Note: Descriptions are shown in the official language in which they were submitted.
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
METHODS AND COMPOSITIONS RELATING TO ELECTRICAL DETECTION
OF NUCLEIC ACID REACTIONS
This is a continuing application of U.S.S.N.s 09/458,501, filed December 9,
1999; 09/459,685, filed
December 13, 1999 and 09/458,533, filed December 9, 1999, all of which are
expressly
incorporated by reference herein.
FIELD OF THE INVENTION
This invention relates to the detection of molecular interactions between
biological molecules.
Specifically, the invention relates to electrical detection of interactions
such as hybridization
between nucleic acids or peptide antigen-antibody interactions using arrays of
peptides or
oligonucleotides. In particular, the invention relates to an apparatus and
methods for detecting
nucleic acid hybridization or peptide binding using electronic methods
including AC impedance. In
some embodiments, no electrochemical or other label moieties are used. In
others,
electrochemically active labels are used to detect reactions on hydrogel
arrays, including
genotyping reactions such as the single base extension reaction.
BACKGROUND OF THE INVENTION
A number of commonly-utilized biological applications, including for example,
diagnoses of genetic
disease, analyses of sequence polymorphisms, and studies of receptor-ligand
interactions, rely on
the ability of analytical technologies to readily detect events related to the
interaction between
probe and target molecules. While these molecular detection technologies have
traditionally
2 0 utilized radioactive isotopes or fluorescent compounds to monitor probe-
target interactions,
methods for the electrical detection of molecular interactions have provided
an attractive
alternative to detection techniques relying on radioactive or fluorescent
labels.
Electrical and electrochemical detection techniques are based on the detection
of alterations in the
electrical properties of an electrode arising from interactions between probe
molecules on the
2 5 surface of the electrode and target molecules in the reaction mixture.
Electrical or electrochemical
1
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
detection eliminates many of the disadvantages inherent in use of radioactive
or fluorescent labels
to discern molecular interactions. This process offers, for example, a
detection technique that is
safe, inexpensive, and sensitive, and is not burdened with complex and onerous
regulatory
requirements.
However, despite these advantages, there are a number of obstacles in using
electrical or
electrochemical detection techniques for analyzing molecular interactions. One
such obstacle is
the requirement, in some methods, of incorporating an electrochemical label
into the target
molecule. For example, labeled target molecules have been used to increase the
signal produced
upon the formation of nucleic acid duplexes during hybridization assays.
For example, Meade et al. (in U.S. Patent Nos. 5,591,578, 5,705,348,
5,770,369, 5,780,234 and
5,824,473) provide methods for the selective covalent modification of nucleic
acids with redox-
active moieties such as transition metal complexes. Meade et al. further
disclose nucleic acid
hybridization assays employing such covalently-modified nucleic acid
molecules. Similar work is
further disclosed in U.S. Patent No. 6,090,933 and WO 98/20162, WO 98/12430,
WO 00/16089,
WO 99/57317, WO 99/67425, WO 99/37819, WO 00/38836, PCT US 00/19889 and WO
99/57319.
Heller et al. (in U.S. Patent Nos. 5,605,662 and 5,632,957) provide methods
for controlling
molecular biological reactions in microscopic formats that utilize a self-
addressable, self-
assembling microelectronic apparatus. Heller et al. further provide an
apparatus in which target
2 0 molecules labeled with fluorescent dyes are transported by free field
electrophoresis to specific
test sites where the target molecules are concentrated thereby, and reacted
with specific probes
bound to that test site. Unbound or non-specifically interacting target
molecules are thereafter
removed by reversing the electric polarity at the test site. Interactions
between probe and target
molecules are subsequently assayed using optical means.
2 5 Certain alternative methods that do not employ labeled target nucleic
acids have been described
in the prior art. For example, Hollis et al. (in U.S. Patent Nos. 5,653,939
and 5,846,708) provide a
meth3d and apparatus for identifying molecular structures within a sample
substance using a
monolithic array of test sites formed on a substrate upon which the sample
substance is applied.
In the method of Hollis et al., changes in the electromagnetic or acoustic
properties - for example,
3 0 the change in resonant frequency - of the test sites following the
addition of the sample substance
are detected in order to determine which probes have interacted with target
molecules in the
sample substance.
In addition, Eggers et al. (in U.S. Patent Nos. 5,532,128, 5,670,322, and
5,891,630) provide a
method and apparatus for identifying molecular structures within a sample
substance. In the
3 5 method of Eggers et al., a plurality of test sites to which probes have
been bound is exposed to a
sample substance and then an electrical signal is applied to the test sites.
The dielectrical
2
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/LTS00/33497
properties of the test sites are subsequently detected to determine which
probes have interacted
with target molecules in the sample substance.
Another obstacle in the development of a simple and cost-effective electrical
and electrochemical
detection apparatus for detecting molecular interactions involves the
attachment of probe
molecules to the microelectrodes or substrate of a microarray. For example,
although the prior art
provides microarrays using polyacrylamide pads for attachment of
oligonucleotide probes to a
solid support, the art has not provided such pads in conjunction with an
electrical or
electrochemical detection apparatus.
Guschin et al., 1997, Anal. Biochem. 250: 203-11 describe a technique for
detecting molecular
interactions between target molecules in a biological sample solution and
polyacrylamide gel-
immobilized probes on a glass substrate. In the technique disclosed by Guschin
et al., molecular
interactions between probes and target molecules are detected using optical
reporters. The
Guschin et al. reference neither teaches nor suggests using electrical or
electrochemical detection
techniques to detect hybridization between target molecules and immobilized
probes.
Guschin et al., 1997, Appl. Environ. Microbiol. 63: 2397-402 also describe the
fabrication of
microarrays through the immobilization of oligonucleotide probes on a
polyacrylamide gel pad
placed in contact with a glass substrate. In this technique disclosed by
Guschin et al., parallel
hybridization between target nucleic acids and immobilized probes is detected
using optical
reporter moieties. This Guschin et al. reference also does not teach or
suggest using electrical or
2 0 electrochemical detection techniques in combination with the
immobilization of probes on
polyacrylamide gel pads.
In addition, Yang et al., 1997, Anal. Chim. Acta 346: 259-75 describe the
fabrication of
microarrays through the immobilization of nucleic acid probes on
polyacrylamide gel pads and
subsequent detection of molecular interactions between probe and target
molecules using optical
2 5 reporter moieties. Yang et al. further describe an alternative technique
in which molecular
interactions between labeled target molecules and nucleic acid probes that
have been directly
attached to solid electrodes are detected using electrical or electrochemical
means. Yang et al.,
however, does not suggest using electrical or electrochemical detection
techniques in combination
with the immobilization of probes on polyacrylamide gel pads.
3 0 The detection of single base mutations and genetic polymorphisms in
nucleic acids is an important
tool in modern diagnostic medicine and biological research. In addition,
nucleic acid-based
assays also play an important role in identifying infectious microorganisms
such as bacteria and
viruses, in assessing levels of both normal and defective gene expression, and
in detecting and
identifying mutant genes associated with disease such as oncogenes.
Improvements in the
3
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/LTS00/33497
speed, efficiency, economy and specificity of such assays are thus significant
needs in the
medical arts.
Ideally, such assays should be sensitive, specific and easily amenable to
automation. Efforts to
improve sensitivity in nucleic acid assays are known in the prior art. For
example, the polymerase
chain reaction (Mullis, U.S. Patent No. 4,683,195, issued July 28, 1987)
provides the capacity to
produce useful amounts (about 1 Ng) of a specific nucleic acid in a sample in
which the original
amount of the specific nucleic acid is substantially smaller (about 1 pg).
However, the prior art has
been much less successful in improving specificity of nucleic acid
hybridization assays.
The specificity of nucleic acid assays is determined by the extent of
molecular complementarity of
hybridization between probe and target sequences. Although it is theoretically
possible to
distinguish complementary targets from one or two mismatched targets under
rigorously-defined
conditions, the dependence of hybridization on target/probe concentration and
hybridization
conditions limits the extent to which hybridization mismatch can be used to
reliably detect, inter
alia. mutations and genetic polymorphisms.
Detection of single base extension has been used for mutation and genetic
polymorphism
detection in the prior art.
U.S. Patent No. 5,925,520 disclosed a method for detecting genetic
polymorphisms using single
base extension and capture groups on oligonucleotide probes using at least two
types of dideoxy,
chain-terminating nucleotide triphosphates, each labeled with a detectable and
distinguishable
2 0 fluorescent labeling group.
U.S. Patent No. 5,710,028 disclosed a method of determining the identity of
nucleotide bases at
specific positions in nucleic acids of interest, using detectably-labeled
chain-terminating
nucleotides, each detectably and distinguishably labeled with a fluorescent
labeling group.
U.S. Patent No. 5,547,839 disclosed a method for determining the identity of
nucleotide bases at
2 5 specfic positions in a nucleic acid of interest, using chain-terminating
nucleotides comprising a
photoremovable protecting group.
U.S. Patent No. 5,534,424 disclosed a method for determining the identity of
nucleotide bases at
specific positions in a nucleic acid of interest, using each of four aliquots
of a target nucleic acid
annealed to an extension primer and extended with one of four chain-
terminating species, and
3 0 then further extended with all four chain-extending nucleotides, whereby
the identity of the
nucleotide at the position of interest is identified by failure of the primer
to be extended more that a
single base.
4
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/LTS00/33497
U.S. Patent No. 4,988,617 disclosed a method for determining the identity of
nucleotide bases at
specific positions in a nucleic acid of interest, by annealing two adjacent
nucleotide primers to a
target nucleic acid and providing a linking agent such as a ligase that
covalently links the two
oligonucleotides to produce a third, combined oligonucleotide only under
circumstances wherein
the two oligonucleotides are perfectly matched to the target nucleic acid at
the 3' extent of the first
oligonucleotide and at the 5' extent of the second oligonucleotide.
U.S. Patent No. 4,656,127 disclosed a method for determining the identity of
nucleotide bases at
specific positions in a nucleic acid of interest, using primer extension with
a chain-terminating or
other nucleotide comprising an exonuclease-resistant linkage, followed by
exonuclease treatment
of the plurality of extension products to detect the resistant species
therein.
One common feature in this prior art is that single base extension has been
detected by
incorporation of fluorescent labels into the extended nucleic acid species.
A significant drawback of single base extension methods based on fluorescent
label detection is
the need for expensive and technically-complex optical components for
detecting the fluorescent
label. Although fluorescent probes used in such methods impart an adequate
level of
discrimination between extended and unextended positions in an oligonucleotide
array, these
methods typically require detection of up to four different fluorescent
labels, each having a unique
excitation and fluorescence emission frequency. As a consequence of these
properties, such
assay systems must be capable of producing and distinguishing light at all of
these different
2 0 excitation and emission frequencies, significantly increasing the cost and
complexity of producing
and operating apparatus used in the practice thereof.
An alternative method for detecting a target nucleic acid molecule is to use
an electrochemical tag
(or label) such as a redox moiety in combination with an electrochemical
detection means such as
cyclic voltammetry, some of which are discussed above.
