U.S. patent application number 10/697908 was filed with the patent office on 2004-07-29 for tissue collection devices containing biosensors.
Invention is credited to Kayyem, Jon Faiz.
Application Number | 20040146899 10/697908 |
Document ID | / |
Family ID | 32737871 |
Filed Date | 2004-07-29 |
United States Patent
Application |
20040146899 |
Kind Code |
A1 |
Kayyem, Jon Faiz |
July 29, 2004 |
Tissue collection devices containing biosensors
Abstract
The invention relates to tissue collection devices such as blood
collection devices that comprise biosensors for the detection of
target analytes such as nucleic acids and proteins, including
antibodies and enzymes.
Inventors: |
Kayyem, Jon Faiz; (Pasadena,
CA) |
Correspondence
Address: |
Robin M. Silva
DORSEY & WHITNEY LLP
Suite 3400
Four Embarcadero Center
San Francisco
CA
94111-4187
US
|
Family ID: |
32737871 |
Appl. No.: |
10/697908 |
Filed: |
October 29, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10697908 |
Oct 29, 2003 |
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09472657 |
Dec 27, 1999 |
|
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60114178 |
Dec 30, 1998 |
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Current U.S.
Class: |
435/6.11 ;
536/24.3 |
Current CPC
Class: |
G01N 33/5438 20130101;
B82Y 15/00 20130101; C12Q 1/001 20130101; B82Y 30/00 20130101 |
Class at
Publication: |
435/006 ;
536/024.3 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
We claim:
1. A method of detecting a target analyte in a sample comprising:
a) applying an initiation signal to a tissue collection device
comprising an electrode comprising: I) a self-assembled monolayer;
II) an assay complex comprising: 1) a capture binding ligand; 2)
said target analyte; and 3) an electron transfer moiety; and b)
detecting electron transfer between said electrode and said
electron transfer moiety.
2. A method according to claim 1 wherein said sample is blood.
3. A method according to claim 1 wherein said self-assembled
monolayer comprises insulators.
4. A method according to claim 1 wherein said self-assembled
monolayer comprises an EFS.
5. A method according to claim 1 wherein said target analyte is
nucleic acid.
6. A method according to claim 1 wherein said capture binding
ligand is a capture probe.
7. A method according to claim 1 wherein said assay complex
comprises a label probe comprising said electron transfer
moiety.
8. A method according to claim 1 wherein said electron transfer
moiety is ferrocene.
Description
[0001] This application is a divisional application of U.S.
application Ser. No. 09/472,657, filed Dec. 27, 1999, which claims
priority to United State Provisional Application No. 60/114,178,
filed Dec. 30, 1998.
FIELD OF THE INVENTION
[0002] The invention relates to tissue collection devices such as
blood collection devices that comprise biosensors for the detection
of target analytes such as nucleic acids and proteins, including
antibodies and enzymes.
BACKGROUND OF THE INVENTION
[0003] The areas of molecular pathology, molecular oncology and
molecular diagnostics are rapidly expanding, with the development
and validation of new molecular techniques of paramount importance
in the clinical setting. With the rapidly accelerating pace of
discovery of new disease genes and a growing awareness of the role
of genetic components of many disease states, the need for rapid,
inexpensive and accurate tests is growing exponentially.
[0004] Generally, these tests can be classified into several
categories, including nucleic acid tests (for pathogens, oncogenes,
genetic diseases, etc.) and immunoassay tests (for a wide variety
of antigens and antibodies). In addition, the growing knowledge
regarding causative mutations in human genes and single nucleotide
polymorphisms (SNPs) leads to the need and desire to test the
population to diagnose and identify those at risk for these genetic
diseases.
[0005] Clinically, these tests require a patient sample, frequently
blood, into a tissue collection device, such as those described in
U.S. Pat. Nos. 4,991,104, 3,974,930, 4,301,936, 4,187,952,
4,186,840, 4,136,794, 4,465,200, 4,111,326, 6,001,087, 6,001,429,
5,975,343 and 5,968,620. The sample is then generally processed,
using a variety of steps, and the assay run.
[0006] Simultaneously, the area of biosensors is rapidly expanding.
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.
[0007] Other assays rely on electronic signals for detection. A
number of systems have been described; see U.S. Pat. Nos.
5,591,578; 5,824,473; 5,770,369; 5,705,348; 5,780,234; and
5,952,172; WO98/20162; WO99/37819; PCT US98/12430, PCT US99/01703;
PCT US98/12082; PCT/US99/14191; PCT/US99/21683; PCT/US99/25464 and
PCT US99/10104; and U.S. Ser. Nos. 09/135,183; 09/295,691 and
09/306,653.
[0008] In general, it would be desirable to provide the ability to
test a tissue sample with a minimum of handling steps. It is an
object of the invention to provide tissue collection devices that
have incorporated biosensors, particularly electrodes, for use in
diagnosis and screening.
SUMMARY OF THE INVENTION
[0009] In accordance with the above objects, the present invention
provides tissue collection devices, particularly blood collection
devices, comprising an electrode. The electrodes may further
comprise self-assembled monolayers and capture binding ligands,
particularly nucleic acid capture probes. The monolayers may
comprise insulators and/or electroconduit-forming species (EFS).
The devices may further comprise at least one reagent, including
anticoagulants, probe nucleic acids, and lysis reagents.
[0010] In a further aspect, the invention provides method of
detecting a target analyte is a sample comprising applying an
initiation signal to a tissue collection device comprising an
electrode. The electrode may comprise a self-assembled monolayer
and an assay complex comprising a capture binding ligand, the
target analyte and an electron transfer moiety. The methods further
comprise detecting electron transfer between the electrode and the
electron transfer moiety. The methods may further comprising
collecting the sample, for example using blood collection
equipment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIGS. 1A, 1B, 1C and 1D depict tissue collection devices
configured with reservoirs. FIG. 1A shows the tube 10 with a cap
20; the cap 20 has a reservoir 30 with reagent(s) 40. FIG. 1B is
similar except that there are two reservoirs 30 and 31 with
reagents 40 and 41. FIG. 1C depicts a similar configuration except
that the reservoir 30 is part of the tube 10 rather than the cap
20. FIG. 1D depicts a configuration that utilizes a membrane 50 at
the end of the tube. It should be noted that none of the systems
depicted in FIG. 1 show the biosensor.
[0012] FIGS. 2A and 2B depict tissue collection devices with
reservoirs for testing of a portion of the sample. FIG. 2A depicts
a device that utilizes a cap 20; as will be appreciated by those in
the art, devices with inlet ports, mermbranes, and other
configurations may be used as well. The reservoir 30 (with optional
reagents 40) is separated from the sample area 70 by a valve or
seal 60. FIG. 2B depicts a similar system except that the reservoir
30 can be detached from the sample chamber 70, using an detachable
union 65. It should be noted that none of the systems depicted in
FIG. 2 show the biosensor.
[0013] FIGS. 3A, 3B and 3C depict some of the possible
configurations of the biosensor and external leads for the
compositions of the invention. FIG. 3A depicts the electrode of the
biosensor 80 in the cap 20 with interconnects 90. FIGS. 3B and 3C
depict the electrode of the biosensor 80 as a component of the tube
10. FIG. 3B depicts a single biosensor; FIG. 3C depicts multiple
biosensors 80. FIG. 3C depicts one set of external leads for all
the electrodes, although as will be appreciated by those in the
art, multiple leads may be used as well. In addition, there may be
physical barriers between compartments of the tube, each with a
biosensor. FIG. 3D depicts a biosensor 80 in a reservoir 30 of a
device 10.
[0014] FIGS. 4A, 4B and 4C 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. 4A 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. As for all the figures and configurations outlined
herein, the capture probes may be the same, or the capture probes
at any particular pad may be different, comprising sequences that
hybridize to different parts of the target. 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. 4B 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. 4C 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.
[0015] FIGS. 5A, 5B, 5C, 5D and 5E 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. 5A-C, the
recruitnient linker is nucleic acid; in FIGS. 5D and E, is 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.
[0016] FIGS. 6A, 6B, 6C, 6D, 6E, 6F and 6G depict some of the
embodiments of the invention. All of the monolayers depicted herein
show the presence of both conductive oligomers 108 and insulators
107 in roughly a 1:1 ratio, although as discussed herein, a variety
of different ratios may be used, or either species may be
completely absent, or other monolayer-forming species may be used.
In addition, as will be appreciated by those in the art, any one of
these structures may be repeated for a particular target sequence;
that is, for long target sequences, there may be multiple assay
complexes formed. Additionally, any of the electrode-attachment
embodiments of FIG. 4 may be used in any of these systems.
[0017] FIGS. 6A, 6B and 6D have the target sequence 120 containing
the ETMs 135; as discussed herein, these may be added
enzymatically, for example during a PCR reaction using nucleotides
modified with ETMs, resulting in essentially random incorporation
throughout the target sequence, or added to the terminus of the
target sequence. FIG. 6D depicts the use of two different capture
probes 100 and 100', that hybridize to different portions of the
target sequence 120. As will be appreciated by those in the art,
the 5'-3' orientation of the two capture probes in this embodiment
is different.
[0018] FIG. 6C depicts the use of label probes 145 that hybridize
directly to the target sequence 120. FIG. 6C shows the use of a
label probe 145, comprising a first portion 141 that hybridizes to
a portion of the target sequence 120, a second portion 142
comprising ETMs 135. Although not depicted, a particularly
preferred embodiment for use in Ussue collection devices is the use
of direct capture of the target sequence to the capture probe, with
at least one label probe hybridizing to a different portion of the
target.
[0019] FIGS. 6E, 6F and 6G depict systems utilizing label probes
145 that do-not hybridize directly to the target, but rather to
amplifier probes that are directly (FIG. 6E) or indirectly (FIGS.
6F and 6G) hybridized to the target sequence. FIG. 6E utilizes an
amplifier probe 150 has a first portion 151 that hybridizes to the
target sequence 120 and at least one second portion 152, i.e. the
amplifier sequence, that hybridizes to the first portion 141 of the
label probe. FIG. 6F is similar, except that a first label extender
probe 160 is used, comprising a first portion 161 that hybridizes
to the target sequence 120 and a second portion 162 that hybridizes
to a first portion 151 of amplifier probe 150. A second portion 152
of the amplifier probe 150 hybridizes to a first portion 141 of the
label probe 140, which also comprises a recruitment linker 142
comprising ETMs 135. FIG. 6G adds a second label extender probe
170, with a first portion 171 that hybridizes to a portion of the
target sequence 120 and a second portion that hybridizes to a
portion of the amplifier probe.
[0020] FIG. 6H depicts a system that utilizes multiple label
probes. The first portion 141 of the label probe 140 can hybridize
to all or part of the recruitment linker 142.
[0021] FIGS. 7A, 7B, 7C, 7D, 7E and 7F depict some "mechanism-1"
detection systems. FIG. 7A shows portions of target sequence 120
hybridized to detection probes 300 linked via conductive oligomers
108 to electrode 105. The hybridization complex comprises an ETM
135 that can be covalently linked either to the target sequence or
the detection probe 300, or non-covalently (i.e. a hybridization
indicator). The monolayer is depicted with insulators 107, although
as outlined herein, any type of passivation agent may be used. FIG.
7B depicts the use of capture probe 100 linked via attachment
linker 106 to electrode 105, although as will be appreciated, since
the label probe 145 hybridizes to the detection probe 300 this can
serve as a type of capture probe. A label probe 145 comprising a
first portion 141 that hybridizes to a portion of the target
sequence 120 and a second portion 142 that hybridizes to the
detection probe 300. FIG. 7C depicts the use of branched amplifier
probe 150 comprising a first portion 151 that hybridizes to the
target sequence 120 (although label extender probes can be used as
well) and amplification sequences 152 that hybridize directly to
the detection probes 300. The target sequence may be additionally
attached to the electrode using capture probes as outlined in FIG.
4. FIG. 7D depicts the same thing utilizing a linear amplification
probe 150. FIG. 7E depicts a similar system, but uses label probes
145 comprising a first portion 141 that hybridizes to the
amplification sequence 152 and a second portion 142 that hybridizes
to a detection probe 300. FIG. 7F depicts the same thing but using
a linear amplification probe 150.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The present invention is directed to the use of biosensors
in a tissue collection device. In general, the ability to collect
the tissue sample, such as a blood sample, in a container that
comprises a biosensor (and optionally assay reagents) allows a
one-step molecular diagnositic assay. This may be used in a variety
of ways. For example, in one embodiment, the donation of blood into
a container that has a biosensor allows the rapid and inexpensive
testing of blood for contaminants, including pathogens such as
viruses (hepatitis strains, HIV, etc.) or bacteria with a minimum
of blood handling. Alternatively, tissues collected specifically
for the purpose of testing, such as blood or vaginal samples for
sexually transmitted diseases, can also be tested rapidly with a
minimum of handling.
