U.S. patent application number 11/982378 was filed with the patent office on 2008-10-09 for detection of target analytes using particles and electrodes.
This patent application is currently assigned to Clinical Micro Sensors, Inc.. Invention is credited to Cynthia C. Bamdad, Robert C. Mucic.
Application Number | 20080248592 11/982378 |
Document ID | / |
Family ID | 22308256 |
Filed Date | 2008-10-09 |
United States Patent
Application |
20080248592 |
Kind Code |
A1 |
Bamdad; Cynthia C. ; et
al. |
October 9, 2008 |
Detection of target analytes using particles and electrodes
Abstract
The invention relates to the use of particles comprising binding
ligands and electron transfer moieties (ETMs). Upon binding of a
target analyte, a particle and a reporter composition are
associated and transported to an electrode surface. The ETMs are
then detected, allowing the presence or absence of the target
analyte to be determined.
Inventors: |
Bamdad; Cynthia C.; (Sharon,
MA) ; Mucic; Robert C.; (Glendale, CA) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS, LLP
ONE MARKET SPEAR STREET TOWER
SAN FRANCISCO
CA
94105
US
|
Assignee: |
Clinical Micro Sensors,
Inc.
Pasadena
CA
|
Family ID: |
22308256 |
Appl. No.: |
11/982378 |
Filed: |
October 31, 2007 |
Related U.S. Patent Documents
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11445815 |
Jun 1, 2006 |
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11982378 |
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10016416 |
Dec 10, 2001 |
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11445815 |
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09428155 |
Oct 27, 1999 |
6541617 |
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10016416 |
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60105875 |
Oct 27, 1998 |
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Current U.S.
Class: |
436/518 |
Current CPC
Class: |
G01N 27/48 20130101;
Y10S 977/92 20130101; C12Q 1/6825 20130101; B82Y 30/00 20130101;
C12Q 2525/197 20130101; C12Q 2565/1015 20130101; C12Q 2525/313
20130101; C12Q 2563/113 20130101; C12Q 2565/1015 20130101; B01J
13/00 20130101; C12Q 2565/101 20130101; G01N 27/3275 20130101; C12Q
1/6825 20130101; B82Y 15/00 20130101; Y10T 436/143333 20150115;
C12Q 1/6825 20130101; Y10S 977/773 20130101; G01N 27/3277
20130101 |
Class at
Publication: |
436/518 |
International
Class: |
G01N 33/543 20060101
G01N033/543 |
Claims
1. A composition comprising a colloid particle comprising at least
one electron transfer moiety (ETM).
2. A composition according to claim 1 wherein said particle further
comprising a self-assembled monolayer (SAM).
3. A composition according to claim 1 wherein said colloid further
comprises at least one covalently attached nucleic acid.
4. A composition comprising a colloid particle comprising a
covalently attached conductive oligomer.
5. A composition according to claim 4 wherein said colloid particle
further comprises at one ETM.
6. A composition comprising a colloid particle comprising: a) a
SAM; b) a capture probe; c) an amplification sequence; and d) a
label probe hybridized to said amplification sequence, wherein said
label probe comprises at least one covalently attached ETM.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the use of particles comprising
binding ligands and electron transfer moieties (ETMs). Upon binding
of a target analyte, a particle and a reporter composition are
associated and transported to an electrode surface. The ETMs are
then detected, allowing the presence or absence of the target
analyte to be determined.
BACKGROUND OF THE INVENTION
[0002] There are a number of assays and sensors for the detection
of the presence and/or concentration of specific substances in
fluids and gases. Many of these rely on specific ligand/antiligand
reactions as the mechanism of detection. That is, pairs of
substances (i.e. the binding pairs or ligand/antiligands) are known
to bind to each other, while binding little or not at all to other
substances. This has been the focus of a number of techniques that
utilize these binding pairs for the detection of the complexes.
These generally are done by labeling one component of the complex
in some way, so as to make the entire complex detectable, using,
for example, radioisotopes, fluorescent and other optically active
molecules, enzymes, etc.
[0003] Other assays rely on electronic signals for detection. Of
particular interest are biosensors. At least two types of
biosensors are known; enzyme-based or metabolic biosensors and
binding or bioaffinity sensors. See for example U.S. Pat. Nos.
4,713,347; 5,192,507; 4,920,047; 3,873,267; and references
disclosed therein. While some of these known sensors use
alternating current (AC) techniques, these techniques are generally
limited to the detection of differences in bulk (or dielectric)
impedance, and rely on the use of mediators in solution to shuttle
the charge to the electrode.
[0004] Recently, there have been several preliminary reports on the
use of very short connections between a binding ligand and the
electrode, for direct detection, i.e. without the use of mediators.
See Lotzbeyer et al., Bioelectrochemistry and Bioenergetics 42:1-6
(1997); Dong et al., Bioelectrochemistry and Bioenergetics 42:7-13
(1997).
[0005] In addition, there are a number of reports of self-assembled
monolayers of conjugated oligomers on surfaces such as gold. See
for example Cygan et al., J. Am. Chem. Soc. 120:2721 (1998).
[0006] In addition, Charych et al. report on the direct colormetric
detection of a receptor-ligand interaction using 3 bilayer assembly
(Science 261:585 (1993).
[0007] PCT applications WO 95/15971, PCT/US96/09769,
PCT/US97/09739, WO96/40712 and PCT/US97/20014 describe novel
compositions comprising nucleic acids containing electron transfer
moieties, including electrodes, which allow for novel detection
methods of nucleic acid hybridization.
[0008] In addition, there are a number of sensors that rely on the
use of particles, including magnetic particles, particularly for
electrochemiluminescence detection. See U.S. Pat. Nos. 5,746,974;
5,770,459; 5,779,976; 4,731,337; 4,115,535; 4,777,145; 4,945,045;
4,978,610; 5,705,402; 4,910,148; 5,512,439; 5,585,241; and
5,609,907; and WO 90/14891; WO 90/05301; WO 92/14139; and WO
90/06044.
[0009] Finally, there are reports that bring particles together
using nucleic acids for archetectural reasons. See Mirkin et al.,
Nature 382:607 (1996); Mirkin et al., Science 277:1078 (1997);
Storhoff et al., J. Am. Chem. Soc. 120:1959 (1998); and WO
98/10289.
SUMMARY OF THE INVENTION
[0010] In accordance with the objects outlined above, the present
invention provides compositions comprising a gold colloid particle
comprising at least one ETM. The colloid can further comprise a
self-assembled monolayer (SAM). The compositions can further
comprise an electrode, that may also contain a SAM.
[0011] In an additional aspect, the invention provides compositions
comprising gold colloid particles comprising a SAM, a capture
probe, an amplification sequence; and a label probe hybridized to
the amplification sequence, wherein the label probe comprises at
least one covalently attached ETM.
[0012] In an additional aspect, the invention provides compositions
comprising a transport composition comprising a first binding
partner that directly or indirectly binds a target analyte, and a
reporter composition. The reporter composition comprises a second
binding partner that directly or indirectly binds the target
analyte and a plurality of electron transfer moieties (ETMs). At
least one of the transport and the reporter compositions is a
particle. Upon introduction of the target analyte, the transport
composition and the reporter composition are associated. These
compositions may also comprise SAMs, and an electrode, optionally
containing a SAM, can be included.
[0013] In a further aspect, the invention provides methods of
detecting a target analyte in a sample. The methods comprise adding
the sample to a composition as outlined above, such that the target
analyte binds to the transport composition and the reporter
composition to form an assay complex. The assay complex is
transported to the electrode, and the presence or absence of the
ETMs is detected.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIGS. 1A-1G depict a variety of different embodiments of the
invention. Many of the figures depict nucleic acids as the binding
ligands, target moieties, and extender moieties, but non-nucleic
acid embodiments can also be used in a similar manner. FIG. 1A
shows an assay complex comprising a transport particle 5 with a
first binding ligand 10. An optional SAM of moieties 45 that can be
either conductive oligomers or insulators may also be present. The
target analyte 20 binds to first binding ligand 10 and the second
binding ligand 25, attached to the reporter particle 30. The
reporter particle 30 comprises a plurality of ETMs 40, preferably
linked to the reporter particle 30 via conductive oligomers 35. A
SAM of moieties 45 may also optionally be present. The binding
ligands 10 and 25 can be attached via attachment linkers 60, not
shown, that can be a conductive oligomer, an insulator, or other
moieties, as outlined herein. FIG. 1B is similar, but utilizes an
extender moiety 50; as will be appreciated by those in the art, the
extender moiety 50 may also be placed between the target 20 and the
first binding ligand 25. In addition, the extender moiety
(sometimes referred to herein as an amplifier probe) may also
contain multiple amplification sequences for attachment of reporter
compositions; thus n is an integer of at least one. FIG. 1C depicts
the use of a capture binding ligand 55 attached to the transport
particle 5 via an attachment linker 60. Additional embodiments for
the attachment of nucleic acids to the electrode surface are shown
in FIG. 2. The capture binding ligand 55 binds to a capture binding
partner 65 attached to electrode 85 via an attachment linker 60.
The electrode comprises conductive oligomers 70 and optional
insulators 80. FIG. 1D depicts the use of a non-particle transport
composition comprising a capture binding ligand 55 attached via an
optional linker 90 (that can be an attachment linker) to the
capture binding ligand 10. FIG. 1E depicts the use of the target
analyte as the capture binding moiety; the target 20 binds to the
capture binding partner 65, attached to the electrode 85 via an
attachment linker 60. The second binding ligand 25 binds to a
portion of the target 20. Depending on the length (i.e. when the
target analyte is a nucleic acid) or size of the target analyte, a
plurality n of reporter compositions can be used; thus n is an
integer of at least one. FIG. 1F depicts the case wherein two
reporter particles are used; what is important in this embodiment
is that aggregation does not occur to an appreciable extent in the
absence of the target 20, and that generally the two second binding
ligands 25 each recognize a different part of the target 20, such
that a single target will bring together at least two reporter
compositions. FIG. 1G depicts the use of a non-particle reporter
composition, using a recruitment linker 90, as outlined herein.
FIG. 1H depicts the use of a reporter particle 30 that has both a
second binding ligand 25 and a recruitment linker 90.
[0015] FIGS. 2A, 2B and 2C depict three preferred embodiments for
attaching a target sequence to the electrode. Although generally
depicted as nucleic acids, non-nucleic acid embodiments are also
useful. FIG. 2A depicts a target sequence 120 hybridized to a
capture probe 100 linked via a attachment linker 106, which as
outlined herein may be either a conductive oligomer or an
insulator. The electrode 105 comprises a monolayer of passivation
agent 107, which can comprise conductive oligomers (herein depicted
as 108) and/or insulators (herein depicted as 109). As for all the
embodiments depicted in the figures, n is an integer of at least 1,
although as will be appreciated by those in the art, the system may
not utilize a capture probe at all (i.e. n is zero), although this
is generally not preferred. The upper limit of n will depend on the
length of the target sequence and the required sensitivity. FIG. 2B
depicts the use of a single capture extender probe 110 with a first
portion 111 that will hybridize to a first portion of the target
sequence 120 and a second portion that will hybridize to the
capture probe 100. FIG. 2C depicts the use of two capture extender
probes 110 and 130. The first capture extender probe 110 has a
first portion 111 that will hybridize to a first portion of the
target sequence 120 and a second portion 112 that will hybridize to
a first portion 102 of the capture probe 100. The second capture
extender probe 130 has a first portion 132 that will hybridize to a
second portion of the target sequence 120 and a second portion 131
that will hybridize to a second portion 101 of the capture probe
100.
[0016] FIGS. 3A, 3B, 3C, 3D and 3E depict different possible
configurations of label probes and attachments of ETMs. The figures
depict a second binding ligand that is a hybridizable nucleic acid,
but as will be appreciated by those in the art, the second binding
ligand can be non-nucleic acid as well. In FIGS. 3A-C, the
recruitment linker is nucleic acid; in FIGS. 3D and E, is not.
A=nucleoside replacement; B=attachment to a base; C=attachment to a
ribose; D=attachment to a phosphate; E=metallocene polymer
(although as described herein, this can be a polymer of other ETMs
as well), attached to a base, ribose or phosphate (or other
backbone analogs); F=dendrimer structure, attached via a base,
ribose or phosphate (or other backbone analogs); G=attachment via a
"branching" structure, through base, ribose or phosphate (or other
backbone analogs); H=attachment of metallocene (or other ETM)
polymers; I=attachment via a dendrimer structure; J=attachment
using standard linkers.
[0017] FIG. 4 depicts a schematic of the synthesis of simultaneous
incorporation of multiple ETMs into a nucleic acid, using the N17
"branch" point nucleoside.
[0018] FIG. 5 depicts a schematic of an alternate method of adding
large numbers of ETMs simultaneously to a nucleic acid using a
"branch" point phosphoramidite, in this case utilizing three branch
points (although two branch points are also possible; see for
example FIG. 1N) as is known in the art. As will be appreciated by
those in the art, each end point can contain any number of
ETMs.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention provides novel analytical biosensors
that can be used to sensitively detect target analytes. In one
embodiment, the system provides two basic components: (1) a first
component (generally but not always a particle) that can bind a
target analyte and that can be used to transport an assay complex
comprising the first component to an electrode, and (2) a reporter
composition (which may or may not be a particle as well) that can
bind a target analyte and comprises electron transfer moieties
(ETMs). The two components are brought together by the direct or
indirect binding of a target analyte. That is, a single target
analyte directly or indirectly binds both a first binding ligand
attached to the first component and a second binding ligand
attached to the reporter composition; this forms an assay complex.
The first component can be used to transport the assay complex to
an electrode for detection of the ETMs. This may be done in a
variety of ways; for example, when the first component is a
particle, transport can occur either magnetically, when the
particle is a magnetic particle, or via gravity or other techniques
based on the specific gravity or density of the particle in
relation to the solution. In some embodiments, both components are
particles and the aggregation of the particles using target
analytes results in transport to the electrode. Alternatively, the
first component may utilize a capture moiety for attachment to an
electrode that comprises a capture binding ligand. Once the assay
complexes are formed, the presence or absence of the ETMs are
detected using the electrode as is described below and in U.S. Pat.
Nos. 5,591,578; 5,824,473; 5,770,369; 5,705,348 and 5,780,234; U.S.
Ser. Nos. 08/911,589; 09/135,183; 09/306,653; 09/134,058;
09/295,691; 09/238,351; 09/245,105, 60/145,912 and 09/338,726; and
PCT applications WO98/20162; PCT US99/01705; PCT US99/01703; PCT
US99/10104, all of which are expressly incorporated herein by
reference in their entirety.
[0020] In general, there are two basic detection mechanisms. In a
preferred embodiment, detection of an ETM is based on electron
transfer through the stacked n-orbitals of double stranded nucleic
acid. This basic mechanism is described in U.S. Pat. Nos.
5,591,578, 5,770,369, 5,705,348, 5,824,473 and 5,780,234 and
WO98/20162, all of which are expressly incorporated by reference,
and is termed "mechanism-1" herein. Briefly, previous work has
shown that electron transfer can proceed rapidly through the
stacked n-orbitals of double stranded nucleic acid, and
significantly more slowly through single-stranded nucleic acid.
Accordingly, this can serve as the basis of an assay. Thus, by
adding ETMs (either covalently to one of the strands or
non-covalently to the hybridization complex through the use of
hybridization indicators, described below) to a nucleic acid that
is attached to a detection electrode via a conductive oligomer,
electron transfer between the ETM and the electrode, through the
nucleic acid and conductive oligomer, may be detected.
[0021] Alternatively, ETMs can be directly detected on a surface of
a monolayer. That is, the electrons from the ETMs need not travel
through the stacked .pi. orbitals in order to generate a signal. As
above, in this embodiment, the detection electrode preferably
comprises a self-assembled monolayer (SAM) that serves to shield
the electrode from redox-active species in the sample. In this
embodiment, the presence of ETMs on the surface of a SAM, that has
been formulated to comprise slight "defects" (sometimes referred to
herein as "microconduits", "nanoconduits" or "electroconduits") can
be directly detected. This basic idea is termed "mechanism-2"
herein. Essentially, the electroconduits allow particular ETMs
access to the surface. Without being bound by theory, it should be
noted that the configuration of the electroconduit depends in part
on the ETM chosen. For example, the use of relatively hydrophobic
ETMs allows the use of hydrophobic electroconduit forming species,
which effectively exclude hydrophilic or charged ETMs. Similarly,
the use of more hydrophilic or charged species in the SAM may serve
to exclude hydrophobic ETMs.
[0022] It should be noted that these defects are to be
distinguished from "holes" that allow direct contact of sample
components with the detection electrode. As is more fully outlined
below, the electroconduits can be generated in several general
ways, including but not limited to the use of rough electrode
surfaces, such as gold electrodes formulated on PC circuit boards;
or the inclusion of at least two different species in the
monolayer, i.e. using a "mixed monolayer", at least one of which is
a electroconduit-forming species (EFS). Thus, upon binding of a
target analyte, a binding ligand comprising an ETM is brought to
the surface, and detection of the ETM can proceed, putatively
through the "electroconduits" to the electrode. Essentially, the
role of the SAM comprising the defects is to allow contact of the
ETM with the electronic surface of the electrode, while still
providing the benefits of shielding the electrode from solution
components and reducing the amount of non-specific binding to the
electrodes. Viewed differently, the role of the binding ligand is
to provide specificity for a recruitment of ETMs to the surface,
where they can be directly detected.
[0023] Thus, in either embodiment, an assay complex is formed that
contains an ETM, which is then detected using the detection
electrode. The invention thus provides assay complexes that
minimally comprise a target analyte. "Assay complex" herein is
meant the collection of binding complexes, e.g. nucleic acids,
including probes and targets, that contains at least one ETM and
thus allows detection. The composition of the assay complex depends
on the use of the different probe components outlined herein.
[0024] Accordingly, the present invention provides methods and
compositions useful in the detection of target analytes in samples.
As will be appreciated by those in the art, the sample solution may
comprise any number of things, including, but not limited to,
bodily fluids (including, but not limited to, blood, urine, serum,
lymph, saliva, anal and vaginal secretions, perspiration and semen,
of virtually any organism, with mammalian samples being preferred
and human samples being particularly preferred); environmental
samples (including, but not limited to, air, agricultural, water
and soil samples); biological warfare agent samples; research
samples (i.e. in the case of nucleic acids, the sample may be the
products of an amplification reaction, including both target and
signal amplification as is generally described in PCT/US99/01705,
such as PCR amplification reaction); purified samples, such as
purified genomic DNA, RNA, proteins, etc.; raw samples (bacteria,
virus, genomic DNA, etc.); as will be appreciated by those in the
art, virtually any experimental manipulation may have been done on
the sample.
[0025] The methods are directed to 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, 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.
[0026] By "proteins" or grammatical equivalents herein is meant
proteins, oligopeptides and peptides, and analogs, including
proteins containing non-naturally occurring amino acids and amino
acid analogs, and peptidomimetic structures.
[0027] As will be appreciated by those in the art, a large number
of analytes may be detected using the present methods; basically,
any target analyte for which a binding ligand, described below, may
be made may be detected using the methods of the invention.
[0028] In a preferred embodiment, the target analytes are nucleic
acids. By "nucleic acid" or "oligonucleotide" or grammatical
equivalents herein means at least two nucleotides covalently linked
together. A nucleic acid of the present invention will generally
contain phosphodiester bonds, although in some cases, as outlined
below, nucleic acid analogs are included that may have alternate
backbones, comprising, for example, phosphoramide (Beaucage et al.,
Tetrahedron 49(10):1925 (1993) and references therein; Letsinger,
J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem.
81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986);
Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem.
Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141
91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437
(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et
al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphosphoroamidite
linkages (see Eckstein, Oligonucleotides and Analogues: A Practical
Approach, Oxford University Press), and peptide nucleic acid
backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895
(1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen,
Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996), all
of which are incorporated by reference). Other analog nucleic acids
include those with positive backbones (Denpcy et al., Proc. Natl.
Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos.
5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863;
Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991);
Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et
al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3,
ASC Symposium Series 580, "Carbohydrate Modifications in Antisense
Research", Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,
Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al.,
J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996))
and non-ribose backbones, including those described in U.S. Pat.
Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium
Series 580, "Carbohydrate Modifications in Antisense Research", Ed.
Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more
carbocyclic sugars are also included within the definition of
nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp
169-176). Several nucleic acid analogs are described in Rawls, C
& E News Jun. 2, 1997 page 35. All of these references are
hereby expressly incorporated by reference. These modifications of
the ribose-phosphate backbone may be done to facilitate the
addition of ETMs, or to increase the stability and half-life of
such molecules in physiological environments.
[0029] As will be appreciated by those in the art, all of these
nucleic acid analogs may find use in the present invention. In
addition, mixtures of naturally occurring nucleic acids and analogs
can be made; for example, at the site of conductive oligomer or ETM
attachment, an analog structure may be used. Alternatively,
mixtures of different nucleic acid analogs, and mixtures of
naturally occurring nucleic acids and analogs may be made.
