbohydrates, microbes, pollutants,
pharmaceuticals, and the like with high sensitivity and specificity
is an active area of research. None of the conventional approaches
has been particularly successful; achieving only modest sensitivity
and specificity mainly due to low binding affinity.
[0004] Antibodies, enzymes, and natural receptors generally have
binding constants in the 10.sup.8-10.sup.12 range, which results in
both nanomolar sensitivity and targeted specificity. By contrast,
conventional artificial receptors typically have binding constants
of about 10.sup.3 to 10.sup.5, with the predictable result of
millimolar sensitivity and limited specificity.
[0005] Several conventional approaches are being pursued in
attempts to achieve highly sensitive and specific artificial
receptors. These approaches include, for example, affinity
isolation, molecular imprinting, and rational and/or combinatorial
design and synthesis of synthetic or semi-synthetic receptors.
[0006] Such rational or combinatorial approaches have been limited
by the relatively small number of receptors which are evaluated
and/or by their reliance on a design strategy which focuses on only
one building block, the homogeneous design strategy. Common
combinatorial approaches form microarrays that include 10,000 or
100,000 distinct spots on a standard microscope slide. However,
such conventional methods for combinatorial synthesis provide a
single molecule per spot. Employing a single building block in each
spot provides only a single possible receptor per spot. Synthesis
of thousands of building blocks would be required to make thousands
of possible receptors.
[0007] Further, these conventional approaches are hampered by the
currently limited understanding of the principals which lead to
efficient binding and the large number of possible structures for
receptors, which makes such an approach problematic.
[0008] There remains a need for methods for detecting ligands and
for detecting compounds that disrupt one or more binding
interactions.
SUMMARY
[0009] An embodiment of a sensor system includes a waveguide, a
detection system that can be operatively coupled to the waveguide,
and a working artificial receptor. The waveguide can be operatively
configured with respect to the working artificial receptor such
that the waveguide is capable of receiving light that from the
viscininty of the working artificial receptor. The detection system
can be configured to detect light from the waveguide.
[0010] An embodiment of an electrochemical sensing system includes
a working electrode, a reference electrode, and a working
artificial receptor that is coupled to the working electrode. The
sensing system can be configured to generate a sensing signal.
[0011] An embodiment of an electrochemical sensing system includes
a field effect transistor, and a working artificial receptor that
is coupled to the field effect transistor. A signal can be
generated by the field effect transistor when a test ligand binds
to a working artificial receptor.
[0012] An embodiment of sensor system includes a detector and a
working artificial receptor that is coupled to the detector. The
detector system can be configured to detect the presence of a test
ligand bound to the working artificial receptor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 schematically illustrates two dimensional
representations of an embodiment of a receptor according to the
present invention that employs 4 different building blocks to make
a ligand binding site.
[0014] FIG. 2 schematically illustrates two and three dimensional
representations of an embodiment of a molecular configuration of 4
building blocks, each building block including a recognition
element, a framework, and a linker coupled to a support
(immobilization/anchor).
[0015] FIG. 3 schematically illustrates an embodiment of the
present methods and artificial receptors employing shuffling and
exchanging building blocks.
[0016] FIG. 4 is a schematic drawing of an electromagnetic
radiation (light) sensor system that includes a wave guide.
[0017] FIG. 5 is a schematic drawing of an optical sensor system
that includes a plurality of fibers.
[0018] FIG. 6 is a schematic drawing of an electrochemical sensor
system.
[0019] FIG. 7 is a schematic drawing of an electrochemical sensor
system that employs a membrane to which the present artificial
receptors can be coupled.
[0020] FIG. 8 is a schematic drawing of a sensor system that uses
both optical and electrochemical sensors.
[0021] FIG. 9 is a cross-sectional view of a microwell and a
protrusion.
[0022] FIG. 10 is an exemplary illustration of a pattern detected
by a sensor array.
[0023] FIG. 11 is a schematic illustration of a fiber bundle to
which the present artificial receptors can be coupled.
[0024] FIGS. 12A-12D are schematic illustrations of field effect
transistor devices to which the present artificial receptors can be
bound.
[0025] FIG. 13 schematically illustrates an embodiment of a method
for evaluating candidate artificial receptors for binding to a test
ligand, such as a molecule or cell.
[0026] FIG. 14 schematically illustrates an embodiment of the
present method employing an array of candidate artificial
receptors.
[0027] FIG. 15 schematically illustrates certain binding patterns
on an array of working artificial receptors.
[0028] FIG. 16 schematically illustrates an embodiment of a method
for developing a method and system for detecting a test ligand.
[0029] FIG. 17 schematically illustrates an embodiment of a method
for detecting an agent that disrupts a binding interaction of a
target molecule.
[0030] FIG. 18 schematically illustrates an embodiment of a method
for detecting an agent that disrupts a binding interaction of a
complex including a target molecule.
[0031] FIG. 19 schematically illustrates an embodiment of a method
of employing the present artificial receptors to produce or as an
affinity support.
[0032] FIG. 20 schematically illustrates a candidate disruptor
disrupting a protein:protein complex.
[0033] FIG. 21 schematically illustrates evaluating an array of
candidate artificial receptors for binding of a test ligand and
selecting one or more working artificial receptors for binding or
operating on a test ligand.
[0034] FIG. 22 schematically illustrates identification of a lead
artificial receptor from among candidate artificial receptors.
[0035] FIG. 23 schematically illustrates a false color fluorescence
image of a labeled microarray according to an embodiment of the
present invention.
[0036] FIG. 24 schematically illustrates a two dimensional plot of
data obtained for candidate artificial receptors contacted with
and/or binding phycoerythrin.
[0037] FIG. 25 schematically illustrates a three dimensional plot
of data obtained for candidate artificial receptors contacted with
and/or binding phycoerythrin.
[0038] FIG. 26 schematically illustrates a two dimensional plot of
data obtained for candidate artificial receptors contacted with
and/or binding a fluorescent derivative of ovalbumin.
[0039] FIG. 27 schematically illustrates a three dimensional plot
of data obtained for candidate artificial receptors contacted with
and/or binding a fluorescent derivative of ovalbumin.
[0040] FIG. 28 schematically illustrates a two dimensional plot of
data obtained for candidate artificial receptors contacted with
and/or binding a fluorescent derivative of bovine serum
albumin.
[0041] FIG. 29 schematically illustrates a three dimensional plot
of data obtained for candidate artificial receptors contacted with
and/or binding a fluorescent derivative of bovine serum
albumin.
[0042] FIG. 30 schematically illustrates a two dimensional plot of
data obtained for candidate artificial receptors contacted with
and/or binding an acetylated horseradish peroxidase.
[0043] FIG. 31 schematically illustrates a three dimensional plot
of data obtained for candidate artificial receptors contacted with
and/or binding an acetylated horseradish peroxidase.
[0044] FIG. 32 schematically illustrates a two dimensional plot of
data obtained for candidate artificial receptors contacted with
and/or binding a TCDD derivative of horseradish peroxidase.
[0045] FIG. 33 schematically illustrates a three dimensional plot
of data obtained for candidate artificial receptors contacted with
and/or binding a TCDD derivative of horseradish peroxidase.
[0046] FIG. 34 schematically illustrates a subset of the data
illustrated in FIG. 25.
[0047] FIG. 35 schematically illustrates a subset of the data
illustrated in FIG. 25.
[0048] FIG. 36 schematically illustrates a subset of the data
illustrated in FIG. 25.
[0049] FIG. 37 schematically illustrates a correlation of binding
data for phycoerythrin against logP for the building blocks making
up the artificial receptor.
[0050] FIG. 38 schematically illustrates a correlation of binding
data for phycoerythrin against logP for the building blocks making
up the artificial receptor.
[0051] FIG. 39 schematically illustrates a two dimensional plot
comparing data obtained for candidate artificial receptors
contacted with and/or binding phycoerythrin to data obtained for
candidate artificial receptors contacted with and/or binding a
fluorescent derivative of bovine serum albumin.
[0052] FIGS. 40, 41, and 42 schematically illustrate subsets of
data from FIGS. 25, 29, and 27, respectively, and demonstrate that
the array of artificial receptors according to the present
invention yields receptors distinguished between three analytes,
phycoerythrin, bovine serum albumin, and ovalbumin.
[0053] FIG. 43 schematically illustrates a gray scale image of the
fluorescence signal from a scan of a control plate which was
prepared by washing off the building blocks with organic solvent
before incubation with the test ligand.
[0054] FIG. 44 schematically illustrates a gray scale image of the
fluorescence signal from a scan of an experimental plate which was
incubated with 1.0 .mu.g/ml Cholera Toxin B at 23.degree. C.
[0055] FIG. 45 schematically illustrates a gray scale image of the
fluorescence signal from a scan of an experimental plate which was
incubated with 1.0 .mu.g/ml Cholera Toxin B at 3.degree. C.
[0056] FIG. 46 schematically illustrates a gray scale image of the
fluorescence signal from a scan of an experimental plate which was
incubated with 1.0 .mu.g/ml Cholera Toxin B at 43.degree. C.
[0057] FIGS. 47-49 schematically illustrate plots of the
fluorescence signals obtained from the candidate artificial
receptors illustrated in FIGS. 44-46.
[0058] FIG. 50 schematically illustrate plots of the fluorescence
signals obtained from the combinations of building blocks employed
in the present studies, when those building blocks are covalently
linked to the support. Binding was conducted at 23.degree. C.
[0059] FIG. 51 schematically illustrates a graph of the changes in
fluorescence signal from individual combinations of building blocks
at 4.degree. C., 23.degree. C., or 44.degree. C.
[0060] FIG. 52 schematically illustrates a graph of the changes in
fluorescence signal from individual combinations of building blocks
at 4.degree. C., 23.degree. C., or 44.degree. C.
[0061] FIG. 53 schematically illustrates the data presented in FIG.
51 (lines marked A) and the data presented in FIG. 52 (lines marked
B).
[0062] FIG. 54 schematically illustrates a graph of the
fluorescence signal at 44.degree. C. divided by the signal at
23.degree. C. against the fluorescence signal obtained from binding
at 23.degree. C. for the artificial receptors with reversibly
immobilized receptors.
[0063] FIG. 55 illustrates fluorescence signals produced by binding
of cholera toxin to a microarray of the present candidate
artificial receptors followed by washing with buffer in an
experiment reported in Example 4.
[0064] FIG. 56 illustrates the fluorescence signals due to cholera
toxin binding that were detected upon competition with GM1 OS (0.34
.mu.M) in an experiment reported in Example 4.
[0065] FIG. 57 illustrates the ratio of the amount bound in the
absence of GM1 OS to the amount bound in competition with GM1 OS
(0.34 .mu.M) in an experiment reported in Example 4.
[0066] FIG. 58 illustrates fluorescence signals produced by binding
of cholera toxin to a microarray of the present candidate
artificial receptors followed by washing with buffer in an
experiment reported in Example 4 and for comparison with
competition experiments using 5.1 .mu.M GM1 OS.
[0067] FIG. 59 illustrates the fluorescence signals due to cholera
toxin binding that were detected upon competition with GM1 OS (5.1
.mu.M) in an experiment reported in Example 4.
[0068] FIG. 60 illustrates the ratio of the amount bound in the
absence of GM1 OS to the amount bound in competition with GM1 OS
(5.1 .mu.M) in an experiment reported in Example 4.
[0069] FIG. 61 illustrates the fluorescence signals produced by
binding of cholera toxin to the microarray of candidate artificial
receptors alone and in competition with each of the three
concentrations of GM1 in the experiment reported in Example 5.
[0070] FIG. 62 illustrates the ratio of the amount bound in the
absence of GM1 OS to the amount bound upon competition with GM1 for
the low concentration of GM1 employed in Example 5.
[0071] FIG. 63 illustrates the fluorescence signals produced by
binding of cholera toxin to the microarray of candidate artificial
receptors without pretreatment with GM1 in the experiment reported
in Example 6.
[0072] FIGS. 64-66 illustrate the fluorescence signals produced by
binding of cholera toxin to the microarray of candidate artificial
receptors with pretreatment with GM1 (100 .mu.g/ml, 10 .mu.g/ml,
and 1 .mu.g/ml GM1, respectively) in the experiment reported in
Example 6.
[0073] FIG. 67 illustrates the ratio of the amount bound in the
presence of 1 .mu.g/ml GM1 to the amount bound in the absence of
GM1 in the experiment reported in Example 6.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Definitions
[0074] As used herein, the term "peptide" refers to a compound
including two or more amino acid residues joined by amide
bond(s).
[0075] As used herein, the terms "polypeptide" and "protein" refer
to a peptide including more than about 20 amino acid residues
connected by peptide linkages.
[0076] As used herein, the term "proteome" refers to the expression
profile of the proteins of an organism, tissue, organ, or cell. The
proteome can be specific to a particular status (e.g., development,
health, etc.) of the organism, tissue, organ, or cell.
[0077] As used herein, the term "support" refers to a solid support
that is, typically, macroscopic.
[0078] As used herein, the term scaffold refers to a molecular
scale structure to which a plurality of building blocks can
covalently bind.
[0079] Reversibly immobilizing building blocks on a support couples
the building blocks to the support through a mechanism that allows
the building blocks to be uncoupled from the support without
destroying or unacceptably degrading the building block or the
support. That is, immobilization can be reversed without destroying
or unacceptably degrading the building block or the support. In an
embodiment, immobilization can be reversed with only negligible or
ineffective levels of degradation of the building block or the
support. Reversible immobilization can employ readily reversible
covalent bonding or noncovalent interactions. Suitable noncovalent
interactions include interactions between ions, hydrogen bonding,
van der Waals interactions, and the like. Readily reversible
covalent bonding refers to covalent bonds that can be formed and
broken under conditions that do not destroy or unacceptably degrade
the building block or the support.
[0080] A combination of building blocks immobilized on, for
example, a support can be a candidate artificial receptor, a lead
artificial receptor, or a working artificial receptor. That is, a
heterogeneous building block spot on a slide or a plurality of
building blocks coated on a tube or well can be a candidate
artificial receptor, a lead artificial receptor, or a working
artificial receptor. A candidate artificial receptor can become a
lead artificial receptor, which can become a working artificial
receptor.
[0081] As used herein the phrase "candidate artificial receptor"
refers to an immobilized combination of building blocks that can be
tested to determine whether or not a particular test ligand binds
to that combination. In an embodiment, the combination includes one
or more reversibly immobilized building blocks. In an embodiment,
the candidate artificial receptor can be a heterogeneous building
block spot on a slide or a plurality of building blocks coated on a
tube or well.
[0082] As used herein the phrase "lead artificial receptor" refers
to an immobilized combination of building blocks that binds a test
ligand at a predetermined concentration of test ligand, for example
at 10, 1, 0.1, or 0.01 .mu.g/ml, or at 1, 0.1, or 0.01 ng/ml. In an
embodiment, the combination includes one or more reversibly
immobilized building blocks. In an embodiment, the lead artificial
receptor can be a heterogeneous building block spot on a slide or a
plurality of building blocks coated on a tube or well.
[0083] As used herein the phrase "working artificial receptor"
refers to a combination of building blocks that binds a test ligand
with a selectivity and/or sensitivity effective for categorizing or
identifying the test ligand. That is, binding to that combination
of building blocks describes the test ligand as belonging to a
category of test ligands or as being a particular test ligand. A
working artificial receptor can, for example, bind the ligand at a
concentration of, for example, 100, 10, 1, 0.1, 0.01, or 0.001
ng/ml. In an embodiment, the combination includes one or more
reversibly immobilized building blocks. In an embodiment, the
working artificial receptor can be a heterogeneous building block
spot on a slide or a plurality of building blocks coated on a tube,
well, slide, or other support or on a scaffold.
[0084] As used herein the phrase "working artificial receptor
complex" refers to a plurality of artificial receptors, each a
combination of building blocks, that binds a test ligand with a
pattern of selectivity and/or sensitivity effective for
categorizing or identifying the test ligand. That is, binding to
the several receptors of the complex describes the test ligand as
belonging to a category of test ligands or as being a particular
test ligand. The individual receptors in the complex can each bind
the ligand at different concentrations or with different
affinities. For example, the individual receptors in the complex
each bind the ligand at concentrations of 100, 10, 1, 0.1, 0.01 or
0.001 ng/ml. In an embodiment, the combination includes one or more
reversibly immobilized building blocks. In an embodiment, the
working artificial receptor complex can be a plurality of
heterogeneous building block spots or regions on a slide; a
plurality of wells, each coated with a different combination of
building blocks; or a plurality of tubes, each coated with a
different combination of building blocks.
[0085] As used herein, the phrase "significant number of candidate
artificial receptors" refers to sufficient candidate artificial
receptors to provide an opportunity to find a working artificial
receptor, working artificial receptor complex, or lead artificial
receptor. As few as about 100 to about 200 candidate artificial
receptors can be a significant number for finding working
artificial receptor complexes suitable for distinguishing two
proteins (e.g., cholera toxin and phycoerythrin). In other
embodiments, a significant number of candidate artificial receptors
can include about 1,000 candidate artificial receptors, about
10,000 candidate artificial receptors, about 100,000 candidate
artificial receptors, or more.
[0086] Although not limiting to the present invention, it is
believed that the significant number of candidate artificial
receptors required to provide an opportunity to find a working
artificial receptor may be larger than the significant number
required to find a working artificial receptor complex. Although
not limiting to the present invention, it is believed that the
significant number of candidate artificial receptors required to
provide an opportunity to find a lead artificial receptor may be
larger than the significant number required to find a working
artificial receptor. Although not limiting to the present
invention, it is believed that the significant number of candidate
artificial receptors required to provide an opportunity to find a
working artificial receptor for a test ligand with few features may
be more than for a test ligand with many features.
[0087] As used herein, the term "building block" refers to a
molecular component of an artificial receptor including portions
that can be envisioned as or that include one or more linkers, one
or more frameworks, and one or more recognition elements. In an
embodiment, the building block includes a linker, a framework, and
one or more recognition elements. In an embodiment, the linker
includes a moiety suitable for reversibly immobilizing the building
block, for example, on a support, surface or lawn. The building
block interacts with the ligand.
[0088] As used herein, the term "linker" refers to a portion of or
functional group on a building block that can be employed to or
that does (e.g., reversibly) couple the building block to a
support, for example, through covalent link, ionic interaction,
electrostatic interaction, or hydrophobic interaction.
[0089] As used herein, the term "framework" refers to a portion of
a building block including the linker or to which the linker is
coupled and to which one or more recognition elements are
coupled.
[0090] As used herein, the term "recognition element" refers to a
portion of a building block coupled to the framework but not
covalently coupled to the support. Although not limiting to the
present invention, the recognition element can provide or form one
or more groups, surfaces, or spaces for interacting with the
ligand.
[0091] As used herein, the phrase "plurality of building blocks"
refers to two or more building blocks of different structure in a
mixture, in a kit, or on a support or scaffold. Each building block
has a particular structure, and use of building blocks in the
plural, or of a plurality of building blocks, refers to more than
one of these particular structures. Building blocks or plurality of
building blocks does not refer to a plurality of molecules each
having the same structure.
[0092] As used herein, the phrase "combination of building blocks"
refers to a plurality of building blocks that together are in a
spot, region, or a candidate, lead, or working artificial receptor.
A combination of building blocks can be a subset of a set of
building blocks. For example, a combination of building blocks can
be one of the possible combinations of 2, 3, 4, 5, or 6 building
blocks from a set of N (e.g., N=10-200) building blocks.
[0093] As used herein, the phrases "homogenous immobilized building
block" and "homogenous immobilized building blocks" refer to a
support or spot having immobilized on or within it only a single
building block.
[0094] As used herein, the phrase "activated building block" refers
to a building block activated to make it ready to form a covalent
bond to a functional group, for example, on a support. A building
block including a carboxyl group can be converted to a building
block including an activated ester group, which is an activated
building block. An activated building block including an activated
ester group can react, for example, with an amine to form a
covalent bond.
[0095] As used herein, the term "naive" used with respect to one or
more building blocks refers to a building block that has not
previously been determined or known to bind to a test ligand of
interest. For example, the recognition element(s) on a naive
building block has not previously been determined or known to bind
to a test ligand of interest. A building block that is or includes
a known ligand (e.g., GM1) for a particular protein (test ligand)
of interest (e.g., cholera toxin) is not naive with respect to that
protein (test ligand).
[0096] As used herein, the term "immobilized" used with respect to
building blocks coupled to a support refers to building blocks
being stably oriented on the support so that they do not migrate on
the support or release from the support. Building blocks can be
immobilized by covalent coupling, by ionic interactions, by
electrostatic interactions, such as ion pairing, or by hydrophobic
interactions, such as van der Waals interactions.
[0097] As used herein a "region" of a support, tube, well, or
surface refers to a contiguous portion of the support, tube, well,
or surface. Building blocks coupled to a region can refer to
building blocks in proximity to one another in that region.
[0098] As used herein, a "bulky" group on a molecule is larger than
a moiety including 7 or 8 carbon atoms.
[0099] As used herein, a "small" group on a molecule is hydrogen,
methyl, or another group smaller than a moiety including 4 carbon
atoms.
[0100] As used herein, the term "lawn" refers to a layer, spot, or
region of functional groups on a support, for example, at a density
sufficient to place coupled building blocks in proximity to one
another. The functional groups can include groups capable of
forming covalent, ionic, electrostatic, or hydrophobic interactions
with building blocks.
[0101] As used herein, the term "alkyl" refers to saturated
aliphatic groups, including straight-chain alkyl groups,
branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl
substituted cycloalkyl groups, and cycloalkyl substituted alkyl
groups. In certain embodiments, a straight chain or branched chain
alkyl has 30 or fewer carbon atoms in its backbone (e.g.,
C.sub.1-C.sub.12 for straight chain, C.sub.1-C.sub.6 for branched
chain). Likewise, cycloalkyls can have from 3-10 carbon atoms in
their ring structure, for example, 5, 6 or 7 carbons in the ring
structure.
[0102] The term "alkyl" as used herein refers to both
"unsubstituted alkyls" and "substituted alkyls", the latter of
which refers to alkyl moieties having substituents replacing a
hydrogen on one or more carbons of the hydrocarbon backbone. Such
substituents can include, for example, a halogen, a hydroxyl, a
carbonyl (such as a carboxyl, an ester, a formyl, or a ketone), a
thiocarbonyl (such as a thioester, a thioacetate, or a
thioformate), an alkoxyl, a phosphoryl, a phosphonate, a
phosphinate, an amino, an amido, an amidine, an imine, a cyano, a
nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a
sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl,
an aryl alkyl, or an aromatic or heteroaromatic moiety. The
moieties substituted on the hydrocarbon chain can themselves be
substituted, if appropriate. For example, the substituents of a
substituted alkyl can include substituted and unsubstituted forms
of the groups listed above.
[0103] The phrase "aryl alkyl", as used herein, refers to an alkyl
group substituted with an aryl group (e.g., an aromatic or
heteroaromatic group).
[0104] As used herein, the terms "alkenyl" and "alkynyl" refer to
unsaturated aliphatic groups analogous in length and optional
substitution to the alkyls groups described above, but that contain
at least one double or triple bond respectively.
[0105] The term "aryl" as used herein includes 5-, 6- and
7-membered single-ring aromatic groups that may include from zero
to four heteroatoms, for example, benzene, pyrrole, furan,
thiophene, imidazole, oxazole, thiazole, triazole, pyrazole,
pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those
aryl groups having heteroatoms in the ring structure may also be
referred to as "aryl heterocycles" or "heteroaromatics". The
aromatic ring can be substituted at one or more ring positions with
such substituents such as those described above for alkyl groups.
The term "aryl" also includes polycyclic ring systems having two or
more cyclic rings in which two or more carbons are common to two
adjoining rings (the rings are "fused rings") wherein at least one
of the rings is aromatic, e.g., the other cyclic ring(s) can be
cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or
heterocyclyls.
[0106] As used herein, the terms "heterocycle" or "heterocyclic
group" refer to 3- to 12-membered ring structures, e.g., 3- to
7-membered rings, whose ring structures include one to four
heteroatoms. Heterocyclyl groups include, for example, thiophene,
thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,
phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,
pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,
indole, indazole, purine, quinolizine, isoquinoline, quinoline,
phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,
pteridine, carbazole, carboline, phenanthridine, acridine,
pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine,
furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole,
piperidine, piperazine, morpholine, lactones, lactams such as
azetidinones and pyrrolidinones, sultams, sultones, and the like.
The heterocyclic ring can be substituted at one or more positions
with such substituents such as those described for alkyl
groups.
[0107] As used herein, the term "heteroatom" as used herein means
an atom of any element other than carbon or hydrogen, such as
nitrogen, oxygen, sulfur and phosphorous.
Overview of the Artificial Receptor
[0108] FIG. 1 schematically illustrates an embodiment employing 4
distinct building blocks in a spot on a microarray to make a ligand
binding site. This Figure illustrates a group of 4 building blocks
at the corners of a square forming a unit cell. A group of four
building blocks can be envisioned as the vertices on any
quadrilateral. FIG. 1 illustrates that spots or regions of building
blocks can be envisioned as multiple unit cells, in this
illustration square unit cells. Groups of unit cells of four
building blocks in the shape of other quadrilaterals can also be
formed on a support.
[0109] Each immobilized building block molecule can provide one or
more "arms" extending from a "framework" and each can include
groups that interact with a ligand or with portions of another
immobilized building block. FIG. 2 illustrates that combinations of
four building blocks, each including a framework with two arms
(called "recognition elements"), provides a molecular configuration
of building blocks that form a site for binding a ligand. Such a
site formed by building blocks such as those exemplified below can
bind a small molecule, such as a drug, metabolite, pollutant, or
the like, and/or can bind a larger ligand such as a macromolecule
or microbe.
[0110] The present artificial receptors can include building blocks
reversibly immobilized on a support or surface. Reversing
immobilization of the building blocks can allow movement of
building blocks to a different location on the support or surface,
or exchange of building blocks onto and off of the surface. For
example, the combinations of building blocks can bind a ligand when
reversibly coupled to or immobilized on the support. Reversing the
coupling or immobilization of the building blocks provides
opportunity for rearranging the building blocks, which can improve
binding of the ligand. Further, the present invention can allow for
adding additional or different building blocks, which can further
improve binding of a ligand.
[0111] FIG. 3 schematically illustrates an embodiment employing an
initial artificial receptor surface (A) with four different
building blocks on the surface, which are represented by shaded
shapes. This initial artificial receptor surface (A) undergoes (1)
binding of a ligand to an artificial receptor and (2) shuffling the
building blocks on the receptor surface to yield a lead artificial
receptor (B). Shuffling refers to reversing the coupling or
immobilization of the building blocks and allowing their
rearrangement on the receptor surface. After forming a lead
artificial receptor, additional building blocks can be (3)
exchanged onto and/or off of the receptor surface (C). Exchanging
refers to building blocks leaving the surface and entering a
solution contacting the surface and/or building blocks leaving a
solution contacting the surface and becoming part of the artificial
receptor. The additional building blocks can be selected for
structural diversity (e.g., randomly) or selected based on the
structure of the building blocks in the lead artificial receptor to
provide additional avenues for improving binding. The original and
additional building blocks can then be (4) shuffled and exchanged
to provide higher affinity artificial receptors on the surface
(D).
Sensors Employing the Artificial Receptors
[0112] The present artificial receptors can be configured in
sensors, sensor systems, and sensing methods. For example, a sensor
system can be can be configured to detect a test ligand in a sample
or an environment based upon a signal received from a sensor
operatively configured to detect a test ligand that binds to the
present artificial receptor.
[0113] In an embodiment, the sensor system includes an optical
sensor system. The optical sensor system can include a waveguide, a
detection system operatively coupled to the waveguide, and an
artificial receptor that binds a test ligand of interest (e.g., a
working artificial receptor). The waveguide can be operatively
configured with respect to the working artificial receptor such
that the waveguide can receive an optical signal (e.g.,
fluorescence, luminescence, or absorbance) from the working
artificial receptor.
[0114] In an embodiment, the sensor system includes an
electrochemical sensor system. An electrochemical sensing system
can include a transducer (e.g., an electrode or CHEMFET), a
detection system operatively coupled to the transducer, and an
artificial receptor that binds a test ligand of interest (e.g., a
working artificial receptor). The transducer can be operatively
configured with respect to the working artificial receptor such
that the transducer can detect changes in electrical charge,
potential, or current (e.g., conductance, capacitance, or
impedence) from the working artificial receptor. In an embodiment,
the transducer includes at least one electrode. An electrochemical
sensing system can include a working electrode, a reference
electrode, and a working artificial receptor. In an embodiment, the
present artificial receptors can be coupled to the working
electrode. In an embodiment, the present artificial receptors can
be coupled to a membrane that is configured between the working
electrode and the reference electrode. In an embodiment, the
working electrode and reference electrode can be conventional
electrodes. In an embodiment, the working electrode and reference
electrode can be a source and a drain of a field effect
transistor.
[0115] A sample expected of containing a test ligand can be brought
into contact with an artificial receptor by a variety of
mechanisms. For example, the sample can be carried in a stream of
air or an aerosol. The sample can be a liquid. In an embodiment, it
can be desirable to increase the surface area to which present
artificial receptors are coupled to provide enhanced detection of a
test ligand in a gaseous or liquid flow. In an embodiment, a flow
can be received through a foamed support substance, such as an
aerogel to which present artificial receptors can be coupled.
Supporting Environments for the Artificial Receptors
[0116] The present artificial receptors can be supported in sensors
or sensors systems in a variety of configurations in or on a
variety of support materials. For example, the present artificial
receptors can be supported in solid, gel, and foam environments. To
accommodate various sensing systems, the present artificial
receptors can be configured in an environment that conducts
electrical currents, light, and/or other electromagnetic
radiation.
[0117] In an embodiment, present artificial receptors can be
located at or near a distal surface of a fiber, such an optical
fiber. The distal surface can, for example, be an end of an optical
fiber. In an embodiment, the present artificial receptors can be
bonded or otherwise coupled to the distal surface on the fiber. For
example, the present artificial receptors can be coupled directly
to the end of a fiber. In an embodiment, the present artificial
receptors can be coupled, embedded, or otherwise configured to a
support material or substrate that can be coupled to the fiber. In
an embodiment, the present artificial receptors can be located near
the end of a fiber without being coupled to the fiber. For example,
the present artificial receptors can be coupled to or suspended in
a target object or substrate that can be configured to be
detectable by the fiber. In an embodiment, a receptor-supporting
material can be layered on the object or substrate. In an
embodiment, the target object itself can be the support for the
receptor, i.e. the object or substrate can be formed from the
material to which the present artificial receptors can be
coupled.