2 5 In addition, disclosure of similar methods for detecting biological
molecules such as DNA and
proteins can be found in Ihara et al., 1996, Nucleic Acids Res. 24: 4273-4280;
Livache et al.,
1995, Synthetic Metals 71: 2143-2146; Hashimoto, 1993, Supramolecular Chem. 2:
265-270;
Millan et al., 1993, Anal. Chem. 65: 2317-2323.
However, most of the electrochemical tag-dependent methods known in the prior
art require
3 0 hybridization of the probe/target in the presence of a redox intercalator.
Electrochemical detection
based on redox intercalators are generally not as reproducible as redox tags
that are covalently
bound to an incorporated moiety. Redox intercalator methods are exceedingly
dependent on
washing conditions to remove excess label while not reducing the actual
signal. As a
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/LTS00/33497
consequence, false positives are often obtained using these methods. The
specificity of redox
intercalator methods is often much worse than can be achieved with covalently-
bound redox tags.
There remains a need in this art for simple, economical, and efficient ways to
detect single base
extension products of nucleic acid assays for detecting mutation and genetic
polymorphisms in
biological samples containing a nucleic acid of interest.
Similarly, there remains a need in the art to develop alternatives to current
detection methods
used to detect interactions between biological molecules, particularly nucleic
acids and peptides.
In particular, there is a need in the art to develop electrical or
electrochemical methods for
detecting interactions between biological molecules that do not require
modifying target or probe
molecules with reporter labels. The development of such methods has wide
applications in the
medical, genetic, and molecular biological arts. There further remains a need
in the art to develop
alternatives for the attaching such biological molecules to the
microelectrodes or substrate of an
electrical or electrochemical device.
SUMMARY OF THE INVENTION
The present invention provides an apparatus and methods, using cations in an
electrolyte solution,
for detecting the nature and extent of molecular interactions between probe
and target molecules.
The most preferred embodiments of the methods of the invention utilize AC
impedance for said
detection. The apparatus and methods of the present invention have the
advantage of providing
electrical detection without any additional requirement that the target
molecule be labeled with a
2 0 reporter molecule.
In preferred embodiments of the present invention, the apparatus and methods
are useful for
detecting molecular interactions such as nucleic acid hybridization between
oligonucleotide probe
molecules bound to defined regions of an ordered array and nucleic acid target
molecules which
are permitted to interact with the probe molecules. In other embodiments of
the present invention,
the apparatus and methods are useful for detecting interactions between
peptides (e.g., receptor-
ligand binding or antibody recognition of antigens).
In more preferred embodiments, the apparatus of the present invention
comprises a supporting
substrate, an array of microelectrodes in contact with the supporting
substrate to which probes are
immobilized, at least one counter-electrode in electrochemical contact with
the supporting
3 0 substrate, a means for producing electrical impedance at each
microelectrode, a means for
detecting changes in impedance at each microelectrode in the presence or
absence of a target
molecule, and an electrolyte solution in contact with the plurality of
microelectrodes.
In alternative preferred embodiments, the apparatus of the present invention
comprises a
6
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
supporting substrate, an array of microelectrodes in contact with the
supporting substrate, a
plurality of polyacrylamide gel pads in contact with microelectrodes and to
which probes are
immobilized, at least one counter-electrode in electrochemical contact with
the supporting
substrate, a means for producing electrical impedance at each microelectrode,
a means for
detecting changes in impedance at each microelectrode in the presence or
absence of a target
molecule, and an electrolyte solution in contact with the plurality of
microelectrodes. Alternatively,
multiple electrodes can be defined on a substrate and covered with a
continuous, unpatterned
layer of polyacrylamide or other polymer.
In preferred embodiments of the present invention, microelectrodes are
prepared from, but not
limited to, metals such as dense or porous films of gold, platinum, titanium,
or copper, metal
oxides, metal nitrides, metal carbides, or carbon.
In a preferred embodiment of the invention, the electrolyte solution comprises
metal cations or
polymerized cations that are ion conductive and capable of reacting with
probes or probe-target
complexes. In a more preferred embodiment, the electrolyte solution comprises
a salt of a lithium
cation, most preferably LiC104.
The apparatus of the present invention may further comprise at least one
reference electrode. In
an alternative embodiment of the present invention, the apparatus further
comprises a plurality of
wells each of which encompasses at least one microelectrode and at least one
counter-electrode
that is sufficient to interrogate the entire array.
2 0 In a preferred method of the present invention, an electrolyte solution as
described above is
placed in contact with a plurality of microelectrodes to which nucleic acid
probes have been
immobilized, preferably having a neutral polypyrrole layer there between. AC
impedance of the
microelectrodes is first measured in the absence of added target nucleic acid.
Thereafter, the
microelectrodes are contacted with a biological sample substance containing
target nucleic acid
2 5 molecules, most preferably by adding the sample to the electrolyte
solution or replacing the
electrolyte solution with the sample contained in or diluted in the
electrolyte solution. The probes
and target molecules are allowed to interact, preferably by hybridization, and
the AC impedance
measured thereafter.
In another embodiment of the methods of the present invention, an electrolyte
solution as
3 0 described above is placed in contact with a plurality of microelectrodes
and polyacrylamide gel
pads to which nucleic acid probes have been immobilized. AC impedance of the
microelectrodes
is first measured in the absence of added target nucleic acid. Thereafter, the
microelectrodes are
contacted with a biological sample substance containing target nucleic acid
molecules, most
preferably by adding the sample to the electrolyte solution or replacing the
electrolyte solution with
3 5 the sample contained in or diluted in the electrolyte solution. The probes
and target molecules are
7
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
allowed to interact, preferably by hybridization, and the AC impedance
measured thereafter.
In a preferred embodiment of the methods of the present invention, the AC
impedance is
measured at different frequencies in order to increase the sensitivity of the
method. Probe-target
interactions are detected by differences in the AC impedance signals at
individual microelectrodes
before and after such interactions. Most preferably, the method is used to
discern the difference
between hybridization between an immobilized oligonucleotide probe on a
microelectrode and a
complimentary target nucleic acid ("complete" hybridization), and
hybridization between the
immobilized oligonucleotide and a mismatched target nucleic acid ("mismatch"
hybridization).
Information about the nucleotide sequence of the oligonucleotides immobilized
at each
microelectrode is then used in conjunction with "complete" or "mismatch"
hybridization as detected
by the method of the invention to determine the presence or absence of a
particular target nucleic
acid in the sample.
In an alternate embodiment, the present invention provides an apparatus and
methods for the
electric or electrochemical detection of the nature and extent of molecular
interactions between
probe molecules and electrochemically active reporter-labeled target
molecules. In preferred
embodiments of the present invention, the apparatus and methods are useful for
detecting
molecular interactions such as nucleic acid hybridization between
oligonucleotide probe molecules
bound to defined regions of an ordered array and electrochemically active
reporter-labeled nucleic
acid target molecules which are permitted to interact with the probe
molecules. In other
2 0 embodiments of the present invention, the apparatus and methods are useful
for detecting
interactions between peptides (e.g., receptor-ligand binding or antibody
recognition of antigens).
In more preferred embodiments, the apparatus of the present invention
comprises a supporting
substrate, an array of microelectrodes in contact with the supporting
substrate, a plurality of
polymeric hydrogel pads in contact with the microelectrodes and to which
probes are immobilized,
2 5 at least one counter-electrode in electrochemical contact with the
supporting substrate, a means
for producing an electrical signal at each microelectrode, a means for
detecting changes in the
electrtcal signal at each microelectrode in the presence or absence of an
electrochemically active
reporter-labeled target molecule, and a electrolyte solution in contact with
the plurality of hydrogel
porous microelectrodes and counter-electrode. Alternatively, multiple
electrodes can be defined
3 0 on a substrate and covered with a continuous, unpatterned layer of
polymeric hydrogel.
In preferred embodiments of the present invention, microelectrodes are
prepared from metals
such as dense or porous films of gold, platinum, titanium, or copper, metal
oxides, metal nitrides,
metal carbides, or graphite carbon.
In some embodiments of the present invention, the probes are oligonucleotide
probes having a
3 5 sequence comprising from about 10 to about 100 nucleotide residues, and
said probes are
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
attached to the polyacrylamide gel pads using techniques known to those with
skill in the art. In
other embodiments, the probes are peptides, such as receptors, ligands,
antibodies, antigens, or
synthetic peptides, and said probes are attached to the polymeric hydrogel
pads using techniques
known to those with skill in the art.
The apparatus of the present invention may further comprise at least one
reference electrode. In
an alternative embodiment of the present invention, the apparatus further
comprises a plurality of
wells each of which encompasses at least one hydrogel porous microelectrode
and at least one
counter-electrode that is sufficient to interrogate the entire array.
In the method of the present invention, molecular interactions between probe
molecules and
electrochemically active reporter-labeled target molecules are detected by
applying conventional
electric or electrochemical detection methods, such as, for example, AC
impedance. In one
embodiment, the AC impedance of a plurality of hydrogel porous microelectrodes
to which nucleic
acid probes have been immobilized is first measured in the absence of an
electrochemically-
labeled target nucleic acid. Thereafter, the hydrogel porous microelectrodes
are contacted with a
biological sample substance containing electrochemically active reporter-
labeled target molecules.
The probes and target molecules are allowed to interact, preferably by
hybridization, and AC
impedance measured thereafter.
In a preferred embodiment of the methods of the present invention, the AC
impedance is
measured at different frequencies in order to increase the sensitivity of the
method. Interactions
2 0 between probe molecules and electrochemically-labeled target molecules are
detected by
differences in the AC impedance signals at individual hydrogel porous
microelectrodes before and
prior to such interactions. Most preferably, the method is used to discern the
difference between
hybridization between an immobilized oligonucleotide probe on a hydrogel
porous microelectrode
and a complimentary target nucleic acid ("complete" hybridization), and
hybridization between the
2 S immobilized oligonucleotide and a mismatched target nucleic acid
("mismatch" hybridization).
Information about the nucleotide sequence of the oligonucleotides immobilized
at each hydrogel
porous microelectrode is then used in conjunction with "complete" or
"mismatch" hybridization as
detected by the method of the invention to determine the presence or absence
of a particular
target nucleic acid in the sample.
3 0 In other embodiments of the present invention, other electric and/or
electrochemical methods can
be used to detect molecular interactions between probe molecules and
electrochemically-labeled
target molecules, including, but not limited to, cyclic voltammetry, stripping
voltammetry, pulse
voltammetry, square wave voltammetry, AC voltammetry, hydrodynamic modulation
voltammetry,
potential step method, potentiometric measurements, amperometric measurements,
current step
3 5 method, and combinations thereof.
9
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
The invention further comprises methods and apparatus for detecting mutations
and genetic
polymorphisms in a biological sample containing a nucleic acid of interest.