[0030] Particularly preferred are peptide nucleic acids (PNA) which
includes peptide nucleic acid analogs. These backbones are
substantially non-ionic under neutral conditions, in contrast to
the highly charged phosphodiester backbone of naturally occurring
nucleic acids. This results in two advantages. First, the PNA
backbone exhibits improved hybridization kinetics. PNAs have larger
changes in the melting temperature (Tm) for mismatched versus
perfectly matched basepairs. DNA and RNA typically exhibit a
2-4.degree. C. drop in Tm for an internal mismatch. With the
non-ionic PNA backbone, the drop is closer to 7-9.degree. C.
Similarly, due to their non-ionic nature, hybridization of the
bases attached to these backbones is relatively insensitive to salt
concentration. This is particularly advantageous in the systems of
the present invention, as a reduced salt hybridization solution has
a lower Faradaic current than a physiological salt solution (in the
range of 150 mM).
[0031] The nucleic acids may be single stranded or double stranded,
as specified, or contain portions of both double stranded or single
stranded sequence. The nucleic acid may be DNA, both genomic and
cDNA, RNA or a hybrid, where the nucleic acid contains any
combination of deoxyribo- and ribo-nucleotides, and any combination
of bases, including uracil, adenine, thymine, cytosine, guanine,
inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc. A
preferred embodiment utilizes isocytosine and isoguanine in nucleic
acids designed to be complementary to other probes, rather than
target sequences, as this reduces non-specific hybridization, as is
generally described in U.S. Pat. No. 5,681,702. As used herein, the
term "nucleoside" includes nucleotides as well as nucleoside and
nucleotide analogs, and modified nucleosides such as amino modified
nucleosides. In addition, "nucleoside" includes non-naturally
occurring analog structures. Thus for example the individual units
of a peptide nucleic acid, each containing a base, are referred to
herein as a nucleoside.
[0032] Thus, in a preferred embodiment, the target analyte is a
target sequence. The term "target sequence" or "target nucleic
acid" or grammatical equivalents herein means a nucleic acid
sequence on a single strand of nucleic acid. The target sequence
may be a portion of a gene, a regulatory sequence, genomic DNA,
cDNA, RNA including mRNA and rRNA, or others. As is outlined
herein, the target sequence may be a target sequence from a sample,
or a secondary target such as a product of an amplification
reaction, etc. It may be any length, with the understanding that
longer sequences are more specific. As will be appreciated by those
in the art, the complementary target sequence may take many forms.
For example, it may be contained within a larger nucleic acid
sequence, i.e. all or part of a gene or mRNA, a restriction
fragment of a plasmid or genomic DNA, among others. As is outlined
more fully below, probes are made to hybridize to target sequences
to determine the presence or absence of the target sequence in a
sample. Generally speaking, this term will be understood by those
skilled in the art. The target sequence may also be comprised of
different target domains; for example, a first target domain of the
sample target sequence may hybridize to a capture probe or a
portion of capture extender probe, a second target domain may
hybridize to a portion of an amplifier probe, a label probe, or a
different capture or capture extender probe, etc. The target
domains may be adjacent or separated as indicated. Unless
specified, the terms "first" and "second" are not meant to confer
an orientation of the sequences with respect to the 5'-3'
orientation of the target sequence. For example, assuming a 5'-3'
orientation of the complementary target sequence, the first target
domain may be located either 5' to the second domain, or 3' to the
second domain.
[0033] In a preferred embodiment, the methods of the invention are
used to detect pathogens such as bacteria. In this embodiment,
preferred target sequences include rRNA, as is generally described
in U.S. Pat. Nos. 4,851,330; 5,288,611; 5,723,597; 6,641,632;
5,738,987; 5,830,654; 5,763,163; 5,738,989; 5,738,988; 5,723,597;
5,714,324; 5,582,975; 5,747,252; 5,567,587; 5,558,990; 5,622,827;
5,514,551; 5,501,951; 5,656,427; 5,352,579; 5,683,870; 5,374,718;
5,292,874; 5,780,219; 5,030,557; and 5,541,308, all of which are
expressly incorporated by reference.
[0034] As will be appreciated by those in the art, a large number
of analytes may be detected using the present methods; basically,
any target analyte for which a binding ligand, described below, may
be made may be detected using the methods of the invention. While
many of the techniques described below exemplify nucleic acids as
the target analyte, those of skill in the art will recognize that
other target analytes can be detected using the same systems.
[0035] If required, the target analyte is prepared using known
techniques. For example, the sample may be treated to lyse the
cells, using known lysis buffers, electroporation, etc., with
purification and/or amplification as needed, as will be appreciated
by those in the art. When the target analyte is a nucleic acid, the
target sequence may be amplified as required; suitable
amplification techniques are outlined in PCT US99101705, hereby
expressly incorporated by reference. In addition, techniques to
increase the amount or rate of hybridization can also be used; see
for example U.S. Ser. No. 09/338,726, filed Jun. 23, 1999, hereby
incorporated by reference.
[0036] All of these techniques rely on the formation of assay
complexes on a surface, frequently an electrode, as is described
herein. The assay complex preferably includes at least one
particle, generally a gold colloid particle, that is used either as
a transport particle or a reporter particle, or both. The assay
complex further comprises at least one electron transfer moiety
(ETM), that is also either directly or indirectly attached to the
assay complex. Once the assay complexes are formed, the presence or
absence of the ETMs are detected as is described below and in U.S.
Pat. Nos. 5,591,578; 5,824,473; 5,770,369; 5,705,348 and 5,780,234;
U.S. Ser. Nos. 08/911,589; 09/135,183; 09/306,653; 09/134,058;
09/295,691; 09/238,351; 60/145,912 and 09/245,105; and PCT
applications WO98/20162; PCT US99/01705; PCT US99/01703; PCT
US99/14191; PCT US99/10104, all of which are expressly incorporated
herein by reference in their entirety.
[0037] Accordingly, the present invention provides methods and
compositions useful in the detection of target analytes, including
nucleic acids. As will be appreciated by those in the art, the
compositions of the invention can take on a wide variety of
configurations, as is generally outlined in the Figures and
described below. It should be noted that while the discussion below
focuses on the detection of nucleic acids, other target analytes
can be used in any of the systems described herein.
[0038] Thus, in a preferred embodiment, the compositions comprise
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. Alternatively an electrode
can be defined as a composition which can apply a potential to
and/or pass electrons to or from species in the solution. Thus, an
electrode is an ETM as described herein. 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 (Mo.sub.2O.sub.6),
tungsten oxide (WO.sub.3) 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.
[0039] 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 SAMs comprising conductive oligomers
and nucleic acids 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.
[0040] In addition, the geometry of the system may alter with the
transport mechanism used. For example, when aggregation of the
particles is used to transport the ETMs to the electrode surface,
the electrode surface needs to be at the bottom. However, other
systems utilizing different transport mechanisms can have the
electrode surface be anywhere.
[0041] In a preferred embodiment, the detection electrodes are
formed on a substrate. In addition, the discussion herein is
generally directed to the formation 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
will be appreciated by those in the art, with printed circuit board
(PCB) materials being particularly preferred. Thus, in general, the
suitable substrates include, but are not limited to, fiberglass,
teflon, ceramics, glass, silicon, mica, plastic (including
acrylics, polystyrene and copolymers of styrene and other
materials, polypropylene, polyethylene, polybutylene,
polycarbonate, polyurethanes, Teflon.TM., and derivatives thereof,
etc.), GETEK (a blend of polypropylene oxide and fiberglass),
etc.
[0042] 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) 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.
[0043] 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.
[0044] The substrates can be part of a larger device comprising a
detection chamber that exposes a given volume of sample to the
detection electrode. Generally, the detection chamber ranges from
about 1 nL to 1 ml, with about 10 .mu.L to 500 .mu.L being
preferred. As will be appreciated by those in the art, depending on
the experimental conditions and assay, smaller or larger volumes
may be used.
[0045] In some embodiments, the detection chamber and electrode are
part of a cartridge that can be placed into a device comprising
electronic components (an AC/DC voltage source, an ammeter, a
processor, a read-out display, temperature controller, light
source, etc.). In this embodiment, the interconnections from each
electrode are positioned such that upon insertion of the cartridge
into the device, connections between the electrodes and the
electronic components are established.
[0046] Detection electrodes on circuit board material (or other
substrates) are generally prepared in a wide variety of ways. In
general, high purity gold is used, and it may be deposited on a
surface via vacuum deposition processes (sputtering and
evaporation) or solution deposition (electroplating or electroless
processes). When electroplating is done, the substrate must
initially comprise a conductive material; fiberglass circuit boards
are frequently provided with copper foil. Frequently, depending on
the substrate, an adhesion layer between the substrate and the gold
in order to insure good mechanical stability is used. Thus,
preferred embodiments utilize a deposition layer of an adhesion
metal such as chromium, titanium, titanium/tungsten, tantalum,
nickel or palladium, which can be deposited as above for the gold.
When electroplated metal (either the adhesion metal or the
electrode metal) is used, grain refining additives, frequently
referred to in the trade as brighteners, can optionally be added to
alter surface deposition properties. Preferred brighteners are
mixtures of organic and inorganic species, with cobalt and nickel
being preferred.
[0047] In general, the adhesion layer is from about 100 .ANG. thick
to about 25 microns (1000 microinches). The If the adhesion metal
is electrochemically active, the electrode metal must be coated at
a thickness that prevents "bleed-through"; if the adhesion metal is
not electrochemically active, the electrode metal may be thinner.
Generally, the electrode metal (preferably gold) is deposited at
thicknesses ranging from about 500 .ANG. to about 5 microns (200
microinches), with from about 30 microinches to about 50
microinches being preferred. In general, the gold is deposited to
make electrodes ranging in size from about 5 microns to about 5 mm
in diameter, with about 100 to 250 microns being preferred. The
detection electrodes thus formed are then preferably cleaned and
SAMs added, as is discussed below.
[0048] Thus, the present invention provides methods of making a
substrate comprising a plurality of gold electrodes. The methods
first comprise coating an adhesion metal, such as nickel or
palladium (optionally with brightener), onto the substrate.
Electroplating is preferred. The electrode metal, preferably gold,
is then coated (again, with electroplating preferred) onto the
adhesion metal. Then the patterns of the device, comprising the
electrodes and their associated interconnections are made using
lithographic techniques, particularly photolithographic techniques
as are known in the art, and wet chemical etching. Frequently, a
non-conductive chemically resistive insulating material such as
solder mask or plastic is laid down using these photolithographic
techniques, leaving only the electrodes and a connection point to
the leads exposed; the leads themselves are generally coated.
[0049] The methods continue with the addition of SAMs as are
described below. In a preferred embodiment, drop deposition
techniques are used to add the required chemistry, i.e. the
monolayer forming species, one of which is preferably a capture
ligand comprising species. Drop deposition techniques are well
known for making "spot" arrays. This is done to add a different
composition to each electrode, i.e. to make an array comprising
different capture ligands. Alternatively, the SAM species may be
identical for each electrode, and this may be accomplished using a
drop deposition technique or the immersion of the entire substrate
or a surface of the substrate into the solution.
[0050] Thus, in a preferred embodiment, the electrode comprises a
monolayer, comprising electroconduit forming species (EFS). As
outlined herein, the efficiency of target analyte binding (for
example, oligonucleotide hybridization) may increase when the
analyte is at a distance from the electrode. Similarly,
non-specific binding of biomolecules, including the target
analytes, to an electrode is generally reduced when a monolayer is
present. Thus, a monolayer facilitates the maintenance of the
analyte away from the electrode surface. In addition, a monolayer
serves to keep charged species away from the surface of the
electrode. Thus, this layer helps to prevent electrical contact
between the electrodes and the ETMs, 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.
[0051] 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.
[0052] In general, the SAMs of the invention can be generated in a
number of ways and comprise a number of different components,
depending on the electrode surface and the system used. For
"mechanism-1" embodiments, preferred embodiments utilize two
monolayer forming species: a monolayer forming species (including
insulators or conductive oligomers) and a conductive oligomer
species comprising the capture binding ligand, although as will be
appreciated by those in the art, additional monolayer forming
species can be included as well. For "mechanism-2" systems, the
composition of the SAM depends on the detection electrode surface.
In general, two basic "mechanism-2" systems are described;
detection electrodes comprising "smooth" surfaces, such as gold
ball electrodes, and those comprising "rough" surfaces, such as
those that are made using commercial processes on PC circuit
boards. In general, without being bound by theory, it appears that
monolayers made on imperfect surfaces, i.e. "rough" surfaces,
spontaneously form monolayers containing enough electroconduits
even in the absence of EFS, probably due to the fact that the
formation of a uniform monolayer on a rough surface is difficult.
"Smoother" surfaces, however, may require the inclusion of
sufficient numbers of EFS to generate the electroconduits, as the
uniform surfaces allow a more uniform monolayer to form. Again,
without being bound by theory, the inclusion of species that
disturb the uniformity of the monolayer, for example by including a
rigid molecule in a background of more flexible ones, causes
electroconduits. Thus "smooth" surfaces comprise monolayers
comprising three components: an insulator species, a EFS, and a
species comprising the capture ligand, although in some
circumstances, for example when the capture ligand species is
included at high density, the capture ligand species can serve as
the EFS. "Smoothness" in this context is not measured physically
but rather as a function of an increase in the measured signal when
EFS are included. That is, the signal from a detection electrode
coated with monolayer forming species is compared to a signal from
a detection electrode coated with monolayer forming species
including a EFS. An increase indicates that the surface is
relatively smooth, since the inclusion of a EFS served to
facilitate the access of the ETM to the electrode. It should also
be noted that while the discussion herein is mainly directed to
gold electrodes and thiol-containing monolayer forming species,
other types of electrodes and monolayer-forming species can be
used.
[0053] It should be noted that the "electroconduits" of mechanism-2
systems do not result in direct contact of sample components with
the electrode surface; that is, the electroconduits are not large
pores or holes that allow physical access to the electrode. Rather,
without being bound by theory, it appears that the electroconduits
allow certain types of ETMs, particularly hydrophobic ETMs, to
penetrate sufficiently into the monolayer to allow detection.
However, other types of redox active species, including some
hydrophilic species, do not penetrate into the monolayer, even with
electroconduits present. Thus, in general, redox active species
that may be present in the sample do not give substantial signals
as a result of the electroconduits. While the exact system will
vary with the composition of the SAM and the choice of the ETM, in
general, the test for a suitable SAM to reduce non-specific binding
that also has sufficient electroconduits for ETM detection is to
add either ferrocene or ferrocyanide to the SAM; the former should
give a signal and the latter should not.
[0054] Accordingly, in mechanism-1 systems, the monolayer comprises
a first species comprising a conductive oligomer comprising the
capture binding ligand, as is more fully outlined below, and a
second species comprising a monolayer forming species, including
either or both insulators or conductive oligomers.
[0055] In a preferred embodiment, the monolayer comprises
electroconduit-forming species. By "electroconduit-forming species"
or "EFS" herein is meant a molecule that is capable of generating
sufficient electroconduits in a monolayer, generally of insulators
such as alkyl groups, to allow detection of ETMs at the surface. In
general, EFS have one or more of the following qualities: they may
be relatively rigid molecules, for example as compared to an alkyl
chain; they may attach to the electrode surface with a geometry
different from the other monolayer forming species (for example,
alkyl chains attached to gold surfaces with thiol groups are
thought to attach at roughly 45.degree. angles, and
phenyl-acetylene chains attached to gold via thiols are thought to
go down at 90.degree. angles); they may have a structure that
sterically interferes or interrupts the formation of a tightly
packed monolayer, for example through the inclusion of branching
groups such as alkyl groups, or the inclusion of highly flexible
species, such as polyethylene glycol units; or they may be capable
of being activated to form electroconduits; for example,
photoactivatable species that can be selectively removed from the
surface upon photoactivation, leaving electroconduits.
[0056] Preferred EFS include conductive oligomers, as defined
below, and phenyl-acetylene-polyethylene glycol species. However,
in some embodiments, the EFS is not a conductive oligomer.
[0057] In a preferred embodiment, 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
n-orbitals, i.e. conjugated n-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 ETM.
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.
[0058] In a preferred embodiment, the conductive oligomers have a
conductivity, S, of from between about 10.sup.-6 to about 10.sup.4
.OMEGA..sup.-1cm.sup.-1, with from about 10.sup.-5 to about
10.sup.-3 .OMEGA..sup.-1cm.sup.-1 being preferred, with these S
values being calculated for molecules ranging from about 20 .ANG.
to about 200 .ANG.. As described below, insulators have a
conductivity S of about 10.sup.-7 .OMEGA..sup.-1cm.sup.-1 or lower,
with less than about 10.sup.-8 .OMEGA..sup.-1cm.sup.-1 being
preferred. See generally Gardner et al., Sensors and Actuators A 51
(1995) 57-66, incorporated herein by reference.
[0059] Desired characteristics of a conductive oligomer include
high conductivity, sufficient solubility in organic solvents and/or
water for synthesis and use of the compositions of the invention,
and preferably chemical resistance to reactions that occur i)
during synthesis of the components of the system, ii) during the
attachment of the conductive oligomer to an electrode, or iii)
during detection assays. In addition, conductive oligomers that
will promote the formation of self-assembled monolayers are
preferred.
[0060] The oligomers of the invention comprise at least two
monomeric subunits, as described herein. As is described more fully
below, oligomers include homo- and hetero-oligomers, and include
polymers.
[0061] In a preferred embodiment, the conductive oligomer has the
structure depicted in Structure 1:
Y B .sub.gD .sub.e .sub.n Y .sub.m Structure 1
[0062] As will be understood by those in the art, all of the
structures depicted herein may have additional atoms or structures;
i.e. the conductive oligomer of Structure 1 may be attached to
ETMs, such as electrodes, transition metal complexes, organic ETMs,
and metallocenes, and to capture binding ligands, or to several of
these. Unless otherwise noted, the conductive oligomers depicted
herein will be attached at the left side to an electrode; that is,
as depicted in Structure 1, the left "Y" is connected to the
electrode as described herein. If the conductive oligomer is to be
attached to a nucleic acid, the right "Y", if present, is attached
to the nucleic acid, either directly or through the use of a
linker, as is described herein.
[0063] In this embodiment, Y is an aromatic group, n is an integer
from 1 to 50, g is either 1 or zero, e is an integer from zero to
10, and m is zero or 1. When g is 1, B-D is a bond able to
conjugate with neighboring bonds (herein referred to as a
"conjugated bond"), preferably selected from acetylene, B-D is a
conjugated bond, preferably selected from acetylene, alkene,
substituted alkene, amide, azo, --C.dbd.N-- (including --N.dbd.C--,
--CR.dbd.N-- and --N.dbd.CR--), --Si.dbd.Si--, and --Si.dbd.C--
(including --C.dbd.Si--, --Si.dbd.CR-- and --CR.dbd.Si--). When g
is zero, e is preferably 1, D is preferably carbonyl, or a
heteroatom moiety, wherein the heteroatom is selected from oxygen,
sulfur, nitrogen, silicon or phosphorus. Thus, suitable heteroatom
moieties include, but are not limited to, --NH and --NR, wherein R
is as defined herein; substituted sulfur; sulfonyl (--SO.sub.2--)
sulfoxide (--SO--); phosphine oxide (--PO-- and --RPO--); and
thiophosphine (--PS-- and --RPS--). However, when the conductive
oligomer is to be attached to a gold electrode, as outlined below,
sulfur derivatives are not preferred.
[0064] By "aromatic group" or grammatical equivalents herein is
meant an aromatic monocyclic or polycyclic hydrocarbon moiety
generally containing 5 to 14 carbon atoms (although larger
polycyclic rings structures may be made) and any carbocylic ketone
or thioketone derivative thereof, wherein the carbon atom with the
free valence is a member of an aromatic ring. Aromatic groups
include arylene groups and aromatic groups with more than two atoms
removed. For the purposes of this application aromatic includes
heterocycle. "Heterocycle" or "heteroaryl" means an aromatic group
wherein 1 to 5 of the indicated carbon atoms are replaced by a
heteroatom chosen from nitrogen, oxygen, sulfur, phosphorus, boron
and silicon wherein the atom with the free valence is a member of
an aromatic ring, and any heterocyclic ketone and thioketone
derivative thereof. Thus, heterocycle includes thienyl, furyl,
pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl, quinolyl,
isoquinolyl, thiazolyl, imidozyl, etc.
[0065] Importantly, the Y aromatic groups of the conductive
oligomer may be different, i.e. the conductive oligomer may be a
heterooligomer. That is, a conductive oligomer may comprise a
oligomer of a single type of Y groups, or of multiple types of Y
groups.