[0118] In an embodiment, a sol-gel can be a support for the present
artificial receptors. For example, the present artificial receptors
can be coupled to or suspended in a sol-gel that can be coupled to
a fiber or electrode. The sol-gel process involves the transition
of a system from a liquid "sol" into a solid "gel" phase. Through a
sol-gel process, it is possible to fabricate ceramic or glass
materials in a wide variety of forms. Dopants can be added to alter
the glass properties. For example, ultra-fine or spherical shaped
powders, thin film coatings, ceramic fibers, microporous inorganic
membranes, monolithic ceramics and glasses, or extremely porous
aerogel materials can be developed using a sol gel process. In one
process, a sol gel is a colloidal suspension of silica particles
that is gelled to form a solid. The resulting porous gel can be
chemically purified and consolidated at high temperatures into high
purity silica.
[0119] A sol-gel can be coated or otherwise applied to an object,
or alternatively formed into an object itself. In an embodiment, a
sol-gel can be applied to the surface of a fiber by dip coating,
and the current artificial receptors can be coupled to the sol gel.
In an embodiment, the present artificial receptors can be coupled
to a sol gel substrate that is configured to be detectable by a
fiber but is not connected to the fiber. For example, sol gel can
be applied to a plate or other substrate, and the optical fiber can
be aligned to detect a portion of the plate.
[0120] In addition to dip-coating, sol-gel can be applied through
other processes such as spraying, casting, and spin coating. Other
forms of sol gels, such as aerogels, powders, and thin films formed
from xerogels can also be configured for use with the present
artificial receptors. Sol gel processes are described in U.S. Pat.
No. 5,774,603. Dual-layer sensors and sensors that utilize
aerosol-generated sol gels are described in U.S. Pat. No.
6,016,689.
[0121] In an embodiment, a microsphere (or "bead") can be
configured as a support for the present artificial receptors. A
sensor incorporating an optical fiber and a solid porous inorganic
microsphere is described in U.S. Pat. No. 5,496,997. Microspheres
having a polymeric shell with consistent shell thickness are
described in U.S. Pat. No. 6,720,007. Microspheres are also
discussed in U.S. Pat. Nos. 6,327,410 and 6,266,459 and Published
U.S. Patent Application Nos. 20030016897, 20020122612, 20010029049.
A plurality or multiplicity of microspheres can be configured to
allow for multiplexed detection (binding of multiple analytes on
multiple sets of coated beads). The present artificial receptors
can also be incorporated into a nanosphere.
[0122] In an embodiment, a support, such as a microsphere can be
encoded with an optical signature. While other support environments
and structures can be encoded, microspheres will be referenced
herein for descriptive purposes. A structure such as a microsphere
can be encoded, for example, by use of a dye that can be entrapped
within the bead. The dye can, for example, be a fluorescent dye.
The dye can also be a chromophore or phosphor, or other
optically-detectable substance. In an embodiment, two or more dyes
can be used to provide a multi-parametric code. In an embodiment,
the code can correspond to a chemical functionality or sensitivity
that is associated with the microsphere. Fiber optic sensors with
encoded microspheres are described in U.S. Pat. No. 6,023,540.
[0123] In an embodiment, the present artificial receptors can be
configured for use in conjunction with a capillary tube. For
example, an optical fiber (or an electrode) can be supported within
a capillary tube. A fluid sample can be introduced into the space
between the fiber (or electrode) and the tube. A fluid can be drawn
into and supported in the space by capillary action. The present
artificial receptors can be coupled to the optical fiber (or
electrode), the capillary tube, or an object in the capillary tube,
and configured to detect a test ligand in the fluid sample. A
fluorescent immunoassay employing an optical fiber in a capillary
tube is described in U.S. Pat. No. 4,447,546.
[0124] The present artificial receptors can also be configured in a
cavity. As used herein, cavity is intended to refer to a microwell,
pit, cavity, depression, void, or similar structure. For purposes
of the present discussion, a microwell will be described, although
the present application also applies to other forms of cavities. In
an embodiment, a microwell can be formed in an end of an optical
fiber. A microwell can also be formed on a substrate that can be
configured to be detectable by an optical fiber. Microwell-based
sensors are described in U.S. Pat. Nos. 6,667,159, 6,377,721, and
6,210,910. In an embodiment, a plurality of microwells can be
formed at the distal end of individual fibers within a
randomly-ordered addressable sensor arrays, as further described in
U.S. Pat. No. 6,667,159.
[0125] In an embodiment, a microwell receives a bead, to which
receptors can be coupled. In an embodiment, the microwell and bead
can be configured on an end surface of a fiber. In an embodiment,
the bead and depression structure can mimic the shape of mammalian
taste buds. The bead can, for example, be constructed of a
polymer.
[0126] FIG. 9 shows a cross-section of a depression 730 in a
substance 720 which can be, for example, a substrate, waveguide, or
electrode. The depression can be, for example, a microwell. A
protrusion 740, which can be a sphere or bead, can reside in the
depression 730. The present working electrodes 710 can be coupled
to the protrusion 740. In an embodiment, the present working
electrodes can be coupled to the depression surface.
[0127] In an embodiment, an array of micron-sized wells can be
formed at the distal tip of an optical imaging fiber, and the
present artificial receptors can be coupled in the wells. In an
embodiment, a well can be created using a wet-etch process. Where
there are differences in core and clad etch rates, a well can be
created between core and clad material. For example, where fiber
cores etch faster than the cladding, the core etches away to
produce a well that is defined by the remaining clad. In an
embodiment, the present artificial receptors can be coupled to the
cells. In an embodiment, a living cell can be received into a well
and the present artificial receptors can be configured to detect
the presence and/or behavior of the living cells.
[0128] In an embodiment, cells can be plated onto a fiber and the
present artificial receptors can be configured to facilitate
detection of the cells.
[0129] In an embodiment, sensors employing the present artificial
receptors can be configured to make optical analytical measurements
at remote locations. U.S. Pat. No. 5,814,524 describes an apparatus
that employs an imaging fiber comprising a fiber optic array and a
Gradient Index lens and utilizes a remotely-positioned solid
substrate having light energy absorbing indicator ligands on an
external surface for reactive contact with individual species of
analytes when present in a fluid sample.
[0130] The present artificial receptors can also be supported in a
carbon paste. In an embodiment, a hybrid optical/electrochemical
sensor can be provided, where the carbon paste can be electrically
coupled to a circuit that can be configured to detect electrical
behaviors or properties of the sample/and or receptors.
[0131] The present artificial receptors can also be configured in a
microchannel. In an embodiment, a microchannel includes a
longitudinal recess on a surface and the present artificial
receptors can be coupled to the recessed surface and configured to
detect a test ligand in a fluid in the microchannel.
[0132] The present artificial receptors can also be supported on a
membrane. In an embodiment, the present artificial receptors can be
coupled to a membrane which can be placed in front of the tip of an
optical fiber. In an embodiment, the membrane can be removable. In
an embodiment, the present artificial receptor can be coupled to a
membrane that is positioned between two electrodes in an
electrochemical sensor.
[0133] In an embodiment, the present artificial receptors can be
coupled to a hexamethyldisiloxane plasma-polymerized film.
[0134] Other materials, including molecularly imprinted polymers
and zeolite, can be configured as a support for the present
artificial receptors. The present artificial receptors can also be
configured for use with disposable screen printing technology.
Fiber Optic Sensors
[0135] Fiber optic technology can be utilized with the present
artificial receptors. Fiber optic systems can be configured to
allow for detection of modulation in the quality or quantity of
electromagnetic radiation, such as visible light, infrared
radiation, or ultraviolet radiation. For example, the intensity
(energy), polarization state, phase and wavelength of radiation can
be detected and measured. Embodiments of fiber optic-based sensors
are described in U.S. Pat. Nos. 5,690,894 and 6,680,206.
[0136] A fiber optic detection system can be configured with a
fiber arranged to detect radiation and/or optical properties in the
vicinity of the present artificial receptors. Embodiments of fiber
optic sensing systems can be designed to provide remote,
distributed and multiplexed sensing. An embodiment of an optical
sensing system that uses receptors is shown and described in U.S.
Pat. No. 5,512,490. In an embodiment, sensors can be employed with
micromechanical systems. Sensors can also be designed to operate in
hostile and hazardous environments. In an embodiment, a fiber can
be covered by a protective cladding layer to protect the fiber.
[0137] Fiber optic fibers can be interfaced with filters and light
sources to facilitate detection of particular properties or
behaviors of a substance that contains the present artificial
receptors or is coupled to the present artificial receptors. The
presence of a test ligand coupled to a receptor in a sample can be
detected, for example, by detection of the emission, absorption, or
refractive index of the sample. Detectors can be configured to
measure a variety of types of radiation, including, for example,
white light, ultraviolet light or fluorescence. In an embodiment,
an emitter and a receiver can be coupled to separate fibers and the
reflected, refracted, or conducted light (or other radiation) can
be detected. Other sources of radiation can also be used.
[0138] FIG. 4 is a schematic illustration of an embodiment of a
sensor system 200 that can be configured to detect an optical
signal. The present artificial receptors 210 can be coupled to a
substrate 220. An light source 230, such as a laser or lamp,
directs light toward the artificial receptors. A waveguide 240 can
be configured to detect electromagnetic radiation, i.e. light. The
waveguide 240 can be configured to detect, for example, reflected
radiation, conducted radiation, or fluorescent radiation. The
waveguide can be, for example, a planar waveguide. A fiber 250, for
example an optical fiber, can be coupled to the waveguide 240 and
to an electromagnetic detection system 260, such as a charge
coupled device (CCD). The detection system can be coupled to a
processing system 270, such as a computer system, which can be
configured to process and save data received from the detection
system. Alternatively, the detection system can be configured to be
directly present or save data, without use of a separate processing
system.
[0139] FIG. 5 shows an embodiment of a sensing system 300 in which
a fiber 320 can be configured for use as a waveguide. As shown in
FIG. 5, multiple fibers can be configured together to detect light
or other radiation reflected, refracted, conducted, or fluoresced
by receptors 310. Data received from multiple fibers can be
processed by a reception system 330 to allow for observation of
different locations on a substrate. In the embodiment shown in FIG.
5, the substrate 340 is not coupled to the fibers. In an
alternative embodiment, a substrate can be coupled to a distal end
of a fiber, and receptors can be coupled to the substrate. In an
embodiment, the present artificial receptors can be coupled
directly to the fiber.
[0140] Various types of optical sensors can be provided, such as
intensity-based sensors, interference-based sensors,
polarization-based sensors, wavelength-based sensors, nonlinear
optics-based sensors, and multiphoton-based sensors. Other
detectors such as photomultiplier tubes, photo diodes, photodiode
arrays, and microchannel plates can also be provided. Bundled
fibers and fiber arrays can also be configured to detect a
plurality of the present artificial sensors.
[0141] Fiber optic detection systems can operate based upon direct
or indirect detection. Directly detectable labels provide a
directly detectable signal without interaction with one or more
additional chemical agents. Suitable directly detectable labels
include colorimetric labels, fluorescent labels, and the like.
Indirectly detectable labels interact with one or more additional
agents to provide a detectable signal. Suitable indirect labels
include a labeled antibody and the like.
[0142] A detection system can be configured to detect the intrinsic
optical properties of a sample, such as the refractive index, the
color, or the emission (e.g. fluorescence) or absorption (e.g.
quench fluorescence) properties of a sample. In an embodiment, the
presence of a test ligand bound to a receptor can be detected based
upon variations in one or more properties that are associated with
the presence of the test ligand. In an embodiment, the
concentration of a test ligand or a label in a sensed region is a
function of the concentration of the analyte that binds to a
particular receptor, which can be determined from the optical
properties or behavior of the sample. In an embodiment, a label can
be bound to the analyte. In an embodiment, a label can be bound to
a blocking analyte. Use of labels is further described in U.S. Pat.
No. 5,690,894.
[0143] Fiber optic systems can allow flexibility in detection
configurations. For example, a fiber optic system can be configured
to allow detection in locations that can be otherwise difficult to
access. Detection at a distance is also possible through use of one
or more fibers. A group of fibers can be configured to collect an
array or data, as further described below. Detection at multiple
points along a fiber or a bundle of fibers is also possible.
[0144] A fiber optic detection system can be configured in a
sensing or a probing configuration. A sensing configuration allows
for a stream of data. For example, a sensing system can be
configured to sense continuously. A probing configuration takes a
sample of data (i.e. a "snapshot") at a particular point in time. A
fiber optic probe can be configured to take a single data samples,
or multiple samples. In an embodiment, for example, a fiber optic
probe can be configured to take periodic data samples.
[0145] In an embodiment, the present artificial receptors can be
configured in conjunction with an optical imaging fiber. An optical
imaging fiber can be formed from a multiplicity of individual
fibers that are melted and drawn together in a coherent manner such
that an image can be carried from one end of the fiber to the
other. In an embodiment, an optical imaging fiber can be configured
to detect the presence, condition or behavior of a ligand (e.g. a
test ligand) that binds to the present artificial receptors. Such
imaging fibers can also be configured in conjunction with
chemically sensitive polymer matrices to combine imaging and
chemical sensing. This allows for simultaneous optical detection
and measurement of chemical dynamics occurring in a sample.
[0146] In an embodiment, the present artificial receptors can be
configured for use in optical tweezers. An optical tweezer traps
particles using focused laser beams to form optical traps. Optical
tweezers are described in published U.S. Patent Application No.
20030032204.
[0147] While fiber optic waveguides have been described in many of
the above applications, waveguides having other geometries (e.g.
planar or strip) can also be configured for use with the receptors.
Various mode structures (e.g. single-mode or multi-mode),
refractive index distributions (step or gradient index) and
material compositions (e.g. glass, polymer, semiconductor) can be
employed. In an embodiment, receptors can be coupled to a surface
of a planar wave guide. Signals can be captured and conveyed
through a fiber where they can be analyzed by a computer system. A
fluorometric-based sensor system that uses a planar waveguide is
described in United States Published Patent Application No.
2002/0160535.
Optical Signals
[0148] A detectable optical signal can be produced by the
interaction of a ligand with the present artificial receptor or
through binding of a labeled moiety to the ligand. Fiber optic
detection systems can be configured to detect a variety of optical
signals, including those produced by physical, chemical, or
biological. For example, the detection systems can be configured to
detect one or more of refractive index, color, emission,
absorption, fluorescence, or fluorescence quenching properties of a
sample, and/or changes to those properties.
[0149] The presence of a test ligand binding to the present
artificial receptor can affect the quality or quantity of optical
signal that is detected through a fiber. The presence of two or
more test ligands that are bound to the present artificial
receptors (or to each other) can also generate detectable changes
in an optical signal or property.
[0150] In an embodiment, a change in fluorescence or quenching of
fluorescence can be detected. Fluorescence is the emission of
radiation following the absorption of radiation of a different
wavelength. For example, an embodiment of a fluorescing substance
can emit visible light after being exposed to ultraviolet light. In
an embodiment, the present artificial receptor can include a
fluorescent molecule whose fluorescent properties (e.g., emission
intensity, emission wavelength, or lifetime) change upon analyte
binding. Sensors employing the present artificial receptors can be
configured to detect fluorescent decay time.
[0151] Sensors employing the present artificial receptors can be
configured to detect the quenching of fluorescence. The quenching
of fluorescence can occur in a variety of forms. Dynamic quenching
can occur where a collisional encounter between the quencher and
the excited state is involved. The lifetime and intensity of the
emission can be decreased by dynamic quenching. Concentration
quenching can occur where a molecule quenches its own fluorescence
at high concentration. `Static` quenching can occur where an
interaction between the fluorophore and quencher is involved.
Color-quenching can occur where photons that are emitted are
reabsorbed by a strongly colored component of the sample. Color
quenching is often accompanied by another quenching process based
on Fluorescent Resonance Energy Transfer (`FRET`). This is a
radiation-less process where excited species transfer excitation
energy to a neighbor having an absorption that overlaps the
fluorophore's emission spectrum.
[0152] Sensors configured with the present artificial receptors can
also be configured to detect fluorescence polarization.
Fluorescence polarization refers to the fact that fluorescent
molecules in solution which are excited with plane-polarized light
will emit light back into a fixed plane if the molecules remain
stationary during the excitation of the fluorophore. If the
molecule rotates during the excited state, light is emitted in a
different plane. The intensity of light emitted can be monitored in
a plane to monitor the rotation of fluorescing molecules. Large
molecules tend to rotate slower than small molecules. Fluorescence
polarization measurements can thus be used to study molecular
interactions. For example, the bound/free ratio of fluorescing
molecules can be detected. A sensor that detects the fluorescence
polarization of a label is described in U.S. Pat. No.
6,555,326.
[0153] Sensors employing the present artificial receptors can be
configured to detect evanescent wave properties. Evanescent
wave-based sensors are described by U.S. Pat. Nos. 5,525,800,
5,639,668, and 6,731,827.
[0154] Sensors employing the present artificial receptors can be
configured to detect phenomena such as chemiluminescence,
bioluminescence, or chemibioluminescence. Chemiluminescence is the
generation of electromagnetic radiation as light by the release of
energy from a chemical reaction. Chemical reactions using synthetic
compounds and usually involving a highly oxidized species such as a
peroxide are commonly termed chemiluminescent reactions.
Light-emitting reactions arising from a living organism, such as
the firefly or jellyfish, are commonly termed bioluminescent
reactions. Light-emitting reactions which take place by the use of
electrical current are designated electrochemiluminescent
reactions.
[0155] Sensors employing the present artificial receptors can be
configured to use multi-parametric techniques. For example,
multi-parametric fluorescence techniques can be performed based on
based on spectral change, intensity, lifetime and polarization of
radiation. In an embodiment, for example, refractive index,
emission, and abortion properties can also be monitored
concurrently. Other combinations are possible.
[0156] In an embodiment, a charge-coupled device (CCD) camera can
be configured to detect optical signals from a system that
incorporates the present artificial receptors. In an embodiment, a
long-duration exposure can be used to gather data over a period of
time. Filters can also be configured to separate signals by
wavelength.
[0157] In an embodiment, a near field array can be created on
optical fibers and configured to observe an object to which the
present artificial receptor can be coupled.
Optical Sensor Arrays
[0158] In an embodiment, fiber optic sensors can be arranged to
provide an array of data. For example, in an embodiment, present
artificial receptors can be arranged on a surface in an array. The
array of present artificial receptors can be detected by a single
fiber, or by a group of fibers. In an embodiment, an array of
present artificial receptors can be positioned at the end of a
fiber. For example, the present artificial receptors can be coupled
to the end of the fiber in an array pattern. Alternatively, present
artificial receptors can be bonded, suspended, or otherwise
positioned on an object, such as a glass plate, with a fiber
operatively arranged to allow detection with respect to the
plate.
[0159] In an embodiment, an array of fibers can be configured with
the present artificial sensors to gather a data array. In an
embodiment, an array of fibers can be positioned to provide data
samples from a variety of positions. In an embodiment, a variety of
present artificial receptors which can be arranged in a pattern
allow for simultaneous monitoring for a plurality of test ligands.
Other variations or combinations of these sensor techniques are
possible. Optical fiber sensor arrays are further described in U.S.
Pat. Nos. 5,690,894, 6,667,159, and 6,680,206.
[0160] In an embodiment, a plurality of fibers can be configured in
a bundle 1100, as shown in FIG. 11. Particular receptors can be
associated with particular fibers, or groups of fibers. For
example, in an embodiment, particular receptors can be coupled to
fibers at a first end 1110 of a bundle, and particular test ligands
can be identified based upon signals received at a second end 1120
of the bundle.
[0161] Techniques for analyzing a sample array include
time-resolved spectroscopy, spatially-resolved spectroscopy,
evanescent wave spectroscopy, laser-assisted spectroscopy, surface
plasmon spectroscopy, and multi-dimensional data acquisition. Fiber
bundles can be configured to form a sensor array, which can be used
for imaging or for data acquisition. In an embodiment, an
artificial neural network can be configured to process data from a
fiber bundle array. An artificial neural network deconvolutes a
signal to allow association of data signal patterns with the
presence of a particular test ligand or group of test ligands. For
example, it may be determined that when a particular test ligand is
present, a particular combination of signals can be detected
through the fiber array. As data is collected, detection of a new
test ligand or combination of test ligands can be determined.
[0162] In an embodiment, sensors based on fiber optic technology in
combination with the present artificial receptors can be configured
to continuously measure the concentration (or concentration
changes) of various components of biological and environmental
samples.
Electrochemical Sensors
[0163] The present artificial receptors can also be configured for
use in electrochemical sensors. The present artificial receptors
can be configured for example with a variety of electrochemical
sensors, including chemically modified electrode sensors and
microelectrodes. Sensing schemes include, for example, voltametric
and potentiometric methods. Electrochemical sensors can be
configured to detect any of a variety of test ligands.
[0164] In an embodiment, an electrochemical sensor includes the
present artificial receptors coupled to or near an electrode. In an
embodiment, if a test ligand binds to a receptor, the presence of
the test ligand can be detected based on electrical monitoring. For
example, the presence of a particular test ligand can create an
electrical charge. The electrical charge can create a detectable
current or voltage differential. In an embodiment, when a
chemically reactive gas is either oxidized (accepts oxygen and/or
gives up electrons) or reduced (gives up oxygen and/or accepts
electrons), a potential difference can be created, which can cause
a current to flow. Non-reducible anions can be detected based upon
electrochemical interaction between the anions and the receptors or
redox active moieties operatively coupled to the receptors.
[0165] In an embodiment, an electrochemical sensor includes a
working electrode (or "sensing" electrode), a reference electrode,
and the present artificial receptor. The working electrode is
exposed to a test ligand, and the reference electrode is not
exposed to the test ligand. When the test ligand is present at the
working electrode, an electrochemical reaction can occur.
Typically, either an oxidation or reduction occurs, depending on
the type of test ligand, but other reactions are possible. An
oxidation reaction results in the flow of electrons from the
working electrode to the reference electrode through an external
circuit. A reduction reaction results in flow of electrons from the
reference electrode to the working electrode. This flow of
electrons in the electric current is proportional to the test
ligand concentration.
[0166] In an embodiment, present artificial receptors can be
coupled to a working electrode. The receptors can be exposed to a
sample suspected of containing a test ligand of interest. A
chemical reaction can cause electrons to flow to or from the
working electrode. For example, in an embodiment, the receptors can
be exposed to the sample, and a test ligand in the sample can bind
to the receptors. The receptors and bound test ligand from the
sample can be exposed to a reactive substance such as an
electrolyte, which can cause a chemical reaction, which in turn can
cause a current to flow.
[0167] FIG. 6 shows an embodiment of an electrochemical sensor 400.
The present working receptors 410 can be coupled to a working
electrode 420. Working electrode 420 and a reference electrode 440
can be electrically coupled to an electrical sensing device 430,
which can be configured to detect, for example, voltage or current.
While receptors 410 are shown coupled directly to working electrode
420, the receptors can alternatively be coupled to a substrate that
can be electrically coupled to the working electrode.
[0168] In an embodiment, an electrochemical sensor includes a
membrane. The present artificial receptors can be attached or
otherwise coupled to the membrane. FIG. 7 shows an embodiment of an
electrochemical sensor 500 where the present working receptors 510
can be coupled to a membrane 520 between a working electrode 530
and a reference electrode 540, which can be coupled to an
electrical sensing device 550. In an embodiment, the membrane is
positioned between a working electrode and a test ligand.
[0169] In an embodiment, the present artificial receptors can be
coupled to a conductive polymer film. For example, in an
embodiment, the present artificial receptors can be coupled to a
conductive polymer film which can be coupled to the surface of a
platinum electrode or other electrode. In an embodiment, the
present artificial receptors can be coupled to a conducting
polypyrrole film.
[0170] In an embodiment, a thick-film electrode can be integrated
in or on a glass substrate. In an embodiment, a thin-film Ag/AgCl
electrode can be integrated on a glass substrate. Platinum
electrodes can also be integrated in or on the glass substrate. A
multi-electrode system can also be provided, with multiple
electrodes being integrated in a single glass substrate.
[0171] The present artificial receptors can also be coupled to a
semiconductor-based sensor. In an embodiment, a semiconductor-based
sensor can be based on a electroadsorptive effect: An electrical
field applied on a sensitive layer of a semiconductor alters the
adsorption characteristics of the material.
[0172] The present artificial receptors can be configured with a
field effect transistor, such as a MOSFET, CHEMFET, ISFET, SAFET,
or SGFET. For example, the present artificial receptors can be
coupled the FET device, or to an object in an environment in the
viscinity of a FET device. In an embodiment, the present artificial
receptors can be coupled to a layer of a metal-oxide-semiconductor
(MOS) field effect transistor (FET), and the binding of a substance
to an artificial receptor can be detected through the MOSTFET.
Schematic illustrations of a ISFET, ChemFET, SAFET, and SGFET are
provided in FIGS. 12A-12D respectively.
[0173] In an embodiment, the present artificial receptors can be
configured with an ion selective field effect transistor (ISFET). A
schematic illustration of an embodiment of an ISFET device 1200 is
shown in FIG. 12A. In an embodiment, an ISFET operates based on
effectsostatic effects caused by a test ligand binding to the
present artificial receptors. In an embodiment, a chemically
sensitive membrane 1205 or insulator layer can be configured to
extend between a source 1210 and a drain 1215. In an embodiment,
the present artificial receptors 1220 can be coupled to the
chemically sensitive membrane or layer. Charges from chemicals that
couple to the receptors and/or the membrane can be amplified
through the operation of the FET signal the presence and/or
identity of a chemical that is bound to the receptors.
[0174] In an embodiment, the present artificial receptors can be
coupled to a chemically modified field effect transistor (ChemFET).
A schematic illustration of a ChemFET 1225 is shown in FIG. 12B. In
an embodiment, an oxide layer 1235 can extend between a drain 1245
and a source 1240, and a chemically sensitive gate 1250 can be
layered on top of the oxide layer. In an embodiment, the present
artificial receptors can be coupled to or embedded in the oxide
layer 1235 or the gate layer 1250. In an embodiment, the layer or
layers can be a gel or a membrane. In an embodiment, both a gel and
a membrane can be layered on a gate surface.
[0175] In an embodiment, the present artificial receptors can be
coupled to a surface accessible field effect transisitor (SAFET).
FIG. 12C shows a schematic illustration of a SAFET device 1255 that
includes a source 1275 and a drain 1280. In an embodiment, the
present artificial receptors 1260 can be coupled to an insulator
structure 1265 or a gate 1270.
[0176] In an embodiment, the present artificial receptors can be
coupled to a suspended gate field effect transisitor (SGFET). FIG.
12D shows a schematic illustration of a SGFET 1285. In an
embodiment, a chemically sensitive insulator structure 1290 can be
layered on a drain 1295 and a source 1300, and a gate 1305 can be
layered on the insulator structure 1290. A chemically sensitive
mesh 1310 can be layered on the gate 1305. The present artificial
receptors 1315 can be coupled to or embedded in one or more of the
chemically sensitive mesh 1310, the gate 1305, and the insulator
structure 1290.
[0177] In an embodiment, the present artificial receptors can be
coupled to a carbon paste that is electrically coupled to an
electrode. In an embodiment, electrochemical sensors that
incorporate the present artificial receptors can be based on carbon
paste screen-printed electrodes. In an embodiment, these sensors
incorporate the conducting polymer polyaniline
(PANI)/poly(vinylsulphonic acid) (PVSA). Such sensors can be useful
in detecting and quantifying a redox active test ligand.
[0178] The present artificial receptors can also be configured with
electrochemical impedance spectroscopy techniques. In an
embodiment, the presence of a test ligand on a receptor can be
detected through a variation in electrical properties at an
electrode interface. For example, a shift in impedance, a change in
capacitance, or a change in admittance (or resistance) can be
detected.
[0179] In an embodiment, the present artificial receptors can be
configured in reagentless sensors. For example, the present
artificial receptors can be configured for use in reagentless
amperometric immunosensors. In an embodiment, a present artificial
receptor is fluorescent, and the fluorescent properties of the
receptor change upon analyte binding. A reagentless assay kit is
described in U.S. Pat. No. 6,660,532.
[0180] In an embodiment, optical and electrochemical sensors can be
configured to simultaneously gather data regarding a sample. A thin
film fiber optic sensor array and apparatus for concurrent viewing
and chemical sensing of a sample is described in U.S. Pat. No.
5,298,741.
[0181] The present artificial receptor can also be configured with
a multiple-element microelectrode array sensor. Such array sensors
can be configured, for example, to detect a variety of different
gasses or test ligands, or can monitor for a single test ligand at
a variety of locations.
[0182] FIG. 8 shows an embodiment of a sensing system 600 where an
array of electrochemical sensors 620 and an array of fiber optic
sensors in a fiber cable 630 can be configured to detect signals
from a multiplicity of present artificial receptors 610 that can be
coupled to a substrate 640. A processing system 650 receives data
from the array of electrochemical sensors and from the optical
sensors. An example of a data pattern 800 received from an array of
fibers is shown in FIG. 10. Locations 810 where a test ligand has
bound to a present artificial receptor can be detected.
Sensor Applications
[0183] Sensors, sensor systems, and methods that employ the present
artificial receptors can be configured to detect a variety of test
ligands or measure a variety of properties or behaviors.
Artificial Sense of Smell or Taste
[0184] In an embodiment, a sensor system that mimics a human or
mammal sense of smell or taste can be provided using the present
artificial receptors. Such systems are sometimes referred to as an
artificial tongue or artificial nose. In an embodiment, for
example, a sample can be tested for bacteria, toxins, or poisons.
In an embodiment, a system can be configured to detect, for
example, a pathogenic microorganism, a cancerous cell, a pollutant
in water, an airborne pollutant, an explosive-related vapor,
protein, and/or a polynucleotide.
[0185] In an embodiment, the sample can be a food or beverage
product. In an embodiment, an optical system can be configured to
detect a change in optical properties of a sample, such as a change
in the intrinsic fluorescence of the sample. Data obtained through
a fiber or fiber bundle can be deconvoluted or otherwise processed.
Complex mixtures of analytes can be identified and quantified. For
example, in an embodiment, a data pattern obtained from a sample
can be interpreted to identify and/or quantify the presence of a
particular test ligand.
[0186] In an embodiment, the present artificial receptors can be
configured in an artificial nose. For example, in an embodiment,
the present artificial receptors can be configured to detect
explosives vapor, for example to facilitate landmine detection. In
an embodiment, an artificial nose includes a light source (or
source of other electromagnetic radiation), an object to which the
present artificial sensors can be coupled, and a CCD camera. In an
embodiment, a vapor delivery system and a vapor removal system can
also be provided. In an embodiment, vapors can be delivered to a
sensing region of an artificial nose in pulses. To identify and
quantify a test ligand in a sample, optical characteristics and
behaviors such as change in fluorescence and/or shift in wavelength
can be detected.
[0187] In an embodiment of an artificial nose, pattern recognition
and/or neural network analysis can be configured to discriminate
between explosives vapors and other organic vapors (e.g. background
vapors.) In an embodiment, an array of optical sensors can be used.
