U.S. patent application number 14/593318 was filed with the patent office on 2015-07-09 for novel chemistry used in biosensors.
This patent application is currently assigned to OHMX CORPORATION. The applicant listed for this patent is OHMX CORPORATION. Invention is credited to MICHAEL J. AHRENS, PAUL A. BERTIN, HARRY B. GRAY, THOMAS J. MEADE.
Application Number | 20150192538 14/593318 |
Document ID | / |
Family ID | 45497165 |
Filed Date | 2015-07-09 |
United States Patent
Application |
20150192538 |
Kind Code |
A1 |
AHRENS; MICHAEL J. ; et
al. |
July 9, 2015 |
NOVEL CHEMISTRY USED IN BIOSENSORS
Abstract
The invention relates to novel compositions of disubstituted
bipyridyl osmium complexes useful for the synthesis of labeled
proteins, nucleic acids, and for the modification of
electrodes.
Inventors: |
AHRENS; MICHAEL J.; (WEST
LAFAYETTE, IN) ; BERTIN; PAUL A.; (WESTERN SPRINGS,
IL) ; GRAY; HARRY B.; (PASADENA, CA) ; MEADE;
THOMAS J.; (EVANSTON, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OHMX CORPORATION |
EVANSTON |
IL |
US |
|
|
Assignee: |
OHMX CORPORATION
EVANSTON
IL
|
Family ID: |
45497165 |
Appl. No.: |
14/593318 |
Filed: |
January 9, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13187142 |
Jul 20, 2011 |
8951400 |
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14593318 |
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12253875 |
Oct 17, 2008 |
8734631 |
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13187142 |
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61366013 |
Jul 20, 2010 |
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61087102 |
Aug 7, 2008 |
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61087094 |
Aug 7, 2008 |
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60980733 |
Oct 17, 2007 |
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Current U.S.
Class: |
205/777.5 ;
29/874; 530/300; 530/391.1; 536/23.1; 536/55; 540/541; 546/12 |
Current CPC
Class: |
Y10T 29/49204 20150115;
C12Q 1/003 20130101; G01N 27/327 20130101; C07F 15/002 20130101;
G01N 33/5438 20130101; Y10T 29/49117 20150115; G01N 33/54353
20130101 |
International
Class: |
G01N 27/327 20060101
G01N027/327; C07F 15/00 20060101 C07F015/00 |
Claims
1. A compound of chemical formula R.sup.1,R.sup.2-bpy-Os(CN).sub.4
##STR00028## wherein R.sup.1 and R.sup.2 may be the same or
different and are chosen from the group consisting of CH.sub.3,
CH.sub.3(CH.sub.2).sub.m, and NH.sub.2(CH.sub.2).sub.n wherein m
and n are independently chosen from the group consisting of 0, 1,
2, 3, 4, 5, and 6.
2-3. (canceled)
4. A compound of formula R.sup.1,R.sup.2-bpy-Os(CN).sub.4
##STR00029## wherein R.sup.1 and R.sup.2 are independently
substituted alkyl groups wherein the substituents are chosen from
the group consisting of an electrophile and a nucleophile.
5-25. (canceled)
26. A method of detecting a target enzyme comprising: a) contacting
a sample with a composition comprising an electrode comprising: i)
an electroactive moiety (EAM) comprising a compound of claim 1 with
a first E.sup.0; ii) a capture ligand; under conditions whereby
said target enzyme, if present, alters said capture ligand such
that said EAM has a second E.sup.0; and b) measuring said second
E.sup.0.
27. The method according to claim 26, wherein said composition
comprises a support comprising a plurality of electrodes each
comprising: i) an electroactive moiety (EAM) comprising a compound
of claim 1 with a first E.sup.04); ii) a capture ligand.
28. A method of detecting a target enzyme comprising: a) contacting
a sample with a composition comprising a REAMC comprising: i) an
electroactive moiety (EAM) comprising a compound of claim 1 with a
first E.sup.0; ii) a capture ligand; under conditions whereby said
target enzyme, if present, alters said capture ligand such that
said EAM has a second E.sup.0; and b) measuring said second
E.sup.0.
29. A composition comprising an electrode comprising: a) a first
species comprising a functional group; and b) an electroactive
complex (EAM), said EAM comprising a compound of claim 1.
30. The composition according to claim 29, wherein said functional
group is a maleimide group.
31. A method of making a biosensor comprising: a) providing an
electrode comprising: i) a first species comprising a first
functional group; and ii) an electroactive complex (EAM), said EAM
comprising a compound of claim 1; c) contacting said electrode with
a biomolecule comprising a second functional group to form a
covalent bond between said first species and said biomolecule.
32. The method according to claim 31, wherein said first functional
group is selected from the group consisting of moieties comprising
a maleimide, imidoester, N-hydroxysuccinimidyl, alkyl halide, aryl
halide, alpha-haloacyl and pryidyl disulfide.
33. The method according to claim 31, wherein said first functional
group comprises a maleimide and said biomolecule is a protein
comprising a cysteine amino acid.
34. The method according to claim 33, wherein said protein is a
peptide.
35. A compound having the formula: Anchor-Spacer 1-EAM-(Spacer
2).sub.n-CL (I) wherein said anchor comprises a cyclic-disulfide
group, EAM is a compound of claim 1, CL is a capture ligand, Spacer
1 is a SAM forming species, and n=0 or 1.
36. The compound according to claim 35, wherein said
cyclic-disulfide group comprises
pyridyl-oligophenylethylnyl-[1,2,5]-dithiazepane.
37. A composition comprising an electrode comprising a
self-assembled monolayer (SAM), wherein said SAM comprises a
compound having the formula: Anchor-Spacer 1-EAM-Spacer 2-CL (II),
wherein said anchor is linked to said electrode group through a
disulfide group, EAM is a compound of claim 1, CL is a capture
ligand, Spacer 1 is either insulating or conducting, and Spacer 2
is optional.
38. A method, comprising: a) providing a compound having the
formula: Anchor-Spacer 1-EAM-(Spacer 2).sub.n-CL (I), wherein said
anchor comprises a cyclic-disulfide group, EAM is a compound of
claim 1, CL is a capture ligand, Spacer 1 is either insulating or
conduction, and n=0 or 1; and contacting said compound to an
electrode by opening said cyclic disulfide to form an attachment of
said anchor to said electrode.
39. The method according to claim 38, wherein said electrode
further comprises a self-assembled monolayer (SAM).
40. A method of detecting a target analyte in a test sample,
comprising: b) providing an electrode comprises a compound having
the formula: Anchor-Spacer 1-EAM-(Spacer 2).sub.n-CL (I), wherein
said anchor comprises a cyclic-disulfide group, EAM is a compound
of claim 1, CL is a capture ligand, Spacer 1 is either insulating
or conduction, and n=0 or 1; and contacting said electrode with
said test sample; and determining the presence of said target
analyte by measuring the reorganization energy of said EAM.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/187,142, filed Jul. 20, 2011, which claims the benefit of
U.S. provisional application No. 61/366,013, filed Jul. 20, 2010
and is a continuation-in-part of U.S. patent application Ser. No.
12/253,875, filed Oct. 17, 2008 which in turn claims the benefit of
priority to U.S. Provisional Patent Application Ser. Nos.
60/980,733, filed on Oct. 17, 2007, and 61/087,094 and 61/087,102,
filed on Aug. 7, 2008, the entire contents and disclosures of each
of which are herein incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates to novel compositions and methods for
the detection of analytes using change in E.sup.0 of target
analytes.
BACKGROUND OF THE INVENTION
[0003] Electron transfer reactions are crucial steps in a wide
variety of biological transformations ranging from photosynthesis
or aerobic respiration. Studies of electron transfer reactions in
both chemical and biological systems have led to the development of
a large body of knowledge and a strong theoretical base, which
describes the rate of electron transfer in terms of a small number
of parameters.
[0004] Electronic tunneling in proteins and other biological
molecules occurs in reactions where the electronic interaction of
the redox centers is relatively weak. Semiclassical theory reaction
predicts that the reaction rate for electron transfer depends on
the driving force (-.DELTA.G.sup.0), a nuclear reorganization
parameter (.lamda.), and the electronic-coupling strength between
the reactants and products at the transition state (H.sub.AB),
according to the following equation:
k.sub.ET=(4.pi..sup.3/h.sup.2.lamda.k.sub.BT).sup.1/2(H.sub.AB).sup.2exp-
[(-.DELTA.G.sup.0+.lamda.)2/.lamda.k.sub.BT]
[0005] The nuclear reorganization energy, .lamda., in the equation
above is defined as the energy of the reactants at the equilibrium
nuclear configuration of the products. For electron transfer
reactions in polar solvents, the dominant contribution to .lamda.
arises from the reorientation of solvent molecules in response to
the change in charge distribution of the reactants. The second
component of .lamda. comes from the changes in bond lengths and
angles due to changes in the oxidation state of the donors and
acceptors.
[0006] Previous work describes using change in reorganization
energy, .lamda., as the basis of novel sensors. See for example,
U.S. Pat. Nos. 6,013,459, 6,013,170, 6,248,229, and 7,267,939, all
herein incorporated by reference in their entirety. The methods
generally comprise binding an analyte to or near a redox active
complex. The redox active complex comprises at least one
electroactive molecule and a capture ligand which will bind the
target analyte, and the complex is bound to an electrode. Upon
analyte binding, the reorganization energy of the redox active
molecule is altered, thus changing the E.sup.0, and allowing
detection.
[0007] It is an object of the present invention to provide
composition and methods for the detection of target analytes using
alterations in the solvent reorganization energy, such as utilizing
cyano ligands with the transition metals of the biosensor,
corresponding to changes in the E.sup.0 of redox active
molecules.
SUMMARY OF THE INVENTION
[0008] The present invention provides methods and compositions
relating to biosensors for use in the detection of target
analytes.
[0009] In one aspect, the invention provides compositions
comprising a solid support (sometimes
[0010] referred to herein as a "substrate") comprising an electrode
comprising a covalently attached electroactive complex (EAM) with a
particular E.sup.0. The substrates can optionally comprise an array
of electrodes. The electrode(s) each comprise an EAM, that
optionally can be part of a ReAMC. Suitable transition metals
include iron, ruthenium and osmium, as well as others outlined
herein. In some embodiments, the EAMs comprise at least one cyano
ligand, with 2, 3, 4 and 5 also finding use in the invention. The
EAMs (as well as the ReAMCs and diluent SAM forming species) can be
linked to the electrodes using attachment linkers, including alkyl
groups (including substituted alkyl groups).
[0011] In a further aspect, the electrodes optionally comprise self
assembled monolayer (SAM) species.
[0012] In an additional aspect, the EAM/ReAMCs of the invention are
attached to the electrode using an anchor ligand, which can be
"unipodal" or "multipodal", for example including the use of
bipodal attachments such as two sulfur atoms or cyclic disulfide
anchor groups.
[0013] In a further aspect, the EAM is part of a redox active
capture complex (REAMC) comprising said EAM and a capture ligand.
In one aspect, the capture ligand provides a coordination atom for
the transition metal. In additional aspects, the capture ligand is
separate from the EAM, such that the electrode comprises a first
species comprising the EAM and a second species comprising a
capture ligand.
[0014] In one aspect, the capture ligand is a protein, including
peptides, or a carbohydrate.
[0015] In an additional aspect, the invention provides methods of
detecting a target analytes comprising contacting a sample with a
composition comprising an electrode as outlined herein. The binding
of the target analyte to the capture ligand alters the E.sup.0 of
the EAM, e.g. creating a second E.sup.0, which is measured to
determine the presence or absence of the target analyte.
[0016] In a further aspect, the invention provides methods of
making a biosensor comprising providing an electrode comprising a
first species (usually a SAM forming species) comprising a first
functional group. The electrode is contacted with a biomolecule
(which will become the capture ligand) comprising a second
functional group to form a covalent bond between the first species
and the biomolecule. The electrode also comprises an electroactive
complex (EAM), to form the biochips of the invention. In some
aspects the functional groups on each molecule are selected from
the group consisting of moieties comprising a maleimide,
imidoester, N-hydroxysuccinimidyl, alkyl halide, aryl halide,
alpha-haloacyl and pryidyl disulfide and cysteines (e.g. the first
functional group comprises a maleimide and the biomolecule is a
protein (e.g. peptide) comprising a cysteine amino acid.
[0017] In an additional aspect, the invention comprises compounds
having the formula:
Anchor-Spacer 1-EAM-(Spacer 2).sub.n-CL (I),
wherein said anchor comprises a cyclic-disulfide group,
[0018] EAM is an electroactive moiety comprises a solvent
accessible redox compound,
[0019] CL is a capture ligand,
[0020] Spacer 1 is a SAM forming species, and
[0021] n=0 or 1.
[0022] In an additional aspect, the invention comprises compounds
having the formula:
Anchor-Spacer 1-EAM-(Spacer 2).sub.n-CL (I),
wherein EAM is an electroactive moiety comprising a transition
metal and at least one charge-neutralizing ligand. The charge
neutralizing ligand can be selected from the group consisting of:
dithiocarbamate, benzenedithiolate, a Schiff base, EDTA, DTPA,
carboxylate, amine, thiolate, phosphine, imidazole, pyridine,
bipyridine, terpyridine, tacn, salen, acacen, Cp, pincer,
scorpionates and pentaammine.
[0023] In one aspect, the invention provides compositions which are
EAMs comprising osmium metal atoms having four cyano ligands and
one bipyridyl ligand wherein the bipyridyl ligand is substituted
independently with two moieties chosen from the group consisting of
alkyl moieties and aminoalkyl moieties.
[0024] In a further aspect the EAMs have the chemical formula
R.sup.1,R.sup.2-bpy-Os(CN).sub.4 wherein bpy is bipyridyl and
R.sup.1 and R.sup.2 are independently chosen from the group alkyl
moiety and aminoalkyl moiety. The structural Formula (IV) of
R.sup.1,R.sup.2-bpy-Os(CN).sub.4 is as follows:
##STR00001##
[0025] In a further embodiment of the invention the bipyridyl group
of R.sup.1,R.sup.2-bpy-Os(CN).sub.4 is either 4,4' disubstituted
(Formula IVa) or 5,5' disubstituted (Formula IVb).
##STR00002##
[0026] In yet another embodiment of the invention, R.sup.1 and
R.sup.2 are independently moieties comprising substituted alkyl
groups wherein the alkyls are substituted with moieties chosen from
the group consisting of carboxylic acid, amine, maleimide,
succinimide, N-hydroxysuccinimide, epoxide, and anhydride. As
defined herein, the term "alkyl group" generally refers to linear
or branched C.sub.1-C.sub.20, C.sub.1-C.sub.10, or
C.sub.1-C.sub.6.
[0027] In a further embodiment of the invention, R.sup.1 and
R.sup.2 are independently moieties comprising an electrophile
and/or a nucleophile wherein the electrophile is chosen from the
group consisting of activated ester, acyl azide, acyl halide, acyl
nitrile, aldehydes, alkyl halide, alkyl halide, alkyl sulfonate,
anhydride, aryl halide, aziridine, boronate, carboxylic acid,
carbodiimide, diazoalkane, epoxide, haloacetamide, halotriazine,
imido ester, isocyanate, isothiocyanate, ketone, maleimide,
phosphoramidite, silyl halide, sulfonate ester, and sulfonyl halide
and the nucleophile is chosen from the group consisting of amine,
aniline, alcohol, phenol, hydrazine, hydroxylamine, carboxylic
acid, thiol, and glycol.