[0023] Accordingly, the present invention provides tissue
collection devices. By "tissue" or grammatical equivalents herein
is meant any tissue for which a bioassay can be done, including,
but not limited to, blood, lymph, saliva, vaginal and anal
secretions, urine, feces, perspiration, tears, and other bodily
fluids. By "tissue collection device" herein is meant any
container, generally capable of being sealed, that can be used to
collect, contain or store tissues, including bodily fluids. Tissue
collection devices are generally sterile. Specifically included
herein are blood collection devices, such as are well known in the
art, including blood collection tubes and bags, including
VACUTAINER.RTM. brand evacuated tubes. These may be formatted in a
variety of configurations; see for example U.S. Pat. Nos.
4,991,104, 3,974,930, 4,301,936, 4,187,952, 4,186,840, 4,136,794,
4,465,200, 4,111,326, 6,001,087, 6,001,429, 5,975,343 and
5,968,620, all of which are incorporated herein by reference in
their entirety.
[0024] As will be appreciated by those in the art and outlined
herein, the tissue collection devices of the invention can be
configured in a variety of ways. In one embodiment, the device has
a single compartment for receiving sample, that may include
optional reagents prior to collection. Alternatively, the tissue
collection device is configured to have more than one compartment.
This may be done in a variety of ways, depending on the use of the
device. For example, when the entire sample is to be used for
assay, the compartments may be used for storage of assay reagents.
Alternatively, when only a portion of the collected sample is to be
assayed, the compartments may be used to segregate the portion for
subsequent assay, or both.
[0025] In a preferred embodiment, the entire sample is used in the
assay. In this embodiment, it may be desirable to have one or more
storage compartments for reagents. For example, U.S. Pat. No.
6,001,087 describes a collection assembly wherein the cap of the
device comprises a reservoir containing reagents as defined below.
The insertion of a needle, such as is done during blood drawing,
results in the rupture of the reservoir and the addition of the
reagents to the sample. This is generally shown in FIGS. 1A and 1B,
showing one and multiple compartments for rupture in this manner.
For example, different membranes may be used, such that a first
centrifugation at a first speed ruptures the first membrane,
causing the first step of an assay; a second centrifugation at a
higher speed will rupture the second membrane, resulting in the
second step of the assay; etc. In addition, the use of different
density materials can generate liquid "compartments" or "phases"
that can be used to separate different components of the assay.
Similarly, the reservoir may be part of the tube, rather than the
cap, as is generally depicted in FIG. 1C. Alternatively, the
reservoir may be at the end of the tube, and utilize a membrane
that can rupture upon centrifugation, as is shown in FIG. 1D.
Alternatively, a gel barrier may be used, as is used in
VACUTAINER.RTM. PPT.TM. systems.
[0026] In a preferred embodiment, only a portion of the collected
sample is used in the assay. In this embodiment, the device can
have a compartment to allow the testing of only a portion of the
sample. For example, as shown in FIG. 2A, there may be a separate
reservoir, optionally comprising reagents, with a seal or valve,
particularly one way valves such as check valves, allowing a
portion of the sample access to the reservoir. This separate
reservoir may be detachable from the sample compartment as well, to
allow for a single collection from a patient, followed by
segregation of the portion to be tested separately, as is depicted
schematically in FIG. 2B.
[0027] In a preferred embodiment, the tissue collection tube
comprises reagents. These include reagents used in tissue handling,
such as anticoagulants for blood, as well as a variety of other
reagents including salts, buffers, neutral proteins, e.g. albumin,
detergents, etc which may be used to facilitate optimal binding of
the target analyte to the sensor composition 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. For example, there may be reagents and other components
used to accelerate the binding of the target analytes to the
biosensors, such as are generally described in PCT US99/14191,
hereby incorporated by reference in its entirety.
[0028] In addition, other reagents comprising components of the
bioassay, including for example amplification probes, label probes,
capture extender probes, etc., as is generally outlined in U.S.
Pat. Nos. 5,591,578; 5,824,473; 5,770,369; 5,705,348; 5,780,234;
and 5,952,172; WO98/20162; WO99/37819; PCT US98/12430, PCT
US99/01703; PCT US98/12082; PCT/US99/14191; PCT/US99/21683;
PCT/US99/25464; and PCT US99/10104; and U.S. Ser. Nos. 09/135,183;
09/295,691 and 09/306,653, can be placed in the tube prior to,
after or during the introduction of the sample tissue.
[0029] In a preferred embodiment, the reagent is a cell lysis agent
for the lysis of cells (including patient cells, bacteria, etc.) or
other pathogens such as virus. Suitable lysis buffers include, but
are not limited to, guanidium chloride, chaotropic salts, enzymes
such as lysozymes, etc. In some embodiments, for example for blood
cells, a simple dilution with water or buffer can result in
hypotonic lysis. The lysis agent may be in solution form or solid
form that is taken up in solution upon introduction of the
sample.
[0030] In a preferred embodiment, one or more reservoirs of the
tissue collection device comprises the reagents.
[0031] The tissue collection device further comprises a biosensor
as outlined herein. As is more fully outlined below, the biosensor
may comprise a single substrate with a single electrode, multiple
substrates each with one or more electrodes, or a single substrate
with a plurality of electrodes in an array format.
[0032] As will be appreciated by those in the art, the placement of
the biosensor in the device can be done in a variety of ways, as is
generally depicted in FIG. 3. In a preferred embodiment, the
biosensor is in the cap or stopper of the device as shown in FIG.
3A. In this embodiment, there are leads or interconnects on the
outside of the cap, with the biosensor contacting the sample on the
inside of the device. As in all the different configurations, what
is important is that the biosensor have some contact with some or
all of the sample to be assayed.
[0033] In a preferred embodiment, the placement of the biosensor(s)
is on a surface internal to the sample compartment, as is generally
depicted in FIGS. 3B and 3C. In this embodiment, the external leads
may be on the sample compartment or on the cap. As will be
appreciated by those in the art, if the biosensor is in the cap,
the device may need to be inverted for assay. In this embodiment,
different biosensors may be located in different sections of the
device. For example, detection of genomic sequences may be done in
one section of the tube, for example near the end, where the
heavier blood cells are after centrifugation, and detection of
pathogenic (particularly viral) sequences may be done in a
different section, for example where the plasma resides after
centrifugation.
[0034] Alternatively, different biosensors may be located in
different physical compartments of the device; for example, the use
of gel barriers or other membranes may allow certain cell types to
be compartmentalized in the sample surface, thus reducing the
complexity of the sample in any one compartment.
[0035] The present invention provides tissue collection devices
comprising biosensors. By "biosensors" herein is meant a sensor
capable of detecting the presence of a target analyte in a sample.
Biosensors in this context includes electrochemical sensors,
optical fiber sensors, evanescent wave sensors, surface plasmon
resonance sensors and acoustic wave sensors.
[0036] Thus, 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.
[0037] By "proteins" or grammatical equivalents herein is meant
proteins, oligopeptides and peptides, and analogs, including
proteins containing non-naturally occuring amino acids and amino
acid analogs, and peptidomimetic structures.
[0038] 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.
[0039] 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. Left. 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. 11 1:2321 (1989), O-methylphophoroamidite
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. Left. 4:395 (1994); Jeffs et al.,
J. Biomolecular NMR 34:17 (1994); Tetrahedron Left. 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)
pp169-176). 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.
[0040] 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 surface species or ETM
attachment, an analog structure may be used. Alternatively,
mixtures of different nucleic acid analogs, and mixtures of
naturally occuring nucleic acids and analogs may be made.
[0041] 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 C
drop in Tm for an internal mismatch. With the non-ionic PNA
backbone, the drop is closer to 7-9 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).
[0042] 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
occuring analog structures. Thus for example the individual units
of a peptide nucleic acid, each containing a base, are referred to
herein as a nucleoside.
[0043] 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.
[0044] 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.
[0045] In addition, in a preferred embodiment, the compositions of
the invention comprising biosensors with assay complexes in tissue
collection devices can be used in a number of standard diagnostic
tests. See for example the Reference Guide for Diagnostic Molecular
Pathology/Flow Cytometry: Fascicle VII, 1996 College of American
Pathologists, ISBN 0-930304-63-2, hereby incorporated by reference
in its entirety. These assays include, but are not limited to,
detection of: Adenosine Deaminase Gene; Adenovirus;
Adrenoleukodystrophy Gene; Adult Polycystic Kidney Disease;
Aeromonas spp.; Pleisiomonas spp.; Alpha.sub.1-Antitrypsin
Deficiency; .alpha.-Thalassemia Detection; Alzheimer's Disease;
Amebae; Androgen Receptor Gene; Antimicrobial Resistance
Determination; Arylsulfatase A Gene; Aspartoacylase Gene;
.beta.-thalassemia; Babesia microti; Bartonella; Basal Cell
Carcinoma; bcl-2 Gene Rearrangements; bcr Gene Rearrangements;
Bordetella spp.; Borrelia burgdorferi; B/T Cell Gene
Rearrangements; c-myc; c-src Proto-Oncogene; Campylobacter spp.;
Arcobacter spp.; Helicobacter spp.; Candida albicans; Carbonic
Anhydrase II Gene; Carcinoembryonic Antigen; Charcot-Marie-Tooth
Neuropathies; Chlamydia trachomatis; Clostridium difficile;
Colorectal Cancer Genetic Profile; Copper-Transporting P-Type
ATPase Genes; Cystic Fibrosis Gene; Cytomegalovirus; DNA Repair
Disorders; DNA Storage Bank; Down Syndrome Gene; Dystrophin Gene;
Ehrlichia; Enterococcus spp.; Enterovirulent Escherschia coli;
Enteroviruses; Epstein-Barr Virus; Factor V.sub.leiden; Factor
VIIIc Gene; Factor IX Gene; Familial Breast Cancer; Familial
Hypertrophic Cardiomyopathy Genes; Family Filarioidea;
Ferrochelatase Gene; Fibrillin Gene; Forensic Testing; Fragile X
Syndrome; G-protein Genes; Gardnerella vaginalis; Gaucher's
Disease; Giardia spp.; Glucokinase Gene; Glucose Transporter Gene;
Haemophilus spp.; Hepatitis A Virus; Hepatitis B Virus; Hepatitis C
Virus; Hepatitis delta Virus; Hepatitis E Virus; HER-2/neu Gene
Amplification; Hereditary Amyloidosis; Herpes Simplex Virus;
Hexosaminidase Alpha Subunit Gene; Histoplasma capsulatum; HIV I;
HLA Testing; Human Herpesvirus-6 Types A and B; Human
Papillomavirus; Huntington's Disease Genes; Hurler/Scheie Syndrome
Gene; Insulin Gene; Insulin Receptor Gene; Laminin/Laminin
Receptor; Leber's Hereditary Optic Nueropathy Genes; Legionella
pneumophila; Leishmania spp.; Leukemia/Lymphoma Molecular
Prognostic Markers; Low Density Lipoprotein Receptor and Ligands
Apolipoproteins B and E; Luteinizing Hormone Gene; Medium-Chain
Acyl-Coenzyme A Dehydrogenase Gene; Minimal Residual Disease
Detection; Mitrochondrial ATPase 6 Gene; Mitochondrial DNA;
Mitrochondrial DNA Deletions; Mitrochondrial DNA Depletions;
Mitochondrial tRNA Genes; Molecular Epidemiology of Nosocomial
Infections; Multi-Drug Resistance Genes; Multiple Endocrine
Neoplasia Type 1; Multiple Endocrine Neoplasia Type 2A and 2B;
Mycobacterium leprae; Mycobacterium spp.; Mycoplasma spp; Myotonic
Dystrophy Gene; Neisseria gonorrhoeae; N-myc Gene Amplification;
Omithine Transcarbamylase Gene; p53 Tumor-Suppressor Gene;
Parvovirus B 19; Paternity Testing; Plasmodium spp; Porphobilinogen
Deaminase Gene; Porphobilinogen Synthase Gene; Post-Transfusion
Graft Versus Host Disease; Protein C Gene; Pyruvate D hydrogenase
Gene; Rabies Virus; ras Family of Oncogenes; Respiratory Syncytial
Virus; Retinoblastoma Gene; Retinoic Acid Receptor Gene; RhD
Genotyping; Rotavirus; Rubella Virus; Schistosoma spp; Sickle Cell
Disease; Thyrotropin Receptor Gene; Toxoplasma gondii; Treponema
pallidum; Trichomonadidae, Family; Trypanosoma spp; Tumor
Metastasis Suppressor Genel--NM23; Uroporphyrinogen III Synthase
Gene; Varicella-Zoster Virus; von Willebrand's Disease and Wilms'
Tumor.
[0046] 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.