In an embodiment, the present artificial receptors can be coupled
to a microbead that can be coupled to a microwell. In an
embodiment, fluorescent signals can be processed and a signature
can be mapped to allow identification, for example, of
nitroamoratic compounds. In an embodiment, signals can be detected
over a period of time and signal data can be processed to identify
and/or quantify the test ligand(s) in a sample. In an embodiment,
an artificial nose based on the present artificial receptors can be
incorporated into a portable system that can be configured for use
in the field, for example to detect land mines.
Microfluidic Systems
[0188] The present artificial receptors can be configured for use
with microfluidic systems. A biosensor incorporating a microfluidic
system is described in U.S. Pat. No. 6,716,620. Microfluidic
systems are also described in U.S. Pat. Nos. 6,773,567; 6,756,019;
6,709,559; 6,670,153; 6,670,133; 6,635,487; 6,632,655; 6,534,013;
6,498,353; 6,488,895; and 6,465,257, and Published U.S. Patent
Application Nos. 20040048360 and 20040028567. In an embodiment, an
optical sensor can be utilized to detect the presence of a test
ligand in a portion of a microfluidic system. In an embodiment, the
present artificial receptors can be employed to extract a test
ligand from a flow in a microfluidic system. Microfluidic systems
can also be configured with indicator components using the present
artificial receptors. Indicator components for microfluidic systems
are described in Published U.S. Patent Application No.
20040141884.
Nanostructures
[0189] In an embodiment, the present artificial receptors can be
configured for use in nanostructures. In an embodiment, the present
artificial receptors can be configured for use in a colloidal
assembly process. In an embodiment, the receptors are coupled to a
larger particle. The colloidal assembly process can be performed
such that smaller particles assemble around larger ones. In an
embodiment, the larger particle can be etched away to produce a
hollow sphere.
[0190] In an embodiment, the present artificial receptors can be
coupled to cantilevers, which can for example be micron-scale arms.
When a test ligand, a strand of DNA for example, bonds to the
artificial receptors, a surface stress is induced which bends the
cantilever. The bending of the cantilever arm can be detected,
thereby allowing for detection of the presence of a test ligand
bound to the present artificial receptor. In an embodiment, an
array of cantilevers can be provided. In an embodiment, different
test ligands can be detected (different genes for example) by
coupling different artificial receptors to cantilevers or groups of
canitilevers.
[0191] In an embodiment, the present artificial receptors can be
configured with nanotubes. For example, the present artificial
receptors can be coupled to a semiconducting carbon nanotube to
facilitate detection of gasses.
[0192] In an embodiment, the present artificial receptors can be
configured with semiconducting nanotubes that are configured to
change electrical resistance when exposed to a particular test
ligand, such as a gas. In an embodiment, the present artificial
receptors are configured to block the binding of one or more test
ligands with the a nanotube to allow detection of other substances
by the nanotube.
[0193] In an embodiment, the present artificial receptors can be
coupled to the surface of a semiconductor nanowire. For example the
present artificial receptors can be coupled to a nanowire field
effect transistor. In an embodiment, the nanowire field effect
transistor can be coupled to or integrated into a silicon chip.
Sensor Coupled Communications Systems
[0194] Embodiments of the present artificial receptors can be
configured with a sensor that is operatively coupled to a
communication system. For example, a sensor employing the present
artificial receptors can be coupled to a communications network
using wired or wireless technology. The communications can, for
example, be the Internet. A processing system that is also coupled
to the communications network can monitor one or more signals from
one or more sensors. In an embodiment of a responsive system,
corrective action can be taken as necessary in response to signals
received from a sensor.
[0195] Various types of sensors can be configured with a network.
For example, electrochemical and fiber optic sensors can be
configured with a network. In an embodiment, electrochemical,
potentiometric, voltametric, and/or amperometric sensors that
incorporate the present artificial receptors can be coupled to a
data network. Absorbance and/or fluorescence-based sensors that
incorporate the present artificial receptors can also be coupled to
a data network. Other types of sensors that can include the present
artificial receptors can also be coupled to a network. Systems can
also be configured to gather and transmit data from a variety of
sensors or sensor arrays.
[0196] In an embodiment, a sensor system can be coupled to a
digital system that receives signals from the sensor system. In an
embodiment, the digital system can also be configured to make
adjustments to an environment. For example, an actuator can be
activated to make an adjustment. In an embodiment, an item for
which particular properties are detected can be removed from a
product flow. For example a contaminated food product can be
removed from an assembly line. In an embodiment, a system can be
configured to detect a particular test ligand in a mail package,
and the package can be isolated for processing or further analysis.
In an embodiment, items such as aircraft luggage can be monitored
for explosive vapors and responsive action can be initiated as
necessary. Other embodiments are possible.
Methods Employing the Artificial Receptors
[0197] Working artificial receptors can be generated to be specific
to a given test ligand or specific to a particular part of a given
test ligand. Heterogeneous and immobilized combinations of building
block molecules form the working artificial receptors. For example,
combinations of 2, 3, 4, or 5 distinct building block molecules
immobilized in proximity to one another on a support provide
molecular structures that serve as candidate and working artificial
receptors. The building blocks can be naive to the test ligand.
Once a plurality of candidate artificial receptors are generated,
they can be tested to determine which are specific or useful for a
given ligand.
[0198] The specific or working artificial receptor or receptor
complex can then be used in a variety of different methods and
systems. For example, the receptors can be employed in methods
and/or devices for binding or detecting a test ligand. By way of
further example, the receptors can be employed in methods and/or
devices for chemical synthesis. Methods and systems for chemical
synthesis can include methods and systems for regiospecific and
stereospecific chemical synthesis. The receptors can also be
employed for developing compounds that disrupt or model binding
interactions. Methods and systems for developing therapeutic agents
can include methods and systems for pharmaceutical and vaccine
development.
[0199] In an embodiment, methods and systems of the present
invention can be employed for detecting a plurality of ligands of
interest. For example, an unknown biological sample can be
characterized by the presence of a combination of specific ligands.
Such a method can be useful in assays for detecting specific
pathogens or disease states. By way of further example, such an
embodiment can be used for determining the genetic profile of a
subject. For example, cancerous tissue can be detected or a genetic
disposition to cancer can be detected.
[0200] The present artificial receptors can be part of products
used in: analyzing a genome and/or proteome (protein isolation and
characterization); pharmaceutical development (such as
identification of sequence specific small molecule leads,
characterization of protein to protein interactions); detectors for
a test ligand; drug of abuse diagnostics or therapy (such as
clinical or field analysis of cocaine or other drugs of abuse);
hazardous waste analysis or remediation; chemical exposure alert or
intervention; disease diagnostics or therapy; cancer diagnostics or
therapy (such as clinical analysis of prostate specific antigen);
biological agent alert or intervention; food chain contamination
analysis or remediation and clinical analysis of food contaminants;
and the like.
Methods of Binding or Detecting Test Ligands
[0201] In an embodiment, the invention can include methods and/or
devices for binding or detecting a test ligand. For example, the
present artificial receptors can be used for a variety of assays
that presently employ an antibody. The present artificial receptors
can be specific for a given ligand, such as an antigen or an
immunogen. Thus, the present artificial receptors can be used in
formats analogous to enzyme immunoassay, enzyme-linked immunoassay,
immunodiffusion, immunoelectrophoresis, latex agglutination, and
the like. Test ligands that can be detected in such a method
include a drug of abuse, a biological agent (such as a hazardous
agent), a marker for a biological agent, a marker for a disease
state, etc. Methods and systems for detection can include methods
and systems for clinical chemistry, environmental analysis, and
diagnostic assays of all types.
[0202] For example, the artificial receptor can be contacted with a
sample including or suspected of including at least one test
ligand. The building blocks making up the artificial receptors can
be naive to the test ligand. Then, binding of one or more of the
test ligands to the artificial receptors can be detected. Next, the
binding results can be interpreted to provide information about the
sample. In an embodiment, the invention includes a method for
detecting a test ligand in a sample including contacting an
artificial receptor specific to the test ligand with a sample
suspected of containing the test ligand. The method can also
include detecting or quantitating binding of the test ligand to the
artificial receptor. For example, an artificial receptor that binds
(e.g., tightly) the molecule, cell, or microbe under appropriate
conditions can be employed in a format where binding itself is
sufficient to indicate presence of the molecule or organism. Such a
format can also include artificial receptors to be probed with
positive and control samples.
[0203] FIG. 14 schematically illustrates an embodiment of a method
for evaluating candidate artificial receptors for binding to a test
ligand, such as a molecule or cell. The method can include making
an array of candidate artificial receptors. Working artificial
receptors can be identified by contacting the array with test
ligand and identifying which receptors bind the test ligand. The
building blocks making up the artificial receptors can be naive to
the test ligand. Such a method can employ a labeled test ligand.
The method can include producing an array or device including the
working artificial receptor or receptor complex. In an embodiment,
the method can include employing the array or device for detecting
or characterizing the test ligand in a sample, such as a
biological, laboratory, or environmental sample.
[0204] FIG. 15 schematically illustrates an embodiment of the
present method employing an array of candidate artificial
receptors. This embodiment of the method can employ an array
including a significant number of the present artificial receptors
to produce an assay or system for characterizing or detecting a
test ligand. The method can include evaluating an array including a
significant number of candidate artificial receptors for binding to
a test ligand, e.g., a molecule or cell. The building blocks making
up the artificial receptors can be naive to the test ligand. The
molecule or cell can exhibit characteristic binding to one or
several of the candidate artificial receptors from that array. The
one or several artificial receptors can be selected as an
artificial receptor (e.g., a working artificial receptor or a
working artificial receptor complex) that can be employed in
methods for characterizing a biological sample, or characterizing
or detecting the molecule or cell.
[0205] As illustrated in FIG. 15, a test ligand can be identified
by a method employing a single or a plurality of lead or working
artificial receptors. The plurality of lead or working artificial
receptors suitable for identifying a test ligand can be employed in
an array format test. A single lead or working artificial receptor
can be configured on a support as a strip together with positive
and/or negative control receptors, which can also be configured as
strips.
[0206] In an embodiment, the method can include producing or
employing the selected working artificial receptor or receptor
complex on a substrate. The substrate can include working
artificial receptors for a single test ligand or working artificial
receptors for a plurality of test ligands. For example, a method
can include contacting the artificial receptors with a sample. A
substrate including working artificial receptors for a single test
ligand can be employed in a method or system for detecting that
test ligand. Binding to the working artificial receptors indicates
that the sample includes the test ligand. A substrate including
working artificial receptors for a plurality of test ligands can be
employed in a method or system for detecting one, several, or all
of the test ligands. Binding to the working artificial receptors
for a particular test ligand or ligands indicates that the sample
includes such test ligand or ligands.
[0207] The working artificial receptors or receptor complexes can
be configured to provide a pattern indicative of the presence of
one or more of the test ligands. The method can include detecting
the binding pattern of the sample and comparing it with binding
patterns from known samples. FIG. 16 schematically illustrates
certain binding patterns on an array of working artificial
receptors. In an embodiment, all artificial receptors for one test
ligand can be arranged in a line across the substrate. Referring to
FIG. 16, receptors that are specific for IL-2 are in a line 12 on
the array 10 of working artificial receptor complexes. Working
artificial receptors that have bound a test ligand (e.g., IL-2) are
indicated as shaded 24. Working artificial receptors that have not
bound a test ligand are illustrated as open circles 26.
[0208] A method employing the illustrated array can include
detecting binding on line 12 of working artificial receptors
through fluorescence or another means described herein. In the
illustrated embodiment, detecting binding on line 12 of working
artificial receptors indicates that the sample contains IL-2.
Further, lack of signal from the other working artificial receptors
in array 10 indicates that the sample does not contain IFN-gamma,
IL-10, TGF-beta, IL-12, or TGF-alpha. Thus, a method employing such
an array can determine whether a sample is a particular type of
biological sample or contains a particular type of molecule or
cell.
[0209] When designed for use with a field assay kit, the device 30
can have spots arranged such that a positive result creates an
easily recognizable pattern 36, such as a plus sign. The readily
recognizable pattern can thus indicate that a particular test
ligand is present in the sample. Alternatively, the artificial
receptors or spots for a particular target 42 can be arranged
randomly on third array 40. In this manner, when the detection
device or array is used, the results of the test may not be
immediately apparent to an observer but will be readily read by a
machine which can be programmed to correlate binding to receptors
or spots in different positions with the identity of a particular
biological sample, molecule, or cell.
[0210] In an embodiment, the invention includes a method for
detecting or characterizing a biological sample, a molecule, or
cell. This embodiment of the method can include selecting an
artificial receptor that binds the biological sample, molecule, or
cell from an array of artificial receptors, contacting the
artificial receptor with a test composition, and detecting binding
of the artificial receptor to the test composition. In such an
embodiment, binding indicates the presence of the biological
sample, molecule, or cell in the test composition. In an
embodiment, the invention includes a method for detecting or
characterizing a biological sample, molecule, or cell. This
embodiment of the method can include contacting an array of
artificial receptors with a test composition and detecting binding
to the artificial receptors. Binding indicates the presence of the
biological sample, molecule, or cell in the test composition.
[0211] The present method can develop or employ a plurality of
working receptors specific for a particular test ligand, e.g.,
biological sample, molecule, or cell. That is, the working
receptors can be specific for a particular test ligand, but
different receptors can interact with different distinct antigens
(e.g., proteins or carbohydrates), ligands, functional groups, or
structural features of the test ligand. Such a method can provide a
robust test for the presence of a test ligand. For example, such a
robust test can reduce the chances of a false-positive or
false-negative result in comparison with an assay that relies upon
a single unique receptor to detect a given test ligand. Further,
this embodiment of the method can develop or employ working
receptors that demonstrate higher binding affinity due to
interaction with multiple antigens or ligands on the same test
ligand (e.g., multivalent binding).
[0212] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a
polynucleotide, e.g., DNA or RNA. The method can include evaluating
an array including a significant number of candidate artificial
receptors for binding to the polynucleotide, e.g., DNA or RNA. The
building blocks making up the artificial receptors can be naive to
the DNA or RNA. The polynucleotide, e.g., DNA or RNA, can exhibit
characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a
biological sample, or characterizing or detecting the
polynucleotide, e.g., DNA or RNA.
[0213] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a polypeptide or
peptide. The method can include evaluating an array including a
significant number of candidate artificial receptors for binding to
the polypeptide or peptide. The building blocks making up the
artificial receptors can be naive to the polypeptide or peptide.
The polypeptide or peptide can exhibit characteristic binding to
one or several of the candidate artificial receptors from that
array. The one or several artificial receptors can be selected as
an artificial receptor (e.g., a working artificial receptor or a
working artificial receptor complex) that can be employed in
methods for characterizing a biological sample, or characterizing
or detecting the polypeptide or peptide.
[0214] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a oligo- or
polysaccharide. The method can include evaluating an array
including a significant number of candidate artificial receptors
for binding to the oligo- or polysaccharide. The building blocks
making up the artificial receptors can be naive to the oligo- or
polysaccharide. The oligo- or polysaccharide can exhibit
characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a
biological sample, or characterizing or detecting the oligo- or
polysaccharide.
[0215] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a cell, e.g., a
hepatocyte. The method can include evaluating an array including a
significant number of candidate artificial receptors for binding to
the cell, e.g., a hepatocyte. The building blocks making up the
artificial receptors can be naive to the cell. The cell, e.g., a
hepatocyte, can exhibit characteristic binding to one or several of
the candidate artificial receptors from that array. The one or
several artificial receptors can be selected as an artificial
receptor (e.g., a working artificial receptor or a working
artificial receptor complex) that can be employed in methods for
characterizing a biological sample, or characterizing or detecting
the cell, e.g., a hepatocyte.
Methods of Binding or Detecting Drugs of Abuse
[0216] In an embodiment, the invention can include methods and/or
devices for binding or detecting a drug of abuse. Methods and
systems for detection can include methods and systems for clinical
chemistry, field analysis, and diagnostic assays of all types. For
example, the artificial receptor can be contacted with a sample
including or suspected of including at least one drug of abuse.
Then, binding of one or more of the drugs of abuse to the
artificial receptors can be detected. Next, the binding results can
be interpreted to provide information about the sample. In an
embodiment, the invention includes a method for detecting a drug of
abuse in a sample including contacting an artificial receptor
specific to the drug of abuse with a sample suspected of containing
the drug of abuse. The method can also include detecting or
quantitating binding of the drug of abuse to the artificial
receptor.
[0217] FIG. 14 schematically illustrates an embodiment of a method
for evaluating candidate artificial receptors for binding to a test
ligand. This embodiment of the present method can be employed for
detecting a test ligand such as a drug of abuse. The method can
include making an array of candidate artificial receptors. The
building blocks making up the artificial receptors can be naive to
the test ligand. Working artificial receptors can be identified by
contacting the array with a drug of abuse and identifying which
receptors bind the drug of abuse. The method can include producing
an array or device including the working artificial receptor or
receptor complex. In an embodiment, the method can include
employing the array or device for detecting or characterizing the
drug of abuse in a sample, such as a biological, laboratory, or
evidence sample.
[0218] FIG. 15 schematically illustrates an embodiment of the
present method employing an array of candidate artificial
receptors. This embodiment of the method can employ an array
including a significant number of the present artificial receptors
to produce an assay or system for characterizing or detecting a
drug of abuse. The method can include evaluating an array including
a significant number of candidate artificial receptors for binding
to a drug of abuse. The building blocks making up the artificial
receptors can be naive to the drug of abuse. The drug of abuse can
exhibit characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a
biological or field sample, or characterizing or detecting the drug
of abuse.
[0219] In an embodiment, the method can include producing or
employing the selected working artificial receptor or receptor
complex on a substrate. The substrate can include working
artificial receptors for a single drug of abuse or working
artificial receptors for a plurality of drugs of abuse. For
example, a method can include contacting the artificial receptors
with a sample. A substrate including working artificial receptors
for a single drug of abuse can be employed in a method or system
for detecting that drug of abuse. Binding to the working artificial
receptors indicates that the sample includes the drug of abuse. A
substrate including working artificial receptors for a plurality of
drugs of abuse can be employed in a method or system for detecting
one, several, or all of the drugs of abuse. Binding to the working
artificial receptors for a particular drug of abuse or drugs of
abuse indicates that the sample includes such a drug of abuse or
drugs of abuse.
[0220] The working artificial receptors or receptor complexes can
be configured to provide a pattern indicative of the presence of
one or more of the drugs of abuse. The method can include detecting
the binding pattern of the sample and comparing it with binding
patterns from known samples. FIG. 16 schematically illustrates
binding patterns on an array of working artificial receptors. Such
patterns and schemes can be employed for identifying a variety of
test ligands including drugs of abuse.
[0221] The present method can develop or employ a plurality of
working receptors specific for a particular drug of abuse or
feature on the drug of abuse. That is, the working receptors can be
specific for a particular drug of abuse, but different receptors
can interact with different distinct ligands, functional groups, or
structural features of the drug of abuse. Such a method can provide
a robust test for the presence of a drug of abuse. For example,
such a robust test can reduce the chances of a false-positive or
false-negative result in comparison with an assay that relies upon
a single unique receptor to detect a given drug of abuse. Further,
this embodiment of the method can develop or employ working
receptors that demonstrate higher binding affinity due to
interaction with multiple ligands or features on the same drug of
abuse (e.g., multivalent binding).
[0222] Suitable drugs of abuse include cannabinoids (e.g., hashish
and marijuana), depressants (e.g., barbiturates, benzodiazepines,
gamma-hydroxy butyrate, methaqualone), dissociative anesthetics
(e.g., ketamine, PCP, and PCP analogs), hallucinogens (e.g., LSD,
mescaline, psilocybin), opiates or opioids (e.g., codeine,
fentanyl, fentanyl analogs, heroin, morphine, opium, oxycodone HCL,
hydrocodone bitartrate), stimulants (e.g., amphetamine, cocaine,
methylenedioxy-methamphetamine, methamphetamine, methylphenidate,
nicotine), inhalants (e.g., solvents), and the like.
[0223] Suitable drugs of abuse include performance enhancing
agents, such as stimulants and beta-blockers, anabolic agents,
oxygen carrier enhancers, masking agents, and inhalants. Suitable
stimulants include caffeine and amphetamines. Suitable
beta-blockers include salbutamol (used in asthma inhalers) and the
like. Suitable anabolic agents include steroids (e.g., anabolic
steroids), steroid analogs, and growth hormone. Suitable oxygen
carrier enhancers include erythropoietin and the like.
Methods of Binding or Detecting Isomers
[0224] In an embodiment, the invention can include methods and/or
devices for binding or detecting an isomer or isomers of a
compound. Methods and systems for detection can include methods and
systems for clinical chemistry, environmental analysis, and
diagnostic assays of all types. For example, the artificial
receptor can be contacted with a sample including or suspected of
including at least one isomer of a compound. Then, binding of one
or more of the isomers of a compound to the artificial receptors
can be detected. Next, the binding results can be interpreted to
provide information about the isomers. In an embodiment, the
invention includes a method for detecting an isomer of a compound
in a sample including contacting an artificial receptor specific to
the isomer with a sample suspected of containing the isomer. The
method can also include detecting or quantitating binding of the
isomer to the artificial receptor.
[0225] The present method can be applied to isomers such as
stereoisomers (e.g., geometric isomers or optical isomers), optical
isomers (e.g., enantiomers and diastereomers), geometric isomers
(e.g., cis- and trans-isomers). The present method can be employed
to develop working or lead artificial receptors or working
artificial complexes that can bind to one or more isomers of a
compound (e.g., enantioselective receptor environments). For
example, the artificial receptor or complex can bind to one
stereoisomer of a compound but bind only weakly or not at all
another stereoisomer of the compound. For example, the artificial
receptor or complex can bind one geometric isomer of a compound but
bind only weakly or not at all another geometric isomer. For
example, the artificial receptor or complex can bind one optical
isomer of a compound but bind only weakly or not at all another
optical isomer. For example, the artificial receptor or complex can
bind one enantiomer of a compound but bind only weakly or not at
all another enantiomer. For example, the artificial receptor or
complex can bind one diastereomer of a compound but bind only
weakly or not at all another diastereomer.
[0226] FIG. 14 schematically illustrates an embodiment of a method
for evaluating candidate artificial receptors for binding to a test
ligand. This embodiment of the present method can be employed for
detecting a test ligand such as an isomer of a compound. The method
can include making an array of candidate artificial receptors. The
building blocks making up the artificial receptors can be naive to
the test ligand. Working artificial receptors can be identified by
contacting the array with an isomer and identifying which receptors
bind the isomer. The method can include producing an array or
device including the working artificial receptor or receptor
complex. In an embodiment, the method can include employing the
array or device for detecting or characterizing the isomer in a
sample, such as a biological, laboratory, or clinical sample.
[0227] FIG. 15 schematically illustrates an embodiment of the
present method employing an array of candidate artificial
receptors. This embodiment of the method can employ an array
including a significant number of the present artificial receptors
to produce an assay or system for characterizing or detecting an
isomer. The method can include evaluating an array including a
significant number of candidate artificial receptors for binding to
an isomer. The building blocks making up the artificial receptors
can be naive to the isomer. The isomer can exhibit characteristic
binding to one or several of the candidate artificial receptors
from that array. The one or several artificial receptors can be
selected as an artificial receptor (e.g., a working artificial
receptor or a working artificial receptor complex) that can be
employed in methods for characterizing a biological, clinical, or
laboratory sample, or characterizing or detecting the isomer.
[0228] In an embodiment, the method can include producing or
employing the selected working artificial receptor or receptor
complex on a substrate. The substrate can include working
artificial receptors for a single isomer or working artificial
receptors for a plurality of isomers. For example, a method can
include contacting the artificial receptors with a sample. A
substrate including working artificial receptors for a single
isomer can be employed in a method or system for detecting that
isomer. Binding to the working artificial receptors indicates that
the sample includes the isomer. A substrate including working
artificial receptors for a plurality of isomers can be employed in
a method or system for detecting one, several, or all of the
isomers. Binding to the working artificial receptors for a
particular isomer or isomers indicates that the sample includes
such an isomer or isomers.
[0229] The working artificial receptors or receptor complexes can
be configured to provide a pattern indicative of the presence of
one or more of the isomers. The method can include detecting the
binding pattern of the sample and comparing it with binding
patterns from known samples. FIG. 16 schematically illustrates
binding patterns on an array of working artificial receptors. Such
patterns and schemes can be employed for identifying a variety of
test ligands including isomers.
[0230] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a stereoisomer.
The method can include evaluating an array including a significant
number of candidate artificial receptors for binding to the
stereoisomer. The building blocks making up the artificial
receptors can be naive to the stereoisomer. The stereoisomer can
exhibit characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a lab
or clinical sample or characterizing or detecting the
stereoisomer.
[0231] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a geometric
isomer (e.g., cis- and trans-isomers). The method can include
evaluating an array including a significant number of candidate
artificial receptors for binding to the geometric isomer (e.g.,
cis- and trans-isomers). The building blocks making up the
artificial receptors can be naive to the geometric isomer. The
geometric isomer can exhibit characteristic binding to one or
several of the candidate artificial receptors from that array. The
one or several artificial receptors can be selected as an
artificial receptor (e.g., a working artificial receptor or a
working artificial receptor complex) that can be employed in
methods for characterizing a lab or clinical sample or
characterizing or detecting the geometric isomer (e.g., cis- and
trans-isomers).
[0232] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting an optical
isomer. The method can include evaluating an array including a
significant number of candidate artificial receptors for binding to
the optical isomer. The building blocks making up the artificial
receptors can be naive to the optical isomer. The optical isomer
can exhibit characteristic binding to one or several of the
candidate artificial receptors from that array. The one or several
artificial receptors can be selected as an artificial receptor
(e.g., a working artificial receptor or a working artificial
receptor complex) that can be employed in methods for
characterizing a lab or clinical sample or characterizing or
detecting the optical isomer.
[0233] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting an enantiomer.
The method can include evaluating an array including a significant
number of candidate artificial receptors for binding to the
enantiomer. The enantiomer can exhibit characteristic binding to
one or several of the candidate artificial receptors from that
array. The one or several artificial receptors can be selected as
an artificial receptor (e.g., a working artificial receptor or a
working artificial receptor complex) that can be employed in
methods for characterizing a lab or clinical sample or
characterizing or detecting the enantiomer.
[0234] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a diastereomer.
The method can include evaluating an array including a significant
number of candidate artificial receptors for binding to the
diastereomer. The building blocks making up the artificial
receptors can be naive to the diastereomer. The diastereomer can
exhibit characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a lab
or clinical sample or characterizing or detecting the
diastereomer.
Methods for Binding or Detecting Peptides
[0235] In an embodiment, the invention can include methods and/or
devices for binding or detecting a peptide. Methods and systems for
detection can include methods and systems for clinical chemistry,
environmental analysis, and diagnostic assays of all types. For
example, the artificial receptor can be contacted with a sample
including or suspected of including at least one peptide. Then,
binding of one or more of the peptides to the artificial receptors
can be detected. Next, the binding results can be interpreted to
provide information about the sample. In an embodiment, the
invention includes a method for detecting a peptide in a sample
including contacting an artificial receptor specific to the peptide
with a sample suspected of containing the peptide. The method can
also include detecting or quantitating binding of the peptide to
the artificial receptor.
[0236] FIG. 14 schematically illustrates an embodiment of a method
for evaluating candidate artificial receptors for binding to a test
ligand. This embodiment of the present method can be employed for
detecting a test ligand such as a peptide. The method can include
making an array of candidate artificial receptors. The building
blocks making up the artificial receptors can be naive to the test
ligand. Working artificial receptors can be identified by
contacting the array with a peptide and identifying which receptors
bind the peptide. The method can include producing an array or
device including the working artificial receptor or receptor
complex. In an embodiment, the method can include employing the
array or device for detecting or characterizing the peptide in a
sample, such as a biological, laboratory, or clinical sample.
[0237] FIG. 15 schematically illustrates an embodiment of the
present method employing an array of candidate artificial
receptors. This embodiment of the method can employ an array
including a significant number of the present artificial receptors
to produce an assay or system for characterizing or detecting a
peptide. The method can include evaluating an array including a
significant number of candidate artificial receptors for binding to
a peptide. The building blocks making up the artificial receptors
can be naive to the peptide. The peptide can exhibit characteristic
binding to one or several of the candidate artificial receptors
from that array. The one or several artificial receptors can be
selected as an artificial receptor (e.g., a working artificial
receptor or a working artificial receptor complex) that can be
employed in methods for characterizing a biological or
environmental sample, or characterizing or detecting the
peptide.
[0238] FIG. 17 schematically illustrates an embodiment of a method
for developing a method and system for detecting a test ligand,
such as a peptide or mixture of peptides. This embodiment of the
present method includes evaluating a plurality (e.g. array) of
candidate artificial receptors for binding to each of a plurality
of peptides. The building blocks making up the artificial receptors
can be naive to one or more of the peptides. The plurality of
peptides can include the peptides found in a cell or organism. The
method can include detecting binding of individual peptides to a
subset of the plurality or array of candidate artificial receptors.
The method can include detecting binding of the peptides found in a
cell or organism to a subset of or all of the plurality or array of
candidate artificial receptors. This can be envisioned as
developing a working artificial receptor or artificial receptor
complex for each peptide or mixture of peptides.
[0239] Thus, each peptide or mixture of peptides can provide a
pattern of bound receptors in the plurality or array. The pattern
of bound receptors can be characteristic of the peptide or mixture
of peptides or a sample including the peptide or mixture of
peptides. The method can include storing a representation of the
binding pattern as an image or a data structure. The representation
of the binding pattern can be evaluated either by an operator or
data processing system. The method can include such evaluating. A
binding pattern from an unknown sample that matches the binding
pattern for a particular peptide then characterizes the unknown
sample as containing that peptide. A binding pattern from an
unknown sample that matches the binding pattern for a particular
mixture of peptides then characterizes the unknown sample as
including or being that mixture of peptides or as including or
being the organism or cell containing that mixture of peptides. A
plurality of binding patterns can be stored as a database.
[0240] An embodiment of the illustrated method can include creating
an array of artificial receptors. This embodiment can also include
compiling a database of the binding patterns of a specific peptide
or mixture of peptides, for example, by probing the array with a
plurality of individual peptides or the peptides found in a cell or
organism. Contacting the array with an unidentified peptide or
mixture of peptides can create a test binding pattern. The method
can then compare the test binding pattern with the binding patterns
of known peptides or mixtures of peptides in the database in order
to characterize or classify the unidentified peptide, mixture of
peptides, or cell or organism. In an embodiment, the database and
the array of receptors has already been constructed and the method
involves probing the array with an unknown peptide or mixture of
peptides to create a test binding pattern and then comparing this
binding pattern with the binding patterns in the database in order
to characterize or classify the unidentified peptide, mixture of
peptides, or cell or organism.