[0028] In yet another embodiment of the invention, R.sup.1 and
R.sup.2 are independently moieties comprising substituted alkyl
groups wherein alkyls are substituted with moieties chosen from the
group consisting of carboxylic acid, amine, maleimide, succinimide,
N-hydroxysuccinimide, epoxide, and anhydride. As defined herein,
the term "alkyl group" generally refers to linear or branched
C.sub.1-C.sub.20, C.sub.1-C.sub.10, or C.sub.1-C.sub.6.
[0029] In yet another embodiment of the invention, R.sup.1 and
R.sup.2 are independently substituted alkyl groups wherein the
substituents are chosen from the group consisting of an
electrophile and a nucleophile.
[0030] In another aspect, the invention provides compositions which
are EAMs comprising a chemical compound having the chemical formula
R.sup.1,R.sup.2-bpy-Os(CN).sub.4 (See structural Formula IV)
wherein R.sup.1 and R.sup.2 may be the same or different and are
chosen from the group consisting of CH.sub.3,
CH.sub.3(CH.sub.2).sub.m, and NH.sub.2(CH.sub.2).sub.n wherein m
and n are independently chosen from the group consisting of 0, 1,
2, 3, 4, 5, and 6. The bpy may be chosen from the group consisting
of 4,4' substituted bpy (Formula IVa) and 5,5' substituted bpy
(Formula IVb).
[0031] In another aspect, the invention comprises representative
compounds of the formula R.sup.1,R.sup.2-bpy-Os(CN).sub.4 wherein
R.sup.1 and R.sup.2 are as defined above selected from the group
consisting of:
##STR00003## ##STR00004##
[0032] In another aspect. the invention provides a composition
comprising a compound of formula R.sup.1,R.sup.2-bpy-Os(CN).sub.4
wherein R.sup.1 and R.sup.2 are as defined above.
[0033] In another aspect, the invention provides compositions
comprising an electrode comprising a covalently attached
electroactive complex (EAM), said EAM comprising a compound of
formula R.sup.1,R.sup.2-bpy-Os(CN).sub.4 wherein R.sup.1 and
R.sup.2 are as defined above.
[0034] In another aspect, the invention provides a method of
detecting a target enzyme comprising:
[0035] a) contacting a sample with a composition comprising an
electrode comprising: [0036] i) an electroactive moiety (EAM)
comprising a compound of of formula
R.sup.1,R.sup.2-bpy-Os(CN).sub.4 wherein R.sup.1 and R.sup.2 are as
defined above. with a first E.sup.0; [0037] ii) a capture ligand;
[0038] under conditions whereby said target enzyme, if present,
alters said capture ligand such that said EAM has a second E.sup.0;
and
[0039] b) measuring said second E.sup.0.
[0040] In one embodiment of the invention, the composition
comprises a support comprising a plurality of electrodes each
comprising:
[0041] i) an electroactive moiety (EAM) comprising a compound of
formula R.sup.1,R.sup.2-bpy-Os(CN).sub.4 wherein R.sup.1 and
R.sup.2 are as defined above.
with a first E0;
[0042] ii) a capture ligand.
[0043] In another aspect, the invention provides a method of
detecting a target enzyme comprising:
[0044] a) contacting a sample with a composition comprising a REAMC
comprising: [0045] i) an electroactive moiety (EAM) comprising a
compound of formula R.sup.1,R.sup.2-bpy-Os(CN).sub.4 wherein
R.sup.1 and R.sup.2 are as defined above. with a first E.sup.0;
[0046] ii) a capture ligand; [0047] under conditions whereby said
target enzyme, if present, alters said capture ligand such that
said EAM has a second E.sup.0; and
[0048] b) measuring said second E.sup.0.
[0049] In another aspect, the invention provides a composition
comprising an electrode comprising: [0050] a) a first species
comprising a functional group; and [0051] b) an electroactive
complex (EAM), said EAM comprising a compound of formula
R.sup.1,R.sup.2-bpy-Os(CN).sub.4 wherein R.sup.1 and R.sup.2 are as
defined above.
[0052] In one aspect, the functional group is a maleimide
group.
[0053] In another aspect, the invention provides a method of making
a biosensor comprising: [0054] a) providing an electrode
comprising: [0055] i) a first species comprising a first functional
group; and [0056] ii) an electroactive complex (EAM), said EAM
comprising a compound of formula R.sup.1,R.sup.2-bpy-Os(CN).sub.4
wherein R.sup.1 and R.sup.2 are as defined above, [0057] c)
contacting said electrode with a biomolecule comprising a second
functional group to form a covalent bond between said first species
and said biomolecule.
[0058] In one embodiment of the invention, the first functional
group is selected from the group consisting of moieties comprising
a maleimide, imidoester, N-hydroxysuccinimidyl, alkyl halide, aryl
halide, alpha-haloacyl and pryidyl disulfide.
[0059] In another embodiment of the invention, the first functional
group comprises a maleimide and said biomolecule is a protein
comprising a cysteine amino acid. The protein may be a peptide.
[0060] In another aspect, a compound is provided, the compound
having the formula:
Anchor-Spacer 1-EAM-(Spacer 2).sub.n-CL (I)
wherein said anchor comprises a cyclic-disulfide group, EAM is
compound of formula R.sup.1,R.sup.2-bpy-Os(CN).sub.4 wherein
R.sup.1 and R.sup.2 are as defined above. [0061] CL is a capture
ligand, [0062] Spacer 1 is a SAM forming species, and [0063] n=0 or
1.
[0064] In one embodiment of the invention, the cyclic-disulfide
group comprises
pyridyl-oligophenylethylnyl-[1,2,5]-dithiazepane.
[0065] In another aspect, the invention provides a composition
comprising an electrode comprising a self-assembled monolayer
(SAM), wherein said SAM comprises a compound having the
formula:
Anchor-Spacer 1-EAM-Spacer 2-CL (II),
wherein said anchor is linked to said electrode group through a
disulfide group, [0066] EAM is a compound of formula
R.sup.1,R.sup.2-bpy-Os(CN).sub.4 wherein R.sup.1 and R.sup.2 are as
defined above, [0067] CL is a capture ligand, [0068] Spacer 1 is
either insulating or conducting, and [0069] Spacer 2 is
optional.
[0070] In another aspect, the invention provides a method
comprising:
[0071] a) providing a compound having the formula:
Anchor-Spacer 1-EAM-(Spacer 2).sub.n-CL (II)
wherein said anchor comprises a cyclic-disulfide group,
[0072] EAM is a compound of formula
R.sup.1,R.sup.2-bpy-Os(CN).sub.4 wherein R.sup.1 and R.sup.2 are as
defined above, CL is a capture ligand,
[0073] Spacer 1 is either insulating or conduction, and [0074] n=0
or 1; and [0075] b) contacting said compound to an electrode by
opening said cyclic disulfide to form an attachment of said anchor
to said electrode.
[0076] An one embodiment of the invention, an electrode further
comprises a self-assembled monolayer (SAM).
[0077] In another aspect, the invention provides a method of
detecting a target analyte in a test sample, comprising: [0078] c)
providing an electrode comprises a compound having the formula:
[0078] Anchor-Spacer 1-EAM-(Spacer 2).sub.n-CL (I),
wherein said anchor comprises a cyclic-disulfide group, [0079] EAM
is compound of formula R.sup.1,R.sup.2-bpy-Os(CN).sub.4 wherein
R.sup.1 and R.sup.2 are as defined above, [0080] CL is a capture
ligand, [0081] Spacer 1 is either insulating or conduction, and
[0082] n=0 or 1; and [0083] d) contacting said electrode with said
test sample; and [0084] e) determining the presence of said target
analyte by measuring the reorganization energy of said EAM.
[0085] These and other aspects of the invention will be apparent in
light of the description below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0086] FIGS. 1A-1C depicts several compounds of the invention. FIG.
1A shows a compound comprising a capture ligand at one end that is
linked to a redox here (here is shown to contain a Ruthenium
example) through a spacer. The compound also comprises an anchor,
through which the compound is attached to the surface of an
electrode. Also shown are insulators (110) that are attached to the
surface of the electrode as well. FIGS. 1B and 1C depicts a
compound comprises multiple metals. The geometries such as the one
shown, where CL is "capture ligand", will attract a single protein
and have it interact with two metal centers simultaneously giving a
larger change in potential.
[0087] FIG. 2 depicts [BIM-Ru(NH3)4L]2+ complexes with BPAI anchors
and [Ru(NH3)5L]2+ complexes with BPA anchors.
[0088] FIG. 3 depicts BIPOD based compounds.
[0089] FIG. 4A depicts [BIM-Ru(NH.sub.3).sub.4L].sup.2+ complexes
with alkylthiol anchors. FIG. 4B depicts
[Ru(NH.sub.3).sub.5L].sup.2+ complexes with conjugated thiol
anchors.
[0090] FIG. 5A depicts new architectures for Ru--N based complexes.
FIG. 5B depicts examples of Ru--N based complexes.
[0091] FIG. 6A schematically depicts modified Prussian blue surface
for detection with amplification. FIG. 6B depicts the use of crown
ether coordination to enhance potential shift.
[0092] FIG. 7 depicts the effect of second-sphere coordination, the
adduct formation between Ru(NH.sub.3).sub.5L and 18-C-6.
[0093] FIGS. 8A, 8B-1, and 8B-2 depict ligands used in the "single"
and "side-by-side" arrangement when multiple metals are used.
[0094] FIGS. 9A-B depict some of the building blocks for generating
the compound for detection of analyte.
[0095] FIGS. 10A and 10B depict some exemplary compounds.
[0096] FIG. 11 depicts some exemplary compounds.
[0097] FIGS. 12A-C and 13 depict several schematics of suitable
geometries of the present invention. FIGS. 12A and C depict the
situation where a linker is attached at one end to the electrode
and the other end terminates in a ligand (L) that provides a
coordination atom for the transition metal (TM). The capture
substrate (CS) provides an additional ligand (not depicted), and a
plurality of other ligands provide the remaining coordination
atoms. Upon action by the enzyme, the capture substrate results in
a leaving group (X). It should be noted that these Figures depicts
a situation where the transition metal utilizes 6 coordination
atoms, but other numbers of coordination atoms can be used,
depending on the metal. Similarly, these Figures depicts the use of
ligands that provide a single coordination atom, but fewer ligands
providing multiple coordination atoms (e.g. multidentate) ligands
can be used as well. FIG. 12B depicts the situation where the
capture substrate and the EAM are attached separately to the
electrode. FIG. 12C depicts a similar situation to FIG. 3A, except
the capture substrate does not provide a coordination atom to the
transition metal. It should be appreciated that solution phase
systems can be similar to FIGS. 12 and 14, in that the
electrochemical potential of the EAM in solution can be altered as
a result of the enzymatic activity of the target enzyme.
[0098] FIG. 14 depicts a general scheme for producing the biochips
of the invention.
[0099] FIG. 15 shows a specific example of the production of FIG.
14.
[0100] FIGS. 16A, B, C and D depict some exemplary compounds.
[0101] FIGS. 17A, B, C and D depict some exemplary compounds.
Similar compounds can be constructed with different anchors, such
as disulfide cyclic anchor groups, for example, or different
spacers.
[0102] FIGS. 18A and B depict some exemplary compounds. Similar
compounds can be constructed with different anchors, such as
disulfide cyclic anchor groups, for example, or different
spacers.
[0103] FIG. 19 depicts a general scheme of synthesis.
[0104] FIG. 20 depicts a general detection scheme.
[0105] FIGS. 21A, B, C, D and E depict some exemplary compounds.
Similar compounds can be constructed with different anchors, such
as disulfide cyclic anchor groups, for example, or different
spacers.
[0106] FIG. 22 depicts a general scheme of synthesis.
[0107] FIG. 23 depicts a general scheme of synthesis for an assay,
also described in the U.S. Ser. No. 12/253,875, filed Oct. 17,
2008, entitled "Novel Chemistry used in Biosensors", hereby
expressly incorporated by reference in its entirety.
[0108] FIG. 24 depict capture ligands. For the carboxylic acid
reactant capture ligands: Anthrax Peptide is SEQ ID NO: 1 and SEB
Peptide is SEQ ID NO: 2. For the maleimide reactive capture
ligands: E. coli Peptide is SEQ ID NO: 3, PSA Peptide is SEQ ID NO:
4; SEB Peptide is SEQ ID NO: 5, and Anthrax Peptide is SEQ ID NO:
6.
[0109] FIGS. 25A, B, C and D depict some exemplary compounds.
Similar compounds can be constructed with different anchors, such
as disulfide cyclic anchor groups, for example, or different
spacers.
[0110] FIGS. 26A, B, C and D depict some exemplary compounds using
ferrocene as the EAM. Similar compounds can be constructed with
different anchors, such as disulfide cyclic anchor groups, for
example, or different spacers.
[0111] FIGS. 27 A and B depict some exemplary compounds. Similar
compounds can be constructed with different anchors, such as
disulfide cyclic anchor groups, for example, or different
spacers.
[0112] FIG. 28 shows the electronic spectra for Compounds 301, 302,
303, 304, 305, and 306.
[0113] FIG. 29 shows the electronic spectra for Compounds 301, 302,
303, 304, 305, and 306 in water at pH 1.5 (gray line), pH 5.5 (thin
black line) and pH 12 (thick black line).
[0114] FIG. 30 shows exemplary cyclic voltammograms for Compound
305 at pH 1.5 (top), pH 5.5 (center) and pH 12 (bottom).
DETAILED DESCRIPTION OF THE INVENTION
[0115] The present invention is directed to improvements in
electrochemical biosensors that rely on changes in the
reorganization energy, .lamda., upon interaction of the target
analyte and the biosensor, as evidenced by alterations in the
observed E.sup.0. As shown previously, biosensors have been
described that rely on changes in reorganization energy. The
present invention has shown surprising improvements such as
utilizing cyano ligands for the transition metal of the
electroactive moieties (EAMs). The cyano ligands provide a
surprising increase in the change of the E.sup.0; e.g., the delta
in the E.sup.0 is higher than seen for other charged ligands.
I. Overview of Reorganization Enemy
[0116] The present invention provides methods and compositions for
the detection of target analytes using changes in the
reorganization energy of redox active molecules upon binding of the
analytes, to facilitate or hinder electron transfer between the
redox active molecule and an electrode. This invention is based on
the fact that when a redox active molecule, such as a transition
metal ion, is either oxidized (losing an electron) or reduced
(gaining an electron), changes occur in the molecular structure as
well as in its immediate solvent environment. These changes in the
molecules structure (bond lengths and angles) and in the
organization of the solvent molecules surrounding the molecule
serve to stabilize the new oxidation state energetically. The sum
of these changes constitute the reorganization energy, .lamda., of
a redox reaction. The intramolecular changes are termed the
inner-sphere reorganization energy, .lamda., and the changes in the
solvent and environment are termed the outer-sphere or solvent
reorganization energy, .lamda..sub.o.
[0117] For the purposes of this invention, the primary focus is on
changes in the solvent reorganization energy although changes in
the inner-sphere reorganization will also be considered in several
embodiments of the invention. It is the intent of this invention to
capitalize on changes in reorganization energy of a redox reaction
when an electroactive molecule (EAM) is attached to a capture
ligand (CL) which can selectively bind to an analyte of interest
(e.g., protein or bacteria). Binding of the EAM-CL to the analyte
results in a change in the solvent environment of the EAM so that
the reorganization energy for a redox reaction involving the EAM is
changed. For the case where the redox reaction involves electron
transfer between an electrode and the EAM, the standard potential,
E.sup.0, is changed. Thus, a change in E.sup.0 for an EAM-CL
complex is an indication that it is bound to the analyte. It is the
intent of this invention to detect the change in E.sup.0 as an
indicator of binding and, consequently, the presence or absence of
the analyte.