Detection of single
base extension using the methods and apparatus of the invention is achieved by
sequence-
specific incorporation of chain-terminating nucleotide species chemically
labeled with an
electrochemical species. In preferred embodiments, single base extension is
performed using
hybridization to an oligonucleotide array, most preferably an addressable
array wherein the
sequence of each oligonucleotide in the array is known and associated with a
particular address in
the array. In additional preferred embodiments, single base extension is
detected using extension
products labeled with electrochemical reporter groups, wherein the
electrochemical reporter
groups comprise a transition metal complex, most preferably containing a
transition metal ion that
is ruthenium, cobalt, iron or osmium.
In the practice of the methods of the invention, the invention provides an
array of oligonucleotide
probes immobilized to a surface that defines a first electrode. Preferably,
the sequence of each
oligonucleotide at each particular identified position (or "address") in the
array is known and at
least one of said oligonucleotides is complementary to a sequence in a nucleic
acid contained in
the biological sample to be assayed (termed the "target" or "target nucleic
acid"). In one preferred
embodiment, the sequence of at least one oligonucleotide is selected to
hybridize to a position
immediately adjacent to the nucleotide position in the sample nucleic acid
that is to be
interrogated, i.e., for mutation or genetic polymorphism. The term "adjacent"
in this context is
2 0 intended to encompass positions that are one nucleotide base upstream of
base to be
interrogated, i.e. in the 3' direction with respect to the template strand of
the target DNA.
Hybridization of the oligonucleotides in the array to nucleic acid in the
sample is performed in a
reaction chamber and in a hybridization buffer for a time and at a temperature
that permits
hybridization to occur between nucleic acid in the sample and the
oligonucleotides in the array
2 5 complementary thereto. Single base extension is performed using a
polymerase, most preferably
a thermally stable polymerase, in the presence of chain-terminating primer
extension units that are
covalently linked to an electrochemical label. In a preferred embodiment, each
chain-terminating
nucleotide species (for example, dideoxy(dd)ATP, ddGTP, ddCTP and ddTTP) is
labeled with a
different electrochemical label, most preferably having a different, distinct
and differentially-
3 0 detectable reduction/oxidation potential. Single base extension is
detected by applying
conventional electrochemical detection methods, such as cyclic voltammetry or
stripping
voltammetry. Other electric or/and electrochemical methods that may also be
used, include, but
are not limited to, AC impedance, pulse voltammetry, square wave voltammetry,
AC voltammetry
(ACV), hydrodynamic modulation voltammetry, potential step method,
potentiometric
3 5 measurements, amperometric measurements, current step method, and
combinations thereof.
In alternative embodiments, the sequence of at least one oligonucleotide is
selected to hybridize
to the target nucleic acid at a position whereby the 3' residue of the
oligonucleotide hybridizes to
the nucleotide position in the sample nucleic acid that is to be interrogated
for mutation or genetic
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
polymorphism. In the array, oligonucleotides having sequence identity to the
oligonucleotide that
hybridizes to the target nucleic acid at it's 3' residue will also hybridize
to the target, but the 3'
residue of such oligonucleotides will produce a "mismatch" with the target and
will not hybridize at
the 3' residue. Single base extension is performed with a polymerase that will
not recognize the
mismatch, so that only the oligonucleotide that hybridizes to the target
including at its 3' residue
will be extended. In these embodiments of the invention, only a single chain-
terminating species
labeled with an electrochemical species can be employed, or the same
electrochemical species
can be used for all four chain-terminating species, provided that the
nucleotide sequence of each
oligonucleotide in the array is known and properly associated with its
position in the array. The
detection of an electrochemical signal from the redox species using
conventional electrochemical
detection methods, such as cyclic voltammetry, at a particular position in the
array thus provides
the identity of the 3'residue of the probe and hence the identity of the
complementary nucleotide at
the corresponding position in the target nucleic acid.
In the practice of a preferred embodiment of the methods and use of the
apparatus of the
invention, electric current is recorded as a function of sweeping voltage to
the first electrode
specific for each particular chain-terminating nucleotide species labeled with
an electrochemically-
active reporter. In preferred embodiments, current flow at each specific
potential is detected at
each address in the array where single base extension has occurred with the
corresponding
chain-terminating nucleotide species labeled with a particular electrochemical
reporter group. The
2 0 detection of the electrical signal at a particular position in the array
wherein the nucleotide
sequence of the oligonucleotide occupying that position is known enables the
identity of the
extended nucleotide, and therefore the mutation or genetic polymorphism, to be
determined.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A and 1 B illustrate a schematic representation of the structure of a
hydrogel porous
2 5 microelectrode (Figure 1 A) and a schematic representation of the
structure of the tip of a hydrogel
porous microelectrode (Figure 1 B);
Figures 2A and 2B illustrate the electrochemical oxidation of pyrrole (Figure
2A) and the
neutralization of polypyrrole (Figure 2B);
Figures 3A and 3B illustrate the Frequency Complex curves obtained from
polypyrrole
3 0 microelectrodes before and after the hybridization of a 15-mer
oligonucleotide probe and
complementary nucleic acid target molecule (Figure 3A) and the Frequency
Complex curve
obtained in the high frequency zone from polypyrrole microelectrodes before
and after the
hybridization of a 15-mer oligonucleotide probe and complementary target
molecule (Figure 3B);
Figures 4A and 4B illustrate a plot of low frequency resistance versus vv "2
(Figure 4A) and the plot
11
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
of high frequency resistance versus ~ni "2 (Figure 4B);
Figures 5A and 5B illustrate the Frequency Complex curve obtained for the
hybridization of an
oligonucleotide probe and a fully complementary nucleic acid target molecule
(Figure 5A) and the
Frequency Complex curve obtained for the hybridization of an oligonucleotide
probe and a nucleic
acid target molecule possessing three mismatches (Figure 5B; curve 1 was
obtained before
hybridization of the target molecule to the probe, curve 2 was obtained
following hybridization of
probe and target molecules for 48 hours, curve 3 was obtained following
washing of hybridized
molecules for 30 min. at 37°C, and curve 4 was obtained following
washing of hybridized
molecules for 30 min. at 38°C);
Figure 6 illustrates a plot of low frequency resistance versus ~ni "2 obtained
for the hybridization of
an oligonucleotide probe and a nucleic acid target molecule possessing three
mismatches (curve
1 was obtained before hybridization of the target molecule to the probe, curve
2 was obtained
following hybridization of probe and target molecules for 48 hours, curve 3
was obtained following
washing of hybridized molecules for 30 min. at 37°C, and curve 4 was
obtained following washing
of hybridized molecules for 30 min. at 38°C)
Figure 7 illustrates the Frequency Complex curve obtained from polypyrrole
microelectrodes
before and after the hybridization of a 15-mer oligonucleotide probe and
complementary nucleic
acid target molecule in an electrolyte containing 0.1 M LiCl04;
2 0 Figures 8A through 8C illustrate a schematic representation of the circuit
(Figure 8A), the AC
impedance response for a polypyrrole microelectrode with an attached single-
strand nucleic acid
probe before hybridization to a target molecule (Figure 8B), and a schematic
representation of the
circuit for a polypyrrole microelectrode with an attached single-strand
nucleic acid probe after
hybridization to a target molecule (Figure 8C);
2 5 Figure 9 illustrates a plot of capacitance versus frequency for a
polypyrrole microelectrode with an
attached single-strand nucleic acid probe after hybridization to a target
molecule;
Figure 10 illustrates a plot of resistance versus frequency for a polypyrrole
microelectrode with an
attached single-strand nucleic acid probe after hybridization to a target
molecule;
Figure 11 illustrates a hydrogel porous microelectrode;
3 0 Figure 12 illustrates the Frequency Complex curves obtained from a
hydrogel porous
microelectrode with attached 15-mer oligonucleotide probe in the absence of a
complementary
target molecule (curve 1 ), following incubation with 2 pM of a complementary
target molecule
(curve 2), and following incubation with 300 nM of a mismatched target
molecule (curve 3);
12
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
Figure 13 illustrates a plot of capacitance versus frequency for a hydrogel
porous microelectrode
with an attached single-strand nucleic acid probe after hybridization to a
target molecule;
Figure 14 illustrates a plot of resistance versus frequency for a hydrogel
porous microelectrode
with an attached single-strand nucleic acid probe after hybridization to a
target molecule.
Figure 15 illustrates single base extension using chain-terminating nucleotdie
species labeled with
an electrochemical reporter group (ECA label).
DETAILED DESCRIPTION OF THE INVENTION
The present invention is direc;ed to a variety of electronic and
electrochemical techniques that
may be used to detect the presence of target analytes, particularly nucleic
acids, in samples. The
methods generally rely on the molecular interactions such as nucleic acid
hybridization or protein-
protein binding reactions done on solid supports with arrays of capture
binding ligands. As a
result of these interactions, some electronic property of the system changes,
and detection is
achieved.
This may be done in a variety of ways. In a preferred embodiment, the methods
of the invention
utilize AC impedance for the detection. In some embodiments, the apparatus and
methods of the
present invention have the advantage of providing electrical detection without
any additional
requirement that the target molecule be labeled with a reporter molecule. That
is, the electrical
impedance of the system changes as a result of the specific binding of a
target analyte to its
corresponding capture binding ligand (frequently referred to herein as
"capture probes" when the
2 0 analyte is a nucleic acid).
Alternatively, the use of electrochemically active labels allows the detection
of specific
interactions, in a manner similar to known fluorescent systems. In this
embodiment, either the
target can be labeled with an electrochemically active (ECA) label, for
example during an
amplfication reaction such as PCR when the target is a nucleic acid, or
through the use of
2 5 secondary labeling systems.
In a preferred embodiment, when the target is a nucleic acid, these ECA labels
are exploited to
allow the identification of specific bases within a target sequence, as is
generally outlined below.
The methods and compositions of the invention are used to detect target
analytes in samples. By
"target analyte" or "analyte" or grammatical equivalents herein is meant any
molecule, compound
3 0 or particle to be detected. As outlined below, target analytes preferably
bind to binding ligands, as
is more fully described above. 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
13
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
binding ligand, described herein, may be made may be detected using the
methods of the
invention.
Suitable analytes include organic and inorganic molecules, including
biomolecules. In a preferred
embodiment, the analyte may be an environmental pollutant (including
pesticides, insecticides,
toxins, etc.); a chemical (including solvents, polymers, organic materials,
etc.); therapeutic
molecules (including therapeutic and abused drugs, antibiotics, etc.);
biomolecules (including
hormones, cytokines, proteins, lipids, carbohydrates, cellular membrane
antigens and receptors
(neural, hormonal, nutrient, and cell surface receptors) or their ligands,
etc); whole cells (including
procaryotic (such as pathogenic bacteria) and eukaryotic cells, including
mammalian tumor cells);
viruses (including retroviruses, herpesviruses, adenoviruses, lentiviruses,
etc.); and spores; etc.
Particularly preferred analytes are environmental pollutants; nucleic acids;
proteins (including
enzymes, antibodies, antigens, growth factors, cytokines, etc); therapeutic
and abused drugs;
cells; and viruses.
In a preferred embodiment, the target analyte is a nucleic acid. 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);
2 0 Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl.
Acids Res. 14:3487 (1986);
Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc.
110:4470 (1988); and
Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al.,
Nucleic Acids Res.