[0241] An array constructed for distinguishing mixtures of peptides
can be contacted with samples from an organism, cell, or tissue of
interest. Peptides that bind to the array can characterize or
detect the organism, cell or tissue; can indicate a disorder caused
by the organism or affecting the cell or tissue; can indicate
successful therapy of a disorder caused by the organism or
affecting the cell or tissue; characterize disease processes;
identify therapeutic leads or strategies; or the like.
[0242] In an embodiment, the method can include producing or
employing the selected working artificial receptor or receptor
complex on a substrate. The substrate can include working
artificial receptors for a single peptide or working artificial
receptors for a plurality of peptides. For example, a method can
include contacting the artificial receptors with a sample. A
substrate including working artificial receptors for a single
peptide can be employed in a method or system for detecting that
peptide. Binding to the working artificial receptors indicates that
the sample includes the peptide. A substrate including working
artificial receptors for a plurality of peptides can be employed in
a method or system for detecting one, several, or all of the
peptides. Binding to the working artificial receptors for a
particular peptide or peptides indicates that the sample includes
such a peptide or peptides.
[0243] The working artificial receptors or receptor complexes can
be configured to provide a pattern indicative of the presence of
one or more of the peptides. The method can include detecting the
binding pattern of the sample and comparing it with binding
patterns from known samples. FIG. 16 schematically illustrates
binding patterns on an array of working artificial receptors. Such
patterns and schemes can be employed for identifying a variety of
test ligands including peptides.
[0244] The present method can develop or employ a plurality of
working receptors specific for a particular peptide or feature on
the peptide. That is, the working receptors can be specific for a
particular peptide, but different receptors can interact with
different distinct ligands, functional groups, or structural
features of the peptide. Such a method can provide a robust test
for the presence of a peptide. For example, such a robust test can
reduce the chances of a false-positive or false-negative result in
comparison with an assay that relies upon a single unique receptor
to detect a given peptide. Further, this embodiment of the method
can develop or employ working receptors that demonstrate higher
binding affinity due to interaction with multiple ligands or
features on the same peptide (e.g., multivalent binding).
[0245] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a peptide. The
method can include evaluating an array including a significant
number of candidate artificial receptors for binding to the
peptide. The building blocks making up the artificial receptors can
be naive to the test ligand. The peptide can exhibit characteristic
binding to one or several of the candidate artificial receptors
from that array. The one or several artificial receptors can be
selected as an artificial receptor (e.g., a working artificial
receptor or a working artificial receptor complex) that can be
employed in methods for characterizing a biological sample or
characterizing or detecting the peptide.
[0246] The present method can include selecting artificial
receptors that bind a particular peptide and/or the building blocks
making up these receptors (e.g., bound to a scaffold molecule) as
leads for pharmaceutical development or as active agents for
modulating an activity of that peptide. The artificial receptor or
building blocks making up that artificial receptor can be selected
to bind to a portion of a peptide required for its interaction with
an other macromolecule (e.g. carbohydrate, protein, or
polynucleotide), thus disrupting this interaction.
Methods for Binding or Detecting Protein or Proteome
[0247] In an embodiment, the invention can include methods and/or
devices for binding or detecting a protein, one or more of a
plurality of proteins, or a proteome. Methods and systems for
detection can include methods and systems for clinical chemistry,
environmental analysis, diagnostic assays, and for proteome
analysis. For example, the artificial receptor can be contacted
with a sample including at least one protein or one proteome. The
building blocks making up the artificial receptors can be naive to
the test ligand. Then, binding of one or more proteins to the
artificial receptors can be detected. Next, the binding results can
be interpreted to provide information about the sample, e.g., the
proteome. In an embodiment, the invention includes a method for
detecting a protein in a sample including contacting an artificial
receptor specific to the protein with a sample suspected of
containing the protein. The method can also include detecting or
quantitating binding of the protein to the artificial receptor.
[0248] FIG. 14 schematically illustrates an embodiment of a method
for evaluating candidate artificial receptors for binding to a test
ligand. This embodiment of the present method can be employed for
detecting a test ligand such as one or more proteins. The method
can include making an array of candidate artificial receptors. The
building blocks making up the artificial receptors can be naive to
the test ligand. Working artificial receptors can be identified by
contacting the array with a protein and identifying which receptors
bind the protein. The method can include producing an array or
device including the working artificial receptor or receptor
complex. In an embodiment, the method can include employing the
array or device for detecting or characterizing the protein in a
sample, such as a biological, laboratory, or environmental
sample.
[0249] In an embodiment, the method can include producing or
employing the selected working artificial receptor or receptor
complex on a substrate. The substrate can include working
artificial receptors for a single protein or working artificial
receptors for a plurality of proteins. For example, a method can
include contacting the artificial receptors with a sample. A
substrate including working artificial receptors for a single
protein can be employed in a method or system for detecting that
protein. Binding to the working artificial receptors indicates that
the sample includes the protein. A substrate including working
artificial receptors for a plurality of proteins can be employed in
a method or system for detecting one, several, or all of the
proteins. Binding to the working artificial receptors for a
particular protein or protein indicates that the sample includes
such a protein or protein.
[0250] The working artificial receptors or receptor complexes can
be configured to provide a pattern indicative of the presence of
one or more of the proteins. The method can include detecting the
binding pattern of the sample and comparing it with binding
patterns from known samples. FIG. 16 schematically illustrates
binding patterns on an array of working artificial receptors. Such
patterns and schemes can be employed for identifying a variety of
test ligands including proteins.
[0251] FIG. 17 schematically illustrates an embodiment of a method
for developing a method and system for detecting a test ligand,
such as a protein or proteome. This embodiment of the present
method includes evaluating a plurality (e.g. array) of candidate
artificial receptors for binding to each of a plurality of test
ligands. The building blocks making up the artificial receptors can
be naive to the test ligand. The plurality of test ligands can
include a plurality of proteins. The plurality of test ligands can
include the proteins making up the proteome of a cell or organism.
The method can include detecting binding of individual proteins to
a subset of the plurality or array of candidate artificial
receptors. The method can include detecting binding of proteins
making up the proteome to a subset of or all of the plurality or
array of candidate artificial receptors. This can be envisioned as
developing a working artificial receptor or artificial receptor
complex for each protein or for the proteome.
[0252] Thus, each protein or proteome can provide a pattern of
bound receptors in the plurality or array. The pattern of bound
receptors can be characteristic of the protein or proteome or a
sample including the protein or proteome. The method can include
storing a representation of the binding pattern as an image or a
data structure. The representation of the binding pattern can be
evaluated either by an operator or data processing system. The
method can include such evaluating. A binding pattern from an
unknown sample that matches the binding pattern for a particular
protein then characterizes the unknown sample as containing that
protein. A binding pattern from an unknown sample that matches the
binding pattern for a particular proteome then characterizes the
unknown sample as including or being that proteome or as including
or being the organism or cell having that proteome. Similarly, a
binding pattern from an unknown sample can be evaluated against the
patterns of a plurality of particular proteins or proteomes and the
sample can be characterized as containing one or more of the
proteins or proteomes. A plurality of binding patterns can be
stored as a database.
[0253] An embodiment of the illustrated method can include creating
an array of artificial receptors. This embodiment can also include
compiling a database of the binding patterns of specific proteins
or proteomes, for example, by probing the array with a plurality of
individual proteins or proteomes. Contacting the array with
unidentified proteins or proteomes can create a test binding
pattern. The method can then compare the test binding pattern with
the binding patterns of known proteins or proteomes in the database
in order to characterize or classify the unidentified protein,
proteome, or cell or organism. In an embodiment, the database and
the array of receptors has already been constructed and the method
involves probing the array with an unknown protein or proteome to
create a test binding pattern and then comparing this binding
pattern with the binding patterns in the database in order to
characterize or classify the unidentified protein, proteome, or
cell or organism.
[0254] A proteome array can be contacted with samples from an
organism, cell, or tissue of interest. Proteins that bind to the
proteome array can characterize or detect the organism, cell or
tissue; can indicate a disorder caused by the organism or affecting
the cell or tissue; can indicate successful therapy of a disorder
caused by the organism or affecting the cell or tissue;
characterize disease processes; identify therapeutic leads or
strategies; or the like.
[0255] The present method can develop or employ a plurality of
working receptors specific for a particular protein or feature on
the protein. That is, the working receptors can be specific for a
particular protein, but different receptors can interact with
different distinct ligands, functional groups, or structural
features of the protein. Such a method can provide a robust test
for the presence of a protein. For example, such a robust test can
reduce the chances of a false-positive or false-negative result in
comparison with an assay that relies upon a single unique receptor
to detect a given protein. Further, this embodiment of the method
can develop or employ working receptors that demonstrate higher
binding affinity due to interaction with multiple ligands or
features on the same protein (e.g., multivalent binding).
[0256] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a protein. The
method can include evaluating an array including a significant
number of candidate artificial receptors for binding to the
protein. The building blocks making up the artificial receptors can
be naive to the test ligand. The protein can exhibit characteristic
binding to one or several of the candidate artificial receptors
from that array. The one or several artificial receptors can be
selected as an artificial receptor (e.g., a working artificial
receptor or a working artificial receptor complex) that can be
employed in methods for characterizing a biological sample or
characterizing or detecting the protein.
[0257] The present method can include selecting artificial
receptors that bind a particular protein and/or the building blocks
making up these receptors (e.g., bound to a scaffold molecule) as
leads for pharmaceutical development or as active agents for
modulating an activity of that protein. The artificial receptor or
building blocks making up that artificial receptor can be selected
to bind to or disrupt the activity of the active site of an enzyme
or the ligand binding site of a receptor. The artificial receptor
or building blocks making up that artificial receptor can be
selected to bind to a portion of a protein required for its
interaction with an other macromolecule (e.g. carbohydrate,
protein, or polynucleotide), thus disrupting this interaction. The
artificial receptor or building blocks making up that artificial
receptor can be selected to bind to the binding site of a receptor
and act as an agonist of that receptor.
[0258] The present method can include selecting working artificial
receptors that bind a preselected protein for use in a system for
proteome analysis. The working artificial receptors for the
preselected protein can be provided on a substrate and the protein
bound to the receptors. In an embodiment, selecting and binding
employ a plurality of different working artificial receptors for
the preselected protein. The plurality of artificial receptors may
bind to different features on the preselected protein and leave
free different features on the preselected protein. This embodiment
of the method includes contacting the working receptors with bound
preselected protein to at least one candidate binding partner for
the preselected protein. The method can include detecting binding
or absence of binding of the candidate binding partner to the
preselected protein. A candidate binding partner that binds to the
preselected protein can be considered a lead binding partner.
[0259] In an embodiment, the method includes contacting the working
receptors with bound preselected protein with a proteome of a cell
or organism serving as the source of candidate binding partners.
The method can then recover from the proteome one or more lead
binding partners. This can then characterize the proteome as
containing or not a binding partner for the preselected
protein.
[0260] In an embodiment, the present artificial receptors can be
employed in studies of proteomics. In such an embodiment, an array
of candidate or working artificial receptors can be contacted with
a mixture of peptides, polypeptides, and/or proteins. Each mixture
can produce a characteristic fingerprint of binding to the array.
In addition, identification of a specific receptor environment for
a target peptide, polypeptide, and/or protein can be utilized for
isolation and analysis of the target. That is, in yet another
embodiment, a particular receptor surface can be employed for
affinity purification methods, e.g. affinity chromatography.
[0261] In an embodiment, the present candidate artificial receptors
can be employed to find receptor surfaces that bind proteins in a
preferred configuration or orientation. Many proteins (e.g.
antibodies, enzymes, receptors) are stable and/or active in
specific environments. Defined receptor surfaces can be used to
produce binding environments that selectively retain or orient the
protein for maximum stability and/or activity. In an embodiment,
the present artificial receptors can be employed to form bioactive
surfaces. For example, receptor surfaces can be used to
specifically bind the active conformation of an antibody or
enzyme.
[0262] In an embodiment, the present method can include labeling a
protein while it remains bound to an artificial receptor. The
resulting protein will be labeled on its portions accessible to the
labeling reagent but not on those portions bound to the artificial
receptor. The method can include releasing the labeled protein from
the artificial receptor. Determining the distribution of labels on
the protein indicates which portion of the protein was bound to the
receptor.
[0263] In certain embodiments, the present artificial receptors can
be employed to distinguish between two conformations of a single
protein. Certain proteins exist in two or more stable
conformations. In an embodiment, the present working artificial
receptor or complex can bind a first conformation of a protein. In
an embodiment, the present working artificial receptor or complex
can bind a second conformation of a protein. In an embodiment, the
present working artificial receptor or complex can bind a first
conformation of a protein, but not a second conformation of the
same protein. In an embodiment, the present working artificial
receptor or complex can bind a second conformation of a protein,
but not a first conformation of the same protein.
[0264] For example, in an embodiment, the present working
artificial receptor or complex can bind a first or non-infectious
conformation of a prion, but not its second or infectious
conformation. For example, in an embodiment, the present working
artificial receptor or complex can bind the second or infectious
conformation of a prion, but not its first or non-infectious
conformation. For example, in an embodiment, the present working
artificial receptor or complex can bind a first or
non-plaque-forming conformation of .beta.-amyloid, but not its
second or plaque-forming conformation. For example, in an
embodiment, the present working artificial receptor or complex can
bind a second or plaque-forming conformation of .beta.-amyloid, but
not its second or non-plaque-forming conformation.
[0265] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a desired
conformation of a protein. The method can include evaluating an
array including a significant number of candidate artificial
receptors for binding to the desired conformation of the protein.
The building blocks making up the artificial receptors can be naive
to the protein or its desired conformation. The desired
conformation of the protein can exhibit characteristic binding to
one or several of the candidate artificial receptors from that
array. The one or several artificial receptors can be selected as
an artificial receptor (e.g., a working artificial receptor or a
working artificial receptor complex) that can be employed in
methods for characterizing a biological sample or characterizing or
detecting the desired conformation of the protein.
[0266] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a first or
non-infectious conformation of a prion. The method can include
evaluating an array including a significant number of candidate
artificial receptors for binding to the first or non-infectious
conformation of a prion. The building blocks making up the
artificial receptors can be naive to the prion. The first or
non-infectious conformation of a prion can exhibit characteristic
binding to one or several of the candidate artificial receptors
from that array. The one or several artificial receptors can be
selected as an artificial receptor (e.g., a working artificial
receptor or a working artificial receptor complex) that can be
employed in methods for characterizing a biological sample or
characterizing or detecting the first or non-infectious
conformation of a prion.
[0267] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a second or
infectious conformation of a prion. The method can include
evaluating an array including a significant number of candidate
artificial receptors for binding to the second or infectious
conformation of a prion. The building blocks making up the
artificial receptors can be naive to the test ligand. The second or
infectious conformation of a prion can exhibit characteristic
binding to one or several of the candidate artificial receptors
from that array. The one or several artificial receptors can be
selected as an artificial receptor (e.g., a working artificial
receptor or a working artificial receptor complex) that can be
employed in methods for characterizing a biological sample or
characterizing or detecting the second or infectious conformation
of a prion.
[0268] In an embodiment, the present method includes developing
receptors or a receptor system that can distinguish between the
first or non-infectious conformation of a prion and the second or
infectious conformation of the prion. Such a method can include
selecting a working artificial receptor or complex can that bind
the first or non-infectious conformation of a prion, but not the
second or infectious conformation of the prion. This embodiment can
include selecting a working artificial receptor or complex can that
bind the second or infectious conformation of a prion, but not the
first or non-infectious conformation of the prion. Employed
together, these two sets of working artificial receptors or systems
can characterize a biological sample as containing one or both
forms of the prion.
[0269] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a first or
non-plaque-forming conformation of a .beta.-amyloid. The method can
include evaluating an array including a significant number of
candidate artificial receptors for binding to the first or
non-plaque-forming conformation of the .beta.-amyloid. The building
blocks making up the artificial receptors can be naive to the
.beta.-amyloid. The first or non-plaque-forming conformation of the
.beta.-amyloid can exhibit characteristic binding to one or several
of the candidate artificial receptors from that array. The one or
several artificial receptors can be selected as an artificial
receptor (e.g., a working artificial receptor or a working
artificial receptor complex) that can be employed in methods for
characterizing a biological sample or characterizing or detecting
the first or non-plaque-forming conformation of the
.beta.-amyloid.
[0270] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a second or
plaque-forming conformation of a .beta.-amyloid. The method can
include evaluating an array including a significant number of
candidate artificial receptors for binding to the second or
plaque-forming conformation of a .beta.-amyloid. The building
blocks making up the artificial receptors can be naive to the
.beta.-amyloid. The second or plaque-forming conformation of the
.beta.-amyloid can exhibit characteristic binding to one or several
of the candidate artificial receptors from that array. The one or
several artificial receptors can be selected as an artificial
receptor (e.g., a working artificial receptor or a working
artificial receptor complex) that can be employed in methods for
characterizing a biological sample or characterizing or detecting
the second or plaque-forming conformation of the
.beta.-amyloid.
[0271] In an embodiment, the present method includes developing
receptors or a receptor system that can distinguish between the
first or non-plaque-forming conformation of .beta.-amyloid and the
second or plaque-forming conformation of the .beta.-amyloid. Such a
method can include selecting a working artificial receptor or
complex can that bind the first or non-plaque-forming conformation
of .beta.-amyloid, but not the second or plaque-forming
conformation of the .beta.-amyloid. This embodiment can include
selecting a working artificial receptor or complex can that bind
the second or plaque-forming conformation of the .beta.-amyloid,
but not the first or non-plaque-forming conformation of
.beta.-amyloid. Employed together, these two sets of working
artificial receptors or systems can characterize a biological
sample as containing one or both forms of the .beta.-amyloid.
[0272] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting cholera toxin.
The method can include evaluating an array including a significant
number of candidate artificial receptors for binding to the cholera
toxin. The building blocks making up the artificial receptors can
be naive to the test ligand. The cholera toxin can exhibit
characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a
biological sample, or characterizing or detecting cholera
toxin.
[0273] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting at least one
protein of a cancer cell. The method can include evaluating an
array including a significant number of candidate artificial
receptors for binding to the cancer cell protein. The building
blocks making up the artificial receptors can be naive to the test
ligand. The cancer cell protein can exhibit characteristic binding
to one or several of the candidate artificial receptors from that
array. The one or several artificial receptors can be selected as
an artificial receptor (e.g., a working artificial receptor or a
working artificial receptor complex) that can be employed in
methods for characterizing a biological sample or characterizing or
detecting the cancer cell protein.
[0274] In an embodiment, the present method can include contacting
a working artificial receptor or array with a sample from cells or
tissues suspected of being cancerous or including a tumor. The
sample can be serum. Binding of at least one protein to the working
artificial receptor or array can indicate or characterize the
presence of the particular cancer or tumor, such as by
characterizing the pattern of proteins present.
[0275] Cancers that can be detected or characterized by such a
method include, for example, bladder cancer, breast cancer, colon
cancer, kidney cancer, liver cancer, lung cancer, including small
cell lung cancer, esophageal cancer, gall-bladder cancer, ovarian
cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid
cancer, prostate cancer, and skin cancer, including squamous cell
carcinoma; hematopoietic tumors of lymphoid lineage, including
leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia,
B-cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin's
lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopoietic
tumors of myeloid lineage, including acute and chronic myelogenous
leukemias, myelodysplastic syndrome and promyelocytic leukemia;
tumors of mesenchymal origin, including fibrosarcoma and
rhabdomyosarcoma; tumors of the central and peripheral nervous
system, including astrocytoma, neuroblastoma, glioma and
schwannomas; other tumors, including melanoma, seminoma,
teratocarcinoma, osteosarcoma, xeroderoma pigmentosum,
keratoctanthoma, thyroid follicular cancer, Kaposi's sarcoma, and
the like.
Methods of Binding or Detecting Microbes
[0276] In an embodiment, the invention can include methods and/or
devices for binding or detecting a microbe, e.g., cell or virus.
Methods and systems for detection can include methods and systems
for clinical chemistry, environmental analysis, and diagnostic
assays of all types. For example, the artificial receptor can be
contacted with a sample including or suspected of including at
least one microbe, e.g., cell or virus. The building blocks making
up the artificial receptors can be naive to the test ligand. Then,
binding of one or more of the microbes to the artificial receptors
can be detected. Next, the binding results can be interpreted to
provide information about the sample. In an embodiment, the
invention includes a method for detecting a microbe, e.g., cell or
virus, in a sample including contacting an artificial receptor
specific to the microbe, e.g., cell or virus, with a sample
suspected of containing the microbe, e.g., cell or virus. The
method can also include detecting or quantitating binding of the
microbe, e.g., cell or virus, to the artificial receptor.
[0277] FIG. 14 schematically illustrates an embodiment of a method
for evaluating candidate artificial receptors for binding to a test
ligand. This embodiment of the present method can be employed for
detecting a test ligand such as a microbe, e.g., cell or virus. The
method can include making an array of candidate artificial
receptors. The building blocks making up the artificial receptors
can be naive to the test ligand. Working artificial receptors can
be identified by contacting the array with a microbe, e.g., cell or
virus, and identifying which receptors bind the microbe. The method
can include producing an array or device including the working
artificial receptor or receptor complex. In an embodiment, the
method can include employing the array or device for detecting or
characterizing the microbe, e.g., cell or virus, in a sample, such
as a biological, laboratory, or environmental sample.
[0278] FIG. 15 schematically illustrates an embodiment of the
present method employing an array of candidate artificial
receptors. This embodiment of the method can employ an array
including a significant number of the present artificial receptors
to produce an assay or system for characterizing or detecting a
microbe, e.g., cell or virus. The method can include evaluating an
array including a significant number of candidate artificial
receptors for binding to a microbe, e.g., cell or virus. The
building blocks making up the artificial receptors can be naive to
the test ligand. The microbe can exhibit characteristic binding to
one or several of the candidate artificial receptors from that
array. The one or several artificial receptors can be selected as
an artificial receptor (e.g., a working artificial receptor or a
working artificial receptor complex) that can be employed in
methods for characterizing a biological sample, or characterizing
or detecting the microbe, e.g., cell or virus.
[0279] FIG. 17 schematically illustrates an embodiment of a method
for developing a method and system for detecting a test ligand,
such as a disease causing organism. This embodiment of the present
method includes evaluating a plurality (e.g. array) of candidate
artificial receptors for binding to each of a plurality of test
ligands, such as disease causing organisms. The building blocks
making up the artificial receptors can be naive to the test
ligands. The method can include detecting binding of each test
ligand (e.g., disease causing organism) to a subset of the
plurality or array of candidate artificial receptors. This can be
envisioned as developing a working artificial receptor or
artificial receptor complex for each of the plurality of test
ligands.
[0280] Thus, each test ligand (e.g., disease causing organism) can
provide a pattern of bound receptors in the plurality or array. The
pattern of bound receptors can be characteristic of the test ligand
or a sample including the test ligand. The method can include
storing a representation of the binding pattern as an image or a
data structure. The representation of the binding pattern can be
evaluated either by an operator or data processing system. The
method can include such evaluating. A binding pattern from an
unknown sample that matches the binding pattern for a particular
test ligand (e.g., disease causing organism) then characterizes the
unknown sample as containing that test ligand. Similarly, a binding
pattern from an unknown sample can be evaluated against the
patterns of a plurality of particular test ligands and the sample
can be characterized as containing one or more of the test ligands.
A plurality of binding patterns can be stored as a database.
[0281] An embodiment of the illustrated method can include creating
an array of artificial receptors. This embodiment can also include
compiling a database of the binding patterns of specific disease
causing organisms, for example, by probing the array with a
plurality of individual organisms. Contacting the array with an
unidentified organism can create a test binding pattern. The method
can then compare the test binding pattern with the binding patterns
of known organisms in the database in order to characterize or
classify the unidentified organism. In an embodiment, the database
and the array of receptors has already been constructed and the
method involves probing the array with an unknown organism to
create a test binding pattern and then comparing this binding
pattern with the binding patterns in the database in order to
characterize or classify the unidentified organism.
[0282] In an embodiment, the method can include producing or
employing the selected working artificial receptor or receptor
complex on a substrate. The substrate can include working
artificial receptors for a single microbe, e.g., cell or virus, or
working artificial receptors for a plurality of microbes, e.g.,
cells or viruses. For example, a method can include contacting the
artificial receptors with a sample. A substrate including working
artificial receptors for a single microbe, e.g., cell or virus, can
be employed in a method or system for detecting that microbe.
Binding to the working artificial receptors indicates that the
sample includes the microbe. A substrate including working
artificial receptors for a plurality of microbes, e.g., cells or
viruses can be employed in a method or system for detecting one,
several, or all of the microbes. Binding to the working artificial
receptors for a particular microbe or microbes indicates that the
sample includes such a microbe or microbes.
[0283] The working artificial receptors or receptor complexes can
be configured to provide a pattern indicative of the presence of
one or more of the microbes, e.g., cells or viruses. The method can
include detecting the binding pattern of the sample and comparing
it with binding patterns from known samples. FIG. 16 schematically
illustrates binding patterns on an array of working artificial
receptors. Such patterns and schemes can be employed for
identifying a variety of test ligands including microbes.
[0284] The present method can develop or employ a plurality of
working receptors specific for a particular microbe or feature on
the microbe. That is, the working receptors can be specific for a
particular microbe, but different receptors can interact with
different distinct antigens (e.g., proteins or carbohydrates),
ligands, or features of the microbe. Such a method can provide a
robust test for the presence of a microbe. For example, such a
robust test can reduce the chances of a false-positive or
false-negative result in comparison with an assay that relies upon
a single unique receptor to detect a given microbe. Further, this
embodiment of the method can develop or employ working receptors
that demonstrate higher binding affinity due to interaction with
multiple antigens or ligands on the same microbe (e.g., multivalent
binding).
[0285] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a bacterium. The
method can include evaluating an array including a significant
number of candidate artificial receptors for binding to the
bacterium. The building blocks making up the artificial receptors
can be naive to the test ligand. The bacterium can exhibit
characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a
biological sample or characterizing or detecting the bacterium.
[0286] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a virus
particle. The method can include evaluating an array including a
significant number of candidate artificial receptors for binding to
the virus particle. The building blocks making up the artificial
receptors can be naive to the test ligand. The virus particle can
exhibit characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a
biological sample or for characterizing or detecting the virus
particle.
[0287] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a biohazard. The
method can include evaluating an array including a significant
number of candidate artificial receptors for binding to the
biohazard. The building blocks making up the artificial receptors
can be naive to the test ligand. The biohazard can exhibit
characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a
biological sample, or characterizing or detecting the
biohazard.
[0288] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting the Vibrio
cholerae. The method can include evaluating an array including a
significant number of candidate artificial receptors for binding to
V. cholerae. The building blocks making up the artificial receptors
can be naive to the V. cholerae. The V. cholerae can exhibit
characteristic binding to one or several of the candidate
artificial receptors from that array. The one or several artificial
receptors can be selected as an artificial receptor (e.g., a
working artificial receptor or a working artificial receptor
complex) that can be employed in methods for characterizing a
biological sample, or characterizing or detecting V. cholerae.
[0289] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a microbe. The
method can include evaluating an array including a significant
number of candidate artificial receptors for binding to the
microbe. The building blocks making up the artificial receptors can
be naive to the test ligand. One or more artificial receptors that
bind the microbe under appropriate conditions can be selected for
use on an affinity support that can bind that microbe. One or more
artificial receptors that bind the microbe or cell sufficiently
tightly under appropriate conditions can be selected and its
building blocks incorporated onto a scaffold molecule. An
embodiment of the method can employ a support with the one or more
artificial receptors or scaffold-receptors on its surface for
binding or immobilizing the microbe. A support with a plurality of
artificial receptors, each binding to a different portion of the
microbe, on its surface can be employed for multivalent capture or
immobilization of the microbe.
[0290] The present method can include selecting artificial
receptors that bind a particular microbe and/or the building blocks
making up these receptors (e.g., bound to a scaffold molecule) as
leads for pharmaceutical development or as active agents for
modulating an activity of the microbe or as an antibiotic against
that microbe.
[0291] In an embodiment, the method can employ an array including a
significant number of the present artificial receptors to produce
an assay or system for characterizing or detecting a microbe of
clinical or environmental interest. The method can include
evaluating an array including a significant number of candidate
artificial receptors for binding to the microbe of clinical or
environmental interest. The building blocks making up the
artificial receptors can be naive to the test ligand. The microbe
of clinical or environmental interest can exhibit characteristic
binding to one or several of the candidate artificial receptors
from that array. The one or several artificial receptors can be
selected as an artificial receptor (e.g., a working artificial
receptor or a working artificial receptor complex) that can be
employed in methods for characterizing a biological sample, or
characterizing or detecting the microbe of clinical or
environmental interest.
[0292] Suitable microbes of clinical or environmental interest
include bacteria, mycoplasma, fungus, rickettsia, or virus.
Suitable bacteria or mycoplasma of clinical or environmental
interest include Escherichia coli (e.g., E. coli H157:07), Vibrio
cholerae, Acinetobacter caicoaceticus, Haemophilus influenzae,
Actinobacillus actinoides, Haemophilus parahaemolyticus,
Actinobacillus lignieresii, Haemophilus parainfluenzae,
Actinobacillus suis, Legionella pneumophila, Actinomyces bovis,
Leptospira interrogans, Actinomyces israelli, Mima polymorphs,
Aeromonas hydrophile, Moraxella lacunata, Arachnia propionica,
Burkholderia mallei, Burkholderia pseudomallei, Moraxella
osioensis, Arizona hinshawii, Mycobacterium osioensis, Bacillus
cereus, Mycobacterium leprae, Bacteroides spp, Mycobacterium spp,
Bartonella bacilliformis, Plesiomonas shigelloides, Bordetella
bronchiseptica, Proteus spp, Clostridium difficile, Pseudomonas
aeruginosa, Clostridium sordellii, Salmonella cholerasuis,
Clostridium tetani, Salmonella enteritidis, Corynebacterium
diphtheriae, Salmonella typhi, Edwardsiella tarda, Serratia
marcescens, Enterobacter aerogenes, Shigella spp, Staphylococcus
epidermidis, Francisella novicida, Vibrio parahaemolyticus,
Haemophilus ducreyi, Haemophilus gallinarum, Haemophilus
haemolyticus, Bacillus anthracis, Mycobacterium bovis, Bordetella
pertussis, Mycobacterium tuberculosis, Borrella burgdorfii,
Mycoplasma pneumoniae, Borrella spp, Neisseria gonorrhoeae,
Campylobacter, Neisseria meningitides, Chlamydia psittaci, Nocardia
asteroids, Chlamydia trachomatis, Nocardia brasillensis,
Clostridium botulinum, Pasteurella haemolytica, Clostridium
chauvoei, Pasteurelia multocida, Clostridium haemolyticus,
Pasteurella pneumotropica, Clostridium histolyticum, Pseudomonas
pseudomallei, Clostridium novyl, Staphylococcus aureus, Clostridium
perfringens, Streptobacillus moniliformis, Clostridium septicum,
Cyclospora cayatanensis, Streptococcus agalacetiae, Erysipelothrix
insidiosa, Streptococcus pneumoniae, Klebsiella pneumoniae,
Streptococcus pyogenes, Listeria manocytogenes, Yersinia pestis,
Yersinia pseudotuberculosis, Yersinia enterocolitica, Brucella
abortus, Brucella canis, Brucella melitensis, Brucella suis, and
Francisella tularensis.
