U.S. patent application number 10/823502 was filed with the patent office on 2005-01-06 for target analyte detection using asymmetrical self-assembled monolayers.
Invention is credited to Tao, Chunlin, Yu, Changjun.
Application Number | 20050003398 10/823502 |
Document ID | / |
Family ID | 26896313 |
Filed Date | 2005-01-06 |
United States Patent
Application |
20050003398 |
Kind Code |
A1 |
Tao, Chunlin ; et
al. |
January 6, 2005 |
Target analyte detection using asymmetrical self-assembled
monolayers
Abstract
The present invention relates to the use asymmetric monolayer
forming species and electroconduit forming species to detect target
analytes.
Inventors: |
Tao, Chunlin; (Beverly
Hills, CA) ; Yu, Changjun; (Pasadena, CA) |
Correspondence
Address: |
DORSEY & WHITNEY LLP
INTELLECTUAL PROPERTY DEPARTMENT
4 EMBARCADERO CENTER
SUITE 3400
SAN FRANCISCO
CA
94111
US
|
Family ID: |
26896313 |
Appl. No.: |
10/823502 |
Filed: |
April 12, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10823502 |
Apr 12, 2004 |
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09847113 |
May 1, 2001 |
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6753143 |
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10823502 |
Apr 12, 2004 |
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09626096 |
Jul 26, 2000 |
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60201026 |
May 1, 2000 |
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Current U.S.
Class: |
435/6.11 ;
436/525 |
Current CPC
Class: |
B82Y 15/00 20130101;
G01N 2610/00 20130101; B82Y 40/00 20130101; G01N 33/5438 20130101;
B82Y 5/00 20130101; G01N 33/553 20130101; B82Y 30/00 20130101 |
Class at
Publication: |
435/006 ;
436/525 |
International
Class: |
C12Q 001/68; G01N
033/553 |
Claims
We claim:
1. A method of modifying a metallic surface comprising contacting
the metallic surface with an asymmetric monolayer forming species
having the formula: 41wherein A is an attachment linker moiety; MFS
is a monlayer forming species; and AG is an electroconduit forming
species.
2. A method according to claim 1 further comprising contacting said
metallic surface with a biological species having the formula:
A-MFS-capture binding ligand wherein A is an attachment linker; and
MFS is a monolayer forming species.
3. A method according to claim 2 wherein said capture binding
ligand is a nucleic acid.
4. A method according to claim 2 wherein said capture binding
ligand is a n protein.
5. A method according to claim 1 wherein A is sulfur.
6. A method according to claim 1 wherein said metallic surface is
gold.
7. A method according to claim 1 wherein said MFS is an
insulator.
8. A method according to claim 7 wherein said insulator comprises
an alkyl group from about 7 to 20 carbons.
9. A method according to claim 8 wherein said alkyl group comprises
a heteroalkyl.
10. A method according to claim 8 wherein said alkyl group
comprises a substituted alkyl.
11. A method according to claim 1 wherein said AG comprises an
alkyl group from about 1 to 6 carbons.
12. A method according to claim 1 or 11 wherein said AG is
branched, having the formula: 42wherein R.sub.3 through R.sub.5 are
independently selected from the group consisting of hydrogen,
alkyl, aryl, alcohol, amine, amido, nitro, ether, ester, ketone,
imino, aldehyde, alkoxy, carbonyl, halogen, sulfur containing
moiety and phosphorus containing moiety;
13. A method according to claim 12 wherein said AG is attached to
said attachment linker via a (CH.sub.2).sub.n group, wherein n is
an integer from 0 to 4.
14. A method according to claim 12 wherein said AG is attached
directly to said attachment linker.
Description
[0001] This application is a continuation of U.S. Ser. No.:
09/847,113, filed May 1, 2001, which claims the benefit of U.S.
Ser. No. 60/201,026, filed May 1, 2000 and is a
continuation-in-part application of U.S. Ser. No. 09/626,096, filed
Jul. 26, 2000.
FIELD OF THE INVENTION
[0002] The present invention relates to the use asymmetric
monolayer forming species and electroconduit forming species to
detect target analytes.
BACKGROUND OF THE INVENTION
[0003] There are a number of assays and sensors for the detection
of the presence and/or concentration of specific substances in
fluids and gases. Many of these rely on specific ligand/antiligand
reactions as the mechanism of detection. That is, pairs of
substances (i.e. the binding pairs or ligand/antiligands) are known
to bind to each other, while binding little or not at all to other
substances. This has been the focus of a number of techniques that
utilize these binding pairs for the detection of the complexes.
These generally are done by labeling one component of the complex
in some way, so as to make the entire complex detectable, using,
for example, radioisotopes, fluorescent and other optically active
molecules, enzymes, etc.
[0004] Other assays rely on electronic signals for detection. Of
particular interest are biosensors. At least two types of
biosensors are known; enzyme-based or metabolic biosensors and
binding or bioaffinity sensors. See for example U.S. Pat. Nos.
4,713,347; 5,192,507; 4,920,047; 3,873,267; and references
disclosed therein. While some of these known sensors use
alternating current (AC) techniques, these techniques are generally
limited to the detection of differences in bulk (or dielectric)
impedance.
[0005] The use of self-assembled monolayers (SAMs) on surfaces for
binding and detection of biological molecules has recently been
explored. See for example WO98/20162; PCT US98/12430; PCT
US98/12082; PCT US99/01705; PCT/US99/21683; PCT/US99/10104;
PCT/US99/01703; PCT/US00/31233; U.S. Pat. Nos. 5,620,850;
6,197,515; 6,013,459; 6,013,170; and 6,065,573; and references
cited therein.
[0006] Accordingly, it is an object of the invention to provide
novel methods and compositions for the electronic detection of
target analytes using self-assembled monolayers.
SUMMARY OF THE INVENTION
[0007] In accordance with the objects outlined above, the present
invention provides compositions comprising metallic surfaces
comprising asymmetric monolayer forming species comprising two
components. One of the components is a standard monolayer forming
species, such an alkyl chain. The other component is an
electroconduit forming species. Electroconduit forming species are
short chain alkyl groups, which may be branched.
[0008] In a further embodiment, the invention provides methods of
detecting a target analyte in a test sample comprising attaching
said target analyte to a metallic surface comprising asymmetric
monolayer forming species via binding to a capture binding ligand.
Recruitment linkers, or label probes are directly or indirectly
attached to the target analyte to form an assay complex. The method
further comprises detecting electron transfer between an electron
transfer moiety and an electrode.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIGS. 1A-1R depict a number of different compositions of the
invention. FIG. 1A depicts I, also referred to as P290. FIG. 1B
depicts II, also referred to as P291. FIG. 1C depicts III, also
referred to as W31. FIG. 1D depicts IV, also referred to as N6.
FIG. 1E depicts V, also referred to as P292. FIG. 1F depicts II,
also referred to as C23. FIG. 1G depicts VII, also referred to as
C15. FIG. 1H depicts VIII, also referred to as C95. FIG. 1I depicts
Y63. FIG. 1J depicts another compound of the invention. FIG. 1K
depicts N11. FIG. 1L depicts C131, with a phosphoramidite group and
a DMT protecting group. FIG. 1M depicts W38, also with a
phosphoramidite group and a DMT protecting group. FIG. 1N depicts
the commercially available moiety that enables "branching" to
occur, as its incorporation into a growing oligonucleotide chain
results in addition at both the DMT protected oxygens. FIG. 10
depicts gIen, also with a phosphoramidite group and a DMT
protecting group, that serves as a non-nucleic acid linker. FIGS.
1A to 1G and 1J are shown without the phosphoramidite and
protecting groups (i.e. DMT) that are readily added.
[0010] FIGS. 2A, 2B and 2C depict some useful disulfide
embodiments. FIG. 2A depicts one example of a general class of an
asymmetric monolayer forming species. FIG. 2B depicts two
embodiments that were used to generate the data shown in FIG. 2C.
In FIG. 2C, M44 is a standard monolayer forming species; the
structure of M44 is shown in Figure
[0011] FIGS. 3A, 3B, 3C, 3D, 3E, 3F and 3G depict the synthesis of
some disulfide embodiments. FIG. 3A depicts the general synthesis;
with R, R' and R" being C1 to C20 alkyl or aromatic derivatives and
B being any base such as HaOH, KOH, LiOH or MOR, with M being a
metal. FIG. 3B shows the synthesis of H-phosphonate, FIGS. 3C and
3D show the synthesis of the CPG derivative, and FIG. 3E shows the
synthesis of the insulator CT105. 3F and 3G depict some cyclic
disulfide embodiments.
[0012] FIGS. 4A, 4B and 4C depict three preferred embodiments for
attaching a target nucleic acid sequence to the electrode. 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. The electrode 105
comprises a monolayer 107 comprising asymmetric monolayer forming
species, i.e., alkyl chains 400 and electroconduit forming species
410 (which may be short branched alkyl chains, short alkyl chains,
or a mixture of branched and short alkyl chains). 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. As will be appreciated by those in the art, while these
systems depict nucleic acid targets, these attachment
configurations may be used with non-nucleic acid capture binding
ligands.
[0013] FIGS. 5A, 5B, 5C and 5D depict several embodiments of the
invention. FIG. 5A is directed to the use of a capture binding
ligand 200 attached via an attachment linker 106 to the electrode
105. Target analyte 210 binds to the capture binding ligand 200,
and a solution binding ligand 22 with a directly attached
recruitment linker 230 with ETMs 135. FIG. 5B depicts a similar
embodiment using an indirectly attached recruitment linker 145 that
binds to a second portion 240 of the solution binding ligand 220.
FIG. 5C depicts the use of an anchor ligand 100 (referred to herein
as an anchor probe when the ligand comprises nucleic acid) to bind
the capture binding ligand 200 comprising a portion 120 that will
bind to the anchor probe 100. As will be appreciated by those in
the art, any of the FIG. 4 embodiments may be used here as well.
FIG. 5D depicts the use of an amplifier probe 145.
[0014] FIGS. 6A and 6B show two competitive type assays of the
invention. FIG. 6A utilizes the replacement of a target analyte 210
with a target analyte analog 310 comprising a directly attached
recruitment linker 145. As will be appreciated by those in the art,
an indirectly attached recruitment linker can also be used, as
shown in FIG. 6B. FIG. 6B shows a competitive assay wherein the
target analyte 210 and the target analyte analog 310 attached to
the surface compete for binding of a solution binding ligand 220
with a directly attached recruitment linker 145 (again, an
indirectly attached recruitment linker can also be used, as shown
in FIG. 5B). In this case, a loss of signal may be seen.
[0015] FIGS. 7A-7R depict nucleic acid detection systems. FIGS. 7A
and 7B have the target sequence 5 containing the ETMs 6; 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. 7A
shows attachment of a capture probe 10 to the electrode 20 via a
linker 15, which as discussed herein can be either a conductive
oligomer or an insulator. The target sequence 5 contains ETMs 6.
FIG. 7B depicts the use of a capture extender probe 11, comprising
a first portion 12 that hybridizes to a portion of the target
sequence and a second portion 13 that hybridizes to the capture
probe 10.
[0016] FIG. 7C depicts the use of two different capture probes 10
and 10', that hybridize to different portions of the target
sequence 5. As will be appreciated by those in the art, the 5'-3'
orientation of the two capture probes in this embodiment is
different.
[0017] FIGS. 7D to 7H depict the use of label probes 40 that
hybridize directly to the target sequence 5. FIG. 7D shows the use
of a label probe 40, comprising a first portion 41 that hybridizes
to a portion of the target sequence 5, a second portion 42 that
hybridizes to the capture probe 10 and a recruitment linker 50
comprising ETMs 6. A similar embodiment is shown in FIG. 7E, where
the label probe 40 has an additional recruitment linker 50. FIG. 7F
depicts a label probe 40 comprising a first portion 41 that
hybridizes to a portion of the target sequence 5 and a recruitment
linker 50 with attached ETMs 6. The parentheses highlight that for
any particular target sequence 5 more than one label probe 40 may
be used, with n being an integer of at least 1. FIG. 7G depicts the
use of the FIG. 7E label probe structures but includes the use of a
single capture extender probe 11, with a first portion 12 that
hybridizes to a portion of the target sequence and a second portion
13 that hybridizes to the capture probe 10. FIG. 7H depicts the use
of the FIG. 7F label probe structures but utilizes two capture
extender probes 11 and 16. The first capture extender probe 11 has
a first portion 12 that hybridizes to a portion of the target
sequence 5 and a second portion 13 that hybridizes to a first
portion 14 of the capture probe 10. The second capture extender
probe 16 has a first portion 18 that hybridizes to a second portion
of the target sequence 5 and a second portion 17 that hybridizes to
a second portion 19 of the capture probe 10.
[0018] FIGS. 7I, 7J and 7K depict systems utilizing label probes 40
that do not hybridize directly to the target, but rather to
amplifier probes. Thus the amplifier probe 60 has a first portion
65 that hybridizes to the target sequence 5 and at least one second
portion 70, i.e. the amplifier sequence, that hybridizes to the
first portion 41 of the label probe.
[0019] FIGS. 7L, 7M and 7N depict systems that utilize a first
label extender probe 80. In these embodiments, the label extender
probe 80 has a first portion 81 that hybridizes to a portion of the
target sequence 5, and a second portion 82 that hybridizes to the
first portion 65 of the amplifier probe 60.
[0020] FIG. 7O depicts the use of two label extender probes 80 and
90. The first label extender probe 80 has a first portion 81 that
hybridizes to a portion of the target sequence 5, and a second
portion 82 that hybridizes to a first portion 62 of the amplifier
probe 60. The second label extender probe 90 has a first portion 91
that hybridizes to a second portion of the target sequence 5 and a
second portion 92 that hybridizes to a second portion 61 of the
amplifier probe 60.
[0021] FIG. 7P depicts a system utilizing a label probe 40
hybridizing to the terminus of a target sequence 5.