19:1437 (1991 ); and U.S. Patent No. 5,644,048), phosphorodithioate (Briu et
al., J. Am. Chem.
Soc. 111:2321 (1989), O-methylphophoroamidite linkages (see Eckstein,
Oligonucleotides and
2 5 Analogues: A Practical Approach, Oxford University Press), and peptide
nucleic acid backbones
and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al.,
Chem. Int. Ed. Engl.
31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature
380:207 (1996), all of
which are incorporated by reference). Other analog nucleic acids include those
with positive
backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic
backbones (U.S.
3 0 Patent Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863;
Kiedrowshi et al., Angew.
Chem. Intl. Ed. English 30:423 (1991 ); Letsinger et al., J. Am. Chem. Soc.
110:4470 (1988);
Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3,
ASC Symposium
Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y.S.
Sanghui and P. Dan
Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs
et al., J.
35 Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-
ribose backbones,
including those described in U.S. Patent 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
14
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
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 June 2,
1997 page 35.
Nucleic acid analogs also include "locked nucleic acids". 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 electron transfer moieties, or to
increase the stability and half-
life of such molecules in physiological environments.
As will be appreciated by those in the art, all of these nucleic acid analogs
may find use in the
present invention. In addition, mixtures of naturally occurring nucleic acids
and analogs can be
made; for example, at the site of conductive oligomer or electron transfer
moiety attachment, an
analog structure may be used. Alternatively, mixtures of different nucleic
acid analogs, and
mixtures of naturally occurring nucleic acids and analogs may be made.
As outlined herein, 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. As
used herein, the term "nucleoside" includes nucleotides and 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
2 0 nucleic acid, each containing a base, are referred to herein as
nucleosides.
In a preferred embodiment, the present invention provides methods of detecting
target nucleic
acids. By "target nucleic acid" or "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.
2 5 It may be any length, with the understanding that longer sequences are
more specific. In some
embodiments, it may be desirable to fragment or cleave the sample nucleic acid
into fragments of
100 to 10,000 basepairs, with fragments of roughly 500 basepairs being
preferred in some
embodiments. 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
3 0 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 (including primers) 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, which
may be adjacent
3 5 (I.e. contiguous) or separated. For example, when ligation chain reaction
(LCR) techniques are
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
used, a first primer may hybridize to a first target domain and a second
primer may hybridize to a
second target domain; either the domains are adjacent, or they may be
separated by one or more
nucleotides, coupled with the use of a polymerise and dNTPs, as is more fully
outlined below.
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.
In a preferred embodiment, the target analyte is a protein. As will be
appreciated by those in the
art, there are a large number of possible proteinaceous target analytes that
may be detected using
the present invention. By "proteins" or grammatical equivalents herein is
meant proteins,
oligopeptides and peptides, derivatives and analogs, including proteins
containing non-naturally
occurring amino acids and amino acid analogs, and peptidomimetic structures.
The side chains
may be in either the (R) or the (S) configuration. In a preferred embodiment,
the amino acids are
in the (S) or L-configuration. As discussed below, when the protein is used as
a binding ligand, it
may be desirable to utilize protein analogs to retard degradation by sample
contaminants.
Suitable protein target analytes include, but are not limited to, (1 )
immunoglobulins, particularly
IgEs, IgGs and IgMs, and particularly therapeutically or diagnostically
relevant antibodies,
including but not limited to, for example, antibodies to human albumin,
apolipoproteins (including
apolipoprotein E), human chorionic gonadotropin, cortisol, a-fetoprotein,
thyroxin, thyroid
2 0 stimulating hormone (TSH), antithrombin, antibodies to pharmaceuticals
(including antieptileptic
drugs (phenytoin, primidone, carbariezepin, ethosuximide, valproic acid, and
phenobarbitol),
cardioactive drugs (digoxin, lidocaine, procainamide, and disopyramide),
bronchodilators
theophylline), antibiotics (chloramphenicol, sulfonamides), antidepressants,
immunosuppresants,
abused drugs (amphetamine, methamphetamine, cannabinoids, cocaine and opiates)
and
2 5 antibodies to any number of viruses (including orthomyxoviruses, (e.g.
influenza virus),
paramyxoviruses (e.g respiratory syncytial virus, mumps virus, measles virus),
adenoviruses,
rhinoviruses, coronaviruses, reoviruses, togaviruses (e.g. rubella virus),
parvoviruses, poxviruses
(e.g.Dariola virus, vaccinia virus), enteroviruses (e.g. poliovirus,
coxsackievirus), hepatitis viruses
(including A, B and C), herpesviruses (e.g. Herpes simplex virus, varicella-
zoster virus,
3 0 cytomegalovirus, Epstein-Barr virus), rotaviruses, Norwalk viruses,
hantavirus, arenavirus,
rhabdovirus (e.g. rabies virus), retroviruses (including HIV, HTLV-I and -II),
papovaviruses (e.g.
papillomavirus), polyomaviruses, and picornaviruses, and the like), and
bacteria (including a wide
variety of pathogenic and non-pathogenic prokaryotes of interest including
Bacillus; Vibrio, e.g. V.
cholerae; Escherichia, e.g. Enterotoxigenic E. coli, Shigella, e.g. S.
dysenteriae; Salmonella, e.g.
3 5 S. typhi; Mycobacterium e.g. M. tuberculosis, M. leprae; Clostridium, e.g.
C. botulinum, C. tetani,
C. difficile, C.perfringens; Cornyebacterium, e.g. C. diphtheriae;
Streptococcus, S. pyogenes, S.
pneumoniae; Staphylococcus, e.g. S. aureus; Haemophilus, e.g. H. influenzae;
Neisseria, e.g. N.
meningitides, N. gonorrhoeae; Yersinia, e.g. G. IambIiaY. pesos, Pseudomonas,
e.g. P.
16
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00133497
aeruginosa, P. putida; Chlamydia, e.g. C. frachomatis; Bordetella, e.g. 8,
pertussis; Treponema,
e.g. T. palladium; and the like); (2) enzymes (and other proteins), including
but not limited to,
enzymes used as indicators of or treatment for heart disease, including
creative kinase, lactate
dehydrogenase, aspartate amino transferase, troponin T, myoglobin, fibrinogen,
cholesterol,
triglycerides, thrombin, tissue plasminogen activator (tPA); pancreatic
disease indicators including
amylase, lipase, chymotrypsin and trypsin; liver function enzymes and proteins
including
cholinesterase, bilirubin, and alkaline phosphotase; aldolase, prostatic acid
phosphatase, terminal
deoxynucleotidyl transferase, and bacterial and viral enzymes such as HIV
protease; (3)
hormones and cytokines (many of which serve as ligands for cellular receptors)
such as
erythropoietin (EPO), thrombopoietin (TPO), the interleukins (including IL-1
through IL-17), insulin,
insulin-like growth factors (including IGF-1 and -2), epidermal growth factor
(EGF), transforming
growth factors (including TGF-a and TGF-~3), human growth hormone,
transferrin, epidermal
growth factor (EGF), low density lipoprotein, high density lipoprotein,
leptin, VEGF, PDGF, ciliary
neurotrophic factor, prolactin, adrenocorticotropic hormone (ACTH),
calcitonin, human chorionic
gonadotropin, cotrisol, estradiol, follicle stimulating hormone (FSH), thyroid-
stimulating hormone
(TSH), leutinzing hormone (LH), progeterone and testosterone; and (4) other
proteins (including
oc-fetoprotein, carcinoembryonic antigen CEA, cancer markers, etc.).
In addition, any of the biomolecules for which antibodies may be detected may
be detected
directly as well; that is, detection of virus or bacterial cells, therapeutic
and abused drugs, etc.,
2 0 may be done directly.
Suitable target analytes include carbohydrates, including but not limited to,
markers for breast
cancer (CA15-3, CA 549, CA 27.29), mucin-like carcinoma associated antigen
(MCA), ovarian
cancer (CA125), pancreatic cancer (DE-PAN-2), prostate cancer (PSA), CEA, and
colorectal and
pancreatic cancer (CA 19, CA 50, CA242).
2 5 Suitable target analytes include metal ions, particularly heavy and/or
toxic metals, including but
not limited to, aluminum, arsenic, cadmium, selenium, cobalt, copper,
chromium, lead, silver and
nickel
These target analytes may be present in any number of different sample types,
including, but not
limited to, bodily fluids including blood, lymph, saliva, vaginal and anal
secretions, urine, feces,
3 0 perspiration and tears, and solid tissues, including liver, spleen, bone
marrow, lung, muscle, brain,
etc.
Accordingly, the present invention provides devices for the detection of
target analytes comprising
a solid substrate. The solid substrate can be made of a wide variety of
materials and can be
configured in a large number of ways, as is discussed herein and will be
apparent to one of skill in
3 5 the art. In addition, a single device may be comprises of more than one
substrate; for example,
there may be a "sample treatment" cassette that interfaces with a separate
"detection" cassette; a
17
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
raw sample is added to the sample treatment cassette and is manipulated to
prepare the sample
for detection, which is removed from the sample treatment cassette and added
to the detection
cassette. There may be an additional functional cassette into which the device
fits; for example, a
heating element which is placed in contact with the sample cassette to effect
reactions such as
PCR. In some cases, a portion of the substrate may be removable; for example,
the sample
cassette may have a detachable detection cassette, such that the entire sample
cassette is not
contacted with the detection apparatus. See for example U.S. Patent No.
5,603,351 and PCT
US96/17116, hereby incorporated by reference.
The composition of the solid substrate will depend on a variety of factors,
including the techniques
used to create the device, the use of the device, the composition of the
sample, the analyte to be
detected, the size of the wells and microchannels, the presence or absence of
electronic
components, etc. Generally, the devices of the invention should be easily
sterilizable as well.
In a preferred embodiment, the solid substrate can be made from a wide variety
of materials.
Preferred embodiments utilize ceramic components as the solid substrate, as is
more generally
outlined below, although as will be appreciated by those in the art, the
devices of the invention
may include other materials. These include, but are not limited to, silicon
such as silicon wafers,
silcon dioxide, silicon nitride, glass and fused silica, gallium arsenide,
indium phosphide,
aluminum, ceramics, polyimide, quartz, plastics, resins and polymers including
polymethylmethacrylate, acrylics, polyethylene, polyethylene terepthalate,
polycarbonate,
2 0 polystyrene and other styrene copolymers, polypropylene,
polytetrafluoroethylene, superalloys,
zircaloy, steel, gold, silver, copper, tungsten, molybdeumn, tantalum, KOVAR,
KEVLAR,
KAPTON, MYLAR, brass, sapphire, etc. High quality glasses such as high melting
borosilicate or
fused silicas may be preferred for their UV transmission properties when any
of the sample
manipulation steps require light based technologies. In addition, as outlined
herein, portions of the
2 5 internal surfaces of the device may be coated with a variety of coatings
as needed, to reduce non-
specific binding, to allow the attachment of binding ligands, for
biocompatibility, for flow resistance,
etc.