[0066] The aromatic group may be substituted with a substitution
group, generally depicted herein as R. R groups may be added as
necessary to affect the packing of the conductive oligomers, i.e. R
groups may be used to alter the association of the oligomers in the
monolayer. R groups may also be added to 1) alter the solubility of
the oligomer or of compositions containing the oligomers; 2) alter
the conjugation or electrochemical potential of the system; and 3)
alter the charge or characteristics at the surface of the
monolayer.
[0067] In a preferred embodiment, when the conductive oligomer is
greater than three subunits, R groups are preferred to increase
solubility when solution synthesis is done. However, the R groups,
and their positions, are chosen to minimally effect the packing of
the conductive oligomers on a surface, particularly within a
monolayer, as described below. In general, only small R groups are
used within the monolayer, with larger R groups generally above the
surface of the monolayer. Thus for example the attachment of methyl
groups to the portion of the conductive oligomer within the
monolayer to increase solubility is preferred, with attachment of
longer alkoxy groups, for example, C3 to C10, is preferably done
above the monolayer surface. In general, for the systems described
herein, this generally means that attachment of sterically
significant R groups is not done on any of the first two or three
oligomer subunits, depending on the average length of the molecules
making up the monolayer.
[0068] Suitable R groups include, but are not limited to, hydrogen,
alkyl, alcohol, aromatic, amino, amido, nitro, ethers, esters,
aldehydes, sulfonyl, silicon moieties, halogens, sulfur containing
moieties, phosphorus containing moieties, and ethylene glycols. In
the structures depicted herein, R is hydrogen when the position is
unsubstituted. It should be noted that some positions may allow two
substitution groups, R and R', in which case the R and R' groups
may be either the same or different.
[0069] By "alkyl group" or grammatical equivalents herein is meant
a straight or branched chain alkyl group, with straight chain alkyl
groups being preferred. If branched, it may be branched at one or
more positions, and unless specified, at any position. The alkyl
group may range from about 1 to about 30 carbon atoms (C1-C30),
with a preferred embodiment utilizing from about 1 to about 20
carbon atoms (C1-C20), with about C1 through about C12 to about C15
being preferred, and C1 to C5 being particularly preferred,
although in some embodiments the alkyl group may be much larger.
Also included within the definition of an alkyl group are
cycloalkyl groups such as C5 and C6 rings, and heterocyclic rings
with nitrogen, oxygen, sulfur or phosphorus. Alkyl also includes
heteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen, and
silicone being preferred. Alkyl includes substituted alkyl groups.
By "substituted alkyl group" herein is meant an alkyl group further
comprising one or more substitution moieties "R", as defined
above.
[0070] By "amino groups" or grammatical equivalents herein is meant
--NH.sub.2, --NHR and --NR.sub.2 groups, with R being as defined
herein.
[0071] By "nitro group" herein is meant an --NO.sub.2 group.
[0072] By "sulfur containing moieties" herein is meant compounds
containing sulfur atoms, including but not limited to, thia-, thio-
and sulfo-compounds, thiols (--SH and --SR), and sulfides
(--RSR--). By "phosphorus containing moieties" herein is meant
compounds containing phosphorus, including, but not limited to,
phosphines and phosphates. By "silicon containing moieties" herein
is meant compounds containing silicon.
[0073] By "ether" herein is meant an --O--R group. Preferred ethers
include alkoxy groups, with --O--(CH.sub.2).sub.2CH.sub.3 and
--O--(CH.sub.2).sub.4CH.sub.3 being preferred.
[0074] By "ester" herein is meant a --COOR group.
[0075] By "halogen" herein is meant bromine, iodine, chlorine, or
fluorine. Preferred substituted alkyls are partially or fully
halogenated alkyls such as CF.sub.3, etc.
[0076] By "aldehyde" herein is meant --RCHO groups.
[0077] By "alcohol" herein is meant --OH groups, and alkyl alcohols
--ROH.
[0078] By "amido" herein is meant --RCONH-- or RCONR-- groups.
[0079] By "ethylene glycol" or "(poly)ethylene glycol" herein is
meant a --(O--CH.sub.2--CH.sub.2).sub.n-- group, although each
carbon atom of the ethylene group may also be singly or doubly
substituted, i.e. --(O--CR.sub.2--CR.sub.2).sub.n--, with R as
described above. Ethylene glycol derivatives with other heteroatoms
in place of oxygen (i.e. --(N--CH.sub.2--CH.sub.2).sub.n-- or
--(S--CH.sub.2--CH.sub.2).sub.n--, or with substitution groups) are
also preferred.
[0080] Preferred substitution groups include, but are not limited
to, methyl, ethyl, propyl, alkoxy groups such as
--O--(CH.sub.2).sub.2CH.sub.3 and --O--(CH.sub.2).sub.4CH.sub.3 and
ethylene glycol and derivatives thereof.
[0081] Preferred aromatic groups include, but are not limited to,
phenyl, naphthyl, naphthalene, anthracene, phenanthroline, pyrole,
pyridine, thiophene, porphyrins, and substituted derivatives of
each of these, included fused ring derivatives.
[0082] In the conductive oligomers depicted herein, when g is 1,
B-D is a bond linking two atoms or chemical moieties. In a
preferred embodiment, B-D is a conjugated bond, containing
overlapping or conjugated n-orbitals.
[0083] Preferred B-D bonds are selected from acetylene
(--C.ident.C--, also called alkyne or ethylene), alkene
(--CH.dbd.CH--, also called ethylene), substituted alkene
(--CR.dbd.CR--, --CH.dbd.CR-- and --CR.dbd.CH--), amide (--NH--CO--
and --NR--CO-- or --CO--NH-- and --CO--NR--), azo (--N.dbd.N--),
esters and thioesters (--CO--O--, --O--CO--, --CS--O-- and
--O--CS--) and other conjugated bonds such as (--CH.dbd.N--,
--CR.dbd.N--, --N.dbd.CH-- and --N.dbd.CR--), (--SiH.dbd.SiH--,
--SiR.dbd.SiH--, --SiR.dbd.SiH--, and --SiR.dbd.SiR--),
(--SiH.dbd.CH--, --SiR.dbd.CH--, --SiH.dbd.CR--, --SiR.dbd.CR--,
--CH.dbd.SiH--, --CR.dbd.SiH--, --CH.dbd.SiR--, and
--CR.dbd.SiR--). Particularly preferred B-D bonds are acetylene,
alkene, amide, and substituted derivatives of these three, and azo.
Especially preferred B-D bonds are acetylene, alkene and amide. The
oligomer components attached to double bonds may be in the trans or
cis conformation, or mixtures. Thus, either B or D may include
carbon, nitrogen or silicon. The substitution groups are as defined
as above for R.
[0084] When g=0 in the Structure 1 conductive oligomer, e is
preferably 1 and the D moiety may be carbonyl or a heteroatom
moiety as defined above.
[0085] As above for the Y rings, within any single conductive
oligomer, the B-D bonds (or D moieties, when g= 0) may be all the
same, or at least one may be different. For example, when m is
zero, the terminal B-D bond may be an amide bond, and the rest of
the B-D bonds may be acetylene bonds. Generally, when amide bonds
are present, as few amide bonds as possible are preferable, but in
some embodiments all the B-D bonds are amide bonds. Thus, as
outlined above for the Y rings, one type of B-D bond may be present
in the conductive oligomer within a monolayer as described below,
and another type above the monolayer level, for example to give
greater flexibility for nucleic acid hybridization when the nucleic
acid is attached via a conductive oligomer.
[0086] In the structures depicted herein, n is an integer from 1 to
50, although longer oligomers may also be used (see for example
Schumm et al., Angew. Chem. Int. Ed. Engl. 1994 33(13):1360).
Without being bound by theory, it appears that for efficient
hybridization of nucleic acids on a surface, the hybridization
should occur at a distance from the surface, i.e. the kinetics of
hybridization increase as a function of the distance from the
surface, particularly for long oligonucleotides of 200 to 300
basepairs. Accordingly, when a nucleic acid is attached via a
conductive oligomer, as is more fully described below, the length
of the conductive oligomer is such that the closest nucleotide of
the nucleic acid is positioned from about 6 .ANG. to about 100
.ANG. (although distances of up to 500 .ANG. may be used) from the
electrode surface, with from about 15 .ANG. to about 60 .ANG. being
preferred and from about 25 .ANG. to about 60 .ANG. also being
preferred. Accordingly, n will depend on the size of the aromatic
group, but generally will be from about 1 to about 20, with from
about 2 to about 15 being preferred and from about 3 to about 10
being especially preferred.
[0087] In the structures depicted herein, m is either 0 or 1. That
is, when m is 0, the conductive oligomer may terminate in the B-D
bond or D moiety, i.e. the D atom is attached to the nucleic acid
either directly or via a linker. In some embodiments, for example
when the conductive oligomer is attached to a phosphate of the
ribose-phosphate backbone of a nucleic acid, there may be
additional atoms, such as a linker, attached between the conductive
oligomer and the nucleic acid. Additionally, as outlined below, the
D atom may be the nitrogen atom of the amino-modified ribose.
Alternatively, when m is 1, the conductive oligomer may terminate
in Y, an aromatic group, i.e. the aromatic group is attached to the
nucleic acid or linker.
[0088] As will be appreciated by those in the art, a large number
of possible conductive oligomers may be utilized. These include
conductive oligomers falling within the Structure 1 and Structure 8
formulas, as well as other conductive oligomers, as are generally
known in the art, including for example, compounds comprising fused
aromatic rings or Teflon.RTM.-like oligomers, such as
--(CF.sub.2).sub.n--, --(CHF).sub.n-- and --(CFR).sub.n--. See for
example, Schumm et al., Angew. Chem. Intl. Ed. Engl. 33:1361
(1994); Grosshenny et al., Platinum Metals Rev. 40(1):26-35 (1996);
Tour, Chem. Rev. 96:537-553 (1996); Hsung et al., Organometallics
14:4808-4815 (1995; and references cited therein, all of which are
expressly incorporated by reference.
[0089] Particularly preferred conductive oligomers of this
embodiment are depicted below:
Y B-D .sub.e .sub.n Y .sub.m Structure 2
[0090] Structure 2 is Structure 1 when g is 1. Preferred
embodiments of Structure 2 include: e is zero, Y is pyrole or
substituted pyrole; e is zero, Y is thiophene or substituted
thiophene; e is zero, Y is furan or substituted furan; e is zero, Y
is phenyl or substituted phenyl; e is zero, Y is pyridine or
substituted pyridine; e is 1, B-D is acetylene and Y is phenyl or
substituted phenyl (see Structure 4 below). A preferred embodiment
of Structure 2 is also when e is one, depicted as Structure 3
below:
Y--B-D .sub.n Y .sub.m Structure 3
[0091] Preferred embodiments of Structure 3 are: Y is phenyl or
substituted phenyl and B-D is azo; Y is phenyl or substituted
phenyl and B-D is acetylene; Y is phenyl or substituted phenyl and
B-D is alkene; Y is pyridine or substituted pyridine and B-D is
acetylene; Y is thiophene or substituted thiophene and B-D is
acetylene; Y is furan or substituted furan and B-D is acetylene; Y
is thiophene or furan (or substituted thiophene or furan) and B-D
are alternating alkene and acetylene bonds.
[0092] Most of the structures depicted herein utilize a Structure 3
conductive oligomer. However, any Structure 3 oligomers may be
substituted with any of the other structures depicted herein, i.e.
Structure 1 or 8 oligomer, or other conducting oligomer, and the
use of such Structure 3 depiction is not meant to limit the scope
of the invention.
[0093] Particularly preferred embodiments of Structure 3 include
Structures 4, 5, 6 and 7, depicted below:
##STR00001##
[0094] Particularly preferred embodiments of Structure 4 include: n
is two, m is one, and R is hydrogen; n is three, m is zero, and R
is hydrogen; and the use of R groups to increase solubility.
##STR00002##
[0095] When the B-D bond is an amide bond, as in Structure 5, the
conductive oligomers are pseudopeptide oligomers. Although the
amide bond in Structure 5 is depicted with the carbonyl to the
left, i.e. --CONH--, the reverse may also be used, i.e. --NHCO--.
Particularly preferred embodiments of Structure 5 include: n is
two, m is one, and R is hydrogen; n is three, m is zero, and R is
hydrogen (in this embodiment, the terminal nitrogen (the D atom)
may be the nitrogen of the amino-modified ribose); and the use of R
groups to increase solubility.
##STR00003##
[0096] Preferred embodiments of Structure 6 include the first n is
two, second n is one, m is zero, and all R groups are hydrogen, or
the use of R groups to increase solubility.
##STR00004##
[0097] Preferred embodiments of Structure 7 include: the first n is
three, the second n is from 1-3, with m being either 0 or 1, and
the use of R groups to increase solubility.
[0098] In a preferred embodiment, the conductive oligomer has the
structure depicted in Structure 8:
C-G-C .sub.n J .sub.m Structure 8
[0099] In this embodiment, C are carbon atoms, n is an integer from
1 to 50, m is 0 or 1, J is a heteroatom selected from the group
consisting of oxygen, nitrogen, silicon, phosphorus, sulfur,
carbonyl or sulfoxide, and G is a bond selected from alkane, alkene
or acetylene, such that together with the two carbon atoms the
C-G-C group is an alkene (--CH.dbd.CH--), substituted alkene
(--CR.dbd.CR--) or mixtures thereof (--CH.dbd.CR-- or
--CR.dbd.CH--), acetylene (--C.dbd.C--), or alkane
(--CR.sub.2--CR.sub.2--, with R being either hydrogen or a
substitution group as described herein). The G bond of each subunit
may be the same or different than the G bonds of other subunits;
that is, alternating oligomers of alkene and acetylene bonds could
be used, etc. However, when G is an alkane bond, the number of
alkane bonds in the oligomer should be kept to a minimum, with
about six or less sigma bonds per conductive oligomer being
preferred. Alkene bonds are preferred, and are generally depicted
herein, although alkane and acetylene bonds may be substituted in
any structure or embodiment described herein as will be appreciated
by those in the art.
[0100] In some embodiments, for example when ETMs are not present,
if m=0 then at least one of the G bonds is not an alkane bond.
[0101] In a preferred embodiment, the m of Structure 8 is zero. In
a particularly preferred embodiment, m is zero and G is an alkene
bond, as is depicted in Structure 9:
##STR00005##
[0102] The alkene oligomer of structure 9, and others depicted
herein, are generally depicted in the preferred trans
configuration, although oligomers of cis or mixtures of trans and
cis may also be used. As above, R groups may be added to alter the
packing of the compositions on an electrode, the hydrophilicity or
hydrophobicity of the oligomer, and the flexibility, i.e. the
rotational, torsional or longitudinal flexibility of the oligomer.
n is as defined above.
[0103] In a preferred embodiment, R is hydrogen, although R may be
also alkyl groups and polyethylene glycols or derivatives.
[0104] In an alternative embodiment, the conductive oligomer may be
a mixture of different types of oligomers, for example of
structures 1 and 8.
[0105] In addition, the terminus of at least some of the conductive
oligomers in the monolayer are electronically exposed. By
"electronically exposed" herein is meant that upon the placement of
an ETM in close proximity to the terminus, and after initiation
with the appropriate signal, a signal dependent on the presence of
the ETM 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 one of the groups depicted in Structures 1 to 9; for example,
a B-D bond such as an acetylene bond. Alternatively, in a preferred
embodiment, a terminal group is added, sometimes depicted herein as
"Q". A terminal group may be used for several reasons; for example,
to contribute to the electronic availability of the conductive
oligomer for detection of ETMs, 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 nucleic acid
is DNA or RNA the nucleic acid is repelled or prevented from lying
down on the surface, to facilitate hybridization. Preferred
terminal groups include --NH.sub.2, --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.
[0106] 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.
[0107] It will be appreciated that the monolayer may comprise
different conductive oligomer species, although preferably the
different species are chosen such that a reasonably uniform SAM can
be formed. Thus, for example, when nucleic acids are covalently
attached to the electrode using conductive oligomers, it is
possible to have one type of conductive oligomer used to attach the
nucleic acid, and another type functioning to detect the ETM.
Similarly, it may be desirable to have mixtures of different
lengths of conductive oligomers in the monolayer, to help reduce
non-specific signals. Thus, for example, preferred embodiments
utilize conductive oligomers that terminate below the surface of
the rest of the monolayer, i.e. below the insulator layer, if used,
or below some fraction of the other conductive oligomers.
Similarly, the use of different conductive oligomers may be done to
facilitate monolayer formation, or to make monolayers with altered
properties.
[0108] In a preferred embodiment, the monolayer forming species are
"interrupted" conductive oligomers, containing an alkyl portion in
the middle of the conductive oligomer.
[0109] In a preferred embodiment, the monolayer comprises
photoactivatable species as EFS. Photoactivatable species are known
in the art, and include 4,5-dimethoxy-2-nitrobenzyl ester, which
can be photolyzed at 365 nm for 2 hours.
[0110] In a preferred embodiment, the monolayer may further
comprise insulator moieties. By "insulator" herein is meant a
substantially nonconducting oligomer, preferably linear. By
"substantially nonconducting" herein is meant that the insulator
will not transfer electrons at 100 Hz. The rate of electron
transfer through the insulator is preferrably slower than the rate
through the conductive oligomers described herein.
[0111] In a preferred embodiment, the insulators have a
conductivity, S, of about 10.sup.-7 .OMEGA..sup.-1cm.sup.-1 or
lower, with less than about 10.sup.-8 .OMEGA..sup.-1cm.sup.-1 being
preferred. See generally Gardner et al., supra.
[0112] 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.
[0113] 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).
[0114] As for the conductive oligomers, the 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. Similarly, the insulators may contain terminal groups,
as outlined above, particularly to influence the surface of the
monolayer.
[0115] The length of the species making up the monolayer will vary
as needed. As outlined above, it appears that hybridization is more
efficient at a distance from the surface. The species to which
nucleic acids are attached (as outlined below, these can be either
insulators or conductive oligomers) may be basically the same
length as the monolayer forming species or longer than them,
resulting in the nucleic acids being more accessible to the solvent
for hybridization. In some embodiments, the conductive oligomers to
which the nucleic acids are attached may be shorter than the
monolayer.
[0116] As will be appreciated by those in the art, the actual
combinations and ratios of the different species making up the
monolayer can vary widely, and will depend on whether mechanism-1
or -2 is used. Generally, three component systems are preferred for
mechanism-2 systems, with the first species comprising a capture
probe containing species, attached to the electrode via either an
insulator or a conductive oligomer. The second species are EFS,
preferably conductive oligomers, and the third species are
insulators. In this embodiment, the first species can comprise from
about 90% to about 1%, with from about 20% to about 40% being
preferred. For nucleic acids, from about 30% to about 40% is
especially preferred for short oligonucleotide targets and from
about 10% to about 20% is preferred for longer targets. The second
species can comprise from about 1% to about 90%, with from about
20% to about 90% being preferred, and from about 40% to about 60%
being especially preferred. The third species can comprise from
about 1% to about 90%, with from about 20% to about 40% being
preferred, and from about 15% to about 30% being especially
preferred. To achieve these approximate proportions, preferred
ratios of first:second:third species in SAM formation solvents are
2:2:1 for short targets, 1:3:1 for longer targets, with total thiol
concentration (when used to attach these species, as is more fully
outlined below) in the 500 .mu.M to 1 mM range, and 833 .mu.M being
preferred.
[0117] Alternatively, two component systems can be used. In one
embodiment, for use in either mechanism-1 or mechanism-2 systems,
the two components are the first and second species. In this
embodiment, the first species can comprise from about 1% to about
90%, with from about 1% to about 40% being preferred, and from
about 10% to about 40% being especially preferred. The second
species can comprise from about 1% to about 90%, with from about
10% to about 60% being preferred, and from about 20% to about 40%
being especially preferred. Alternatively, for mechanism-1 systems,
the two components are the first and the third species. In this
embodiment, the first species can comprise from about 1% to about
90%, with from about 1% to about 40% being preferred, and from
about 10% to about 40% being especially preferred. The second
species can comprise from about 1% to about 90%, with from about
10% to about 60% being preferred, and from about 20% to about 40%
being especially preferred.
[0118] In a preferred embodiment, the deposition of the SAM is done
using aqueous solvents. As is generally described in Steel et al.,
Anal. Chem. 70:4670 (1998), Herne et al., J. Am. Chem. Soc.
119:8916 (1997), and Finklea, Electrochemistry of Organized
Monolayers of Thiols and Related Molecules on Electrodes, from A.
J. Bard, Electroanalytical Chemistry: A Series of Advances, Vol.
20, Dekker N.Y. 1966-, all of which are expressly incorporated by
reference, the deposition of the SAM-forming species can be done
out of aqueous solutions, frequently comprising salt.