[0293] Suitable fungus include Absidia, Piedraia hortae,
Aspergillus, Prototheca, Candida, Paecilomyces, Cryptococcus
neoformans, Cryptosporidium parvum, Phialaphora, Dermatophilus
congolensis, Rhizopus, Epidermophyton, Scopulariopsis, Exophiala,
Sporothrix schenkii, Fusarium, Trichophyton, Madurella mycetomi,
Toxoplasma, Trichosporon, Microsporum, Microsporidia, Wangiella
dermatitidis, Mucor, Blastomyces dermatitidis, Giardia lamblia,
Entamoeba histolytica, Coccidioides immitis, and Histoplasma
capsulatum.
[0294] Suitable rickettsia or viruses of clinical or environmental
interest include Coronaviruses, Hepatitis viruses, Hepatitis A
virus, Myxo-Paramyxoviruses (Influenza viruses, Measles virus,
Mumps virus, Newcastle disease virus), Picornavirus (Coxsackie
viruses, Echoviruses, Poliomyelitis virus), Rickettsia akari,
Rochalimaea Quintana, Rochalimaea vinsonii, Norwalk Agent,
Adenoviruses, Arenaviruses (Lymphocytic choriomenigitis,
Viscerotrophic strains), Herpesvirus Group (Herpesvirus hominis,
Cytomegalovirus, Epstein-Barr virus, Caliciviruses, Pseudo-rabies
virus, Varicella virus), Human Immunodeficiency Virus,
Parainfluenza viruses (Respiratory syncytial virus, Subsclerosing
panencephalitis virus), Picornaviruses (Poliomyelitis virus),
Poxviruses Variola, Cowpox virus (Molluscum contagiosum virus,
Monkeypox virus, Orf virus, Paravaccinia virus, Tanapox virus,
Vaccinia virus, Yabapox virus), Papovaviruses (SV 40 virus,
B-K-virus), Spongiform Encephalopathy Viruses (Creutzfeld-Jacob
agent, Kuru agent, BSE), Rhabdoviruses (Rabies virus), Tobaviruses
(Rubella virus), Coxiella burnetii, Rickettsia canada, Rickettsia
prowazekii, Rickettsia rickettsii, Rickettsia Tsutsugamushi,
Rickettsia typhi (R. mooseri), Spotted Fever Group Agents,
Vesicular Stomatis Virus (VSV), and Toga, Arena (e.g., LCM, Junin,
Lassa, Marchupo, Guanarito, etc.), Bunya (e.g., hantavirus, Rift
Valley Fever, etc.), Flaviruses (Dengue), and Filoviruses (e.g.,
Ebola, Marburg, etc.) of all types, Nipah virus, viral encephalitis
agents, LaCrosse, Kyasanur Forest virus, Yellow fever, and West
Nile virus.
[0295] Suitable microbes of clinical or environmental interest
include Variola Viruses, Congo-Crimean hemorrhagic fever,
Tick-borne encephalitis virus complex (Absettarov, Hanzalova, Hypr,
Kumlinge, Kyasanur Forest disease, Omsk hemorrhagic fever, and
Russian Spring-Summer Encephalitis), Marburg, Ebola, Junin, Lassa,
Machupo, Herpesvirus simiae, Bluetongue, Louping III, Rift Valley
fever (Zing a), Wesselsbron, Foot and Mouth Disease, Newcastle
Disease, African Swine Fever, Vesicular exanthema, Swine vesicular
disease, Rinderpest, African horse sickness, Avian influenza, and
Sheep pox. Other components of interest include Ricinus
communis.
Methods for Making and Using Affinity Supports
[0296] In an embodiment, a working artificial receptor or receptor
complex can be employed to produce or as an affinity support for
any of the test ligands described herein. For example, the present
method can include a method for producing an affinity support for a
test ligand. This method can include selecting a working artificial
receptor or receptor complex that binds to the test ligand. This
method can also include coupling the working artificial receptor or
receptor complex to a support. FIG. 20 schematically illustrates an
embodiment of such a method. The support can be suitable for use as
an affinity support for, for example, chromatography, membrane
filtration, electrophoresis (e.g., 1 or 2 dimensional
electrophoresis), or the like.
[0297] The present method can include selecting artificial
receptors that bind a particular test ligand and/or the building
blocks making up these receptors (e.g., bound to a scaffold
molecule) for isolation or analysis of a particular test ligand.
The building blocks making up the artificial receptors can be naive
to the test ligand. For example, the artificial receptor can be
employed as a receptor surface that can bind the test ligand and
remove (e.g., purify) it from a mixture or biological sample.
[0298] Such a method can include contacting one or more candidate
artificial receptors with a test ligand of interest. The building
blocks making up the artificial receptors can be naive to the test
ligand. The method can include selecting one or more of the
candidate artificial receptors that bind the test ligand as working
artificial receptor(s). The method can then include employing the
working artificial receptor(s) to make a receptor surface. Making a
receptor surface can include coupling the building blocks making up
the working artificial receptor(s) to a support. The support can
have sufficient area to bind a significant quantity of the test
ligand of interest. The support can be a chromatography support or
medium. The support can be a plate, tube, or membrane. In an
embodiment, binding of the test ligand of interest to the support
can be followed by eluting the test ligand of interest from the
support. Eluting can employ a wash with a pH, buffer, solvent, salt
concentration, or ligand concentration effective to elute the test
ligand of interest from the support.
[0299] FIG. 21 schematically illustrates evaluating an array of
candidate artificial receptors for binding of a test ligand and
selecting one or more working artificial receptors. The building
blocks making up the artificial receptors can be naive to the test
ligand. FIG. 21 illustrates that a receptor surface employing such
a working artificial receptor can be employed for binding a
protein, immobilizing an antibody, binding a single enantiomer, or
protecting a structural feature (e.g., a functional group) on a
compound. In an embodiment, the receptor surface can bind more than
one structural feature on the protein. In an embodiment, the
working artificial receptor can be selected to bind the constant
portion, rather than the variable portions, of an antibody. In an
embodiment, the receptor surface can include a catalytic moiety
that can catalyze a reaction of a functional group of the bound
test ligand. Such a catalytic moiety can be a building block, for
example, an organometallic building block.
[0300] The present method can include selecting artificial
receptors that bind a particular isomer of a compound and/or the
building blocks making up these receptors (e.g., bound to a
scaffold molecule) for isolation or analysis of a particular
isomer. For example, the artificial receptor can be employed as a
receptor surface that can bind the isomer and remove (e.g., purify)
it from a mixture or biological sample.
[0301] Such a method can include contacting one or more candidate
artificial receptors with an isomer of interest. The building
blocks making up the artificial receptors can be naive to the test
ligand. The method can include selecting one or more of the
candidate artificial receptors that bind the isomer as working
artificial receptor(s). The method can then include employing the
working artificial receptor(s) to make a receptor surface. Making a
receptor surface can include coupling the building blocks making up
the working artificial receptor(s) to a support. The support can
have sufficient area to bind a significant quantity of the isomer
of interest. The support can be a chromatography support or medium.
The support can be a plate, tube, or membrane. In an embodiment,
binding of the isomer of interest to the support can be followed by
eluting the isomer of interest from the support. Eluting can employ
a wash with a pH, buffer, solvent, salt concentration, or ligand
concentration effective to elute the isomer of interest from the
support.
[0302] The present method can include selecting artificial
receptors that bind or protect a particular structural feature of a
compound and/or the building blocks making up these receptors
(e.g., bound to a scaffold molecule) for isolation or analysis of
the compound including the structural feature. For example, the
artificial receptor can be employed as a receptor surface that can
bind or protect the structural feature of the compound. Binding of
the structural feature can be determined by lack of binding of an
analogous compound the lacking the structural feature. Protection
of the structural feature can be evaluated by that structural
feature being unavailable to a, for example, solution phase
reactive species when the compound is bound to the receptor
surface.
[0303] Such a method can include contacting one or more candidate
artificial receptors with a compound of interest. The building
blocks making up the artificial receptors can be naive to the test
ligand. The method can include selecting one or more of the
candidate artificial receptors that bind the structural feature of
the compound as lead artificial receptor(s). The lead artificial
receptor can be evaluated for protecting the structural feature.
The method can then include employing the working artificial
receptor(s) to make a receptor surface. Making a receptor surface
can include coupling the building blocks making up the working
artificial receptor(s) to a support. The support can have
sufficient area to bind a significant quantity of the compound of
interest. In an embodiment, binding of the compound of interest to
the support can be followed by reacting a portion of the compound
that is not the bound or protected structure feature.
[0304] The present method can include selecting artificial
receptors that bind a particular peptide or protein and/or the
building blocks making up these receptors (e.g., bound to a
scaffold molecule) for isolation or analysis of a particular
peptide or protein. For example, the artificial receptor can be
employed as a receptor surface that can bind the peptide or protein
and remove (e.g., purify) it from a mixture or biological
sample.
[0305] Such a method can include contacting one or more candidate
artificial receptors with a peptide or protein of interest. The
building blocks making up the artificial receptors can be naive to
the test ligand. The method can include selecting one or more of
the candidate artificial receptors that bind the peptide or protein
as working artificial receptor(s). The method can then include
employing the working artificial receptor(s) to make a receptor
surface. Making a receptor surface can include coupling the
building blocks making up the working artificial receptor(s) to a
support. The support can have sufficient area to bind a significant
quantity of the peptide or protein of interest. The support can be
a chromatography support or medium. The support can be a plate,
tube, or membrane. In an embodiment, binding of the peptide or
protein of interest to the support can be followed by eluting the
peptide or protein of interest from the support. Eluting can employ
a wash with a pH, buffer, salt concentration, or ligand
concentration effective to elute the peptide or protein of interest
from the support.
[0306] In an embodiment, the present artificial receptors can be
employed to form selective membranes. Such a selective membrane can
be based on a molecular gate including an artificial receptor
surface. For example, an artificial receptor surface can line the
walls of pores in the membrane and either allow or block a target
molecule from passing through the pores. For example, an artificial
receptor surface can line the walls of pores in the membrane and
act as "gatekeepers" on e.g. micro cantilevers/molecular
cantilevers to allow gate opening or closing on binding of the
target. The artificial receptors to be used in the selective
membranes can be identified by exposing the target molecule to a
plurality of distinct artificial receptors and then determining
which ones it binds to. For example, the binding can be detected
through any of the techniques described herein, including
fluorescence.
[0307] In certain embodiments, the method can include producing one
or more receptor surfaces, each receptor surface including building
blocks from a working receptor for a particular test ligand. Such a
method can include employing the receptor surface for
chromatography of the test ligand. Chromatographing the test ligand
against a plurality of such receptor surfaces can rank the affinity
of the surfaces for the test ligand. Under a given set of
conditions, the receptor surface that retains the chromatographed
test ligand the longest exhibits the greatest affinity for the test
ligand. The method can include selecting the receptor surface with
suitable (e.g., the greatest) affinity for use as an affinity
support for the test ligand.
[0308] Any of a variety of supports can be employed as the affinity
support. In certain embodiments, the affinity support can be a
dish, a tube, a well, a bead, a chromatography support, a
microchannel, or the like. The artificial receptor affinity support
can be used in various applications, such as chromatography,
microchannel devices, as an immunoassay support, or the like. A
microchannel with the artificial receptor on its surface can be
employed as an analytical device. In an embodiment, the present
artificial receptors can be employed to form bioactive surfaces.
For example, receptor surfaces can be used to specifically bind
antibodies or enzymes.
Methods for Making and Using Reaction Supports
[0309] In an embodiment, a working artificial receptor or receptor
complex can be employed to produce or as a reaction support for any
of the test ligands described herein. For example, the present
method can include a method for producing a reaction support for at
least one test ligand. This method can include selecting a working
artificial receptor or receptor complex that binds to the test
ligand under conditions suitable for a desired reaction with that
test ligand. This method can also include coupling the working
artificial receptor or receptor complex to a support. FIG. 20
schematically illustrates an embodiment of such a method. The
support can be suitable for use as a reaction support for, for
example, oxidation, reduction, substitution, or displacement
reactions.
[0310] The present method can include selecting artificial
receptors that bind a particular test ligand and/or the building
blocks making up these receptors (e.g., bound to a scaffold
molecule) for reaction of a particular test ligand. For example,
the artificial receptor can be employed as a receptor surface that
can bind the test ligand and position it for reaction at a
particular prochiral group, functional group, or orientation.
[0311] Such a method can include contacting one or more candidate
artificial receptors with a test ligand of interest. The building
blocks making up the artificial receptors can be naive to the test
ligand. The method can include selecting one or more of the
candidate artificial receptors that bind the test ligand as working
artificial receptor(s). The method can then include employing the
working artificial receptor(s) to make a receptor surface. Making a
receptor surface can include coupling the building blocks making up
the working artificial receptor(s) to a support. The support can
have sufficient area to bind a desired quantity of the test ligand
of interest. The support can be a chromatography support or medium.
The support can be a plate, bead, tube, or membrane.
[0312] The method also includes contacting the support including
bound test ligand with a reactant for the desired reaction.
Suitable reactants include reducing agent, oxidizing agent,
nucleophile, electrophile, solvent (e.g., aqueous or organic
solvent), or the like. The method can include contacting with one
or more reactants and selecting reactant or reactants suitable for
participating in the desired reaction. This embodiment of the
method includes reacting the test ligand with the reactant. In an
embodiment, reacting can be followed by washing the reactant or
side products from the support. In an embodiment, reacting can be
followed by eluting the product (e.g., reacted test ligand) from
the support. Eluting can employ a wash with a pH, buffer, solvent,
salt concentration, or ligand concentration effective to elute the
product from the support.
[0313] FIG. 21 schematically illustrates evaluating an array of
candidate artificial receptors for binding of a test ligand and
selecting one or more working artificial receptors. The building
blocks making up the artificial receptors can be naive to the test
ligand. FIG. 21 illustrates that a receptor surface employing such
a working artificial receptor can be employed for binding a test
ligand. The test ligand can be bound in an orientation that leaves
a reactive moiety available for reaction with a reactant placed
into contact with the receptor surface. In an embodiment, the test
ligand can be bound in an orientation that occludes or protects a
second reactive moiety from reacting with the reactant. This
embodiment includes reacting the test ligand and release of the
reacted test ligand from the receptor surface. Specifically, the
illustration shows the reduction of an aldehyde with sodium
borohydride to produce an alcohol. In an embodiment, the receptor
surface can include a catalytic moiety that can catalyze a reaction
of a functional group of the bound to test ligand the catalytic
reaction can also employ the reactant. Such a catalytic moiety can
be a building block, for example, an organometallic building
block.
[0314] The present method can include selecting artificial
receptors that bind or protect a particular structural feature of a
compound and/or the building blocks making up these receptors
(e.g., bound to a scaffold molecule) for reacting the compound
including the structural feature. For example, the artificial
receptor can be employed as a receptor surface that can bind and
protect the structural feature of the compound while another
feature of the compound reacts with a reactant. Protection of the
structural feature can be evaluated by that structural feature not
reacting with the reactant. For example, a substrate (e.g. a
steroid) can be stereospecifically bound to the artificial receptor
and present a particular moiety/sub-structure/"face" for reaction
with a reagent in solution.
[0315] In an embodiment, a first side of a molecule (or a
functional group) is bound to a receptor surface while a second
side is left exposed. Then a reagent is added that could react with
either side (or group) but is hindered from reacting with the first
side of the molecule since it is bound to the receptor surface,
accordingly the reagent reacts with the second side of the molecule
only.
[0316] Such a method can include contacting one or more candidate
artificial receptors with a compound of interest. The method can
include selecting one or more of the candidate artificial receptors
that bind the structural feature of the compound as lead artificial
receptor(s). The lead artificial receptor can be evaluated for
protecting the structural feature. The method can then include
employing the working artificial receptor(s) to make a receptor
surface. Making a receptor surface can include coupling the
building blocks making up the working artificial receptor(s) to a
support. The support can have sufficient area to bind a desired
quantity of the compound of interest. In an embodiment, binding of
the compound of interest to the support can be followed by reacting
a portion of the compound that is not the bound or protected
structure feature.
[0317] Conventional synthesis of a chiral compound generally
requires complicated procedures. In an embodiment, the present
candidate artificial receptors can be employed to find receptor
surfaces that provide a spatially oriented binding surface for a
stereospecific reaction. For example, an artificial receptor
surface can bind a small molecule so that particular functional
groups are exposed to the environment, and others are obscured by
the receptor. In this manner, the stereospecificity of the reaction
can be controlled. Therefore, an artificial receptor surface can be
employed in synthesis including chiral induction. Similarly,
regiospecificity can also be controlled using receptors of the
present invention.
[0318] The present method can include selecting artificial
receptors that bind a first reaction ligand and a second reaction
ligand or the building blocks making up these receptors (e.g.,
bound to a scaffold molecule) for a reaction including the first
and second reaction ligands. For example, the artificial receptor
can be employed as a receptor surface that can bind the first
reaction ligand and the second reaction ligand at a distance or
orientation at which these ligands can react with one another. The
reaction can optionally include one or more reactants not bound to
the receptor surface.
[0319] Such a method can include contacting one or more candidate
artificial receptors with a first reaction ligand and a second
reaction ligand. The building blocks making up the artificial
receptors can be naive to the ligands. The method can include
selecting one or more of the candidate artificial receptors that
bind both of the reaction ligands as working artificial
receptor(s). The method can then include employing the working
artificial receptor(s) to make a receptor surface. Making a
receptor surface can include coupling the building blocks making up
the working artificial receptor(s) to a support. The support can
have sufficient area to bind a desired quantity of the first and
second reaction ligands. The support can be a chromatography
support or medium. The support can be a plate, bead, tube, or
membrane. The first and second reaction ligands can be bound to the
support at one or several molar ratios, the reaction evaluated, and
a molar ratio selected for conducting the reaction.
[0320] The method can also include contacting the support including
bound reaction ligands with a reactant for the desired reaction.
Suitable reactants include reducing agent, oxidizing agent,
nucleophile, electrophile, or the like. This embodiment of the
method includes reacting the test ligand with the reactant. In an
embodiment, reacting can be followed by washing the reactant or
side products from the support. In an embodiment, the first and
second reaction ligands react without the reactant. In an
embodiment, reacting can be followed by eluting the product (e.g.,
reacted test ligand) from the support. Eluting can employ a wash
with a pH, buffer, solvent, salt concentration, or ligand
concentration effective to elute the product from the support.
[0321] In an embodiment, the present candidate artificial receptors
can be employed to find receptor surfaces that provide a spatially
oriented binding surface for a stereospecific reaction. For
example, an artificial receptor surface can bind a small molecule
with particular functional groups exposed to the environment, and
others obscured by the receptor. Such an artificial receptor
surface can be employed in synthesis including chiral induction.
For example, a substrate (e.g. a steroid) can be stereospecifically
bound to the artificial receptor and present a particular
moiety/sub-structure/"face" for reaction with a reagent in
solution. Similarly, the artificial receptor surface can act as a
protecting group where a reactive moiety of a molecule is
"protected" by binding to the receptor surface so that a different
moiety with similar reactivity can be transformed.
[0322] In an embodiment, the one or more working artificial
receptors that bind a plurality (e.g., 2) of the reactants can be
produced on a substrate and the reactants bound. Each receptor with
a plurality of bound reactants can then be screened against one or
more reagents or conditions (e.g., various molar ratios of the
reactants or various solvents). The artificial receptor allowing or
promoting reaction between the two or more reactants can be
identified. The artificial receptor can then be produced on a
substrate to provide a reactor for the reaction of interest.
[0323] Any of a variety of supports can be employed as the reaction
support. In certain embodiments, the reaction support can be a
dish, a tube, a well, a bead, a chromatography support, a
microchannel, or the like. The artificial receptor reaction support
can be used in various applications, such as a microchannel device.
A microchannel with the artificial receptor on its surface can be
employed as a reactor.
Methods of Making an Artificial Receptor
[0324] The present invention relates to a method of making an
artificial receptor or a candidate artificial receptor. In an
embodiment, this method includes preparing a spot or region on a
support, the spot or region including a plurality of building
blocks immobilized on the support. The method can include forming a
plurality of spots on a solid support, each spot including a
plurality of building blocks, and immobilizing (e.g., reversibly) a
plurality of building blocks on the solid support in each spot. In
an embodiment, an array of such spots is referred to as a
heterogeneous building block array.
[0325] The method can include mixing a plurality of building blocks
and employing the mixture in forming the spot(s). Alternatively,
the method can include spotting individual building blocks on the
support. Coupling building blocks to the support can employ
covalent bonding or noncovalent interactions. Suitable noncovalent
interactions include interactions between ions, hydrogen bonding,
van der Waals interactions, and the like. In an embodiment, the
support can be functionalized with moieties that can engage in
covalent bonding or noncovalent interactions. Forming spots can
yield a microarray of spots of heterogeneous combinations of
building blocks, each of which can be a candidate artificial
receptor. The method can apply or spot building blocks onto a
support in combinations of 2, 3, 4, or more building blocks.
[0326] In an embodiment, the present method can be employed to
produce a solid support having on its surface a plurality of
regions or spots, each region or spot including a plurality of
building blocks. For example, the method can include spotting a
glass slide with a plurality of spots, each spot including a
plurality of building blocks. Such a spot can be referred to as
including heterogeneous building blocks. A plurality of spots of
building blocks can be referred to as an array of spots.
[0327] In an embodiment, the present method includes making a
receptor surface. Making a receptor surface can include forming a
region on a solid support, the region including a plurality of
building blocks, and immobilizing (e.g., reversibly) the plurality
of building blocks to the solid support in the region. The method
can include mixing a plurality of building blocks and employing the
mixture in forming the region or regions. Alternatively, the method
can include applying individual building blocks in a region on the
support. Forming a region on a support can be accomplished, for
example, by soaking a portion of the support with the building
block solution. The resulting coating including building blocks can
be referred to as including heterogeneous building blocks.
[0328] A region including a plurality of building blocks can be
independent and distinct from other regions including a plurality
of building blocks. In an embodiment, one or more regions including
a plurality of building blocks can overlap to produce a region
including the combined pluralities of building blocks. In an
embodiment, two or more regions including a single building block
can overlap to form one or more regions each including a plurality
of building blocks. The overlapping regions can be envisioned, for
example, as portions of overlap in a Ven diagram, or as portions of
overlap in a pattern like a plaid or tweed.
[0329] In an embodiment, the method produces a spot or surface with
a density of building blocks sufficient to provide interactions of
more than one building block with a ligand. That is, the building
blocks can be in proximity to one another. Proximity of different
building blocks can be detected by determining different (e.g.,
greater) binding of a test ligand to a spot or surface including a
plurality of building blocks compared to a spot or surface
including only one of the building blocks.
[0330] In an embodiment, the method includes forming an array of
heterogeneous spots made from combinations of a subset of the total
building blocks and/or smaller groups of the building blocks in
each spot. That is, the method forms spots including only, for
example, 2 or 3 building blocks, rather than 4 or 5. For example,
the method can form spots from combinations of a full set of
building blocks (e.g. 81 of a set of 81) in groups of 2 and/or 3.
For example, the method can form spots from combinations of a
subset of the building blocks (e.g., 25 of the set of 81) in groups
of 4 or 5. For example, the method can form spots from combinations
of a subset of the building blocks (e.g., 25 of a set of 81) in
groups of 2 or 3. The method can include forming additional arrays
incorporating building blocks, lead artificial receptors, or
structurally similar building blocks.
[0331] In an embodiment, the method includes forming an array
including one or more spots that function as controls for
validating or evaluating binding to artificial receptors of the
present invention. In an embodiment, the method includes forming
one or more regions, tubes, or wells that function as controls for
validating or evaluating binding to artificial receptors of the
present invention. Such a control spot, region, tube, or well can
include no building block, only a single building block, only
functionalized lawn, or combinations thereof.
[0332] The method can immobilize (e.g., reversibly) building blocks
on supports using known methods for immobilizing compounds of the
types employed as building blocks. Coupling building blocks to the
support can employ covalent bonding or noncovalent interactions.
Suitable noncovalent interactions include interactions between
ions, hydrogen bonding, van der Waals interactions, and the like.
In an embodiment, the support can be functionalized with moieties
that can engage in reversible covalent bonding, moieties that can
engage in noncovalent interactions, a mixture of these moieties, or
the like.
[0333] In an embodiment, the support can be functionalized with
moieties that can engage in covalent bonding, e.g., reversible
covalent bonding. The present invention can employ any of a variety
of the numerous known functional groups, reagents, and reactions
for forming reversible covalent bonds. Suitable reagents for
forming reversible covalent bonds include those described in Green,
T W; Wuts, P G M (1999), Protective Groups in Organic Synthesis
Third Edition, Wiley-Interscience, New York, 779 pp. For example,
the support can include functional groups such as a carbonyl group,
a carboxyl group, a silane group, boric acid or ester, an amine
group (e.g., a primary, secondary, or tertiary amine, a
hydroxylamine, a hydrazine, or the like), a thiol group, an alcohol
group (e.g., primary, secondary, or tertiary alcohol), a diol group
(e.g., a 1,2 diol or a 1,3 diol), a phenol group, a catechol group,
or the like. These functional groups can form groups with
reversible covalent bonds, such as ether (e.g., alkyl ether, silyl
ether, thioether, or the like), ester (e.g., alkyl ester, phenol
ester, cyclic ester, thioester, or the like), acetal (e.g., cyclic
acetal), ketal (e.g., cyclic ketal), silyl derivative (e.g., silyl
ether), boronate (e.g., cyclic boronate), amide, hydrazide, imine,
carbamate, or the like. Such a functional group can be referred to
as a covalent bonding moiety, e.g., a first covalent bonding
moiety.
[0334] A carbonyl group on the support and an amine group on a
building block can form an imine or Schiff's base. The same is true
of an amine group on the support and a carbonyl group on a building
block. A carbonyl group on the support and an alcohol group on a
building block can form an acetal or ketal. The same is true of an
alcohol group on the support and a carbonyl group on a building
block. A thiol (e.g., a first thiol) on the support and a thiol
(e.g., a second thiol) on the building block can form a
disulfide.
[0335] A carboxyl group on the support and an alcohol group on a
building block can form an ester. The same is true of an alcohol
group on the support and a carboxyl group on a building block. Any
of a variety of alcohols and carboxylic acids can form esters that
provide covalent bonding that can be reversed in the context of the
present invention. For example, reversible ester linkages can be
formed from alcohols such as phenols with electron withdrawing
groups on the aryl ring, other alcohols with electron withdrawing
groups acting on the hydroxyl-bearing carbon, other alcohols, or
the like; and/or carboxyl groups such as those with electron
withdrawing groups acting on the acyl carbon (e.g., nitrobenzylic
acid, R--CF.sub.2--COOH, R--CCl.sub.2--COOH, and the like), other
carboxylic acids, or the like.
[0336] In an embodiment, the support, matrix, or lawn can be
functionalized with moieties that can engage in noncovalent
interactions. For example, the support can include functional
groups such as an ionic group, a group that can hydrogen bond, or a
group that can engage in van der Waals or other hydrophobic
interactions. Such functional groups can include cationic groups,
anionic groups, lipophilic groups, amphiphilic groups, and the
like.
[0337] In an embodiment, the support, matrix, or lawn includes a
charged moiety (e.g., a first charged moiety). Suitable charged
moieties include positively charged moieties and negatively charged
moieties. Suitable positively charged moieties (e.g., at neutral pH
in aqueous compositions) include amines, quaternary ammonium
moieties, ferrocene, or the like. Suitable negatively charged
moieties (e.g., at neutral pH in aqueous compositions) include
carboxylates, phenols substituted with strongly electron
withdrawing groups (e.g., tetrachlorophenols), phosphates,
phosphonates, phosphinates, sulphates, sulphonates,
thiocarboxylates, hydroxamic acids, or the like.
[0338] In an embodiment, the support, matrix, or lawn includes
groups that can hydrogen bond (e.g., a first hydrogen bonding
group), either as donors or acceptors. The support, matrix, or lawn
can include a surface or region with groups that can hydrogen bond.
For example, the support, matrix, or lawn can include a surface or
region including one or more carboxyl groups, amine groups,
hydroxyl groups, carbonyl groups, or the like. Ionic groups can
also participate in hydrogen bonding.
[0339] In an embodiment, the support, matrix, or lawn includes a
lipophilic moiety (e.g., a first lipophilic moiety). Suitable
lipophilic moieties include branched or straight chain C.sub.6-36
alkyl, C.sub.8-24 alkyl, C.sub.12-24 alkyl, C.sub.12-18 alkyl, or
the like; C.sub.6-36 alkenyl, C.sub.8-24 alkenyl, C.sub.12-24
alkenyl, C.sub.12-18 alkenyl, or the like, with, for example, 1 to
4 double bonds; C.sub.6-36 alkynyl, C.sub.8-24 alkynyl, C.sub.12-24
alkynyl, C.sub.12-18 alkynyl, or the like, with, for example, 1 to
4 triple bonds; chains with 1-4 double or triple bonds; chains
including aryl or substituted aryl moieties (e.g., phenyl or
naphthyl moieties at the end or middle of a chain); polyaromatic
hydrocarbon moieties; cycloalkane or substituted alkane moieties
with numbers of carbons as described for chains; combinations or
mixtures thereof; or the like. The alkyl, alkenyl, or alkynyl group
can include branching; within chain functionality like an ether
group; terminal functionality like alcohol, amide, carboxylate or
the like; or the like. A lipophilic moiety like a quaternary
ammonium lipophilic moiety can also include a positive charge.
Artificial Receptors
[0340] A candidate artificial receptor, a lead artificial receptor,
or a working artificial receptor includes combination of building
blocks immobilized (e.g., reversibly) on, for example, a support.
An individual artificial receptor can be a heterogeneous building
block spot on a slide or a plurality of building blocks coated on a
slide, tube, or well. The building blocks can be immobilized
through any of a variety of interactions, such as covalent,
electrostatic, or hydrophobic interactions. For example, the
building block and support or lawn can each include one or more
functional groups or moieties that can form covalent,
electrostatic, hydrogen bonding, van der Waals, or like
interactions.