In a preferred embodiment, the solid support comprises ceramic materials, such
as are outlined in
U.S.S.N.s 09/235,081; 09/337,086; 09/464,490; 09/492,013; 09/466,325;
09/460,281; 09/460,283;
3 0 09/387,691; 09/438,600; 09/506,178; and 09/458,534; all of which are
expressly incorporated by
reference in their entirety. In this embodiment, the devices are made from
layers of green-sheet
that have been laminated and sintered together to form a substantially
monolithic structure.
Green-sheet is a composite material that includes inorganic particles of
glass, glass-ceramic,
ceramic, or mixtures thereof, dispersed in a polymer binder, and may also
include additives such
3 5 as plasticizers and dispersants. The green-sheet is preferably in the form
of sheets that are 50 to
250 microns thick. The ceramic particles are typically metal oxides, such as
aluminum oxide or
zirconium oxide. An example of such a green-sheet that includes glass-ceramic
particles is
18
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
"AX951" that is sold by E.I. Du Pont de Nemours and Company. An example of a
green-sheet
that includes aluminum oxide particles is "Ferro Alumina" that is sold by
Ferro Corp. The
composition of the green-sheet may also be custom formulated to meet
particular applications.
The green-sheet layers are laminated together and then fired to form a
substantially monolithic
multilayered structure. The manufacturing, processing, and applications of
ceramic green-sheets
are described generally in Richard E. Mistier, "Tape Casting: The Basic
Process for Meeting the
Needs of the Electronics Industry," Ceramic Bulletin, vol. 69, no. 6, pp. 1022-
26 (1990), and in
U.S. Patent No. 3,991,029, which are incorporated herein by reference.
The method for fabricating devices (such as those depicted in Figures 27-30 as
devices 100 and
200) begins with providing sheets of green-sheet that are preferably 50 to 250
microns thick. The
sheets of green-sheet are cut to the desired size, typically 6 inches by 6
inches for conventional
processing, although smaller or larger devices may be used as needed. Each
green-sheet layer
may then be textured using various techniques to form desired structures, such
as vias, channels,
or cavities, in the finished multilayered structure.
Various techniques may be used to texture a green-sheet layer. For example,
portions of a green-
sheet layer may be punched out to form vias or channels. This operation may be
accomplished
using conventional multilayer ceramic punches, such as the Pacific Trinetics
Corp. Model APS-
8718 Automated Punch System. Instead of punching out part of the material,
features, such as
channels and wells may be embossed into the surface of the green-sheet by
pressing the green-
2 0 sheet against an embossing plate that has a negative image of the desired
structure. Texturing
may also be accomplished by laser tooling with a laser via system, such as the
Pacific Trinetics
LVS-3012.
Next, a wide variety of materials may be applied, preferably in the form of
thick-film pastes, to
each textured green-sheet layer. For example, electrically conductive pathways
may be provided
2 5 by depositing metal-containing thick-film pastes onto the green-sheet
layers. Thick-film pastes
typically include the desired material, which may be either a metal or a
dielectric, in the form of a
powder dispersed in an organic vehicle, and the pastes are designed to have
the viscosity
appropriate for the desired deposition technique, such as screen-printing. The
organic vehicle
may include resins, solvents, surfactants, and flow-control agents. The thick-
film paste may also
3 0 include a small amount of a flux, such as a glass frit, to facilitate
sintering. Thick-film technology is
further described in J.D. Provance, "Performance Review of Thick Film
Materials,"
InsulationlCircuits (April, 1977) and in Morton L. Topfer, Thick Film
Microelectronics, Fabrication,
Design, and Applications (1977), pp. 41-59, which are incorporated herein by
reference.
The porosity of the resulting thick-film can be adjusted by adjusting the
amount of organic vehicle
3 5 present in the thick-film paste. Specifically, the porosity of the thick-
film can be increased by
increased the percentage of organic vehicle in the thick-film paste.
Similarly, the porosity of a
19
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
green-sheet layer can be increased by increasing the proportion of organic
binder. Another way
of increasing porosity in thick-films and green-sheet layers is to disperse
within the organic
vehicle, or the organic binder, another organic phase that is not soluble in
the organic vehicle.
Polymer microspheres can be used advantageously for this purpose.
To add electrically conductive pathways, the thick film pastes typically
include metal particles,
such as silver, platinum, palladium, gold, copper, tungsten, nickel, tin, or
alloys thereof. Silver
pastes are preferred. Examples of suitable silver pastes are silver conductor
composition
numbers 7025 and 7713 sold by E.I. Du Pont de Nemours and Company.
The thick-film pastes are preferably applied to a green-sheet layer by screen-
printing. In the
screen-printing process, the thick-film paste is forced through a patterned
silk screen so as to be
deposited onto the green-sheet layer in a corresponding pattern. Typically,
the silk screen pattern
is created photographically by exposure to a mask. In this way, conductive
traces may be applied
to a surface of a green-sheet layer. Vias present in the green-sheet layer may
also be filled with
thick-film pastes. If filled with thick-filled pastes containing electrically
conductive materials, the
vias can serve to provide electrical connections between layers.
After the desired structures are formed in each layer of green-sheet,
preferably a layer of adhesive
is applied to either surface of the green-sheet. Preferably, the adhesive is a
room-temperature
adhesive. Such room-temperature adhesives have glass transition temperatures
below room
temperature, i.e., below about 20° C, so that they can bind substrates
together at room
2 0 temperature. Moreover, rather than undergoing a chemical change or
chemically reacting with or
dissolving components of the substrates, such room-temperature adhesives bind
substrates
together by penetrating into the surfaces of the substrates. Sometimes such
room-temperature
adhesives are referred to as "pressure-sensitive adhesives." Suitable room-
temperature
adhesives are typically supplied as water-based emulsions and are available
from Rohm and
2 5 Haas, Inc. and from Air Products, Inc. For example, a material sold by Air
Products, Inc. as
"Flexcryl 1653" has been found to work well.
The room-temperature adhesive may be applied to the green-sheet by
conventional coating
techniques. To facilitate coating, it is often desirable to dilute the
supplied pressure-sensitive
adhesive in water, depending on the coating technique used and on the
viscosity and solids
3 0 loading of the starting material. After coating, the room-temperature
adhesive is allowed to dry.
The dried thickness of the film of room-temperature adhesive is preferably in
the range of 1 to 10
microns, and the thickness should be uniform over the entire surface of the
green-sheet. Film
thicknesses that exceed 15 microns are undesirable. With such thick films of
adhesive voiding or
delamination can occur during firing, due to the large quantity of organic
material that must be
3 5 removed. Films that are less than about 0.5 microns thick when dried are
too thin because they
provide insufficient adhesion between the layers.
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
From among conventional coating techniques, spin-coating and spraying are the
preferred
methods. If spin-coating is used, it is preferable to add 1 gram of deionized
water for every 10
grams of "Flexcryl 1653." If spraying is used, a higher dilution level is
preferred to facilitate ease
of spraying. Additionally, when room-temperature adhesive is sprayed on, it is
preferable to hold
the green-sheet at an elevated temperature, e.g., about 60 to 70° C, so
that the material dries
nearly instantaneously as it is deposited onto the green-sheet. The
instantaneous drying results in
a more uniform and homogeneous film of adhesive.
After the room-temperature adhesive has been applied to the green-sheet
layers, the layers are
stacked together to form a multilayered green-sheet structure. Preferably, the
layers are stacked
in an alignment die, so as to maintain the desired registration between the
structures of each
layer. When an alignment die is used, alignment holes must be added to each
green-sheet layer.
Typically, the stacking process alone is sufficient to bind the green-sheet
layers together when a
room-temperature adhesive is used. In other words, little or no pressure is
required to bind the
layers together. However, in order to effect a more secure binding of the
layers, the layers are
preferably laminated together after they are stacked.
The lamination process involves the application of pressure to the stacked
layers. For example, in
the conventional lamination process, a uniaxiat pressure of about 1000 to 1500
psi is applied to
the stacked green-sheet layers that is then followed by an application of an
isostatic pressure of
about 3000 to 5000 psi for about 10 to 15 minutes at an elevated temperature,
such as 70° C.
2 0 Adhesives do not need to be applied to bind the green-sheet layers
together when the
conventional lamination process is used.
However, pressures less than 2500 psi are preferable in order to achieve good
control over the
dimensions of such structures as internal or external cavities and channels.
Even tower pressures
are more desirable to allow the formation of larger structures, such as
cavities and channels. For
2 5 example, if a lamination pressure of 2500 psi is used, the size of well-
formed internal cavities and
channels is typically limited to no larger than roughly 20 microns.
Accordingly, pressures less
than X000 psi are more preferred, as such pressures generally enable
structures having sizes
greater than about 100 microns to be formed with some measure of dimensional
control.
Pressures of less than 300 psi are even more preferred, as such pressures
typically allow
3 0 structures with sizes greater than 250 microns to be formed with some
degree of dimensional
control. Pressures less than 100 psi, which are referred to herein as "near-
zero pressures," are
most preferred, because at such pressures few limits exist on the size of
internal and external
cavities and channels that can be formed in the multilayered structure.
The pressure is preferably applied in the lamination process by means of a
uniaxial press.
3 5 Alternatively, pressures less than about 100 psi may be applied by hand.
21
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
As with semiconductor device fabrication, many devices may be present on each
sheet.
Accordingly, after lamination the multilayered structure may be diced using
conventional green-
sheet dicing or sawing apparatus to separate the individual devices. The high
level of peel and
shear resistance provided by the room-temperature adhesive results in the
occurrence of very
little edge delamination during the dicing process. If some layers become
separated around the
edges after dicing, the layers may be easily re-laminated by applying pressure
to the affected
edges by hand, without adversely affecting the rest of the device.
The final processing step is firing to convert the laminated multilayered
green-sheet structure from
its "green" state to form the finished, substantially monolithic, multilayered
structure. The firing
process occurs in two important stages as the temperature is raised. The first
important stage is
the binder burnout stage that occurs in the temperature range of about 250 to
500° C, during
which the other organic materials, such as the binder in the green-sheet
layers and the organic
components in any applied thick-film pastes, are removed from the structure.
In the next important stage, the sintering stage, which occurs at a higher
temperature, the
inorganic particles sinter together so that the multilayered structure is
densified and becomes
substantially monolithic. The sintering temperature used depends on the nature
of the inorganic
particles present in the green-sheet. For many types of ceramics, appropriate
sintering
temperatures range from about 950 to about 1600° C, depending on the
material. For example,
for green-sheet containing aluminum oxide, sintering temperatures between 1400
and 1600° C are
2 0 typical. Other ceramic materials, such as silicon nitride, aluminum
nitride, and silicon carbide,
require higher sintering temperatures, namely 1700 to 2200° C. For
green-sheet with glass-
ceramic particles, a sintering temperature in the range of 750 to 950°
C is typical. Glass particles
generally require sintering temperatures in the range of only about 350 to
700° C. Finally, metal
particles may require sintering temperatures anywhere from 550 to 1700°
C, depending on the
2 5 metal.