[0022] FIGS. 7Q and 7R depict systems that utilizes multiple label
probes. The first portion 41 of the label probe 40 can hybridize to
all (FIG. 7R) or part (FIG. 7Q) of the recruitment linker 50. FIG.
8 depicts a detection system with a label probe labeled with
multiple ETMs, in which a first portion hybridizes to a portion of
a target sequence and a capture probe that hybridizes to a
different portion of the target sequence.
[0023] FIG. 9 depicts the chemical structures of a standard
monolayer forming species, M44 and two asymmetrical monolayer
forming species, CT99 and CT105.
[0024] FIG. 10 depicts an example of a layout for an array chip
with sensor pads. FIG. 11 depicts the electrochemical response of
asymmetric monolayer forming species vs a standard monolayer
forming species in a direct assay using a 2 N6 ferrocene signaling
probe.
[0025] FIG. 12 depicts the electrochemical response of asymmetric
monolayer forming species vs a standard monolayer forming species a
sandwich assay using 8 N6 ferrocene.
[0026] FIG. 13 depicts the electrochemical response of asymmetric
monolayer forming species versus a standard monolayer forming
species a sandwich assay using a 20 C23 type ferrocene.
[0027] FIG. 14 depicts the electrochemical response of asymmetric
monolayer forming species vs a standard monolayer forming species a
sandwich assay using a 54 C23 type ferrocene.
[0028] FIG. 15 depicts the nonspecific binding of asymmetric
monolayer forming species versus a standard monolayer forming
species in a direct assay at 1000 Hz and 4.sup.th harmonics.
[0029] FIG. 16 depicts the nonspecific binding of asymmetric
monolayer forming species versus a standard monolayer forming
species in a sandwich assay at 1000 Hz and 4.sup.th harmonics.
[0030] FIG. 17 depicts a monolayer comprising insulators only (i.e.
M44) and a monolayer comprising asymmetric monolayer forming
species (i.e. CT105).
[0031] FIG. 18 depicts the frequency response for D1085 of two N6
ferrocenes.
[0032] FIG. 19 depicts the frequency response for a sandwich assay
of an 8 ferrocene system.
[0033] FIG. 20 depicts the frequency response for a sandwich assay
of an 20 ferrocene system.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention is directed to the electronic
detection of analytes. Previous work, described in PCT US97/20014,
is directed to the detection of nucleic acids, and utilizes nucleic
acids covalently attached to electrodes using conductive oligomers,
i.e. chemical "wires". Upon formation of double stranded nucleic
acids containing electron transfer moieties (ETMs), electron
transfer can proceed through the stacked ri-orbitals of the
heterocyclic bases to the electrode, thus enabling electronic
detection of target nucleic acids (termed "mechanism 1"). In the
absence of the stacked n-orbitals, i.e. when the target strand is
not present, electron transfer is negligible, thus allowing the use
of the system as an assay. This previous work also reported on the
use of self-assembled monolayers (SAMs) to electronically shield
the electrodes from solution components and significantly decrease
the amount of non-specific binding to the electrodes.
[0035] Alternatively, the ETM can be detected, not necessarily via
electron transfer through nucleic acid, but rather can be directly
detected on an electrode comprising a SAM; that is, the electrons
from the ETMs need not travel through the stacked n 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. Upon
binding of a target analyte to a binding species on the surface, a
recruitment linker or label probe comprising at least one ETM is
brought to the surface, and detection of the ETM can proceed. Thus,
the role of the target analyte and-binding species is to provide
specificity for a recruitment of ETMs to the surface, where they
can be detected using the electrode. The role of the asymmetric
monolayer species 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. See, for example, WO98/20162; PCT US98/12430; PCT
US98/12082; PCT US99/01705; PCT/US99/21683; PCT/US99/10104;
PCT/US99/01703; PCT/US00/20476; PCT/US00/31233; U.S. Pat. Nos.
5,620,850; 6,197,515; 6,013,459; 6,013,170; and 6,065,573; and U.S.
Ser. Nos. 09/660,374; and, 09/135,183 and references cited
therein.
[0036] Thus, the present invention is directed to novel
compositions of monolayer forming species that are thought to form
electroconduits; that is, the present invention is directed to the
use of monolayers comprising asymmetric monolayer forming species
("AMFS"). As described more fully below, AMFS comprise two
components, usually linked by a disulfide bridge, at least one of
which is a standard monolayer forming species such as an alkyl
chain, and the other is a shorter species, for example a shorter
alkyl chain or a short branched chain. These two elements are put
down together, for example by attaching them as a disulfide moiety
that then is used to form a monolayer on a metallic surface such as
gold. The "shorter" element thus, is thought to form an
electroconduit, and protect the surface from redox-active species
in solution.
[0037] 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.
[0038] Asymmetric monolayer forming species preferably comprise a
mixture of insulators and electroconduit forming species, although
conductive oligomers as either component, also may be included.
Preferably, the insulators are long chain alkyl groups from about 7
to 20 carbons in length which are covalently attached to a metallic
surface via a linker moiety such as sulfur. Electroconduit forming
species include alkyl groups, phenyl-acetylene-polyethylene glycol
species, and branched alkyl groups. In addition asymmetric
monolayer forming species include asymmetrical SAM-forming
disulfide species such as depicted in FIG. 3.
[0039] The invention can be generally described as follows, with a
number of possible embodiments depicted in the Figures. In a
preferred embodiment, as depicted in FIG. 5, an electrode
comprising an asymmetric monolayer forming species comprising
insulators (preferably a long chain alkyl group), an electroconduit
forming species, and a covalently attached target analyte binding
ligand (frequently referred to herein as a "capture binding
ligand") is made. The target analyte is added, which binds to the
support-bound binding ligand. A solution binding ligand is added,
which may be the same or different from the first binding ligand,
which can also bind to the target analyte, forming a "sandwich" of
sorts. The solution binding ligand either comprises a recruitment
linker containing ETMs, or comprises a portion that will either
directly or indirectly bind a recruitment linker containing the
ETMs. This "recruitment" of ETMs to the surface of the monolayer
allows electronic detection via electron transfer between the ETM
and the electrode. In the absence of the target analyte, the
recruitment linker is either washed away or not in sufficient
proximity to the surface to allow detection.
[0040] For example, when both the target analyte and the capture
binding ligand (generally referred to herein as a "capture probe"
when it is a nucleic acid) are nucleic acids, a preferred
embodiment is shown in FIG. 8. In this embodiment, the surface
comprises an AMFS and a capture probe. A first portion of the
target sequence hybridizes to the capture probe, and a label probe,
comprising a recruitment linker comprising ETMs, hybridizes to a
second portion of the target sequence.
[0041] In an alternate preferred embodiment, as depicted in FIG. 6,
a competitive binding type assay is run. In this embodiment, the
target analyte in the sample is replaced by a target analyte analog
as is described below and generally known in the art. The analog
comprises a directly or indirectly attached recruitment linker
comprising at least one ETM. The binding of the analog to the
capture binding ligand recruits the ETM to the surface and allows
detection based on electron transfer between the ETM and the
electrode.
[0042] In an additional preferred embodiment, as depicted in FIG.
6B, a competitive assay wherein the target analyte and a target
analyte analog attached to the surface compete for binding of a
solution binding ligand with a directly or indirectly attached
recruitment linker. In this case, a loss of signal may be seen.
[0043] Accordingly, the present invention provides methods and
compositions useful in the detection of target analytes. As will be
appreciated by those in the art, the sample solution may comprise
any number of things, including, but not limited to, bodily fluids
(including, but not limited to, blood, urine, serum, lymph, saliva,
anal and vaginal secretions, perspiration and semen, of virtually
any organism, with mammalian samples being preferred and human
samples being particularly preferred); environmental samples
(including, but not limited to, air, agricultural, water and soil
samples); biological warfare agent samples; research samples (i.e.
in the case of nucleic acids, the sample may be the products of an
amplification reaction, including both target and signal
amplification as is generally described in PCT/US99/01705, such as
PCR amplification reaction); purified samples, such as purified
genomic DNA, RNA, proteins, etc.; raw samples (bacteria, virus,
genomic DNA, etc.). As will be appreciated by those in the art,
virtually any experimental manipulation may have been done on the
sample.
[0044] 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.
[0045] By "proteins" or grammatical equivalents herein is meant
proteins, oligopeptides and peptides, and analogs, including
proteins containing non-naturally occurring amino acids and amino
acid analogs, and peptidomimetic structures.
[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.
[0047] By "nucleic acid" or "oligonucleotide" or grammatical
equivalents herein means at least two nucleotides covalently linked
together. A nucleic acid of the present invention will generally
contain phosphodiester bonds, although in some cases, as outlined
below, nucleic acid analogs are included that may have alternate
backbones, comprising, for example, phosphoramide (Beaucage et al.,
Tetrahedron 49(10):1925 (1993) and references therein; Letsinger,
J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem.
81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986);
Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem.
Soc. 1 10:4470 (1988); and Pauwels et al., Chemica Scripta 26:141
91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437
(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et
al., J. Am. Chem. Soc. 111:2321 (1989), O-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); those with bicyclic structures
including locked nucleic acids, Koshkin et al., J. Am. Chem. Soc.
120:13252-3 (1998); 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.
[0048] 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. Alternatively, mixtures of different nucleic acid
analogs, and mixtures of naturally occurring nucleic acids and
analogs may be made.
[0049] Particularly preferred are peptide nucleic acids (PNA) which
includes peptide nucleic acid analogs. These backbones are
substantially non-ionic under neutral conditions, in contrast to
the highly charged phosphodiester backbone of naturally occurring
nucleic acids. This results in two advantages. First, the PNA
backbone exhibits improved hybridization kinetics. PNAs have larger
changes in the melting temperature (Tm) for mismatched versus
perfectly matched basepairs. DNA and RNA typically exhibit a
2-4.degree. C. drop in Tm for an internal mismatch. With the
non-ionic PNA backbone, the drop is closer to 7-9.degree. C.
Similarly, due to their non-ionic nature, hybridization of the
bases attached to these backbones is relatively insensitive to salt
concentration. This is particularly advantageous in the systems of
the present invention, as a reduced salt hybridization solution has
a lower Faradaic current than a physiological salt solution (in the
range of 150 mM).
[0050] 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.
[0051] 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.
[0052] Accordingly, the present invention provides methods and
compositions useful in the detection of target analytes. In a
preferred embodiment, the compositions comprise a substrate
comprising a metallic surface comprising an asymmetric monolayer
forming species.
[0053] By "substrate" or "solid support" or other grammatical
equivalents herein is meant any material that can be modified to
contain discrete individual sites appropriate for the attachment or
association of target analytes. The substrate can comprise a wide
variety of materials, as will be appreciated by those in the art,
with printed circuit board (PCB) materials being particularly
preferred. Other suitable substrates include, but are not limited
to, metal surfaces such as gold, electrodes as defined below, glass
and modified or functionalized glass, plastics (including acrylics,
polystyrene and copolymers of styrene and other materials,
polypropylene, polyethylene, polybutylene, polyurethanes),
Teflon.TM., fiberglass, GETEK (a blend of polypropylene oxide and
fiberglass), etc.), polysaccharides, nylon or nitrocellulose,
resins, ceramics, mica, silica or silica-based materials including
silicon and modified silicon, carbon, metals, inorganic glasses,
and a variety of other polymers.
[0054] In general, preferred materials include printed circuit
board materials. Circuit board materials are those that comprise an
insulating substrate that is coated with a conducting layer and
processed using lithography techniques, particularly
photolithography techniques, to form the patterns of electrodes and
interconnects (sometimes referred to in the art as interconnections
or leads). The insulating substrate is generally, but not always, a
polymer. As is known in the art, one or a plurality of layers may
be used, to make either "two dimensional" (e.g. all electrodes and
interconnections in a plane) or "three dimensional" (wherein the
electrodes are on one surface and the interconnects may go through
the board to the other side) boards. Three dimensional systems
frequently rely on the use of drilling or etching, followed by
electroplating with a metal such as copper, such that the "through
board" interconnections are made. Circuit board materials are often
provided with a foil already attached to the substrate, such as a
copper foil, with additional copper added as needed (for example
for interconnections), for example by electroplating. The copper
surface may then need to be roughened, for example through etching,
to allow attachment of the adhesion layer.
[0055] The substrates comprise one or more metallic surfaces,
preferably electrodes. By "metallic surface" or other grammatical
equivalents herein is meant any material that can be modified to
contain discrete individual sites appropriate for the attachment or
association of target analytes. Preferred metallic surfaces
include, but are not limited to, gold, indium tin oxide, and
electrodes.
[0056] In a preferred embodiment, the metallic surface is an
electrode. By "electrode" herein is meant a composition, which,
when connected to an electronic device, is able to sense a current
or charge and convert it to a signal. Thus, an electrode is an ETM
as described herein. Preferred electrodes are known in the art and
include, but are not limited to, certain metals and their oxides,
including gold; platinum; palladium; silicon; aluminum; metal oxide
electrodes including platinum oxide, titanium oxide, tin oxide,
indium tin oxide, palladium oxide, silicon oxide, aluminum oxide,
molybdenum oxide (Mo.sub.2O.sub.6), tungsten oxide (WO.sub.3) and
ruthenium oxides; and carbon (including glassy carbon electrodes,
graphite and carbon paste). Preferred electrodes include gold,
silicon, carbon and metal oxide electrodes, with gold being
particularly preferred.
[0057] The substrate may also include arrays, i.e. wherein there is
a matrix of addressable detection electrodes (herein generally
referred to "pads", "addresses" or "micro-locations"). By "array"
herein is meant a plurality of capture ligands in an array format;
the size of the array will depend on the composition and end use of
the array. Arrays containing from about 2 different capture 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.