[0341] An array of candidate artificial receptors can be a
commercial product sold to parties interested in using the
candidate artificial receptors as implements in developing
receptors for test ligands of interest. In an embodiment, a useful
array of candidate artificial receptors includes at least one glass
slide, the at least one glass slide including spots of a
predetermined number of combinations of members of a set of
building blocks, each combination including a predetermined number
of building blocks.
[0342] One or more lead artificial receptors can be developed from
a plurality of candidate artificial receptors. In an embodiment, a
lead artificial receptor includes a combination of building blocks
and binds detectable quantities of test ligand upon exposure to,
for example, several picomoles of test ligand at a concentration of
1, 0.1, or 0.01 .mu.g/ml, or at 1, 0.1, or 0.01 ng/ml test ligand;
at a concentration of 0.01 .mu.g/ml, or at 1, 0.1, or 0.01 ng/ml
test ligand; or a concentration of 1, 0.1, or 0.01 ng/ml test
ligand.
[0343] Artificial receptors, particularly candidate or lead
artificial receptors, can be in the form of an array of artificial
receptors. Such an array can include, for example, 1.66 million
spots, each spot including one combination of 4 building blocks
from a set of 81 building blocks. Such an array can include, for
example, 28,000 spots, each spot including one combination of 2, 3,
or 4 building blocks from a set of 29 building blocks. Each spot is
a candidate artificial receptor and a combination of building
blocks. The array can also be constructed to include lead
artificial receptors. For example, the array of artificial
receptors can include combinations of fewer building blocks and/or
a subset of the building blocks.
[0344] In an embodiment, an array of candidate artificial receptors
includes building blocks of general Formula 2 (shown hereinbelow),
with RE.sub.1 being B1, B2, B3, B3a, B4, B5, B6, B7, B8, or B9
(shown hereinbelow) and with RE.sub.2 being A1, A2, A3, A3a, A4,
A5, A6, A7, A8, or A9 (shown hereinbelow). In an embodiment, the
framework is tyrosine.
[0345] One or more working artificial receptors can be developed
from one or more lead artificial receptors. In an embodiment, a
working artificial receptor includes a combination of building
blocks and binds categorizing or identifying quantities of test
ligand upon exposure to, for example, several picomoles of test
ligand at a concentration of 100, 10, 1, 0.1, 0.01, or 0.001 ng/ml
test ligand; at a concentration of 10, 1, 0.1, 0.01, or 0.001 ng/ml
test ligand; or a concentration of 1, 0.1, 0.01, or 0.001 ng/ml
test ligand.
[0346] In an embodiment, the artificial receptor of the invention
includes a plurality of building blocks coupled to a support. In an
embodiment, the plurality of building blocks can include or be
building blocks of Formula 2 (shown below). An abbreviation for the
building block including a linker, a tyrosine framework, and
recognition elements AxBy is TyrAxBy. In an embodiment, a candidate
artificial receptor can include combinations of building blocks of
formula TyrA1B1, TyrA2B2, TyrA2B4, TyrA2B6, TyrA2B8, TyrA3B3,
TyrA4B2, TyrA4B4, TyrA4B6, TyrA4B8, TyrA5B5, TyrA6B2, TyrA6B4,
TyrA6B6, TyrA6B8, TyrA7B7, TyrA8B2, TyrA8B4, TyrA8B6, or
TyrA8B8.
[0347] The present artificial receptors can employ any of a variety
of supports to which building blocks or other array materials can
be coupled. For example, the support can be glass or plastic; a
slide, a tube, or a well; an optical fiber; a nanotube or a
buckyball, a nanodevice; a dendrimer, or a scaffold; or the
like.
[0348] The present artificial receptors can include a signal
element that produces a detectable signal when a test ligand is
bound to the receptor. In an embodiment, the signal element can
produce an optical signal or a electrochemical signal. Suitable
optical signals include chemiluminescence or fluorescence. The
signal element can be a fluorescent moiety. The fluorescent
molecule can be one that is quenched by binding to the artificial
receptor. For example, the signal element can be a molecule that
fluoresces only when binding occurs. Suitable electrochemical
signal elements include those that give rise to current or a
potential. Suitable electrochemical signal elements include phenols
and anilines, such as those with substitutents oriented ortho or
para to one another, polynuclear aromatic hydrocarbons,
sulfide-disulfide, sulfide-sulfoxide-sulfone, polyenes,
polyeneynes, and the like. Suitable electrochemical signal elements
include quinones and ferrocenes.
Building Blocks
[0349] The present invention relates to building blocks for making
or forming candidate artificial receptors. Building blocks can be
designed, made, and selected to provide a variety of structural
characteristics among a small number of compounds. A building block
can provide one or more structural characteristics such as positive
charge, negative charge, acid, base, electron acceptor, electron
donor, hydrogen bond donor, hydrogen bond acceptor, free electron
pair, .pi. electrons, charge polarization, hydrophilicity,
hydrophobicity, and the like. A building block can be bulky or it
can be small.
[0350] A building block can be visualized as including several
components, such as one or more frameworks, one or more linkers,
and/or one or more recognition elements. The framework can be
covalently coupled to each of the other building block components.
The linker can be covalently coupled to the framework. The linker
can be coupled to a support through one or more of covalent,
electrostatic, hydrogen bonding, van der Waals, or like
interactions. The recognition element can be covalently coupled to
the framework. In an embodiment, a building block includes a
framework, a linker, and a recognition element. In an embodiment, a
building block includes a framework, a linker, and two recognition
elements.
[0351] A description of general and specific features and functions
of a variety of building blocks and their synthesis can be found in
copending U.S. patent application Ser. Nos. 10/244,727, filed Sep.
16, 2002, 10/813,568, filed Mar. 29, 2004, and Application No.
PCT/US03/05328, filed Feb. 19, 2003, each entitled "ARTIFICIAL
RECEPTORS, BUILDING BLOCKS, AND METHODS"; U.S. patent application
Ser. Nos. 10/812,850 and 10/813,612, and application No.
PCT/US2004/009649, each filed Mar. 29, 2004 and each entitled
"ARTIFICIAL RECEPTORS INCLUDING REVERSIBLY IMMOBILIZED BUILDING
BLOCKS, THE BUILDING BLOCKS, AND METHODS"; and U.S. Provisional
Patent Application Nos. 60/499,965, filed Sep. 3, 2003, and
60/526,699, filed Dec. 2, 2003, each entitled BUILDING BLOCKS FOR
ARTIFICIAL RECEPTORS; the disclosures of which are incorporated
herein by reference. These patent documents include, in particular,
a detailed written description of: function, structure, and
configuration of building blocks, framework moieties, recognition
elements, synthesis of building blocks, specific embodiments of
building blocks, specific embodiments of recognition elements, and
sets of building blocks.
Framework
[0352] The framework can be selected for functional groups that
provide for coupling to the recognition moiety and for coupling to
or being the linking moiety. The framework can interact with the
ligand as part of the artificial receptor. In an embodiment, the
framework includes multiple reaction sites with orthogonal and
reliable functional groups and with controlled stereochemistry.
Suitable functional groups with orthogonal and reliable chemistries
include, for example, carboxyl, amine, hydroxyl, phenol, carbonyl,
and thiol groups, which can be individually protected, deprotected,
and derivatized. In an embodiment, the framework has two, three, or
four functional groups with orthogonal and reliable chemistries. In
an embodiment, the framework has three functional groups. In such
an embodiment, the three functional groups can be independently
selected, for example, from carboxyl, amine, hydroxyl, phenol,
carbonyl, or thiol group. The framework can include alkyl,
substituted alkyl, cycloalkyl, heterocyclic, substituted
heterocyclic, aryl alkyl, aryl, heteroaryl, heteroaryl alkyl, and
like moieties.
[0353] A general structure for a framework with three functional
groups can be represented by Formula 1a:
##STR00001##
A general structure for a framework with four functional groups can
be represented by Formula 1b:
##STR00002##
In these general structures: R.sub.1 can be a 1-12, a 1-6, or a 1-4
carbon alkyl, substituted alkyl, cycloalkyl, heterocyclic,
substituted heterocyclic, aryl alkyl, aryl, heteroaryl, heteroaryl
alkyl, or like group; and F.sub.1, F.sub.2, F.sub.3, or F.sub.4 can
independently be a carboxyl, amine, hydroxyl, phenol, carbonyl, or
thiol group. F.sub.1, F.sub.2, F.sub.3, or F.sub.4 can
independently be a 1-12, a 1-6, a 1-4 carbon alkyl, substituted
alkyl, cycloalkyl, heterocyclic, substituted heterocyclic, aryl
alkyl, aryl, heteroaryl, heteroaryl alkyl, or inorganic group
substituted with carboxyl, amine, hydroxyl, phenol, carbonyl, or
thiol group. F.sub.3 and/or F.sub.4 can be absent.
[0354] A variety of compounds fit the formulas and text describing
the framework including amino acids, and naturally occurring or
synthetic compounds including, for example, oxygen and sulfur
functional groups. The compounds can be racemic, optically active,
or achiral. For example, the compounds can be natural or synthetic
amino acids, .alpha.-hydroxy acids, thioic acids, and the like.
[0355] Suitable molecules for use as a framework include a natural
or synthetic amino acid, particularly an amino acid with a
functional group (e.g., third functional group) on its side chain.
Amino acids include carboxyl and amine functional groups. The side
chain functional group can include, for natural amino acids, an
amine (e.g., alkyl amine, heteroaryl amine), hydroxyl, phenol,
carboxyl, thiol, thioether, or amidino group. Natural amino acids
suitable for use as frameworks include, for example, serine,
threonine, tyrosine, aspartic acid, glutamic acid, asparagine,
glutamine, cysteine, lysine, arginine, histidine. Synthetic amino
acids can include the naturally occurring side chain functional
groups or synthetic side chain functional groups which modify or
extend the natural amino acids with alkyl, substituted alkyl,
cycloalkyl, heterocyclic, substituted heterocyclic, aryl alkyl,
aryl, heteroaryl, heteroaryl alkyl, and like moieties as framework
and with carboxyl, amine, hydroxyl, phenol, carbonyl, or thiol
functional groups. Suitable synthetic amino acids include
.beta.-amino acids and homo or .beta. analogs of natural amino
acids. In an embodiment, the framework amino acid can be serine,
threonine, or tyrosine, e.g., serine or tyrosine, e.g.,
tyrosine.
[0356] Although not limiting to the present invention, a framework
amino acid, such as serine, threonine, or tyrosine, with a linker
and two recognition elements can be visualized with one of the
recognition elements in a pendant orientation and the other in an
equatorial orientation, relative to the extended carbon chain of
the framework.
[0357] All of the naturally occurring and many synthetic amino
acids are commercially available. Further, forms of these amino
acids derivatized or protected to be suitable for reactions for
coupling to recognition element(s) and/or linkers can be purchased
or made by known methods (see, e.g., Green, T W; Wuts, P G M
(1999), Protective Groups in Organic Synthesis Third Edition,
Wiley-Interscience, New York, 779 pp.; Bodanszky, M.; Bodanszky, A.
(1994), The Practice of Peptide Synthesis Second Edition,
Springer-Verlag, New York, 217 pp.).
Recognition Element
[0358] The recognition element can be selected to provide one or
more structural characteristics to the building block. The
recognition element can interact with the ligand as part of the
artificial receptor. For example, the recognition element can
provide one or more structural characteristics such as positive
charge, negative charge, acid, base, electron acceptor, electron
donor, hydrogen bond donor, hydrogen bond acceptor, free electron
pair, .pi. electrons, charge polarization, hydrophilicity,
hydrophobicity, and the like. A recognition element can be a small
group or it can be bulky.
[0359] In an embodiment the recognition element can be a 1-12, a
1-6, or a 1-4 carbon alkyl, substituted alkyl, cycloalkyl,
heterocyclic, substituted heterocyclic, aryl alkyl, aryl,
heteroaryl, heteroaryl alkyl, or like group. The recognition
element can be substituted with a group that includes or imparts
positive charge, negative charge, acid, base, electron acceptor,
electron donor, hydrogen bond donor, hydrogen bond acceptor, free
electron pair, .pi. electrons, charge polarization, hydrophilicity,
hydrophobicity, and the like.
[0360] Recognition elements with a positive charge (e.g., at
neutral pH in aqueous compositions) include amines, quaternary
ammonium moieties, sulfonium, phosphonium, ferrocene, and the like.
Suitable amines include alkyl amines, alkyl diamines, heteroalkyl
amines, aryl amines, heteroaryl amines, aryl alkyl amines,
pyridines, heterocyclic amines (saturated or unsaturated, the
nitrogen in the ring or not), amidines, hydrazines, and the like.
Alkyl amines generally have 1 to 12 carbons, e.g., 1-8, and rings
can have 3-12 carbons, e.g., 3-8. Suitable alkyl amines include
that of formula B9. Suitable heterocyclic or alkyl heterocyclic
amines include that of formula A9. Suitable pyridines include those
of formulas A5 and B5. Any of the amines can be employed as a
quaternary ammonium compound. Additional suitable quaternary
ammonium moieties include trimethyl alkyl quaternary ammonium
moieties, dimethyl ethyl alkyl quaternary ammonium moieties,
dimethyl alkyl quaternary ammonium moieties, aryl alkyl quaternary
ammonium moieties, pyridinium quaternary ammonium moieties, and the
like.
[0361] Recognition elements with a negative charge (e.g., at
neutral pH in aqueous compositions) include carboxylates, phenols
substituted with strongly electron withdrawing groups (e.g.,
substituted tetrachlorophenols), phosphates, phosphonates,
phosphinates, sulphates, sulphonates, thiocarboxylates, and
hydroxamic acids. Suitable carboxylates include alkyl carboxylates,
aryl carboxylates, and aryl alkyl carboxylates. Suitable phosphates
include phosphate mono-, di-, and tri-esters, and phosphate mono-,
di-, and tri-amides. Suitable phosphonates include phosphonate
mono- and di-esters, and phosphonate mono- and di-amides (e.g.,
phosphonamides). Suitable phosphinates include phosphinate esters
and amides.
[0362] Recognition elements with a negative charge and a positive
charge (at neutral pH in aqueous compositions) include sulfoxides,
betaines, and amine oxides.
[0363] Acidic recognition elements can include carboxylates,
phosphates, sulphates, and phenols. Suitable acidic carboxylates
include thiocarboxylates. Suitable acidic phosphates include the
phosphates listed hereinabove.
[0364] Basic recognition elements include amines. Suitable basic
amines include alkyl amines, aryl amines, aryl alkyl amines,
pyridines, heterocyclic amines (saturated or unsaturated, the
nitrogen in the ring or not), amidines, and any additional amines
listed hereinabove. Suitable alkyl amines include that of formula
B9. Suitable heterocyclic or alkyl heterocyclic amines include that
of formula A9. Suitable pyridines include those of formulas A5 and
B5.
[0365] Recognition elements including a hydrogen bond donor include
amines, amides, carboxyls, protonated phosphates, protonated
phosphonates, protonated phosphinates, protonated sulphates,
protonated sulphinates, alcohols, and thiols. Suitable amines
include alkyl amines, aryl amines, aryl alkyl amines, pyridines,
heterocyclic amines (saturated or unsaturated, the nitrogen in the
ring or not), amidines, ureas, and any other amines listed
hereinabove. Suitable alkyl amines include that of formula B9.
Suitable heterocyclic or alkyl heterocyclic amines include that of
formula A9. Suitable pyridines include those of formulas A5 and B5.
Suitable protonated carboxylates, protonated phosphates include
those listed hereinabove. Suitable amides include those of formulas
A8 and B8. Suitable alcohols include primary alcohols, secondary
alcohols, tertiary alcohols, and aromatic alcohols (e.g., phenols).
Suitable alcohols include those of formulas A7 (a primary alcohol)
and B7 (a secondary alcohol).
[0366] Recognition elements including a hydrogen bond acceptor or
one or more free electron pairs include amines, amides,
carboxylates, carboxyl groups, phosphates, phosphonates,
phosphinates, sulphates, sulphonates, alcohols, ethers, thiols, and
thioethers. Suitable amines include alkyl amines, aryl amines, aryl
alkyl amines, pyridines, heterocyclic amines (saturated or
unsaturated, the nitrogen in the ring or not), amidines, ureas, and
amines as listed hereinabove. Suitable alkyl amines include that of
formula B9. Suitable heterocyclic or alkyl heterocyclic amines
include that of formula A9. Suitable pyridines include those of
formulas A5 and B5. Suitable carboxylates include those listed
hereinabove. Suitable amides include those of formulas A8 and B8.
Suitable phosphates, phosphonates and phosphinates include those
listed hereinabove. Suitable alcohols include primary alcohols,
secondary alcohols, tertiary alcohols, aromatic alcohols, and those
listed hereinabove. Suitable alcohols include those of formulas A7
(a primary alcohol) and B7 (a secondary alcohol). Suitable ethers
include alkyl ethers, aryl alkyl ethers. Suitable alkyl ethers
include that of formula A6. Suitable aryl alkyl ethers include that
of formula A4. Suitable thioethers include that of formula B6.
[0367] Recognition elements including uncharged polar or
hydrophilic groups include amides, alcohols, ethers, thiols,
thioethers, esters, thio esters, boranes, borates, and metal
complexes. Suitable amides include those of formulas A8 and B8.
Suitable alcohols include primary alcohols, secondary alcohols,
tertiary alcohols, aromatic alcohols, and those listed hereinabove.
Suitable alcohols include those of formulas A7 (a primary alcohol)
and B7 (a secondary alcohol). Suitable ethers include those listed
hereinabove. Suitable ethers include that of formula A6. Suitable
aryl alkyl ethers include that of formula A4.
[0368] Recognition elements including uncharged hydrophobic groups
include alkyl (substituted and unsubstituted), alkene (conjugated
and unconjugated), alkyne (conjugated and unconjugated), aromatic.
Suitable alkyl groups include lower alkyl, substituted alkyl,
cycloalkyl, aryl alkyl, and heteroaryl alkyl. Suitable lower alkyl
groups include those of formulas A1, A3, A3a, and B1. Suitable aryl
alkyl groups include those of formulas A3, A3a, A4, B3, B3a, and
B4. Suitable alkyl cycloalkyl groups include that of formula B2.
Suitable alkene groups include lower alkene and aryl alkene.
Suitable aryl alkene groups include that of formula B4. Suitable
aromatic groups include unsubstituted aryl, heteroaryl, substituted
aryl, aryl alkyl, heteroaryl alkyl, alkyl substituted aryl, and
polyaromatic hydrocarbons. Suitable aryl alkyl groups include those
of formulas A3, A3a and B4. Suitable alkyl heteroaryl groups
include those of formulas A5 and B5.
[0369] Spacer (e.g., small) recognition elements include hydrogen,
methyl, ethyl, and the like. Bulky recognition elements include 7
or more carbon or hetero atoms.
[0370] Formulas A1-A9 and B1-B9 are:
##STR00003##
[0371] These A and B recognition elements can be called derivatives
of, according to a standard reference: A1, ethylamine; A2,
isobutylamine; A3, phenethylamine; A4, 4-methoxyphenethylamine;
A5,2-(2-aminoethyl)pyridine; A6,2-methoxyethylamine; A7,
ethanolamine; A8, N-acetylethylenediamine;
A9,1-(2-aminoethyl)pyrrolidine; B1, acetic acid, B2,
cyclopentylpropionic acid; B3,3-chlorophenylacetic acid; B4,
cinnamic acid; B5, 3-pyridinepropionic acid; B6, (methylthio)acetic
acid; B7,3-hydroxybutyric acid; B8, succinamic acid; and
B9,4-(dimethylamino)butyric acid.
[0372] In an embodiment, the recognition elements include one or
more of the structures represented by formulas A1, A2, A3, A3a, A4,
A5, A6, A7, A8, and/or A9 (the A recognition elements) and/or B1,
B2, B3, B3a, B4, B5, B6, B7, B8, and/or B9 (the B recognition
elements). In an embodiment, each building block includes an A
recognition element and a B recognition element. In an embodiment,
a group of 81 such building blocks includes each of the 81 unique
combinations of an A recognition element and a B recognition
element. In an embodiment, the A recognition elements are linked to
a framework at a pendant position. In an embodiment, the B
recognition elements are linked to a framework at an equatorial
position. In an embodiment, the A recognition elements are linked
to a framework at a pendant position and the B recognition elements
are linked to the framework at an equatorial position.
[0373] Although not limiting to the present invention, it is
believed that the A and B recognition elements represent the
assortment of functional groups and geometric configurations
employed by polypeptide receptors. Although not limiting to the
present invention, it is believed that the A recognition elements
represent six advantageous functional groups or configurations and
that the addition of functional groups to several of the aryl
groups increases the range of possible binding interactions.
Although not limiting to the present invention, it is believed that
the B recognition elements represent six advantageous functional
groups, but in different configurations than employed for the A
recognition elements. Although not limiting to the present
invention, it is further believed that this increases the range of
binding interactions and further extends the range of functional
groups and configurations that is explored by molecular
configurations of the building blocks.
[0374] In an embodiment, the building blocks including the A and B
recognition elements can be visualized as occupying a binding space
defined by lipophilicity/hydrophilicity and volume. A volume can be
calculated (using known methods) for each building block including
the various A and B recognition elements. A measure of
lipophilicity/hydrophilicity (logP) can be calculated (using known
methods) for each building block including the various A and B
recognition elements. Negative values of logP show affinity for
water over nonpolar organic solvent and indicate a hydrophilic
nature. A plot of volume versus logP can then show the distribution
of the building blocks through a binding space defined by size and
lipophilicity/hydrophilicity.
[0375] Reagents that form many of the recognition elements are
commercially available. For example, reagents for forming
recognition elements A1, A2, A3, A3a, A4, A5, A6, A7, A8, A9 B1,
B2, B3, B3a, B4, B5, B6, B7, B8, and B9 are commercially
available.
Linkers
[0376] The linker is selected to provide a suitable coupling of the
building block to a support. The framework can interact with the
ligand as part of the artificial receptor. The linker can also
provide bulk, distance from the support, hydrophobicity,
hydrophilicity, and like structural characteristics to the building
block. Coupling building blocks to the support can employ covalent
bonding or noncovalent interactions. Suitable noncovalent
interactions include interactions between ions, hydrogen bonding,
van der Waals interactions, and the like. In an embodiment, the
linker includes moieties that can engage in covalent bonding or
noncovalent interactions. In an embodiment, the linker includes
moieties that can engage in covalent bonding. Suitable groups for
forming covalent and reversible covalent bonds are described
hereinabove.
Linkers for Reversibly Immobilizable Building Blocks
[0377] The linker can be selected to provide suitable reversible
immobilization of the building block on a support or lawn. In an
embodiment, the linker forms a covalent bond with a functional
group on the framework. In an embodiment, the linker also includes
a functional group that can reversibly interact with the support or
lawn, e.g., through reversible covalent bonding or noncovalent
interactions.
[0378] In an embodiment, the linker includes one or more moieties
that can engage in reversible covalent bonding. Suitable groups for
reversible covalent bonding include those described hereinabove. An
artificial receptor can include building blocks reversibly
immobilized on the lawn or support through, for example, imine,
acetal, ketal, disulfide, ester, or like linkages. Such functional
groups can engage in reversible covalent bonding. Such a functional
group can be referred to as a covalent bonding moiety, e.g., a
second covalent bonding moiety.
[0379] In an embodiment, the linker can be functionalized with
moieties that can engage in noncovalent interactions. For example,
the linker can include functional groups such as an ionic group, a
group that can hydrogen bond, or a group that can engage in van der
Waals or other hydrophobic interactions. Such functional groups can
include cationic groups, anionic groups, lipophilic groups,
amphiphilic groups, and the like.
[0380] In an embodiment, the present methods and compositions can
employ a linker including a charged moiety (e.g., a second charged
moiety). Suitable charged moieties include positively charged
moieties and negatively charged moieties. Suitable positively
charged moieties include amines, quaternary ammonium moieties,
sulfonium, phosphonium, ferrocene, and the like. Suitable
negatively charged moieties (e.g., at neutral pH in aqueous
compositions) include carboxylates, phenols substituted with
strongly electron withdrawing groups (e.g., tetrachlorophenols),
phosphates, phosphonates, phosphinates, sulphates, sulphonates,
thiocarboxylates, and hydroxamic acids.
[0381] In an embodiment, the present methods and compositions can
employ a linker including a group that can hydrogen bond, either as
donor or acceptor (e.g., a second hydrogen bonding group). For
example, the linker can include one or more carboxyl groups, amine
groups, hydroxyl groups, carbonyl groups, or the like. Ionic groups
can also participate in hydrogen bonding.
[0382] In an embodiment, the present methods and compositions can
employ a linker including a lipophilic moiety (e.g., a second
lipophilic moiety). Suitable lipophilic moieties include one or
more branched or straight chain C.sub.6-36 alkyl, C.sub.8-24 alkyl,
C.sub.12-24 alkyl, C.sub.12-18 alkyl, or the like; C.sub.6-36
alkenyl, C.sub.8-24 alkenyl, C.sub.12-24 alkenyl, C.sub.12-18
alkenyl, or the like, with, for example, 1 to 4 double bonds;
C.sub.6-36 alkynyl, C.sub.8-24 alkynyl, C.sub.12-24 alkynyl,
C.sub.12-18 alkynyl, or the like, with, for example, 1 to 4 triple
bonds; chains with 1-4 double or triple bonds; chains including
aryl or substituted aryl moieties (e.g., phenyl or naphthyl
moieties at the end or middle of a chain); polyaromatic hydrocarbon
moieties; cycloalkane or substituted alkane moieties with numbers
of carbons as described for chains; combinations or mixtures
thereof; or the like. The alkyl, alkenyl, or alkynyl group can
include branching; within chain functionality like an ether group;
terminal functionality like alcohol, amide, carboxylate or the
like; or the like. In an embodiment the linker includes or is a
lipid, such as a phospholipid. In an embodiment, the lipophilic
moiety includes or is a 12-carbon aliphatic moiety.
[0383] In an embodiment, the linker includes a lipophilic moiety
(e.g., a second lipophilic moiety) and a covalent bonding moiety
(e.g., a second covalent bonding moiety). In an embodiment, the
linker includes a lipophilic moiety (e.g., a second lipophilic
moiety) and a charged moiety (e.g., a second charged moiety).
[0384] In an embodiment, the linker forms or can be visualized as
forming a covalent bond with an alcohol, phenol, thiol, amine,
carbonyl, or like group on the framework. Between the bond to the
framework and the group participating in or formed by the
reversible interaction with the support or lawn, the linker can
include an alkyl, substituted alkyl, cycloalkyl, heterocyclic,
substituted heterocyclic, aryl alkyl, aryl, heteroaryl, heteroaryl
alkyl, ethoxy or propoxy oligomer, a glycoside, or like moiety.
[0385] For example, suitable linkers can include: the functional
group participating in or formed by the bond to the framework, the
functional group or groups participating in or formed by the
reversible interaction with the support or lawn, and a linker
backbone moiety. The linker backbone moiety can include about 4 to
about 48 carbon or heteroatoms, about 8 to about 14 carbon or
heteroatoms, about 12 to about 24 carbon or heteroatoms, about 16
to about 18 carbon or heteroatoms, about 4 to about 12 carbon or
heteroatoms, about 4 to about 8 carbon or heteroatoms, or the like.
The linker backbone can include an alkyl, substituted alkyl,
cycloalkyl, heterocyclic, substituted heterocyclic, aryl alkyl,
aryl, heteroaryl, heteroaryl alkyl, ethoxy or propoxy oligomer, a
glycoside, mixtures thereof, or like moiety.
[0386] In an embodiment, the linker includes a lipophilic moiety,
the functional group participating in or formed by the bond to the
framework, and, optionally, one or more moieties for forming a
reversible covalent bond, a hydrogen bond, or an ionic interaction.
In such an embodiment, the lipophilic moiety can have about 4 to
about 48 carbons, about 8 to about 14 carbons, about 12 to about 24
carbons, about 16 to about 18 carbons, or the like. In such an
embodiment, the linker can include about 1 to about 8 reversible
bond/interaction moieties or about 2 to about 4 reversible
bond/interaction moieties. Suitable linkers have structures such as
(CH.sub.2).sub.1COOH, with n=12-24, n=17-24, or n=16-18.
Additional Embodiments of Linkers
[0387] The linker can be selected to provide a suitable covalent
coupling of the building block to a support. The framework can
interact with the ligand as part of the artificial receptor. The
linker can also provide bulk, distance from the support,
hydrophobicity, hydrophilicity, and like structural characteristics
to the building block. In an embodiment, the linker forms a
covalent bond with a functional group on the framework. In an
embodiment, before attachment to the support the linker also
includes a functional group that can be activated to react with or
that will react with a functional group on the support. In an
embodiment, once attached to the support, the linker forms a
covalent bond with the support and with the framework.
[0388] In an embodiment, the linker forms or can be visualized as
forming a covalent bond with an alcohol, phenol, thiol, amine,
carbonyl, or like group on the framework. The linker can include a
carboxyl, alcohol, phenol, thiol, amine, carbonyl, maleimide, or
like group that can react with or be activated to react with the
support. Between the bond to the framework and the group formed by
the attachment to the support, the linker can include an alkyl,
substituted alkyl, cycloalkyl, heterocyclic, substituted
heterocyclic, aryl alkyl, aryl, heteroaryl, heteroaryl alkyl,
ethoxy or propoxy oligomer, a glycoside, or like moiety.
[0389] The linker can include a good leaving group bonded to, for
example, an alkyl or aryl group. The leaving group being "good"
enough to be displaced by the alcohol, phenol, thiol, amine,
carbonyl, or like group on the framework. Such a linker can include
a moiety represented by the formula: R--X, in which X is a leaving
group such as halogen (e.g., --Cl, --Br or --I), tosylate,
mesylate, triflate, and R is alkyl, substituted alkyl, cycloalkyl,
heterocyclic, substituted heterocyclic, aryl alkyl, aryl,
heteroaryl, heteroaryl alkyl, ethoxy or propoxy oligomer, a
glycoside, or like moiety.
[0390] Suitable linker groups include those of formula:
(CH.sub.2).sub.1COOH, with n=1-16, n=2-8, n=2-6, or n=3. Reagents
that form suitable linkers are commercially available and include
any of a variety of reagents with orthogonal functionality.