[0119] The covalent attachment of the conductive oligomers and
insulators may be accomplished in a variety of ways, depending on
the electrode and the composition of the insulators and conductive
oligomers used. In a preferred embodiment, the attachment linkers
with covalently attached capture binding ligands (e.g. nucleosides
or nucleic acids) as depicted herein are covalently attached to an
electrode. Thus, one end or terminus of the attachment linker is
attached to the nucleoside or nucleic acid, and the other is
attached to an electrode. In some embodiments it may be desirable
to have the attachment linker attached at a position other than a
terminus, or even to have a branched attachment linker that is
attached to an electrode at one terminus and to two or more
nucleosides at other termini, although this is not preferred.
Similarly, the attachment linker may be attached at two sites to
the electrode, as is generally depicted in Structures 11-13.
Generally, some type of linker is used, as depicted below as "A" in
Structure 10, where "X" is the conductive oligomer, "I" is an
insulator and the hatched surface is the electrode:
##STR00006##
[0120] 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. In a preferred embodiment, epoxide type linkages
with redox polymers such as are known in the art are not used.
[0121] Although depicted herein as a single moiety, the insulators
and conductive oligomers 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 11, 12 and 13. As will be
appreciated by those in the art, other such structures can be made.
In Structures 11, 12 and 13, the A moiety is just a sulfur atom,
but substituted sulfur moieties may also be used.
##STR00007##
[0122] It should also be noted that similar to Structure 13, it may
be possible to have a conductive oligomer terminating in a single
carbon atom with three sulfur moities attached to the electrode.
Additionally, although not always depicted herein, the conductive
oligomers and insulators may also comprise a "Q" terminal
group.
[0123] In a preferred embodiment, the electrode is a gold
electrode, and attachment is via a sulfur linkage as is well known
in the art, i.e. the A moiety is a sulfur atom or moiety. Although
the exact characteristics of the gold-sulfur attachment are not
known, this linkage is considered covalent for the purposes of this
invention. A representative structure is depicted in Structure 14,
using the Structure 3 conductive oligomer, although as for all the
structures depicted herein, any of the conductive oligomers, or
combinations of conductive oligomers, may be used. Similarly, any
of the conductive oligomers or insulators may also comprise
terminal groups as described herein. Structure 14 depicts the "A"
linker as comprising just a sulfur atom, although additional atoms
may be present (i.e. linkers from the sulfur to the conductive
oligomer or substitution groups). In addition, Structure 14 shows
the sulfur atom attached to the Y aromatic group, but as will be
appreciated by those in the art, it may be attached to the B-D
group (i.e. an acetylene) as well.
##STR00008##
[0124] In a preferred embodiment, 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. Again, additional atoms may be present, i.e. Z
type linkers and/or terminal groups.
##STR00009##
[0125] In Structure 16, 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).
[0126] The SAMs of the invention can be made in a variety of ways,
including deposition out of organic solutions and deposition out of
aqueous solutions. The methods outlined herein use a gold electrode
as the example, although as will be appreciated by those in the
art, other metals and methods may be used as well. In one preferred
embodiment, indium-tin-oxide (ITO) is used as the electrode.
[0127] In a preferred embodiment, a gold surface is first cleaned.
A variety of cleaning procedures may be employed, including, but
not limited to, chemical cleaning or etchants (including Piranha
solution (hydrogen peroxide/sulfuric acid) or aqua regia
(hydrochloric acid/nitric acid), electrochemical methods, flame
treatment, plasma treatment or combinations thereof.
[0128] Following cleaning, the gold substrate is exposed to the SAM
species. When the electrode is ITO, the SAM species are
phosphonate-containing species. This can also be done in a variety
of ways, including, but not limited to, solution deposition, gas
phase deposition, microcontact printing, spray deposition,
deposition using neat components, etc. A preferred embodiment
utilizes a deposition solution comprising a mixture of various SAM
species in solution, generally thiol-containing species. Mixed
monolayers that contain nucleic acids are usually prepared using a
two step procedure. The thiolated nucleic acid is deposited during
the first deposition step (generally in the presence of at least
one other monolayer-forming species) and the mixed monolayer
formation is completed during the second step in which a second
thiol solution minus nucleic acid is added. The second step
frequently involves mild heating to promote monolayer
reorganization.
[0129] In a preferred embodiment, the deposition solution is an
organic deposition solution. In this embodiment, a clean gold
surface is placed into a clean vial. A binding ligand deposition
solution in organic solvent is prepared in which the total thiol
concentration is between micromolar to saturation; preferred ranges
include from about 1 .mu.M to 10 mM, with from about 400 uM to
about 1.0 mM being especially preferred. In a preferred embodiment,
the deposition solution contains thiol modified DNA (i.e. nucleic
acid attached to an attachment linker) and thiol diluent molecules
(either conductive oligomers or insulators, with the latter being
preferred). The ratio of nucleic acid to diluent (if present) is
usually between 1000:1 to 1:1000, with from about 10:1 to about
1:10 being preferred and 1:1 being especially preferred. The
preferred solvents are tetrahydrofuran (THF), acetonitrile,
dimethylformamide (DMF), ethanol, or mixtures thereof; generally
any solvent of sufficient polarity to dissolve the capture ligand
can be used, as long as the solvent is devoid of functional groups
that will react with the surface. Sufficient nucleic acid
deposition solution is added to the vial so as to completely cover
the electrode surface. The gold substrate is allowed to incubate at
ambient temperature or slightly above ambient temperature for a
period of time ranging from seconds to hours, with 5-30 minutes
being preferred. After the initial incubation, the deposition
solution is removed and a solution of diluent molecule only (from
about 1 .mu.M to 10 mM, with from about 100 uM to about 1.0 mM
being preferred) in organic solvent is added. The gold substrate is
allowed to incubate at room temperature or above room temperature
for a period of time (seconds to days, with from about 10 minutes
to about 24 hours being preferred). The gold sample is removed from
the solution, rinsed in clean solvent and used.
[0130] In a preferred embodiment, an aqueous deposition solution is
used. As above, a clean gold surface is placed into a clean vial. A
nucleic acid deposition solution in water is prepared in which the
total thiol concentration is between about 1 uM and 10 mM, with
from about 1 .mu.M to about 200 uM being preferred. The aqueous
solution frequently has salt present (up to saturation, with
approximately 1M being preferred), however pure water can be used.
The deposition solution contains thiol modified nucleic acid and
often a thiol diluent molecule. The ratio of nucleic acid to
diluent is usually between between 1000:1 to 1:1000, with from
about 10:1 to about 1:10 being preferred and 1:1 being especially
preferred. The nucleic acid deposition solution is added to the
vial in such a volume so as to completely cover the electrode
surface. The gold substrate is allowed to incubate at ambient
temperature or slightly above ambient temperature for 1-30 minutes
with 5 minutes usually being sufficient. After the initial
incubation, the deposition solution is removed and a solution of
diluent molecule only (10 uM-1.0 mM) in either water or organic
solvent is added. The gold substrate is allowed to incubate at room
temperature or above room temperature until a complete monolayer is
formed (10 minutes-24 hours). The gold sample is removed from the
solution, rinsed in clean solvent and used.
[0131] In a preferred embodiment, as outlined herein, a circuit
board is used as the substrate for the gold electrodes. Formation
of the SAMs on the gold surface is generally done by first cleaning
the boards, for example in a 10% sulfuric acid solution for 30
seconds, detergent solutions, aqua regia, plasma, etc., as outlined
herein. Following the sulfuric acid treatment, the boards are
washed, for example via immersion in two Milli-Q water baths for 1
minute each. The boards are then dried, for example under a stream
of nitrogen. Spotting of the deposition solution onto the boards is
done using any number of known spotting systems, generally by
placing the boards on an X-Y table, preferably in a humidity
chamber. The size of the spotting drop will vary with the size of
the electrodes on the boards and the equipment used for delivery of
the solution; for example, for 250 .mu.M size electrodes, a 30
nanoliter drop is used. The volume should be sufficient to cover
the electrode surface completely. The drop is incubated at room
temperature for a period of time (sec to overnight, with 5 minutes
preferred) and then the drop is removed by rinsing in a Milli-Q
water bath. The boards are then preferably treated with a second
deposition solution, generally comprising insulator in organic
solvent, preferably acetonitrile, by immersion in a 45.degree. C.
bath. After 30 minutes, the boards are removed and immersed in an
acetonitrile bath for 30 seconds followed by a milli-Q water bath
for 30 seconds. The boards are dried under a stream of
nitrogen.
[0132] In a preferred embodiment, the electrode comprising the
monolayer further comprises a capture binding ligand covalently
attached to the electrode. This attachment can be via an attachment
linker, which may be a conductive oligomer or an insulator. By
"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. ("Capture probe" or "anchor probe" are particularly used
when the capture binding ligand is a nucleic acid). Generally, the
capture binding 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
binding 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.
[0133] In a preferred embodiment, the binding is specific, and the
binding 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 binding 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.
In some embodiments, for example in the detection of certain
biomolecules, the binding constants of the analyte to the binding
ligand will be at least about 10.sup.4-10.sup.6 M.sup.-1, with at
least about 10.sup.5 to 10.sup.9 M.sup.-1 being preferred and at
least about 10.sup.7-10.sup.9 M.sup.-1 being particularly
preferred.
[0134] As will be appreciated by those in the art, the composition
of the binding ligand will depend on the composition of the target
analyte. Binding 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 binding 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.
[0135] The method of attachment of the capture binding ligand to
the attachment linker 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.
[0136] In this way, capture binding ligands comprising proteins,
lectins, nucleic acids, small organic molecules, carbohydrates,
etc. can be added.
[0137] In a preferred embodiment, the capture binding ligand is
attached directly to the electrode as outlined herein, for example
via an attachment linker. Alternatively, the capture binding ligand
may utilize a capture extender component. In this embodiment, the
capture binding ligand comprises a first portion that will bind the
target analyte and a second portion that can be used for attachment
to the surface. FIG. 2C depicts the use of a nucleic acid component
for binding to the surface, although this can be other binding
partners as well.
[0138] A preferred embodiment utilizes proteinaceous capture
binding ligands. As is known in the art, any number of techniques
may be used to attach a proteinaceous capture binding ligand.
"Protein" in this context includes proteins, polypeptides and
peptides. A wide variety of techniques are known to add moieties to
proteins. One preferred method is outlined in U.S. Pat. No.
5,620,850, hereby incorporated by reference in its entirety. The
attachment of proteins to electrodes is known; see also Heller,
Acc. Chem. Res. 23:128 (1990), and related work.
[0139] A preferred embodiment utilizes nucleic acids as the capture
binding ligand, for example for when the target analyte is a
nucleic acid or a nucleic acid binding protein, or when the nucleic
acid serves as an aptamer for binding a protein; see 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.
In this embodiment, the nucleic acid capture binding ligand is
covalently attached to the electrode, via an "attachment linker",
that can be either a conductive oligomer or via an insulator. Thus,
one end of the attachment linker is attached to a nucleic acid, 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.
[0140] As is more fully outlined below, attachment of the target
sequence to the capture probe may be direct (i.e. the target
sequence hybridizes to the capture probe) or indirect (one or more
capture extender probes are used). In addition, as is more fully
outlined below, the capture probes may have both nucleic and
non-nucleic acid portions. Thus, for example, flexible linkers such
as alkyl groups, including polyethylene glycol linkers, may be used
to get the nucleic acid portion of the capture probe off the
electrode surface. This may be particularly useful when the target
sequences are large, for example when genomic DNA or rRNA is the
target.
[0141] The capture probe nucleic acid is covalently attached to the
electrode, via an "attachment linker", that can be either a
conductive oligomer or via an insulator. Thus, one end of the
attachment linker is attached to a nucleic acid, 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.
Thus, any of structures depicted herein may further comprise a
nucleic acid effectively as a terminal group. Thus, the present
invention provides compositions comprising nucleic acids covalently
attached to electrodes as is generally depicted below in Structure
17:
##STR00010##
[0142] In Structure 17, the hatched marks on the left represent an
electrode. X is a conductive oligomer and I is an insulator as
defined herein. F.sub.1 is a linkage that allows the covalent
attachment of the electrode and the conductive oligomer or
insulator, including bonds, atoms or linkers such as is described
herein, for example as "A", defined below. F.sub.2 is a linkage
that allows the covalent attachment of the conductive oligomer or
insulator to the nucleic acid, and may be a bond, an atom or a
linkage as is herein described. F.sub.2 may be part of the
conductive oligomer, part of the insulator, part of the nucleic
acid, or exogeneous to both, for example, as defined herein for
"Z".
[0143] In a preferred embodiment, the capture probe nucleic acid is
covalently attached to the electrode via a conductive oligomer. The
covalent attachment of the nucleic acid and the conductive oligomer
may be accomplished in several ways. In a preferred embodiment, the
attachment is via attachment to the base of the nucleoside, via
attachment to the backbone of the nucleic acid (either the ribose,
the phosphate, or to an analogous group of a nucleic acid analog
backbone), or via a transition metal ligand, as described below.
The techniques outlined below are generally described for naturally
occurring nucleic acids, although as will be appreciated by those
in the art, similar techniques may be used with nucleic acid
analogs.
[0144] In a preferred embodiment, the conductive oligomer is
attached to the base of a nucleoside of the nucleic acid. This may
be done in several ways, depending on the oligomer, as is described
below. In one embodiment, the oligomer is attached to a terminal
nucleoside, i.e. either the 3' or 5' nucleoside of the nucleic
acid. Alternatively, the conductive oligomer is attached to an
internal nucleoside.
[0145] The point of attachment to the base will vary with the base.
Generally, attachment at any position is possible. In some
embodiments, for example when the probe containing the ETMs may be
used for hybridization, it is preferred to attach at positions not
involved in hydrogen bonding to the complementary base. Thus, for
example, generally attachment is to the 5 or 6 position of
pyrimidines such as uridine, cytosine and thymine. For purines such
as adenine and guanine, the linkage is preferably via the 8
position. Attachment to non-standard bases is preferably done at
the comparable positions.
[0146] In one embodiment, the attachment is direct; that is, there
are no intervening atoms between the conductive oligomer and the
base. In this embodiment, for example, conductive oligomers with
terminal acetylene bonds are attached directly to the base.
Structure 18 is an example of this linkage, using a Structure 3
conductive oligomer and uridine as the base, although other bases
and conductive oligomers can be used as will be appreciated by
those in the art:
##STR00011##
[0147] It should be noted that the pentose structures depicted
herein may have hydrogen, hydroxy, phosphates or other groups such
as amino groups attached. In addition, the pentose and nucleoside
structures depicted herein are depicted non-conventionally, as
mirror images of the normal rendering. In addition, the pentose and
nucleoside structures may also contain additional groups, such as
protecting groups, at any position, for example as needed during
synthesis.
[0148] In addition, the base may contain additional modifications
as needed, i.e. the carbonyl or amine groups may be altered or
protected, for example as is depicted in FIG. 18A of PCT
US97/20014. This may be required to prevent significant
dimerization of conductive oligomers instead of coupling to the
iodinating base. In addition, changing the components of the
palladium reaction may be desirable also. R groups may be preferred
on longer conductive oligomers to increase solubility.
[0149] In an alternative embodiment, the attachment is any number
of different Z linkers, including amide and amine linkages, as is
generally depicted in Structure 19 using uridine as the base and a
Structure 3 oligomer:
##STR00012##
[0150] In this embodiment, Z is a linker. Preferably, Z is a short
linker of about 1 to about 10 atoms, with from 1 to 5 atoms being
preferred, that may or may not contain alkene, alkynyl, amine,
amide, azo, imine, etc., bonds. 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, with propyl, acetylene, and C.sub.2 alkene being
especially preferred. Z may also be a sulfone group, forming
sulfonamide linkages as discussed below.
[0151] In a preferred embodiment, the attachment of the nucleic
acid and the conductive oligomer is done via attachment to the
backbone of the nucleic acid. This may be done in a number of ways,
including attachment to a ribose of the ribose-phosphate backbone,
or to the phosphate of the backbone, or other groups of analogous
backbones.
[0152] As a preliminary matter, it should be understood that the
site of attachment in this embodiment may be to a 3' or 5' terminal
nucleotide, or to an internal nucleotide, as is more fully
described below.
[0153] In a preferred embodiment, the conductive oligomer is
attached to the ribose of the ribose-phosphate backbone. This may
be done in several ways. As is known in the art, nucleosides that
are modified at either the 2' or 3' position of the ribose with
amino groups, sulfur groups, silicone groups, phosphorus groups, or
oxo groups can be made (Imazawa et al., J. Org. Chem., 44:2039
(1979); Hobbs et al., J. Org. Chem. 42(4):714 (1977); Verheyden et
al., J. Org. Chem. 36(2):250 (1971); McGee et al., J. Org. Chem.
61:781-785 (1996); Mikhailopulo et al., Liebigs. Ann. Chem. 513-519
(1993); McGee et al., Nucleosides & Nucleotides 14(6):1329
(1995), all of which are incorporated by reference). These modified
nucleosides are then used to add the conductive oligomers.
[0154] A preferred embodiment utilizes amino-modified nucleosides.
These amino-modified riboses can then be used to form either amide
or amine linkages to the conductive oligomers. In a preferred
embodiment, the amino group is attached directly to the ribose,
although as will be appreciated by those in the art, short linkers
such as those described herein for "Z" may be present between the
amino group and the ribose.
[0155] In a preferred embodiment, an amide linkage is used for
attachment to the ribose. Preferably, if the conductive oligomer of
Structures 1-3 is used, m is zero and thus the conductive oligomer
terminates in the amide bond. In this embodiment, the nitrogen of
the amino group of the amino-modified ribose is the "D" atom of the
conductive oligomer. Thus, a preferred attachment of this
embodiment is depicted in Structure 20 (using the Structure 3
conductive oligomer):
##STR00013##
[0156] As will be appreciated by those in the art, Structure 20 has
the terminal bond fixed as an amide bond.
[0157] In a preferred embodiment, a heteroatom linkage is used,
i.e. oxo, amine, sulfur, etc. A preferred embodiment utilizes an
amine linkage. Again, as outlined above for the amide linkages, for
amine linkages, the nitrogen of the amino-modified ribose may be
the "D" atom of the conductive oligomer when the Structure 3
conductive oligomer is used. Thus, for example, Structures 21 and
22 depict nucleosides with the Structures 3 and 9 conductive
oligomers, respectively, using the nitrogen as the heteroatom,
although other heteroatoms can be used:
##STR00014##
[0158] In Structure 21, preferably both m and t are not zero. A
preferred Z here is a methylene group, or other aliphatic alkyl
linkers. One, two or three carbons in this position are
particularly useful for synthetic reasons; see PCT US97/20014.
##STR00015##
[0159] In Structure 22, Z is as defined above. Suitable linkers
include methylene and ethylene.
[0160] In an alternative embodiment, the conductive oligomer is
covalently attached to the nucleic acid via the phosphate of the
ribose-phosphate backbone (or analog) of a nucleic acid. In this
embodiment, the attachment is direct, utilizes a linker or via an
amide bond. Structure 23 depicts a direct linkage, and Structure 24
depicts linkage via an amide bond (both utilize the Structure 3
conductive oligomer, although Structure 8 conductive oligomers are
also possible). Structures 23 and 24 depict the conductive oligomer
in the 3' position, although the 5' position is also possible.
Furthermore, both Structures 23 and 24 depict naturally occurring
phosphodiester bonds, although as those in the art will appreciate,
non-standard analogs of phosphodiester bonds may also be used.
##STR00016##
[0161] In Structure 23, if the terminal Y is present (i.e. m=1),
then preferably Z is not present (i.e. t=0). If the terminal Y is
not present, then Z is preferably present.
[0162] Structure 24 depicts a preferred embodiment, wherein the
terminal B-D bond is an amide bond, the terminal Y is not present,
and Z is a linker, as defined herein.
##STR00017##
[0163] In a preferred embodiment, the conductive oligomer is
covalently attached to the nucleic acid via a transition metal
ligand. In this embodiment, the conductive oligomer is covalently
attached to a ligand which provides one or more of the coordination
atoms for a transition metal. In one embodiment, the ligand to
which the conductive oligomer is attached also has the nucleic acid
attached, as is generally depicted below in Structure 25.
Alternatively, the conductive oligomer is attached to one ligand,
and the nucleic acid is attached to another ligand, as is generally
depicted below in Structure 26. Thus, in the presence of the
transition metal, the conductive oligomer is covalently attached to
the nucleic acid. Both of these structures depict Structure 3
conductive oligomers, although other oligomers may be utilized.
Structures 25 and 26 depict two representative structures:
##STR00018##
[0164] In the structures depicted herein, M is a metal atom, with
transition metals being preferred. 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, are preferred. Particularly
preferred are ruthenium, rhenium, osmium, platinum, cobalt and
iron.
[0165] 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.
[0166] 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).
[0167] 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 L.sub.m). Suitable nitrogen donating ligands are well
known in the art and include, but are not limited to, NH.sub.2;
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), EDTA, EGTA
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.
[0168] 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 Wilkenson, 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 Wilkenson.
[0169] The oxygen, sulfur, phosphorus and nitrogen-donating ligands
are attached in such a manner as to allow the heteroatoms to serve
as coordination atoms.