[0058] The electrodes described herein are depicted as a flat
surface, which is only one of the possible conformations of the
electrode and is for schematic purposes only. The conformation of
the electrode will vary with the detection method used. For
example, flat planar electrodes may be preferred for optical
detection methods, or when arrays of nucleic acids are made, thus
requiring addressable locations for both synthesis and detection.
Alternatively, for single probe analysis, the electrode may be in
the form of a tube, with the SAMs comprising AMFS and nucleic acids
bound to the inner surface. This allows a maximum of surface area
containing the nucleic acids to be exposed to a small volume of
sample.
[0059] The electrode comprises asymmetric monolayer forming
species. By "monolayer" or "self-assembled monolayer" or "SAM"
herein is meant a relatively ordered assembly of molecules
spontaneously chemisorbed on a surface, in which the molecules are
oriented approximately parallel to each other and roughly
perpendicular to the surface. Each of the molecules includes a
functional group that adheres to the surface, and a portion that
interacts with neighboring molecules in the monolayer to form the
relatively ordered array. A "mixed" monolayer comprises a
heterogeneous monolayer, that is, where at least two different
molecules make up the monolayer. As outlined herein, the use of a
monolayer reduces the amount of non-specific binding of
biomolecules to the surface, and, in the case of nucleic acids,
increases the efficiency of oligonucleotide hybridization as a
result of the distance of the oligonucleotide from the electrode.
Thus, a monolayer facilitates the maintenance of the target analyte
away from the electrode surface. In addition, a monolayer serves to
keep charge carriers away from the surface of the electrode. Thus,
this layer helps to prevent electrical contact between the
electrodes and the ETMs, or between the electrode and charged
species within the solvent. Such contact can result in a direct
"short circuit" or an indirect short circuit via charged species
which may be present in the sample. Accordingly, the monolayer is
preferably tightly packed in a uniform layer on the electrode
surface, such that a minimum of "holes" exist. The monolayer thus
serves as a physical barrier to block solvent accessibility to the
electrode.
[0060] In a preferred embodiment, the AMFS comprises a standard
monolayer forming species comprises a standard monolayer forming
species. By standard monolayer forming species herein is meant an
alkyl chain, preferably linear, from about 7 to 20 carbons in
length.
[0061] In a preferred embodiment, the AFMS 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 or above 100 Hz when an AC voltage is applied. The
rate of electron transfer through the insulator is preferably
slower than the rate through the conductive oligomers described
herein.
[0062] 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.
[0063] 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.
[0064] 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).
[0065] The insulators may be substituted with R groups as defined
below 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.
[0066] The length of the insulator may vary. Preferably the
insulator is a straight chain alkyl group, comprising
(CH.sub.2).sub.n and (OCH.sub.2CH.sub.2).sub.n groups and a
terminal OH group. The integer, n, will vary, but generally will be
from about 7 to 20 for (CH.sub.2).sub.n and from about 0 to 10 for
(OCH.sub.2CH.sub.2).sub.n.
[0067] In a preferred embodiment, the asymmetric monolayer forming
species comprises a disulfide group which links a monolayer forming
species, such as an insulator, and an asymmetric group. Preferably,
the asymmetric group is an electroconduit forming species (EFS). 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,
photoactivatible species that can be selectively removed from the
surface upon photoactivation, leaving electroconduits.
[0068] Preferred EFS include conductive oligomers, as defined
below, and phenyl-acetylene-polyethylene glycol species, and
branched alkyl groups.
[0069] In a preferred embodiment, the EFS is an alkyl group as
defined below. If the EFS is a straight chain alkyl group, 1 to 6
carbon atoms are preferred.
[0070] In a preferred embodiment, the EFS is a branched chain alkyl
group, substituted with one or more substitution moieties "R" as
defined below. It may be branched at one or more positions. The EFS
may be directly attached to an attachment linker as defined below.
Alternatively, the EFS may be attached to the attachment linker via
a (CH.sub.2).sub.n group, wherein n is an integer from 1 to 4.
[0071] In one embodiment, the AFMS has the structure depicted in
Structure 44:
Structure 44
[0072] EFS--S--S--I
[0073] In Structure 44, I represents an insulator moiety as defined
within, EFS is an electroconduit moiety as defined within and S
represents a S atom.
[0074] In a preferred embodiment, the AFMS has the structure
depicted in Structure 45: 1
[0075] In this embodiment, n is an integer from 7-16, m is an
integer from 0-7 and o is an integer from 0 to 4. In Structure 45,
R1, R2 and R3 may each be independently selected from the group
consisting of hydrogen, alkyl groups including cycloalkyl, alchol
groups, amine groups, amido, ester, phosphorus moieties, and aryl
groups including substituted aryl and heteroaryl.
[0076] In a preferred embodiment, the AFMS has the structure
depicted in Structure 46: 2
[0077] In a preferred embodiment, the AFMS has the structure
depicted in Structure 47: 3
[0078] In one embodiment, in addition to the AFMS, the monolayer
comprises insulators.
[0079] In one embodiment, in addition to the AFMS, the monolayer
comprises conductive oligomers. By "conductive oligomer" herein is
meant a substantially conducting oligomer, preferably linear, some
embodiments of which are referred to in the literature as
"molecular wires". By "substantially conducting" herein is meant
that the oligomer is capable of transfering electrons at 100 Hz.
Generally, the conductive oligomer has substantially overlapping
n-orbitals, i.e. conjugated n-orbitals, as between the monomeric
units of the conductive oligomer, although the conductive oligomer
may also contain one or more sigma (a) 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.
[0080] 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 A to
about 200 A. As described below, insulators have a conductivity S
of about 10.sup.-7.OMEGA..sup.-1cm.sup.-1 or lower, with less than
about 10.sup.-8.OMEGA..sup.-1cm.sup.-1 being preferred. See
generally Gardner et al., Sensors and Actuators A 51 (1995) 57-66,
incorporated herein by reference.
[0081] 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 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 hybridization assays. In addition, conductive
oligomers that will promote the formation of self-assembled
monolayers are preferred.
[0082] 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.
[0083] In a preferred embodiment, the conductive oligomer has the
structure depicted in Structure 1: 4
[0084] 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 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 nucleic acid, the right "Y", if present, is attached to the
nucleic acid, either directly or through the use of a linker, as is
described herein.
[0085] In this embodiment, Y is an aromatic group, n is an integer
from 1 to 50, g is either 1 or zero, e is an integer from zero to
10, and m is zero or 1. When g is 1, B-D is a bond able to
conjugate with neighboring bonds (herein referred to as a
"conjugated bond"), preferably selected from acetylene, B-D D is a
conjugated bond, preferably selected from acetylene, alkene,
substituted alkene, amide, azo, --C.dbd.N-- (including --N.dbd.C--,
--CR.dbd.N-- and --N.dbd.CR--), --Si.dbd.Si--, and --Si.dbd.C--
(including --C.dbd.Si--, --Si.dbd.CR-- and --CR.dbd.Si--). When g
is zero, e is preferably 1, D is preferably carbonyl, or a
heteroatom moiety, wherein the heteroatom is selected from oxygen,
sulfur, nitrogen, silicon or phosphorus. Thus, suitable heteroatom
moieties include, but are not limited to, --NH and --NR, wherein R
is as defined herein; substituted sulfur; sulfonyl (--SO.sub.2--)
sulfoxide (--SO--); phosphine oxide (--PO-- and --RPO--); and
thiophosphine (--PS-- and --RPS--). However, when the conductive
oligomer is to be attached to a gold electrode, as outlined below,
sulfur derivatives are not preferred.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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. 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.
[0091] By "amino groups" or grammatical equivalents herein is meant
--NH.sub.2, --NHR and --NR.sub.2 groups, with R being as defined
herein.
[0092] By "nitro group" herein is meant an --NO.sub.2 group.
[0093] 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.
[0094] 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.
[0095] By "ester" herein is meant a --COOR group.
[0096] 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.
[0097] By "aldehyde" herein is meant --RCHO groups.
[0098] By "alcohol" herein is meant --OH groups, and alkyl alcohols
--ROH.
[0099] By "amido" herein is meant --RCONH-- or RCONR-- groups.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] In the conductive oligomers depicted herein, when g is 1,
B-D is a bond linking two atoms or chemical moieties. In a
preferred embodiment, B-D is a conjugated bond, containing
overlapping or conjugated n-orbitals.
[0104] 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.
[0105] 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.
[0106] As above for the Y rings, within any single conductive
oligomer, the B-D bonds (or D moieties, when g=0) may be all the
same, or at least one may be different. For example, when m is
zero, the terminal B-D bond may be an amide bond, and the rest of
the B-D bonds may be acetylene bonds. Generally, when amide bonds
are present, as few amide bonds as possible are preferable, but in
some embodiments all the B-D bonds are amide bonds. Thus, as
outlined above for the Y rings, one type of B-D bond may be present
in the conductive oligomer within a monolayer as described below,
and another type above the monolayer level, for example to give
greater flexibility for nucleic acid hybridization when the nucleic
acid is attached via a conductive oligomer.
[0107] 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 A to about 100 A
(although distances of up to 500 A may be used) from the electrode
surface, with from about 15 A to about 60 A being preferred and
from about 25 A to about 60 A 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.
[0108] 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.
[0109] 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. EngI. 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.
[0110] Particularly preferred conductive oligomers of this
embodiment are depicted below: 5
[0111] 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: 6
[0112] 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.
[0113] 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.
[0114] Particularly preferred embodiments of Structure 3 include
Structures 4, 5, 6 and 7, depicted below: 7
[0115] 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. 8
[0116] When the B-D bond is an amide bond, as in Structure 5, the
conductive oligomers are pseudopeptide oligomers. Although the
amide bond in Structure 5 is depicted with the carbonyl to the
left, i.e. --CONH--, the reverse may also be used, i.e. --NHCO--.
Particularly preferred embodiments of Structure 5 include: n is
two, m is one, and R is hydrogen; n is three, m is zero, and R is
hydrogen (in this embodiment, the terminal nitrogen (the D atom)
may be the nitrogen of the amino-modified ribose); and the use of R
groups to increase solubility. 9
[0117] 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. 10
[0118] 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.
[0119] In a preferred embodiment, the conductive oligomer has the
structure depicted in Structure 8: 11
[0120] 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.
[0121] 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.
[0122] 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: 12
[0123] 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.
[0124] In a preferred embodiment, R is hydrogen, although R may be
also alkyl groups and polyethylene glycols or derivatives.
[0125] In an alternative embodiment, the conductive oligomer may be
a mixture of different types of oligomers, for example of
structures 1 and 8.
[0126] In addition, the terminus of at least some of the conductive
oligomers in the monolayer are electronically exposed. By
"electronically exposed" herein is meant that upon the placement of
an ETM in close proximity to the terminus, and after initiation
with the appropriate signal, a signal dependent on the presence of
the ETM may be detected. The conductive oligomers may or may not
have terminal groups. Thus, in a preferred embodiment, there is no
additional terminal group, and the conductive oligomer terminates
with one of the groups depicted in Structures 1 to 9; for example,
a B-D bond such as an acetylene bond. Alternatively, in a preferred
embodiment, a terminal group is added, sometimes depicted herein as
"Q". A terminal group may be used for several reasons; for example,
to contribute to the electronic availability of the conductive
oligomer for detection of ETMs, or to alter the surface of the SAM
for other reasons, for example to prevent non-specific binding. For
example, there may be negatively charged groups on the terminus to
form a negatively charged surface such that when the nucleic acid
is DNA or RNA the nucleic acid is repelled or prevented from lying
down on the surface, to facilitate hybridization. Preferred
terminal groups include --NH.sub.2, --OH, --COOH, and alkyl groups
such as --CH.sub.3, and (poly)alkyloxides such as (poly)ethylene
glycol, with --OCH.sub.2CH.sub.2OH, --(OCH.sub.2CH.sub.2O).sub.2H,
--(OCH.sub.2CH.sub.2O).sub.3H, and --(OCH.sub.2CH.sub.2O).sub.4H
being preferred.
[0127] 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.
[0128] It will be appreciated that the monolayer may comprise
different conductive oligomer species, although preferably the
different species are chosen such that a reasonably uniform SAM can
be formed. Thus, for example, when nucleic acids are covalently
attached to the electrode using conductive oligomers, it is
possible to have one type of conductive oligomer used to attach the
nucleic acid, and another type functioning to detect the ETM.
Similarly, it may be desirable to have mixtures of different
lengths of conductive oligomers in the monolayer, to help reduce
non-specific signals. Thus, for example, preferred embodiments
utilize conductive oligomers that terminate below the surface of
the rest of the monolayer, i.e. below the insulator layer, if used,
or below some fraction of the other conductive oligomers.
Similarly, the use of different conductive oligomers may be done to
facilitate monolayer formation, or to make monolayers with altered
properties.
[0129] The length of the species making up the monolayer will vary
as needed. As outlined above, it appears that binding 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 nucleic acids 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.
[0130] The covalent attachment of the conductive oligomers and
insulators may be accomplished in a variety of ways, depending on
the electrode and the composition of the insulators and conductive
oligomers used. In a preferred embodiment, the attachment linkers
with covalently attached capture binding ligands as depicted herein
are covalently attached to an electrode. Thus, one end or terminus
of the attachment linker is attached to the capture binding ligand,
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 capture binding ligands at other termini,
although this is not preferred. Similarly, the attachment linker
may be attached at two sites to the electrode, as is generally
depicted in Structures 11-13. Generally, some type of linker is
used, as depicted below as "A" in Structure 10, where "X" is the
conductive oligomer, "I" is an insulator and the hatched surface is
the electrode: 13
[0131] 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.
[0132] Although depicted herein as a single moiety, the insulators
and conductive oligomers may be attached to the electrode with more
than one "A" moiety; the "A" moieties may be the same or different.
Thus, for example, when the electrode is a gold electrode, and "A"
is a sulfur atom or moiety, multiple sulfur atoms may be used to
attach the conductive oligomer to the electrode, such as is
generally depicted below in Structures 11, 12 and 13. As will be
appreciated by those in the art, other such structures can be made.