[0047] The present invention is directed to the detection of target
analytes, preferably using electrodes, in tissue collection
devices. In general, there are two basic detection mechanisms. In a
preferred embodiment, detection of an ETM is based on electron
transfer through the stacked .pi.-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, and PCT US97/20014 and is termed
"mechanism-1" herein. Briefly, previous work has shown that
electron transfer can proceed rapidly through the stacked
.pi.-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.
[0048] This may be done where the target analyte is a nucleic acid;
alternatively, a non-nucleic acid target analyte is used, with an
optional capture binding ligand (to attach the target analyte to
the detection electrode) and a soluble binding ligand that carries
a nucleic acid "tail", that can then bind either directly or
indirectly to a detection probe on the surface to effect
detection.
[0049] Alternatively, the ETM can be detected, not necessarily via
electron transfer through nucleic acid, but rather can be directly
detected on the 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.
[0050] 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 soluble 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.
[0051] 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 exogeneous 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.
[0052] 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 penentrate 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.
[0053] 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. Mechanism-2
system monolayers comprise a first species comprising an attachment
linker comprising the capture binding ligand, a second species
comprising an electroconduit-forming species (EFS), and an optional
third species comprising insulators. In addition, for "rough"
mechanism-2 systems, two component systems are desirable: a first
species comprising an attachment linker comprising the capture
binding ligand and a second species comprising insulators.
[0054] The biosensors of the invention preferably comprise
electrodes. By "electrode" herein is meant a composition, which,
when connected to an electronic device, is able to sense a current
or a potential 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 below.
Preferred electodes 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,
platinum, carbon and metal oxide electrodes, with gold being
particularly preferred.
[0055] 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 bound to the inner surface.
Electrode coils or mesh may be preferred in some embodiments as
well. This allows a maximum of surface area containing the target
analyte to be exposed to a small volume of sample.
[0056] 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 van ety 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.
[0057] In general, preferred substrate 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.
[0058] Accordingly, in a preferred embodiment, the present
invention provides biosensors or biochips (sometimes referred to
herein "chips") that comprise substrates comprising a plurality of
electrodes, preferably gold electrodes. The number of electrodes is
as outined below 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.
[0059] The substrates are generally a component of the tissue
collection device, and can be included as part of a cap or tube, or
as part of a reservoir that exposes a given volume of sample to the
detection electrode. Generally, the detection chamber ranges from
about 1 nL to 10 ml. As will be appreciated by those in the art,
depending on the experimental conditions and assay, smaller or
larger volumes may be used.
[0060] In a preferred embodiment, all or part of the tissue
collection device is configured as a "cartridge" that is 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] The methods continue with the addition of SAMs. 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.
[0065] The present system finds particular utility in array
formats, i.e. wherein there is a matrix of addressable detection
electrodes (herein generally referred to "pads", "addresses" or
"micro-locations"). That is, the biosensors can be made in an array
format. By "array" herein is meant a plurality of micro-locationsi
in an array format; the size of the array will depend on the
composition and end use of the array. Arrays containing from about
2 different capture ligands to many thousands can be made.
Generally, the array will comprise from two to as many as 100,000
or more, depending on the size of the electrodes, as well as the
end use of the array. Preferred ranges are from about 2 to about
10,000, with from about 5 to about 1000 being preferred, and from
about 10 to about 100 being particularly preferred. In some
embodiments, the compositions of the invention may not be in array
format; that is, for some embodiments, compositions comprising a
single capture ligand may be made as well. In addition, in some
arrays, multiple substrates may be used, either of different or
identical compositions. Thus for example, large arrays may comprise
a plurality of smaller substrates, or different substrates may be
positioned in different locations within the tissue collection
device.
[0066] Thus, in a preferred embodiment, the tissue collection
devices of the invention comprise electrodes.
[0067] In a preferred embodiment, the electrodes of the biosensors
comprise a self-assembled monolayer (SAM). By "monolayer" or
"self-assembled monolayer" or "SAM" herein is meant a relatively
ordered assembly of molecules spontaneously chemisorbed on a
surface, in which the molecules have a preferred orientation
relative to each other (e.g. are oriented approximately parallel to
each other) and a preferred orientation relative to the surface
(e.g. roughly perpendicular to it). 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 a variety of
species including, but not limited to, EFS such as conductive
oligomers, insulators, attachment linkers, etc. (collectively
"surface species" or "monolayer-forming species"). In general, all
of the attachment chemistries outlined herein can be used for any
surface species, even if specifically depicted for conductive
oligomers or other species. 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 extraneous electroactive 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 extraneous electroactive 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, in one embodiment, the monolayer is
preferably tightly packed in a uniform layer on the electrode
surface, such that a minimum of "holes" exist. In this embodiment,
the monolayer thus serves as a physical barrier to block solvent
accesibility to the electrode.
[0068] 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,
photoactivauble species that can be selectively removed from the
surface upon photoactivation, leaving electroconduits, as is
generally described in U.S. Ser. No. 60/145,912, hereby expressly
incorporated herein by reference in its entirety.
[0069] 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.
[0070] In a preferred embodiment, the EFS comprise 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 transfering electrons at 100 Hz. Generally, the
conductive oligomer has substantially overlapping .pi.-orbitals,
i.e. conjugated .pi.-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.
[0071] 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.3 .OMEGA..sup.-1cm.sup.-1 being
preferred. See generally Gardner et al., Sensors and Actuators A 51
(1995) 57-66, incorporated herein by reference.
[0072] 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 binding ligand synthesis (i.e. nucleic acid synthesis, such
that nucleosides containing the conductive oligomers may be added
to a nucleic acid synthesizer during the synthesis of the
compositions of the invention, ii) during the attachment of the
conductive oligomer to an electrode, or iii) during binding assays.
In addition, conductive oligomers that will promote the formation
of self-assembled monolayers are preferred.
[0073] 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.
[0074] In a preferred embodiment, the conductive oligomer has the
structure depicted in Structure 1: 1
[0075] 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 binding ligands such as nucleic acids, 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 binding ligand, the right "Y", if
present, is attached to the binding ligand such as a nucleic acid,
either directly or through the us of a linker, as is described
herein.
[0076] 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
Aconjugated 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] By "amino groups" or grammatical equivalents herein is meant
--NH.sub.2, --NHR and --NR.sub.2 groups, with R being as defined
herein.
[0084] By "nitro group" herein is meant an --NO.sub.2 group.
[0085] 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.
[0086] 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.
[0087] By "ester" herein is meant a --COOR group.
[0088] 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.
[0089] By "aldehyde" herein is meant --RCHO groups.
[0090] By "alcohol" herein is meant --OH groups, and alkyl alcohols
--ROH.
[0091] By "amido" herein is meant --RCONH-- or RCONR-- groups.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] 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 .pi.-orbitals.
[0096] Preferred B-D bonds are selected from acetylene
(--C.ident.C--, also called alkyne or ethyne), 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.
[0097] 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.
[0098] 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.
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] Particularly preferred conductive oligomers of this
embodiment are depicted below: 2
[0104] 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: 3
[0105] 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.
[0106] 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.
[0107] Particularly preferred embodiments of Structure 3 include
Structures 4, 5, 6 and 7, depicted below: 4
[0108] 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. 5
[0109] 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 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. 6
[0110] 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. 7
[0111] 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.
[0112] In a preferred embodiment, the conductive oligomer has the
structure depicted in Structure 8: 8
[0113] 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.ident.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.
[0114] 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.
[0115] 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: 9
[0116] 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.
[0117] In a preferred embodiment, R is hydrogen, although R may be
also alkyl groups and polyethylene glycols or derivatives.
[0118] In an alternative embodiment, the conductive oligomer may be
a mixture of different types of oligomers, for example of
structures 1 and 8.
[0119] 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, when the target analyte is a nucleic acid, 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.
[0120] 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.
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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).
[0125] 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.
[0126] The length of the species making up the monolayer will vary
as needed. As outlined above, it appears that binding of target
analytes (for example, hybridization of nucleic acids) is more
efficient at a distance from the surface. The species to which
capture binding ligands 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 capture binding ligands being more accessible to
the solvent for hybridization. In some embodiments, the conductive
oligomers to which the capture binding ligands are attached may be
shorter than the monolayer.
[0127] It will be appreciated that the monolayer may comprise
different surface species, in a variety of configurations, although
preferably the different species are chosen such that a reasonably
uniform SAM can be formed. Thus, for example, when capture binding
ligands such as 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 in the SAM. Similarly, it may be desirable to have
mixtures of different lengths of surface species in the monolayer,
to help reduce non-specific signals. Thus, for example, preferred
embodiments utilize surface species such as EFS including
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 species such as conductive oligomers may be done to
facilitate monolayer formation, or to make monolayers with altered
properties.
[0128] 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, and whether rough or smooth surfaces are used.
Generally, three component systems are preferred for mechanism-2
systems, with the first species comprising a capture binding ligand
containing species (termed a capture probe when the target analyte
is a nucleic acid), attached to the electrode via an attachment
linker (e.g. an insulator or conductive oligomer). The second
species are EFS, 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. When the
target analytes are 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.
[0129] 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
or "rough" mechanism-2 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.
[0130] 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.
[0131] The covalent attachment of the monolayer-forming species and
the capture binding ligand species to the electrode may be
accomplished in a variety of ways, depending on the electrode and
the composition of the moieties. In a preferred embodiment, the
attachment linkers with covalently attached 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 EFS (e.g. conductive oligomer), "I" is an insulator and the
hatched surface is the electrode: 10
[0132] 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.
[0133] Although depicted herein as a single moiety, these moieties
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 species to
the electrode, such as is generally depicted below in Structures
11, 12 and 13 for conductive oligomers. 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. 11
[0134] It should also be noted that similar to Structure 13, it may
be possible to have a species terminating in a single carbon atom
with three sulfur moities attached to the electrode. Additionally,
although not always depicted herein, the species herein may also
comprise a "Q" terminal group.
[0135] 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 surface species may be used.
Similarly, any of the surface species 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. 12
[0136] In general, thiol linkages are preferred.
[0137] 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. 13
[0138] 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 chemisorpton of phosphates to an
indium tin oxide electrode (talk by H. Holden Thorpe, CHI
conference, May 4-5, 1998).
[0139] 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.
[0140] 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.
[0141] 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 target analytes, particularly DNA, are
usually prepared using a two step procedure. The thiolated DNA 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 DNA is added. The second step
frequently involves mild heating to promote monolayer
reorganization.
[0142] 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 DNA 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, dimethylforamide
(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 DNA 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.
[0143] In a preferred embodiment, an aqueous deposition solution is
used. As above, a clean gold surface is placed into a clean vial. A
DNA 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 DNA and often a thiol diluent
molecule. The ratio of DNA 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 DNA 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.
[0144] 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.
[0145] In a preferred embodiment, the detection electrode further
comprises a capture binding ligand, preferably covalently attached.
By "binding ligand" or "binding species" herein is meant a compound
that is used to probe for the presence of the target analyte, that
will bind to the target analyte. In general, for most of the
embodiments described herein, there are at least two binding
ligands used per target analyte molecule; a "capture" or "anchor"
binding ligand (also referred to herein as a "capture probe",
particularly in reference to a nucleic acid binding ligand) that is
attached to the detection electrode as described herein, and a
soluble binding ligand (also referred to herein as a "label probe"
or "signalling probe", when nucleic acids are used), that binds
independently to the target analyte, and either directly or
indirectly comprises at least one ETM. However, as described
herein, in some cases the target analyte may comprise the ETMs and
thus only one capture binding ligand is used.
[0146] Generally, the capture binding ligand allows the attachment
of a target analyte to the biosensor electrode, for the purposes of
detection. As is more fully outlined below, attachment of the
target analyte to the capture binding ligand may be direct (i.e.
the target analyte binds to the capture binding ligand) or indirect
(one or more capture extender ligands may be used).
[0147] 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 that 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. The binding
should be sufficient to allow the analyte 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 to
10.sup.-6 M.sup.-1, with at least about 10.sup.-5 to 10.sup.-9
being preferred and at least about 10.sup.-7 to 10.sup.-9 M.sup.-1
being particularly preferred.
[0148] 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 is
generally a substantially complementary nucleic acid.
Alternatively, as is generally described in U.S. Pat. Nos.