[0118] In conventional methodologies for analyte detection using
electron transfer usually employ the EAM as a label or tag attached
to one member of a binding pair (e.g., antibody and antigen). In
these methods, EAM's are chosen in which the outer sphere solvent
effect is minimal, by using electroactive molecules that have
minimal solvent reorganization upon oxidation or reduction. Such
EAMs generally comprise large hydrophobic ligands which have little
interaction with water. Thus, the ligands for the transition metal
ions traditionally used are non-polar and are generally
hydrophobic, frequently containing organic rings (e.g., bipyridyl
and terpyridyl). Such EAMs are chosen because conventionally
because the magnitude of the total electron transfer reaction is
measured (current) at a predetermined electrode potential.
[0119] Without being bound by theory, it is expected that the redox
molecules best suited for this invention will be those whose redox
reaction has a large solvent reorganization energy in aqueous
environments. Solvent reorganization to stabilize an increase or
decrease in charge can be attributed to several phenomena. In polar
solvents such as water, the charge on a redox molecule is
stabilized by orientation of the polar solvent molecules in the
environment near the redox molecule. Since polar molecules have
slight charge variation on different atoms of the molecule, their
orientation around the redox molecule can help to stabilize it.
Further, some ligands, such as CN.sup.-, themselves are polar and
have partial charges on atoms. These polar ligands can themselves
induce an orientation of surrounding solvent molecules.
Stabilization (or destabilization) of charged redox molecules can
also occur by hydrogen bonding of solvent and/or other molecules to
the ligands of the transition metal in the redox molecule. Solvent
molecules, as well as other molecules in the solvent surrounding a
redox molecules can be characterized and compared based on their
donor number or acceptor number (Neyhart et al., J. Am. Chem. Soc
118 (1996) 3724-29, incorporated herein by reference). The use of a
particular solvent or a particular additive to a solvent of a
molecule having a preferred donor or acceptor number would affect
the solvent reorganization energy of a redox reaction. Further, a
change in the charge of a redox molecule is stabilized by charged
ion in the solvent. Thus, changes in solvent reorganization change
upon analyte binding can be maximized by the proper choice of an
electrolyte, considering the charge on the ions, the concentration
of the ions, the size of the ions, and the hydrophobicity of the
ions.
[0120] Without being bound by theory, it is preferred to maximize
the stabilization of the redox molecule (i.e., maximize its solvent
reorganization energy) in the solvent system of choice in order
that the phenomena which stabilize the redox molecule are disrupted
upon binding of the redox molecule/capture ligand complex, EAM-CL
to the analyte. Under such conditions, one would expect that the
change in reorganization energy, evidenced by a change in E.sup.0,
would be optimum. It is expected that the binding of the CL to the
analyte will "force" the EAM into an environment on the surface or
in a cleft or pocket of the analyte (e.g., a protein) which will be
less favorable to the optimal organization of the solvent
environment. In one embodiment it is expected that binding would
cause a shedding of water molecules near the EAM because of steric
constraints.
[0121] It should be noted, and not being bound by theory, that
whether the solvent reorganization energy increases or decreases
upon binding (and whether E.sup.0 moves to more positive or to more
negative potentials is dependent upon the particular charge of the
EAM. If the EAM redox reaction being monitored results in an
increased charge of the EAM, such as EAM.sup.2+ oxidation to
EAM.sup.3+, then the bound environment of the EAM-CL would be less
stabilized by reorganization than the unbound EAM-CL. Hence, one
would expect the E.sup.0 to move to more positive potentials.
Alternatively, if the EAM redox reaction being monitored results in
a decreased charge of the EAM, such as EAM.sup.2- oxidation to
EAM.sup.-, then the unbound EAM-CL would be less stabilized by
reorganization than the bound EAM-CL. Hence, one would expect the
E.sup.0 to move to less positive potentials.
[0122] Without being bound by theory, there are two general
mechanisms which may be exploited in the present invention. The
first relates to inner sphere change due to the redox label. In
this embodiment, the binding of a target analyte to a capture
ligand which is sterically close to an EAM causes one or more of
the small, polar ligands of the EAM to be replaced by one or more
coordination atoms supplied by the target analyte, causing a change
in the inner-sphere reorganization energy for at least two reasons.
First, the exchange of a small, polar ligand for a putatively
larger ligand will generally exclude more water from the metal,
lowering the required solvent reorganization energy (i.e. an inner
sphere .lamda..sub.i effect). Secondly, the proximity of a
generally large target analyte to the relatively small redox active
molecule will sterically exclude water within the first or second
coordination sphere of the metal ion, also changing the solvent
reorganization energy.
[0123] Alternatively, the invention relies on substitutionally
inert ligand, plus outer sphere effects. In this embodiment
exchange of the polar ligands on the metal ion by a target analyte
coordination atom. Rather, in this embodiment, the polar ligands
are effectively irreversibly bound to the metal ion, and the change
in solvent reorganization energy is obtained as a result of the
exclusion of water in the first or second coordination sphere of
the metal ion as a result of the binding of the target analyte;
essentially the water is excluded (i.e. an outer sphere
.lamda..sub.o effect).
[0124] The present invention provides compounds with novel
architecture and methods of using these compounds for detection of
target analytes.
[0125] In some embodiments, the target analyte binds to the capture
ligand. In some embodiments, the target analyte can be an enzyme,
and the change in E.sup.0 is as a result of an enzymatic event, as
described in U.S. Patent Application No. 61/087,094, hereby
incorporated by reference in its entirety.
[0126] In the embodiments of the invention, there is a change in
the E.sup.0, presumably due to a change in the reorganization
energy, upon the introduction of the target analyte. As discussed
more fully below, the change may be either a positive or negative
shift in E0, depending on a variety of factors. In general, when
cyano ligands are used, the change in E.sup.0 can be a negative
shift in E.sup.0, although depending on the system and the other
ligands used (if any), the effect of interaction of the target
analyte with the capture ligand can result in a positive shift in
E.sup.0. Surprisingly, shifts of greater than about 50 mV, 100 mV,
150 mV, 200 mV, 250 mV and 300 mV can be seen using cyano
ligands.
[0127] CN-- is a good nucleophile and in an example, the Iron-cyano
(-2) complex has a partial charge on the N of a cyano of -0.8767
and has a partial charge on the C of a cyano of 0.5549. That charge
number on the nitrogen is very large that one can consider it
acting like there is a lone pair of electrons. Therefore, the
partial negative charge on the N arranges water molecules with the
protons of water oriented toward the N (the opposite is true for
NH3 ligands). Since the partial charge on the nitrogen is high,
this local orientation effect is strong and therefore the delta
between water nearby (before target capture) and water excluded
(after target capture) is high . . . i.e., the difference in lambda
between the two states is quantitatively higher.
[0128] Unidentate CN-- always binds through the carbon and
therefore that large partial negative charge resides on the N. The
larger that partial negative charge oriented toward the solvent
(water) the larger the observable effect will be. As such, ligands
with partial negative charges on the ligands stabilize high
oxidation state metals, and have a strong impact on the orientation
of water.
[0129] CN being the best because it has the highest net charge on
the N and therefore the strongest interaction with protons of
water:
CN.sup.->NO.sub.2.sup.->SO.sub.3.sup.2->NCS.sup.->NO
equivalent to NCS.sup.- is SCN.sup.-.
[0130] In general, the more positive the Metal center becomes the
higher the potential of the metal. Accordingly, when most if not
all the negative charges are neutralized by interaction with water,
the metal becomes more positive.
II. Geometries of the Sensors
[0131] The present invention is directed to methods and
compositions for detection of target analytes, based on a change of
electrochemical potential, E.sup.0, of a redox active molecule
either on the surface of an electrode, or in some cases, in
solution (while most of the description herein is directed to solid
phase assays, as will be appreciated by those in the art, the
invention can be used in solution as well, and such description
herein is meant to apply as applicable to solution phase assays as
well).
[0132] In general, the invention can be described as follows. A
redox active molecule, generally comprising a transition metal and
at least one ligand (such as one cyano ligand (or more, as
described herein)) that provide coordination atoms for the
transition metal, is attached to the surface of an electrode,
generally through a linker as described herein. In addition, the
electrode may also optionally comprise a self-assembled monolayer
(SAM) as described herein. In the spatial vicinity of the redox
active molecule, a capture ligand is also attached, generally in
one of three ways, as described herein. Introduction and/or binding
of the target analyte results in a change in the electrochemical
potential of the redox active molecule, which is then detected in a
variety of ways as described herein.
[0133] As depicted in FIGS. 12-14, there are three basic geometries
for the sensor, although the descriptions herein are not meant to
be so limited. In one embodiment, as shown in FIG. 12A, an
electroactive moiety (EAM), comprising a transition metal ion and
ligands that provide coordination atoms for the transition metal
(in some embodiments, at least one of which is a cyano ligand), is
attached to an electrode. In addition, a capture ligand (sometimes
also referred to as a "binding ligand") that will specifically bind
the target analyte is also attached to the electrode. Both species
are generally attached to the electrode using an attachment linker
as described herein. The two species are attached to the electrode
in such a manner that they are spatially close, such that the
E.sup.0 of the EAM is altered upon binding of a target analyte. It
should be noted that a third species, comprising a monolayer
forming species, described below, can also be optionally present on
the electrode. In this embodiment, the EAM species can have the
formula (Ia), the capture ligand species can have the formula (Ib)
and the diluent species can have the formula (Ic):
AG-Spacer 1-EAM (Ia)
AG-Spacer 1-CL (Ib)
AG-Spacer 1-TG, (Ic)
wherein AG is an anchor group, EAM is an electroactive moiety
comprises a solvent accessible redox complex, spacer 1 is a SAM
forming species described herein, CL is a capture ligand, and TG is
a terminal group, with n being 0 or 1.
[0134] In a second embodiment, one of the coordination atoms for
the transition metal of the EAM is provided by the capture ligand,
forming a "redox active moiety complex", or ReAMC. In this
embodiment, the coordination atom can be actually part of the
capture ligand (e.g. if the capture ligand is a peptide, an amino
group can provide the coordination atom) or part of a linker used
to attach the capture ligand (e.g. a pyridine linker, etc.). The
ReAMC is attached as a single species, and as above, an additional
species, comprising a monolayer forming species, described below,
can also be optionally present on the electrode. In this
embodiment, the present invention provides a compound having the
formula (II):
AG-Spacer 1-EAM-(Spacer 2).sub.n-CL (II)
wherein AG is an anchor group, EAM is an electroactive moiety
comprises a solvent accessible redox complex, CL is a capture
ligand, spacer 1 is a SAM forming species described herein, and
Spacer 2 is a linker, with n=0 or 1.
[0135] In a third embodiment, as depicted in FIG. 12C, there ReAMC
is a single species, but the capture ligand does not provide a
coordination atom; rather, it is spatially close but distinct from
the EAM of the ReAMC. Again, a third species, comprising a
monolayer forming species, described below, can also be optionally
present on the electrode. In this embodiment, the present invention
provides a compound having the formula (III):
##STR00005##
wherein AG is an anchor group, EAM is an electroactive moiety
comprises a solvent accessible redox complex, CL is a capture
ligand, spacer 1 is a SAM forming species described herein, and
S.sub.2 and S.sub.3 are two linkages that link the EAM and CL
together with the AG to form a branched structure. S.sub.2 and
S.sub.3 can be different or the same.
[0136] One example of this configuration is shown below:
##STR00006##
where M=transitional metal; Ln=coordinating ligand that covalently
connected to the anchor and capture ligand, n=0 or 1; and
L=coordinating ligand.
III. Electrode
[0137] In one aspect, the present invention provides these ligand
architectures attached to an electrode. By "electrode" herein is
meant a composition, which, when connected to an electronic device,
is able to sense a current or charge and convert it to a signal.
Preferred electrodes are known in the art and include, but are not
limited to, certain metals and their oxides, including gold;
platinum; palladium; silicon; aluminum; metal oxide electrodes
including platinum oxide, titanium oxide, tin oxide, indium tin
oxide, palladium oxide, silicon oxide, aluminum oxide, molybdenum
oxide (Mo2O6), tungsten oxide (WO3) and ruthenium oxides; and
carbon (including glassy carbon electrodes, graphite and carbon
paste). Preferred electrodes include gold, silicon, carbon and
metal oxide electrodes, with gold being particularly preferred.
[0138] The electrodes described herein are depicted as a flat
surface, which is only one of the possible conformations of the
electrode and is for schematic purposes only. The conformation of
the electrode will vary With the detection method used. For
example, flat planar electrodes may be preferred for optical
detection methods, or when arrays of nucleic acids are made, thus
requiring addressable locations for both synthesis and detection.
Alternatively, for single probe analysis, the electrode may be in
the form of a tube, with the components of the system such as SAMs,
EAMs and capture ligands bound to the inner surface. This allows a
maximum of surface area containing the nucleic acids to be exposed
to a small volume of sample.
[0139] The electrodes of the invention are generally incorporated
into biochip cartridges and can take a wide variety of
configurations, and can include working and reference electrodes,
interconnects (including "through board" interconnects), and
microfluidic components. See for example U.S. Pat. No. 7,312,087,
incorporated herein by reference in its entirety.
[0140] The biochip cartridges include substrates comprising the
arrays of biomolecules, and can be configured in a variety of ways.
For example, the chips can include reaction chambers with inlet and
outlet ports for the introduction and removal of reagents. In
addition, the cartridges can include caps or lids that have
microfluidic components, such that the sample can be introduced,
reagents added, reactions done, and then the sample is added to the
reaction chamber comprising the array for detection.
[0141] In a preferred embodiment, the biochips comprise substrates
with a plurality of array locations. By "substrate" or "solid
support" or other grammatical equivalents herein is meant any
material that can be modified to contain discrete individual sites
appropriate of the attachment or association of capture ligands.
Suitable substrates include metal surfaces such as gold, electrodes
as defined below, glass and modified or functionalized glass,
fiberglass, Teflon, ceramics, mica, plastic (including acrylics,
polystyrene and copolymers of styrene and other materials,
polypropylene, polyethylene, polybutylene, polyimide,
polycarbonate, polyurethanes, Teflon.TM., and derivatives thereof,
etc.), GETEK (a blend of polypropylene oxide and fiberglass), etc.,
polysaccharides, nylon or nitrocellulose, resins, silica or
silica-based materials including silicon and modified silicon,
carbon, metals, inorganic glasses and a variety of other polymers,
with printed circuit board (PCB) materials being particularly
preferred.
[0142] The present system finds particular utility in array
formats, i.e. wherein there is a matrix of addressable detection
electrodes (herein generally referred to "pads", "addresses" or
"micro-locations"). By "array" herein is meant a plurality of
capture ligands in an array format; the size of the array will
depend on the composition and end use of the array. Arrays
containing from about 2 different capture substrates to many
thousands can be made.
[0143] In a preferred embodiment, the detection electrodes are
formed on a substrate. In addition, the discussion herein is
generally directed to the use of gold electrodes, but as will be
appreciated by those in the art, other electrodes can be used as
well. The substrate can comprise a wide variety of materials, as
outlined herein and in the cited references.
[0144] In general, preferred materials include printed circuit
board materials. Circuit board materials are those that comprise an
insulating substrate that is coated with a conducting layer and
processed using lithography techniques, particularly
photolithography techniques, to form the patterns of electrodes and
interconnects (sometimes referred to in the art as interconnections
or leads). The insulating substrate is generally, but not always, a
polymer. As is known in the art, one or a plurality of layers may
be used, to make either "two dimensional" (e.g. all electrodes and
interconnections in a plane) or "three dimensional" (wherein the
electrodes are on one surface and the interconnects may go through
the board to the other side or wherein electrodes are on a
plurality of surfaces) boards. Three dimensional systems frequently
rely on the use of drilling or etching, followed by electroplating
with a metal such as copper, such that the "through board"
interconnections are made. Circuit board materials are often
provided with a foil already attached to the substrate, such as a
copper foil, with additional copper added as needed (for example
for interconnections), for example by electroplating. The copper
surface may then need to be roughened, for example through etching,
to allow attachment of the adhesion layer.