In Structures 11, 12 and 13, the A moiety is just a sulfur atom,
but substituted sulfur moieties may also be used. 14
[0133] It should also be noted that similar to Structure 13, it may
be possible to have a conductive oligomer terminating in a single
carbon atom with three sulfur moieties attached to the electrode.
Additionally, although not always depicted herein, the conductive
oligomers and insulators may also comprise a "Q" terminal
group.
[0134] 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. Representative structures are depicted in Structure 14
and Structure 48. Structure 14 uses the Structure 3 conductive
oligomer, although as for all the structures depicted herein, any
of the conductive oligomers, or combinations of conductive
oligomers, may be used. Similarly, any of the conductive oligomers
or insulators may also comprise terminal groups as described
herein. Structure 14 depicts the "A" linker as comprising just a
sulfur atom, although additional atoms may be present (i.e. linkers
from the sulfur to the conductive oligomer or substitution groups).
15
[0135] Structure 48 depicts the "A" linker as comprising a cyclic
disulfide to which an insulator (I) is attached. Preferably the
insulator is a standard monolayer forming species as defined
herein, although as will be appreciated by those of skill in the
art, other insulators as are known in the art and conductive
oligomer may be used as well. 16
[0136] 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. 17
[0137] In Structure 16, the oxygen atom is from the oxide of the
metal oxide electrode. The Si atom may also contain other atoms,
i.e. be a silicon moiety containing substitution groups. Other
attachments for SAMs to other electrodes are known in the art; see
for example Napier et al., Langmuir, 1997, for attachment to indium
tin oxide electrodes, and also the chemisorption of phosphates to
an indium tin oxide electrode (talk by H. Holden Thorpe, CHI
conference, May 4-5, 1998).
[0138] 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. Generally, three component systems are
preferred, with the first species comprising a capture binding
ligand containing species (i.e. a capture probe, that can be
attached to the electrode via either an insulator or a conductive
oligomer, as is more fully described below). The second species are
the insulators, and the third species are electroconduit forming
species. 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 capture binding ligands are nucleic acids and
the target is nucleic acid as well, 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. Preferred ratios of first:second:third species are 2:2:1
for short targets, 1:3:1 for longer targets, with total thiol
concentration in the 500 .mu.M to 1 mM range, and 833 .mu.M being
preferred.
[0139] In a preferred embodiment, two component systems are used,
comprising the first and second species. In this embodiment, the
first species can comprise from about 90% to about 1%, with from
about 1% to about 40% being preferred, and from about 10% to about
40% being especially preferred. The second species can comprise
from about 1% to about 90%, with from about 10% to about 60% being
preferred, and from about 20% to about 40% being especially
preferred.
[0140] In a particularly preferred embodiment, two component
systems are used, comprising the first species and wherein the
second species is the AFMS. In this embodiment, the first species
can comprise from about 90% to about 1%, with from about 1% to
about 40% being preferred, and from about 10% to about 40% being
especially preferred. The second species, the AFMS, 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.
[0141] In a particularly preferred embodiment, the aqueous solution
used to form the monolayer also comprises s a pH buffering
component and a zwitterionic hygroscopic agent. Preferably the
buffer is Tris and the hygroscopic agent is betaine. Prefereable,
the concentartion of the buffer is about 1 mM to 1 M and more
preferable about 10 mM to about 200 mM. Also preferable, the
concentration of the hygroscopic agent is from about 1 mM to about
1M and more preferably from about 100 mM to about 800 mM.
[0142] 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.
[0143] 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.
[0144] In a particularly preferred embodiment, the SAM is formed in
only one step and the second step is omitted.
[0145] Following cleaning, the gold substrate is exposed to the SAM
species. When the electrode is ITO, the SAM species are
phosphonate-containing species. This can also be done in a variety
of ways, including, but not limited to, solution deposition, gas
phase deposition, microcontact printing, spray deposition,
deposition using neat components, etc. A preferred embodiment
utilizes a deposition solution comprising a mixture of various SAM
species in solution, generally thiol-containing species. Mixed
monolayers that contain nucleic acids are usually prepared using a
two step procedure. The thiolated nucleic acid is deposited during
the first deposition step (generally in the presence of at least
one other monolayer-forming species) and the mixed monolayer
formation is completed during the second step in which a second
thiol solution minus nucleic acid is added. The second step
frequently involves mild heating to promote monolayer
reorganization.
[0146] In a preferred embodiment, the deposition solution is an
organic deposition solution. In this embodiment, a clean gold
surface is placed into a clean vial. A binding ligand deposition
solution in organic solvent is prepared in which the total thiol
concentration is between micromolar to saturation; preferred ranges
include from about 1 .mu.M to 10 mM, with from about 400 uM to
about 1.0 mM being especially preferred. In a preferred embodiment,
the deposition solution contains thiol modified DNA (i.e. nucleic
acid attached to an attachment linker) and thiol diluent molecules
(either conductive oligomers or insulators, with the latter being
preferred). The ratio of nucleic acid to diluent (if present) is
usually between 1000:1 to 1:1000, with from about 10:1 to about
1:10 being preferred and 1:1 being especially preferred. The
preferred solvents are tetrahydrofuran (THF), acetonitrile,
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 nucleic acid deposition
solution is added to the vial so as to completely cover the
electrode surface. The gold substrate is allowed to incubate at
ambient temperature or slightly above ambient temperature for a
period of time ranging from seconds to hours, with 5-30 minutes
being preferred. After the initial incubation, the deposition
solution is removed and a solution of diluent molecule only (from
about 1 .mu.M to 10 mM, with from about 100 uM to about 1.0 mM
being preferred) in organic solvent is added. The gold substrate is
allowed to incubate at room temperature or above room temperature
for a period of time (seconds to days, with from about 10 minutes
to about 24 hours being preferred). The gold sample is removed from
the solution, rinsed in clean solvent and used.
[0147] In a preferred embodiment, an aqueous deposition solution is
used. As above, a clean gold surface is placed into a clean vial. A
nucleic acid deposition solution in water is prepared in which the
total thiol concentration is between about 1 uM and 10 mM, with
from about 1 .mu.M to about 200 uM being preferred. The aqueous
solution frequently has salt present (up to saturation, with
approximately 1M being preferred), however pure water can be used.
The deposition solution contains thiol modified nucleic acid and
often a thiol diluent molecule. The ratio of nucleic acid to
diluent is usually between between 1000:1 to 1:1000, with from
about 10:1 to about 1:10 being preferred and 1:1 being especially
preferred. The nucleic acid deposition solution is added to the
vial in such a volume so as to completely cover the electrode
surface. The gold substrate is allowed to incubate at ambient
temperature or slightly above ambient temperature for 1-30 minutes
with 5 minutes usually being sufficient. After the initial
incubation, the deposition solution is removed and a solution of
diluent molecule only (10 uM-1.0 mM) in either water or organic
solvent is added. The gold substrate is allowed to incubate at room
temperature or above room temperature until a complete monolayer is
formed (10 minutes-24 hours). The gold sample is removed from the
solution, rinsed in clean solvent and used.
[0148] In a preferred embodiment, the deposition solution comprises
a zwitterionic compound, preferably betaine. Preferred embodiments
utilize betain and Tris-HCl buffers.
[0149] 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.
[0150] In a preferred embodiment, the electrode comprising the
monolayer including conductive oligomers further comprises a
capture binding ligand. By "capture binding ligand" or "capture
binding species" or "capture probe" herein is meant a compound that
is used to probe for the presence of the target analyte, that will
bind to the target analyte. Generally, the capture binding ligand
allows the attachment of a target analyte to the electrode, for the
purposes of detection. As is more fully outlined below, attachment
of the target analyte to the capture probe may be direct (i.e. the
target analyte binds to the capture binding ligand) or indirect
(one or more capture extender ligands are used). By "covalently
attached" herein is meant that two moieties are attached by at
least one bond, including sigma bonds, pi bonds and coordination
bonds.
[0151] In a preferred embodiment, the binding is specific, and the
binding ligand is part of a binding pair. By "specifically bind"
herein is meant that the ligand binds the analyte, with specificity
sufficient to differentiate between the analyte and other
components or contaminants of the test sample. However, as will be
appreciated by those in the art, it will be possible to detect
analytes using binding which is not highly specific; for example,
the systems may use different binding ligands, for example an array
of different ligands, and detection of any particular analyte is
via its "signature" of binding to a panel of binding ligands,
similar to the manner in which "electronic noses" work. This finds
particular utility in the detection of chemical analytes. The
binding should be sufficient to remain bound under the conditions
of the assay, including wash steps to remove non-specific binding.
In some embodiments, for example in the detection of certain
biomolecules, the binding constants of the analyte to the binding
ligand will be at least about 10.sup.4-10.sup.6 M.sup.-1, with at
least about 10.sup.5 to 10.sup.9 M.sup.-1 being preferred and at
least about 10.sup.7-10.sup.9M.sup.-1 being particularly
preferred.
[0152] As will be appreciated by those in the art, the composition
of the binding ligand will depend on the composition of the target
analyte. Binding ligands to a wide variety of analytes are known or
can be readily found using known techniques. For example, when the
analyte is a single-stranded nucleic acid, the binding ligand may
be a complementary nucleic acid. Similarly, the analyte may be a
nucleic acid binding protein and the capture binding ligand is
either single-stranded or double stranded nucleic acid;
alternatively, the binding ligand may be a nucleic acid-binding
protein when the analyte is a single or double-stranded nucleic
acid. When the analyte is a protein, the binding ligands include
proteins or small molecules. Preferred binding ligand proteins
include peptides. For example, when the analyte is an enzyme,
suitable binding ligands include substrates and inhibitors. As will
be appreciated by those in the art, any two molecules that will
associate may be used, either as an analyte or as the binding
ligand. Suitable analyte/binding ligand pairs include, but are not
limited to, antibodies/antigens, receptors/ligands,
proteins/nucleic acid, enzymes/substrates and/or inhibitors,
carbohydrates (including glycoproteins and glycolipids)/lectins,
proteins/proteins, proteins/small molecules; and carbohydrates and
their binding partners are also suitable analyte-binding ligand
pairs. These may be wild-type or derivative sequences. In a
preferred embodiment, the binding ligands are portions
(particularly the extracellular portions) of cell surface receptors
that are known to multimerize, such as the growth hormone receptor,
glucose transporters (particularly GLUT 4 receptor), transferrin
receptor, epidermal growth factor receptor, low density lipoprotein
receptor, high density lipoprotein receptor, epidermal growth
factor receptor, leptin receptor, interleukin receptors including
IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12,
IL-13, IL-15, and IL-17 receptors, human growth hormone receptor,
VEGF receptor, PDGF receptor, EPO receptor, TPO receptor, ciliary
neurotrophic factor receptor, prolactin receptor, and T-cell
receptors.
[0153] The method of attachment of the capture binding ligand to
the attachment linker will generally be done as is known in the
art, and will depend on the composition of the attachment linker
and the capture binding ligand. In general, the capture binding
ligands are attached to the attachment linker through the use of
functional groups on each that can then be used for attachment.
Preferred functional groups for attachment are amino groups,
carboxy groups, oxo groups and thiol groups. These functional
groups can then be attached, either directly or through the use of
a linker, sometimes depicted herein as "Z". Linkers are known in
the art; for example, homo-or hetero-bifunctional linkers as are
well known (see 1994 Pierce Chemical Company catalog, technical
section on cross-linkers, pages 155-200, incorporated herein by
reference). Preferred Z linkers include, but are not limited to,
alkyl groups (including substituted alkyl groups and alkyl groups
containing heteroatom moieties), with short alkyl groups, esters,
amide, amine, epoxy groups and ethylene glycol and derivatives
being preferred. Z may also be a sulfone group, forming
sulfonamide.
[0154] In this way, capture binding ligands comprising proteins,
lectins, nucleic acids, small organic molecules, carbohydrates,
etc. can be added.
[0155] In a preferred embodiment, the capture binding ligand is
attached directly to the electrode as outlined herein, for example
via an attachment linker. Alternatively, the capture binding ligand
may utilize a capture extender component, such as depicted in FIG.
7G. In this embodiment, the capture binding ligand comprises a
first portion that will bind the target analyte and a second
portion that can be used for attachment to the surface. FIGS. 7A-7R
depict the use of a nucleic acid component for binding to the
surface, although this can be other binding partners as well.
[0156] A preferred embodiment utilizes proteinaceous capture
binding ligands. As is known in the art, any number of techniques
may be used to attach a proteinaceous capture binding ligand.
"Protein" in this context includes proteins, polypeptides and
peptides. A wide variety of techniques are known to add moieties to
proteins. One preferred method is outlined in U.S. Pat. No.
5,620,850, hereby incorporated by reference in its entirety. The
attachment of proteins to electrodes is known; see also Heller,
Acc. Chem. Res. 23:128 (1990), and related work.
[0157] A preferred embodiment utilizes nucleic acids as the capture
binding ligand, for example for when the target analyte is a
nucleic acid or a nucleic acid binding protein, or when the nucleic
acid serves as an aptamer for binding a protein; see U.S. Pat. Nos.
5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867,
5,705,337, and related patents, hereby incorporated by reference.
In this embodiment, the nucleic acid capture binding ligand is
covalently attached to the electrode, via an "attachment linker"
that can be either a conductive oligomer or via an insulator. Thus,
one end of the attachment linker is attached to a nucleic acid, and
the other end (although as will be appreciated by those in the art,
it need not be the exact terminus for either) is attached to the
electrode. Thus, any of structures 1-16 may further comprise a
nucleic acid effectively as a terminal group. Thus, the present
invention provides compositions comprising binding ligands
covalently attached to electrodes as is generally depicted below in
Structure 17 for a nucleic acid: 18
[0158] In Structure 17, the hatched marks on the left represent an
electrode. X is a conductive oligomer and I is an insulator as
defined herein. F.sub.1 is a linkage that allows the covalent
attachment of the electrode and the conductive oligomer or
insulator, including bonds, atoms or linkers such as is described
herein, for example as "A", defined below. F.sub.2 is a linkage
that allows the covalent attachment of the conductive oligomer or
insulator to the binding ligand, a nucleic acid in Structure 17,
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 binding ligand, or exogeneous to both, for
example, as defined herein for "Z".