5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867,
5,705,337, and related patents, hereby incorporated by reference,
nucleic acid "aptomers" can be developed for binding to virtually
any target analyte. Similarly the analyte may be a nucleic acid
binding protein and the capture binding ligand is either a
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
(particularly including antibodies or fragments thereof (FAbs,
etc.)), small molecules, or aptamers, described above. Preferred
binding ligand proteins include peptides. For example, when the
analyte is an enzyme, suitable binding ligands include substrates,
inhibitors, and other proteins that bind the enzyme, i.e.
components of a multi-enzyme (or protein) complex. As will be
appreciated by those in the art, any two molecules that will
associate, preferably specifically, may be used, either as the
analyte or the binding ligand. Suitable analyte/binding ligand
pairs include, but are not limited to, antibodies/antigens,
receptors/ligand, proteins/nucleic acids; nucleic acids/nucleic
acids, enzymes/substrates and/or inhibitors, carbohydrates
(including glycoproteins and glycolipids)/lectins, carbohydrates
and other binding partners, proteins/proteins; and protein/small
molecules. 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 GLUT4 receptor), transferrin
receptor, epidermal growth factor receptor, low density lipoprotein
receptor, high density lipoprotein 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,
VEGF receptor, PDGF receptor, EPO receptor, TPO receptor, ciliary
neurotrophic factor receptor, prolactin receptor, and T-cell
receptors. Similarly, there is a wide body of literature relating
to the development of binding partners based on combinatorial
chemistry methods.
[0149] In this embodiment, when the binding ligand is a nucleic
acid, preferred compositions and techniques are outlined in U.S.
Pat. Nos. 5,591,578; 5,824,473; 5,770,369; 5,705,348; 5,780,234;
and 5,952,172; WO98/20162; WO99/37819; PCT US98/12430, PCT
US99/01703; PCT US98/12082; PCT/US99/14191; PCT/US99/21683;
PCT/US99/25464; PCT US99/10104; and U.S. Ser. Nos. 09/135,183;
09/295,691 and 09/306,653, all of which are hereby expressly
incorporated by reference.
[0150] The method of attachment of the capture binding ligands to
the attachment linker (either an insulator or conductive oligomer)
will generally be done as is known in the art, and will depend on
both 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 indirectly through the use of a
linker, sometimes depicted herein as "Z". Linkers are well 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.
[0151] In this way, capture binding ligands comprising proteins,
lectins, nucleic acids, small organic molecules, carbohydrates,
etc. can be added.
[0152] 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 to an
attachment linker. A wide variety of techniques are known to add
moieties to proteins.
[0153] A preferred embodiment utilizes nucleic acids as the capture
binding ligand. While most of the following discussion focuses on
nucleic acids, as will be appreciated by those in the art, many of
the techniques outlined below apply in a similar manner to
non-nucleic acid systems as well.
[0154] The capture probe nucleic acid is covalently attached to the
electrode, via an "attachment linker", that can be any surface
species, including either a conductive oligomer (required for
mechanism-1 systems) or an insulator. By "covalently attached"
herein is meant that two moieties are attached by at least one
bond, including sigma bonds, pi bonds and coordination bonds.
[0155] Thus, one end of the attachment linker is attached to a
nucleic acid (or other binding ligand), and the other end (although
as will be appreciated by those in the art, it need not be the
exact terminus for either) is attached to the electrode. 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:
14
[0156] 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; again, other surface species can be used. 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 surface species 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".
[0157] In a preferred embodiment, for example for "mechanism-1"
systems, 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 occuring
nucleic acids, although as will be appreciated by those in the art,
similar techniques may be used with nucleic acid analogs, and in
some cases with other binding ligands.
[0158] In a preferred embodiment, the attachment linkers are
attached to the base of a nucleoside of the nucleic acid. This may
be done in several ways, depending on the attachment linker, as is
described below. In one embodiment, the attachment linker is
attached to a terminal nucleoside, i.e. either the 3' or 5'
nucleoside of the nucleic acid. Alternatively, the attachment
linker is attached to an internal nucleoside.
[0159] 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 (i.e. mechanism-1 systems), 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.
[0160] In one embodiment, the attachment is direct; that is, there
are no intervening atoms between the attachment linker and the
base. In this embodiment, for example, attachment linkers that are
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 or attachment linkers
can be used as will be appreciated by those in the art: 15
[0161] 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.
[0162] In addition, the base may contain additional modifications
as needed, i.e. the carbonyl or amine groups may be altered or
protected.
[0163] 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: 16
[0164] 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.
[0165] In a preferred embodiment, the attachment of the nucleic
acid and the attachment linker 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.
[0166] 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.
[0167] In a preferred embodiment, the attachment linker 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. Orrg.
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 attachment linkers.
[0168] A preferred embodiment utilizes amino-modified nucleosides.
These amino-modified riboses can then be used to form either amide
or amine linkages to the attachment linkers. 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.
[0169] 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): 17
[0170] As will be appreciated by those in the art, Structure 20 has
the terminal bond fixed as an amide bond.
[0171] 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,
athough other heteroatoms can be used: 18
[0172] 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. 19
[0173] In Structure 22, Z is as defined above. Suitable linkers
include methylene and ethylene.
[0174] In an alternative embodiment, the attachment linker 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 and
other attachment linkers 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. 20
[0175] 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.
[0176] 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. 21
[0177] In a preferred embodiment, the attachment linker is
covalently attached to the nucleic acid via a transition metal
ligand. In this embodiment, the attachment linker 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 attachment linker is attached also has the nucleic acid
attached, as is generally depicted below in Structure 25.
Alternatively, the attachment linker is attached to one ligand, and
the nucleic acid is attached to another ligand, as is generally
depicted below in Structure 26 using conductive oligomers as the
attachment linker. Thus, in the presence of the transition metal,
the attachment linker 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: 22
[0178] 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), platinium
(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, platinium, cobalt and
iron.
[0179] 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.
[0180] 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).
[0181] 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 (a) donors) and
organometallic ligands such as metallocene ligands (generally
referred to in the literature as pi (n) 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-azacyclote- tradecane (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 (pp73-98), 21.1
(pp.813-898) and 21.3 (pp 915-957), all of which are hereby
expressly incorporated by reference.
[0182] 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.
[0183] The oxygen, sulfur, phosphorus and nitrogen-donating ligands
are attached in such a manner as to allow the heteroatoms to serve
as coordination atoms.
[0184] 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 conjuction 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.
[0185] 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.
[0186] 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). 23
[0187] In a preferred embodiment, the ligands used in the invention
show altered fluoroscent 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.
[0188] In addition, similar methods can be used to attach proteins
to the biosensor electrode; see for example U.S. Pat. No.
5,620,850, hereby incorporated by reference.
[0189] 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.
[0190] 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 attachment linker, 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.
[0191] In a preferred embodiment, the capture probe nucleic acids
(or other binding ligands) are covalently attached to the electrode
via an insulator. The attachment of nucleic acids (and other
binding ligands) 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.
[0192] 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 (particular in the case of nucleic
acid analytes and binding ligands in mechanism-2 systems) 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.
[0193] Thus the present invention provides tissue collection
devices comprising biosensors with substrates comprising at least
one electrode comprising monolayers and capture binding ligands,
useful in target analyte detection systems of collected
samples.
[0194] In a preferred embodiment, the compositions further comprise
a solution or soluble binding ligand, although as is more fully
described below, for mechanism-1 systems, the ETMs may be added in
the form of non-covalently attached hybridization indicators.
Solution binding ligands are similar to capture binding ligands, in
that they bind, preferably specifically, to target analytes. The
solution binding ligand may be the same or different from the
capture binding ligand. Generally, the solution binding ligands are
not directed attached to the surface, although in some embodiments
they may be.
[0195] The solution binding ligand either directly comprises a
recruitment linker that comprises at least one ETM or the
recruitment linker binds, either directly or indirectly to the
solution binding ligand.
[0196] Thus, "solution binding ligands" or "soluble binding
ligands" or "signalling ligands" or "signal carriers" or "label
probes" or "label binding ligands" with recruitment linkers
comprising covalently attached ETMs are provided. That is, one
portion of the label probe or solution binding ligand directly or
indirectly binds to the target analyte, and one portion comprises a
recruitment linker comprising covalently attached ETMs. In some
systems, for example in mechanism-1 nucleic acid systems, these may
be the same. Similarly, for mechanism-1 systems, the recruitment
linker comprises nucleic acid that will hybridize to detection
probes.
[0197] The terms "electron donor moiety", "electron acceptor
moiety", and "ETMs" (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.
[0198] 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 are listed above.
[0199] The transition metals are complexed with a variety of
ligands, L, defined above, to form suitable transition metal
complexes, as is well known in the art.
[0200] In addition to transition metal complexes, other organic
electron donors and acceptors may be covalently attached to the
nucleic acid 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-def6,5,10-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-ami- no-10-methylphenoxyazine
chloride), methylene blue; Nile blue A
(aminoaphthodiethylaminophenoxazine sulfate),
indigo-5,5',7,7'-tetrasulfo- nic 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
subsitituted derivatives of these compounds.
[0201] 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.
[0202] The choice of the specific ETMs will be influenced by the
type of electron transfer detection used, as is generally outlined
below. Preferred ETMs are metallocenes, with ferrocene, including
derivatives, being particularly preferred.
[0203] In a preferred embodiment, a plurality of ETMs are used. The
use of multiple ETMs provides signal amplification and thus allows
more sensitive detection limits. While the use of multiple ETMs on
nucleic acids that hybridize to complementary strands can cause
decreases in Tms of the hybridization complexes depending on the
number, site of attachment and spacing between the multiple ETMs,
this is not a factor when the ETMs are on the recruitment linker
(i.e. "mechanism-2" systems), since this does not hybridize to a
complementary sequence. Accordingly, pluralities of ETMs are
preferred, with at least about 2 ETMs per recruitment linker being
preferred, and at least about 10 being particularly preferred, and
at least about 20 to 50 being especially preferred. In some
instances, very large numbers of ETMs (50 to 1000) can be used.
[0204] Thus, solution binding ligands, or label probes, with
covalently attached ETMs are provided. The method of attachment of
the ETM to the solution binding ligand will vary depending on the
mode of detection (i.e. mechanism-1 or -2 systems) and the
composition of the solution binding ligand. As is more fully
outlined below, in mechanism-2 systems, the portion of the solution
binding ligand (or label probe) that comprises the ETM is referred
to as a "recruitment linker" and can comprise either nucleic acid
or non-nucleic acid. For mechanism-1 systems, the recruitment
linker must be nucleic acid.
[0205] 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. 5.
[0206] 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.
[0207] In addition, as is described below, when the recruitment
linker is nucleic acid, it may be desirable to use secondary label
probes, that have a first portion that will hybridize to a portion
of the primary label probes and a second portion comprising a
recruitment linker as is defined herein. This is similar to the use
of an arhplifier probe, except that both the primary and the
secondary label probes comprise ETMs.
[0208] In a preferred embodiment, the ETM is attached to the base
of a nucleoside as is generally outlined above for attachment of
the conductive oligomer. Attachment can be to an internal
nucleoside or a terminal nucleoside.
[0209] The covalent attachment to the base will depend in part on
the ETM chosen, but in general is similar to the attachment of
conductive oligomers to bases, as outlined above. Attachment may
generally be done to any position of the base. In a preferred
embodiment, the ETM is a transition metal complex, and thus
attachment of a suitable metal ligand to the base leads to the
covalent attachment of the ETM. Alternatively, similar types of
linkages may be used for the attachment of organic ETMs, as will be
appreciated by those in the art.
[0210] In one embodiment, the C4 attached amino group of cytosine,
the C6 attached amino group of adenine, or the C2 attached amino
group of guanine may be used as a transition metal ligand.
[0211] 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, pp521-549, and pp950-953, hereby incorporated by
reference). Structure 30 depicts a representative structure in the
presence of the metal ion and any other necessary ligands;
Structure 30 depicts uridine, although as for all the structures
herein, any other base may also be used. 24
[0212] 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.
[0213] Similarly, as for the attachment linkers, 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 31, which again uses
uridine as the base, although as above, the other bases may also be
used: 25
[0214] In this embodiment, L is a ligand as defined above, with
L.sub.r and M as defined above as well. Preferably, L is amino,
phen, byp and terpy.
[0215] In a preferred embodiment, the ETM attached to a nucleoside
is a metallocene; i.e. the L and L.sub.r of Structure 31 are both
metallocene ligands, L.sub.m, as described above. Structure 32
depicts a preferred embodiment wherein the metallocene
is-ferrocene, and the base is uridine, although other bases may be
used: 26
[0216] Preliminary data suggest that Structure 32 may cyclize, with
the second acetylene carbon atom attacking the carbonyl oxygen,
forming a furan-like structure. Preferred metallocenes include
ferrocene, cobaltocene and osmiumocene.
[0217] 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, the
conductive oligomers depicted in Structure 13, 14 and 15 may be
replaced by ETMs; alternatively, the ETMs may be added to the free
terminus of the conductive oligomer.
[0218] In a preferred embodiment, a metallocene serves as the ETM,
and is attached via an amide bond as depicted below in Structure
33. The examples outline the synthesis of a preferred compound when
the metallocene is ferrocene. 27
[0219] In a preferred embodiment, amine linkages are used, as is
generally depicted in Structure 34. 28
[0220] 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.