Typically, the devices are fired for a period of about 4 hours to about 12
hours or more, depending
on the material used. Generally, the firing should be of a sufficient duration
so as to remove the
organic materials from the structure and to completely sinter the inorganic
particles. In particular,
polymers are present as a binder in the green-sheet and in the room-
temperature adhesive. The
3 0 firing should be of sufficient temperature and duration to decompose these
polymers and to allow
for their removal from the multilayered structure.
Typically, the multilayered structure undergoes a reduction in volume during
the firing process.
During the binder burnout phase, a small volume reduction of about 0.5 to 1.5%
is normally
observed. At higher temperatures, during the sintering stage, a further volume
reduction of about
3 5 14 to 17% is typically observed.
22
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/CTS00/33497
The volume change due to firing, on the other hand, can be controlled. In
particular, to match
volume changes in two materials, such as green-sheet and thick-film paste, one
should match:
(1 ) the particle sizes; and (2) the percentage of organic components, such as
binders, which are
removed during the firing process. Additionally, volume changes need not be
matched exactly,
but any mismatch will typically result in internal stresses in the device. But
symmetrical
processing, placing the identical material or structure on opposite sides of
the device can, to some
extent, compensate for shrinkage mismatched materials. Too great a mismatch in
either sintering
temperatures or volume changes may result in defects in or failure of some or
all of the device.
For example, the device may separate into its individual layers, or it may
become warped or
distorted.
As noted above, preferably any dissimilar materials added to the green-sheet
layers are co-fired
with them. Such dissimilar materials could be added as thick-film pastes or as
other green-sheet
layers, or added later in the fabrication process, after sintering. The
benefit of co-firing is that the
added materials are sintered to the green-sheet layers and become integral to
the substantially
monolithic microfluidic device. However, to be co-fireable, the added
materials should have
sintering temperatures and volume changes due to firing that are matched with
those of the green-
sheet layers. Sintering temperatures are largely material-dependent, so that
matching sintering
temperatures simply requires proper selection of materials. For example,
although silver is the
preferred metal for providing electrically conductive pathways, if the green-
sheet layers contain
2 0 alumina particles, which require a sintering temperature in the range of
1400 to 1600° C, some
other metal, such as platinum, must be used due to the relatively low melting
point of silver (961 °
C).
Alternatively, the addition of other substrates or joining of two post-
sintered pieces can be done
using any variety of adhesive techniques, including those outlined herein. For
example, two
2 5 "halves" of a device can be glued or fused together. For example, a
particular detection platform,
reagent mixture such as a hydrogel or biological components that are not
stable at high
temperature can be sandwiched in between the two halves. Alternatively,
ceramic devices
comprising open channels or wells can be made, additional substrates or
materials placed into the
devices, and then they may be sealed with other materials.
3 0 The substrates comprise arrays of capture binding ligands. As will be
appreciated by those in the
art, any number of different capture binding ligands, or capture probes (when
the target analyte is
a nucleic acid) can be used, and in a wide variety of formats. Preferred
embodiments utilize
arrays of microelectrodes or hydrogel arrays as are known in the art and
disclosed, for example, in
U.S.S.N.s 09/458,553; 09/458,501; 09/572,187; 09/495,992; 09/344,217;
WO00/31148;
3 5 09/439,889; 09/438,209; 09/344,620; PCTUS00/17422; 09/478,727, all of
which are expressly
incorporated by reference in their entirety.
23
SUBSTITUTE SHEET (RULE 26~
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
In some embodiments of the present invention, the probes are oligonucleotide
probes having a
sequence comprising from about 10 to about 30 nucleotide residues wherein said
probes are
attached to a conjugated polymer or copolymer that is in contact with the
microelectrodes. The
conjugated polymer or copolymer used for probe attachment includes, but is not
limited to,
polypyrrole, polythiphene, polyaniline, polyfuran, polypyridine,
polycarbazole, polyphenylene, poly
(phenylenvinylene), polyfluorene, polyindole, their derivatives, their
copolymers, and combinations
thereof. In a preferred embodiment, the oligonucleotide probes are attached to
the
microelectrodes through a neutral polypyrrole matrix.
In other embodiments of the present invention, the probes are oligonucleotide
probes having a
sequence comprising from about 10 to about 30 nucleotide residues and said
probes are attached
to polyacrylamide gel pads that are in contact with the microelectrodes.
As is known in the art, samples are prepared in a variety of ways and applied
to a device of the
invention. Preferred embodiments are directed to the use of systems that do
not require the use
of ECA labels and are discussed below with reference to Figures 1-14 and
Examples 1-6.
In this embodiment, the apparatus and methods of the present invention are
illustrated herein
using hybridization between oligonucleotide probes immobilized on
microelectrodes and target
nucleic acid molecules contained in a biological sample. The phosphate groups
of nucleic acids
are negatively charged at all biologically relevant pH values. Thus, a nucleic
acid duplex
possesses a high negative charge density. Following electrical perturbation of
the nucleic acid,
2 0 strong interactions, such as the intercalation or binding of metal ions to
the nucleic acid, occur.
These interactions are dependent upon the structure and charge density of the
nucleic acid.
Since the structural and electrical properties of a nucleic acid molecule
(such as a probe) are
altered when the probe is hybridized to a suitable target molecule, the result
of this molecular
interaction is a change in AC impedance. This change is used in the methods
and apparatus of
2 5 the invention to distinguish between "complete" hybridization and
incomplete or "mismatch"
hybridization between the immobilized oligonucleotide probe and target nucleic
acid.
In one embodiment, the apparatus of the present invention comprises a
supporting substrate, a
plurality of microelectrodes in contact with the supporting substrate to which
probes are
immobilized, at least one counter-electrode in contact with the supporting
substrate, a means for
3 0 producing electrical impedance at each microelectrode, a means for
detecting changes in
impedance at each microelectrode in the presence or absence of a target
molecule, and an
electrolyte solution in contact with the plurality of microelectrodes.
In another embodiment, the apparatus of the present invention comprises a
supporting substrate,
a plurality of microelectrodes in contact with the supporting substrate, a
plurality of polyacrylamide
3 5 gel pads in contact with the microelectrodes and to which probes are
immobilized, at least one
24
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCTlLTS00/33497
counter-electrode in contact with the supporting substrate, a means for
producing electrical
impedance at each microelectrode, a means for detecting changes in impedance
at each
microelectrode in the presence or absence of a target molecule, and an
electrolyte solution in
contact with the plurality of microelectrodes.
In one embodiment, the apparatus is a microarray containing at least 5
microelectrodes on a
single substrate to which oligonucleotide probes have been attached.
Alternatively, arrayed
oligonucleotides are attached to polyacrylamide gel pads that are in contact
with the
microelectrodes of the apparatus of the present invention. Most preferably,
oligonucleotides
having a particular nucleotide sequence, or groups of such oligonucleotides
having related (e.g.,
overlapping) nucleotide sequences, are immobilized at each of the plurality of
microelectrodes. In
further preferred embodiments, the nucleotide sequences) of the immobilized
oligonucleotides at
each microelectrode, and the identity and correspondence between a particular
microelectrode
and the nucleotide sequence of the oligonucleotide immobilized thereto, are
known.
In preferred embodiments, the probes are oligonucleotides comprising from
about 10 to about
100, more preferably from about 10 to about 50, and most preferably from about
15 to about 30,
nucleotide residues. In alternative embodiments, the probes are nucleic acids
comprising from
about 10 to about 5000 basepairs, more preferably from about 100 to about 1000
basepairs, and
most preferably from about 200 to about 500 basepairs. In further preferred
embodiments, the
immobilized probes are peptides comprising from about 5 to about 500 amino
acid residues.
2 0 In the preferred embodiment of the apparatus of the present invention, the
substrate is composed
of silicon. In alternative embodiments, the substrate is prepared from
substances including, but not
limited to, glass, plastic, rubber, fabric, or ceramics. The microelectrodes
are embedded within or
placed in contact with the substrate.
In preferred embodiments, microelectrodes are prepared from substances
including, but not
2 5 limited to, metals such as gold, silver, platinum, titanium or copper, in
solid or porous form and
preferably as foils or films, metal oxides, metal nitrides, metal carbides, or
carbon. In certain
preferred embodiments, probes are attached to conjugated polymers or
copolymers including, but
not limited to, polypyrrole, polythiphene, polyaniline, polyfuran,
polypyridine, polycarbazole,
polyphenylene, poly(phenylenvinylene), polyfluorene, polyindole, their
derivatives, their
3 0 copolymers, and combinations thereof. In alternative embodiments, probes
are attached to
polyacrylamide gel pads that are in contact with the microelectrodes.
The substrate of the present invention has a surface area of between 0.01 mm2
and 5 cm2
containing between 1 and 1 x 108 microelectrodes. In one embodiment, the
substrate has a
surface area of 100 mm2 and contains 10' microelectrodes, each microelectrode
having an
3 5 oligonucleotide having a particular sequence immobilized thereto. In
another embodiment, the
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
substrate has a surface area of 100 mm' and contains 104 microelectrodes, each
microelectrode
in contact with a polyacrylamide gel pad to which an oligonucleotide having a
particular sequence
has been immobilized thereto. In preferred embodiments, the microelectrodes
are arranged on
the substrate so as to be separated by a distance of between 0.05 mm to 0.5
mm. Most
preferably, the microelectrodes are regularly spaced on the solid substrate
with a uniform spacing
there between.
In some embodiments of the present invention, the microelectrodes project from
the surface of the
substrate, with such projections extending between 5 x 10'8 and 1 x 10-5 cm
from the surface of the
substrate. In other embodiments, the microelectrodes comprise a flat disk of
conductive material
that is embedded in the substrate and exposed at the substrate surface, with
the substrate acting
as an insulator in the spaces between the microelectrodes.
In the preferred embodiment of the present invention the microelectrodes
comprise a gold
conductor and glass insulator. In alternative embodiments, the microelectrodes
comprise
conductor substances such as solid or porous films of silver, platinum,
titanium, copper, or metal
oxides, metal nitrides, metal carbides, or carbon (graphite). In alternative
embodiments, the
microelectrodes comprise substrate and/or insulator substances such as glass,
silicon, plastic,
rubber, fabric, or ceramics. The microelectrodes of the present invention have
an exposed
conductive surface of between 0.01 mm2 to 0.5 cm2. In the preferred
embodiment, the exposed
conductive material is between 100 to 10,000 mm2. One embodiment of the
present invention is
shown in Figure 1A, wherein the microelectrode comprises a glass capillary
tube 1, containing an
ultra fine platinum wire 2, to which a transition wire 3 has been soldered 6.
The transition wire 3,
is soldered 6 in turn to a hookup wire 4, which protrudes from an epoxy plug 5
that seals the
capillary tube. In one embodiment of the present invention, polyacrylamide gel
material 7 is
packed into a recess etched into the exposed surface of the platinum wire 2.
In some embodiments, oligonucleotide probes are immobilized on the
microelectrodes of the
apparatus of the present invention using a neutral layer between the
oligonucleotides and the
micrd2lectrodes. In a preferred embodiment, this layer comprises neutral
polypyrrole. In
alternative embodiments, this layer comprises such substances as polythiphene,
polyaniline,
polyfuran, polypyridine, polycarbazole, polyphenylene, poly(phenylenvinylene),
polyfluorene,
3 0 polyindole, their derivatives, their copolymers, and combinations thereof.