[0159] In general, the methods, synthetic schemes and compositions
useful for the attachment of capture binding ligands, particularly
nucleic acids, are outlined in WO98/20162, PCT US98/12430, PCT
US98/12082; PCT US99/01705 and PCT US99/01703, all of which are
expressly incorporated herein by reference in their entirety.
[0160] In a preferred embodiment, the capture binding ligand is
covalently attached to the electrode via a conductive oligomer. The
covalent attachment of the binding ligand and the conductive
oligomer may be accomplished in several ways, as will be
appreciated by those in the art.
[0161] In a preferred embodiment, the capture binding ligand is a
nucleic acid, and 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.
[0162] In a preferred embodiment, the conductive oligomer is
attached to the base of a nucleoside of the nucleic acid. This may
be done in several ways, depending on the oligomer, as is described
below. In one embodiment, the oligomer is attached to a terminal
nucleoside, i.e. either the 3' or 5' nucleoside of the nucleic
acid. Alternatively, the conductive oligomer is attached to an
internal nucleoside.
[0163] The point of attachment to the basewill vary with the base.
Generally, attachment at any position is possible. In some
embodiments, for example when the probe containing the ETMs may be
used for hybridization, it is preferred to attach at positions not
involved in hydrogen bonding to the complementary base. Thus, for
example, generally attachment is to the 5 or 6 position of
pyrimidines such as uridine, cytosine and thymine. For purines such
as adenine and guanine, the linkage is preferably via the 8
position. Attachment to non-standard bases is preferably done at
the comparable positions.
[0164] In one embodiment, the attachment is direct; that is, there
are no intervening atoms between the conductive oligomer and the
base. In this embodiment, for example, conductive oligomers with
terminal acetylene bonds are attached directly to the base.
Structure 18 is an example of this linkage, using a Structure 3
conductive oligomer and uridine as the base, although other bases
and conductive oligomers can be used as will be appreciated by
those in the art: 19
[0165] 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.
[0166] In addition, the base may contain additional modifications
as needed, i.e. the carbonyl or amine groups may be altered or
protected.
[0167] 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: 20
[0168] 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.
[0169] In a preferred embodiment, the attachment of the nucleic
acid and the conductive oligomer is done via attachment to the
backbone of the nucleic acid. This may be done in a number of ways,
including attachment to a ribose of the ribose-phosphate backbone,
or to the phosphate of the backbone, or other groups of analogous
backbones.
[0170] 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.
[0171] In a preferred embodiment, the conductive oligomer is
attached to the ribose of the ribose-phosphate backbone. This may
be done in several ways. As is known in the art, nucleosides that
are modified at either the 2' or 3' position of the ribose with
amino groups, sulfur groups, silicone groups, phosphorus groups, or
oxo groups can be made (Imazawa et al., J. Org. Chem., 44:2039
(1979); Hobbs et al., J. Org. Chem. 42(4):714 (1977); Verheyden et
al., J. 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 conductive oligomers.
[0172] A preferred embodiment utilizes amino-modified nucleosides.
These amino-modified riboses can then be used to form either amide
or amine linkages to the conductive oligomers. In a preferred
embodiment, the amino group is attached directly to the ribose,
although as will be appreciated by those in the art, short linkers
such as those described herein for "Z" may be present between the
amino group and the ribose.
[0173] 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): 21
[0174] As will be appreciated by those in the art, Structure 20 has
the terminal bond fixed as an amide bond.
[0175] 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: 22
[0176] 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. 23
[0177] In Structure 22, Z is as defined above. Suitable linkers
include methylene and ethylene.
[0178] In an alternative embodiment, the conductive oligomer is
covalently attached to the nucleic acid via the phosphate of the
ribose-phosphate backbone (or analog) of a nucleic acid. In this
embodiment, the attachment is direct, utilizes a linker or via an
amide bond. Structure 23 depicts a direct linkage, and Structure 24
depicts linkage via an amide bond (both utilize the Structure 3
conductive oligomer, although Structure 8 conductive oligomers are
also possible). Structures 23 and 24 depict the conductive oligomer
in the 3' position, although the 5' position is also possible.
Furthermore, both Structures 23 and 24 depict naturally occurring
phosphodiester bonds, although as those in the art will appreciate,
non-standard analogs of phosphodiester bonds may also be used.
24
[0179] 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.
[0180] 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. 25
[0181] In a preferred embodiment, the conductive oligomer is
covalently attached to the nucleic acid via a transition metal
ligand. In this embodiment, the conductive oligomer is covalently
attached to a ligand which provides one or more of the coordination
atoms for a transition metal. In one embodiment, the ligand to
which the conductive oligomer is attached also has the nucleic acid
attached, as is generally depicted below in Structure 25.
Alternatively, the conductive oligomer is attached to one ligand,
and the nucleic acid is attached to another ligand, as is generally
depicted below in Structure 26. Thus, in the presence of the
transition metal, the conductive oligomer is covalently attached to
the nucleic acid. Both of these structures depict Structure 3
conductive oligomers, although other oligomers may be utilized.
Structures 25 and 26 depict two representative structures for
nucleic acids; as will be appreciated by those in the art, it is
possible to connect other types of capture binding ligands, for
example proteinaceous binding ligands, in a similar manner: 26
[0182] 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.
[0183] 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.
[0184] 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).
[0185] 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.
[0186] 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.
[0187] The oxygen, sulfur, phosphorus and nitrogen-donating ligands
are attached in such a manner as to allow the heteroatoms to serve
as coordination atoms.
[0188] 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 6-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
6-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.
[0189] 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 denvatized.
[0190] 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). 27
[0191] In a preferred embodiment, the ligands used in the invention
show altered fluorescent properties depending on the redox state of
the chelated metal ion. As described below, this thus serves as an
additional mode of detection of electron transfer between the ETM
and the electrode.
[0192] 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.
[0193] The use of metal ions to connect the nucleic acids can serve
as an internal control or calibration of the system, to evaluate
the number of available nucleic acids on the surface. However, as
will be appreciated by those in the art, if metal ions are used to
connect the nucleic acids to the conductive oligomers, it is
generally desirable to have this metal ion complex have a different
redox potential than that of the ETMs used in the rest of the
system, as described below. This is generally true so as to be able
to distinguish the presence of the capture probe from the presence
of the target sequence. This may be useful for identification,
calibration and/or quantification. Thus, the amount of capture
probe on an electrode may be compared to the amount of hybridized
double stranded nucleic acid to quantify the amount of target
sequence in a sample. This is quite significant to serve as an
internal control of the sensor or system. This allows a measurement
either prior to the addition of target or after, on the same
molecules that will be used for detection, rather than rely on a
similar but different control system. Thus, the actual molecules
that will be used for the detection can be quantified prior to any
experiment. This is a significant advantage over prior methods.
[0194] In a preferred embodiment, the capture probe nucleic acids
are covalently attached to the electrode via an insulator. The
attachment of nucleic acids to insulators such as alkyl groups is
well known, and can be done to the base or the backbone, including
the ribose or phosphate for backbones containing these moieties, or
to alternate backbones for nucleic acid analogs.
[0195] In a preferred embodiment, the capture binding ligands are
covalently attached to the electrode via an insulator. The
attachment of a variety of binding ligands such as proteins and
nucleic acids to insulators such as alkyl groups is well known, and
can be done to the nucleic acid bases or the backbone, including
the ribose or phosphate for backbones containing these moieties, or
to alternate backbones for nucleic acid analogs, or to the side
chains or backbone of the amino acids.
[0196] In a preferred embodiment, there may be one or more
different capture binding ligand species (sometimes referred to
herein as "anchor ligands", "anchor probes" or "capture probes"
with the phrase "probe" generally referring to nucleic acid
species) on the surface, as is generally depicted in the Figures.
In some embodiments, there may be one type of capture binding
ligand, or one type of capture binding ligand extender, as is more
fully described below. Alternatively, different capture binding
ligands, or one capture binding ligand with a multiplicity of
different capture extender binding ligands can be used. Similarly,
when nucleic acid systems are used, it may be desirable to use
auxiliary capture probes that comprise relatively short probe
sequences, that can be used to "tack down" components of the
system, for example the recruitment linkers, to increase the
concentration of ETMs at the surface.
[0197] Thus the present invention provides electrodes comprising
monolayers comprising conductive oligomers and capture binding
ligands, useful in target analyte detection systems.
[0198] In a preferred embodiment, the compositions further comprise
a solution binding ligand. Solution binding ligands are similar to
capture binding ligands, in that they bind 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 directly attached to the surface, although as depicted in FIG.
5A they may be. The solution binding ligand either directly
comprises a recruitment linker that comprises at least one ETM, or
the recruitment linker is part of a label probe that will bind to
the solution binding ligand.
[0199] Thus, "recruitment linkers" or "signal carriers" with
covalently attached ETMs are provided. 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.
[0200] 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.
[0201] 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.
[0202] 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-def:6,5,10-d- 'e'f')diisoquinoline
dichloride (ADIQ.sup.2+); porphyrins
([meso-tetrakis(N-methyl-x-pyridinium)porphyrin tetrachloride],
varlamine blue B hydrochloride, Bindschedler's green;
2,6-dichloroindophenol, 2,6-dibromophenolindophenol; Brilliant
crest blue (3-amino-9-dimethyl-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.
[0203] 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.
[0204] 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 being
particularly preferred.
[0205] In a preferred embodiment, a plurality of ETMs are used. As
is shown in the examples, the use of multiple ETMs provides signal
amplification and thus allows more sensitive detection limits.
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
(100 to 1000) can be used.
[0206] As will be appreciated by those in the art, the portion of
the label probe (or target, in some embodiments) that comprises the
ETMs (termed herein a "recruitment linker" or "signal carrier") can
be nucleic acid, or it can be a non-nucleic acid linker that links
the solution binding ligand to the ETMs. 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.
[0207] In a preferred embodiment, the recruitment linker is a
nucleic acid, and comprises covalently attached ETMs. The ETMs may
be attached to nucleosides within the nucleic acid in a variety of
positions. Preferred embodiments include, but are not limited to,
(1) attachment to the base of the nucleoside, (2) attachment of the
ETM as a base replacement, (3) attachment to the backbone of the
nucleic acid, including either to a ribose of the ribose-phosphate
backbone or to a phosphate moiety, or to analogous structures in
nucleic acid analogs, and (4) attachment via metallocene polymers,
with the latter being preferred.
[0208] 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 generally depicted
in FIGS. 7Q and 7R; this is similar to the use of an amplifier
probe, except that both the primary and the secondary label probes
comprise ETMs.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] 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. 28
[0213] 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.
[0214] Similarly, as for the conductive oligomers, the linkage may
be done using a linker, which may utilize an amide linkage (see
generally Telser et al., J. Am. Chem. Soc. 111:7221-7226 (1989);
Telser et al., J. Am. Chem. Soc. 111:7226-7232 (1989), both of
which are expressly incorporated by reference). These structures
are generally depicted below in Structure 31, which again uses
uridine as the base, although as above, the other bases may also be
used: 29
[0215] In this embodiment, L is a ligand as defined above, with Lr
and M as defined above as well. Preferably, L is amino, phen, byp
and terpy.
[0216] 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: 30
[0217] 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.
[0218] 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.
[0219] 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. 31
[0220] In a preferred embodiment, amine linkages are used, as is
generally depicted in Structure 34. 32
[0221] 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.
[0222] In a preferred embodiment, oxo linkages are used, as is
generally depicted in Structure 35. 33
[0223] 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)n, with n from 1 to 10 being preferred, and n=1
to 4 being especially preferred, and n=4 being particularly
preferred.
[0224] Linkages utilizing other heteroatoms are also possible.
[0225] 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. 34
[0226] In Structure 361, 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.
[0227] 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.
[0228] 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. 35
[0229] 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.
36
[0230] 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.
[0231] 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.
[0232] 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 acids having a
backbone comprising at least one metallocene. This is distinguished
from nucleic acids 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.
[0233] 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.
[0234] 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.
[0235] 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.
[0236] 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: 37
[0237] 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.
[0238] Structure 40 depicts the ferrocene derivative: 38
[0239] These phosphoramidite analogs can be added to standard
oligonucleotide syntheses as is known in the art.
[0240] Structure 41 depicts the ferrocene peptide nucleic acid
(PNA) monomer, that can be added to PNA synthesis (or regular
protein synthesis) as is known in the art and as illustrated in
PCT/US99/10104 and PCT/US00/20476, incorporated herein by
reference: 39
[0241] In Structure 41, the PG protecting group is suitable for use
in peptide nucleic acid synthesis, with MMT, boc and Fmoc being
preferred.
[0242] 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.
[0243] In a preferred embodiment, the ETMs are attached as
polymers, for example as metallocene polymers, in a "branched"
configuration similar to the "branched DNA" embodiments herein and
as outlined in U.S. Pat. No. 5,124,246, using modified
functionalized nucleotides. The general idea is as follows. A
modified phosphoramidite nucleotide is generated that can
ultimately contain a free hydroxy group that can be used in the
attachment of phosphoramidite ETMs such as metallocenes. This free
hydroxy group could be on the base or the backbone, such as the
ribose or the phosphate (although as will be appreciated by those
in the art, nucleic acid analogs containing other structures can
also be used). The modified nucleotide is incorporated into a
nucleic acid, and any hydroxy protecting groups are removed, thus
leaving the free hydroxyl. Upon the addition of a phosphoramidite
ETM such as a metallocene, as described above in 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
PCT/US99/10104. In addition, Win some embodiments, it is desirable
to increase the solubility of the polymers by adding a "capping"
group to the terminal ETM in the polymer. 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.