[0221] In a preferred embodiment, oxo linkages are used, as is
generally depicted in Structure 35. 29
[0222] In Structure 35, 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).sub.n and
(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.
[0223] Linkages utilizing other heteroatoms are also possible.
[0224] 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
Structures 23 and 24 may be replaced by ETMs. In a preferred
embodiment, the composition has the structure shown in Structure
36. 30
[0225] In Structure 36, 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.
[0226] In mechanism-2 systems, 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.
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.
[0227] 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 orthongonal 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 37 (nucleic acid with a ribose-phosphate
backbone) and Structure 38 (peptide nucleic acid backbone).
Structures 37 and 38 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. 31
[0228] In Structure 37, 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 37 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.
32
[0229] In Structure 38, 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.
[0230] 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.
[0231] Thus, by inserting a metallocene such as ferrocene (or other
ETM) into the backbone of a nucleic acid, nucleic acid analogs are
made; that is, the invention provides nucleic adds having a
backbone comprising at least one metallocene. This is distinguished
from nucleic adds having metallocenes attached to the backbone,
i.e. via a ribose, a phosphate, etc. That is, two nucleic acids
each made up of a traditional nucleic acid or analog (nucleic acids
in this case including a single nucleoside), may be covalently
attached to each other via a metallocene. Viewed differently, a
metallocene derivative or substituted metallocene is provided,
wherein each of the two aromatic rings of the metallocene has a
nucleic acid substitutent group.
[0232] 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.
[0233] 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). For example, as these nucleoside replacements are generally
part of probe sequences to be hybridized with a substantially
complementary nucleic acid, for example a target sequence or
another probe sequence, it is possible to add substitutent groups
to the metallocene rings to facilitate hydrogen bonding to the base
or bases on the opposite strand. These may be added to any position
on the metallocene rings. Suitable substitutent groups include, but
are not limited to, amide groups, amine groups, carboxylic acids,
and alcohols, including substituted alcohols. In addition, these
substitutent groups can be attached via linkers as well, although
in general this is not preferred.
[0234] In addition, 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, as
is more fully described below, it may be desirable to have
different ETMs attached in different ways (i.e. base or ribose
attachment), on different probes, 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.
[0235] 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 39: 33
[0236] In Structure 39, 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
40 depicts the ferrocene derivative: 34
[0237] These phosphoramidite analogs can be added to standard
oligonucleotide syntheses as is known in the art.
[0238] Structure 41 depicts the ferrocene peptide nucleic acid
(PNA) monomer, that can be added to PNA synthesis as is known in
the art: 35
[0239] In Structure 41, the PG protecting group is suitable for use
in peptide nucleic acid synthesis, with MMT, boc and Fmoc being
preferred.
[0240] 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.
[0241] In a preferred embodiment, particularly for us in
mechanism-2 systems, 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
structures 39 and 40, 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 in FIG. 9 with 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.
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
[0242] In a preferred 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 41, peptide nucleic acid structures containing
metallocene polymers could be generated.
[0243] Thus, the present invention provides recruitment linkers of
nucleic acids comprising "branches" of metallocene polymers.
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.
[0244] In addition, when the recruitment linker is nucleic acid,
any combination of ETM attachments may be done. In general, as
outlined herein, when mechanism-1 systems are used, clusters of
nucleosides, containing ETMs can decrease the Tm of hybridization
of the probe to its target sequence; thus in general, for
mechanism-1 systems, the ETMs are spaced out over the length of the
sequence, or only small numbers of them are used.
[0245] In mechanism-1 systems, non-covalently attached ETMs may be
used. In one embodiment, the ETM is a hybridization indicator.
Hybridization indicators serve as an ETM that will preferentially
associate with double stranded nucleic acid is added, usually
reversibly, similar to the method of Millan et al., Anal. Chem.
65:2317-2323 (1993); Millian et al., Anal. Chem. 662943-2948
(1994), both of which are hereby expressly incorporated by
reference. In this embodiment, increases in the local concentration
of ETMs, due to the association of the ETM hybridization indicator
with double stranded nucleic acid at the surface, can be monitored
using the monolayers comprising the conductive oligomers.
Hybridization indicators include intercalators and minor and/or
major groove binding moieties. In a preferred embodiment,
intercalators may be used; since intercalation generally only
occurs in the presence of double stranded nucleic acid, only in the
presence of double stranded nucleic acid will the ETMs concentrate.
Intercalating transition metal complex ETMs are known in the art.
Similarly, major or minor groove binding moieties, such as
methylene blue, may also be used in this embodiment.
[0246] In addition, the biosensors of the invention may be used in
virtually any method that relies on electrochemical detection of
target analytes, with particular utility in nucleic acid detection.
For example, the methods and compositions of the invention can be
used in nucleic acid detection methods that rely on the detection
of ETMs that are inherent to the target analyte. For example, as is
generally described in Napier et al., Bioconj. Chem. 8:906 (1997),
hereby expressly incorporated by reference, the guanine bases of
nucleic acid can be detected via changes in the redox state, i.e.
guanine oxidation by ruthenium complexes. Similarly, the methods of
the invention find use in detection systems that utilize copper
surfaces as catalytic electrodes to oxidize the riboses of nucleic
acids.
[0247] 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.
[0248] 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.
[0249] 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).
[0250] 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. As outlined above, in some
embodiments, charged recruitment linkers are preferred, for example
when non-charged target analytes are to be detected. 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.
[0251] In a preferred embodiment, the recruitment linker comprises
a metallocene polymer, as is described above.
[0252] The attachment of the recruitment linkers to the first
portion of the label probe, i.e. the portion that binds either
directly or indirectly to the target analyte, will depend on the
composition of the recruitment linker, as will be appreciated by
those in the art. When the recruitment linker is nucleic acid, it
is generally formed during the synthesis of the first portion of
the label probe, with incorporation of nucleosides containing ETMs
as required. Alternatively, the first portion of the label probe
and the recruitment linker may be made separately, and then
attached. For example, there may be an overlapping section of
complementarity, forming a section of double stranded nucleic acid
that can then be chemically crosslinked, for example by using
psoralen as is known in the art.
[0253] 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.
[0254] 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).
[0255] In a preferred embodiment, for example when the target
analyte is a nucleic acid, it is the target sequence itself that
carries the ETMs, rather than the recruitment linker of a label
probe. 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 "nudeoside
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.
[0256] 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.
[0257] 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. Thus the invention provides compositions utilizing
electrodes comprising monolayers with capture probes, and target
sequences that comprises a first portion that is capable of
hybridizing to a component of an assay complex, and a second
portion that does not hybridize to a component of an assay complex
and comprises at least one covalently attached electron transfer
moiety. Similarly, methods utilizing these compositions are also
provided.
[0258] It is also possible to have ETMs connected to probe
sequences, i.e. sequences designed to hybridize to complementary
sequences, i.e. in mechanism-1 sequences, although this may also be
used in mechanism-2 systems. Thus, ETMs may be added to
non-recruitment linkers as well. For example, there may be ETMs
added to sections of label probes that do hybridize to components
of the assay complex, for example the first portion, or to the
target sequence as outlined above. These ETMs may be used for
electron transfer detection in some embodiments, or they may not,
depending on the location and system. For example, in some
embodiments, when for example the target sequence containing
randomly incorporated ETMs is hybridized directly to the capture
probe, there may be ETMs in the portion hybridizing to the capture
probe. If the capture probe is attached to the electrode using a
conductive oligomer, these ETMs can be used to detect electron
transfer as has been previously described. Alternatively, these
ETMs may not be specifically detected.
[0259] Similarly, 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, for example in
the mechanism-2 systems. 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, i.e.
in mechanism-1 systems, 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.
[0260] For nucleic acid systems, the 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.
[0261] Generally, the nucleic acid compositions of the invention
are useful as oligonucleotide probes. As is appreciated by those in
the art, the length of the probe will vary with the length of the
target sequence and the hybridization and wash conditions.
Generally, oligonucleotide probes range from about 8 to about 50
nucleotides, with from about 10 to about 30 being preferred and
from about 12 to about 25 being especially preferred. In some
cases, very long probes may be used, e.g. 50 to 200-300 nucleotides
in length. Thus, in the structures depicted herein, nucleosides may
be replaced with nucleic acids.
[0262] 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 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 C
for short probes (e.g. 10 to 50 nucleotides) and at least about 60
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.
[0263] In another embodiment, less stringent hybridization
conditions are used; for example, moderate or low stringency
conditions may be used, as are known in the art; see Maniatis and
Ausubel, supra, and Tijssen, supra.
[0264] 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.
[0265] As will be appreciated by those in the art, the systems of
the invention may take on a large number of different
configurations, some of which are depicted in FIGS. 6 and 7. In
general, there are three types of systems that can be used: (1)
systems in which the target sequence itself is labeled with ETMs
(see FIG. 6A; this is generally useful for nucleic acid systems);
(2) systems in which label probes directly bind to the target
analytes (see FIG. 6C); and (3) systems in which label probes are
indirectly bound to the target sequences, for example through the
use of amplifier probes (see FIG. 6E).
[0266] In all three of these systems, it is preferred, although not
required, that the target sequence be immobilized on the electrode
surface. This is preferably done using capture probes and
optionally one or more capture extender probes; see FIG. 3 for
representative nucleic acid examples. When only capture probes are
utilized, it is necessary to have unique capture probes for each
target sequence; that is, the surface must be customized to contain
unique capture probes. Alternatively, capture extender probes may
be used, that allow a "universal" surface, i.e. a surface
containing a single type of capture probe that can be used to
detect any target sequence. "Capture extender" probes are generally
depicted in FIG. 6C and other, and have a first portion that will
hybridize to all or part of the capture probe, and a second portion
that will hybridize to a portion of the target sequence. This then
allows the generation of customized soluble probes, which as will
be appreciated by those in the art is generally simpler and less
costly. As shown herein, two capture extender probes may be used.
This has generally been done to stabilize assay complexes (for
example when the target sequence is large, or when large amplifier
probes (particularly branched or dendrimer amplifier probes) are
used.
[0267] While the discussion and figures herein generally depict
nucleic acid embodiments, these same ideas can be used for
non-nucleic acid target analytes. For example, capture extender
ligands can be generated, as will be appreciated by those in the
art. For example, a nucleic acid "tail" can be added to a binding
ligand.
[0268] In a preferred embodiment, the binding ligands are added
after the formation of the SAM ((4) above). This may be done in a
variety of ways, as will be-appreciated by those in the art. In one
embodiment, conductive oligomers with terminal functional groups
are made, with preferred embodiments utilizing activated
carboxylates and isothiocyanates, that will react with primary
amines that are either present or put onto the binding ligand such
as a nucleic acid, using an activated carboxylate. These two
reagents have the advantage of being stable in aqueous solution,
yet react with primary alkylamines. However, the primary aromatic
amines and secondary and tertiary amines of the bases should not
react, thus allowing site specific addition of nucleic acids to the
surface. Similar techniques can be used with non-nucleic acid
components; for example, as outlined above, the attachment of
proteins to SAMs comprising metal chelates is known; see U.S. Pat.
No. 5,620,850. This allows the spotting of probes (either capture
or detection probes, or both) using known methods (ink jet,
spotting, etc.) onto the surface.
[0269] In addition, there are a number of non-nucleic acid methods
that can be used to immobilize a nucleic acid on a surface. For
example, binding partner pairs can be utilized; i.e. one binding
partner is attached to the terminus of the conductive oligomer, and
the other to the end of the nucleic acid. This may also be done
without using a nucleic acid capture probe; that is, one binding
partner serves as the capture probe and the other is attached to
either the target sequence or a capture extender probe. That is,
either the target sequence comprises the binding partner, or a
capture extender probe that will hybridize to the target sequence
comprises the binding partner. Suitable binding partner pairs
include, but are not limited to, hapten pairs such as
biotin/streptavidin; antigens/antibodies; NTA/histidine tags; etc.
In general, smaller binding partners are preferred, such that the
electrons can pass from the nucleic acid into the conductive
oligomer to allow detection.
[0270] In a preferred embodiment, when the target sequence itself
is modified to contain a binding partner, the binding partner is
attached via a modified nucleotide that can be enzymatically
attached to the target sequence, for example during a PCR target
amplification step. Alternatively, the binding partner should be
easily attached to the target sequence.
[0271] Alternatively, a capture extender probe may be utilized that
has a nucleic acid portion for hybridization to the target as well
as a binding partner (for example, the capture extender probe may
comprise a non-nucleic acid portion such as an alkyl linker that is
used to attach a binding partner). In this embodiment, it may be
desirable to cross-link the double-stranded nucleic acid of the
target and capture extender probe for stability, for example using
psoralen as is known in the art.