Embodiments of Building Blocks
[0391] In an embodiment, building blocks can be represented by
Formula 2:
##STR00004##
in which: RE.sub.1 is recognition element 1, RE.sub.2 is
recognition element 2, and L is a linker. X is absent, C.dbd.O,
CH.sub.2, NR, NR.sub.2, NH, NHCONH, SCONH, CH.dbd.N, or
OCH.sub.2NH. In certain embodiments, X is absent or C.dbd.O. Y is
absent, NH, O, CH.sub.2, or NRCO. In certain embodiments, Y is NH
or O. In an embodiment, Y is NH. Z.sub.1 and Z.sub.2 can
independently be CH.sub.2, O, NH, S, CO, NR, NR.sub.2, NHCONH,
SCONH, CH.dbd.N, or OCH.sub.2NH. In an embodiment, Z.sub.i and/or
Z.sub.2 can independently be O. Z.sub.2 is optional. R.sub.2 is H,
CH.sub.3, or another group that confers chirality on the building
block and has size similar to or smaller than a methyl group.
R.sub.3 is CH.sub.2; CH.sub.2-phenyl; CHCH.sub.3; (CH.sub.2).sub.n
with n=2-3; or cyclic alkyl with 3-8 carbons, e.g., 5-6 carbons,
phenyl, naphthyl. In certain embodiments, R.sub.3 is CH.sub.2 or
CH.sub.2-phenyl.
[0392] RE.sub.1 is B1, B2, B3, B3a, B4, B5, B6, B7, B8, B9, A1, A2,
A3, A3a, A4, A5, A6, A7, A8, or A9. In certain embodiments,
RE.sub.1 is B1, B2, B3, B3a, B4, B5, B6, B7, B8, or B9. RE.sub.2 is
A1, A2, A3, A3a, A4, A5, A6, A7, A8, A9, B1, B2, B3, B3a, B4, B5,
B6, B7, B8, or B9. In certain embodiments, RE.sub.2 is A1, A2, A3,
A3a, A4, A5, A6, A7, A8, or A9. In an embodiment, RE.sub.1 can be
B2, B3a, B4, B5, B6, B7, or B8. In an embodiment, RE.sub.2 can be
A2, A3a, A4, A5, A6, A7, or A8.
[0393] In an embodiment, L is the functional group participating in
or formed by the bond to the framework (such groups are described
herein), the functional group or groups participating in or formed
by the reversible interaction with the support or lawn (such groups
are described herein), and a linker backbone moiety. In an
embodiment, the linker backbone moiety is about 4 to about 48
carbon or heteroatom alkyl, substituted alkyl, cycloalkyl,
heterocyclic, substituted heterocyclic, aryl alkyl, aryl,
heteroaryl, heteroaryl alkyl, ethoxy or propoxy oligomer, a
glycoside, or mixtures thereof; or about 8 to about 14 carbon or
heteroatoms, about 12 to about 24 carbon or heteroatoms, about 16
to about 18 carbon or heteroatoms, about 4 to about 12 carbon or
heteroatoms, about 4 to about 8 carbon or heteroatoms.
[0394] In an embodiment, the L is the functional group
participating in or formed by the bond to the framework (such
groups are described herein) and a lipophilic moiety (such groups
are described herein) of about 4 to about 48 carbons, about 8 to
about 14 carbons, about 12 to about 24 carbons, about 16 to about
18 carbons. In an embodiment, this L also includes about 1 to about
8 reversible bond/interaction moieties (such groups are described
herein) or about 2 to about 4 reversible bond/interaction moieties.
In an embodiment, L is (CH.sub.2).sub.nCOOH, with n=12-24, n=17-24,
or n=16-18.
[0395] In an embodiment, L is (CH.sub.2).sub.nCOOH, with n=1-16,
n=2-8, n=4-6, or n=3.
[0396] Building blocks including an A and/or a B recognition
element, a linker, and an amino acid framework can be made by
methods illustrated in general Scheme 1.
Techniques for Using Artificial Receptors
[0397] The present invention includes a method of using artificial
receptors. The present invention includes a method of screening
candidate artificial receptors to find lead artificial receptors
that bind a particular test ligand. Detecting test ligand bound to
a candidate artificial receptor can be accomplished using known
methods for detecting binding to arrays on a slide or to coated
tubes or wells. For example, the method can employ test ligand
labeled with a detectable label, such as a fluorophore or an enzyme
that produces a detectable product. Alternatively, the method can
employ an antibody (or other binding agent) specific for the test
ligand and including a detectable label. One or more of the spots
that are labeled by the test ligand or that are more or most
intensely labeled with the test ligand are selected as lead
artificial receptors. The degree of labeling can be evaluated by
evaluating the signal strength from the label. The amount of signal
can be directly proportional to the amount of label and binding.
FIG. 13 provides a schematic illustration of an embodiment of this
process.
[0398] According to the present method, screening candidate
artificial receptors against a test ligand can yield one or more
lead artificial receptors. One or more lead artificial receptors
can be a working artificial receptor. That is, the one or more lead
artificial receptors can be useful for detecting the ligand of
interest as is. The method can then employ the one or more
artificial receptors as a working artificial receptor for
monitoring or detecting the test ligand. Alternatively, the one or
more lead artificial receptors can be employed in the method for
developing a working artificial receptor. For example, the one or
more lead artificial receptors can provide structural or other
information useful for designing or screening for an improved lead
artificial receptor or a working artificial receptor. Such
designing or screening can include making and testing additional
candidate artificial receptors including combinations of a subset
of building blocks, a different set of building blocks, or a
different number of building blocks.
[0399] The present invention includes a method of screening
candidate artificial receptors to find lead artificial receptors
that bind a particular test ligand. The method can include allowing
movement of the building blocks that make up the artificial
receptors. Movement of building blocks can include mobilizing the
building block to move along or on the support and/or to leave the
support and enter a fluid (e.g., liquid) phase separate from the
support or lawn.
[0400] In an embodiment, building blocks can be mobilized to move
along or on the support (translate or shuffle). Such translation
can be employed, for example, to allow building blocks already
bound to a test ligand to rearrange into a lower energy or tighter
binding configuration still bound to the test ligand. Such
translation can be employed, for example, to allow the ligand
access to building blocks that are on the support but not bound to
the ligand. These building blocks can translate into proximity with
and bind to a test ligand.
[0401] Building blocks can be induced to move along or on the
support or to be reversibly immobilized on the support through any
of a variety of mechanisms. For example, inducing mobility of
building blocks can include altering the conditions of the support
or lawn. That is, altering the conditions can reverse the
immobilization of the building blocks, thus mobilizing them.
Reversibly immobilizing the building blocks after they have moved
can include, for example, returning to the previous conditions.
Suitable alterations of conditions include changing pH, changing
temperature, changing polarity or hydrophobicity, changing ionic
strength, changing nucleophilicity or electrophilicity (e.g. of
solvent or solute), and the like.
[0402] A building block reversibly immobilized by hydrophobic
interactions can be mobilized by increasing the temperature, by
exposing the surface, lawn, or building block to a more hydrophobic
solvent (e.g., an organic solvent or a surfactant), or by reducing
ionic strength around the building block. In an embodiment, the
organic solvent includes acetonitrile, acetic acid, an alcohol,
tetrahydrofuran (THF), dimethylformamide (DMF), hydrocarbons such
as hexane or octane, acetone, chloroform, methylene chloride, or
the like, or mixture thereof. In an embodiment, the surfactant
includes a nonionic surfactant, such as a nonylphenol ethoxylate,
or the like. A building block that is mobile on a support can be
reversibly immobilized by hydrophobic interactions, for example, by
decreasing the temperature, exposing the surface, lawn, or building
block to a more hydrophilic solvent (e.g., an aqueous solvent) or
increased ionic strength.
[0403] A building block reversibly immobilized by hydrogen bonding
can be mobilized by increasing the ionic strength, concentration of
hydrophilic solvent, or concentration of a competing hydrogen
bonder in the environs of the building block. A building block that
is mobile on a support can be reversibly immobilized through an
electrostatic interaction by decreasing ionic strength of the
hydrophilic solvent, or the like.
[0404] A building block reversibly immobilized by an electrostatic
interaction can be mobilized by increasing the ionic strength in
the environs of the building block. Increasing ionic strength can
disrupt electrostatic interactions. A building block that is mobile
on a support can be reversibly immobilized through an electrostatic
interaction by decreasing ionic strength.
[0405] A building block reversibly immobilized by an imine, acetal,
or ketal bond can be mobilized by decreasing the pH or increasing
concentration of a nucleophilic catalyst in the environs of the
building block. In an embodiment, the pH is about 1 to about 4.
Imines, acetals, and ketals undergo acid catalyzed hydrolysis. A
building block that is mobile on a support can be reversibly
immobilized by a reversible covalent interaction, such as by
forming an imine, acetal, or ketal bond, by increasing the pH.
[0406] In an embodiment, building blocks can be mobilized to leave
the support and enter a fluid (e.g., liquid) phase separate from
the support or lawn (exchange). For example, building blocks can be
exchanged onto and/or off of the support. Exchange can be employed,
for example, to allow building blocks on a support but not bound to
a test ligand to be removed from the support. Exchange can be
employed, for example, to add additional building blocks to the
support. The added building blocks can have structures selected
based on knowledge of the structures of the building blocks in
artificial receptors that bind the test ligand. The added building
blocks can have structures selected to provide additional
structural diversity. The added building blocks can include all of
the building blocks.
[0407] A building block reversibly immobilized by hydrophobic
interactions can be released from the support by, for example,
raising the temperature, e.g., of the support and/or artificial
receptor. For example, the hydrophobic interactions (e.g., the
hydrophobic group on the support or lawn and on the building block)
can be selected to provide immobilized building block at about room
temperature or below and release can be accomplished at a
temperature above room temperature. For example, the hydrophobic
interactions can be selected to provide immobilized building block
at about refrigerator temperature (e.g., 4.degree. C.) or below and
release can be accomplished at a temperature of, for example, room
temperature or above. By way of further example, a building block
can be reversibly immobilized by hydrophobic interactions, for
example, by contacting the surface or artificial receptor with a
fluid containing the building block and that is at or below room
temperature.
[0408] A building block reversibly immobilized by hydrophobic
interactions can be released from the support by, for example,
contacting the artificial receptor with a sufficiently hydrophobic
fluid (e.g., an organic solvent or a surfactant). In an embodiment,
the organic solvent includes acetonitrile, acetic acid, an alcohol,
tetrahydrofuran (THF), dimethylformamide (DMF), hydrocarbons such
as hexane or octane, acetone, chloroform, methylene chloride, or
the like, or mixture thereof. In an embodiment, the surfactant
includes a nonionic surfactant, such as a nonylphenol ethoxylate,
or the like. Such reversible immobilization can also be effected by
contacting the surface or artificial receptor with a hydrophilic
solvent and allowing the somewhat lipophilic building block to
partition on to the hydrophobic surface or lawn.
[0409] A building block reversibly immobilized by an imine, acetal,
or ketal bond can be released from the support by, for example,
contacting the artificial receptor with fluid having an acid pH or
including a nucleophilic catalyst. In an embodiment, the pH is
about 1 to about 4. A building block can be reversibly immobilized
by a reversible covalent interaction, such as by forming an imine,
acetal, or ketal bond, by contacting the surface or artificial
receptor with fluid having a neutral or basic pH.
[0410] A building block reversibly immobilized by an electrostatic
interaction can be released by, for example, contacting the
artificial receptor with fluid having sufficiently high ionic
strength to disrupt the electrostatic interaction. A building block
can be reversibly immobilized through an electrostatic interaction
by contacting the surface or artificial receptor with fluid having
ionic strength that promotes electrostatic interaction between the
building block and the support and/or lawn.
Test Ligands
[0411] The test ligand can be any ligand for which binding to an
array or surface can be detected. The test ligand can be a pure
compound, a mixture, or a "dirty" mixture containing a natural
product or pollutant. Such dirty mixtures can be tissue homogenate,
biological fluid, soil sample, water sample, or the like.
[0412] Test ligands include prostate specific antigen, other cancer
markers, insulin, warfarin, other anti-coagulants, cocaine, other
drugs-of-abuse, markers for E. coli, markers for Salmonella sp.,
markers for other food-borne toxins, food-borne toxins, markers for
Smallpox virus, markers for anthrax, markers for other possible
toxic biological agents, pharmaceuticals and medicines, pollutants
and chemicals in hazardous waste, toxic chemical agents, markers of
disease, pharmaceuticals, pollutants, biologically important
cations (e.g., potassium or calcium ion), peptides, carbohydrates,
enzymes, bacteria, viruses, mixtures thereof, and the like. In
certain embodiments, the test ligand can be at least one of small
organic molecules, inorganic/organic complexes, metal ion, mixture
of proteins, protein, nucleic acid, mixture of nucleic acids,
mixtures thereof, and the like.
[0413] Suitable test ligands include any compound or category of
compounds described elsewhere in this document as being a test
ligand, including, for example, the microbes, proteins, cancer
cells, drugs of abuse, and the like described above.
[0414] The present invention may be better understood with
reference to the following examples. These examples are intended to
be representative of specific embodiments of the invention, and are
not intended as limiting the scope of the invention.
EXAMPLES
Example 1
Synthesis of Building Blocks
[0415] Selected building blocks representative of the
alkyl-aromatic-polar span of the an embodiment of the building
blocks were synthesized and demonstrated effectiveness of these
building blocks for making candidate artificial receptors. These
building blocks were made on a framework that can be represented by
tyrosine and included numerous recognition element pairs. These
recognition element pairs were selected along the diagonal of Table
2, and include enough of the range from alkyl, to aromatic, to
polar to represent a significant degree of the interactions and
functional groups of the full set of 81 such building blocks.
Synthesis
[0416] Building block synthesis employed a general procedure
outlined in Scheme 7, which specifically illustrates synthesis of a
building block on a tyrosine framework with recognition element
pair A4B4. This general procedure was employed for synthesis of
building blocks including TyrA1B1 [1-1], TyrA2B2, TyrA2B4, TyrA2B6,
TyrA2B8, TyrA4B2, TyrA4B4, TyrA4B6, TyrA4B8, TyrA6B2, TyrA6B4,
TyrA6B6, TyrA6B8, TyrA8B2, TyrA8B4, TyrA8B6, TyrA8B8, and TyrA9B9,
respectively.
##STR00005##
Results
[0417] Synthesis of the desired building blocks proved to be
generally straightforward. These syntheses illustrate the relative
simplicity of preparing the building blocks with 2 recognition
elements having different structural characteristics or structures
(e.g. A4B2, A6B3, etc.) once the building blocks with corresponding
recognition elements (e.g. A2B2, A4B4, etc) have been prepared via
their X BOC intermediate.
[0418] The conversion of one of these building blocks to a building
block with a lipophilic linker can be accomplished by reacting the
activated building block with, for example, dodecyl amine.
Example 2
Preparation and Evaluation of Microarrays of Candidate Artificial
Receptors
[0419] Microarrays of candidate artificial receptors were made and
evaluated for binding several protein ligands. The results obtained
demonstrate the 1) the simplicity with which microarrays of
candidate artificial receptors can be prepared, 2) binding affinity
and binding pattern reproducibility, 3) significantly improved
binding for building block heterogeneous receptor environments when
compared to the respective homogeneous controls, and 4) ligand
distinctive binding patterns (e.g., working receptor
complexes).
Materials and Methods
[0420] Building blocks were synthesized and activated as described
in Example 1. The building blocks employed in this example were
TyrA1B1 [1-1], TyrA2B2, TyrA2B4, TyrA2B6, TyrA4B2, TyrA4B4,
TyrA4B6, TyrA6B2, TyrA6B4, and TyrA6B6. The abbreviation for the
building block including a linker, a tyrosine framework, and
recognition elements AxBy is TyrAxBy.
[0421] Microarrays for the evaluation of the 130 n=2 and n=3, and
for evaluation of the 273 n=2, n=3, and n=4, candidate receptor
environments were prepared as follows by modifications of known
methods. Briefly: Amine modified (amine "lawn"; SuperAmine
Microarray plates) microarray plates were purchased from Telechem
Inc., Sunnyvale, Calif. (www.arrayit.com). These plates were
manufactured specifically for microarray preparation and had a
nominal amine load of 2-4 amines per square nm according to the
manufacturer. The CAM microarrays were prepared using a pin
microarray spotter instrument from Telechem Inc. (SpotBot.TM.
Arrayer) typically with 200 um diameter spotting pins from Telechem
Inc. (Stealth Micro Spotting Pins, SMP6) and 400-420 um spot
spacing.
[0422] The 9 building blocks were activated in aqueous
dimethylformamide (DMF) solution as described above. For preparing
the 384-well feed plate, the activated building block solutions
were diluted 10-fold with a solution of DMF/H.sub.2O/PEG400
(90/10/10, v/v/v; PEG400 is polyethylene glycol nominal 400 FW,
Aldrich Chemical Co., Milwaukee, Wis.). These stock solutions were
aliquotted (10 .mu.l per aliquot) into the wells of a 384-well
microwell plate (Telechem Inc.). A separate series of controls were
prepared by aliquotting 10 .mu.l of building block with either 10
.mu.l or 20 .mu.l of the activated [1-1] solution. The plate was
covered with aluminum foil and placed on the bed of a rotary shaker
for 15 minutes at 1,000 RPM. This master plate was stored covered
with aluminum foil at -20.degree. C. when not in use.
[0423] For preparing the 384-well SpotBot.TM. plate, a well-to-well
transfer (e.g. A-1 to A-1, A-2 to A-2, etc.) from the feed plate to
a second 384-well plate was performed using a 4 .mu.l transfer
pipette. This plate was stored tightly covered with aluminum foil
at -20.degree. C. when not in use. The SpotBot.TM. was used to
prepare up to 13 microarray plates per run using the 4 .mu.l
microwell plate. The SpotBot.TM. was programmed to spot from each
microwell in quadruplicate. The wash station on the SpotBot.TM.
used a wash solution of EtOH/H.sub.2O (20/80, v/v). This wash
solution was also used to rinse the microarrays on completion of
the SpotBot.TM. printing run. The plates were given a final rinse
with deionized (DI) water, dried using a stream of compressed air,
and stored at room temperature.
[0424] Certain of the microarrays were further modified by reacting
the remaining amines with succinic anhydride to form a carboxylate
lawn in place of the amine lawn.
[0425] The following test ligands and labels were used in these
experiments:
[0426] 1) r-Phycoerythrin, a commercially available and
intrinsically fluorescent protein with a FW of 2,000,000.
[0427] 2) Ovalbumin labeled with the Alexa.TM. fluorophore
(Molecular Probes Inc., Eugene, Oreg.).
[0428] 3) BSA, bovine serum albumin, labeled with activated
Rhodamine (Pierce Chemical, Rockford, Ill.) using the known
activated carboxyl protocol. BSA has a FW of 68,000; the material
used for this study had ca. 1.0 rhodamine per BSA.
[0429] 4) Horseradish peroxidase (HRP) modified with extra amines
and labeled as the acetamide derivative or with a
2,3,7,8-tetrachlorodibenzodixoin derivative were available through
known methods. Fluorescence detection of these HRP conjugates was
based on the Alexa 647-tyramide kit available from Molecular
Probes, Eugene, Oreg.
[0430] 5) Cholera toxin.
[0431] Microarray incubation and analysis was conducted as follows:
For test ligand incubation with the microarrays, solutions (e.g.
500 .mu.l) of the target proteins in PBS-T (PBS with 20 .mu.l/L of
Tween-20) at typical concentrations of 10, 1.0 and 0.1 .mu.g/ml
were placed onto the surface of a microarray and allowed to react
for, e.g., 30 minutes. The microarray was rinsed with PBS-T and DI
water and dried using a stream of compressed air.
[0432] The incubated microarray was scanned using an Axon Model
4200A Fluorescence Microarray Scanner (Axon Instruments, Union
City, Calif.). The Axon scanner and its associated software produce
a false color 16-bit image of the fluorescence intensity of the
plate. This 16-bit data is integrated using the Axon software to
give a Fluorescence Units value (range 0-65,536) for each spot on
the microarray. This data is then exported into an Excel file
(Microsoft) for further analysis including mean, standard deviation
and coefficient of variation calculations.
Results
[0433] The CARA.TM.: Combinatorial Artificial Receptor Array.TM.
concept has been demonstrated using a microarray format. A CARA
microarray based on N=9 building blocks was prepared and evaluated
for binding to several protein and substituted protein ligands.
This microarray included 144 candidate receptors (18 n=1 controls
plus 6 blanks; 36 n=2 candidate receptors; 84 n=3 candidate
receptors). This microarray demonstrated: 1) the simplicity of CARA
microarray preparation, 2) binding affinity and binding pattern
reproducibility, 3) significantly improved binding for building
block heterogeneous receptor environments when compared to the
respective homogeneous controls, and 4) ligand distinctive binding
patterns.
Reading the Arrays
[0434] A typical false color/gray scale image of a microarray that
was incubated with 2.0 .mu.g/ml r-phycoerythrin is shown in FIG.
23. This image illustrates that the processes of both preparing the
microarray and probing it with a protein test ligand produced the
expected range of binding as seen in the visual range of relative
fluorescence from dark to bright spots.
[0435] The starting point in analysis of the data was to take the
integrated fluorescence units data for the array of spots and
normalize to the observed value for the [1-1] building block
control. Subsequent analysis included mean, standard deviation and
coefficient of variation calculations. Additionally, control values
for homogeneous building blocks were obtained from the building
block plus [1-1] data.
First Set of Experiments
[0436] The following protein ligands were evaluated for binding to
the candidate artificial receptors in the microarray. The resulting
Fluorescence Units versus candidate receptor environment data is
presented in both a 2D format where the candidate receptors are
placed along the X-axis and the Fluorescence Units are shown on the
Y-axis and a 3D format where the Candidate Receptors are placed in
an X-Y format and the Fluorescence Units are shown on the Z-axis. A
key for the composition of each spot was developed (not shown). A
key for the building blocks in each of the 2D and 3D
representations of the results was also developed (not shown). The
data presented are for 1-2 .mu.g/ml protein concentrations.
[0437] FIGS. 24 and 25 illustrate binding data for r-phycoerythrin
(intrinsic fluorescence). FIGS. 26 and 27 illustrate binding data
for ovalbumin (commercially available with fluorescence label).
FIGS. 28 and 29 illustrate binding data for bovine serum albumin
(labeled with rhodamine). FIGS. 30 and 31 illustrate binding data
for HRP-NH-Ac (fluorescent tyramide read-out). FIGS. 32 and 33
illustrate binding data for HRP-NH-TCDD (fluorescent tyramide
read-out).
[0438] These results demonstrate not only the application of the
CARA microarray to candidate artificial receptor evaluation but
also a few of the many read-out methods (e.g. intrinsic
fluorescence, fluorescently labeled, in situ fluorescence labeling)
which can be utilized for high throughput candidate receptor
evaluation.
[0439] The evaluation of candidate receptors benefits from
reproducibility. The following results demonstrate that the present
microarrays provided reproducible ligand binding.
[0440] The microarrays were printed with each combination of
building blocks spotted in quadruplicate. Visual inspection of a
direct plot (FIG. 34) of the raw fluorescence data (from the run
illustrated in FIG. 23) for one block of binding data obtained for
r-phycoerythrin demonstrates that the candidate receptor
environment "spots" showed reproducible binding to the test ligand.
Further analysis of the r-phycoerythrin data (FIG. 23) led to only
9 out of 768 spots (1.2%) being deleted as outliers. Analysis of
the r-phycoerythrin quadruplicate data for the entire array gives a
mean standard deviation for each experimental quadruplicate set of
938 fluorescence units, with a mean coefficient of variation of
19.8%.
[0441] Although these values are acceptable, a more realistic
comparison employed the standard deviation and coefficient of
variation of the more strongly bound, more fluorescent receptors.
The overall mean standard deviation unrealistically inflates the
coefficient of variation for the weakly bound, less fluorescent
receptors. The coefficient of variation for the 19 receptors with
greater than 10,000 Fluorescent Units of bound target is 11.1%,
which is well within the range required to produce meaningful
binding data.
[0442] One goal of the CARA approach is the facile preparation of a
significant number of candidate receptors through combinations of
structurally simple building blocks. The following results
establish that both the individual building blocks and combinations
of building blocks have a significant, positive effect on test
ligand binding.
[0443] The binding data illustrated in FIGS. 23-33 demonstrate that
heterogeneous combinations of building blocks (n=2, n=3) are
dramatically superior candidate receptors made from a single
building block (n=1). For example, FIG. 25 illustrate both the
diversity of binding observed for n=2, n=3 candidate receptors with
fluorescent units ranging from 0 to ca. 40,000. These data also
illustrate and the ca. 10-fold improvement in binding affinity
obtained upon going from the homogeneous (n=1) to heterogeneous
(n=2, n=3) receptor environments.
[0444] The effect of heterogeneous building blocks is most easily
observed by comparing selected n=3 receptor environments candidate
receptors including 1 or 2 of those building blocks (their n=2 and
n=1 subsets). FIGS. 35 and 36 illustrate this comparison for two
different n=3 receptor environments using the r-phycoerythrin data.
In these examples, it is clear that progression from the
homogeneous system (n=1) to the heterogeneous systems (n=2, n=3)
produces significantly enhanced binding.
[0445] Although van der Waals interactions are an important part of
molecular recognition, it is important to establish that the
observed binding is not a simple case of hydrophobic/hydrophilic
partitioning. That is, that the observed binding was the result of
specific interactions between the individual building blocks and
the target The simplest way to evaluate the effects of
hydrophobicity and hydrophilicity is to compare building block logP
value with observed binding. LogP is a known and accepted measure
of lipophilicity, which can be measured or calculated by known
methods for each of the building blocks. FIGS. 37 and 38 establish
that the observed target binding, as measured by fluorescence
units, is not directly proportional to building block logP. The
plots in FIGS. 37 and 38 illustrate a non-linear relationship
between binding (fluorescence units) and building block logP.
[0446] One advantage of the present methods and arrays is that the
ability to screen large numbers of candidate receptor environments
will lead to a combination of useful target affinities and to
significant target binding diversity. High target affinity is
useful for specific target binding, isolation, etc. while binding
diversity can provide multiplexed target detection systems. This
example employed a relatively small number of building blocks to
produce ca. 120 binding environments. The following analysis of the
present data clearly demonstrates that even a relatively small
number of binding environments can produce diverse and useful
artificial receptors.
[0447] The target binding experiments performed for this study used
protein concentrations including 0.1 to 10 .mu.g/ml. Considering
the BSA data as representative, it is clear that some of the
receptor environments readily bound 1.0 ug/ml BSA concentrations
near the saturation values for fluorescence units (see, e.g., FIG.
29). Based on these data and the formula weight of 68,000 for BSA,
several of the receptor environments readily bind BSA at ca. 15
picomole/ml or 15 nanomolar concentrations. Additional experiments
using lower concentrations of protein (data not shown) indicate
that, even with a small selection of candidate receptor
environments, femptomole/ml or picomolar detection limits have been
attained.
[0448] One goal of artificial receptor development is the specific
recognition of a particular target. FIG. 39 compares the observed
binding for r-phycoerythrin and BSA. Comparison of the overall
binding pattern indicates some general similarities. However,
comparison of specific features of binding for each receptor
environment demonstrates that the two targets have distinctive
recognition features as indicated by the (*) in FIG. 39.
[0449] One goal of artificial receptor development is to develop
receptors which can be used for the multiplexed detection of
specific targets. Comparison of the r-phycoerythrin, BSA and
ovalbumin data from this study (FIGS. 25, 27, 29) were used to
select representative artificial receptors for each target. FIGS.
40, 41 and 42 employ data obtained in the present example to
illustrate identification of each of these three targets by their
distinctive binding patterns.
Conclusions
[0450] The optimum receptor for a particular target requires
molecular recognition which is greater than the expected sum of the
individual hydrophilic, hydrophobic, ionic, etc. interactions.
Thus, the identification of an optimum (specific, sensitive)
artificial receptor from the limited pool of candidate receptors
explored in this prototype study, was not expected and not likely.
Rather, the goal was to demonstrate that all of the key components
of the CARA: Combinatorial Artificial Receptor Array concept could
be assembled to form a functional receptor microarray. This goal
has been successfully demonstrated.
[0451] This study has conclusively established that CARA
microarrays can be readily prepared and that target binding to the
candidate receptor environments can be used to identify artificial
receptors and test ligands. In addition, these results demonstrate
that there is significant binding enhancement for the building
block heterogeneous (n=2, n=3, or n=4) candidate receptors when
compared to their homogeneous (n=1) counterparts. When combined
with the binding pattern recognition results and the demonstrated
importance of both the heterogeneous receptor elements and
heterogeneous building blocks, these results clearly demonstrate
the significance of the CARA Candidate Artificial Receptor->Lead
Artificial Receptor->Working Artificial Receptor strategy.
Example 3
Preparation and Evaluation of Microarrays of Candidate Artificial
Receptors Including Reversibly Immobilized Building Blocks
[0452] Microarrays of candidate artificial receptors including
building blocks immobilized through van der Waals interactions were
made and evaluated for binding of a protein ligand. The evaluation
was conducted at several temperatures, above and below a phase
transition temperature for the lawn (vide infra).
Materials and Methods
[0453] Building blocks 2-2, 2-4, 2-6, 4-2, 4-4, 4-6, 6-2, 6-4, 6-6
where prepared as described in Example 1. The C12 amide was
prepared using the previously described carbodiimide activation of
the carboxyl followed by addition of dodecylamine. This produced a
building block with a 12 carbon alkyl chain linker for reversible
immobilization in the C18 lawn.
[0454] Amino lawn microarray plates (Telechem) were modified to
produce the C18 lawn by reaction of stearoyl chloride (Aldrich
Chemical Co.) in A) dimethylformamide/PEG 400 solution (90:10, v/v,
PEG 400 is polyethylene glycol average MW 400 (Aldrich Chemical
Co.) or B) methylene chloride/TEA solution (100 ml methylene
chloride, 200 .mu.l triethylamine) using the lawn modification
procedures generally described in Example 2.
[0455] The C18 lawn plates where printed using the SpotBot standard
procedure as described in Example 2. The building blocks were in
printing solutions prepared by solution of ca. 10 mg of each
building block in 300 .mu.l of methylene chloride and 100 .mu.l
methanol. To this stock was added 900 .mu.l of dimethylformamide
and 100 .mu.l of PEG 400. The 36 combinations of the 9 building
blocks taken two at a time (N9:n2, 36 combinations) where prepared
in a 384-well microwell plate which was then used in the SpotBot to
print the microarray in quadruplicate. A random selection of the
print positions contained only print solution.
[0456] The selected microarray was incubated with a 1.0 .mu.g/ml
solution of the test ligand, cholera toxin subunit B labeled with
the Alexa.TM. fluorophore (Molecular Probes Inc., Eugene, Oreg.),
using the following variables: 1) the microarray was washed with
methylene chloride, ethanol and water to create a control plate;
and 2) the microarray was incubated at 4.degree. C., 23.degree. C.,
or 44.degree. C. After incubation, the plate(s) were rinsed with
water, dried and scanned (AXON 4100A). Data analysis was as
described in Example 2.