[0145] Accordingly, in a preferred embodiment, the present
invention provides biochips (sometimes referred to herein "chips")
that comprise substrates comprising a plurality of electrodes,
preferably gold electrodes. The number of electrodes is as outlined
for arrays. Each electrode preferably comprises a self-assembled
monolayer as outlined herein. In a preferred embodiment, one of the
monolayer-forming species comprises a capture ligand as outlined
herein. In addition, each electrode has an interconnection, that is
attached to the electrode at one end and is ultimately attached to
a device that can control the electrode. That is, each electrode is
independently addressable.
[0146] Finally, the compositions of the invention can include a
wide variety of additional components, including microfluidic
components and robotic components (see for example U.S. Pat. Nos.
6,942,771 and 7,312,087 and related cases, both of which are hereby
incorporated by reference in its entirety), and detection systems
including computers utilizing signal processing techniques (see for
example U.S. Pat. No. 6,740,518, hereby incorporated by reference
in its entirety).
A. Self Assembled Monolayer Spacers
[0147] In some embodiments, the electrodes optionally further
comprise a SAM. By "monolayer" or "self-assembled monolayer" or
"SAM" herein is meant a relatively ordered assembly of molecules
spontaneously chemisorbed on a surface, in which the molecules are
oriented approximately parallel to each other and roughly
perpendicular to the surface. Each of the molecules includes a
functional group that adheres to the surface, and a portion that
interacts with neighboring molecules in the monolayer to form the
relatively ordered array. A "mixed" monolayer comprises a
heterogeneous monolayer, that is, where at least two different
molecules make up the monolayer. As outlined herein, the use of a
monolayer reduces the amount of non-specific binding of
biomolecules to the surface, and, in the case of nucleic acids,
increases the efficiency of oligonucleotide hybridization as a
result of the distance of the oligonucleotide from the electrode.
Thus, a monolayer facilitates the maintenance of the target enzyme
away from the electrode surface. In addition, a monolayer serves to
keep charge carriers away from the surface of the electrode. Thus,
this layer helps to prevent electrical contact between the
electrodes and the ReAMs, or between the electrode and charged
species within the solvent. Such contact can result in a direct
"short circuit" or an indirect short circuit via charged species
which may be present in the sample. Accordingly, the monolayer is
preferably tightly packed in a uniform layer on the electrode
surface, such that a minimum of "holes" exist. The monolayer thus
serves as a physical barrier to block solvent accessibility to the
electrode.
[0148] In some embodiments, the monolayer comprises conductive
oligomers. By "conductive oligomer" herein is meant a substantially
conducting oligomer, preferably linear, some embodiments of which
are referred to in the literature as "molecular wires". By
"substantially conducting" herein is meant that the oligomer is
capable of transferring electrons at 100 Hz. Generally, the
conductive oligomer has substantially overlapping Tr-orbitals, i.e.
conjugated Tr-orbitals, as between the monomeric units of the
conductive oligomer, although the conductive oligomer may also
contain one or more sigma (.sigma.) bonds. Additionally, a
conductive oligomer may be defined functionally by its ability to
inject or receive electrons into or from an associated EAM.
Furthermore, the conductive oligomer is more conductive than the
insulators as defined herein. Additionally, the conductive
oligomers of the invention are to be distinguished from
electroactive polymers, that themselves may donate or accept
electrons.
[0149] A more detailed description of conductive oligomers is found
in WO/1999/57317, herein incorporated by reference in its entirety.
In particular, the conductive oligomers as shown in Structures 1 to
9 on page 14 to 21 of WO/1999/57317 find use in the present
invention. In some embodiments, the conductive oligomer has the
following structure:
##STR00007##
[0150] In addition, the terminus of at least some of the conductive
oligomers in the monolayer is electronically exposed. By
"electronically exposed" herein is meant that upon the placement of
an EAM in close proximity to the terminus, and after initiation
with the appropriate signal, a signal dependent on the presence of
the EAM may be detected. The conductive oligomers may or may not
have terminal groups. Thus, in a preferred embodiment, there is no
additional terminal group, and the conductive oligomer terminates
with a terminal group; for example, such as an acetylene bond.
Alternatively, in some embodiments, a terminal group is added,
sometimes depicted herein as "0". A terminal group may be used for
several reasons; for example, to contribute to the electronic
availability of the conductive oligomer for detection of EAMs, or
to alter the surface of the SAM for other reasons, for example to
prevent non-specific binding. For example, there may be negatively
charged groups on the terminus to form a negatively charged surface
such that when the target analyte is nucleic acid such as DNA or
RNA, the nucleic acid is repelled or prevented from lying down on
the surface, to facilitate hybridization. Preferred terminal groups
include --NH, --OH, --COOH, and alkyl groups such as --CH.sub.3,
and (poly)alkyloxides such as (poly)ethylene glycol, with
--OCH.sub.2CH.sub.2OH, --(OCH.sub.2CH.sub.2O).sub.2H,
--(OCH.sub.2CH.sub.2O).sub.3H, and --(OCH.sub.2CH.sub.2O).sub.4H
being preferred.
[0151] In one embodiment, it is possible to use mixtures of
conductive oligomers with different types of terminal groups. Thus,
for example, some of the terminal groups may facilitate detection,
and some may prevent non-specific binding.
[0152] In some embodiments, the electrode further comprises a
passivation agent, preferably in the form of a monolayer on the
electrode surface. For some analytes the efficiency of analyte
binding (i.e. hybridization) may increase when the binding ligand
is at a distance from the electrode. In addition, the presence of a
monolayer can decrease non-specific binding to the surface (which
can be further facilitated by the use of a terminal group, outlined
herein. A passivation agent layer facilitates the maintenance of
the binding ligand and/or analyte away from the electrode surface.
In addition, a passivation agent serves to keep charge carriers
away from the surface of the electrode. Thus, this layer helps to
prevent electrical contact between the electrodes and the electron
transfer moieties, or between the electrode and charged species
within the solvent. Such contact can result in a direct "short
circuit" or an indirect short circuit via charged species which may
be present in the sample. Accordingly, the monolayer of passivation
agents is preferably tightly packed in a uniform layer on the
electrode surface, such that a minimum of "holes" exist.
Alternatively, the passivation agent may not be in the form of a
monolayer, but may be present to help the packing of the conductive
oligomers or other characteristics.
[0153] The passivation agents thus serve as a physical barrier to
block solvent accessibility to the electrode. As such, the
passivation agents themselves may in fact be either (1) conducting
or (2) nonconducting, i.e. insulating, molecules. Thus, in one
embodiment, the passivation agents are conductive oligomers, as
described herein, with or without a terminal group to block or
decrease the transfer of charge to the electrode. Other passivation
agents which may be conductive include oligomers of
--(CF.sub.2).sub.n--, --(CHF).sub.n-- and --(CFR).sub.n--. In a
preferred embodiment, the passivation agents are insulator
moieties.
[0154] In some embodiments, the monolayers comprise insulators. An
"insulator" is a substantially nonconducting oligomer, preferably
linear. By "substantially nonconducting" herein is meant that the
rate of electron transfer through the insulator is slower than the
rate of electron transfer through the conductive oligomer. Stated
differently, the electrical resistance of the insulator is higher
than the electrical resistance of the conductive oligomer. It
should be noted however that even oligomers generally considered to
be insulators, such as --(CH2)16 molecules, still may transfer
electrons, albeit at a slow rate.
[0155] In some embodiments, the insulators have a conductivity, S,
of about 10-7 .OMEGA.-1 cm-1 or lower, with less than about 10-8
.OMEGA.-1 cm-1 being preferred. Gardner et al., Sensors and
Actuators A 51 (1995) 57-66, incorporated herein by reference.
[0156] Generally, insulators are alkyl or heteroalkyl oligomers or
moieties with sigma bonds, although any particular insulator
molecule may contain aromatic groups or one or more conjugated
bonds. By "heteroalkyl" herein is meant an alkyl group that has at
least one heteroatom, i.e. nitrogen, oxygen, sulfur, phosphorus,
silicon or boron included in the chain. Alternatively, the
insulator may be quite similar to a conductive oligomer with the
addition of one or more heteroatoms or bonds that serve to inhibit
or slow, preferably substantially, electron transfer. Preferably,
the alkyl or heteroalkyl chains are from about four to about 18
atoms in length, and more preferably from about six to about 16
atoms in length/
[0157] The passivation agents, including insulators, may be
substituted with R groups as defined herein to alter the packing of
the moieties or conductive oligomers on an electrode, the
hydrophilicity or hydrophobicity of the insulator, and the
flexibility, i.e. the rotational, torsional or longitudinal
flexibility of the insulator. For example, branched alkyl groups
may be used. In addition, the terminus of the passivation agent,
including insulators, may contain an additional group to influence
the exposed surface of the monolayer, sometimes referred to herein
as a terminal group ("TG"). For example, the addition of charged,
neutral or hydrophobic groups may be done to inhibit non-specific
binding from the sample, or to influence the kinetics of binding of
the analyte, etc. For example, there may be charged groups on the
terminus to form a charged surface to encourage or discourage
binding of certain target analytes or to repel or prevent from
lying down on the surface.
[0158] The length of the passivation agent will vary as needed.
Generally, the length of the passivation agents is similar to the
length of the conductive oligomers, as outlined above. In addition,
the conductive oligomers may be basically the same length as the
passivation agents or longer than them, resulting in the binding
ligands being more accessible to the solvent.
[0159] The monolayer may comprise a single type of passivation
agent, including insulators, or different types. Suitable
insulators are known in the art, and include, but are not limited
to, --(CH.sub.2).sub.n--, --(CRH).sub.n--, and
--(CR.sub.2).sub.n--, ethylene glycol or derivatives using other
heteroatoms in place of oxygen, i.e. nitrogen or sulfur (sulfur
derivatives are not preferred when the electrode is gold).
Preferably, insulators are of the form --(CH.sup.2).sub.n-- having
a thiol or disulfide terminus for attachment to gold. Also
preferable, the alternate end of the insulator is terminated in a
hydrophilic group such as oligoethylene glycol, --OH, or
--COOH.
[0160] In some embodiments, the electrode is a metal surface and
need not necessarily have interconnects or the ability to do
electrochemistry.
B. Anchor Groups
[0161] The present invention provides compounds comprising an
anchor group. By "anchor" or "anchor group" herein is meant a
chemical group that attaches the compounds of the invention to an
electrode.
[0162] As will be appreciated by those in the art, the composition
of the anchor group will vary depending on the composition of the
surface to which it is attached. In the case of gold electrodes,
both pyridinyl anchor groups and thiol based anchor groups find
particular use.
[0163] The covalent attachment of the conductive oligomer may be
accomplished in a variety of ways, depending on the electrode and
the conductive oligomer used. Generally, some type of linker is
used, as depicted below as "A" in Structure 1, where X is the
conductive oligomer, and the hatched surface is the electrode:
##STR00008##
[0164] In this embodiment, A is a linker or atom. The choice of "A"
will depend in part on the characteristics of the electrode. Thus,
for example, A may be a sulfur moiety when a gold electrode is
used. Alternatively, when metal oxide electrodes are used, A may be
a silicon (silane) moiety attached to the oxygen of the oxide (see
for example Chen et al., Langmuir 10:3332-3337 (1994); Lenhard et
al., J. Electroanal. Chem. 78:195-201 (1977), both of which are
expressly incorporated by reference). When carbon based electrodes
are used, A may be an amino moiety (preferably a primary amine; see
for example Deinhammer et al., Langmuir 10:1306-1313 (1994)). Thus,
preferred A moieties include, but are not limited to, silane
moieties, sulfur moieties (including alkyl sulfur moieties), and
amino moieties.
[0165] In some embodiments, the electrode is a carbon electrode,
i.e. a glassy carbon electrode, and attachment is via a nitrogen of
an amine group. A representative structure is depicted in Structure
15 of US Patent Application Publication No. 20080248592, hereby
incorporated by reference in its entirety but particularly for
Structures as described therein and the description of different
anchor groups and the accompanying text. Again, additional atoms
may be present, i.e. linkers and/or terminal groups.
[0166] In Structure 16 of US Patent Application Publication No.
20080248592, hereby incorporated by reference as above, the oxygen
atom is from the oxide of the metal oxide electrode. The Si atom
may also contain other atoms, i.e. be a silicon moiety containing
substitution groups. Other attachments for SAMs to other electrodes
are known in the art; see for example Napier et al., Langmuir,
1997, for attachment to indium tin oxide electrodes, and also the
chemisorption of phosphates to an indium tin oxide electrode (talk
by H. Holden Thorpe, CHI conference, May 4-5, 1998).
[0167] In one preferred embodiment, indium-tin-oxide (ITO) is used
as the electrode, and the anchor groups are phosphonate-containing
species.
[0168] 1). Pyridinyl Anchor Groups
[0169] In one aspect, the present invention provides the use of
pyridine and derivatives thereof to attach the compounds of the
invention to the surface.
[0170] In some embodiments, the anchor comprises a pyridyl group,
having the structure of formula (ID:
##STR00009##
where the carbons on the ring can optionally and independently be
substituted, using R groups as defined herein. Pyridine is a
heterocyclic aromatic organic compound that is structurally related
to benzene, wherein one CH group in the six-membered ring is
replaced by a nitrogen atom. Pyridine can be used as a ligand in
coordination chemistry. As a ligand, it is usually abbreviated as
"py." The invention utilizes the ability of the lone electron pair
on the nitrogen atom of the pyridine to bind to metal surfaces. One
advantage of the pyridine based compounds is that they are air
stable. Curtis et al., Inorg. Chem. 24:385-397 (1985); Callahan et
al., Inorg. Chem. 14:1443-1453 (1975); Lavallee and Fleischer, J.
Am. Chem. Soc. 94:2583-2599 (1972); and Jwo et al., J. Am. Chem.
Soc. 101:6189-6197 (1979), all of which are incorporated by
reference.
[0171] In some embodiments, the pyridyl group comprises a bipyridyl
group (Bispyridylacetylene, BPA), comprising two pyridyl groups
separated by an acetylene group, shown below:
##STR00010##
[0172] In this embodiment, the carbons on either ring can be
optionally and independently be substituted, using R groups as
defined herein. One of the rings will contain a linkage to a
spacer, as defined herein, or, as shown in some of the figures,
there may be more than one spacer attached to the pyridyl group
(e.g. n=1 or more, with 2 finding particular use in some
embodiments).
[0173] 2). Sulfur Anchor Groups
[0174] Although depicted in Structure 1 as a single moiety, the
conductive oligomer may be attached to the electrode with more than
one "A" moiety; the "A" moieties may be the same or different.
Thus, for example, when the electrode is a gold electrode, and "A"
is a sulfur atom or moiety, multiple sulfur atoms may be used to
attach the conductive oligomer to the electrode, such as is
generally depicted below in Structures 2, 3 and 4. As will be
appreciated by those in the art, other such structures can be made.
In Structures 2, 3 and 4 the A moiety is just a sulfur atom, but
substituted sulfur moieties may also be used.