[0272] In a preferred embodiment, the target sequence itself
contains the ETMs. As discussed above, this may be done using
target sequences that have ETMs incorporated at any number of
positions, as outlined above. In this embodiment, as for the others
of the system, the 3'-5' orientation of the probes and targets is
chosen to get the ETM-containing structures (i.e. recruitment
linkers or target sequences) as close to the surface of the
monolayer as possible, and in the correct orientation. This may be
done using attachment via insulators or conductive oligomers as is
generally shown in the Figures. In addition, as will be appreciated
by those in the art, multiple capture probes can be utilized,
either in a configuration such as depicted in FIG. 6D, wherein the
5'-3' orientation of the capture probes is different, or where
"loops" of target form-when multiples of capture probes are
used.
[0273] In a preferred embodiment, the label probes directly
hybridize to the target sequences, as is generally depicted in the
figures. In these embodiments, the target sequence is immobilized
on the surface using capture-probes, including capture extender
probes. Label probes are then used to bring the ETMs into proximity
of the surface of the monolayer. In a preferred embodiment,
multiple label probes are used; that is, label probes are designed
such that the portion that hybridizes to the target sequence can be
different for a number of different label probes, such that
amplification of the signal occurs, since multiple label probes can
bind for every target sequence. Thus, as depicted in the figures, n
is an integer of at least one. Depending on the sensitivity
desired, the length of the target sequence, the number of ETMs per
label probe, etc., preferred ranges of n are from 1 to 50, with
from about 1 to about 20 being particularly preferred, and from
about 2 to about 5 being especially preferred. In addition, if
"generic" label probes are desired, label extender probes can be
used as generally described below for use with amplifier
probes.
[0274] As above, generally in this embodiment the configuration of
the system and the label probes are designed to recruit the ETMs as
close as possible to the monolayer surface.
[0275] In a preferred embodiment, the label probes are hybridized
to the target sequence indirectly. That is, the present invention
finds use in novel combinations of signal amplification
technologies and electron transfer detection on electrodes, which
may be particularly useful in sandwich hybridization assays, as
generally depicted in the Figures for nucleic acid embodiments;
similar systems can be developed for non-nucleic acid target
analytes. In these embodiments, the amplifier probes of the
invention are bound to the target sequence in a sample either
directly or indirectly. Since the amplifier probes preferably
contain a relatively large number of amplification sequences that
are available for binding of label probes, the detectable signal is
significantly increased, and allows the detection limits of the
target to be significantly improved. These label and amplifier
probes, and the detection methods described herein, may be used in
essentially any known nucleic acid hybridization formats, such as
those in which the target is bound directly to a solid phase or in
sandwich hybridization assays in which the target is bound to one
or more nucleic acids that are in turn bound to the solid
phase.
[0276] In general, these embodiments may be described as follows
with particular reference to nucleic adds. An amplifier probe is
hybridized to the target sequence, either directly or through the
use of a label extender probe which serves to allow "generic"
amplifier probes to be made. The target sequence is preferably, but
not required to be, immobilized on the electrode using capture
probes. Preferably, the amplifier probe contains a multiplicity of
amplification sequences, although in some embodiments, as described
below, the amplifier probe may contain only a single amplification
sequence. The amplifier probe may take on a number of different
forms; either a branched conformation, a dendrimer conformation, or
a linear "string" of amplification sequences. These amplification
sequences are used to form hybridization complexes with label
probes, and the ETMs can be detected using the electrode.
[0277] Accordingly, the present invention provides assay complexes
comprising at least one amplifier probe. By "amplifier probe" or
"nucleic acid multimer" or "amplification multimer" or grammatical
equivalents herein is meant a nucleic acid probe that is used to
facilitate signal amplification. Amplifier probes comprise at least
a first single-stranded nucleic acid probe sequence, as defined
below, and at least one single-stranded nucleic acid amplification
sequence, with a multiplicity of amplification sequences being
preferred.
[0278] Amplifier probes comprise a first probe sequence that is
used, either directly or indirectly, to hybridize to the target
sequence. That is, the amplifier probe itself may have a first
probe sequence that is substantially complementary to the target
sequence or it has a first probe sequence that is substantially
complementary to a portion of an additional probe, in this case
called a label extender probe, that has a first portion that is
substantially complementary to the target sequence. In a preferred
embodiment, the first probe sequence-of the amplifier probe is
substantially complementary to the target sequence.
[0279] In general, as for all the probes herein, the first probe
sequence is of a length sufficient to give specificity and
stability. Thus generally, the probe sequences of the invention
that are designed to hybridize to another nucleic acid (i.e. probe
sequences, amplification sequences, portions or domains of larger
probes) are at least about 5 nucleosides long, with at least about
10 being preferred and at least about 15 being especially
preferred.
[0280] In a preferred embodiment, the amplifier probes, or any of
the other probes of the invention, may form hairpin stem-loop
structures in the absence of their target. The length of the stem
double-stranded sequence will be selected such that the hairpin
structure is not favored in the presence of target. The use of
these type of probes, in the systems of the invention or in any
nucleic acid detection systems, can result in a significant
decrease in non-specific binding and thus an increase in the signal
to noise ratio.
[0281] Generally, these hairpin structures comprise four
components. The first component is a target binding sequence, i.e.
a region complementary to the target (which may be the sample
target sequence or another probe sequence to which binding is
desired), that is about 10 nucleosides long, with about 15 being
preferred. The second component is a loop sequence, that can
facilitate the formation of nucleic acid loops. Particularly
preferred in this regard are repeats of GTC, which has been
identified in Fragile X Syndrome as forming turns. (When PNA
analogs are used, turns comprising proline residues may be
preferred). Generally, from three to five repeats are used, with
four to five being preferred. The third component is a
self-complementary region, which has a first portion that is
complementary to a portion of the target sequence region and a
second portion that comprises a first portion of the label probe
binding sequence. The fourth component is substantially
complementary to a label probe (or other probe, as the case may
be). The fourth component further comprises a "sticky end", that
is, a portion that does not hybridize to any other portion of the
probe, and preferably contains most, if not all, of the ETMs. As
will be appreciated by those in the art, the any or all of the
probes described herein may be configured to form hairpins in the
absence of their targets, including the amplifier, capture, capture
extender, label and label extender probes.
[0282] In a preferred embodiment, several different amplifier
probes are used, each with first probe sequences that will
hybridize to a different portion of the target sequence. That is,
there is more than one level of amplification; the amplifier probe
provides an amplification of signal due to a multiplicity of
labelling events, and several different amplifier probes, each with
this multiplicity of labels, for each target sequence is used.
Thus, preferred embodiments utilize at least two different pools of
amplifier probes, each pool having a different probe sequence for
hybridization to different portions of the target sequence; the
only real limitation on the number of different amplifier probes
will be the length of the original target sequence. In addition, it
is also possible that the different amplifier probes contain
different amplification sequences, although this is generally not
preferred.
[0283] In a preferred embodiment, the amplifier probe does not
hybridize to the sample target sequence directly, but instead
hybridizes to a first portion of a label extender probe. This is
particularly useful to allow the use of "generic" amplifier probes,
that is, amplifier probes that can be used with a variety of
different targets. This may be desirable since several of the
amplifier probes require special synthesis techniques. Thus, the
addition of a relatively short probe as a label extender probe is
preferred. Thus, the first probe sequence of the amplifier probe is
substantially complementary to a first portion or domain of a first
label extender single-stranded nucleic acid probe. The label
extender probe also contains a second portion or domain that is
substantially complementary to a portion of the target sequence.
Both of these portions are preferably at least about 10 to about 50
nucleotides in length, with a range of about 15 to about 30 being
preferred. 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 or probe sequences. For example, assuming
a 5'-3' orientation of the complementary target sequence, the first
portion may be located either 5' to the second portion, or 3' to
the second portion. For convenience herein, the order of probe
sequences are generally shown from left to right.
[0284] In a preferred embodiment, more than one label extender
probe-amplifier probe pair may be used, tht is, n is more than 1.
That is, a plurality of label extender probes may be used, each
with a portion that is substantially complementary to a different
portion of the target sequence; this can serve as another level of
amplification. Thus, a preferred embodiment utilizes pools of at
least two label extender probes, with the upper limit being set by
the length of the target sequence.
[0285] In a preferred embodiment, more than one label extender
probe is used with a single amplifier probe to reduce non-specific
binding, as is generally outlined in U.S. Pat. No. 5,681,697,
incorporated by reference herein. In this embodiment, a first
portion of the first label extender probe hybridizes to a first
portion of the target sequence, and the second portion of the first
label extender probe hybridizes to a first probe sequence of the
amplifier probe. A first portion of the second label extender probe
hybridizes to a second portion of the target sequence, and the
second portion of the second label extender probe hybridizes to a
second probe sequence of the amplifier probe. These form structures
sometimes referred to as "cruciform" structures or configurations,
and are generally done to confer stability when large branched or
dendrimeric amplifier probes are used.
[0286] In addition, as will be appreciated by those in the art, the
label extender probes may interact with a preamplifier probe,
described below, rather than the amplifier probe directly.
[0287] Similarly, as outlined above, a preferred embodiment
utilizes several different amplifier probes, each with first probe
sequences that will hybridize to a different portion of the label
extender probe. In addition, as outlined above, it is also possible
that the different amplifier probes contain different amplification
sequences, although this is generally not preferred.
[0288] In addition to the first probe sequence, the amplifier probe
also comprises at least one amplification sequence. 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
as is more fully described below. Preferably, the amplifier probe
comprises a multiplicity of amplification sequences, with from
about 3 to about 1000 being preferred, from about 10 to about 100
being particularly preferred, and about 50 being especially
preferred. In some cases, for example when linear amplifier probes
are used, from 1 to about 20 is preferred with from about 5 to
about 10 being particularly preferred.
[0289] The amplification sequences may be linked to each other in a
variety of ways, as will be appreciated by those in the art. They
may be covalently linked directly to each other, or to intervening
sequences or chemical moieties, through nucleic acid linkages such
as phosphodiester bonds, PNA bonds, etc., or through interposed
linking agents such amino acid, carbohydrate or polyol bridges, or
through other cross-linking agents or binding partners. The site(s)
of linkage may be at the ends of a segment, and/or at one or more
internal nucleotides in the strand. In a preferred embodiment, the
amplification sequences are attached via nucleic acid linkages.
[0290] In a preferred embodiment, branched amplifier probes are
used, as are generally described in U.S. Pat. No. 5,124,246, hereby
incorporated by reference. Branched amplifier probes may take on
"fork-like" or "comb-like" conformations. "Fork-like" branched
amplifier probes generally have three or more oligonucleotide
segments emanating from a point of origin to form a branched
structure. The point of origin may be another nucleotide segment or
a multifunctional molecule to whcih at least three segments can be
covalently or tightly bound. "Comb-like" branched amplifier probes
have a linear backbone with a multiplicity of sidechain
oligonucleotides extending from the backbone. In either
conformation, the pendant segments will normally depend from a
modified nucleotide or other organic moiety having the appropriate
functional groups for attachment of oligonudeotides. Furthermore,
in either conformation, a large number of amplification sequences
are available for binding, either directly or indirectly, to
detection probes. In general, these structures are made as is known
in the art, using modified multifunctional nucleotides, as is
described in U.S. Pat. Nos. 5,635,352 and 5,124,246, among
others.
[0291] In a preferred embodiment, dendrimer amplifier probes are
used, as are generally described in U.S. Pat. No. 5,175,270, hereby
expressly incorporated by reference. Dendrimeric amplifier probes
have amplification sequences that are attached via hybridization,
and thus have portions of double-stranded nucleic acid as a
component of their structure. The outer surface of the dendrimer
amplifier probe has a multiplicity of amplification sequences.
[0292] In a preferred embodiment, linear amplifier probes are used,
that have individual amplification sequences linked end-to-end
either directly or with short intervening sequences to form a
polymer. As with the other amplifier configurations, there may be
additional sequences or moieties between the amplification
sequences.
[0293] In one embodiment, the linear amplifier probe has a single
amplification sequence. This may be useful when cycles of
hybridization/disassociation occurs, forming a pool of amplifier
probe that was hybridized to the target and then removed to allow
more probes to bind, or when large numbers of ETMs are used for
each label probe. However, in a preferred embodiment, linear
amplifier probes comprise a multiplicity of amplification
sequences.
[0294] In addition, the amplifier probe may be totally linear,
totally branched, totally dendrimeric, or any combination
thereof.
[0295] 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.
[0296] In addition, the compositions of the invention may include
"preamplifier" molecules, which serves a bridging moiety between
the label extender molecules and the amplifier probes. In this way,
more amplifier and thus more ETMs are ultimately bound to the
detection probes. Preamplifier molecules may be either linear or
branched, and typically contain in the range of about 30-3000
nucleotides.
[0297] The tissue collection devices of the invention may be used
in a variety of ways. Components of the reaction may be added
simultaneously, or sequentially, in any order, with preferred
embodiments outlined below.