[0170] In a preferred embodiment, 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 n-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
[C.sub.5H.sub.5(-1)] and various ring substituted and ring fused
derivatives, such as the indenylide (-1) ion, that yield a class of
bis(cyclopentadieyl) 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
[(C.sub.5H.sub.5).sub.2Fe] 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
n-bonded ligands such as the allyl(-1) ion, or butadiene yield
potentially suitable organometallic compounds, and all such
ligands, in conjunction with other n-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.
[0171] 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
Wilkenson, 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.
[0172] 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. These combinations are depicted in representative
structures using the conductive oligomer of Structure 3 are
depicted in Structures 27 (using phenanthroline and amino as
representative ligands), 28 (using ferrocene as the metal-ligand
combination) and 29 (using cyclopentadienyl and amino as
representative ligands).
##STR00019##
[0173] In addition to serving as attachments for conductive
oligomers and electrodes, the above compositions can also be used
as ETM labels. That is, as is outlined in FIGS. 19 and 20,
transition metals (or other ETMs) attached to conductive oligomers
can be added to the nucleic acids for detection. In this
embodiment, without being bound by theory, the conductive oligomer,
terminating preferably in an F1 linkage (a linkage that allows the
attachment of the conductive oligomer to the surface), will
penetrate the SAM and facilitate electron transfer between the ETM
and the electrode. Without being bound by theory, this appears to
allow rapid electron transfer, similar to a "mechanism-1" system,
by providing a direct pathway for electrons; this is sometimes
referred to herein as "hardwiring".
[0174] Surprisingly, the system appears to work whether or not the
F1 moiety is protected; that is, a direct attachment may not be
required to increase the frequency response of the ETM. Thus, the
conductive oligomer can terminate either in an F1 moiety, an F1
moiety protected with a protecting group (see Greene, supra), or
need not terminate in an F1 moiety at all; terminal groups such as
are used on the surfaces of the SAMs may also be used.
Alternatively, the bare terminus of the conductive oligomer may be
sufficient.
[0175] In this embodiment, a plurality of ETMs per "branch" may be
used. They may be attached as a group, e.g. as a metallocene
polymer, terminating in the conductive oligomer, or may be
substitution groups off of the conductive oligomer. In general,
preferred embodiments utilize electronic conjugation between the
ETMs and the conductive oligomer, to facilitate electron
transfer.
[0176] In general, the length of the conductive oligomer in this
embodiment will vary with the length of the SAM on the electrode,
and preferred embodiments utilize two unit and three unit
oligomers. Preferred conductive oligomers in this embodiment are
the same as those outlined above for attachment of nucleic acids to
electrodes, with phenyl-acetylene oligomers being the most
preferred.
[0177] In this embodiment, the ETM with the attached conductive
oligomer is generally synthesized, and then a phosphoramidite
moiety is made.
[0178] In a preferred embodiment, the ligands used in the invention
show altered fluorescent properties depending on the redox state of
the chelated metal ion. As described below, this thus serves as an
additional mode of detection of electron transfer between the ETM
and the electrode.
[0179] In a preferred embodiment, as is described more fully below,
the ligand attached to the nucleic acid is an amino group attached
to the 2' or 3' position of a ribose of the ribose-phosphate
backbone. This ligand may contain a multiplicity of amino groups so
as to form a polydentate ligand which binds the metal ion. Other
preferred ligands include cyclopentadiene and phenanthroline.
[0180] The use of metal ions to connect the nucleic acids can serve
as an internal control or calibration of the system, to evaluate
the number of available nucleic acids on the surface. However, as
will be appreciated by those in the art, if metal ions are used to
connect the nucleic acids to the conductive oligomers, it is
generally desirable to have this metal ion complex have a different
redox potential than that of the ETMs used in the rest of the
system, as described below. This is generally true so as to be able
to distinguish the presence of the capture probe from the presence
of the target sequence. This may be useful for identification,
calibration and/or quantification. Thus, the amount of capture
probe on an electrode may be compared to the amount of hybridized
double stranded nucleic acid to quantify the amount of target
sequence in a sample. This is quite significant to serve as an
internal control of the sensor or system. This allows a measurement
either prior to the addition of target or after, on the same
molecules that will be used for detection, rather than rely on a
similar but different control system. Thus, the actual molecules
that will be used for the detection can be quantified prior to any
experiment. This is a significant advantage over prior methods.
[0181] In a preferred embodiment, the capture probe nucleic acids
are covalently attached to the electrode via an insulator. The
attachment of nucleic acids to insulators such as alkyl groups is
well known, and can be done to the base or the backbone, including
the ribose or phosphate for backbones containing these moieties, or
to alternate backbones for nucleic acid analogs.
[0182] In a preferred embodiment, there may be one or more
different capture probe species on the surface. In some
embodiments, there may be one type of capture probe, or one type of
capture probe extender, as is more fully described below.
Alternatively, different capture probes, or one capture probes with
a multiplicity of different capture extender probes can be used.
Similarly, it may be desirable to use auxiliary capture probes that
comprise relatively short probe sequences, that can be used to
"tack down" components of the system, for example the recruitment
linkers, to increase the concentration of ETMs at the surface.
[0183] Thus the present invention provides electrodes, preferably
comprising monolayers and capture binding ligands, useful in target
analyte detection systems.
[0184] In a preferred embodiment, the assay complex comprises a
first transport component comprising a first binding partner. The
first transport component is used to transport the assay complex
comprising the first component to the electrode, as is more
described herein.
[0185] In a preferred embodiment, the first component is a
particle. By "particle" or "microparticle" or "nanoparticle" or
"bead" or "microsphere" herein is meant microparticulate matter. As
will be appreciated by those in the art, the first particles can
comprise a wide variety of materials, including, but not limited
to, cross-linked starch, dextrans, cellulose, proteins, organic
polymers including styrene polymers including polystyrene and
methylstyrene as well as other styrene co-polymers, plastics,
glass, ceramics, acrylic polymers, magnetically responsive
materials, colloids, thoria sol, carbon graphite, titanium dioxide,
nylon, latex, and teflon may all be used. "Microsphere Detection
Guide" from Bangs Laboratories, Fishers Ind. is a helpful guide.
Preferred embodiments utilize magnetic particles and colloids.
[0186] The size of the particles will depend on their composition.
The particles need not be spherical; irregular particles may be
used. In addition, the particles may be porous, thus increasing the
surface area of the particle available for attachment of binding
ligands, capture moieties, or ETMs. In general, the size of the
particles will vary with their composition; for example, magnetic
particles are generally bigger than colloid particles. Thus, the
particles have diameters ranging from 1-5 nm (colloids) to 200
.mu.m (magnetic particles).
[0187] In a preferred embodiment, the first particle is a magnetic
particle or a particle that can be induced to display magnetic
properties. By "magnetic" herein is meant that the particle is
attracted in a magnetic field, including ferromagnetic,
paramagnetic, and diamagnetic. In this embodiment, the particles
are preferably from about 0.001 to about 200 .mu.m in diameter,
with from about 0.05 to about 200 .mu.m preferred, from about 0.1
to about 100 .mu.m being particularly preferred, and from about 0.5
to about 10 .mu.m being especially preferred.
[0188] In a preferred embodiment, the first particle is a colloid
particle or nanoparticle. By "colloid" or "nanoparticle" herein is
meant a particle that is small enough to stay suspended in solution
in standard solvents and standard conditions. Generally, a colloid
is a particle in the size range of 1 nm to 1 .mu.m. A suspension of
colloids is distinguishable from non-colloids in that their spatial
distribution is largely unaltered by gravity during the time course
of an experimental observation. As is known in the art, colloids
can be made from a variety of materials; see Schmid, ed. Clusters
and Colloids, VCH, Weinheim, 1994; Hayat ed. Colloidal Gold:
Principles, Methods and Applications (Academic, San Diego, 1991);
Bassell et al., J. Cell Biol. 126:863 1994; and Creighton et al.,
J. Chem. Soc. Faraday II 75:790 (1979), all of which are hereby
incorporated by reference. Preferred colloids include, but are not
limited to, those of Au, Se, Te, Co, Ni, Fe, Cu, Pt and other
transition metals, and other colloids known in the art. It is known
that many colloid particles are charged and thus naturally repel
each other; for example, Au colloid particles are generally
negatively charged. This facilitates non-aggregation except in the
presence of target analyte as described below.
[0189] In some embodiments, the transport composition is not a
particle, and is in solution. In this embodiment, the transport
composition comprises a capture ligand as outlined below.
[0190] The first components comprise a first binding partner. By
"binding partner" or "binding ligand" or grammatical equivalents
herein is meant a compound that is used to probe for the presence
of the target analyte, and that will bind to the target analyte. As
for the capture binding ligands outlined herein, the composition of
the binding ligand will depend on the composition of the target
analyte, as generally described above.
[0191] Generally, the first binding ligands are attached to the
transport component in a variety of ways, depending on whether the
transport component is a particle, the composition of the particle,
and the composition of the binding ligand.
[0192] In a preferred embodiment, the transport component is a
particle, and attachment proceeds on the basis of the composition
of the particle and the composition of the first binding ligand. In
a preferred embodiment, depending on the composition of the
particle, it will contain chemical functional groups for subsequent
attachment of other moieties. For example, when the particle is a
magnetic particle, magnetic particles with chemical functional
groups such as amines are commercially available. Preferred
functional groups for attachment are amino groups, carboxy groups,
oxo groups and thiol groups, with amino groups being particularly
preferred. Using these functional groups, the binding ligands can
be attached using functional groups on the binding ligands. For
example, proteins and nucleic acids containing amino groups can be
attached to particles comprising amino groups, for example using
linkers as 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).
[0193] When the transport particle is a colloid particle,
attachment will again depend on the composition of the colloid and
of the binding ligand, and generally proceeds as outlined above for
attachment to electrodes.
[0194] The density of the binding ligand on the particle can vary
widely, and will depend in part on the presence or absence of the
other components on the particle, including, but not limited to,
capture binding ligands, SAMs including conductive oligomers and
insulators, charged moieties, etc. In general, when large target
analytes are to be detected, such as large proteins or long nucleic
acids, the density of the binding ligand is decreased, to allow
sufficient "space" for the target analyte to bind.
[0195] In one embodiment, the transport component comprises a
capture ligand that can be used for attachment to an electrode that
comprises a capture binding partner. As defined herein for binding
ligands, capture ligands and capture binding partners are pairs
that specifically interact, such that the transport composition can
be attached to the electrode. Capture ligands and capture binding
partners may comprising a large number of different species, as is
outlined above for binding ligands, with proteins and nucleic acids
being particularly preferred. Thus, the capture ligand and capture
binding partner can be used to transport the assay complex to the
electrode surface, as is more fully described below.
[0196] In a preferred embodiment, the transport component is not a
particle, and instead comprises a capture binding ligand and a
first binding ligand. Again, the method of attachment will depend
on the composition of each. In general, a variety of methods are
known for the attachment of two proteins, including the use of
linkers including polymers, and homo- and heterobifunctional
linkers as described herein. In some cases, recombinant DNA
technology can be utilized to make fusion proteins comprising at
least a first domain comprising the first binding ligand and a
second domain comprising the capture binding ligand. Similarly,
when the binding ligand and capture binding ligand are both nucleic
acids, they may be generally contiguous sequences that are
synthesized as such. Preferably in this embodiment, the reporter
composition is a particle, preferably a gold colloid particle with
a SAM including ETMs.
[0197] In a preferred embodiment, when the transport component does
not comprise a particle, it is possible to use polymers, either
linear or branched, that have a plurality of first binding ligands
attached, using techniques known in the art. Upon binding of a
plurality of target molecules and the reporter compositions,
particularly when the reporter compositions comprise particles, it
is possible to effect aggregation of the assay complex, leading to
transport to the electrode surface. Thus, in this embodiment, there
may or may not be capture binding ligands. Preferably in this
embodiment, the reporter composition is a particle, preferably a
gold colloid particle with a SAM including ETMs.
[0198] In a preferred embodiment, the transport particle further
comprises a self-assembled monolayer (SAM), particularly when the
transport particle is a gold colloid particle. 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. The SAM may comprise
attachment linkers, including conductive oligomers and insulators
either together or each separately, and the composition of the SAM
may depend on its location; that is, as is more fully described
below, SAMs on the electrode must comprise at least some conductive
oligomers. As outlined herein, the efficiency of oligonucleotide
hybridization, and other binding events, may increase when the
analyte is at a distance from the surface. Similarly, non-specific
binding of biomolecules, including nucleic acids, to a surface is
generally reduced when a monolayer is present. Thus, a monolayer
facilitates the maintenance of the analyte away from the surface.
In addition, when the SAM is on the electrode, a monolayer serves
to keep charge carriers away from the surface of the electrode.
Thus, this layer helps to prevent direct electrical contact 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.
[0199] As will be appreciated by those in the art, the actual
combinations and ratios of the different species making up the
monolayer can vary widely, and will depend on where the monolayer
is, i.e. on a transport particle, a reporter particle, or on the
electrode. For attachment on a transport particle, generally two,
three or four component systems are preferred. The components are
as follows. The first species comprises a binding ligand containing
species (that is generally attached to the surface via an
attachment linker, i.e. either an insulator or a conductive
oligomer, as is more fully described below). The second species are
the conductive oligomers. The third species are insulators. The
fourth species is a charged species that will prevent the particles
of the invention from aggregating in the absence of the target
analyte; this may or may not be required, depending on the particle
and the binding ligand; that is, in some instances the binding
ligand may comprise a charged species. For example, when the
binding ligand is a nucleic acid, the nucleic acids provide the
required charge and a fourth species is not required. The second,
third and fourth species are optional on a transport particle,
particularly a magnetic particle.
[0200] A representative three component system comprises the first,
second and third species. In this embodiment, the actual ratio of
the components will vary, in part depending on the size of the
binding ligand and target analyte; i.e. for larger ligands or
analytes, a lower amount of first species is desirable. Thus, a
preferred embodiment utilizes a three component system with the
first species comprising from about 90% to about 1%, with from
about 20% to about 40% being preferred, and from about 30% to about
40% being especially preferred for small targets and from about 10%
to about 20% preferred for larger targets. The second species can
comprise from about 1% to about 90%, with from about 20% to about
90% being preferred, and from about 40% to about 60% being
especially preferred. The third species can comprise from about 1%
to about 90%, with from about 20% to about 40% being preferred, and
from about 15% to about 30% being especially preferred. Preferred
ratios of first:second:third species are 2:2:1 for small targets,
1:3:1 for larger targets, with total thiol concentration in the 500
.mu.M to 1 mM range, and 833 .mu.M being preferred.
[0201] In a preferred embodiment, two component systems are used,
comprising the first and second species. In this embodiment, the
first species can comprise from about 90% to about 1%, with from
about 1% to about 40% being preferred, and from about 10% to about
40% being especially preferred. The second species can comprise
from about 1% to about 90%, with from about 10% to about 60% being
preferred, and from about 20% to about 40% being especially
preferred.
[0202] In some embodiments, it is possible to avoid the use of a
first transport component completely. What is important in this
embodiment is that the reporter compositions are unable to
associate with the electrode in the absence of target analyte. This
can be done in two different ways. In one embodiment, two reporter
particles are used; that is, when the aggregation of the particles
is used to transport the assay complex to the electrode as is more
fully outlined below, it is possible to use a first and a second
reporter particle, each containing a binding ligand for a different
portion of the target analyte and each containing ETMs, as is
generally depicted in FIG. 1F. In this embodiment, the specificity
of the system is achieved through the use of a reporter particles
that will not settle in the absence of aggregation. That is, in the
absence of target analyte, the two reporter particles stay
suspended in solution; upon the introduction of the target analyte,
large "cross-linked" aggregation assay complexes are formed that
can settle or be brought into contact with the electrode.
Alternatively, it is possible to use the target analyte as the
transport component; that is, part of the target analyte is used as
a capture ligand, as described above and depicted in FIGS. 1E and
2, with one or more reporter compositions.
[0203] Generally, the first transport component does not comprise
any ETM labels to decrease the amount of background signal.
However, it is possible to use transport components that do
comprise ETMs; in this embodiment, it is important that the first
transport component does not contain the same ETM labels that are
used in the reporter components. For example, ETM labels with
different redox potentials may be used, such that the ETM labels on
the reporter components are distinguishable from the labels on the
transport components. Alternatively, other types of labels, such as
fluorescent labels, may be used on the components. This may find
use in the development of controls and calibration of the
system.
[0204] In addition to the first components, the compositions of the
invention further comprise a reporter composition that is used to
signal the presence of the target analyte.
[0205] In a preferred embodiment, the reporter composition
comprises a second particle. As defined above, the second particle
may comprise a large number of different compositions. However, if
the transport mechanism used is magnetic attraction, it is
preferred in this embodiment that the second particle is
non-magnetic.
[0206] In a preferred embodiment, the second particle is a colloid
particle as defined above. Particularly preferred are gold colloid
particles, due to the ease of attachment of sulfur-containing
moieties.
[0207] In a preferred embodiment, the reporter composition is not a
particle. In this embodiment, the reporter composition comprises a
second binding ligand and at least one ETM, as defined below and
shown in FIGS. 3, 4 and 5. Generally in this embodiment the
reporter composition comprises a recruitment linker that has
attached ETMs.
[0208] The reporter composition comprises a second binding ligand,
as outlined above. The second binding ligand can be the same or
different from the first binding ligand. In addition, the
compositions of the invention (including both the transport and the
reporter compositions), may comprise multiple different binding
ligands.
[0209] The reporter composition further comprises at least one, and
preferably a plurality, of signalling moieties or labels that can
be used to detect the reporter compositions or assay complexes
containing them, particularly reporter particles including
colloids. Suitable signalling moieties include any detectable
labels, including, but not limited to, labels detectable optically,
fluorescently, electronically, electrochemically, radioactively,
labels detectable via chemiluminescence, electrochemiluminesce,
enzymes, fluorescence-resonance energy transfer (FRET), and RAMAN
techniques. Thus, signalling moieties include, but are not limited
to, electron transfer moieties, fluorescent moieties, radioisotopic
moieties, optical dyes, RAMAN labels, etc.
[0210] In a preferred embodiment, the signalling moieties are
electron transfer moieties. The terms "electron donor moiety",
"electron acceptor moiety", and "electron transfer moieties" (ETMs)
or grammatical equivalents herein refers to molecules capable of
electron transfer under certain conditions. 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. It is to be understood that the
number of possible electron donor moieties and electron acceptor
moieties 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. Preferred ETMs include, but are not limited
to, transition metal complexes, organic ETMs, and electrodes.
[0211] In a preferred embodiment, the ETMs are transition metal
complexes. Transition metals are those whose atoms have a partial
or complete d 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, are preferred. Particularly
preferred are ruthenium, rhenium, osmium, platinum, cobalt and
iron.
[0212] The transition metals are complexed with a variety of
ligands, L, to form suitable transition metal complexes, as is well
known in the art. The ligands provide the coordination atoms for
the binding of the metal ion. 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 L.sub.m). Suitable nitrogen
donating ligands are well known in the art and include, but are not
limited to, NH.sub.2; 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), EDTA, EGTA
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.
[0213] 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 Wilkenson, 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 Wilkenson.
[0214] The oxygen, sulfur, phosphorus and nitrogen-donating ligands
are attached in such a manner as to allow the heteroatoms to serve
as coordination atoms.
[0215] In a preferred embodiment, 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 n-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
[C.sub.5H.sub.5(-1)] and various ring substituted and ring fused
derivatives, such as the indenylide (-1) ion, that yield a class of
bis(cyclopentadieyl) 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
[(C.sub.5H.sub.5).sub.2Fe] 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
n-bonded ligands such as the allyl(-1) ion, or butadiene yield
potentially suitable organometallic compounds, and all such
ligands, in conjunction with other n-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.
[0216] 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
Wilkenson, 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.
[0217] 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.
[0218] In addition to transition metal complexes, other organic
electron donors and acceptors may be attached for use in the
invention. These organic molecules include, but are not limited to,
riboflavin, xanthene dyes, azine dyes, acridine orange,
N,N'-dimethyl-2,7-diazapyrenium dichloride (DAP.sup.2+),
methylviologen, ethidium bromide, quinones such as
N,N'-dimethylanthra(2,1,9-def:6,5,10-d'e'f)diisoquinoline
dichloride (ADIQ.sup.2+); porphyrins
([meso-tetrakis(N-methyl-x-pyridinium)porphyrin tetrachloride],
varlamine blue B hydrochloride, Bindschedler's green;
2,6-dichloroindophenol, 2,6-dibromophenolindophenol; Brilliant
crest blue (3-amino-9-dimethyl-amino-10-methylphenoxyazine
chloride), methylene blue; Nile blue A
(aminoaphthodiethylaminophenoxazine sulfate),
indigo-5,5',7,7'-tetrasulfonic acid, indigo-5,5',7-trisulfonic
acid; phenosafranine, indigo-5-monosulfonic acid; safranine T;
bis(dimethylglyoximato)-iron(II) chloride; induline scarlet,
neutral red, anthracene, coronene, pyrene, 9-phenylanthracene,
rubrene, binaphthyl, DPA, phenothiazene, fluoranthene,
phenanthrene, chrysene, 1,8-diphenyl-1,3,5,7-octatetracene,
naphthalene, acenaphthalene, perylene, TMPD and analogs and
substituted derivatives of these compounds.