[0244] Briefly, 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.
[0245] 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.
[0246] In addition, when the recruitment linker is nucleic acid,
any combination of ETM attachments may be done.
[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. 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 either the
solution binding ligand or the first portion of the label probe
will depend on the composition of the recruitment linker and of the
label and/or binding ligand, as will be appreciated by those in the
art. When either the label probe or the binding ligand is nucleic
acid, nucleic acid recruitment linkers are generally formed during
the synthesis of the first species, with incorporation of
nucleosides containing ETMs as required. Alternatively, the first
portion of the label probe or the binding ligand and the
recruitment linker may be made separately, and then attached. When
they are both nucleic acid, 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, 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 "nucleoside
replacement" embodiments and putatively some of the phosphate
attachments, may or may not be recognized by the enzymes to allow
incorporation into a growing nucleic acid. Therefore, preferred
attachments in this embodiment are to the base or ribose of the
nucleotide.
[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.
[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 of conductive oligomers and
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. 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 and depicted in FIG. 7R. 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, as is depicted in
FIGS. 7A and 7B, 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. 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 Tm may be
decreased, depending on the site of attachment and the
characteristics of the ETM. Thus, in general, when multiple ETMs
are used on hybridizing strands, generally there are less than
about 5, with less than about 3 being preferred, or alternatively
the ETMs should be spaced sufficiently far apart that the
intervening nucleotides can sufficiently hybridize to allow good
kinetics.
[0260] In one embodiment, when nucleic acid targets and/or binding
ligands and/or recruitment linkers are used, 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); Millan 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.
[0261] Similarly, the systems of the invention may utilize
non-covalently attached ETMs, as is generally described in Napier
et al., Bioconj. Chem. 8:906 (1997), hereby expressly incorporated
by reference. In this embodiment, changes in the redox state of
certain molecules as a result of the presence of DNA (i.e. guanine
oxidation by ruthenium complexes) can be detected using the SAMs
comprising conductive oligomers as well.
[0262] Thus, the present invention provides electrodes comprising
monolayers comprising conductive oligomers, generally including
capture binding ligands, and either binding ligands or label probes
that will bind to the binding ligands comprising recruitment
linkers containing ETMs.
[0263] In a preferred embodiment, the compositions of the invention
are used to detect target analytes in a sample. In a preferred
embodiment, the target analyte is a nucleic acid, and thus
detection of target sequences is done. The term "target sequence"
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. 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. 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.
[0264] If required, the target analyte is prepared using known
techniques. For example, the sample may be treated to lyse the
cells, using known lysis buffers, electroporation, etc., with
purification occuring as needed, as will be appreciated by those in
the art. In a preferred embodiment, when the target analyte is
nucleic acid, amplification may be done, including PCR and other
amplification techniques as outlined in PCT US99/01705,
incorporated herein by reference in its entirety. When the target
analyte is a nucleic acid, 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.
[0265] 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.
[0266] 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. 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.
[0267] When gold electrodes are used, a C6 insulator, comprising
6-mercaptohexanol, is included in the hybridization buffer.
[0268] As will be appreciated by those in the art, the nucleic acid
systems of the invention may take on a large number of different
configurations, as is generally depicted in the figures. In
general, there are three types of systems that can be used: (1)
systems in which the target analyte itself is labeled with ETMs
(i.e. the use of a target analyte analog, for non-nucleic acid
systems, or, for nucleic acid systems, the target sequence is
labeled); (2) systems in which label probes (or capture binding
ligands with recruitment linkers) directly bind (i.e. hybridize for
nucleic acids) to the target analytes; and (3) systems in which
label probes comprising recruitment linkers are indirectly bound to
the target analytes, for example through the use of amplifier
probes.
[0269] In all three of these systems, it is preferred, although not
required, that the target analyte be immobilized on the electrode
surface. This is preferably done using capture binding ligands and
optionally one or more capture extender ligands. When only capture
binding ligands are utilized, it is necessary to have unique
capture binding ligands for each target analyte; that is, the
surface must be customized to contain unique capture binding
ligands. Alternatively, the use of capture extender ligands,
particularly when the capture extender ligands are capture extender
probes (i.e. nucleic acids) 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.
[0270] In a preferred embodiment, the capture 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 put onto the binding ligand, using an activated
carboxylate and nucleic acid, although other capture ligands may be
attached in this way as well. These two reagents have the advantage
of being stable in aqueous solution, yet react with primary
alkylamines. This allows the spotting of probes (either capture or
detection probes, or both) using known methods (ink jet, spotting,
etc.) onto the surface.
[0271] In addition, there are a number of non-nucleic acid methods
that can be used to immobilize a capture binding ligand 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 binding ligand. 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.
[0272] 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.
[0273] 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.
[0274] In one embodiment, the target is not bound to the electrode
surface using capture binding ligands. In this embodiment, what is
important, as for all the assays herein, is that excess label
probes be removed prior to detection and that the assay complex
(comprising the recruitment linker) be in proximity to the surface.
As will be appreciated by those in the art, this may be
accomplished in other ways. For example, the assay complex may be
present on beads that are added to the electrode comprising the
monolayer. The recruitment linkers comprising the ETMs may be
placed in proximity to the conductive oligomer surface using
techniques well known in the art, including gravity settling of the
beads on the surface, electrostatic or magnetic interactions
between bead components and the surface, using binding partner
attachment as outlined above. Alternatively, after the removal of
excess reagents such as excess label probes, the assay complex may
be driven down to the surface, for example by pulsing the system
with a voltage sufficient to drive the assay complex to the
surface.
[0275] However, preferred embodiments utilize assay complexes
attached via capture binding ligands.
[0276] For nucleic acid systems, a preferred embodiments utilize
the target sequence itself containing 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. In addition, as will be appreciated by those
in the art, multiple capture probes can be utilized, either in a
configuration wherein the 5'-3' orientation of the capture probes
is different, or where "loops" of target form when multiples of
capture probes are used.
[0277] For nucleic acid systems, a preferred embodiments utilize
the label probes directly hybridizing to the target sequences, as
is generally depicted in FIGS. 7D-7I. In these embodiments, the
target sequence is preferably, but not required to be, 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 comprising conductive oligomers. 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 (labeled 41 in the figures) 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.
[0278] As above, generally in this embodiment the configuration of
the system and the label probes (recruitment linkers) are designed
to recruit the ETMs as close as possible to the monolayer
surface.
[0279] In a preferred embodiment, the label probes are bound to the
target analyte 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, for nucleic
acid detection. 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.
[0280] In general, these embodiments may be described as follows.
An amplifier probe is hybridized to the target sequence, either
directly (e.g. FIG. 7I), or through the use of a label extender
probe (e.g. FIGS. 7N and 7O), 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.
[0281] 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. In some embodiments, it is possible to use amplifier
binding ligands, that are non-nucleic acid based but that comprise
a plurality of binding sites for the later association/binding of
label ligands comprising recruitment linkers. However, amplifier
probes are preferred in nucleic acid systems.
[0282] 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 (e.g. FIG. 7I), 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 (e.g.
FIG. 7N). In a preferred embodiment, the first probe sequence of
the amplifier probe is substantially complementary to the target
sequence, as is generally depicted in FIG. 7I.
[0283] 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.
[0284] 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.
[0285] 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.
[0286] 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.
[0287] 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, as is
generally depicted in FIG. 7L. 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.
[0288] In a preferred embodiment, more than one label extender
probe-amplifier probe pair may be used, that 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.
[0289] In a preferred embodiment, more than one label extender
probe is used with a single amplifier probe to reduce non-specific
binding, as is depicted in FIG. 7O and 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.
[0290] 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.
[0291] 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.
[0292] 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. Again, when non-nucleic acid
amplifier moieties are used, the amplification segment can bind
label ligands.
[0293] 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.
[0294] 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 oligonucleotides. 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.
[0295] 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.
[0296] 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. In addition, as outlined herein, linear amplification
probes may form hairpin stem-loop structures.
[0297] 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.
[0298] In addition, the amplifier probe may be totally linear,
totally branched, totally dendrimeric, or any combination
thereof.
[0299] 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.
[0300] 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. The reactions outlined below may be accomplished in a
variety of ways, as will be appreciated by those in the art.
Components of the reaction may be added simultaneously, or
sequentially, in any order, with preferred embodiments outlined
below. In addition, the reaction may include a variety of other
reagents may be included in the assays. These include reagents like
salts, buffers, neutral proteins, e.g. albumin, detergents, etc
which may be used to facilitate optimal hybridization and
detection, and/or reduce non-specific or background interactions.
Also reagents that otherwise improve the efficiency of the assay,
such as protease inhibitors, nuclease inhibitors, anti-microbial
agents, etc., may be used, depending on the sample preparation
methods and purity of the target.
[0301] Generally, the methods are as follows. In a preferred
embodiment, the target is initially immobilized or attached to the
electrode. For nucleic acids, this is done by forming a
hybridization complex between a capture probe and a portion of the
target sequence. A preferred embodiment utilizes capture extender
probes; in this embodiment, a hybridization complex is formed
between a portion of the target sequence and a first portion of a
capture extender probe, and an additional hybridization complex
between a second portion of the capture extender probe and a
portion of the capture probe. Additional preferred embodiments
utilize additional capture probes, thus forming a hybridization
complex between a portion of the target sequence and a first
portion of a second capture extender probe, and an additional
hybridization complex between a second portion of the second
capture extender probe and a second portion of the capture probe.
Non-nucleic acid embodiments utilize capture binding ligands and
optional capture extender ligands.
[0302] Alternatively, the attachment of the target sequence to the
electrode is done simultaneously with the other reactions.
[0303] The method proceeds with the introduction of amplifier
probes, if utilized. In a preferred embodiment, the amplifier probe
comprises a first probe sequence that is substantially
complementary to a portion of the target sequence, and at least one
amplification sequence.
[0304] In one embodiment, the first probe sequence of the amplifier
probe is hybridized to the target sequence, and any unhybridized
amplifier probe is removed. This will generally be done as is known
in the art, and depends on the type of assay. When the target
sequence is immobilized on a surface such as an electrode, the
removal of excess reagents generally is done via one or more
washing steps, as will be appreciated by those in the art. In this
embodiment, the target may be immobilized on any solid support.
When the target sequence is not immobilized on a surface, the
removal of excess reagents such as the probes of the invention may
be done by adding beads (i.e. solid support particles) that contain
complementary sequences to the probes, such that the excess probes
bind to the beads. The beads can then be removed, for example by
centrifugation, filtration, the application of magnetic or
electrostatic fields, etc.
[0305] The reaction mixture is then subjected to conditions
(temperature, high salt, changes in pH, etc.) under which the
amplifier probe disassociates from the target sequence, and the
amplifier probe is collected. The amplifier probe may then be added
to an electrode comprising capture probes for the amplifier probes,
label probes added, and detection is achieved.
[0306] In a preferred embodiment, a larger pool of probe is
generated by adding more amplifier probe to the target sequence and
the hybridization/disassociation reactions are repeated, to
generate a larger pool of amplifier probe. This pool of amplifier
probe is then added to an electrode comprising amplifier capture
probes, label probes added, and detection proceeds.
[0307] In this embodiment, it is preferred that the target analyte
be immobilized on a solid support, including an electrode, using
the methods described herein; although as will be appreciated by
those in the art, alternate solid support attachment technologies
may be used, such as attachment to glass, polymers, etc. It is
possible to do the reaction on one solid support and then add the
pooled amplifier probe to an electrode for detection.
[0308] In a preferred embodiment, the amplifier probe comprises a
multiplicity of amplification sequences.
[0309] In one embodiment, the first probe sequence of the amplifier
probe is hybridized to the target sequence, and any unhybridized
amplifier probe is removed. Again, preferred embodiments utilize
immobilized target sequences, wherein the target sequences are
immobilized by hybridization with capture probes that are attached
to the electrode, or hybridization to capture extender probes that
in turn hybridize with immobilized capture probes as is described
herein. Generally, in these embodiments, the capture probes and the
detection probes are immobilized on the electrode, generally at the
same "address".
[0310] In a preferred embodiment, the first probe sequence of the
amplifier probe is hybridized to a first portion of at least one
label extender probe, and a second portion of the label extender
probe is hybridized to a portion of the target sequence. Other
preferred embodiments utilize more than one label extender probe,
as is generally shown in FIG. 7O.
[0311] In a preferred embodiment, the amplification sequences of
the amplifier probe are used directly for detection, by hybridizing
at least one label probe sequence.
[0312] The invention thus provides assay complexes that minimally
comprise a target sequence and a label probe. "Assay complex"
herein is meant the collection of binding complexes comprising
capture binding ligands, target analytes (or analogs, as described
below) and label moieties comprising recruitment linkers that
allows detection. The composition of the assay complex depends on
the use of the different components outlined herein. Thus, the
assay complex comprises the capture probe and the target sequence.
The assay complexes may also include capture extender ligands
(including probes), label extender ligands, and amplifier ligands,
as outlined herein, depending on the configuration used.
[0313] The assays are generally run under conditions which allows
formation of the assay 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.
[0314] These parameters may also be used to control non-specific
binding for nucleic acids, 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.
[0315] In a preferred embodiment, when all of the components
outlined herein are used, a preferred method for nucleic acid
detection 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.
[0316] 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.
[0317] 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 to the monolayer surface. Thus for example, 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.
[0318] 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.
[0319] Other embodiments of the invention utilize different steps.
For example, competitive assays may be run. In this embodiment, the
target analyte in a sample may be replaced by a target analyte
analog comprising a portion that either comprises a recruitment
linker or can indirectly bind a recruitment linker. This may be
done as is known in the art, for example by using affinity
chromatography techniques that exchange the analog for the analyte,
leaving the analyte bound and the analog free to interact with the
capture binding ligands on the electrode surface. This is generally
depicted in FIG. 6A.