[0175] Thus, for example, when the electrode is a gold electrode,
and "A" is a sulfur atom or moiety, such as generally depicted
below in Structure 6, multiple sulfur atoms may be used to attach
the conductive oligomer to the electrode, such as is generally
depicted below in Structures 2, 3 and 4. As will be appreciated by
those in the art, other such structures can be made. In Structures
2, 3 and 4, the A moiety is just a sulfur atom, but substituted
sulfur moieties may also be used.
##STR00011##
[0176] It should also be noted that similar to Structure 4, it may
be possible to have a conductive oligomer terminating in a single
carbon atom with three sulfur moieties attached to the
electrode.
[0177] In another aspect, the present invention provide anchor
comprise conjugated thiols.
[0178] Some exemplary complexes with conjugated thiol anchors are
shown in FIG. 4. In some embodiments, the anchor comprises an
alkylthiol group. Some of the examples are shown in FIGS. 4A and
4B. The two compounds depicts in FIG. 4B are based on carbene and
4-pyridylalanine, respectively.
[0179] In another aspect, the present invention provides conjugated
multipodal thio-containing compounds that serve as anchoring groups
in the construction of electroactive moieties for analyte detection
on electrodes, such as gold electrodes. That is, spacer groups
(which can be attached to EAMs, ReAMCs, or an "empty" monolayer
forming species) are attached using two or more sulfur atoms. These
multipodal anchor groups can be linear or cyclic, as described
herein.
[0180] In some embodiments, the anchor groups are "bipedal",
containing two sulfur atoms that will attach to the gold surface,
and linear, although in some cases it can be possible to include
systems with other multipodalities (e.g. "tripodal"). Such a
multipodal anchoring group display increased stability and/or allow
a greater footprint for preparing SAMs from thiol-containing
anchors with sterically demanding head groups.
[0181] In some embodiments, the anchor comprises cyclic disulfides
("bipod"). Although in some cases it can be possible to include
ring system anchor groups with other multipodalities (e.g.
"tripodal"). The number of the atoms of the ring can vary, for
example from 5 to 10, and also includes multicyclic anchor groups,
as discussed below
[0182] In some embodiments, the anchor groups comprise a
[1,2,5]-dithiazepane unit which is seven-membered ring with an apex
nitrogen atom and a intramolecular disulfide bond as shown
below:
##STR00012##
[0183] In Structure (IIIa), it should also be noted that the carbon
atoms of the ring can additionally be substituted. As will be
appreciated by those in the art, other membered rings are also
included. In addition, multicyclic ring structures can be used,
which can include cyclic heteroalkanes such as the
[1,2,5]-dithiazepane shown above substituted with other cyclic
alkanes (including cyclic heteroalkanes) or aromatic ring
structures.
[0184] In some embodiments, the anchor group and part of the spacer
has the structure shown below
##STR00013##
[0185] The "R" group herein can be any substitution group,
including a conjugated oligophenylethynylene unit with terminal
coordinating ligand for the transition metal component of the
EAM.
[0186] The anchors are synthesized from a bipodal intermediate (I)
(the compound as formula III where R=I), which is described in Li
et al., Org. Lett. 4:3631-3634 (2002), herein incorporated by
reference. See also Wei et al, J. Org, Chem. 69:1461-1469 (2004),
herein incorporated by reference.
[0187] The number of sulfur atoms can vary as outlined herein, with
particular embodiments utilizing one, two, and three per
spacer.
C. Electroactive Moieties
[0188] In addition to anchor groups, the present invention provides
compound comprising electroactive moieties. By "electroactive
moiety (EAM)" or "transition metal complex" or "redox active
molecule" or "electron transfer moiety (ETM)" herein is meant a
metal-containing compound which is capable of reversibly or
semi-reversibly transferring one or more electrons. It is to be
understood that electron donor and acceptor capabilities are
relative; that is, a molecule which can lose an electron under
certain experimental conditions will be able to accept an electron
under different experimental conditions.
[0189] It is to be understood that the number of possible
transition metal complexes is very large, and that one skilled in
the art of electron transfer compounds will be able to utilize a
number of compounds in the present invention. By "transitional
metal" herein is meant metals whose atoms have a partial or
completed shell of electrons. Suitable transition metals for use in
the invention include, but are not limited to, cadmium (Cd), copper
(Cu), cobalt (Co), palladium (Pd), zinc (Zn), iron (Fe), ruthenium
(Ru), rhodium (Rh), osmium (Os), rhenium (Re), platinum (Pt),
scandium (Sc), titanium (Ti), Vanadium (V), chromium (Cr),
manganese (Mn), nickel (Ni), Molybdenum (Mo), technetium (Tc),
tungsten (W), and iridium (Ir). That is, the first series of
transition metals, the platinum metals (Ru, Rh, Pd, Os, Ir and Pt),
along with Fe, Re, W, Mo and Tc, find particular use in the present
invention. Particularly preferred are metals that do not change the
number of coordination sites upon a change in oxidation state,
including ruthenium, osmium, iron, platinum and palladium, with
osmium, ruthenium and iron being especially preferred, and osmium
finding particular use in many embodiments. In some embodiments,
iron is not preferred. Generally, transition metals are depicted
herein as TM or M.
[0190] The transitional metal and the coordinating ligands form a
metal complex. By "ligand" or "coordinating ligand" (depicted
herein in the figures as "L") herein is meant an atom, ion,
molecule, or functional group that generally donates one or more of
its electrons through a coordinate covalent bond to, or shares its
electrons through a covalent bond with, one or more central atoms
or ions.
[0191] The other coordination sites of the metal are used for
attachment of the transition metal complex to either a capture
ligand (directly or indirectly using a linker), or to the electrode
(frequently using a spacer, as is more fully described below), or
both. Thus for example, when the transition metal complex is
directly joined to a binding ligand, one, two or more of the
coordination sites of the metal ion may be occupied by coordination
atoms supplied by the binding ligand (or by the linker, if
indirectly joined). In addition, or alternatively, one or more of
the coordination sites of the metal ion may be occupied by a spacer
used to attach the transition metal complex to the electrode. For
example, when the transition metal complex is attached to the
electrode separately from the binding ligand as is more fully
described below, all of the coordination sites of the metal (n)
except 1 (n-1) may contain polar ligands.
[0192] Suitable small polar ligands, generally depicted herein as
"L", fall into two general categories, as is more fully described
herein. In one embodiment, the small polar ligands will be
effectively irreversibly bound to the metal ion, due to their
characteristics as generally poor leaving groups or as good sigma
donors, and the identity of the metal. These ligands may be
referred to as "substitutionally inert". Alternatively, as is more
fully described below, the small polar ligands may be reversibly
bound to the metal ion, such that upon binding of a target analyte,
the analyte may provide one or more coordination atoms for the
metal, effectively replacing the small polar ligands, due to their
good leaving group properties or poor sigma donor properties. These
ligands may be referred to as "substitutionally labile". The
ligands preferably form dipoles, since this will contribute to a
high solvent reorganization energy.
Some of the structures of transitional metal complexes are shown
below:
##STR00014##
[0193] L are the co-ligands, that provide the coordination atoms
for the binding of the metal ion. As will be appreciated by those
in the art, the number and nature of the co-ligands will depend on
the coordination number of the metal ion. Mono-, di- or polydentate
co-ligands may be used at any position. Thus, for example, when the
metal has a coordination number of six, the L from the terminus of
the conductive oligomer, the L contributed from the nucleic acid,
and r, add up to six. Thus, when the metal has a coordination
number of six, r may range from zero (when all coordination atoms
are provided by the other two ligands) to four, when all the
co-ligands are monodentate. Thus generally, r will be from 0 to 8,
depending on the coordination number of the metal ion and the
choice of the other ligands.
[0194] In one embodiment, the metal ion has a coordination number
of six and both the ligand attached to the conductive oligomer and
the ligand attached to the nucleic acid are at least bidentate;
that is, r is preferably zero, one (i.e. the remaining co-ligand is
bidentate) or two (two monodentate co-ligands are used). As will be
appreciated in the art, the co-ligands can be the same or
different. Suitable ligands fall into two categories: ligands which
use nitrogen, oxygen, sulfur, carbon or phosphorus atoms (depending
on the metal ion) as the coordination atoms (generally referred to
in the literature as sigma (.sigma.) donors) and organometallic
ligands such as metallocene ligands (generally referred to in the
literature as pi (.pi.) donors, and depicted herein as Lm).
Suitable nitrogen donating ligands are well known in the art and
include, but are not limited to, cyano (C.ident.N), NH2; NHR; NRR';
pyridine; pyrazine; isonicotinamide; imidazole; bipyridine and
substituted derivatives of bipyridine; terpyridine and substituted
derivatives; phenanthrolines, particularly 1,10-phenanthroline
(abbreviated phen) and substituted derivatives of phenanthrolines
such as 4,7-dimethylphenanthroline and
dipyridol[3,2-a:2',3'-c]phenazine (abbreviated dppz);
dipyridophenazine; 1,4,5,8,9,12-hexaazatriphenylene (abbreviated
hat); 9,10-phenanthrenequinone diimine (abbreviated phi);
1,4,5,8-tetraazaphenanthrene (abbreviated tap);
1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam) and
isocyanide. Substituted derivatives, including fused derivatives,
may also be used. In some embodiments, porphyrins and substituted
derivatives of the porphyrin family may be used. See for example,
Comprehensive Coordination Chemistry, Ed. Wilkinson et al.,
Pergammon Press, 1987, Chapters 13.2 (pp 73-98), 21.1 (pp. 813-898)
and 21.3 (pp 915-957), all of which are hereby expressly
incorporated by reference. As will be appreciated in the art, any
ligand donor(1)-bridge-donor(2) where donor (1) binds to the metal
and donor(2) is available for interaction with the surrounding
medium (solvent, protein, etc.) can be used in the present
invention, especially if donor(1) and donor(2) are coupled through
a pi system, as in cyanos (C is donor(1), N is donor(2), pi system
is the CN triple bond). One example is bipyrimidine, which looks
much like bipyridine but has N donors on the "back side" for
interactions with the medium. Additional co-ligands include, but
are not limited to cyanates, isocyanates (--N.dbd.C.dbd.O),
thiocyanates, isonitrile, N2, O2, carbonyl, halides, alkoxyide,
thiolates, amides, phosphides, and sulfur containing compound such
as sulfino, sulfonyl, sulfoamino, and sulfamoyl.
[0195] In some embodiments, multiple cyanos are used as co-ligand
to complex with different metals. For example, seven cyanos bind
Re(III); eight bind Mo(IV) and W(IV). Thus at Re(III) with 6 or
less cyanos and one or more L, or Mo(IV) or W(IV) with 7 or less
cyanos and one or more L can be used in the present invention. The
EAM with W(IV) system has particular advantages over the others
because it is more inert, easier to prepare, more favorable
reduction potential. Generally that a larger CN/L ratio will give
larger shifts. Suitable sigma donating ligands using carbon,
oxygen, sulfur and phosphorus are known in the art. For example,
suitable sigma carbon donors are found in Cotton and Wilkinson,
Advanced Organic Chemistry, 5th Edition, John Wiley & Sons,
1988, hereby incorporated by reference; see page 38, for example.
Similarly, suitable oxygen ligands include crown ethers, water and
others known in the art. Phosphines and substituted phosphines are
also suitable; see page 38 of Cotton and Wilkinson. The oxygen,
sulfur, phosphorus and nitrogen-donating ligands are attached in
such a manner as to allow the heteroatoms to serve as coordination
atoms.
[0196] In some embodiments, organometallic ligands are used. In
addition to purely organic compounds for use as redox moieties, and
various transition metal coordination complexes with .delta.-bonded
organic ligand with donor atoms as heterocyclic or exocyclic
substituents, there is available a wide variety of transition metal
organometallic compounds with .pi.-bonded organic ligands (see
Advanced Inorganic Chemistry, 5th Ed., Cotton & Wilkinson, John
Wiley & Sons, 1988, chapter 26; Organometallics, A Concise
Introduction, Elschenbroich et al., 2nd Ed., 1992, VCH; and
Comprehensive Organometallic Chemistry II, A Review of the
Literature 1982-1994, Abel et al. Ed., Vol. 7, chapters 7, 8, 10
& 11, Pergamon Press, hereby expressly incorporated by
reference). Such organometallic ligands include cyclic aromatic
compounds such as the cyclopentadienide ion [C5H5 (-1)] and various
ring substituted and ring fused derivatives, such as the indenylide
(-1) ion, that yield a class of bis(cyclopentadienyl)metal
compounds, (i.e. the metallocenes); see for example Robins et al.,
J. Am. Chem. Soc. 104:1882-1893 (1982); and Gassman et al., J. Am.
Chem. Soc. 108:4228-4229 (1986), incorporated by reference. Of
these, ferrocene [(C5H5)2 Fe] and its derivatives are prototypical
examples which have been used in a wide variety of chemical
(Connelly et al., Chem. Rev. 96:877-910 (1996), incorporated by
reference) and electrochemical (Geiger et al., Advances in
Organometallic Chemistry 23:1-93; and Geiger et al., Advances in
Organometallic Chemistry 24:87, incorporated by reference) electron
transfer or "redox" reactions. Metallocene derivatives of a variety
of the first, second and third row transition metals are potential
candidates as redox moieties that are covalently attached to either
the ribose ring or the nucleoside base of nucleic acid. Other
potentially suitable organometallic ligands include cyclic arenes
such as benzene, to yield bis(arene)metal compounds and their ring
substituted and ring fused derivatives, of which
bis(benzene)chromium is a prototypical example. Other acyclic
.pi.-bonded ligands such as the allyl(-1) ion, or butadiene yield
potentially suitable organometallic compounds, and all such
ligands, in conduction with other .pi.-bonded and .delta.-bonded
ligands constitute the general class of organometallic compounds in
which there is a metal to carbon bond. Electrochemical studies of
various dimers and oligomers of such compounds with bridging
organic ligands, and additional non-bridging ligands, as well as
with and without metal-metal bonds are potential candidate redox
moieties in nucleic acid analysis. When one or more of the
co-ligands is an organometallic ligand, the ligand is generally
attached via one of the carbon atoms of the organometallic ligand,
although attachment may be via other atoms for heterocyclic
ligands. Preferred organometallic ligands include metallocene
ligands, including substituted derivatives and the
metalloceneophanes (see page 1174 of Cotton and Wilkinson, supra).
For example, derivatives of metallocene ligands such as
methylcyclopentadienyl, with multiple methyl groups being
preferred, such as pentamethylcyclopentadienyl, can be used to
increase the stability of the metallocene. In a preferred
embodiment, only one of the two metallocene ligands of a
metallocene are derivatized. As described herein, any combination
of ligands may be used. Preferred combinations include: a) all
ligands are nitrogen donating ligands; b) all ligands are
organometallic ligands; and c) the ligand at the terminus of the
conductive oligomer is a metallocene ligand and the ligand provided
by the nucleic acid is a nitrogen donating ligand, with the other
ligands, if needed, are either nitrogen donating ligands or
metallocene ligands, or a mixture. As a general rule, EAM
comprising non-macrocyclic chelators are bound to metal ions to
form non-macrocyclic chelate compounds, since the presence of the
metal allows for multiple proligands to bind together to give
multiple oxidation states.
[0197] In some embodiments, nitrogen donating proligands are used.