[0298] The invention thus provides assay complexes that generally
minimally comprise a target sequence, a capture probe and a label
probe (or in the case where the target analyte is labeled with the
ETMs, the assay complex comprises a target sequence and a capture
probe). "Assay complex" herein is meant the collection of
attachment or hybridization complexes comprising analytes,
including binding ligands and targets and at least one ETM, that
allows detection. The composition of the assay complex depends on
the use of the different probe component outlined herein. Thus, in
FIG. 6A, the assay complex comprises the capture probe and the
target sequence. A preferred embodiment utilizes a capture probe, a
target sequence and a label probe. The assay complexes may also
include capture extender probes, label extender probes, and
amplifier probes, as outlined herein, depending on the
configuration used.
[0299] The assays are generally run under stringency conditions
which allows formation of the label probe attachment 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. Stringency may also include the use of an
electrophoretic step to drive non-specific (i.e. low stringency)
materials away from the detection electrode.
[0300] 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; for example, when an initial hybridization step is done
between the target sequence and the label extender and capture
extender probes. Running this step at conditions which favor
specific binding can allow the reduction of non-specific
binding.
[0301] In a preferred nucleic acid embodiment, when all of the
components outlined herein are used, a preferred method is as
follows. Single-stranded target sequence is incubated under
hybridization conditions with the capture extender probes and the
label extender probes. A preferred embodiment does this reaction in
the presence of the electrode with immobilized capture probes,
although this may also be done in two steps, with the initial
incubation and the subsequent addition to the electrode. Excess
reagents are washed off, and-amplifier probes are then added. If
preamplifier probes are used, they may be added either prior to the
amplifier probes or simultaneously with the amplifier probes.
Excess reagents are washed off, and label probes are then added.
Excess reagents are washed off, and detection proceeds as outlined
below.
[0302] In one embodiment, a number of capture probes (or capture
probes and capture extender probes) that are each substantially
complementary to a different portion of the target sequence are
used.
[0303] Again, as outlined herein, when amplifier probes are used,
the system is generally configured such that upon label probe
binding, the recruitment linkers comprising the ETMs are placed in
proximity either to the monolayer surface containing conductive
oligomers (mechanism-2) or in proximity to detection probes. Thus
for example, for mechanism-2 systems, when the ETMs are attached
via "dendrimer" type structures as outlined herein, the length of
the linkers from the nucleic acid point of attachment to the ETMs
may vary, particularly with the length of the capture probe when
capture extender probes are used. That is, longer capture probes,
with capture extenders, can result in the target sequences being
"held" further away from the surface than for shorter capture
probes. Adding extra linking sequences between the probe nucleic
acid and the ETMs can result in the ETMs being spatially closer to
the surface, giving better results. Similarly, for mechanism-1
systems, the length of the recruitment linker, the length of the
detection probe, and their distance, may be optimized.
[0304] In addition, if desirable, nucleic acids utilized in the
invention may also be ligated together prior to detection, if
applicable, by using standard molecular biology techniques such as
the use of a ligase. Similarly, if desirable for stability,
cross-linking agents may be added to hold the structures
stable.
[0305] As will be appreciated by those in the art, while described
for nucleic acids, the systems outlined herein can be used for
other target analytes as well.
[0306] The compositions of the invention are generally synthesized
as outlined below and in U.S. Pat. Nos. 5,591,578; 5,824,473;
5,770,369; 5,705,348; 5,780,234; and 5,952,172; WO98/20162;
WO99/37819; PCT US98/12430, PCT US99/01703; PCT US98/12082;
PCT/US99/14191; PCT/US99/21683; PCT/US99/25464; PCT US99/10104; and
U.S. Ser. Nos. 09/135,183; 09/295,691 and 09/306,653, all of which
are expressly incorporated by reference, and utilizing techniques
well known in the art. As will be appreciated by those in the art,
many of the techniques outlined below are directed to nucleic acids
containing a ribose-phosphate backbone. However, as outlined above,
many alternate nucleic acid analogs may be utilized, some of which
may not contain either ribose or phosphate in the backbone. In
these embodiments, for attachment at positions other than the base,
attachment is done as will be appreciated by those in the art,
depending on the backbone. Thus, for example, attachment can be
made at the carbon atoms of the PNA backbone, as is described
below, or at either terminus of the PNA. In addition, systems
utilizing non-nucleic acid components can be made, as is generally
outlined in the references cited above.
[0307] The compositions may be made in several ways and will depend
on the components as well as the mechanism used. A preferred method
first synthesizes an attachment linker attached to a nucleoside,
with addition of additional nucleosides to form the capture probe
followed by attachment to the electrode. Alternatively, the whole
capture probe may be made and then the completed attachment linker
added, followed by attachment to the electrode. Alternatively, a
monolayer comprising attachment linkers (some of which have
functional groups for attachment of capture probes) is attached to
the electrode first, followed by attachment of the capture probe.
The latter two methods may be preferred when attachment linkers are
used which are not stable in the solvents and under the conditions
used in traditional nucleic acid synthesis.
[0308] In a preferred embodiment, the compositions of the invention
are made by first forming the attachment linker covalently attached
to the nucleoside, followed by the addition of additional
nucleosides to form a capture probe nucleic acid, with the last
step comprising the addition of the attachment linker to the
electrode.
[0309] The attachment of the attachment linker to the nucleoside
may be done in several ways. In a preferred embodiment, all or part
of the attachment linker is synthesized first (generally with a
functional group on the end for attachment to the electrode), which
is then attached to the nucleoside. Additional nucleosides are then
added as required, with the last step generally being attachment to
the electrode. Alternatively, for example when the attachment
linker is a conductive oligomer, oligomer units are added one at a
time to the nucleoside, with addition of additional nucleosides and
attachment to the electrode. A number of representative syntheses
are shown in the Figures of WO 98/20162; PCT/US98/12430;
PCT/US98/12082; PCT/US99/01705; PCT/US99/01703; and U.S. Ser. Nos.
09/135,183; 60/105,875; and 09/295,691, all of which are
incorporated by reference.
[0310] The attachment linker is then attached to a nucleoside that
may contain one (or more) of the oligomer units, attached as
depicted herein.
[0311] In a preferred embodiment, attachment is to a ribose of the
ribose-phosphate backbone, including amide and amine linkages. In a
preferred embodiment, there is at least a methylene group or other
short aliphatic alkyl groups (as a Z group) between the nitrogen
attached to the ribose and the aromatic ring of the conductive
oligomer.
[0312] Alternatively, attachment is via a phosphate of the
ribose-phosphate backbone, as generally outlined in PCT
US97/20014.
[0313] In a preferred embodiment, attachment is via the base. In a
preferred embodiment, protecting groups may be added to the base
prior to addition of the conductive oligomers, as is generally
known in the art. In addition, the palladium cross-coupling
reactions may be altered to prevent dimerization problems; i.e. two
conductive oligomers dimerizing, rather than coupling to the
base.
[0314] Alternatively, attachment to the base may be done by making
the nucleoside with one unit of the oligomer, followed by the
addition of others.
[0315] Once the modified nucleosides are prepared, protected and
activated, prior to attachment to the electrode, they may be
incorporated into a growing oligonucleotide by standard synthetic
techniques (Gait, Oligonucleotide Synthesis: A Practical Approach,
IRL Press, Oxford, UK 1984; Eckstein) in several ways.
[0316] In one embodiment, one or more modified nucleosides are
converted to the triphosphate form and incorporated into a growing
oligonucleotide chain by using standard molecular biology
techniques such as with the use of the enzyme DNA polymerase 1, T4
DNA polymerase, T7 DNA polymerase, Taq DNA polymerase, reverse
transcriptase, and RNA polymerases. For the incorporation of a 3'
modified nucleoside to a nucleic acid, terminal
deoxynucleotidyltransferase may be used. (Ratliff, Terminal
deoxynucleotidyltransferase. In The Enzymes, Vol 14A. P. D. Boyer
ed. pp 105-118. Academic Press, San Diego, Calif. 1981). Thus, the
present invention provides deoxyribonucleoside triphosphates
comprising a covalently attached ETM. Preferred embodiments utilize
ETM attachment to the base or the backbone, such as the ribose
(preferably in the 2' position), as is generally depicted below in
Structures 42 and 43: 36
[0317] Thus, in some embodiments, it may be possible to generate
the nucleic acids comprising ETMs in situ. For example, a target
sequence can hybridize to a capture probe (for example on the
surface) in such a way that the terminus of the target sequence is
exposed, i.e. unhybridized. The addition of enzyme and triphosphate
nucleotides labelled with ETMs allows the in situ creation of the
label. Similarly, using labeled nucleotides recognized by
polymerases can allow simultaneous PCR and detection; that is, the
target sequences are generated in situ.
[0318] In a preferred embodiment, the modified nucleoside is
converted to the phosphoramidite or H-phosphonate form, which are
then used in solid-phase or solution syntheses of oligonucleotides.
In this way the modified nucleoside, either for attachment at the
ribose (i.e. amino- or thiol-modified nucleosides) or the base, is
incorporated into the oligonucleotide at either an internal
position or the 5' terminus. This is generally done in one of two
ways. First, the 5' position of the ribose is protected with
4',4-dimethoxytrityl (DMT) followed by reaction with either
2-cyanoethoxy-bis-diisopropylaminophosphine in the presence of
diisopropylammonium tetrazolide, or by reaction with
chlorodiisopropylamino 2'-cyanoethyoxyphosphine, to give the
phosphoramidite as is known in the art; although other techniques
may be used as will be appreciated by those in the art. See Gait,
supra; Caruthers, Science 230:281 (1985), both of which are
expressly incorporated herein by reference.
[0319] For attachment of a group to the 3' terminus, a preferred
method utilizes the attachment of the modified nucleoside (or the
nucleoside replacement) to controlled pore glass (CPG) or other
oligomeric supports. In this embodiment, the modified nucleoside is
protected at the 5' end with DMT, and then reacted with succinic
anhydride with activation. The resulting succinyl compound is
attached to CPG or other oligomeric supports as is known in the
art. Further phosphoramidite nucleosides are added, either modified
or not, to the 5' end after deprotection. Thus, the present
invention provides conductive oligomers or insulators covalently
attached to nucleosides attached to solid oligomeric supports such
as CPG, and phosphoramidite derivatives of the nucleosides of the
invention.
[0320] The invention further provides methods of making label
probes with recruitment linkers comprising ETMs. These synthetic
reactions will depend on the character of the recruitment linker
and the method of attachment of the ETM, as will be appreciated by
those in the art. For nucleic acid recruitment linkers, the label
probes are generally made as outlined herein with the incorporation
of ETMs at one or more positions. When a transition metal complex
is used as the ETM, synthesis may occur in several ways. In a
preferred embodiment, the ligand(s) are added to a nucleoside,
followed by the transition metal ion, and then the nucleoside with
the transition metal complex attached is added to an
oligonucleotide, i.e. by addition to the nucleic acid synthesizer.
Alternatively, the ligand(s) may be attached, followed by
incorportation into a growing oligonucleotide chain, followed by
the addition of the metal ion.
[0321] In a preferred embodiment, ETMs are attached to a ribose of
the ribose-phosphate backbone. This is generally done as is
outlined herein for conductive oligomers, as described herein, and
in PCT publication WO 95/15971, using amino-modified or
oxo-modified nucleosides, at either the 2' or 3' position of the
ribose. The amino group may then be used either as a ligand, for
example as a transition metal ligand for attachment of the metal
ion, or as a chemically functional group that can be used for
attachment of other ligands or organic ETMs, for example via amide
linkages, as will be appreciated by those in the art. For example,
the examples describe the synthesis of nucleosides with a variety
of ETMs attached via the ribose.
[0322] In a preferred embodiment, ETMs are attached to a phosphate
of the ribose-phosphate-backbone. As outlined herein, this may be
done using phosphodiester analogs such as phosphoramidite bonds,
see generally PCT publication WO 95/15971, or can be done in a
similar manner to that described in PCT US97/20014, where the
conductive oligomer is replaced by a transition metal ligand or
complex or an organic ETM.
[0323] Attachment to alternate backbones, for example peptide
nucleic acids or alternate phosphate linkages will be done as will
be appreciated by those in the art.
[0324] In a preferred embodiment, ETMs are attached to a base of
the nucleoside. This may be done in a variety of ways. In one
embodiment, amino groups of the base, either naturally occurring or
added as is described herein (see the fiigures, for example), are
used either as ligands for transition metal complexes or as a
chemically functional group that can be used to add other ligands,
for example via an amide linkage, or organic ETMs. This is done as
will be appreciated by those in the art. Alternatively, nucleosides
containing halogen atoms attached to the heterocyclic ring are
commercially available. Acetylene linked ligands may be added using
the halogenated bases, as is generally known; see for example,
Tzalis et al., Tetrahedron Left. 36(34):6017-6020 (1995); Tzalis et
al., Tetrahedron Left. 36(2):3489-3490 (1995); and Tzalis et al.,
Chem. Communications (in press) 1996, all of which are hereby
expressly incorporated by reference. See also the figures and the
examples, which describes the synthesis of metallocenes (in this
case, ferrocene) attached via acetylene linkages to the bases.