[0320] Alternatively, a preferred embodiment utilizes a competitive
binding assay when the solution binding ligand comprises a directly
or indirectly associated recruitment linker comprising ETMs. In
this embodiment, a target analyte or target analyte analog that
will bind the solution binding ligand is attached to the surface.
The solution binding ligand will bind to the surface bound analyte
and give a signal. Upon introduction of the target analyte of the
sample, a proportion of the solution binding ligand will dissociate
from the surface bound target and bind to the incoming target
analyte. Thus, a loss of signal proportional to the amount of
target analyte in the sample is seen.
[0321] The compositions of the invention are generally synthesized
as outlined below, generally 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.
[0322] The compositions may be made in several ways. A preferred
method first synthesizes a conductive oligomer 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 conductive oligomer added, followed by attachment to the
electrode. Alternatively, a monolayer of conductive oligomer (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
conductive oligomers are used which are not stable in the solvents
and under the conditions used in traditional nucleic acid
synthesis.
[0323] In a preferred embodiment, the compositions of the invention
are made by first forming the conductive oligomer 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 conductive oligomer to the
electrode.
[0324] The attachment of the conductive oligomer to the nucleoside
may be done in several ways. In a preferred embodiment, all or part
of the conductive oligomer 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, 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
US99/01705 and PCT US99/01703, all of which are expressly
incorporated by reference.
[0325] The conductive oligomer is then attached to a nucleoside
that may contain one (or more) of the oligomer units, attached as
depicted herein.
[0326] In a preferred embodiment, attachment is to a ribose of the
ribose-phosphate backbone in a number of ways, including attachment
via 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.
[0327] Alternatively, attachment is via a phosphate of the
ribose-phosphate backbone.
[0328] In a preferred embodiment, attachment is via the base, and
can include acetylene linkages and amide linkages. In a preferred
embodiment, protecting groups may be added to the base prior to
addition of the conductive oligomers. 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.
[0329] Alternatively, attachment to the base may be done by making
the nucleoside with one unit of the oligomer, followed by the
addition of others.
[0330] 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.
[0331] 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 I, 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: 40
[0332] 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.
[0333] 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.
[0334] 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.
[0335] 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.
[0336] 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.
[0337] 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 the figures.
[0338] 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.
[0339] 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 figures, 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 Lett. 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 PCT/US99/10104 which
describes the synthesis of metallocenes (in this case, ferrocene)
attached via acetylene linkages to the bases.
[0340] 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.
[0341] 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.
[0342] 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. 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.
[0343] 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.
[0344] 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.
[0345] 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.
[0346] 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.
[0347] The above methods may also be used to attach insulator
molecules to a gold electrode, and moieties comprising capture
binding ligands.
[0348] In a preferred embodiment, a monolayer comprising conductive
oligomers (and preferably 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 theattachment 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).
[0349] 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. The bases
can then be incorporated into monomeric subunits.
[0350] 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 a-carbon atoms, either to a terminal monomeric subunit
or an internal one.
[0351] 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.
[0352] 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.
[0353] As will be appreciated by those in the art, electrodes may
be made that have any combination of nucleic acids, conductive
oligomers and insulators.
[0354] The compositions of the invention may additionally contain
one or more 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, and colored or fluorescent dyes. 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.
[0355] Once made, the compositions find use in a number of
applications, as described herein. In particular, the compositions
of the invention find use in target analyte assays. As will be
appreciated by those in the art, electrodes can be made that have a
single species of binding ligands such as nucleic acid, i.e. a
single binding ligand, or multiple binding ligand species.
[0356] In addition, as outlined herein, the use of a solid support
such as an electrode enables the use of these probes in an array
form. The use of oligonucleotide arrays are well known in the art,
and the methods and compositions herein allow the use of array
formats for other target analytes as well. In addition, techniques
are known for "addressing" locations within an electrode and for
the surface modification of electrodes. Thus, in a preferred
embodiment, arrays of different binding ligands are laid down on
the electrode, each of which are covalently attached to the
electrode via a conductive linker. In this embodiment, the number
of different species may vary widely, from one to thousands, with
from about 4 to about 100,000 being preferred, and from about 10 to
about 10,000 being particularly preferred.
[0357] The invention finds use in the screening of candidate
bioactive agents for therapeutic agents that can alter the binding
of the analyte to the binding ligand, and thus may be involved in
biological function. The term "agent" or "exogeneous compound" as
used herein describes any molecule, e.g., protein, oligopeptide,
small organic molecule, polysaccharide, polynucleotide, etc., with
the capability of directly or indirectly altering target analyte
binding. Generally a plurality of assay mixtures are run in
parallel with different agent concentrations to obtain a
differential response to the various concentrations. Typically, one
of these concentrations serves as a negative control, i.e., at zero
concentration or below the level of detection.
[0358] Candidate agents encompass numerous chemical classes, though
typically they are organic molecules, preferably small organic
compounds having a molecular weight of more than 100 and less than
about 2,500 daltons. Candidate agents comprise functional groups
necessary for structural interaction with proteins, particularly
hydrogen bonding, and typically include at least an amine,
carbonyl, hydroxyl or carboxyl group, preferably at least two of
the functional chemical groups. The candidate agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof. Particularly preferred are peptides.
[0359] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides. Altematively, libraries
of natural compounds in the form of bacterial, fungal, plant and
animal extracts are available or readily produced. Additionally,
natural or synthetically produced libraries and compounds are
readily modified through conventional chemical, physical and
biochemical means. Known pharmacological agents may be subjected to
directed or random chemical modifications, such as acylation,
alkylation, esterification, amidification to produce structural
analogs.
[0360] Candidate agents may be added either before or after the
target analyte.
[0361] Once the assay complexes of the invention are made, that
minimally comprise a target sequence and a label probe, detection
proceeds with electronic initiation. Without being limited by the
mechanism or theory, detection is based on the transfer of
electrons from the ETM to the electrode.
[0362] 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.
[0363] 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.
[0364] 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.
[0365] 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.
[0366] 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.
[0367] 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.
[0368] Suitable electron source molecules are well known in the
art, and include, but are not limited to, ferricyanide, and
luminol.
[0369] 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.
[0370] 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 fluoroscence.
[0371] 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 ppl 97-202).
These differences can be monitored using a spectrophotometer or
simple photomultiplier tube device.
[0372] 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.
[0373] 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.
[0374] 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.
[0375] In addition, scanning fluorescence detectors such as the
Fluorlmager 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.
[0376] 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.
[0377] 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.
[0378] 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.
[0379] 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.
[0380] 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.
[0381] 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.
[0382] 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.
[0383] 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
determinaton 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.
[0384] 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 AC ] cosh [ nF RT E AC ] + cosh [ nF
RT ( E DC - E O ) ] Equation 1
[0385] In Equation 1, n is the number of electrons oxidized or
reduced per redox molecule, f is the applied frequency, F is
Faraday's constant, N.sub.total is the total number of redox
molecules, E.sub.O is the formal potential of the redox molecule, R
is the gas constant, T is the temperature in degrees Kelvin, and
E.sub.DC is the electrode potential. The model fits the
experimental data very well. In some cases the current is smaller
than predicted, however this has been shown to be caused by
ferrocene degradation which may be remedied in a number of
ways.
[0386] In addition, the faradaic current can also be expressed as a
function of time, as shown in Equation 2:
Equation 2
[0387] I.sub.F is the Faradaic current and q.sub.e is the
elementary charge.
[0388] 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
[0389] i.sub.AC=f(Nemst factors)f(k.sub.ET)f(instrument
factors)
[0390] 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.
[0391] 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.
[0392] 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.
[0393] 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.
[0394] The use of combinations of AC and DC signals gives a variety
of advantages, including surprising sensitivity and signal
maximization.
[0395] 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.
[0396] 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.
[0397] 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
[0398] 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.
[0399] 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.
[0400] 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.
[0401] 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.
[0402] 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.
[0403] 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.
[0404] 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.
[0405] 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.
[0406] 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.
[0407] 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.
[0408] The output signal is characteristic of the presence of the
ETM; that is, the output signal is characteristic of the presence
of the target-specific assay complex comprising label probes and
ETMs. In a preferred embodiment, the basis of the detection is a
difference in the faradaic impedance of the system as a result of
the formation of the assay complex. Faradaic impedance is the
impedance of the system between the electrode and the ETM. Faradaic
impedance is quite different from the bulk or dielectric impedance,
which is the impedance of the bulk solution between the electrodes.
Many factors may change the faradaic impedance which may not effect
the bulk impedance, and vice versa. Thus, the assay complexes
comprising the nucleic acids in this system have a certain faradaic
impedance, that will depend on the distance between the ETM and the
electrode, their electronic properties, and the composition of the
intervening medium, among other things. Of importance in the
methods of the invention is that the faradaic impedance between the
ETM and the electrode is signficantly different depending on
whether the label probes containing the ETMs are specifically or
non-specifically bound to the electrode.
[0409] Accordingly, the present invention further provides
apparatus for the detection of nucleic acids using AC detection
methods. The apparatus includes a test chamber which has at least a
first measuring or sample electrode, and a second measuring or
counter electrode. Three electrode systems are also useful. The
first and second measuring electrodes are in contact with a test
sample receiving region, such that in the presence of a liquid test
sample, the two electrodes may be in electrical contact.
[0410] In a preferred embodiment, the first measuring electrode
comprises a single stranded nucleic acid capture probe covalently
attached via an attachment linker, and a monolayer comprising
conductive oligomers, such as are described herein.
[0411] 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.
[0412] 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.
[0413] Thus, the compositions of the present invention may be used
in a variety of research, clinical, quality control, or field
testing settings.
[0414] In a preferred embodiment, the probes are used in genetic
diagnosis. For example, probes can be made using the techniques
disclosed herein to detect target sequences such as the gene for
nonpolyposis colon cancer, the BRCA1 breast cancer gene, P53, which
is a gene associated with a variety of cancers, the Apo E4 gene
that indicates a greater risk of Alzheimer's disease, allowing for
easy presymptomatic screening of patients, mutations in the cystic
fibrosis gene, or any of the others well known in the art.
[0415] In an additional embodiment, viral and bacterial detection
is done using the complexes of the invention. In this embodiment,
probes are designed to detect target sequences from a variety of
bacteria and viruses. For example, current blood-screening
techniques rely on the detection of anti-HIV antibodies. The
methods disclosed herein allow for direct screening of clinical
samples to detect HIV nucleic acid sequences, particularly highly
conserved HIV sequences. In addition, this allows direct monitoring
of circulating virus within a patient as an improved method of
assessing the efficacy of anti-viral therapies. Similarly, viruses
associated with leukemia, HTLV-I and HTLV-II, may be detected in
this way. Bacterial infections such as tuberculosis, clymidia and
other sexually transmitted diseases, may also be detected, for
example using ribosomal RNA (rRNA) as the target sequences.
[0416] In a preferred embodiment, the nucleic acids of the
invention find use as probes for toxic bacteria in the screening of
water and food samples. For example, samples may be treated to lyse
the bacteria to release its nucleic acid (particularly rRNA), and
then probes designed to recognize bacterial strains, including, but
not limited to, such pathogenic strains as, Salmonella,
Campylobacter, Vibrio cholerae, Leishmania, enterotoxic strains of
E. coli and Legionnaire's disease bacteria. Similarly,
bioremediation strategies may be evaluated using the compositions
of the invention.
[0417] In a further embodiment, the probes are used for forensic
"DNA fingerprinting" to match crime-scene DNA against samples taken
from victims and suspects.
[0418] In an additional embodiment, the probes in an array are used
for sequencing by hybridization.
[0419] Thus, the present invention provides for extremely specific
and sensitive probes, which may, in some embodiments, detect target
sequences without removal of unhybridized probe. This will be
useful in the generation of automated gene probe assays.
[0420] Alternatively, the compositions of the invention are useful
to detect successful gene amplification in PCR, thus allowing
successful PCR reactions to be an indication of the presence or
absence of a target sequence. PCR may be used in this manner in
several ways. For example, in one embodiment, the PCR reaction is
done as is known in the art, and then added to a composition of the
invention comprising the target nucleic acid with a ETM, covalently
attached to an electrode via a conductive oligomer with subsequent
detection of the target sequence. Alternatively, PCR is done using
nucleotides labelled with a ETM, either in the presence of, or with
subsequent addition to, an electrode with a conductive oligomer and
a target nucleic acid. Binding of the PCR product containing ETMs
to the electrode composition will allow detection via electron
transfer. Finally, the nucleic acid attached to the electrode via a
conductive polymer may be one PCR primer, with addition of a second
primer labelled with an ETM. Elongation results in double stranded
nucleic acid with a ETM
[0421] and electrode covalently attached. In this way, the present
invention is used for PCR detection of target sequences.
[0422] In a preferred embodiment, the arrays are used for mRNA
detection. A preferred embodiment utilizes either capture probes or
capture extender probes that hybridize close to the 3'
polyadenylation tail of the mRNAs. This allows the use of one
species of target binding probe for detection, i.e. the probe
contains a poly-T portion that will bind to the poly-A tail of the
mRNA target. Generally, the probe will contain a second portion,
preferably non-poly-T, that will bind to the detection probe (or
other probe). This allows one target-binding probe to be made, and
thus decreases the amount of different probe synthesis that is
done.
[0423] In a preferred embodiment, the use of restriction enzymes
and ligation methods allows the creation of "universal" arrays. In
this embodiment, monolayers comprising capture probes that comprise
restriction endonuclease ends, as is generally depicted in FIG. 7
of PCT US97/20014 . By utilizing complementary portions of nucleic
acid, while leaving "sticky ends", an array comprising any number
of restriction endonuclease sites is made. Treating a target sample
with one or more of these restriction endonucleases allows the
targets to bind to the array. This can be done without knowing the
sequence of the target. The target sequences can be ligated, as
desired, using standard methods such as ligases, and the target
sequence detected, using either standard labels or the methods of
the invention.