Suitable nitrogen donating proligands are well known in the art and
include, but are not limited to, NH2; NHR; NRR'; pyridine;
pyrazine; isonicotinamide; imidazole; bipyridine and substituted
derivatives of bipyridine; terpyridine and substituted derivatives;
phenanthrolines, particularly 1,10-phenanthroline (abbreviated
phen) and substituted derivatives of phenanthrolines such as
4,7-dimethylphenanthroline and dipyridol[3,2-a:2',3'-c]phenazine
(abbreviated dppz); dipyridophenazine;
1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat);
9,10-phenanthrenequinone diimine (abbreviated phi);
1,4,5,8-tetraazaphenanthrene (abbreviated tap);
1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam) and
isocyanide. Substituted derivatives, including fused derivatives,
may also be used. It should be noted that macrocylic ligands that
do not coordinatively saturate the metal ion, and which require the
addition of another proligand, are considered non-macrocyclic for
this purpose. As will be appreciated by those in the art, it is
possible to covalent attach a number of "non-macrocyclic" ligands
to form a coordinatively saturated compound, but that is lacking a
cyclic skeleton.
[0198] In some embodiments, a mixture of monodentate (e.g. at least
one cyano ligand), bi-dentate, tri-dentate, and polydentate ligands
(till to saturate) can be used in the construction of EAMs
Generally, it is the composition or characteristics of the ligands
that determine whether a transition metal complex is solvent
accessible. By "solvent accessible transition metal complex" or
grammatical equivalents herein is meant a transition metal complex
that has at least one, preferably two, and more preferably three,
four or more small polar ligands. The actual number of polar
ligands will depend on the coordination number (n) of the metal
ion. Preferred numbers of polar ligands are (n-1) and (n-2). For
example, for hexacoordinate metals, such as Fe, Ru, and Os, solvent
accessible transition metal complexes preferably have one to five
small polar ligands, with two to five being preferred, and three to
five being particularly preferred, depending on the requirement for
the other sites, as is more fully described below. Tetracoordinate
metals such as Pt and Pd preferably have one, two or three small
polar ligands. It should be understood that "solvent accessible"
and "solvent inhibited" are relative terms. That is, at high
applied energy, even a solvent accessible transition metal complex
may be induced to transfer an electron.
Some examples of EAMs are described herein.
[0199] 1). Cyano-Based Complexes
[0200] In one aspect, the present invention provides EAMs with a
transition metal and at least one cyano (--C.ident.N) ligand.
Depending on the valency of the metal and the configuration of the
system (e.g. capture ligand contributing a coordination atom,
etc.), 1, 2, 3, 4 or 5 cyano ligands can be used. In general,
embodiments which use the most cyano ligands are preferred; again,
this depends on the configuration of the system. For example, as
depicted in FIG. 15, an EAM using a hexadentate metal such as
osmium, separately attached from the capture ligand, allows 5 cyano
ligands, with the 6th coordination site being occupied by the
terminus of the attachment linker. When a hexadentate metal has
both an attachment linker and a capture ligand providing
coordination atoms, there can be four cyano ligands.
[0201] In some embodiments, such as depicted in the Figures, the
attachment linker and/or the capture ligand can provide more than a
single coordination atom. Thus, for example, in FIG. 17, the
attachment linker comprises a bipyridine which contributes two
coordination atoms. Compounds 301, 302, 303, 304, 305, and 306 are
additional examples of EAMs comprising bipyridine (also denoted
bipyridyl) ligands.
[0202] In some embodiments, ligands other than cyano ligands are
used in combination with at least one cyano ligand.
[0203] 2). Ru--N Based Complexes
[0204] In one aspect, the resent invention provides new
architectures for Ru--N based complexes, where the coordination
could be monodentate, bidentate, tridentate, or multidendate. Thus
the number of coordination ligand L (which covalently connected to
the anchor and capture ligand) can be 1, 2, 3, or 4. Some of the
examples are shown in FIG. 5A.
[0205] The charge-neutralizing ligands can be any suitable ligand
known in the art, such as dithiocarbamate, benzenedithiolate, or
Schiff base as described herein. The capture ligand and the anchor
can be on the same framework or separate.
[0206] In another aspect of the present invention, each component
of the EAM ligand architecture is connected through covalent bonds
rather than Ru coordination chemistry. The construction of the
architectures provide herein relies on modern synthetic organic
chemical methodology. An important design consideration includes
the necessary orthogonal reactivity of the functional groups
present in the anchor and capture ligand component versus the
coordinating ligand component. Preferably, the entire compound can
be synthesized and the redox active transitional metal coordinated
to the ligand near the last step of the synthesis. The coordinating
ligands provided herein rely on well-established inorganic
methodologies for ruthenium pentaamine precursors. See Gerhardt and
Weck, J. Org. Chem. 71:6336-6341 (2006); Sizova et al., Inorg.
Chim. Acta, 357:354-360 (2004); and Scott and Nolan, Eur. J. Inorg.
Chem. 1815-1828 (2005), all herein incorporated by reference. Some
examples of EAM architectures with Ru-pentaamine complexes are
shown below in FIG. 5B. As can be understood by those skilled in
the art, the anchor components of the compounds provided herein
could be interchanged between alkyl and multipodal-based
thiols.
[0207] 3). Ferrocene-Based EAMs
[0208] In some embodiments, the EAMs comprise substituted
ferrocenes. Ferrocene is air-stable. It can be easily substituted
with both capture ligand and anchoring group. Upon binding of the
target protein to the capture ligand on the ferrocene which will
not only change the environment around the ferrocene, but also
prevent the cyclopentadienyl rings from spinning, which will change
the energy by approximately 4 kJ/mol. WO/1998/57159; Heinze and
Schlenker, Eur. J. Inorg. Chem. 2974-2988 (2004); Heinze and
Schlenker, Eur. J. Inorg. Chem. 66-71 (2005); and Holleman-Wiberg,
Inorganic Chemistry, Academic Press 34th Ed, at 1620, all
incorporated by reference.
##STR00015##
[0209] In some embodiments the anchor and capture ligands are
attached to the same ligand for easier synthesis. In some
embodiments the anchor and capture ligand are attached to different
ligands.
[0210] There are many ligands that can be used to build the new
architecture disclosed herein. They include but not limited to
carboxylate, amine, thiolate, phosphine, imidazole, pyridine,
bipyridine, terpyridine, tacn (1,4,7-Triazacyclononane), salen
(N,N'-bis(salicylidene) ethylenediamine), acacen
(N,N'-Ethylenebis(acetylacetoniminate(-)), EDTA (ethylenediamine
tetraacetic acid), DTPA (diethylene triamine pentaacetic acid), Cp
(cyclopentadienyl), pincer ligands, and scorpionates. In some
embodiments, the preferred ligand is pentaamine.
[0211] Pincer ligands are a specific type of chelating ligand. A
pincer ligand wraps itself around the metal center to create bonds
on opposite sides of the metal as well as one in between. The
effects pincer ligand chemistry on the metal core electrons is
similar to amines, phosphines, and mixed donor ligands. This
creates a unique chemical situation where the activity of the metal
can be tailored. For example, since there is such a high demand on
the sterics of the complex in order to accommodate a pincer ligand,
the reactions that the metal can participate in is limited and
selective.
[0212] Scorpionate ligand refers to a tridentate ligand which would
bind to a metal in a fac manner. The most popular class of
scorpionates are the tris(pyrazolyl)hydroborates or Tp ligands. A
Cp ligand is isolobal to Tp
[0213] In some embodiments, the following restraints are desirable:
the metal complex should have small polar ligands that allow close
contact with the solvent.
[0214] 4). Osmium-Based EAMs
[0215] In some embodiments, the EAMs comprise substituted osmium
complexes defined by the chemical formula
R.sup.1,R.sup.2-bpy-Os(CN).sub.4 wherein R.sup.1 and R.sup.2 are as
described herein. The structural formula IV of
R.sup.1,R.sup.2-bpy-Os(CN).sub.4 is shown below. The preferred 4,4'
and 5,5' osmium complex isomers are also shown below as structural
formulae IVa and IVb. Osmium complexes appear to be attractive
candidates for use in electrochemical biosensor platforms due to
their extraordinary redox sensitivity to solvation environment,
good optical absorption in the visible region and as strong
reductants in both the ground and excited states, relative to
ferrocene-based complexes. However, osmium complexes are less air
stable, relative to ferrocene complexes. Moreover, while Olabe and
co-workers had described preparation and characterization of
unsubstituted Os.sup.II(bipy)(CN).sub.4.sup.2- complexes,
unsubstituted osmium complexes however lack sufficient synthetic
functionality that would allow them to be incorporated into more
elaborate systems. See, for instance, Ward et al. Dalton Trans.,
2010, Vol. 39, pp. 8851-8867; Baca et al. Inorg. Chem. 2007, Vol.
46, pp. 9770-9789; and Baca et al., Inorg. Chem, 2008, Vol. 47, pp.
3736-3747. Because osmium complexes can be less chemically stable,
air stable, water stable, and/or because prior reported
Os.sup.II(bpy)(CN).sub.4.sup.2- complexes lack sufficient synthetic
functionality, the commercial applicability of
Os.sup.II(bpy)(CN).sub.4.sup.2- complexes as probes for biomolecule
ligand-receptor interactions have not been explored.
##STR00016##
[0216] The invention provides the first examples of a new series of
mono- and di-amine-functionalized osmium complexes defined by
chemical formula R.sup.1,R.sup.2-bpy-Os(CN).sub.4 wherein R.sup.1
and R.sup.2 are as described herein. Representative osmium
complexes of the aforementioned formula corresponding to compounds
301 to 306 are described herein. The synthesis and electrochemical
characterization of each complex in solution as described in the
Examples demonstrates the surprising and unexpected stability of
the Os.sup.II/Os.sup.III redox couple and the ability to modify the
oxidation potential based on pH. Furthermore, the amine
functionality can serve as synthetic handles for further
functionalization or as basic sites for pH-induced modification of
complex properties.
[0217] 5). Charge-Neutralizing Ligands
[0218] In another aspect, the present invention provides
compositions having metal complexes comprising charged ligands. The
reorganization energy for a system that changes from neutral to
charged or from charged to neutral (e.g.
M(L).sub.n.sup.+<->M(L).sub.n.sup.0;
M(L).sub.n<->M(L).sub.n.sup.0) may be larger than that for a
system in which the charge simply changes (e.g.
M(L).sub.n.sup.2+<->M(L).sub.n.sup.3+) because the water
molecules and surrounding ions have to "reorganize" more to
accommodate the change to or from an uncharged state.
[0219] In some embodiments, charged ligand anionic compounds can be
used to attach the anchor and the capture ligand to the metal
center. A metal complex containing a halide ion X in the inner
complex sphere reacts with charged ligands, including but not
limited to, thiols (R--SH), thiolates (RS-E; E=leaving group, i.e.,
trimethylsilyl-group), carbonic acids, dithiols, carbonates,
acetylacetonates, salicylates, cysteine,
3-mercapto-2-(mercaptomethyl) propanoic acid. The driving force for
this reaction is the formation of HX or EX. If the anionic ligand
contains both capture ligand and anchor, one substitution reaction
is required, and therefore the metal complex, with which it is
reacted, needs to have one halide ligand in the inner sphere. If
the anchor and capture ligand are introduced separately the
starting material generally needs to contain two halide in the
inner coordination sphere. Seidel et al., Inorg. Chem 37:6587-6596
(1998); Kathari and Busch, Inorg. Chem. 8:2276-2280 (1978); Isied
and Kuehn J. Am. Chem. Soc. 100:6752-6754; and Volkers et al., Eur.
J. Inorg. Chem. 4793-4799 (2006), all herein incorporated by
reference.
[0220] Examples for suitable metal complexes are the following (it
should be noted that the structures depicted below show multiple
unidentate ligands, and multidentate ligands can be substituted for
or combined with unidentate ligands such as cyano ligands):
##STR00017##
[0221] In some embodiments, dithiocarbamate is used as a
charge-neutralizing ligand, such as the following example:
##STR00018##
[0222] In some embodiments, benzenedithiolate is used as
charge-neutralizing ligand, such as the following example:
##STR00019##
[0223] In the above depicted structures, Ln is coordinate ligand
and n=0 or 1.
[0224] In some embodiments, the EAM comprises Schiff base type
complexes. By "Schiff base" or "azomethine" herein is meant a
functional group that contains a carbon-nitrogen double bond with
the nitrogen atom connected to an aryl or alkyl group--but not
hydrogen. Schiff bases are of the general formula
R.sub.1R.sub.2C.dbd.N--R.sub.3, where R.sub.3 is a phenyl or alkyl
group that makes the Schiff base a stable imine. Schiff bases can
be synthesized from an aromatic amine and a carbonyl compound by
nucleophilic addition forming a hemiaminal, followed by a
dehydration to generate an imine.
[0225] Acacen is a small planar tetradentate ligand that can form
hydrogen bonds to surrounding water molecules through its nitrogen
and oxygen atoms, which would enhance the reorganization energy
effect. It can be modified with many functionalities, including but
not limited to, carboxylic acid and halides, which can be used to
couple the acacen-ligand to the capture ligand and to the anchoring
group. This system allows a large variety of different metal
centers to be utilized in the EAMs. Since the ligand binds with its
two oxygen and two nitrogen atoms, only four coordination sites are
occupied. This leaves two additional coordination sites open,
depending on the metal center. These coordination sites can be
occupied by a large variety of organic and inorganic ligands. These
additional open sites can be used for inner-sphere substitution
(e.g. labile H.sub.2O or NH.sub.3 can be displaced by protein
binding) or outer-sphere influence (e.g. CO, CN can for H-bonds) to
optimize the shift of potentials upon binding of the capture ligand
to the target. WO/1998/057158, WO/1997/21431, Louie et al., PNAS
95:6663-6668 (1999), and Bottcher et al., Inorg. Chem. 36:2498-2504
(1997), herein all incorporated by reference.
[0226] In some embodiments, salen-complexes are used as well.
Syamal et al., Reactive and Functional Polymers 39:27-35
(1999).
[0227] The structures of some acacen-based complexes and
salen-based complexes are shown below, where positions on the
ligand that are suitable for functionalization with the capture
ligand and/or the anchor are marked with an asterisk.
##STR00020##
[0228] One example of using acacen as ligand to form a cobalt
complex is the following:
##STR00021##
wherein is A and B are substitute groups, Ln is coordinating ligand
and n=0 or 1, and Y is a counterion.
[0229] 6). Sulfato Ligands
[0230] In some embodiments, the EAM comprises sulfato complexes,
including but not limited to,
[L-Ru(III)(NH.sub.3).sub.4SO.sub.4].sup.+ and
[L-Ru(III)(NH.sub.3).sub.4SO2.sub.2].sup.2+. The
SO.sub.4--Ru(III)-complexes are air stable. The ligand L comprises
a capture ligand and anchor. The sulfate ligand is more polar than
amine and negatively charged. The surface complexes therefore will
have a larger reorganization energy contribution from surrounding
water molecules than both the [L-Ru(NH.sub.3).sub.4-L'] and
[L-Ru(NH.sub.3).sub.5].sup.2+. Isied and Taube, Inorg. Chem.
13:1545-1551 (1974), herein incorporated by reference.
##STR00022##
[0231] 7). EAM With Multiple Metals
[0232] In one aspect of the present invention multiple metal
centers are incorporated to a single ligand complex and thereby
increases the signal. This arrangement increases the ratio of
metals per target, resulting in a higher sensitivity.
[0233] In some embodiments, multiple metal centers are present
close to the capture ligand of an anchoring moiety to enable larger
interaction with the analyte of interest (target). Having more than
one reporter metal per analyte could boost the signal to noise
ratio, increasing the sensitivity of the device. One of such
examples is shown in FIG. 19.