The layer is preferably
at least about 0.001 to 50 mm thick, more preferably at least about 0.01 to 10
mm thick and most
preferably at least about 0.5 mm thick.
In other embodiments, oligonucleotide probes are immobilized on polyacrylamide
gel pads in
contact with the microelectrodes of the apparatus of the present invention. In
a preferred
3 5 embodiment, the polyacrylamide gel pad is embedded into a recess etched
into the surface of the
microelectrode. The polyacrylamide gel pad is preferably at least about 0.1 to
30 mm thick, more
26
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
preferably at least about 0.5 to 10 mm thick, and most preferably about 0.5 mm
thick
The apparatus of the present invention comprises at least one counter-
electrode. In the preferred
embodiment of the present invention the counter-electrode comprises a
conductive material, with
an exposed surface that is significantly larger than that of the individual
microelectrodes. In a
preferred embodiment, the counter electrode comprises platinum. In alternative
embodiments, the
counter electrode comprises solid or porous films of silver, gold, platinum,
titanium, copper, or
metal oxides, metal nitrides, metal carbides, or carbon.
In other embodiments of the present invention, the apparatus comprises at
least one reference
electrode. The reference electrode is used in assays where the further
quantification of target
molecules is desired. In preferred embodiments, the reference electrode
comprises a silver/ silver
chloride electrode. In alternative embodiments, the reference electrode
comprises solid or porous
films of gold, platinum, titanium, or copper, metal oxides, metal nitrides,
metal carbides, or carbon.
The electrolyte solution comprising the apparatus of the present invention is
any electrolyte
solution comprising at least one salt containing metal or polymerized cations
that are ion-
conductive and can react with biological molecules, most preferably nucleic
acids or peptides.
Most preferably, the salt further comprises anions that exhibit a reduced
specific adsorption for the
surface of the microelectrode, thereby reducing the noise during the detection
of molecular
interactions between probe and target molecules.
In a preferred embodiment of the present invention, the electrolyte solution
used for the detection
2 0 of nucleic acid hybridization contains 0.1 M LiClO,. This electrolyte is
preferred since CIOQ- is not
specifically adsorbed on the electrode surface and thus generates a low
background noise. In
addition, Li' is preferred since its small size facilitates intercalation of
the Li' cations into the
nucleic acid duplex and has less diffusion resistance. However, in other
embodiments, the AC
impedance is measured in hybridization buffers such as 1 X SSC following
molecular interactions
2 5 between probe and target molecules.
In the apparatus of the present invention the means for producing electrical
impedance at each
microelectrode can be accomplished using a model 1260 Impedance/Gain-Phase
Analyser with
model 1287 Electrochemical Interface (Solartron Inc., Houston, TX). Other
electrical impedance
measurement means include, but are not limited to, transient methods with AC
signal perturbation
3 0 superimposed upon a DC potential applied to an electrochemical cell such
as AC bridge and AC
voltammetry. The measurements can be conducted at certain frequency determined
by scanning
frequencies to ascertain the frequency producing the highest signal. The means
for detecting
changes in impedance at each microelectrode in the presence or absence of a
target molecule
can be accomplished by using one of the above-described instruments.
3 5 In still further alternative embodiments of the present invention, the
apparatus further comprises a
27
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/LTS00/33497
plurality of wells each of which encompasses at least one microelectrode and
at least one counter-
electrode. The term "wells" is used herein in its conventional sense, to
describe a portion of the
substrate in which the microelectrode and at least one counter-electrode are
contained in a
defined volume.
The present invention provides an apparatus and methods for detecting
molecular interactions by
detecting cation interactions associated with nucleic acid hybridization. The
detection method
used is most preferably AC impedance, but encompasses any detection methods
that do not
employ or require a reporter-labeled moiety to obtain measurable signals. The
impedance is
measured at different frequencies in order to obtain a "signature" of the
hybridization reaction that
is sensitive enough to permit mismatch hybridization between the
oligonucleotide probe and target
molecules to be detected. The inventive methods disclosed herein are useful
for electrical
detection of molecular interactions between probe molecules bound to defined
regions of an
ordered array (conventionally termed "a biochip array") and target molecules
in a sample which
are permitted to interact with the probe molecules. By arraying
microelectrodes to which individual
probe molecules have been attached on a biochip, parallel measurements of many
probes can be
performed in a single assay.
The present invention further provides an apparatus and methods for detecting
cation interactions
associated with peptide binding using AC impedance, but without the use of
reporter-labeled
target to obtain measurable signals. The methods are used for electrical
detection of molecular
2 0 interactions between probe molecules bound to defined regions of an
ordered peptide array and
target molecules in a sample which are permitted to interact with the probe
molecules. By
arraying microelectrodes to which individual probe molecules have been
attached on a biochip,
parallel measurements of many probes can be performed in a single assay.
The apparatus and methods of the present invention can be adapted further to
be used with
2 5 arrays of any substance that can participate in a molecular interaction
that can be interrogated
with cations, most preferably lithium cations. Such interactions include
ligand-receptor
inter2Ctions, enzyme-inhibitor interactions, and antigen-antibody
interactions.
An important advantage of the apparatus and methods of the present invention
is that they are not
dependent on labeling the target molecule. By removing the labeling step, the
cost of the assay is
3 0 reduced as well as simplified, thereby making electrical detection easier
and more cost-effective to
use. Furthermore, by not requiring target molecules to be labeled, the range
of assays for which a
method of the present invention may be employed is extended. For example, the
present
invention enables one to perform high sensitivity, high resolution
measurements of RNA
concentrations in gene expression studies without having to label the
chemically-labile RNA or to
3 5 convert the RNA into cDNA. The methods of the present invention may also
enable new types of
assays to be developed.
28
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCTfUS00/33497
In an additional embodiment, the invention relies on the use of ECA labels and
detection on
hydrogel arrays. The apparatus and methods of the present invention are
illustrated herein using
hybridization between oligonucleotide probes immobilized to a polymeric
hydrogel pad that is
placed in contact with a microelectrode (a hydrogel porous microelectrode) and
electrochemically-
labeled target nucleic acid molecules contained in a biological sample.
In a preferred embodiment, the apparatus of the present invention comprises a
supporting
substrate, a plurality of microelectrodes in contact with the supporting
substrate, a plurality of
polyacrylamide gel pads in contact with the microelectrodes and to which
probes are immobilized,
at least one counter-electrode in contact with the supporting substrate, a
means for producing an
electrical signal at each microelectrode, a means for detecting changes in the
electrical signal at
each microelectrode in the presence or absence of an electrochemically active
reporter-labeled
target molecule, and an electrolyte solution in contact with the plurality of
microelectrodes and
polymeric hydrogel pads and the counter-electrode. The polymeric hydrogel is
constructed from
hydrophilic polymeric materials including but not limited to polyacrylamide,
agarose gel,
polyethylene glycol, cellular, and sol gels.
In one embodiment, the apparatus is a microarray containing at least 103
hydrogel porous
microelectrodes to which oligonucleotide probes have been attached. Most
preferably,
oligonucleotides having a particular nucleotide sequence, or groups of such
oligonucleotides having
related (e.g., overlapping) nucleotide sequences, are immobilized at each of
the plurality of hydrogel
2 0 porous microelectrodes. In further preferred embodiments, the nucleotide
sequences) of the
immobilized oligonucleotides at each hydrogel porous microelectrode, and the
identity and
correspondence between a particular hydrogel porous microelectrode and the
nucleotide sequence
of the oligonucleotide immobilized thereto, are known.
In preferred embodiments, the probes are oligonucleotides comprising from
about 10 to about 100,
2 5 more preferably from about 10 to about 50, and most preferably from about
15 to about 30, nucleotide
residues. In alternative embodiments, the probes are nucleic acids comprising
from about 10 to about
5000~asepairs, more preferably from about 100 to about 1000 basepairs, and
most preferably from
about 200 to about 500 basepairs. In further preferred embodiments, the
immobilized probes are
peptides comprising from about 5 to about 500 amino acid residues.
3 0 In the preferred embodiment of the apparatus of the present invention, the
substrate is composed of
silicon. In alternative embodiments, the substrate is prepared from substances
including, but not
limited to, glass, plastic, rubber, fabric, or ceramics. The hydrogel porous
microelectrodes are
embedded within or placed in contact with the substrate.
In preferred embodiments, microelectrodes are prepared from substances
including, but not limited
3 5 to, metals such as gold, silver, platinum, titanium or copper, in solid or
porous form and preferably as
29
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
foils or films, metal oxides, metal nitrides, metal carbides, or carbon. In
the apparatus of the present
invention, the probes are attached to polyacrylamide gel pads that are placed
in contact with the
microelectrodes.
The substrate of the present invention has a surface area of between 0.01 mm'
and 5 cm2 containing
between 1 and 1 x 108 hydrogel porous microelectrodes. In one embodiment, the
substrate has a
surface area of 10,000 mm2 and contains 104 hydrogel porous microelectrodes,
each of which has
an oligonucleotide having a particular sequence immobilized thereto. In
preferred embodiments, the
hydrogel porous microelectrodes are arranged on the substrate so as to be
separated by a distance
of between 0.05 mm to 0.5 mm. Most preferably, the hydrogel porous
microelectrodes are regularly
spaced on the solid substrate with a uniform spacing there between.
In some embodiments of the present invention, the hydrogel porous
microelectrodes project from the
surface of the substrate, with such projections extending between 5 x 10'8 and
1 x 10-5 cm from the
surface of the substrate. In other embodiments, the hydrogel porous
microelectrodes comprise a flat
disk of conductive material that is embedded in the substrate and exposed at
the substrate surface,
with the substrate acting as an insulator in the spaces between the hydrogel
porous microelectrodes.
In a preferred embodiment of the present invention the microelectrodes
comprise a gold conductor
and glass insulator. In alternative embodiments, the microelectrodes comprise
conductor substances
such as solid or porous films of silver, platinum, titanium, or copper, metal
oxides, metal nitrides, metal
carbides, or carbon. In alternative embodiments, the microelectrodes comprise
substrate and/or
2 0 insulator substances such as glass, silicon, plastic, rubber, fabric, or
ceramics. The microelectrodes
of the present invention have an exposed conductive surface of between 0.01
mm2 to 0.5 cmz. In the
preferred embodiment, the exposed conductive material is between 100 to
160,000 mm2.
One embodiment of the present invention is shown in Figure 1A, wherein the
microelectrode
comprises a glass capillary tube 1, containing an ultra fine platinum wire 2,
to which a transition wire
2 5 3 has been soldered 6. The transition wire 3, is soldered 6 in turn to a
hookup wire 4, which protrudes
from an epoxy plug 5 that seals the capillary tube. In one embodiment of the
present invention,
polyfi~rylamide gel material 7 is packed into a recess etched into the exposed
surface of the platinum
wire 2. In a preferred embodiment, the polyacrylamide gel pad is embedded into
a recess etched into
the surface of the microelectrode. The polymerix hydrogel pad is preferably at
least about 0.1 to 30
3 0 mm thick, more preferably at least about 0.5 to 10 mm thick, and most
preferably about 0.5 mm thick.