[0219] In one embodiment, the electron donors and acceptors are
redox proteins as are known in the art. However, redox proteins in
many embodiments are not preferred.
[0220] The number of ETMs per reporter composition can vary, and
can depend on the composition of the reporter composition (i.e.
whether it is a particle) and the method of attachment, as is more
fully described below. In a preferred embodiment, a plurality of
ETMs are used. As is shown in the examples, the use of multiple
ETMs provides signal amplification and thus allows more sensitive
detection limits. Generally from one to millions or more can be
used.
[0221] The ETMs may be attached to the reporter compositions in a
variety of ways, including methods described in WO 98/20162 and
U.S. Ser. No. 09/135,183, filed Aug. 17, 1998, both of which are
hereby incorporated by reference in their entirety. In a preferred
embodiment, as is more fully outlined below, it is desirable to
attach the ETMs to the reporter composition in a manner that may
maximize "cross-talk" as between different ETMs; that is, to
maximize the possible conjugation of the system. This generally
involves using either closely packed ETMs, as is generally depicted
in FIGS. 4 and 5, or, when using colloid particles, conductive
oligomers to attach the ETMs to the colloid particle, as is
generally depicted in FIG. 1. Without being bound by theory, it
appears that the use of ETMs attached to the metallic colloid
particle via conductive oligomers allows electron transfer between
ETMs on the entire particle rather than just those in spatial
proximity to the electrode; that is, the system is conjugated,
allowing "access" of most or all of the ETMs on a particular
reporter composition.
[0222] Accordingly, in a preferred embodiment, the ETM is attached
to the reporter composition in a variety of ways.
[0223] In a preferred embodiment, the ETM is attached directly to
the reporter composition, i.e. directly to the particle. This may
be done as is generally outlined above for the attachment of
binding ligands to magnetic particles or other particles, using
functional groups on both the particle and the ETMs for
attachment.
[0224] In a preferred embodiment, the reporter composition is a
particle, and the ETM is attached via an attachment linker. The
method of attachment will depend on the composition of the ETM and
the attachment linker as will be appreciated by those in the art.
In a preferred embodiment, the reporter composition is a particle
and the attachment linker is a conductive oligomer. Preferred
methods of attachment for this embodiment when the binding ligand
is a nucleic acid are outlined in WO 98/20162, hereby incorporated
by reference in its entirety; see particularly structures
31-34.
[0225] In a preferred embodiment, the ETM is attached to the
terminus of the attachment linker, as depicted below in Structures
17, 18, 19 and 20. These structures depict the preferred embodiment
of conductive oligomers as the attachment linkers, although
insulators are useful as well. In addition, these structures depict
metallocenes as the ETMs, although as will be appreciated by those
in the art, other ETMs may be used as well. These structures depict
the use of a single ETM per attachment linker, although as will be
appreciated by those in the art, "branched" attachment linkers may
also be used to result in multiple ETMs at the termini of the
linker; in addition, ETMs may be attached as substitution groups
along the length of the linker as well. In this embodiment, the
attachment linkers basically form a SAM.
##STR00020##
[0226] Structure 17 utilizes a Structure 10 conductive oligomer,
although as will be appreciated by those in the art, other
conductive oligomers may be used. Preferred embodiments of
Structure 17 are depicted below.
##STR00021##
[0227] Preferred R groups of Structure 19 are hydrogen.
##STR00022##
[0228] These compositions are synthesized as follows. The
conductive oligomer linked to the metallocene is made as described
herein; see also, Hsung et al., Organometallics 14:4808-4815
(1995); and Bumm et al., Science 271:1705 (1996), both of which are
expressly incorporated herein by reference. The conductive oligomer
is then attached to the electrode using the novel ethylpyridine
protecting group, as outlined herein.
[0229] Alternatively, the ETMs can be attached as a plurality of
ETMs as is generally outlined below for non-particle reporter
compositions, using components as is generally outlined in FIG.
3.
[0230] In a preferred embodiment, the reporter composition is a
particle and the attachment linker is an insulator. This is
generally done using the above techniques as will be appreciated by
those in the art. In a preferred embodiment, the reporter
composition is not a particle and the binding ligand is linked
either directly or indirectly to at least one ETM. This can be done
directly, by attaching the binding ligand directly to the ETM,
again generally by using chemical functionalities on each, as is
generally described above. Similarly, linkers can be used,
including polymers and homo- and heterobifunctional linkers as
described herein.
[0231] In a preferred embodiment, a recruitment linker comprising a
plurality of ETMs is used, either for attachment to the binding
ligand or for attachment to a particle comprising the binding
ligand. This is particularly preferred when a particle is not used
to allow more signal per target analyte, although as outlined
above, these techniques can be used with particles as well. In this
embodiment, the recruitment linker can be virtually any polymer,
with nucleic acid being preferred, particularly when a particle is
not used and the ETMs are to be linked directly to a nucleic acid
binding ligand; this allows the synthesis of the reporter
composition to be done in one step. In some embodiments, as is more
fully outlined below, the recruitment linker may comprise
double-stranded portions.
[0232] Thus, as will be appreciated by those in the art, there are
a variety of configurations that can be used. In a preferred
embodiment, the recruitment linker is nucleic acid (including
analogs), and attachment of the ETMs can be via (1) a base; (2) the
backbone, including the ribose, the phosphate, or comparable
structures in nucleic acid analogs; (3) nucleoside replacement,
described below; or (4) metallocene polymers, as described below.
In a preferred embodiment, the recruitment linker is non-nucleic
acid, and can be either a metallocene polymer or an alkyl-type
polymer (including heteroalkyl, as is more fully described below)
containing ETM substitution groups. These options are generally
depicted in FIG. 3.
[0233] In a preferred embodiment, the recruitment linker is a
nucleic acid, and comprises covalently attached ETMs. The ETMs may
be attached to nucleosides within the nucleic acid in a variety of
positions. Preferred embodiments include, but are not limited to,
(1) attachment to the base of the nucleoside, (2) attachment of the
ETM as a base replacement, (3) attachment to the backbone of the
nucleic acid, including either to a ribose of the ribose-phosphate
backbone or to a phosphate moiety, or to analogous structures in
nucleic acid analogs, and (4) attachment via metallocene polymers,
with the latter being preferred.
[0234] In a preferred embodiment, the ETM is attached to the base
of a nucleoside as is generally outlined herein and in WO 98/20162
for the attachment of conductive oligomers. Attachment can be to an
internal nucleoside or a terminal nucleoside, or combinations of
these.
[0235] Ligands containing aromatic groups can be attached via
acetylene linkages as is known in the art (see Comprehensive
Organic Synthesis, Trost et al., Ed., Pergamon Press, Chapter 2.4:
Coupling Reactions Between sp.sup.2 and sp Carbon Centers,
Sonogashira, pp 521-549, and pp 950-953, hereby incorporated by
reference). Structure 21 depicts a representative structure in the
presence of the metal ion and any other necessary ligands;
Structure 21 depicts uridine, although as for all the structures
herein, any other base may also be used.
##STR00023##
[0236] L.sub.a is a ligand, which may include nitrogen, oxygen,
sulfur or phosphorus donating ligands or organometallic ligands
such as metallocene ligands. Suitable L.sub.a ligands include, but
not limited to, phenanthroline, imidazole, bpy and terpy. L.sub.r
and M are as defined above. Again, it will be appreciated by those
in the art, a linker ("Z") may be included between the nucleoside
and the ETM.
[0237] Similarly, as for the conductive oligomers, the linkage may
be done using a linker, which may utilize an amide linkage (see
generally Telser et al., J. Am. Chem. Soc. 111:7221-7226 (1989);
Telser et al., J. Am. Chem. Soc. 111:7226-7232 (1989), both of
which are expressly incorporated by reference). These structures
are generally depicted below in Structure 22, which again uses
uridine as the base, although as above, the other bases may also be
used:
##STR00024##
[0238] In this embodiment, L is a ligand as defined above, with L,
and M as defined above as well. Preferably, L is amino, phen, byp
and terpy.
[0239] In a preferred embodiment, the ETM attached to a nucleoside
is a metallocene; i.e. the L and L.sub.r of Structure 22 are both
metallocene ligands, L.sub.m, as described above. Structure 23
depicts a preferred embodiment wherein the metallocene is
ferrocene, and the base is uridine, although other bases may be
used:
##STR00025##
[0240] Preliminary data suggest that Structure 23 may cyclize, with
the second acetylene carbon atom attacking the carbonyl oxygen,
forming a furan-like structure. Preferred metallocenes include
ferrocene, cobaltocene and osmiumocene.
[0241] In a preferred embodiment, the ETM is attached to a ribose
at any position of the ribose-phosphate backbone of the nucleic
acid, i.e. either the 5' or 3' terminus or any internal nucleoside.
Ribose in this case can include ribose analogs. As is known in the
art, nucleosides that are modified at either the 2' or 3' position
of the ribose can be made, with nitrogen, oxygen, sulfur and
phosphorus-containing modifications possible. Amino-modified and
oxygen-modified ribose is preferred. See generally PCT publication
WO 95/15971, incorporated herein by reference. These modification
groups may be used as a transition metal ligand, or as a chemically
functional moiety for attachment of other transition metal ligands
and organometallic ligands, or organic electron donor moieties as
will be appreciated by those in the art. In this embodiment, a
linker such as depicted herein for "Z" may be used as well, or a
conductive oligomer between the ribose and the ETM. Preferred
embodiments utilize attachment at the 2' or 3' position of the
ribose, with the 2' position being preferred. Thus for example,
conductive oligomers may be replaced by ETMs; alternatively, the
ETMs may be added to the free terminus of the conductive
oligomer.
[0242] In a preferred embodiment, a metallocene serves as the ETM,
and is attached via an amide bond as depicted below in Structure
24. The examples outline the synthesis of a preferred compound when
the metallocene is ferrocene.
##STR00026##
[0243] In a preferred embodiment, amine linkages are used, as is
generally depicted in Structure 25.
##STR00027##
[0244] Z is a linker, as defined herein, with 1-16 atoms being
preferred, and 2-4 atoms being particularly preferred, and t is
either one or zero.
[0245] In a preferred embodiment, oxo linkages are used, as is
generally depicted in Structure 26.
##STR00028##
[0246] In Structure 26, Z is a linker, as defined herein, and t is
either one or zero. Preferred Z linkers include alkyl groups
including heteroalkyl groups such as (CH.sub.2)n and
(CH.sub.2CH.sub.2O)n, with n from 1 to 10 being preferred, and n=1
to 4 being especially preferred, and n=4 being particularly
preferred.
[0247] Linkages utilizing other heteroatoms are also possible.
[0248] In a preferred embodiment, an ETM is attached to a phosphate
at any position of the ribose-phosphate backbone of the nucleic
acid. This may be done in a variety of ways. In one embodiment,
phosphodiester bond analogs such as phosphoramide or
phosphoramidite linkages may be incorporated into a nucleic acid,
where the heteroatom (i.e. nitrogen) serves as a transition metal
ligand (see PCT publication WO 95/15971, incorporated by
reference). Alternatively, the conductive oligomers depicted in the
structures may be replaced by ETMs. In a preferred embodiment, the
composition has the structure shown in Structure 25.
##STR00029##
[0249] In Structure 25, the ETM is attached via a phosphate
linkage, generally through the use of a linker, Z. Preferred Z
linkers include alkyl groups, including heteroalkyl groups such as
(CH.sub.2).sub.n, (CH.sub.2CH.sub.2O).sub.n, with n from 1 to 10
being preferred, and n=1 to 4 being especially preferred, and n=4
being particularly preferred.
[0250] When the ETM is attached to the base or the backbone of the
nucleoside, it is possible to attach the ETMs via "dendrimer"
structures, as is more fully outlined below. As is generally
depicted in the Figures, alkyl-based linkers can be used to create
multiple branching structures comprising one or more ETMs at the
terminus of each branch. Generally, this is done by creating branch
points containing multiple hydroxy groups, which optionally can
then be used to add additional branch points. The terminal hydroxy
groups can then be used in phosphoramidite reactions to add ETMs,
as is generally done below for the nucleoside replacement and
metallocene polymer reactions.
[0251] In a preferred embodiment, an ETM such as a metallocene is
used as a "nucleoside replacement", serving as an ETM. For example,
the distance between the two cyclopentadiene rings of ferrocene is
similar to the orthogonal distance between two bases in a double
stranded nucleic acid. Other metallocenes in addition to ferrocene
may be used, for example, air stable metallocenes such as those
containing cobalt or ruthenium. Thus, metallocene moieties may be
incorporated into the backbone of a nucleic acid, as is generally
depicted in Structure 26 (nucleic acid with a ribose-phosphate
backbone) and Structure 27 (peptide nucleic acid backbone).
Structures 26 and 27 depict ferrocene, although as will be
appreciated by those in the art, other metallocenes may be used as
well. In general, air stable metallocenes are preferred, including
metallocenes utilizing ruthenium and cobalt as the metal.
##STR00030##
[0252] In Structure 26, Z is a linker as defined above, with
generally short, alkyl groups, including heteroatoms such as oxygen
being preferred. Generally, what is important is the length of the
linker, such that minimal perturbations of a double stranded
nucleic acid is effected, as is more fully described below. Thus,
methylene, ethylene, ethylene glycols, propylene and butylene are
all preferred, with ethylene and ethylene glycol being particularly
preferred. In addition, each Z linker may be the same or different.
Structure 26 depicts a ribose-phosphate backbone, although as will
be appreciated by those in the art, nucleic acid analogs may also
be used, including ribose analogs and phosphate bond analogs.
##STR00031##
[0253] In Structure 27, preferred Z groups are as listed above, and
again, each Z linker can be the same or different. As above, other
nucleic acid analogs may be used as well.
[0254] In addition, although the structures and discussion above
depicts metallocenes, and particularly ferrocene, this same general
idea can be used to add ETMs in addition to metallocenes, as
nucleoside replacements or in polymer embodiments, described below.
Thus, for example, when the ETM is a transition metal complex other
than a metallocene, comprising one, two or three (or more) ligands,
the ligands can be functionalized as depicted for the ferrocene to
allow the addition of phosphoramidite groups. Particularly
preferred in this embodiment are complexes comprising at least two
ring (for example, aryl and substituted aryl) ligands, where each
of the ligands comprises functional groups for attachment via
phosphoramidite chemistry. As will be appreciated by those in the
art, this type of reaction, creating polymers of ETMs either as a
portion of the backbone of the nucleic acid or as "side groups" of
the nucleic acids, to allow amplification of the signals generated
herein, can be done with virtually any ETM that can be
functionalized to contain the correct chemical groups.
[0255] In addition, as is more fully outlined below, it is possible
to incorporate more than one metallocene into the backbone, either
with nucleotides in between and/or with adjacent metallocenes. When
adjacent metallocenes are added to the backbone, this is similar to
the process described below as "metallocene polymers"; that is,
there are areas of metallocene polymers within the backbone.
[0256] In addition to the nucleic acid substitutent groups, it is
also desirable in some instances to add additional substituent
groups to one or both of the aromatic rings of the metallocene (or
ETM). Substituent groups on an ETM, particularly metallocenes such
as ferrocene, may be added to alter the redox properties of the
ETM. Thus, for example, in some embodiments, it may be desirable to
have different ETMs attached in different ways (i.e. base or ribose
attachment), on different components, or for different purposes
(for example, calibration or as an internal standard). Thus, the
addition of substituent groups on the metallocene may allow two
different ETMs to be distinguished.
[0257] In order to generate these metallocene-backbone nucleic acid
analogs, the intermediate components are also provided. Thus, in a
preferred embodiment, the invention provides phosphoramidite
metallocenes, as generally depicted in Structure 28:
##STR00032##
[0258] In Structure 28, PG is a protecting group, generally
suitable for use in nucleic acid synthesis, with DMT, MMT and TMT
all being preferred. The aromatic rings can either be the rings of
the metallocene, or aromatic rings of ligands for transition metal
complexes or other organic ETMs. The aromatic rings may be the same
or different, and may be substituted as discussed herein. Structure
29 depicts the ferrocene derivative:
##STR00033##
[0259] These phosphoramidite analogs can be added to standard
oligonucleotide syntheses as is known in the art.
[0260] Structure 30 depicts the ferrocene peptide nucleic acid
(PNA) monomer, that can be added to PNA synthesis as is known in
the art and depicted within the Figures and Examples:
##STR00034##
[0261] In Structure 30, the PG protecting group is suitable for use
in peptide nucleic acid synthesis, with MMT, boc and Fmoc being
preferred.
[0262] These same intermediate compounds can be used to form ETM or
metallocene polymers, which are added to the nucleic acids, rather
than as backbone replacements, as is more fully described
below.
[0263] In a preferred embodiment, the ETMs are attached as
polymers, for example as metallocene polymers, in a "branched"
configuration similar to the "branched DNA" embodiments herein and
as outlined in U.S. Pat. No. 5,124,246, using modified
functionalized nucleotides. The general idea is as follows. A
modified phosphoramidite nucleotide is generated that can
ultimately contain a free hydroxy group that can be used in the
attachment of phosphoramidite ETMs such as metallocenes. This free
hydroxy group could be on the base or the backbone, such as the
ribose or the phosphate (although as will be appreciated by those
in the art, nucleic acid analogs containing other structures can
also be used). The modified nucleotide is incorporated into a
nucleic acid, and any hydroxy protecting groups are removed, thus
leaving the free hydroxyl. Upon the addition of a phosphoramidite
ETM such as a metallocene, as described above in the Structures,
ETMs, such as metallocene ETMs, are added. Additional
phosphoramidite ETMs such as metallocenes can be added, to form
"ETM polymers", including "metallocene polymers" as depicted
herein, particularly for ferrocene. In addition, in some
embodiments, it is desirable to increase the solubility of the
polymers by adding a "capping" group to the terminal ETM in the
polymer, for example a final phosphate group to the metallocene as
is generally depicted in FIG. 4. Other suitable solubility
enhancing "capping" groups will be appreciated by those in the art.
It should be noted that these solubility enhancing groups can be
added to the polymers in other places, including to the ligand
rings, for example on the metallocenes as discussed herein.
[0264] A preferred embodiment of this general idea is outlined in
the Figures. In this embodiment, the 2' position of a ribose of a
phosphoramidite nucleotide is first functionalized to contain a
protected hydroxy group, in this case via an oxo-linkage, although
any number of linkers can be used, as is generally described herein
for Z linkers. The protected modified nucleotide is then
incorporated via standard phosphoramidite chemistry into a growing
nucleic acid. The protecting group is removed, and the free hydroxy
group is used, again using standard phosphoramidite chemistry to
add a phosphoramidite metallocene such as ferrocene. A similar
reaction is possible for nucleic acid analogs. For example, using
peptide nucleic acids and the metallocene monomer shown in
Structure 30, peptide nucleic acid structures containing
metallocene polymers could be generated.
[0265] Thus, the present invention provides recruitment linkers of
nucleic acids comprising "branches" of metallocene polymers as is
generally depicted in FIGS. 3, 4 and 5. Preferred embodiments also
utilize metallocene polymers from one to about 50 metallocenes in
length, with from about 5 to about 20 being preferred and from
about 5 to about 10 being especially preferred.
[0266] In addition, when the recruitment linker is nucleic acid,
any combination of ETM attachments may be done.
[0267] In a preferred embodiment, the recruitment linker is not
nucleic acid, and instead may be any sort of linker or polymer. As
will be appreciated by those in the art, generally any linker or
polymer that can be modified to contain ETMs can be used. In
general, the polymers or linkers should be reasonably soluble and
contain suitable functional groups for the addition of ETMs.
[0268] As used herein, a "recruitment polymer" comprises at least
two or three subunits, which are covalently attached. At least some
portion of the monomeric subunits contain functional groups for the
covalent attachment of ETMs. In some embodiments coupling moieties
are used to covalently link the subunits with the ETMs. Preferred
functional groups for attachment are amino groups, carboxy groups,
oxo groups and thiol groups, with amino groups being particularly
preferred. As will be appreciated by those in the art, a wide
variety of recruitment polymers are possible.
[0269] Suitable linkers include, but are not limited to, alkyl
linkers (including heteroalkyl (including (poly)ethylene
glycol-type structures), substituted alkyl, aryalkyl linkers, etc.
As above for the polymers, the linkers will comprise one or more
functional groups for the attachment of ETMs, which will be done as
will be appreciated by those in the art, for example through the
use 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).