[0234] In some embodiments Prussian blue (PB) is used. Prussian
blue is an inorganic, three-dimensional polymer (see below) that
can be formed chemically or electrochemically from simple iron
cyanide precursors. Other metals such as Mn and V and Ru have also
been incorporated into PB-like films. Films of 50-100 nm thickness
are formed quickly. An iron (or other similar metal) complex with a
capture ligand can be incorporated on the surface of the film by
combining this complex with the precursors during the formation of
the film:
Fe(H.sub.2O).sub.6.sup.3++Fe(CL)(H.sub.2O).sub.5.sup.3++Fe(CN).sub.6.sup-
.4-.fwdarw.PB with Fe--CL on surface
[0235] When the target analyte (such as a protein) binds, the
hydrogen bonding of water to cyano groups and other water molecules
on the surface will be disrupted, and will affect more metals than
just the one with the capture ligand. The electrochemical signal
will be drastically changed due to this amplification. See FIG. 6A.
In some embodiments a background subtraction of the signal before
protein binding may be advantageous.
##STR00023##
[0236] In the example shown above, the axial positions on the iron
metal are functionalized with capture ligands arranged orthogonal
to the surface. The binding of a single target to the
functionalized surface will impact the metal that is directly
attached as well as adjacent metal centers. The Fe--CL complex may
be added after initial formation of the film so that it will be
incorporated on the surface only, and not cause excessive defects.
The film thickness can be controlled by the time the chemical
reaction is allowed to proceed or by how much current is applied to
the solution if formed electrochemically. Alternatively, islands of
PB can be grown between areas of alkane or conjugated SAMs with or
without the capture ligand. This would require nanopatterning and
would help prevent electrochemical signals from extraneous species
in the sample solution. The metal can be used include, but not
limited to, ruthenium, iron, rhenium, and osmium, with the
appropriate ligand structure associated with each. When there are
multiple metals in the same complex the connectivity between the
multiple metal centers generally should not allow "cross-talk"
between the metals; but should rather be insulating.
[0237] 8). Crown Ether Ligands
[0238] In one aspect, the present invention provides compositions
where polar groups, such as crown ethers (CEs), are introduced in
the vicinity to the metal center. This could increase the potential
shift upon binding of a target analyte (e.g. a protein) to an EAM
and therefore increase the sensitivity of the probe. Crown ethers
are heterocyclic chemical compounds that, in their simplest form,
are cyclic oligomers of ethylene oxide. The essential repeating
unit of any simple crown ether is ethyleneoxy, i.e.,
--CH.sub.2CH.sub.2O--, which repeats twice in dioxane and six times
in 18-crown-6. In general, macrocycles of the
(--CH.sub.2CH.sub.2O--).sub.n type in which n 4 are referred to as
"crown" ethers rather than by their lengthier systematic names: for
instance, the systematic name of 18-crown-6 is
"1,4,7,10,13,16-hexaoxacyclooctadecane." The first number in a
crown ether's name refers to the number of atoms in the cycle, and
the second number refers to the number of those atoms which are
oxygen. Also envisioned by the present invention are crown ether
derivatives. This embodiment of the is based on having a
second-sphere moiety bound to the ruthenium center of the EAM prior
to the protein binding event. It has been shown that having a crown
ether hydrogen bonded to pentaammine ruthenium complexes shifts the
redox potential significantly (up to -100 mV) negative in
acetonitrile. See Ando, Coordination Chemistry Reviews, 248:185-203
(2004), and references therein; Ando et al., Polyhedron,
11:2335-2340 (1992); Zang et al., Inorg. Chem., 4738-4743 (1994);
Todd et al. Inorg. Chem., 32:2001-2001 (1993); Dong et al., J. Am.
Chem. Soc., 115, 4379-4380 (1993), all herein incorporated by
reference. The applicants have observed a positive shift in redox
potential upon protein binding to the EAM provided herein, thus the
use of crown ether would amplify this effect. By having some
electron donating moiety (i.e. crown ether) bound to the metal
center prior to protein binding "stacks the deck" for us by moving
our initial potential more negative such that upon protein binding
the crown ether is displaced (changing the second sphere
coordination) giving a larger positive potential shift. Without
being bound by theory, the reason for the increased potential shift
is likely the following: CEs form hydrogen bonds to surrounding
water molecules. CEs are known to bind to alkali metal ions (e.g.
Na.sup.+, K.sup.+ in electrolyte) which bind to the oxygen atoms of
the CE. In water, the CE, the ion and the counterion (e.g. CD are
hydrated with surrounding water molecules. Upon binding of the
target to the capture ligand the water molecules surrounding the
transition metal complex are replaced as well as the water
molecules hydrating the CE, the alkali metal ion and the counterion
of the alkali metal ion. See FIGS. 6A and 6B. The fact that many
more water molecules are replaced will increase the shift in
potential observed in a binding event. In some cases the change in
environment from hydrophilic to more hydrophobic actually expels
the alkali metal salt (such as K.sup.+ and Na.sup.+ ions) from the
CE as well as the water molecules, and a potential shift of as much
as 1.0V can be expected. Electrochimica Acta 2001, 2733, herein
incorporated by reference. See. e.g. FIG. 7. Some of the examples
are:
##STR00024##
wherein S.sup.1 and S.sup.2 are spacers, CL=capture ligand and
AG=anchoring group.
[0239] 9). Pyridine-Thioether/Ether-Ligands
[0240] In some embodiments, pyridine-thioether/ether-ligands are
used in the synthesis of EAM. These ligand systems will be able to
bind to various metal centers via the pyridine-nitrogen and the
thioether/ether functionality. Without being bound by the theory,
there is the possibility that upon binding of the EAM to the
target, the metal-thioether/ether bond is getting cleaved and e.g.
halide binds to the metal center, which would be an inner-sphere
effect, leading to a large shift in the electrochemical
potential.
One of the examples for such complexes is shown below:
##STR00025##
[0241] where Ln=coordinate ligand of metal center, L=0 or 1, and
X=O or S;
[0242] In some embodiments, bipyridines and other
multidentate-nitrogen-based ligands, such as 1,10 phenanthrolines
or terpyridines, are used. Examples for these type of ligands are
shown below:
##STR00026##
D. Spacer Groups
[0243] In some embodiments, the EAM or ReAMC is covalently attached
to the anchor group (which is attached to the electrode) via an
attachment linker or spacer ("Spacer 1"), that further generally
includes a functional moiety that allows the association of the
attachment linker to the electrode. See for example U.S. Pat. No.
7,384,749, incorporated herein by reference in its entirety and
specifically for the discussion of attachment linkers). It should
be noted in the case of a gold electrode, a sulfur atom can be used
as the functional group (this attachment is considered covalent for
the purposes of this invention). By "spacer" or "attachment linker"
herein is meant a moiety which holds the redox active complex off
the surface of the electrode. In some embodiments, the spacer is a
conductive oligomer as outlined herein, although suitable spacer
moieties include passivation agents and insulators as outlined
below. In some cases, the spacer molecules are SAM forming species.
The spacer moieties may be substantially non-conductive, although
preferably (but not required) is that the electron coupling between
the redox active molecule and the electrode (HAB) does not limit
the rate in electron transfer.
[0244] In addition, attachment linkers can be used to between the
coordination atom of the capture ligand and the capture ligand
itself, in the case when ReAMCs are utilized. Similarly, attachment
linkers can be branched, such as shown in FIGS. 12-14. In addition,
attachment linkers can be used to attach capture ligands to the
electrode when they are not associated in a ReAMC. One end of the
attachment linker is linked to the EAM/ReAMC/capture ligand, and
the other end (although as will be appreciated by those in the art,
it need not be the exact terminus for either) is attached to the
electrode. The covalent attachment of the conductive oligomer
containing the redox active molecule (and the attachment of other
spacer molecules) may be accomplished in a variety of ways,
depending on the electrode and the conductive oligomer used. See
for example Structures 12-19 and the accompanying text in U.S.
Patent Publication No. 20020009810, hereby incorporated by
reference in its entirety.
[0245] In general, the length of the spacer is as outlined for
conductive polymers and passivation agents in U.S. Pat. Nos.
6,013,459, 6,013,170, and 6,248,229, as well as U.S. Pat. No.
7,384,749 all herein incorporated by reference in their entireties.
As will be appreciated by those in the art, if the spacer becomes
too long, the electronic coupling between the redox active molecule
and the electrode will decrease rapidly.
E. Capture Ligands
[0246] A variety of molecules can be used in the present invention
as capture ligands. By "capture ligand" or "binding ligand" or
"capture binding ligand" or "capture binding species" or "capture
probe" herein is meant a compound that is used to probe for the
presence of the target analyte that will bind to the target
analyte. Generally, the capture ligand allows the attachment of a
target analyte to the electrode, for the purposes of detection. As
is more fully outlined below, attachment of the target analyte to
the capture probe may be direct (i.e. the target analyte binds to
the capture ligand) or indirect (one or more capture extender
ligands are used). By "covalently attached" herein is meant that
two moieties are attached by at least one bond, including sigma
bonds, pi bonds and coordination bonds.
[0247] In some embodiments, the binding is specific, and the
capture ligand is part of a binding pair. By "specifically bind"
herein is meant that the ligand binds the analyte, with specificity
sufficient to differentiate between the analyte and other
components or contaminants of the test sample. However, as will be
appreciated by those in the art, it will be possible to detect
analytes using binding which is not highly specific; for example,
the systems may use different capture ligands, for example an array
of different ligands, and detection of any particular analyte is
via its "signature" of binding to a panel of binding ligands,
similar to the manner in which "electronic noses" work. This finds
particular utility in the detection of chemical analytes. The
binding should be sufficient to remain bound under the conditions
of the assay, including wash steps to remove non-specific binding.
This binding should be sufficient to remain bound under the
conditions of the assay, including wash steps to remove
non-specific binding. Generally, the disassociation constants of
the analyte to the binding ligand will be in the range of at least
10-4-10-6 M-1, with a preferred range being 10-5 to 10-9 M-1 and a
particularly preferred range being 10-7-10-9 M-1. As will be
appreciated by those in the art, the composition of the capture
ligand will depend on the composition of the target analyte.
Capture ligands to a wide variety of analytes are known or can be
readily found using known techniques. For example, when the analyte
is a single-stranded nucleic acid, the capture ligand may be a
complementary nucleic acid. Similarly, the analyte may be a nucleic
acid binding protein and the capture binding ligand is either
single-stranded or double stranded nucleic acid; alternatively, the
binding ligand may be a nucleic acid-binding protein when the
analyte is a single or double-stranded nucleic acid. When the
analyte is a protein, the binding ligands include proteins or small
molecules. Preferred binding ligand proteins include peptides. For
example, when the analyte is an enzyme, suitable binding ligands
include substrates and inhibitors. As will be appreciated by those
in the art, any two molecules that will associate may be used,
either as an analyte or as the binding ligand. Suitable
analyte/binding ligand pairs include, but are not limited to,
antibodies/antigens, receptors/ligands, proteins/nucleic acid,
enzymes/substrates and/or inhibitors, carbohydrates (including
glycoproteins and glycolipids)/lectins, proteins/proteins,
proteins/small molecules; and carbohydrates and their binding
partners are also suitable analyte-binding ligand pairs. These may
be wild-type or derivative sequences. In a preferred embodiment,
the binding ligands are portions (particularly the extracellular
portions) of cell surface receptors that are known to multimerize,
such as the growth hormone receptor, glucose transporters
(particularly GLUT 4 receptor), transferrin receptor, epidermal
growth factor receptor, low density lipoprotein receptor, high
density lipoprotein receptor, epidermal growth factor receptor,
leptin receptor, interleukin receptors including IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15, and
IL-17 receptors, human growth hormone receptor, VEGF receptor, PDGF
receptor, EPO receptor, TPO receptor, ciliary neurotrophic factor
receptor, prolactin receptor, and T-cell receptors. As described
herein, the capture ligand can be attached to the coordinating
ligand and/or anchor via a covalent bond. The method of attachment
of the capture binding ligand will generally be done as is known in
the art, and will depend on the composition of the attachment
linker and the capture binding ligand. In general, the capture
binding ligands are attached to the attachment linker through the
use of functional groups on each that can then be used for
attachment. Preferred functional groups for attachment are amino
groups, carboxy groups, oxo groups and thiol groups. These
functional groups can then be attached, either directly or through
the use of a linker, sometimes depicted herein as "Z". Linkers are
known in the art; for example, homo- or hetero-bifunctional linkers
as are well known (see 1994 Pierce Chemical Company catalog,
technical section on cross-linkers, pages 155-200, incorporated
herein by reference). Preferred Z linkers include, but are not
limited to, alkyl groups (including substituted alkyl groups and
alkyl groups containing heteroatom moieties), with short alkyl
groups, esters, amide, amine, epoxy groups and ethylene glycol and
derivatives being preferred. Z may also be a sulfone group, forming
sulfonamide.
[0248] In this way, capture binding ligands comprising proteins,
lectins, nucleic acids, small organic molecules, carbohydrates,
etc. can be added.
[0249] In some embodiment, antibodies or a fragment thereof are
used as capture ligands. By "antibody" herein is meant a member of
a family of glycosylated proteins called immunoglobulins, which can
specifically combine with an antigen. The term "antibody" includes
full-length as well antibody fragments, as are known in the art,
including Fab, Fab2, single chain antibodies (Fv for example),
chimeric antibodies, humanized and human antibodies, etc., either
produced by the modification of whole antibodies or those
synthesized de novo using recombinant DNA technologies, and
derivatives thereof. However, in some embodiments, whole antibodies
are not preferred. This is because antibodies could be too bulky,
leads to interference with transducer. Thus in some embodiments,
antibody fragments and mimitopes are used as capture ligands. By
"epitope" herein is meant the actual site of antibody recognition
of the antigen. The term is also used interchangeably with
"antigenic determinant" or "antigenic determinant site". By
"mimitopes" or "mimotope" herein is meant a peptide which has the
spatial structure of a biologically important site, e.g., an
epitope, or an enzyme active site, or a receptor binding site.
[0250] In some embodiments, the capture ligand comprises antibody
alternatives, including but not limited to avimer. By "avimer"
herein is meant proteins that are evolved from a large family of
human extracellular receptor domains by in vitro exon shuffling and
phage display. It is generally a multidomain protein with binding
and inhibitory properties. See Silverman et al., Nature
Biotechnology 23:1556-1561 (2005), herein incorporated by
reference.
[0251] In some embodiments, the capture ligand comprises oligomeric
peptides. These peptides can be obtained using techniques known in
the art, including but not limited to phage display, Sidhu et al.,
Methods Enzymol., 328, 333-363 (2000), and one bead one peptide.
For example, the peptide can be obtained using Biopanning. Giodano
et al., Nat Med. 7:1249-53 (2001); herein incorporated by
reference. The capture ligand may be nucleic acid, when the target
analyte is a nucleic acid or nucleic acid binding proteins;
alternatively, as is generally described in U.S. Pat. Nos.
5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459,
5,683,867,5,705,337, and related patents, hereby incorporated by
reference, nucleic acid "aptamers" can be developed for binding to
virtually any target analyte. Similarly, there is a wide body of
literature relating to the development of binding partners based on
combinatorial chemistry methods. In this embodiment, when the
capture ligand is a nucleic acid, preferred compositions and
techniques are outlined in PCT US97/20014, hereby incorporated by
reference.