Results
[0457] A control array from which the building blocks had been
removed by washing with organic solvent did not bind cholera toxin
(FIG. 43). FIGS. 44-46 illustrate fluorescence signals from arrays
printed identically, but incubated with cholera toxin at 4.degree.
C., 23.degree. C., or 44.degree. C., respectively. Spots of
fluorescence can be seen in each array, with very pronounced spots
produced by incubation at 44.degree. C. The fluorescence values for
the spots in each of these three arrays are shown in FIGS. 47-49.
Fluorescence signal generally increases with temperature, with many
nearly equally large signals observed after incubation at
44.degree. C. Linear increases with temperature can reflect
expected improvements in binding with temperature. Nonlinear
increases reflect rearrangement of the building blocks on the
surface to achieve improved binding, which occurred above the phase
transition for the lipid surface (vide infra).
[0458] FIG. 50 can be compared to FIG. 48. The fluorescence signals
plotted in FIG. 48 resulted from binding to reversibly immobilized
building blocks on a support at 23.degree. C. The fluorescence
signals plotted in FIG. 50 resulted from binding to covalently
immobilized building blocks on a support at 23.degree. C. These
figures compare the same combinations of building blocks in the
same relative positions, but immobilized in two different ways.
[0459] The binding to covalently immobilized building blocks was
also evaluated at 4.degree. C., 23.degree. C., or 44.degree. C.
FIG. 51 illustrates the changes in fluorescence signal from
individual combinations of covalently immobilized building blocks
at 4.degree. C., 23.degree. C., or 44.degree. C. Binding increased
modestly with temperature. The mean increase in binding was
1.3-fold. A plot of the fluorescence signal for each of the
covalently immobilized artificial receptors at 23.degree. C.
against its signal at 44.degree. C. (not shown) yields a linear
correlation with a correlation coefficient of 0.75. This linear
correlation indicates that the mean 1.3-fold increase in binding is
a thermodynamic effect and not optimization of binding.
[0460] FIG. 52 illustrates the changes in fluorescence signal from
individual combinations of reversibly immobilized building blocks
at 4.degree. C., 23.degree. C., or 44.degree. C. This graph
illustrates that at least one combination of building blocks
(candidate artificial receptor) exhibited a signal that remained
constant as temperature increased. At least one candidate
artificial receptor exhibited an approximately linear increase in
signal as temperature increased. Such a linear increase indicates
normal temperature effects on binding. The candidate artificial
receptor with the lowest binding signal at 4.degree. C. became one
of the best binders at 44.degree. C. This indicates that
rearrangement of the building blocks of this receptor above the
phase transition for the lawn, which increases the building blocks'
mobility, produced increased binding. Other receptors characterized
by greater changes in binding between 23.degree. C. and 44.degree.
C. (compared to between 4.degree. C. and 23.degree. C.) also
underwent dynamic affinity optimization.
[0461] FIG. 53 illustrates the data presented in FIG. 51 (lines
marked A) and the data presented in FIG. 52 (lines marked B). The
increases in binding observed with the reversibly immobilized
building blocks are significantly greater than the increases
observed with covalently bound building blocks. Binding to
reversibly immobilized building blocks increased from 23.degree. C.
and 44.degree. C. by a median value of 6.1-fold and a mean value of
24-fold. This confirms that movement of the reversibly immobilized
building blocks within the receptors increased binding (i.e., the
receptor underwent dynamic affinity optimization).
[0462] A plot of the fluorescence signal for each of the reversibly
immobilized artificial receptors at 23.degree. C. against its
signal at 44.degree. C. (not shown) yields no correlation
(correlation coefficient of 0.004). A plot of the fluorescence
signal for each of the reversibly immobilized artificial receptors
at 44.degree. C. against the signal for the corresponding
covalently immobilized receptor (not shown) also yields no
correlation (correlation coefficient 0.004). This lack of
correlation provides further evidence that movement of the
reversibly immobilized building blocks within the receptors
increased binding.
[0463] FIG. 54 illustrates a graph of the fluorescence signal at
44.degree. C. divided by the signal at 23.degree. C. against the
fluorescence signal obtained from binding at 23.degree. C. for the
artificial receptors with reversibly immobilized receptors. This
comparison indicates that the binding enhancement is independent of
the initial affinity of the receptor for the test ligand.
[0464] Table 1 identifies the reversibly immobilized building
blocks making up each of the artificial receptors, lists the
fluorescence signal (binding strength) at 44.degree. C. and
23.degree. C., and the ratios of the observed binding at these two
temperatures. These data illustrate that each artificial receptor
reflects a unique attribute for each combination of building blocks
relative to the role of each individual building block.
TABLE-US-00001 TABLE 1 Building Blocks Making Up Signal at Ratio of
Signals, Receptor 44.degree. C. Signal at 23.degree. C. 44.degree.
C./23.degree. C. 22 24 24136 4611 5.23 22 26 16660 43 387.44 22 42
17287 -167 -103.51 22 44 16726 275 60.82 22 46 25016 3903 6.41 22
62 13990 3068 4.56 22 64 15294 3062 4.99 22 66 11980 3627 3.30 24
26 22688 1291 17.57 24 42 26808 -662 -40.50 24 44 23154 904 25.61
24 46 42197 2814 15.00 24 62 19374 2567 7.55 24 64 27599 262 105.34
24 66 16238 5334 3.04 26 42 22282 4974 4.48 26 44 26240 530 49.51
26 46 23144 4273 5.42 26 62 29022 4920 5.90 26 64 23416 5551 4.22
26 66 19553 5353 3.65 42 44 29093 6555 4.44 42 46 18637 3039 6.13
42 62 22643 4853 4.67 42 64 20836 6343 3.28 42 66 14391 9220 1.56
44 46 25600 3266 7.84 44 62 15544 4771 3.26 44 64 25842 3073 8.41
44 66 22471 5142 4.37 46 62 32764 8522 3.84 46 64 21901 3343 6.55
46 66 23516 3742 6.28 62 64 24069 7149 3.37 62 66 15831 2424 6.53
64 66 21310 2746 7.76
Conclusions
[0465] This experiment demonstrated that an array including
reversibly immobilized building blocks binds a protein substrate,
like an array with covalently immobilized building blocks. The
binding increased nonlinearly as temperature increased, indicating
that movement of the building blocks increased binding. Many of the
candidate artificial receptors demonstrated improved binding upon
mobilization of the building blocks.
Example 4
The Oligosaccharide Portion of GM1 Competes with Artificial
Receptors for Binding to Cholera Toxin
[0466] Microarrays of candidate artificial receptors were made and
evaluated for binding of cholera toxin. The arrays were also
evaluated for disrupting that binding. Disrupting of binding
employed a compound that binds to cholera toxin, the
oligosaccharide moiety from GM1 (GM1 OS). The results obtained
demonstrate that a ligand of a protein specifically disrupted
binding of the protein to the microarray.
Materials and Methods
[0467] Building blocks were synthesized and activated as described
in Example 1. The building blocks employed in this example were
TyrA1B1 [1-1], TyrA2B2, TyrA2B4, TyrA2B6, TyrA2B8, TyrA3B3,
TyrA3B5, TyrA3B7, TyrA4B2, TyrA4B4, TyrA4B6, TyrA4B8, TyrA5B3,
TyrA5B5, TyrA5B7, TyrA6B2, TyrA6B4, TyrA6B6, TyrA6B8, TyrA7B3,
TyrA7B5, TyrA7B7, TyrA8B2, TyrA8B4, TyrA8B6, and TyrA8B8. The
abbreviation for the building block including a linker, a tyrosine
framework, and recognition elements AxBy is TyrAxBy.
[0468] Microarrays for the evaluation of the 171 n=2 candidate
receptor environments were prepared as follows by modifications of
known methods. An "n=2" receptor environment includes two different
building blocks. Briefly: Amine modified (amine "lawn"; SuperAmine
Microarray plates) microarray plates were purchased from Telechem
Inc., Sunnyvale, Calif. These plates were manufactured specifically
for microarray preparation and had a nominal amine load of 2-4
amines per square nm according to the manufacturer. The microarrays
were prepared using a pin microarray spotter instrument from
Telechem Inc. (SpotBot.TM. Arrayer) typically with 200 .mu.m
diameter spotting pins from Telechem Inc. (Stealth Micro Spotting
Pins, SMP6) and 400-420 .mu.m spot spacing.
[0469] The 19 building blocks were activated in aqueous
dimethylformamide (DMF) solution as described above. For preparing
the 384-well feed plate, the activated building block solutions
were diluted 10-fold with a solution of DMF/H.sub.2O/PEG400
(90/10/10, v/v/v; PEG400 is polyethylene glycol nominal 400 FW,
Aldrich Chemical Co., Milwaukee, Wis.). These stock solutions were
aliquotted (10 .mu.l per aliquot) into the wells of a 384-well
microwell plate (Telechem Inc.). Control spots included the
building block [1-1]. The plate was covered with aluminum foil and
placed on the bed of a rotary shaker for 15 minutes at 1,000 RPM.
This master plate was stored covered with aluminum foil at
-20.degree. C. when not in use.
[0470] For preparing the 384-well SpotBot.TM. plate, a well-to-well
transfer (e.g. A-1 to A-1, A-2 to A-2, etc.) from the feed plate to
a second 384-well plate was performed using a 4 .mu.l transfer
pipette. This plate was stored tightly covered with aluminum foil
at -20.degree. C. when not in use. The SpotBot.TM. was used to
prepare up to 13 microarray plates per run using the 4 .mu.l
microwell plate. The SpotBot.TM. was programmed to spot from each
microwell in quadruplicate. The wash station on the SpotBot.TM.
used a wash solution of EtOH/H.sub.2O (20/80, v/v). This wash
solution was adjusted to pH 4 with 1 M HCl and used to rinse the
microarrays on completion of the SpotBot.TM. printing run. The
plates were given a final rinse with deionized (DI) water, dried
using a stream of compressed air, and stored at room temperature.
The microarrays were further modified by reacting the remaining
amines with acetic anhydride to form an acetamide lawn in place of
the amine lawn.
[0471] The test ligand employed in these experiments was cholera
toxin labeled with the Alexa.TM. fluorophore (Molecular Probes
Inc., Eugene, Oreg.). The candidate disruptor employed in these
experiments was GM1 OS (GM1 oligosaccharide), a known ligand for
cholera toxin.
[0472] Microarray incubation and analysis was conducted as follows:
For control incubations with the microarrays, solutions (e.g. 500
.mu.l) of the cholera toxin in PBS-T (PBS with 20 .mu.l/L of
Tween-20) at a concentrations of 1.7 pmol/ml (0.1 .mu.g/ml) was
placed onto the surface of a microarray and allowed to react for 30
minutes. For disruptor incubations with the microarrays, solutions
(e.g. 500 .mu.l) of the cholera toxin (1.7 pmol/ml, 0.1 .mu.g/ml)
and the desired concentration of GM1 OS in PBS-T (PBS with 20
.mu.l/L of Tween-20) was placed onto the surface of a microarray
and allowed to react for 30 minutes. GM1 OS was added at 0.34 and
at 5.1 .mu.M in separate experiments. After either of these
incubations, the microarray was rinsed with PBS-T and DI water and
dried using a stream of compressed air.
[0473] The incubated microarray was scanned using an Axon Model
4200A Fluorescence Microarray Scanner (Axon Instruments, Union
City, Calif.). The Axon scanner and its associated software produce
a false color 16-bit image of the fluorescence intensity of the
plate. This 16-bit data is integrated using the Axon software to
give a Fluorescence Units value (range 0-65,536) for each spot on
the microarray. This data is then exported into an Excel file
(Microsoft) for further analysis including mean, standard deviation
and coefficient of variation calculations.
[0474] Table 2 identifies the building blocks in each of the first
150 receptor environments.
TABLE-US-00002 TABLE 2 Building Blocks 1 22 24 2 22 28 3 22 42 4 22
46 5 22 55 6 22 64 7 22 68 8 22 82 9 22 86 10 24 26 11 24 33 12 24
44 13 26 77 14 26 84 15 26 88 16 28 42 17 22 26 18 22 33 19 22 44
20 22 48 21 22 62 22 22 66 23 22 77 24 22 84 25 22 88 26 24 28 27
24 42 28 26 82 29 26 85 30 28 33 31 28 44 32 28 46 33 28 55 34 28
64 35 28 68 36 28 82 37 28 86 38 33 42 39 33 46 40 42 88 41 44 48
42 44 62 43 44 66 44 44 77 45 44 84 46 44 88 47 46 55 48 28 48 49
28 62 50 28 66 51 28 77 52 28 84 53 28 88 54 33 44 55 44 46 56 44
55 57 44 64 58 44 68 59 44 82 60 44 86 61 46 48 62 46 62 63 24 46
64 24 55 65 24 64 66 24 68 67 24 82 68 24 86 69 26 28 70 26 42 71
26 46 72 26 55 73 26 64 74 26 68 75 33 48 76 33 63 77 33 66 78 33
77 79 24 48 80 24 62 81 24 66 82 24 77 83 24 84 84 24 88 85 26 33
86 26 44 87 26 48 88 26 62 89 26 66 90 33 55 91 33 64 92 33 68 93
33 82 94 33 84 95 33 88 96 42 46 97 42 55 98 42 64 99 42 68 100 42
82 101 42 86 102 46 88 103 48 62 104 48 66 105 46 77 106 48 84 107
48 88 108 55 64 109 55 68 110 33 86 111 42 44 112 42 48 113 42 62
114 42 66 115 42 77 116 42 84 117 48 55 118 48 64 119 48 68 120 48
82 121 48 86 122 55 62 123 55 66 124 55 77 125 46 64 126 46 68 127
46 82 128 46 86 129 62 77 130 62 84 131 62 88 132 64 68 133 64 82
134 64 86 135 66 68 136 66 82 137 66 86 138 68 77 139 68 84 140 68
88 141 46 66 142 46 77 143 46 84 144 62 82 145 62 86 146 64 66 147
64 77 148 64 84 149 64 88 150 66 77
Results
Low Concentration of GM1 OS
[0475] FIG. 55 illustrates binding of cholera toxin to the
microarray of candidate artificial receptors followed by washing
with buffer produced fluorescence signals. These fluorescence
signals demonstrate that the cholera toxin bound strongly to
certain receptor environments, weakly to others, and undetectably
to some. Comparison to experiments including those reported in
Example 2 indicates that cholera toxin binding was reproducible
from array to array and from month to month.
[0476] Binding of cholera toxin was also conducted with competition
from GM1 OS (0.34 .mu.M). FIG. 56 illustrates the fluorescence
signals due to cholera toxin binding that were detected after this
competition. Notably, many of the signals illustrated in FIG. 56
are significantly smaller than the corresponding signals recorded
in FIG. 55. The small signals observed in FIG. 56 represent less
cholera toxin bound to the array. GM1 OS significantly disrupted
binding of cholera toxin to many of the receptor environments.
[0477] The disruption in cholera toxin binding caused by GM1 OS can
be visualized as the ratio of the amount bound in the absence of
GM1 OS to the amount bound in competition with GM1 OS. This ratio
is illustrated in FIG. 57. The larger the ratio, the less cholera
toxin remained bound to the artificial receptor after competition
with GM1 OS. The ratio can be as large as about 30. The ratios are
independent of the quantity bound in the control.
High Concentration of GM1 OS
[0478] Binding of cholera toxin to the microarray of candidate
artificial receptors followed by washing with buffer produced
fluorescence signals illustrated in FIG. 58. As before, cholera
toxin was reproducible and it bound strongly to certain receptor
environments, weakly to others, and undetectably to some. FIG. 59
illustrates the fluorescence signals detected due to cholera toxin
binding that were detected upon competition with GM1 OS at 5.1
.mu.M. Again, GM1 OS significantly disrupted binding of cholera
toxin to many of the receptor environments.
[0479] This disruption is presented as the ratio of the amount
bound in the absence of GM1 OS to the amount bound after contacting
with GM1 OS in FIG. 60. The ratios range up to about 18 and are
independent of the quantity bound in the control.
Conclusions
[0480] This experiment demonstrated that binding of a test ligand
to an artificial receptor of the present invention can be
diminished (e.g., competed) by a candidate disruptor molecule. In
this case the test ligand was the protein cholera toxin and the
candidate disruptor was a compound known to bind to cholera toxin,
GM1 OS. The degree to which binding of the test ligand was
disrupted was independent of the degree to which the test ligand
bound to the artificial receptor.
Example 5
GM1 Competes With Artificial Receptors for Binding to Cholera
Toxin
[0481] Microarrays of candidate artificial receptors were made and
evaluated for binding of cholera toxin. The arrays were also
evaluated for disrupting that binding. Disrupting of binding
employed a compound that binds to cholera toxin, the liposaccharide
GM1. The results obtained demonstrate that a ligand of a protein
specifically disrupts binding of the protein to the microarray.
Materials and Methods
[0482] Building blocks were synthesized and activated as described
in Example 1. The building blocks employed in this example were
TyrA1B1 [1-1], TyrA2B2, TyrA2B4, TyrA2B6, TyrA4B2, TyrA4B4,
TyrA4B6, TyrA6B2, TyrA6B4, and TyrA6B6 in groups of 4 building
blocks per artificial receptor. The abbreviation for the building
block including a linker, a tyrosine framework, and recognition
elements AxBy is TyrAxBy.
[0483] Microarrays for the evaluation of the 126 n=4 candidate
receptor environments were prepared as described above for Example
4. The test ligand employed in these experiments was cholera toxin
labeled with the Alexa.TM. fluorophore (Molecular Probes Inc.,
Eugene, Oreg.). Cholera toxin was employed at 5.3 nM in both the
control and the competition experiments. The candidate disruptor
employed in these experiments was GM1, a known ligand for cholera
toxin, which competed at concentrations of 0.042, 0.42, and 8.4
.mu.M. Microarray incubation and analysis was conducted as
described for Example 4.
[0484] Table 3 identifies the building blocks in each receptor
environment.
TABLE-US-00003 TABLE 3 Building Blocks 1 22 24 26 42 2 22 24 26 44
3 22 24 26 46 4 22 24 26 61 5 22 24 26 64 6 22 24 26 66 7 22 24 42
44 8 22 24 42 46 9 22 24 42 62 10 22 24 42 46 11 22 24 42 66 12 22
24 44 46 13 22 24 44 62 14 22 24 44 64 15 22 24 44 66 16 22 24 46
62 17 22 24 46 64 18 22 24 46 66 19 22 24 62 64 20 22 24 62 66 21
22 24 64 66 22 22 26 42 44 23 22 26 42 46 24 22 26 42 62 25 22 26
42 64 26 22 26 42 66 27 22 26 44 46 28 22 26 44 62 29 22 26 44 64
30 22 26 44 66 31 22 26 46 62 32 22 26 46 64 33 22 26 46 66 34 22
26 62 64 35 22 26 62 66 36 22 26 64 66 37 22 42 44 46 38 22 42 44
62 39 22 42 44 64 40 22 42 44 66 41 22 42 46 62 42 22 42 46 64 43
22 42 46 66 44 22 42 62 64 45 22 42 62 66 46 22 42 64 66 47 22 44
46 62 48 22 44 46 64 49 22 44 46 66 50 22 44 62 64 51 22 44 62 66
52 22 44 64 66 53 22 46 62 64 54 22 46 62 66 55 22 46 64 66 56 22
62 64 66 57 24 26 42 44 58 24 26 42 46 59 24 26 42 62 60 24 26 42
64 61 24 26 42 66 62 24 26 44 46 63 24 26 44 62 64 24 26 44 64 65
24 26 44 66 66 24 26 46 62 67 24 26 46 64 68 24 26 46 66 69 24 26
62 64 70 24 26 62 66 71 24 26 64 66 72 24 42 44 46 73 24 42 44 62
74 24 42 44 64 75 24 42 44 66 76 24 42 46 62 77 24 42 46 64 78 24
42 46 66 79 24 42 62 64 80 24 42 62 66 81 24 42 64 66 82 24 44 46
62 83 24 44 46 64 84 24 44 46 66 85 24 44 62 64 86 24 44 62 66 87
24 44 64 66 88 24 46 62 64 89 24 46 62 66 90 24 46 64 66 91 24 62
64 66 92 26 42 44 46 93 26 42 44 62 94 26 42 44 64 95 26 42 44 66
96 26 42 46 62 97 26 42 46 64 98 26 42 46 66 99 26 42 62 64 100 26
42 62 66 101 26 42 64 66 102 26 44 46 62 103 26 44 46 64 104 26 44
46 66 105 26 44 62 64 106 26 44 62 66 107 26 44 64 66 108 26 46 62
64 109 26 46 62 66 110 26 46 64 66 111 26 62 64 66 112 42 44 46 62
113 42 44 46 64 114 42 44 46 66 115 42 44 62 64 116 42 44 62 66 117
42 44 64 66 118 42 46 62 64 119 42 46 62 66 120 42 46 64 66 121 42
62 64 66 122 44 46 62 64 123 44 46 62 66 124 44 46 64 66 125 44 62
64 66 126 46 62 64 66
Results
[0485] FIG. 61 illustrates the fluorescence signals produced by
binding of cholera toxin to the microarray of candidate artificial
receptors alone and in competition with each of the three
concentrations of GM1. The magnitude of the fluorescence signal
decreases steadily with increasing concentration of GM1. The amount
of decrease is not quantitatively identical for all of the
receptors, but each receptor experienced decreased binding of
cholera toxin. These decreases indicate that GM1 competed with the
artificial receptor for binding to the cholera toxin.
[0486] The decreases show a pattern of relative competition for the
binding site on cholera toxin. This can be demonstrated through
graphs of fluorescence signal obtained at a particular
concentration of GM1 against fluorescence signal in the absence of
GM1 (not shown). Certain of the receptors appear at similar
relative positions on these plots as concentration of GM1
increases.
[0487] The disruption in cholera toxin binding caused by GM1 can be
visualized as the ratio of the amount bound in the absence of GM1
OS to the amount bound upon competition with GM1. This ratio is
illustrated in FIG. 62. The larger the ratio, the more cholera
toxin remained bound to the artificial receptor upon competition
with GM1. The ratio can be as large as about 14. The ratios are
independent of the quantity bound in the control.
[0488] Interestingly, in several instances minor changes in
structure to the artificial receptor caused significant changes in
the ratio. For example, the artificial receptor including building
blocks 24, 26, 46, and 66 differs from that including 24, 42, 46,
and 66 by only substitution of a single building block. (xy
indicates building block TyrAxBy.) The substitution of building
block 42 for 26 increased binding in the presence of GM1 by about
14-fold.
[0489] By way of further example, the artificial receptor including
building blocks 22, 24, 46, and 64 differs from that including 22,
46, 62, and 64 by only substitution of a single building block. The
substitution of building block 24 for 62 increased binding in the
presence of GM1 by about 3-fold.
[0490] Even substitution of a single recognition element affected
binding. The artificial receptor including building blocks 22, 24,
42, and 44 differs from that including 22, 24, 42, and 46 by only
substitution of a single recognition element. The substitution of
building block 44 for 46 (a change of recognition element B6 to B4)
increased binding in the presence of GM1 by about 3-fold.
Conclusions
[0491] This experiment demonstrated that binding of a test ligand
to an artificial receptor of the present invention can be
diminished (e.g., competed) by a candidate disruptor molecule. In
this case the test ligand was the protein cholera toxin and the
candidate disruptor was a compound known to bind to cholera toxin,
GM1. Minor changes in structure of the building blocks making up
the artificial receptor caused significant changes in the
competition.
Example 6
GM1 Employed as a Building Block Alters Binding of Cholera Toxin to
the Present Artificial Receptors
[0492] Microarrays of candidate artificial receptors were made, GM1
was bound to the arrays, and they were evaluated for binding of
cholera toxin. The results obtained demonstrate that adding GM1 as
a building block in an array of artificial receptors can increase
binding to certain of the receptors.
Materials and Methods
[0493] Building blocks were synthesized and activated as described
in Example 1. The building blocks employed in this example were
those described in Example 4. Microarrays for the evaluation of the
171 n=2 candidate receptor environments were prepared as described
above for Example 4. The test ligand employed in these experiments
was cholera toxin labeled with the Alexa.TM. fluorophore (Molecular
Probes Inc., Eugene, Oreg.). Cholera toxin was employed at 0.01
ug/ml (0.17 pM) or 0.1 ug/ml (1.7 pM) in both the control and the
competition experiments. GM1 was employed as a test ligand for the
artificial receptors and became a building block for receptors used
to bind cholera toxin. The arrays were contacted with GM1 at either
100 .mu.g/ml, 10 .mu.g/ml, or 1 .mu.g/ml as described above for
cholera toxin and then rinsed with deionized water. The arrays were
then contacted with cholera toxin under the conditions described
above. Microarray analysis was conducted as described for Example
4. Table 2 identifies the building blocks in each receptor
environment.
Results
[0494] FIG. 63 illustrates the fluorescence signals produced by
binding of cholera toxin to the microarray of candidate artificial
receptors without pretreatment with GM1. Binding of GM1 to the
microarray of candidate artificial receptors followed by binding of
cholera toxin produced fluorescence signals illustrated in FIGS.
64, 65, and 66 (100 .mu.g/ml, 10 .mu.g/ml, and 1 .mu.g/ml GM1,
respectively).
[0495] The enhancement of cholera toxin binding caused by
pretreatment with GM1 can be visualized as the ratio of the amount
bound in the presence of GM1 to the amount bound in the absence of
GM1. This ratio is illustrated in FIG. 67 for 1 .mu.g/ml GM1. The
larger the ratio, the more cholera toxin bound to the artificial
receptor after pretreatment with GM1. The ratio can be as large as
about 16.
[0496] In several instances minor changes in structure to the
artificial receptor caused significant changes in the ratio. For
example, the artificial receptor including building blocks 46 and
48 differs from that including 46 and 88 by only substitution of a
single recognition element on a single building block. (xy
indicates building block TyrAxBy.) The substitution of building
block 48 for 88 (a change of recognition element A8 to A4)
increased the ratio representing increased binding the presence of
GM1 building block from about 0.5 to about 16. Similarly, the
artificial receptor including building blocks 42 and 77 differs
from that including 24 and 77 by only substitution of a single
building block. The substitution of building block 42 for 24
increased the ratio representing increased binding the presence of
GM1 building block from about 2 to about 14.
[0497] Interestingly, several building blocks that exhibited high
levels of binding of cholera toxin (signals of 45,000 to 65,000
fluorescence units) and that include the building block 33 were not
strongly affected by the presence of GM1 as a building block.
Conclusions
[0498] This experiment demonstrated that binding of GM1 to an
artificial receptor of the present invention can significantly
increase binding by cholera toxin. Minor changes in structure of
the building blocks making up the artificial receptor caused
significant changes in the degree to which GM1 enhanced binding of
cholera toxin.
Discussion of Examples 4-6
[0499] We have previously demonstrated that an array of working
artificial receptors bind to a protein target in a manner which is
complementary to the specific environment presented by each region
of the proteins surface topology. Thus the pattern of binding of a
protein target to an array of working artificial receptors
describes the proteins surface topology; including surface
structures which participate in e.g., protein.about.small molecule,
protein.about.peptide, protein-protein, protein.about.carbohydrate,
protein.about.DNA, etc. interactions. It is thus possible to use
the binding of a selected protein to a working artificial receptor
array to characterize these protein.about.small molecule,
protein.about.peptide, protein-protein, protein.about.carbohydrate,
protein.about.DNA, etc. interactions. Moreover, it is possible to
utilize the protein to array interactions to define "leads" for the
disruption of these interactions.
[0500] Cholera Toxin B sub-unit binds to GM1 on the cell surface
(structure of GM1). Studies to identify competitors to this binding
event have shown that competitors to the cholera toxin: GM1 binding
interaction (binding site) can utilize both a sugar and an
alkyl/aromatic functionality (Pickens, et al., Chemistry and
Biology, vol. 9, pp 215-224 (2002)). We have previously
demonstrated that fluorescently labeled Cholera Toxin B sub-unit
binds to arrays of working artificial receptors to give a defined
binding pattern which (vida infra) reflects cholera toxin B's
surface topology. For this study, we sought to demonstrate that the
binding of the cholera toxin to at least some members of the array
could be disrupted using cholera toxins natural ligand, GM1.
[0501] The results presented in the figures clearly demonstrate
that these goals have been achieved. Specifically, competition
between the GM1 OS pentasaccharide or GM1 and a working artificial
receptor array for cholera binding clearly gave a binding pattern
which was distinct from the cholera binding pattern control.
Moreover, these results demonstrated the complementarity between
several of the working artificial receptors which contained a
naphthyl moiety when compared to working artificial receptors which
only contained phenyl functionality. These results are in keeping
with the active site competition studies in Pickens, et al. and
indicate that the naphthyl and phenyl derivatives represent good
mimics/probes for the cholera to GM1 interaction. The specificity
of these interactions was particularly demonstrated by the
observation that the change of a single building block out of 4 in
a combination of 4 building blocks system changed a non-competitive
to a significantly competitive environment. These results also
indicated that selected working artificial receptors can be used to
develop a high-throughput screen for the further evaluation of the
cholera: GM1 interaction.
[0502] Additionally, we sought to demonstrate that an affinity
support/membrane mimic could be prepared by pre-incubating an array
of artificial receptors with GM1 which would then bind/capture
cholera toxin in a binding pattern which could be used to select a
working artificial receptor(s) for, for example, the
high-throughput screen of lead compounds which will disrupt the
"cholera: membrane.about.GM1 mimic". The GM1 pre-incubation studies
clearly demonstrated that several of the working artificial
receptors which were poor cholera binders significantly increased
their cholera binding, presumably through an affinity interaction
between the cholera toxin and BOTH the immobilized GM1
pentasaccharide moiety and the working artificial receptor building
block environment.
[0503] It should be noted that, as used in this specification and
the appended claims, the singular forms "a," "an," and "the"
include plural referents unless the content clearly dictates
otherwise. Thus, for example, reference to a composition containing
"a compound" includes a mixture of two or more compounds. It should
also be noted that the term "or" is generally employed in its sense
including "and/or" unless the content clearly dictates
otherwise.
[0504] It should also be noted that, as used in this specification
and the appended claims, the phrase "adapted and configured"
describes a system, apparatus, or other structure that is
constructed or configured to perform a particular task or adopt a
particular configuration. The phrase "adapted and configured" can
be used interchangeably with other similar phrases such as arranged
and configured, constructed and arranged, adapted, constructed,
manufactured and arranged, and the like.
[0505] All publications and patent applications in this
specification are indicative of the level of ordinary skill in the
art to which this invention pertains. All of the U.S. patents and
published U.S. patent applications referenced in this application
are incorporated by reference as if fully reproduced herein.
[0506] The invention has been described with reference to various
specific and preferred embodiments and techniques. However, it
should be understood that many variations and modifications may be
made while remaining within the spirit and scope of the
invention.
* * * * *