[0270] Suitable recruitment polymers include, but are not limited
to, functionalized styrenes, such as amino styrene, functionalized
dextrans, and polyamino acids. Preferred polymers are polyamino
acids (both poly-D-amino acids and poly-L-amino acids), such as
polylysine, and polymers containing lysine and other amino acids
being particularly preferred. Other suitable polyamino acids are
polyglutamic acid, polyaspartic acid, co-polymers of lysine and
glutamic or aspartic acid, co-polymers of lysine with alanine,
tyrosine, phenylalanine, serine, tryptophan, and/or proline.
[0271] In a preferred embodiment, the recruitment linker comprises
a metallocene polymer, as is described above.
[0272] When non-nucleic acid recruitment linkers are used,
attachment of the linker/polymer of the recruitment linker will be
done generally using standard chemical techniques, such as will be
appreciated by those in the art. For example, when alkyl-based
linkers are used, attachment can be similar to the attachment of
insulators to nucleic acids.
[0273] In addition, it is possible to have recruitment linkers that
are mixtures of nucleic acids and non-nucleic acids, either in a
linear form (i.e. nucleic acid segments linked together with alkyl
linkers) or in branched forms (nucleic acids with alkyl "branches"
that may contain ETMs and may be additionally branched).
[0274] In a preferred embodiment, it is the target sequence itself
that carries the ETMs. For example, as is more fully described
below, it is possible to enzymatically add triphosphate nucleotides
comprising the ETMs of the invention to a growing nucleic acid, for
example during a polymerase chain reaction (PCR). As will be
recognized by those in the art, while several enzymes have been
shown to generally tolerate modified nucleotides, some of the
modified nucleotides of the invention, for example the "nucleoside
replacement" embodiments and putatively some of the phosphate
attachments, may or may not be recognized by the enzymes to allow
incorporation into a growing nucleic acid. Therefore, preferred
attachments in this embodiment are to the base or ribose of the
nucleotide.
[0275] Thus, for example, PCR amplification of a target sequence,
as is well known in the art, will result in target sequences
comprising ETMs, generally randomly incorporated into the sequence.
The system of the invention can then be configured to allow
detection using these ETMs.
[0276] Alternatively, as outlined more fully below, it is possible
to enzymatically add nucleotides comprising ETMs to the terminus of
a nucleic acid, for example a target nucleic acid. In this
embodiment, an effective "recruitment linker" is added to the
terminus of the target sequence, that can then be used for
detection.
[0277] In some embodiments, when the recruitment linker is nucleic
acid, it may be desirable in some instances to have some or all of
the recruitment linker be double stranded. In one embodiment, there
may be a second recruitment linker, substantially complementary to
the first recruitment linker, that can hybridize to the first
recruitment linker. In a preferred embodiment, the first
recruitment linker comprises the covalently attached ETMs. In an
alternative embodiment, the second recruitment linker contains the
ETMs, and the first recruitment linker does not, and the ETMs are
recruited to the surface by hybridization of the second recruitment
linker to the first. In yet another embodiment, both the first and
second recruitment linkers comprise ETMs. It should be noted, as
discussed above, that nucleic acids comprising a large number of
ETMs may not hybridize as well, i.e. the T.sub.m may be decreased,
depending on the site of attachment and the characteristics of the
ETM. Thus, in general, when multiple ETMs are used on hybridizing
strands, generally there are less than about 5, with less than
about 3 being preferred, or alternatively the ETMs should be spaced
sufficiently far apart that the intervening nucleotides can
sufficiently hybridize to allow good kinetics.
[0278] In a preferred embodiment, the reporter particle does not
comprise directly attached ETMs. Rather, the reporter particle
comprises two different components: a capture binding partner (used
to bind to the assay complex) and at least one amplification
sequences, to which label probes will bind. This embodiment is
generally depicted in FIG. 2H.
[0279] In this embodiment, the reporter particle comprises a
capture binding partner as outlined above. In addition, the
reporter particle comprises nucleic acid probes comprising
amplification sequences, to which label probes can bind. Again, for
each of these, binding may be direct or indirect. An "amplification
sequence" or "amplification segment" or grammatical equivalents
herein is meant a sequence that is used, either directly or
indirectly, to bind to a first portion of a label probe; the label
probe comprises the ETMs. Preferably, the reporter particle
comprises a multiplicity of amplification sequences.
[0280] The amplification sequences of the amplifier probe are used,
either directly or indirectly, to bind to a label probe to allow
detection. In a preferred embodiment, the amplification sequences
of the amplifier probe are substantially complementary to a first
portion of a label probe. Alternatively, amplifier extender probes
are used, that have a first portion that binds to the amplification
sequence and a second portion that binds to the first portion of
the label probe.
[0281] Thus, label probes are either substantially complementary to
an amplification sequence or to a portion of the target sequence.
Accordingly, the label probes can be configured in a variety of
ways, as is generally described herein, depending on whether a
"mechanism-1" or "mechanism-2" detection system is utilized, as
described below.
[0282] In a preferred embodiment, the reporter particle further
comprises a SAM, as is outlined above for the transport particle
and/or electrode. In general, this is a mixed monolayer comprising
one, two, three or four components, as outlined above for
electrodes and/or transport particles.
[0283] The transport moiety and the reporter composition may be
added together in a wide variety of ratios, depending on the
composition of the moieties (for example, whether they are both
particles), the relative size of the moieties, the density of the
binding partners on the particles, etc. For example, when a
magnetic first particle is used and a colloid particle as the
reporter particle, the reporter particles are generally added in
excess. What is important is that neither the transport composition
nor the reporter composition is added in such excess that a single
type of particle or composition will carry all the target analytes;
the ratios are important to allow both the transport and reporter
compositions to bind. However, in general, depending on the size of
the particles, generally an excess of reporter particles are added,
particularly when large magnetic particles are used with smaller
reporter particles.
[0284] Accordingly, the methods of the invention provide for the
detection of target analytes in a test sample. In general, the
sample is added to the compositions of the invention comprising
transport and reporter compositions. The sample is added to the
compositions under conditions whereby the target analyte, if
present, can bind to the first and second binding ligands to form
an assay complex. "Assay complex" herein is meant the collection of
moieties, including the target analyte and the binding ligands that
contains at least one ETM and thus allows detection. The
composition of the assay complex depends on the use of the
different components outlined herein. The assay complex is then
transported to the electrode surface, comprising the conductive
oligomers, and the presence or absence of the ETMs is detected.
[0285] Transport of the assay complex using the transport
composition may be done in a wide variety of ways, as will be
appreciated by those in the art, and will depend in part on the
components of the system. Thus, for example, when magnetic
particles are used as the transport particles, the application of a
magnetic field to bring the assay complexes and extra magnetic
transport particles to the surface can be done. When the transport
particles are not magnetic, transport can proceed via physical
aggregation of the assay complexes with gravitational settling of
the assay complexes on the electrode. Alternate transport
mechanisms include, but are not limited to, electrophoretic
transport of the assay complexes, centrifugation, etc. It should be
noted that the use of different sized colloids may allow the use of
differential electrophoretic transport based on different charge
densities; that is, when a larger transport particle is used with
smaller (charged) reporter particles, i.e. the transport particle
can be "covered" or "coated" with the smaller particles, and thus
the charge densities of the transport particle and the assay
complex will allow differential electrophoretic transport.
[0286] Probes of the present invention are designed to be
complementary to a target sequence (either the target sequence of
the sample or to other probe sequences, as is described below),
such that hybridization of the target sequence and the probes of
the present invention occurs. As outlined below, this
complementarity need not be perfect; there may be any number of
base pair mismatches which will interfere with hybridization
between the target sequence and the single stranded nucleic acids
of the present invention. However, if the number of mutations is so
great that no hybridization can occur under even the least
stringent of hybridization conditions, the sequence is not a
complementary target sequence. Thus, by "substantially
complementary" herein is meant that the probes are sufficiently
complementary to the target sequences to hybridize under normal
reaction conditions.
[0287] A variety of hybridization conditions may be used in the
present invention, including high, moderate and low stringency
conditions; see for example Maniatis et al., Molecular Cloning: A
Laboratory Manual, 2d Edition, 1989, and Short Protocols in
Molecular Biology, ed. Ausubel, et al, hereby incorporated by
reference. Stringent conditions are sequence-dependent and will be
different in different circumstances. Longer sequences hybridize
specifically at higher temperatures. An extensive guide to the
hybridization of nucleic acids is found in Tijssen, Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic Acid
Probes, "Overview of principles of hybridization and the strategy
of nucleic acid assays" (1993). Generally, stringent conditions are
selected to be about 5-10.degree. C. lower than the thermal melting
point (Tm) for the specific sequence at a defined ionic strength
pH. The Tm is the temperature (under defined ionic strength, pH and
nucleic acid concentration) at which 50% of the probes
complementary to the target hybridize to the target sequence at
equilibrium (as the target sequences are present in excess, at Tm,
50% of the probes are occupied at equilibrium). Stringent
conditions will be those in which the salt concentration is less
than about 1.0 sodium ion, typically about 0.01 to 1.0 M sodium ion
concentration (or other salts) at pH 7.0 to 8.3 and the temperature
is at least about 30.degree. C. for short probes (e.g. 10 to 50
nucleotides) and at least about 60.degree. C. for long probes (e.g.
greater than 50 nucleotides). Stringent conditions may also be
achieved with the addition of destabilizing agents such as
formamide. The hybridization conditions may also vary when a
non-ionic backbone, i.e. PNA is used, as is known in the art. In
addition, cross-linking agents may be added after target binding to
cross-link, i.e. covalently attach, the two strands of the
hybridization complex.
[0288] Thus, the assays are generally run under stringency
conditions which allows formation of the label probe hybridization
complex only in the presence of target. Stringency can be
controlled by altering a step parameter that is a thermodynamic
variable, including, but not limited to, temperature, formamide
concentration, salt concentration, chaotropic salt concentration
pH, organic solvent concentration, etc.
[0289] These parameters may also be used to control non-specific
binding, as is generally outlined in U.S. Pat. No. 5,681,697. Thus
it may be desirable to perform certain steps at higher stringency
conditions to reduce non-specific binding.
[0290] The reactions outlined herein may be accomplished in a
variety of ways, as will be appreciated by those in the art.
Components of the reaction may be added simultaneously, or
sequentially, in any order, with preferred embodiments outlined
below. In addition, the reaction may include a variety of other
reagents may be included in the assays. These include reagents like
salts, buffers, neutral proteins, e.g. albumin, detergents, etc
which may be used to facilitate optimal hybridization and
detection, and/or reduce non-specific or background interactions.
Also reagents that otherwise improve the efficiency of the assay,
such as protease inhibitors, nuclease inhibitors, anti-microbial
agents, etc., may be used, depending on the sample preparation
methods and purity of the target.
[0291] Once the assay complexes of the invention are made, that
minimally comprise a target analyte and at least one ETM, detection
proceeds with electronic initiation. Without being limited by the
mechanism or theory, detection is based on the transfer of
electrons from the ETM to the electrode.
[0292] Detection of electron transfer, i.e. the presence of the
ETMs, is generally initiated electronically, with voltage being
preferred. A potential is applied to the assay complex. Precise
control and variations in the applied potential can be via a
potentiostat and either a three electrode system (one reference,
one sample (or working) and one counter electrode) or a two
electrode system (one sample and one counter electrode). This
allows matching of applied potential to peak potential of the
system which depends in part on the choice of ETMs and in part on
the conductive oligomer used, the composition and integrity of the
monolayer, and what type of reference electrode is used. As
described herein, ferrocene is a preferred ETM.
[0293] Once the assay complexes of the invention are made, that
minimally comprise a target sequence and a label probe, detection
proceeds with electronic initiation. Without being limited by the
mechanism or theory, detection is based on the transfer of
electrons from the ETM to the electrode.
[0294] Detection of electron transfer, i.e. the presence of the
ETMs, is generally initiated electronically, with voltage being
preferred. A potential is applied to the assay complex. Precise
control and variations in the applied potential can be via a
potentiostat and either a three electrode system (one reference,
one sample (or working) and one counter electrode) or a two
electrode system (one sample and one counter electrode). This
allows matching of applied potential to peak potential of the
system which depends in part on the choice of ETMs and in part on
the conductive oligomer used, the composition and integrity of the
monolayer, and what type of reference electrode is used. As
described herein, ferrocene is a preferred ETM.
[0295] In a preferred embodiment, a co-reductant or co-oxidant
(collectively, co-redoxant) is used, as an additional electron
source or sink. See generally Sato et al., Bull. Chem. Soc. Jpn
66:1032 (1993); Uosaki et al., Electrochimica Acta 36:1799 (1991);
and Alleman et al., J. Phys. Chem 100:17050 (1996); all of which
are incorporated by reference.
[0296] In a preferred embodiment, an input electron source in
solution is used in the initiation of electron transfer, preferably
when initiation and detection are being done using DC current or at
AC frequencies where diffusion is not limiting. In general, as will
be appreciated by those in the art, preferred embodiments utilize
monolayers that contain a minimum of "holes", such that
short-circuiting of the system is avoided. This may be done in
several general ways. In a preferred embodiment, an input electron
source is used that has a lower or similar redox potential than the
ETM of the label probe. Thus, at voltages above the redox potential
of the input electron source, both the ETM and the input electron
source are oxidized and can thus donate electrons; the ETM donates
an electron to the electrode and the input source donates to the
ETM. For example, ferrocene, as a ETM attached to the compositions
of the invention as described in the examples, has a redox
potential of roughly 200 mV in aqueous solution (which can change
significantly depending on what the ferrocene is bound to, the
manner of the linkage and the presence of any substitution groups).
Ferrocyanide, an electron source, has a redox potential of roughly
200 mV as well (in aqueous solution). Accordingly, at or above
voltages of roughly 200 mV, ferrocene is converted to ferricenium,
which then transfers an electron to the electrode. Now the
ferricyanide can be oxidized to transfer an electron to the ETM. In
this way, the electron source (or co-reductant) serves to amplify
the signal generated in the system, as the electron source
molecules rapidly and repeatedly donate electrons to the ETM
attached to the nucleic acid. The rate of electron donation or
acceptance will be limited by the rate of diffusion of the
co-reductant, the electron transfer between the co-reductant and
the ETM, which in turn is affected by the concentration and size,
etc.
[0297] Alternatively, input electron sources that have lower redox
potentials than the ETM are used. At voltages less than the redox
potential of the ETM, but higher than the redox potential of the
electron source, the input source such as ferrocyanide is unable to
be oxided and thus is unable to donate an electron to the ETM; i.e.
no electron transfer occurs. Once ferrocene is oxidized, then there
is a pathway for electron transfer.
[0298] In an alternate preferred embodiment, an input electron
source is used that has a higher redox potential than the ETM of
the label probe. For example, luminol, an electron source, has a
redox potential of roughly 720 mV. At voltages higher than the
redox potential of the ETM, but lower than the redox potential of
the electron source, i.e. 200-720 mV, the ferrocene is oxided, and
transfers a single electron to the electrode via the conductive
oligomer. However, the ETM is unable to accept any electrons from
the luminol electron source, since the voltages are less than the
redox potential of the luminol. However, at or above the redox
potential of luminol, the luminol then transfers an electron to the
ETM, allowing rapid and repeated electron transfer. In this way,
the electron source (or co-reductant) serves to amplify the signal
generated in the system, as the electron source molecules rapidly
and repeatedly donate electrons to the ETM of the label probe.
[0299] Luminol has the added benefit of becoming a chemiluminescent
species upon oxidation (see Jirka et al., Analytica Chimica Acta
284:345 (1993)), thus allowing photo-detection of electron transfer
from the ETM to the electrode. Thus, as long as the luminol is
unable to contact the electrode directly, i.e. in the presence of
the SAM such that there is no efficient electron transfer pathway
to the electrode, luminol can only be oxidized by transferring an
electron to the ETM on the label probe. When the ETM is not
present, i.e. when the target sequence is not hybridized to the
composition of the invention, luminol is not significantly
oxidized, resulting in a low photon emission and thus a low (if
any) signal from the luminol. In the presence of the target, a much
larger signal is generated. Thus, the measure of luminol oxidation
by photon emission is an indirect measurement of the ability of the
ETM to donate electrons to the electrode. Furthermore, since photon
detection is generally more sensitive than electronic detection,
the sensitivity of the system may be increased. Initial results
suggest that luminescence may depend on hydrogen peroxide
concentration, pH, and luminol concentration, the latter of which
appears to be non-linear.
[0300] Suitable electron source molecules are well known in the
art, and include, but are not limited to, ferricyanide, and
luminol.
[0301] Alternatively, output electron acceptors or sinks could be
used, i.e. the above reactions could be run in reverse, with the
ETM such as a metallocene receiving an electron from the electrode,
converting it to the metallicenium, with the output electron
acceptor then accepting the electron rapidly and repeatedly. In
this embodiment, cobalticenium is the preferred ETM.
[0302] The presence of the ETMs at the surface of the monolayer can
be detected in a variety of ways. A variety of detection methods
may be used, including, but not limited to, optical detection (as a
result of spectral changes upon changes in redox states), which
includes fluorescence, phosphorescence, luminiscence,
chemiluminescence, electrochemiluminescence, and refractive index;
and electronic detection, including, but not limited to,
amperommetry, voltammetry, capacitance and impedance. These methods
include time or frequency dependent methods based on AC or DC
currents, pulsed methods, lock-in techniques, filtering (high pass,
low pass, band pass), and time-resolved techniques including
time-resolved fluorescence.
[0303] In one embodiment, the efficient transfer of electrons from
the ETM to the electrode results in stereotyped changes in the
redox state of the ETM. With many ETMs including the complexes of
ruthenium containing bipyridine, pyridine and imidazole rings,
these changes in redox state are associated with changes in
spectral properties. Significant differences in absorbance are
observed between reduced and oxidized states for these molecules.
See for example Fabbrizzi et al., Chem. Soc. Rev. 1995 pp 197-202).
These differences can be monitored using a spectrophotometer or
simple photomultiplier tube device.
[0304] In this embodiment, possible electron donors and acceptors
include all the derivatives listed above for photoactivation or
initiation. Preferred electron donors and acceptors have
characteristically large spectral changes upon oxidation and
reduction resulting in highly sensitive monitoring of electron
transfer. Such examples include Ru(NH.sub.3).sub.4py and
Ru(bpy).sub.2im as preferred examples. It should be understood that
only the donor or acceptor that is being monitored by absorbance
need have ideal spectral characteristics.
[0305] In a preferred embodiment, the electron transfer is detected
fluorometrically. Numerous transition metal complexes, including
those of ruthenium, have distinct fluorescence properties.
Therefore, the change in redox state of the electron donors and
electron acceptors attached to the nucleic acid can be monitored
very sensitively using fluorescence, for example with
Ru(4,7-biphenyl.sub.2-phenanthroline).sub.3.sup.2+. The production
of this compound can be easily measured using standard fluorescence
assay techniques. For example, laser induced fluorescence can be
recorded in a standard single cell fluorimeter, a flow through
"on-line" fluorimeter (such as those attached to a chromatography
system) or a multi-sample "plate-reader" similar to those marketed
for 96-well immuno assays.
[0306] Alternatively, fluorescence can be measured using fiber
optic sensors with nucleic acid probes in solution or attached to
the fiber optic. Fluorescence is monitored using a photomultiplier
tube or other light detection instrument attached to the fiber
optic. The advantage of this system is the extremely small volumes
of sample that can be assayed.
[0307] In addition, scanning fluorescence detectors such as the
FluorImager sold by Molecular Dynamics are ideally suited to
monitoring the fluorescence of modified nucleic acid molecules
arrayed on solid surfaces. The advantage of this system is the
large number of electron transfer probes that can be scanned at
once using chips covered with thousands of distinct nucleic acid
probes.
[0308] Many transition metal complexes display fluorescence with
large Stokes shifts. Suitable examples include bis- and
trisphenanthroline complexes and bis- and trisbipyridyl complexes
of transition metals such as ruthenium (see Juris, A., Balzani, V.,
et. al. Coord. Chem. Rev., V. 84, p. 85-277, 1988). Preferred
examples display efficient fluorescence (reasonably high quantum
yields) as well as low reorganization energies. These include
Ru(4,7-biphenyl.sub.2-phenanthroline).sub.3.sup.2+,
Ru(4,4'-diphenyl-2,2'-bipyridine).sub.3.sup.2+ and platinum
complexes (see Cummings et al., J. Am. Chem. Soc. 118:1949-1960
(1996), incorporated by reference). Alternatively, a reduction in
fluorescence associated with hybridization can be measured using
these systems.
[0309] In a further embodiment, electrochemiluminescence is used as
the basis of the electron transfer detection. With some ETMs such
as R.sup.2+(bpy).sub.3, direct luminescence accompanies excited
state decay. Changes in this property are associated with nucleic
acid hybridization and can be monitored with a simple
photomultiplier tube arrangement (see Blackburn, G. F. Clin. Chem.