[0252] In some embodiments, the capture ligand comprises an
aptamer. By "aptamer" herein is meant a single-stranded, partially
single-stranded, partially double-stranded or double-stranded
nucleotide sequence, advantageously a replicatable nucleotide
sequence, capable of specifically recognizing a selected
non-oligonucleotide molecule or group of molecules by a mechanism
other than Watson-Crick base pairing or triplex formation. Aptamers
disclosed herein include, without limitation, defined sequence
segments and sequences comprising nucleotides, ribonucleotides,
deoxyribonucleotides, nucleotide analogs, modified nucleotides and
nucleotides comprising backbone modifications, branch points and
non-nucleotide residues, groups or bridges. Aptamers of the
invention include partially and fully single-stranded and
double-stranded nucleotide molecules and sequences, synthetic RNA,
DNA and chimeric nucleotides, hybrids, duplexes, heteroduplexes,
and any ribonucleotide, deoxyribonucleotide or chimeric counterpart
thereof and/or corresponding complementary sequence, promoter or
primer-annealing sequence needed to amplify, transcribe or
replicate all or part of the aptamer molecule or sequence. Aptamers
can specifically bind to soluble, insoluble or immobilized selected
molecules (e.g., ligands, receptors and effector molecules).
Alternatively, the term "aptamer" includes nucleotides capable of
shape-specific recognition of chemically bland surfaces by a
mechanism distinctly different from specific binding. Aptamers of
the instant invention may be selected to specifically recognize a
structural shape or surface feature comprising a chemically bland
surface (e.g., a silicon chip or carbon nanostructure) rather than
the chemical identity of a selected target molecule (e.g., a ligand
or receptor). An aptamer may be a molecule unto itself or a
sequence segment comprising a nucleotide molecule or group of
molecules, e.g., a defined sequence segment or aptameric sequence
comprising a synthetic heteropolymer, multivalent heteropolymeric
hybrid structure or aptameric multimolecular device.
IV. Method of Making the Compositions of the Invention
[0253] As will be appreciated by those in the art, the compositions
can be made using a variety of techniques known in the art. See for
example the disclosures of U.S. Pat. Nos. 6,013,459, 6,248,229,
7,018,523, 7,267,939, U.S. patent application Ser. Nos. 09/096,593
and 60/980,733, and U.S. Provisional Application titled
"Electrochemical Assay for the Detection of Enzymes" which is
hereby incorporated herein by reference.
[0254] In one embodiment, the compositions of the invention are
made as depicted in FIG. 3. In this embodiment, the electrodes
comprising a species including a functional group for the
attachment of the capture ligand is used, and after the composition
is made, a capture ligand with a complementary functional group is
added, resulting in essentially spontaneous addition of the capture
ligand to the surface. As will be appreciated by those in the art,
there are a wide variety of functional groups/complementary
functional groups that can be used. Suitable functional groups
include, but are not limited to, maleimide, imidoesters,
N-hydroxysuccinimidyls, alkyl halides, aryl halides,
alpha-haloacyls and pryidyl disulfides. In general, the
corresponding/complementary functional groups sulfhydryls, amines,
amines, sulfhydryls, sulfhydryls, sulfhydryls and sulfhydryls,
respectively. As will be appreciated by those in the art, it is
also possible to switch the orientation of these functional groups,
e.g. the sulfhydryl is present on the attachment linker and the
maleimide is added to the biomolecule to be used as the capture
ligand. As noted herein, the methods of attaching are dependent
upon the reactive groups present on the two components. In an
exemplary embodiment, the reactive functional group of the haptens
of the invention and the functional group of the reactive part
comprise electrophiles and nucleophiles that can generate a
covalent linkage between them. Alternatively, the reactive
functional group comprises a photoactivatable group, which becomes
chemically reactive only after illumination with light of an
appropriate wavelength. Typically, the conjugation reaction between
the reactive functional group and the reactive partner results in
one or more atoms of the reactive functional group or the reactive
partner being incorporated into a new linkage attaching the two
components. Selected examples of functional groups and linkages are
shown in Table 1, where the reaction of an electrophilic group and
a nucleophilic group yields a covalent linkage.
TABLE-US-00001 TABLE 1 Examples of some routes to useful covalent
linkages with electrophile and nucleophile reactive groups
Electrophilic Group Nucleophilic Group Resulting Covalent Linkage
activated esters* amines/anilines carboxamides acyl azides**
amines/anilines carboxamides acyl halides amines/anilines
carboxamides acyl halides alcohols/phenols esters acyl nitriles
alcohols/phenols esters acyl nitriles amines/anilines carboxamides
aldehydes amines/anilines imines aldehydes or ketones hydrazines
hydrazones aldehydes or ketones hydroxylamines oximes alkyl halides
amines/anilines alkyl amines alkyl halides carboxylic acids esters
alkyl halides thiols thioethers alkyl halides alcohols/phenols
ethers alkyl sulfonates thiols thioethers alkyl sulfonates
carboxylic acids esters alkyl sulfonates alcohols/phenols ethers
anhydrides alcohols/phenols esters anhydrides amines/anilines
carboxamides aryl halides thiols thiophenols aryl halides amines
aryl amines aziridines thiols thioethers boronates glycols boronate
esters carboxylic acids amines/anilines carboxam ides carboxylic
acids alcohols esters carboxylic acids hydrazines hydrazides
carbodiimides carboxylic acids N-acylureas or anhydrides
diazoalkanes carboxylic acids esters epoxides thiols thioethers
haloacetamides thiols thioethers halotriazines amines/anilines
aminotriazines halotriazines alcohols/phenols triazinyl ethers
imido esters amines/anilines amidines isocyanates amines/anilines
ureas isocyanates alcohols/phenols urethanes isothiocyanates
amines/anilines thioureas maleimides thiols thioethers
phosphoramidites alcohols phosphite esters silyl halides alcohols
silyl ethers sulfonate esters amines/anilines alkyl amines
sulfonate esters thiols thioethers sulfonate esters carboxylic
acids esters sulfonate esters alcohols ethers sulfonyl halides
amines/anilines sulfonamides sulfonyl halides phenols/alcohols
sulfonate esters *Activated esters, as understood in the art,
generally have the formula--CO.OMEGA., where .OMEGA. is a good
leaving group (e.g. oxysuccinimidyl (--OC.sub.4H.sub.4O.sub.2)
oxysulfosuccinimidyl
(--OC.sub.4H.sub.3O.sub.2--SO.sub.3H),-1-oxybenzotriazolyl
(--OC.sub.6H.sub.4N.sub.3); or an aryloxy group or aryloxy
substituted one or more times by electron withdrawing substituents
such as nitro, fluoro, chloro, cyano, or trifluoromethyl, or
combinations thereof, used to form activated aryl esters; or a
carboxylic acid activated by a carbodiimide to form an anhydride or
mixed anhydride--OCOR.sup.a or --OCNR.sup.aNHR.sup.b, where R.sup.a
and R.sup.b, which may be the same or different, are
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 perfluoroalkyl, or
C.sub.1-C.sub.6 alkoxy; or cyclohexyl, 3-dimethylaminopropyl, or
N-morpholinoethyl). **Acyl azides can also rearrange to
isocyanates
[0255] The functional groups and complementary functional groups
can also include linkers, for flexibility or steric rigidity as the
case may be, or other reasons.
[0256] It should be noted that while the figures depict the
presence of a functional group and the complementary functional
group, in some cases the addition results in the loss of atoms from
these groups, and thus this is not meant to depict a situation when
the entire functional group and complementary functional group is
present in the final composition.
[0257] In addition, the figures depict the use of "monofunctional"
linkers, e.g. a maleimide. It is also possible to include
additional steps that utilize either homo- or heterobifunctional
groups, (see 1994 Pierce Chemical Company catalog, technical
section on cross linkers, pages 155-200, incorporated herein by
reference). For example, an attachment linker comprising a sulfur
atom on one terminus and an amino group on the other end could be
reacted with a bifunctional linker that reacts with amines, and
then subsequently a capture ligand comprising an amino group can be
added.
[0258] In another embodiment, the compositions of the invention are
made by synthesizing each component and adding them to the
electrode, generally simultaneously. That is, in the embodiment of
FIG. 12A, for example, the REAMC comprising the attachment linker
(with the attachment functional moiety such as a sulfur atom), the
ligands, the transition metal and the binding ligand is made, and
added (optionally with a SAM forming species) to the electrode.
Similarly, a two or three component system is done in FIG. 1B, with
a first species comprising the EAM with the attachment linker and
attachment functional group, a second species comprising an
attachment linker with the capture ligand, and the optional third
species of a SAM forming species, which are added, against
generally simultaneously, to the electrode. In some cases, the
components can be added sequentially, and in some cases, a post
synthesis step done of adding extra SAM forming species (and/or
other components) with optional heating can be done to ensure good
packing on the electrode.
[0259] In some embodiments, the ligands can have functionalities
that allow the anchor and capture ligand to be added to it after
the metal complex is formed.
[0260] In some embodiments, the compound is synthesized stepwise.
Thus, the capture ligand and the anchor are added to the ligand of
the EAM sequentially.
[0261] In some embodiments, the capture ligand and the anchor are
added to the EAM concomitantly.
[0262] In some embodiments, "clip" are added to the EAM first and
the capture ligand and anchor groups are added to the "clips"
later. By "clips" herein is meant a group or moiety that can be
attached to an EAM covalently, and on to which the capture ligand
and/or the anchor group could be added.
[0263] One of the clips is shown below:
##STR00027##
[0264] wherein PG=protection group.
[0265] In some embodiments, wherein pentaammine is used as
coordinating ligands, the capture ligand can be added first, and
the anchor group is added to the capture ligand. The compositions
of the present invention may be used in a variety of research,
clinical, quality control, or field testing settings. The examples
provided herein are for illustration purposes only and are in no
means to limit the scope the present invention. Further, all
references cited herein are incorporated by reference for all the
relevant contents therein.
V. Method of Using the Composition of the Invention
A. Target Analyte and Sample
[0266] In one aspect, the present invention provides methods and
compositions useful in the detection of target analytes. By "target
analyte" or "analyte" or grammatical equivalents herein is meant
any molecule or compound to be detected and that can bind to a
binding species, e.g. a capture ligand, defined below. Suitable
analytes include, but not limited to, small chemical molecules such
as environmental or clinical chemical or pollutant or biomolecule,
including, but not limited to, pesticides, insecticides, toxins,
therapeutic and abused drugs, hormones, antibiotics, antibodies,
organic materials, etc. Suitable biomolecules include, but are not
limited to, proteins (including enzymes, immunoglobulins and
glycoproteins), nucleic acids, lipids, lectins, carbohydrates,
hormones, whole cells (including procaryotic (such as pathogenic
bacteria) and eucaryotic cells, including mammalian tumor cells),
viruses, spores, etc. Particularly preferred analytes are proteins
including enzymes; drugs, cells; antibodies; antigens; cellular
membrane antigens and receptors (neural, hormonal, nutrient, and
cell surface receptors) or their ligands.
[0267] In some embodiments, the target analyte is cytochrome P450,
avidin/streptavdin, SEB, PSA- (protease), tryprin/chymotrypin
(protease), anthrax spore and E. col. 0157:H7.
[0268] In some embodiments, the target analyte is a protein. As
will be appreciated by those in the art, there are a large number
of possible proteinaceous target analytes that may be detected
using the present invention. By "proteins" or grammatical
equivalents herein is meant proteins, oligopeptides and peptides,
derivatives and analogs, including proteins containing
non-naturally occurring amino acids and amino acid analogs, and
peptidomimetic structures. The side chains may be in either the (R)
or the (S) configuration. In a preferred embodiment, the amino
acids are in the (S) or L-configuration. As discussed below, when
the protein is used as a capture ligand, it may be desirable to
utilize protein analogs to retard degradation by sample
contaminants. Suitable protein target analytes include, but are not
limited to, (1) immunoglobulins, particularly IgEs, IgGs and IgMs,
and particularly therapeutically or diagnostically relevant
antibodies, including but not limited to, for example, antibodies
to human albumin, apolipoproteins (including apolipoprotein E),
human chorionic gonadotropin, cortisol, .alpha.-fetoprotein,
thyroxin, thyroid stimulating hormone (TSH), antithrombin,
antibodies to pharmaceuticals (including antieptileptic drugs
(phenytoin, primidone, carbariezepin, ethosuximide, valproic acid,
and phenobarbitol), cardioactive drugs (digoxin, lidocaine,
procainamide, and disopyramide), bronchodilators (theophylline),
antibiotics (chloramphenicol, sulfonamides), antidepressants,
immunosuppresants, abused drugs (amphetamine, methamphetamine,
cannabinoids, cocaine and opiates) and antibodies to any number of
viruses (including orthomyxoviruses, (e.g. influenza virus),
paramyxoviruses (e.g respiratory syncytial virus, mumps virus,
measles virus), adenoviruses, rhinoviruses, coronaviruses,
reoviruses, togaviruses (e.g. rubella virus), parvoviruses,
poxviruses (e.g. variola virus, vaccinia virus), enteroviruses
(e.g. poliovirus, coxsackievirus), hepatitis viruses (including A,
B and C), herpesviruses (e.g. Herpes simplex virus,
varicella-zoster virus, cytomegalovirus, Epstein-Barr virus),
rotaviruses, Norwalk viruses, hantavirus, arenavirus, rhabdovirus
(e.g. rabies virus), retroviruses (including HIV, HTLV-I and -II),
papovaviruses (e.g. papillomavirus), polyomaviruses, and
picornaviruses, and the like), and bacteria (including a wide
variety of pathogenic and non-pathogenic prokaryotes of interest
including Bacillus; Vibrio, e.g. V. cholerae; Escherichia, e.g.
Enterotoxigenic E. coli, Shigella, e.g. S. dysenteriae; Salmonella,
e.g. S. typhi; Mycobacterium e.g. M. tuberculosis, M. leprae;
Clostridium, e.g. C. botulinum, C. tetani, C. difficile, C.
perfringens; Cornyebacterium, e.g. C. diphtheriae; Streptococcus,
S. pyogenes, S. pneumoniae; Staphylococcus, e.g. S. aureus;
Haemophilus, e.g. H. influenzae; Neisseria, e.g. N. meningitidis,
N. gonorrhoeae; Yersinia, e.g. G. lamblia Y. pestis, Pseudomonas,
e.g. P. aeruginosa, P. putida; Chlamydia, e.g. C. trachomatis;
Bordetella, e.g. B. pertussis; Treponema, e.g. T. palladium; and
the like); (2) enzymes (and other proteins), including but not
limited to, enzymes used as indicators of or treatment for heart
disease, including creatine kinase, lactate dehydrogenase,
aspartate amino transferase, troponin T, myoglobin, fibrinogen,
cholesterol, triglycerides, thrombin, tissue plasminogen activator
(tPA); pancreatic disease indicators including amylase, lipase,
chymotrypsin and trypsin; liver function enzymes and proteins
including cholinesterase, bilirubin, and alkaline phosphotase;
aldolase, prostatic acid phosphatase, terminal deoxynucleotidyl
transferase, and bacterial and viral enzymes such as HIV protease;
(3) hormones and cytokines (many of which serve as ligands for
cellular receptors) such as erythropoietin (EPO), thrombopoietin
(TPO), the interleukins (including IL-1 through IL-17), insulin,
insulin-like growth factors (including IGF-1 and -2), epidermal
growth factor (EGF), transforming
Sequence CWU 1
1
618PRTArtificial SequenceSynthetic 1Ala Thr Tyr Pro Leu Pro Ile Arg
1 5 27PRTArtificial SequenceSynthetic 2Tyr Tyr Trp Leu His His Cys
1 5 316PRTArtificial SequenceSynthetic 3Leu His Ile His Arg Thr Leu
Ser Ile Gln Gly Gly Gly Gly Ser Cys 1 5 10 15 47PRTArtificial
SequenceSynthetic 4His Ser Ser Lys Leu Gln Cys 1 5 57PRTArtificial
SequenceSynthetic 5Tyr Tyr Trp Leu His His Cys 1 5 68PRTArtificial
SequenceSynthetic 6Ala Thr Tyr Pro Leu Pro Ile Arg 1 5
* * * * *