[0325] In one embodiment, the nucleosides are made with transition
metal ligands, incorporated into a nucleic acid, and then the
transition metal ion and any remaining necessary ligands are added
as is known in the art. In an alternative embodiment, the
transition metal ion and additional ligands are added prior to
incorporation into the nucleic acid.
[0326] Once the nucleic acids of the invention are made, with a
covalently attached attachment linker (i.e. either an insulator or
a conductive oligomer), the attachment linker is attached to the
electrode. The method will vary depending on the type of electrode
used. As is described herein, the attachment linkers are generally
made with a terminal "A" linker to facilitate attachment to the
electrode. For the purposes of this application, a sulfur-gold
attachment is considered a covalent attachment.
[0327] In a preferred embodiment, conductive oligomers, insulators,
and attachment linkers are covalently attached via sulfur linkages
to the electrode. However, surprisingly, traditional protecting
groups for use of attaching molecules to gold electrodes are
generally not ideal for use in both synthesis of the compositions
described herein and inclusion in oligonucleotide synthetic
reactions. Accordingly, the present invention provides novel
methods for the attachment of conductive oligomers to gold
electrodes, utilizing unusual protecting groups, including
ethylpyridine, and trimethylsilylethyl as is depicted in the
Figures. However, as will be appreciated by those in the art, when
the conductive oligomers do not contain nucleic acids, traditional
protecting groups such as acetyl groups and others may be used. See
Greene et al., supra.
[0328] This may be done in several ways. In a preferred embodiment,
the subunit of the conductive oligomer which contains the sulfur
atom for attachment to the electrode is protected with an
ethyl-pyridine or trimethylsilylethyl group. For the former, this
is generally done by contacting the subunit containing the sulfur
atom (preferably in the form of a sulfhydryl) with a vinyl pyridine
group or vinyl trimethylsilylethyl group under conditions whereby
an ethylpyridine group or trimethylsilylethyl group is added to the
sulfur atom.
[0329] This subunit also generally contains a functional moiety for
attachment of additional subunits, and thus additional subunits are
attached to form the conductive oligomer. The conductive oligomer
is then attached to a nucleoside, and additional nucleosides
attached. The protecting group is then removed and the sulfur-gold
covalent attachment is made. Alternatively, all or part of the
conductive oligomer is made, and then either a subunit containing a
protected sulfur atom is added, or a sulfur atom is added and then
protected. The conductive oligomer is then attached to a
nucleoside, and additional nucleosides attached. Alternatively, the
conductive oligomer attached to a nucleic acid is made, and then
either a subunit containing a protected sulfur atom is added, or a
sulfur atom is added and then protected. Alternatively, the ethyl
pyridine protecting group may be used as above, but removed after
one or more steps and replaced with a standard protecting group
like a disulfide. Thus, the ethyl pyridine or trimethylsilylethyl
group may serve as the protecting group for some of the synthetic
reactions, and then removed and replaced with a traditional
protecting group.
[0330] By "subunit" of a conductive polymer herein is meant at
least the moiety of the conductive oligomer to which the sulfur
atom is attached, although additional atoms may be present,
including either functional groups which allow the addition of
additional components of the conductive oligomer, or additional
components of the conductive oligomer. Thus, for example, when
Structure 1 oligomers are used, a subunit comprises at least the
first Y group.
[0331] A preferred method comprises 1) adding an ethyl pyridine or
trimethylsilylethyl protecting group to a sulfur atom attached to a
first subunit of a conductive oligomer, generally done by adding a
vinyl pyridine or trimethylsilylethyl group to a sulfhydryl; 2)
adding additional subunits to form the conductive oligomer; 3)
adding at least a first nucleoside to the conductive oligomer; 4)
adding additional nucleosides to the first nucleoside to form a
nucleic acid; 5) attaching the conductive oligomer to the gold
electrode. This may also be done in the absence of nucleosides, as
is described in the Examples.
[0332] The above method may also be used to attach insulator
molecules to a gold electrode.
[0333] In a preferred embodiment, a monolayer comprising surface
species (including conductive oligomers and insulators) is added to
the electrode. Generally, the chemistry of addition is similar to
or the same as the addition of conductive oligomers to the
electrode, i.e. using a sulfur atom for attachment to a gold
electrode, etc. Compositions comprising monolayers in addition to
the conductive oligomers covalently attached to nucleic acids may
be made in at least one of five ways: (1) addition of the
monolayer, followed by subsequent addition of the
attachment-linker-nucleic acid complex; (2) addition of the
attachment linker-nucleic acid complex followed by addition of the
monolayer; (3) simultaneous addition of the monolayer and
attachment linker-nucleic acid complex; (4) formation of a
monolayer (using any of 1, 2 or 3) which includes attachment
linkers which terminate in a functional moiety suitable for
attachment of a completed nucleic acid; or (5) formation of a
monolayer which includes attachment linkers which terminate in a
functional moiety suitable for nucleic acid synthesis, i.e. the
nucleic acid is synthesized on the surface of the monolayer as is
known in the art. Such suitable functional moieties include, but
are not limited to, nucleosides, amino groups, carboxyl groups,
protected sulfur moieties, or hydroxyl groups for phosphoramidite
additions. The examples describe the formation of a monolayer on a
gold electrode using the preferred method (1).
[0334] In a preferred embodiment, the nucleic acid is a peptide
nucleic acid or analog. In this embodiment, the invention provides
peptide nucleic acids with at least one covalently attached ETM or
attachment linker. In a preferred embodiment, these moieties are
covalently attached to an monomeric subunit of the PNA. By
"monomeric subunit of PNA" herein is meant the
--NH--CH.sub.2CH.sub.2--N(COCH.sub.2-Base)-CH.sub.2--CO-- monomer,
or derivatives (herein included within the definition of
"nucleoside") of PNA. For example, the number of carbon atoms in
the PNA backbone may be altered; see generally Nielsen et al.,
Chem. Soc. Rev. 1997 page 73, which discloses a number of PNA
derivatives, herein expressly incorporated by reference. Similarly,
the amide bond linking the base to the backbone may be altered;
phosphoramide and sulfuramide bonds may be used. Alternatively, the
moieties are attached to an internal monomeric subunit. By
"internal" herein is meant that the monomeric subunit is not either
the N-terminal monomeric subunit or the C-terminal monomeric
subunit. In this embodiment, the moieties can be attached either to
a base or to the backbone of the monomeric subunit. Attachment to
the base is done as outlined herein or known in the literature. In
general, the moieties are added to a base which is then
incorporated into a PNA as outlined herein. The base may be either
protected, as required for incorporation into the PNA synthetic
reaction, or derivatized, to allow incorporation, either prior to
the addition of the chemical substituent or afterwards. Protection
and derivatization of the bases is shown in PCT US97/20014. The
bases can then be incorporated into monomeric subunits.
[0335] In a preferred embodiment, the moieties are covalently
attached to the backbone of the PNA monomer. The attachment is
generally to one of the unsubstituted carbon atoms of the monomeric
subunit, preferably the .alpha.-carbon of the backbone, although
attachment at either of the carbon 1 or 2 positions, or the
.alpha.-carbon of the amide bond linking the base to the backbone
may be done. In the case of PNA analogs, other carbons or atoms may
be substituted as well. In a preferred embodiment, moieties are
added at the .alpha.-carbon atoms, either to a terminal monomeric
subunit or an internal one.
[0336] In this embodiment, a modified monomeric subunit is
synthesized with an ETM or an attachment linker, or a functional
group for its attachment, and then the base is added and the
modified monomer can be incorporated into a growing PNA chain.
[0337] Once generated, the monomeric subunits with covalently
attached moieties are incorporated into a PNA using the techniques
outlined in Will et al., Tetrahedron 51(44):12069-12082 (1995), and
Vanderlaan et al., Tett. Let. 38:2249-2252 (1997), both of which
are hereby expressly incorporated in their entirety. These
procedures allow the addition of chemical substituents to peptide
nucleic acids without destroying the chemical substituents.
[0338] As will be appreciated by those in the art, electrodes may
be made that have any combination of nucleic acids, conductive
oligomers and insulators.
[0339] The compositions of the invention may additionally contain,
in addition to ETMs, one or more other labels at any position. By
"label" herein is meant an element (e.g. an isotope) or chemical
compound that is attached to enable the detection of the compound.
Preferred labels are radioactive isotopic labels, colored or
fluorescent dyes and electrochemical labels such as ETMs. The
labels may be incorporated into the compound at any position. In
addition, the compositions of the invention may also contain other
moieties such as cross-linking agents to facilitate cross-linking
of the target-probe complex. See for example, Lukhtanov et al.,
Nucl. Acids. Res. 24(4):683 (1996) and Tabone et al., Biochem.
33:375 (1994), both of which are expressly incorporated by
reference.
[0340] Once made, the compositions find use in a number of
applications, as described herein. In particular, the compositions
of the invention find use in binding assays for the detection of
target analytes, in particular nucleic acid target sequences. As
will be appreciated by those in the art, electrodes can be made
that have a single species of binding ligand, or multiple binding
ligand species, i.e. in an array format.
[0341] In addition, as outlined above, the use of a solid support
such as an electrode enables the use of these assays in an array
form.
[0342] Once the assay complexes of the invention are made, that
generally minimally comprise a capture probe, a target analyte 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,
including via the ".pi.-way".
[0343] 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.
[0344] 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.
[0345] 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.
[0346] 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.
[0347] 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.
[0348] Luminol has the added benefit of becoming a chemiluminiscent
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.
[0349] Suitable electron source molecules are well known in the
art, and include, but are not limited to, ferricyanide, and
luminol.
[0350] 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.
[0351] 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 impedence. 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.
[0352] 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 pp197-202).
These differences can be monitored using a spectrophotometer or
simple photomultiplier tube device.
[0353] 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.
[0354] 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.
[0355] 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.
[0356] 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.
[0357] 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.
[0358] In a further embodiment, electrochemiluminescence is used as
the basis of the electron transfer detection. With some ETMs such
as Ru.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.
[0359] In a preferred embodiment, electronic detection is used,
including amperommetry, voltammetry, capacitance, and impedence.
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.
[0360] 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.
[0361] 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.
[0362] 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 capicitance) 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.
[0363] 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.
[0364] 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 (I) 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.
[0365] 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: 1 i avg =
2 nfFN total sinh [ n F RT E A C ] cosh [ n F RT E A C ] + cosh [ n
F RT ( E D C - E O ) ] Equation 1 37
[0366] 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.0 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.
[0367] In addition, the faradaic current can also be expressed as a
function of time, as shown in Equation 2: 2 I f ( t ) = q e N total
n F 2 RT ( cosh [ n F RT ( V ( t ) - E 0 ) ] + 1 ) - V ( t ) t
Equation 2
[0368] <.vertline.<BOX45>.vertline.>.vertline..sub.F is
the Faradaic current and q.sub.e is the elementary charge.
[0369] 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.
Equation 3
i.sub.AC=f(Nernst factors)f(k.sub.ET)f(instrument factors)
[0370] 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.
[0371] 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.
[0372] 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.
[0373] 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.
[0374] The use of combinations of AC and DC signals gives a variety
of advantages, including surprising sensitivity and signal
maximization.
[0375] 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.
[0376] 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.
[0377] 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.
[0378] 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.
[0379] In a preferred embodiment, measurements of the system are
taken at 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.
[0380] 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.
[0381] 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.
[0382] 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.
[0383] In a preferred embodiment, measurements of the system are
taken at 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 at
least two, preferably at least about five, and more preferably at
least about ten frequencies.
[0384] 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.
[0385] 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.
[0386] 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.
[0387] 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.
[0388] 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 adds 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 signficantly different depending on
whether the label probes containing the ETMs are specifically or
non-specifically bound to the electrode.
[0389] Accordingly, the present invention further provides
electronic devices or apparatus for the detection of analytes using
the compositions of the invention. The apparatus includes a test
chamber for receiving a sample solution 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 electrophoresis electrodes may be in electrical
contact.
[0390] In a preferred embodiment, the apparatus also includes
detection electrodes comprising a single stranded nucleic acid
capture probe covalently attached via an attachment linker, and a
monolayer comprising conductive oligomers, such as are described
herein.
[0391] 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.
[0392] 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.
[0393] Thus, the compositions of the present invention may be used
in a variety of research, clinical, quality control, or field
testing settings as outlined herein.
[0394] All references cited herein are incorporated by reference in
their entirety.
* * * * *