37: 1534-1539 (1991); and Juris et al., supra.
[0310] In a preferred embodiment, electronic detection is used,
including amperommetry, voltammetry, capacitance, and impedance.
Suitable techniques include, but are not limited to,
electrogravimetry; coulometry (including controlled potential
coulometry and constant current coulometry); voltametry (cyclic
voltametry, pulse voltametry (normal pulse voltametry, square wave
voltametry, differential pulse voltametry, Osteryoung square wave
voltametry, and coulostatic pulse techniques); stripping analysis
(aniodic stripping analysis, cathiodic stripping analysis, square
wave stripping voltammetry); conductance measurements (electrolytic
conductance, direct analysis); time-dependent electrochemical
analyses (chronoamperometry, chronopotentiometry, cyclic
chronopotentiometry and amperometry, AC polography,
chronogalvametry, and chronocoulometry); AC impedance measurement;
capacitance measurement; AC voltametry; and
photoelectrochemistry.
[0311] In a preferred embodiment, monitoring electron transfer is
via amperometric detection. This method of detection involves
applying a potential (as compared to a separate reference
electrode) between the nucleic acid-conjugated electrode and a
reference (counter) electrode in the sample containing target genes
of interest. Electron transfer of differing efficiencies is induced
in samples in the presence or absence of target nucleic acid; that
is, the presence or absence of the target nucleic acid, and thus
the label probe, can result in different currents.
[0312] The device for measuring electron transfer amperometrically
involves sensitive current detection and includes a means of
controlling the voltage potential, usually a potentiostat. This
voltage is optimized with reference to the potential of the
electron donating complex on the label probe. Possible electron
donating complexes include those previously mentioned with
complexes of iron, osmium, platinum, cobalt, rhenium and ruthenium
being preferred and complexes of iron being most preferred.
[0313] In a preferred embodiment, alternative electron detection
modes are utilized. For example, potentiometric (or voltammetric)
measurements involve non-faradaic (no net current flow) processes
and are utilized traditionally in pH and other ion detectors.
Similar sensors are used to monitor electron transfer between the
ETM and the electrode. In addition, other properties of insulators
(such as resistance) and of conductors (such as conductivity,
impedance and capacitance) could be used to monitor electron
transfer between ETM and the electrode. Finally, any system that
generates a current (such as electron transfer) also generates a
small magnetic field, which may be monitored in some
embodiments.
[0314] It should be understood that one benefit of the fast rates
of electron transfer observed in the compositions of the invention
is that time resolution can greatly enhance the signal-to-noise
results of monitors based on absorbance, fluorescence and
electronic current. The fast rates of electron transfer of the
present invention result both in high signals and stereotyped
delays between electron transfer initiation and completion. By
amplifying signals of particular delays, such as through the use of
pulsed initiation of electron transfer and "lock-in" amplifiers of
detection, and Fourier transforms.
[0315] In a preferred embodiment, electron transfer is initiated
using alternating current (AC) methods. Without being bound by
theory, it appears that ETMs, bound to an electrode, generally
respond similarly to an AC voltage across a circuit containing
resistors and capacitors. Basically, any methods which enable the
determination of the nature of these complexes, which act as a
resistor and capacitor, can be used as the basis of detection.
Surprisingly, traditional electrochemical theory, such as
exemplified in Laviron et al., J. Electroanal. Chem. 97:135 (1979)
and Laviron et al., J. Electroanal. Chem. 105:35 (1979), both of
which are incorporated by reference, do not accurately model the
systems described herein, except for very small E.sub.AC (less than
10 mV) and relatively large numbers of molecules. That is, the AC
current (1) is not accurately described by Laviron's equation. This
may be due in part to the fact that this theory assumes an
unlimited source and sink of electrons, which is not true in the
present systems.
[0316] The AC voltametry theory that models these systems well is
outlined in O'Connor et al., J. Electroanal. Chem. 466(2):197-202
(1999), hereby expressly incorporated by reference. The equation
that predicts these systems is shown below as Equation 1:
i avg = 2 nfFN total sin h [ n F RT E AC ] cos h [ n F RT E AC ] +
cos h [ n F RT ( E DC - E O ) ] Equation 1 ##EQU00001##
[0317] In Equation 1, n is the number of electrons oxidized or
reduced per redox molecule, f is the applied frequency, F is
Faraday's constant, N.sub.total is the total number of redox
molecules, E.sub.O is the formal potential of the redox molecule, R
is the gas constant, T is the temperature in degrees Kelvin, and
E.sub.DC is the electrode potential. The model fits the
experimental data very well. In some cases the current is smaller
than predicted, however this has been shown to be caused by
ferrocene degradation which may be remedied in a number of
ways.
[0318] In addition, the faradaic current can also be expressed as a
function of time, as shown in Equation 2:
I f ( t ) = q e N total n F 2 RT ( cos h [ n F RT ( V ( t ) - E 0 )
] + 1 ) V ( t ) t Equation 2 ##EQU00002##
[0319] I.sub.F is the Faradaic current and q.sub.e is the
elementary charge.
[0320] However, Equation 1 does not incorporate the effect of
electron transfer rate nor of instrument factors. Electron transfer
rate is important when the rate is close to or lower than the
applied frequency. Thus, the true i.sub.AC should be a function of
all three, as depicted in Equation 3.
i.sub.AC=f(Nernst factors)f(k.sub.ET)f(instrument factors) Equation
3
[0321] These equations can be used to model and predict the
expected AC currents in systems which use input signals comprising
both AC and DC components. As outlined above, traditional theory
surprisingly does not model these systems at all, except for very
low voltages.
[0322] In general, non-specifically bound label probes/ETMs show
differences in impedance (i.e. higher impedances) than when the
label probes containing the ETMs are specifically bound in the
correct orientation. In a preferred embodiment, the
non-specifically bound material is washed away, resulting in an
effective impedance of infinity. Thus, AC detection gives several
advantages as is generally discussed below, including an increase
in sensitivity, and the ability to "filter out" background noise.
In particular, changes in impedance (including, for example, bulk
impedance) as between non-specific binding of ETM-containing probes
and target-specific assay complex formation may be monitored.
[0323] Accordingly, when using AC initiation and detection methods,
the frequency response of the system changes as a result of the
presence of the ETM. By "frequency response" herein is meant a
modification of signals as a result of electron transfer between
the electrode and the ETM. This modification is different depending
on signal frequency. A frequency response includes AC currents at
one or more frequencies, phase shifts, DC offset voltages, faradaic
impedance, etc.
[0324] Once the assay complex including the target sequence and
label probe is made, a first input electrical signal is then
applied to the system, preferably via at least the sample electrode
(containing the complexes of the invention) and the counter
electrode, to initiate electron transfer between the electrode and
the ETM. Three electrode systems may also be used, with the voltage
applied to the reference and working electrodes. The first input
signal comprises at least an AC component. The AC component may be
of variable amplitude and frequency. Generally, for use in the
present methods, the AC amplitude ranges from about 1 mV to about
1.1 V, with from about 10 mV to about 800 mV being preferred, and
from about 10 mV to about 500 mV being especially preferred. The AC
frequency ranges from about 0.01 Hz to about 100 MHz, with from
about 10 Hz to about 10 MHz being preferred, and from about 100 Hz
to about 20 MHz being especially preferred.
[0325] The use of combinations of AC and DC signals gives a variety
of advantages, including surprising sensitivity and signal
maximization.
[0326] In a preferred embodiment, the first input signal comprises
a DC component and an AC component. That is, a DC offset voltage
between the sample and counter electrodes is swept through the
electrochemical potential of the ETM (for example, when ferrocene
is used, the sweep is generally from 0 to 500 mV) (or
alternatively, the working electrode is grounded and the reference
electrode is swept from 0 to -500 mV). The sweep is used to
identify the DC voltage at which the maximum response of the system
is seen. This is generally at or about the electrochemical
potential of the ETM. Once this voltage is determined, either a
sweep or one or more uniform DC offset voltages may be used. DC
offset voltages of from about -1 V to about +1.1 V are preferred,
with from about -500 mV to about +800 mV being especially
preferred, and from about -300 mV to about 500 mV being
particularly preferred. In a preferred embodiment, the DC offset
voltage is not zero. On top of the DC offset voltage, an AC signal
component of variable amplitude and frequency is applied. If the
ETM is present, and can respond to the AC perturbation, an AC
current will be produced due to electron transfer between the
electrode and the ETM.
[0327] For defined systems, it may be sufficient to apply a single
input signal to differentiate between the presence and absence of
the ETM (i.e. the presence of the target sequence) nucleic acid.
Alternatively, a plurality of input signals are applied. As
outlined herein, this may take a variety of forms, including using
multiple frequencies, multiple DC offset voltages, or multiple AC
amplitudes, or combinations of any or all of these.
[0328] Thus, in a preferred embodiment, multiple DC offset voltages
are used, although as outlined above, DC voltage sweeps are
preferred. This may be done at a single frequency, or at two or
more frequencies.
[0329] In a preferred embodiment, the AC amplitude is varied.
Without being bound by theory, it appears that increasing the
amplitude increases the driving force. Thus, higher amplitudes,
which result in higher overpotentials give faster rates of electron
transfer. Thus, generally, the same system gives an improved
response (i.e. higher output signals) at any single frequency
through the use of higher overpotentials at that frequency. Thus,
the amplitude may be increased at high frequencies to increase the
rate of electron transfer through the system, resulting in greater
sensitivity. In addition, this may be used, for example, to induce
responses in slower systems such as those that do not possess
optimal spacing configurations.
[0330] In a preferred embodiment, measurements of the system are
taken at least two separate amplitudes or overpotentials, with
measurements at a plurality of amplitudes being preferred. As noted
above, changes in response as a result of changes in amplitude may
form the basis of identification, calibration and quantification of
the system. In addition, one or more AC frequencies can be used as
well.
[0331] In a preferred embodiment, the AC frequency is varied. At
different frequencies, different molecules respond in different
ways. As will be appreciated by those in the art, increasing the
frequency generally increases the output current. However, when the
frequency is greater than the rate at which electrons may travel
between the electrode and the ETM, higher frequencies result in a
loss or decrease of output signal. At some point, the frequency
will be greater than the rate of electron transfer between the ETM
and the electrode, and then the output signal will also drop.
[0332] In one embodiment, detection utilizes a single measurement
of output signal at a single frequency. That is, the frequency
response of the system in the absence of target sequence, and thus
the absence of label probe containing ETMs, can be previously
determined to be very low at a particular high frequency. Using
this information, any response at a particular frequency, will show
the presence of the assay complex. That is, any response at a
particular frequency is characteristic of the assay complex. Thus,
it may only be necessary to use a single input high frequency, and
any changes in frequency response is an indication that the ETM is
present, and thus that the target sequence is present.
[0333] In addition, the use of AC techniques allows the significant
reduction of background signals at any single frequency due to
entities other than the ETMs, i.e. "locking out" or "filtering"
unwanted signals. That is, the frequency response of a charge
carrier or redox active molecule in solution will be limited by its
diffusion coefficient and charge transfer coefficient. Accordingly,
at high frequencies, a charge carrier may not diffuse rapidly
enough to transfer its charge to the electrode, and/or the charge
transfer kinetics may not be fast enough. This is particularly
significant in embodiments that do not have good monolayers, i.e.
have partial or insufficient monolayers, i.e. where the solvent is
accessible to the electrode. As outlined above, in DC techniques,
the presence of "holes" where the electrode is accessible to the
solvent can result in solvent charge carriers "short circuiting"
the system, i.e. the reach the electrode and generate background
signal. However, using the present AC techniques, one or more
frequencies can be chosen that prevent a frequency response of one
or more charge carriers in solution, whether or not a monolayer is
present. This is particularly significant since many biological
fluids such as blood contain significant amounts of redox active
molecules which can interfere with amperometric detection
methods.
[0334] In a preferred embodiment, measurements of the system are
taken at least two separate frequencies, with measurements at a
plurality of frequencies being preferred. A plurality of
frequencies includes a scan. For example, measuring the output
signal, e.g., the AC current, at a low input frequency such as 1-20
Hz, and comparing the response to the output signal at high
frequency such as 10-100 kHz will show a frequency response
difference between the presence and absence of the ETM. In a
preferred embodiment, the frequency response is determined at least
two, preferably at least about five, and more preferably at least
about ten frequencies.
[0335] After transmitting the input signal to initiate electron
transfer, an output signal is received or detected. The presence
and magnitude of the output signal will depend on a number of
factors, including the overpotential/amplitude of the input signal;
the frequency of the input AC signal; the composition of the
intervening medium; the DC offset; the environment of the system;
the nature of the ETM; the solvent; and the type and concentration
of salt. At a given input signal, the presence and magnitude of the
output signal will depend in general on the presence or absence of
the ETM, the placement and distance of the ETM from the surface of
the monolayer and the character of the input signal. In some
embodiments, it may be possible to distinguish between non-specific
binding of label probes and the formation of target specific assay
complexes containing label probes, on the basis of impedance.
[0336] In a preferred embodiment, the output signal comprises an AC
current. As outlined above, the magnitude of the output current
will depend on a number of parameters. By varying these parameters,
the system may be optimized in a number of ways.
[0337] In general, AC currents generated in the present invention
range from about 1 femptoamp to about 1 milliamp, with currents
from about 50 femptoamps to about 100 microamps being preferred,
and from about 1 picoamp to about 1 microamp being especially
preferred.
[0338] In a preferred embodiment, the output signal is phase
shifted in the AC component relative to the input signal. Without
being bound by theory, it appears that the systems of the present
invention may be sufficiently uniform to allow phase-shifting based
detection. That is, the complex biomolecules of the invention
through which electron transfer occurs react to the AC input in a
homogeneous manner, similar to standard electronic components, such
that a phase shift can be determined. This may serve as the basis
of detection between the presence and absence of the ETM, and/or
differences between the presence of target-specific assay complexes
comprising label probes and non-specific binding of the label
probes to the system components.
[0339] The output signal is characteristic of the presence of the
ETM; that is, the output signal is characteristic of the presence
of the target-specific assay complex comprising label probes and
ETMs. In a preferred embodiment, the basis of the detection is a
difference in the faradaic impedance of the system as a result of
the formation of the assay complex. Faradaic impedance is the
impedance of the system between the electrode and the ETM. Faradaic
impedance is quite different from the bulk or dielectric impedance,
which is the impedance of the bulk solution between the electrodes.
Many factors may change the faradaic impedance which may not effect
the bulk impedance, and vice versa. Thus, the assay complexes
comprising the nucleic acids in this system have a certain faradaic
impedance, that will depend on the distance between the ETM and the
electrode, their electronic properties, and the composition of the
intervening medium, among other things. Of importance in the
methods of the invention is that the faradaic impedance between the
ETM and the electrode is significantly different depending on
whether the label probes containing the ETMs are specifically or
non-specifically bound to the electrode.
[0340] Accordingly, the present invention further provides
apparatus for the detection of nucleic acids using AC detection
methods. The apparatus includes a test chamber which has at least a
first measuring or sample electrode, and a second measuring or
counter electrode. Three electrode systems are also useful. The
first and second measuring electrodes are in contact with a test
sample receiving region, such that in the presence of a liquid test
sample, the two electrodes may be in electrical contact.
[0341] In a preferred embodiment, the first measuring electrode
comprises a single stranded nucleic acid capture probe covalently
attached via an attachment linker, and a monolayer comprising
conductive oligomers, such as are described herein.
[0342] The apparatus further comprises an AC voltage source
electrically connected to the test chamber; that is, to the
measuring electrodes. Preferably, the AC voltage source is capable
of delivering DC offset voltage as well.
[0343] In a preferred embodiment, the apparatus further comprises a
processor capable of comparing the input signal and the output
signal. The processor is coupled to the electrodes and configured
to receive an output signal, and thus detect the presence of the
target nucleic acid.
[0344] Thus, the compositions of the present invention may be used
in a variety of research, clinical, quality control, or field
testing settings.
[0345] In a preferred embodiment, the probes are used in genetic
diagnosis. For example, probes can be made using the techniques
disclosed herein to detect target sequences such as the gene for
nonpolyposis colon cancer, the BRCA1 breast cancer gene, P53, which
is a gene associated with a variety of cancers, the Apo E4 gene
that indicates a greater risk of Alzheimer's disease, allowing for
easy presymptomatic screening of patients, mutations in the cystic
fibrosis gene, or any of the others well known in the art.
[0346] In an additional embodiment, viral and bacterial detection
is done using the complexes of the invention. In this embodiment,
probes are designed to detect target sequences from a variety of
bacteria and viruses. For example, current blood-screening
techniques rely on the detection of anti-HIV antibodies. The
methods disclosed herein allow for direct screening of clinical
samples to detect HIV nucleic acid sequences, particularly highly
conserved HIV sequences. In addition, this allows direct monitoring
of circulating virus within a patient as an improved method of
assessing the efficacy of anti-viral therapies. Similarly, viruses
associated with leukemia, HTLV-I and HTLV-II, may be detected in
this way. Bacterial infections such as tuberculosis, clymidia and
other sexually transmitted diseases, may also be detected, for
example using ribosomal RNA (rRNA) as the target sequences.
[0347] In a preferred embodiment, the nucleic acids of the
invention find use as probes for toxic bacteria in the screening of
water and food samples. For example, samples may be treated to lyse
the bacteria to release its nucleic acid (particularly rRNA), and
then probes designed to recognize bacterial strains, including, but
not limited to, such pathogenic strains as, Salmonella,
Campylobacter, Vibrio cholerae, Leishmania, enterotoxic strains of
E. coli, and Legionnaire's disease bacteria. Similarly,
bioremediation strategies may be evaluated using the compositions
of the invention.
[0348] In a further embodiment, the probes are used for forensic
"DNA fingerprinting" to match crime-scene DNA against samples taken
from victims and suspects.
[0349] In an additional embodiment, the probes in an array are used
for sequencing by hybridization.
[0350] Thus, the present invention provides for extremely specific
and sensitive probes, which may, in some embodiments, detect target
sequences without removal of unhybridized probe. This will be
useful in the generation of automated gene probe assays.
[0351] Alternatively, the compositions of the invention are useful
to detect successful gene amplification in PCR, thus allowing
successful PCR reactions to be an indication of the presence or
absence of a target sequence. PCR may be used in this manner in
several ways. For example, in one embodiment, the PCR reaction is
done as is known in the art, and then added to a composition of the
invention comprising the target nucleic acid with a ETM, covalently
attached to an electrode via a conductive oligomer with subsequent
detection of the target sequence. Alternatively, PCR is done using
nucleotides labelled with a ETM, either in the presence of, or with
subsequent addition to, an electrode with a conductive oligomer and
a target nucleic acid. Binding of the PCR product containing ETMs
to the electrode composition will allow detection via electron
transfer. Finally, the nucleic acid attached to the electrode via a
conductive polymer may be one PCR primer, with addition of a second
primer labelled with an ETM. Elongation results in double stranded
nucleic acid with a ETM and electrode covalently attached. In this
way, the present invention is used for PCR detection of target
sequences.
[0352] In a preferred embodiment, the arrays are used for mRNA
detection. A preferred embodiment utilizes either capture probes or
capture extender probes that hybridize close to the 3'
polyadenylation tail of the mRNAs. This allows the use of one
species of target binding probe for detection, i.e. the probe
contains a poly-T portion that will bind to the poly-A tail of the
mRNA target. Generally, the probe will contain a second portion,
preferably non-poly-T, that will bind to the detection probe (or
other probe). This allows one target-binding probe to be made, and
thus decreases the amount of different probe synthesis that is
done.
[0353] In a preferred embodiment, the use of restriction enzymes
and ligation methods allows the creation of "universal" arrays. In
this embodiment, monolayers comprising capture probes that comprise
restriction endonuclease ends, as is generally depicted in FIG. 7
of PCT US97/20014. By utilizing complementary portions of nucleic
acid, while leaving "sticky ends", an array comprising any number
of restriction endonuclease sites is made. Treating a target sample
with one or more of these restriction endonucleases allows the
targets to bind to the array. This can be done without knowing the
sequence of the target. The target sequences can be ligated, as
desired, using standard methods such as ligases, and the target
sequence detected, using either standard labels or the methods of
the invention.
[0354] The present invention provides methods which can result in
sensitive detection of nucleic acids. In a preferred embodiment,
less than about 10.times.10.sup.6 molecules are detected, with less
than about 10.times.10.sup.5 being preferred, less than
10.times.10.sup.4 being particularly preferred, less than about
10.times.10.sup.3 being especially preferred, and less than about
10.times.10.sup.2 being most preferred. As will be appreciated by
those in the art, this assumes a 1:1 correlation between target
sequences and reporter molecules; if more than one reporter
molecule (i.e. electron transfer moeity) is used for each target
sequence, the sensitivity will go up.
[0355] All references cited herein are incorporated by reference in
their entirety.
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