[0424] The present invention provides methods which can result in
sensitive detection of nucleic acids. In a preferred embodiment,
less than about 10.times.10.sup.6 molecules are detected, with less
than about 10.times.10.sup.5 being preferred, less than
10.times.10.sup.4 being particularly preferred, less than about
10.times.10.sup.3 being especially preferred, and less than about
10.times.10.sup.2 being most preferred. As will be appreciated by
those in the art, this assumes a 1:1 correlation between target
sequences and reporter molecules; if more than one reporter
molecule (i.e. electron transfer moeity) is used for each target
sequence, the sensitivity will go up.
[0425] While the limits of detection are currently being evaluated,
based on the published electron transfer rate through DNA, which is
roughly 10.times.10.sup.6 electrons/sec/duplex for an 8 base pair
separation (see Meade et al., Angw. Chem. Eng. Ed., 34:352 (1995))
and high driving forces, AC frequencies of about 100 kHz should be
possible. As the preliminary results show, electron transfer
through these systems is quite efficient, resulting in nearly
100.times.10.sup.3 electrons/sec, resulting in potential femptoamp
sensitivity for very few molecules.
[0426] The following examples serve to more fully describe the
manner of using the above-described invention, as well as to set
forth the best modes contemplated for carrying out various aspects
of the invention. It is understood that these examples in no way
serve to limit the true scope of this invention, but rather are
presented for illustrative purposes. All references cited herein
are incorporated by reference.
EXAMPLES
Example 1
Preparation and Evaluation of Asymmetric Monolayers
[0427] PreDaration of Asymmetric Monolayers
[0428] Synthesis of Symmetrical Disulfide
[0429] Symmetrical disulfides may be prepared from neopentyl
alcohol or from neopentyl iodide in the presence of thiol acetic
acid. Preferably, the reaction is carried out using neopentyl
alcohol in the presence of PPh3 and DIPAD. The resulting disulfide
is recrystallized from hexane, purified on a chromatographic
column, and treated with sodium hydroxide to yield a symmetrical
disulfide (See FIG. 3A).
[0430] Synthesis of CT103
[0431] The strategy for the synthesis of a precursor for CPG with
an AMFS was based on the chemistry established for exchanging an
symmetrical disulfide with acetyl thiol moiety in the presence of a
base as shown in FIG. 3C. First, CT100 was obtained from
dimethyloxetane and thiolacetic acide. Next, the symmetrical
disulfide CT101 was obtained from CT100 by treating CT100 with NaOH
and dioxane. Treatment of K133 and CT101 in the presence of NaOH
and Dioxane yielded the asymmetrical monolayer forming species
CT102. Further treatment of CT102 with succinic anhydride
[0432] Synthesis of CT105
[0433] The synthesis of CT105 is depicted in FIG. 3E and involves
the following steps:
[0434] Synthesis of M41. To a flask was added sodium hydride (6.68
g, 60% in mineral oil) to a solution of triethylene glycol (83.7 g)
in 1 L anhydrous DMF. The reaction mixture was stirred for 45 min
at room temperature, then cool to 0.degree. C. in an ice bath. To
the mixture was added 1 1-bromo-1-undecene (26 g) in 100 mL of DMF
dropwise within 30 min at 0.degree. C. and then the mixture was
vigorously stirred for 1 hour at 0.degree. C. After removing the
cooling bath, the reaction mixture was vigorously stirred overnight
at room temperature. To the mixture was add 50 mL of water, then
the solution was concentrated on a high vacuum rotavapor. To the
residue was add 400 mL of ethyl acetate and 600 mL of water and the
organic layer was separated, and the aqueous layer was extracted
with 30% ethyl acetate/70% hexane solution (3.times.300 mL). The
combined organic layers were washed with water (2.times.300 ml),
dried over anhydrous sodium sulfate, filtered and concentrated. T
he residue was purified with silica gel chromatography eluted with
10%-70% ethyl acetate in hexane to provide the desired product.
[0435] Synthesis of M42. To a Shlenk tube were added M41 (7.6 g),
thioacetic acid (2.2 mL), and t-butyl peroxide (0.48 mL). Then the
solution was flushed with argon for 5 min. After the cap was
closed, the tube was heated at 100.degree. C. overnight in an oil
bath. The tube was cooled to room temperature, and the reaction
mixture was diluted with 400 mL of dichloromethane. The organic
solution was washed with 200 mL of 5% sodium bicarbonate. Then the
aqueous layer was extracted with dichloromethane (2.times.300 mL).
The combined organic layers, was washed with brine, dried over
anhydrous sodium sulfate, filtered and concentrated. The residue
was purified with silica gel chromatography eluted with 30%-50%
ethyl acetate in hexane to provide the desired product.
[0436] Synthesis of CT101. To a flask was added 3,3-dimethyloxetane
(8.25 g, 96 mmol) and thiol acetic acid (13.06 g, 172 mmol). The
reaction mixture was heated to 65.degree. C. in an oil bath for 40
hours. Then the non-consumed starting material was removed by
distillation under vacuum (20-30 mmHg) at 65.degree. C. The crude
product was dissolved in 60 mL of methanol in a round bottom flask
and potassium carbonate (15.6 g) was added. The reaction mixture
was vigorously stirred at room temperature in open air for 24
hours. The reaction mixture was filtered through a bed of Celite
and washed with a mixture of methanol and dichloromethane (2:1,
3.times.50 mL). Then the filtrate was concentrated. The residue was
purified with silica gel chromatography eluted with 1% -5% methanol
in dichloromethane to provide the desired product. .sup.1H NMR (300
MHz, CDCl.sub.3).sub.--3.48 (s, 2H, CH.sub.2O), 2.89 (s, 2H,
CH.sub.2S), 1.01 (s, 6H, 2.times.CH.sub.3).
[0437] Synthesis of CT105. To a flask containing M42 (0.64 g) and
CT101(1.0 g) in THF (10 mL) and methanol (10 mL) was added NaOH
solution (1.0 mL, 8 M). The mixture was stirred in the air for six
hours. Then the solvents were removed on a rotavapor at 40.degree.
C. and residue was dissolved in 150 mL of dichloromethane. The
mixture was extracted with water (2.times.50 mL) and the organic
layer dried over anhydrous sodium sulfate, filtered and
concentrated. The residue was purified with silica gel
chromatography eluted with 50% -90% ethyl acetate in hexane to
provide the desired product as colorless viscous oil. .sup.1H NMR
(300 MHz, CDCl.sub.3).sub.--3.80-3.62 (m, 12H, 6.times.OCH.sub.2),
3.47 (m, 4H, 2.times.CH.sub.2OH), 2.85 (s, 2H, SCH.sub.2CMe.sub.2),
2.74 (t, J=7.2 Hz, 2H, SCH.sub.2), 1.77-1.63 (m, 4H,
2.times.CH.sub.2), 1.32 (m, 16H), 1.02 (s, 6H, CMe.sub.2). Anal.
calcd. for (C.sub.22H.sub.46O.sub.5S.sub.2 +Na).sup.+: 477. Found:
477.
[0438] Electrochemical Evaluation of New Insulators
[0439] The following oligonucleotides were used to evaluate the new
insulators:
[0440] 1) Capture oligonucleotides:
1 D1650: 5'-TCA TTG ATG GTC TCT TTT AAC A(N152) D1678: 5'-GAC TGA
CTC GTA CTA(N152)
[0441] 2) Direct assay signaling probe:
2 D1085: 5'-TCT ACA G(N6) C(N6) TGT TAA AAG AGA CCA TCA ATG A
[0442] 3) Sandwich Assay target and signaling probes:
3 D765: 5'-GAC ATC AAG CAG CCA TGC AAA TGT TAA AAG AGA CCA TCA ATG
AGG AAG CTG CAG AAT GGG ATA GAG TGC ATC CAG T D772: 5'-(N6) C(N6)
G(N6) C(N6) GCT TA(N6) C(N6) G(N6) C(N6) G(C131) TTT GCA TGG CTG
CTT GAT GTC D1156: 5'-CAC AGT GGG GGG ACA TCA AGC AGC CAT GCA AAT
GTT AAA AGA GAC CAT CAA TGA GGA AGC TGC AGA ATG GGA TAG AGT CAT CCA
GT
[0443] D1355 (20 Fc), D1356 (30 Fc) and D1357 (36 Fc) have the
similar sequence as D1358 (54 (Fc):
4 D1358: 5'-(C23)(C23)(C23)(C23)(C23)(C23)(N87)(N87) (N87)(N87) ATC
(C140)(N87)(N 87)(N87)(N87)(C140) TTT GCA TGG CTG CTT GAT GTC CCC
CCA CTG TG D998: 5'-TGT GCA GTT GAC GTG GAT TGT TAA AAG AGA CCA TCA
ATG AGG AAG CTG GAG AAT GGG ATA GAG TCA TCC AGT D1055 (20Fc):
5'-(C23)(C23)(C23)(C23)(- N87)(N87) (N87)(N87)(C140) ATC CAC GTG
AAC TGG AGA
[0444] Chip preparation and deposition solution.
[0445] Chips were made on spotting machine. To diminish the effect
of chip materials, the chips with different insulators were made
from the same circuit board. An array chip (lot # DC228, DC229, and
DC231, Type CB37-4) with sensor pads containing self assembled
monolayer according to
[0446] the pattern shown in FIG. 10.
[0447] The pad surfaces were respectively treated with the
deposition solutions. To reduce the influence of the concentration
of capture oligonucleotides, a stock deposition solution was
prepared according tothe standard procedure without insulator.
Next, the stock solution was divided into three portions, into
which the insulators, M44, CT99 and CT105, were added respectively.
The final deposition solutions consisted of a mixture of
DNA/H6-two-unit wire/insulator (with ratio of 1/10/5) with a total
thiol concentration of 53 .mu.M. The pads are deposited using the
spotting machine and post treated with insulator. As shown in FIG.
10, the chip has two rows of capture pads (D1650), a row of
negative control pads (D1678) non-specific binding pads (D1678),
and a row of insulator. The DNA-probe of D1650 is complementary to
the target oligonucleotide D1085, D765, D998, and D1156. D1678 is
non-complementary to either target or signaling
oligonucleotides.
[0448] Hybridization solution and testing.
[0449] For direct assays, the solution consists of 200 nM of
signaling probe and 41% H.sub.2O, 25% 4000 mM NaClO.sub.4 with 80
mM Tris (pH 6.5) 1.0 mM C6 insulator, 10% FCS, and 24% lysis
buffer.
[0450] For sandwich assays, the hybridization solution consisted of
10 nM target oligonucleotides and 30 nM signaling molecule in the
mixture of 41% H.sub.2O, 25% 4000 nM NaClO.sub.4 with 80 mM Tris,
pH 6.5, 1.0 mM C6 insulator, 10% FCS, and 24% lysis buffer.
[0451] The hybridization solution is injected into the cartridge of
3 chips. The solution is allowed to hybridize at room temperature
for 2 hours for direct assays or for overnight for sandwich assays.
The chip is then plugged into the reader and scanned in 4th
harmonics at different frequencies.
[0452] All peaks are calculated using the auto peak finder. Ip of
each data set (at least three chips) and the standard deviation at
all of the frequencies are calculated. The normalized frequency
response is determined by Ip (high frequency)/Ip(10 Hz).times.folds
increase in frequency).
[0453] As shown in FIG. 11 (direct assay of 2 N6 ferrocene
signaling probe), FIG. 12 (sandwich assay of 8 N6 ferrocene), FIG.
13 (sandwich assay of 20 C23 type ferrocene) and FIG. 14 (sandwich
assay for 54 C23 type ferrocene), the new insulators, CT99 and
CT105, gave much better electrochemical response than control
insulator M44. All data were collected for 1000 Hz at 4.sup.th
harmonics.
[0454] However, the nonspecific binding of three different
insulators is similar. FIG. 15 and FIG. 16, illustrate nonspecific
binding for direct and sandwich assays at 1000 Hz and .sub.4th
harmonics. Non-specific signaling on the pads with insulator alone
was higher for the new insulators, CT99 and CT105, but there were
no difference on pads containing non complementary DNA.
[0455] FIG. 17 depicts a monolayer comprising insulators only (i.e.
M44) and a monolayer comprising asymmetric monolayer forming
species (i.e. CT105).
[0456] In order to further evaluate the behavior of the new
insulators, a frequency response study was carried out at 10 Hz,
100 Hz and 100 Hz. As shown in FIGS. 18-20, the new insulators gave
better frequency response than the control insulator M44. FIG. 18
is the frequency response for D1085 of two N6 ferrocenes, while
FIG. 19 and FIG. 20,respectively are the frequency response for the
sandwich assays of 8 and 20 ferrocene systems.
Sequence CWU 1
1
9 1 22 DNA Artificial Synthetic 1 tcattgatgg tctcttttaa ca 22 2 15
DNA Artificial Synthetic 2 gactgactcg tacta 15 3 22 DNA Artificial
Synthetic 3 tgttaaaaga gaccatcaat ga 22 4 76 DNA Artificial
Synthetic 4 gacatcaagc agccatgcaa atgttaaaag agaccatcaa tgaggaagct
gcagaatggg 60 atagagtgca tccagt 76 5 21 DNA Artificial Synthetic 5
tttgcatggc tgcttgatgt c 21 6 86 DNA Artificial Synthetic 6
cacagtgggg ggacatcaag cagccatgca aatgttaaaa gagaccatca atgaggaagc
60 tgcagaatgg gatagagtca tccagt 86 7 32 DNA Artificial Synthetic 7
tttgcatggc tgcttgatgt ccccccactg tg 32 8 72 DNA Artificial
Synthetic 8 tgtgcagttg acgtggattg ttaaaagaga ccatcaatga ggaagctgca
gaatgggata 60 gagtcatcca gt 72 9 18 DNA Artificial Synthetic 9
atccacgtca actgcaca 18
* * * * *