In one embodiment of the hydrogel porous microelectrode of the present
invention, oligonucleotide
probes are immobilized on the polyacrylamide gel.
The apparatus of the present invention comprises at least one counter-
electrode. In the preferred
embodiment of the present invention the counter-electrode comprises a
conductive material, with an
3 5 exposed surface that is significantly larger than that of the individual
microelectrodes. In a preferred
embodiment, the counter electrode comprises platinum. In alternative
embodiments, the counter
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
electrode comprises solid or porous films of silver, gold, platinum, titanium,
or copper, metal oxides,
metal nitrides, metal carbides, or carbon.
In other embodiments of the present invention, the apparatus comprises at
least one reference
electrode. The reference electrode is used in assays where the further
quantification of target
molecules is desired. In preferred embodiments, the reference electrode
comprises a silver/ silver
chloride electrode. In alternative embodiments, the reference electrode
comprises solid or porous
films of gold, platinum, titanium, or copper, metal oxides, metal nitrides,
metal carbides, or carbon.
In still further alternative embodiments of the present invention, the
apparatus further comprises a
plurality of wells each of which encompasses at least one hydrogel porous
microelectrode and at least
one counter-electrode. The term "wells" is used herein in its conventional
sense, to describe a portion
of the substrate in which the hydrogel porous microelectrode and at least one
counter-electrode are
contained in a defined volume.
In the method of the present invention, molecular interactions between probe
molecules bound to
hydrogel porous microelectrodes and electrochemically-labeled target molecules
are detected.
Electrochemically-labeled target molecules useful in the methods of the
present invention may be
prepared by labeling suitable target molecules with any electrochemically-
distinctive redox reporter
which does not interfere with the molecular interaction to be detected. In
preferred embodiments of
the method of the present invention, target molecules are labeled with
electrochemical reporter groups
comprising a transition metal complex, most preferably containing a transition
metal ion that is
2 0 ruthenium, cobalt, iron, or osmium.
In other embodiments of the present invention, target molecules may be labeled
with the following
non-limiting examples of electrochemically-active moieties:
Redox moieties useful against an aqueous saturated calomel reference electrode
include, but are not
limited to, transition metal complexes, 1,4-benzoquinone, ferrocene,
tetracyanoquinodimethane,
2 5 N,N,N',N'-tetramethyl-p-phenylenediamine, or tetrathiafulvalene.
Redox moieities useful against an Ag/AgCI reference electrode include: 9-
aminoacridine, acridine
orange, aclarubicin, daunomycin, doxorubicin, pirarubicin, ethidium bromide,
ethidium monoazide,
chlortetracycline, tetracycline, minocycline, Hoechst 33258, Hoechst 33342, 7-
aminoactinomycin D,
Chromomycin A3, mithramycin A, Vinblastine, Rifampicin,
Os(bipyridine)z(dipyridophenazine)z',
3 0 Co(bipyridine)33', or Fe-bleomycin.
The electrochemically-active moiety comprising the electrochemically active
reporter-labeled target
molecule of the method of the present invention is optionally linked to the
target molecule through a
linker, preferably having a length of from about 10 to about 20 Angstroms. The
linker can be an
organic moiety such as a hydrocrabon chain (CHZ)~, or can comprise an ether,
ester, carboxyamide,
31
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
or thioether moiety, or a combination thereof. The linker can also be an
inorganic moiety such as
siloxane (O-Si-O). The length of the linker is selected so that the
electrochemically-active moiety
does not interfere with the molecular interaction to be detected.
Electrochemical contact is advantageously provided using an electrolyte
solution in contact with each
of the hydrogel porous microelectrodes of the invention. Electrolyte solutions
useful in the apparatus
and methods of the invention include any electrolyte solution at
physiologically-relevant ionic strength
(equivalent to about 0.15 M NaCI) and neutral pH. Examples of electrolyte
solutions useful with the
apparatus and methods of the invention include but are not limited to
phosphate buffered saline,
HEPES buffered solutions, and sodium bicarbonate buffered solutions.
In preferred embodiments, the present invention provides an apparatus and
methods for detecting
molecular interactions by detecting changes in AC impedance. The impedance is
measured at
different frequencies in order to obtain a "signature" of the hybridization
reaction that is sensitive
enough to permit mismatch hybridization between the oligonucleotide probe and
target molecules to
be detected. The inventive methods disclosed herein are useful for
electrochemical detection of
molecular interactions between probe molecules bound to defined regions of an
ordered array
(conventionally termed "a biochip array") and electrochemically-labeled target
molecules in a sample
which are permitted to interact with the probe molecules. By arraying
microelectrodes to which
individual probe molecules have been attached on a biochip, parallel
measurements of many probes
can be performed in a single assay.
2 0 In one embodiment of the apparatus of the present invention, the means for
producing electrical
impedance at each microelectrode is accomplished using a model 1260
Impedance/Gain-Phase
Analyser with model 1287 Electrochemical Interface (Solartron Inc., Houston,
TX). Other electrical
impedance measurement means include, but are not limited to, transient methods
with AC signal
perturbation superimposed upon a DC potential applied to an electrochemical
cell such as AC bridge
2 5 and AC voltammetry. The measurements can be conducted at certain frequency
determined by
scanning frequencies to ascertain the frequency producing the highest signal.
The means for
detec~ng changes in impedance at each microelectrode in the presence or
absence of a
electrochemically-labeled target molecule can be accomplished by using one of
the above-described
instruments.
3 0 In other embodiments of the present invention, other electric and/or
electrochemical methods can be
used to detect molecular interactions between probe molecules and
electrochemically-labeled target
molecules, including, but not limited to, cyclic voltammetry, stripping
voltammetry, pulse voltammetry,
square wave voltammetry, AC voltammetry, hydrodynamic modulation voltammetry,
potential step
method, potentiometric measurements, amperometric measurements, current step
method, and
3 5 combinations thereof. In these embodiments, the electrical signal is
current flow in response to an
applied voltage at the redox potential of the electrochemical label.
32
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
The present invention also provides an apparatus and methods for detecting
single nucleotide
polymorphisms (SNP) in a nucleic acid sample comprising a specific target
nucleic acid.
The devices of the invention are particularly useful for analyzing target
nucleic acid for the diagnosis
of infectious and genetic disease. The target nucleic acid is generally a
portion of a gene having a
known nucleotide sequence that is associated with an infectious agent or
genetic disease; more
specifically, the disease is caused by a single nucleotide (or point)
mutation. The device incorporates
a nucleic acid oligonucleotide array specific for the target gene, and means
for detecting and
determining the identity of a specific single base in the target sequence
adjacent to the hybridization
site of at least one probe in the oligonucleotide array (termed the "3' offset
method") or encompassing
the 3' residue of at least one oligonucleotide probe in the array (termed the
"3' inclusive method").
The present invention provides an array of oligonucleotide primers or probes
immobilized to a surface
that defines a first electrode. Preferably, the sequence of each
oligonucleotide at each address in the
array is known and at least one oligonucleotide in said oligonucleotide array
is complementary to part
of a sequence in a nucleic acid in the sample to be assayed. The sequence of
at least one
oligonucleotide is most preferably selected to extend to a position
immediately adjacent to the
nucleotide position in the sample nucleic acid that is to be interrogated,
i.e., for mutation or genetic
polymorphism. Alternatively, the oligonucleotide is selected to encompass the
site of mutation or
genetic polymorphism; in these latter embodiments, it is generally preferred
to provide a multiplicity
of oligonucleotides having one of each possible nucleotide at the polymorphic
position to ensure
2 0 hybridization of at least one of the oligonucleotides in the array to
nucleic acid in the sample.
Hybridization and extension reactions are performed in a reaction chamber and
in a hybridization
buffer for a time and at temperature that permits hybridization to occur
between nucleic acid in the
sample and the oligonucleotides in the array complementary thereto.
In one embodiment, the apparatus of the present invention comprises a
supporting substrate, a
2 5 plurality of a first electrode (or an array of microelectrodes) in contact
with the supporting substrate
to which probes are immobilized, at least one counter-electrode and optionally
a reference electrode,
and an electrolyte solution in contact with the plurality of microelectrodes,
counter electrode and
reference electrode.
In another embodiment, the apparatus of the present invention comprises a
supporting substrate, a
3 0 plurality of first electrodes (or an array of microelectrodes) in contact
with the supporting substrate,
a plurality of polyacrylamide gel pads in contact with the microelectrodes and
to which probes are
immobilized, at least one counter-electrode and optionally a reference
electrode, and an electrolyte
solution in contact with the plurality of microelectrodes, counter electrode
and reference electrode.
In the preferred embodiment of the apparatus of the present invention, the
substrate is composed of
3 5 silicon. In alternative embodiments, the substrate is prepared from
substances including, but not
33
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
limited to, glass, plastic, rubber, fabric, or ceramics.
The electrode comprising the first surface to which the oligonucleotide or
array thereof is attached is
made of at least one of the following materials: metals such as gold, silver,
platinum, copper, and
electrically-conductive alloys thereof; conductive metal oxides such as indium
oxide, indium-tin oxide,
zinc oxide; and other conductive materials such carbon black, conductive
epoxy.
In preferred embodiments, microelectrodes are prepared from substances
including, but not limited
to, metals such as gold, silver, platinum, titanium or copper, in solid or
porous form and preferably as
foils or films, metal oxides, metal nitrides, metal carbides, or carbon. In
certain preferred
embodiments, probes are attached to conjugated polymers or copolymers
including, but not limited
to, polypyrrole, polythiophene, polyaniline, polyfuran, polypyridine,
polycarbazole, polyphenylene,
poly(phenylenvinylene), polyfiluorene, polyindole, their derivatives, their
copolymers, and combinations
thereof. In alternative embodiments, probes are attached to polyacrylamide gel
pads that are in
contact with the microelectrodes.
The substrate of the present invention has a surface area of between 0.01 mm2
and 5 cm2 containing
between 1 and 1 x 108 microelectrodes. In one embodiment, the substrate has a
surface area of 100
mmz and contains 10° microelectrodes, each microelectrode having an
oligonucleotide having a
particular sequence immobilized thereto. In another embodiment, the substrate
has a surface area
of 100 mmZ and contains 10° microelectrodes, each microelectrode in
contact with a polyacrylamide
gel pad to which an oligonucleotide having a particular sequence has been
immobilized thereto. In
2 0 preferred embodiments, the microelectrodes are arranged on the substrate
so as to be separated by
a distance of between 0.05 mmz to 0.5 mm. Most preferably, the microelectrodes
are regularly
spaced on the solid substrate with a uniform spacing there between.
In one embodiment, the apparatus comprises a microarray containing at least
103 microelectrodes
on a single substrate to which oligonucleotide probes have been attached.
Alternatively, arrayed
2 5 oligonucleotides are attached to polyacrylamide gel pads that are in
contact with the microelectrodes
of th>s apparatus of the present invention. Most preferably, oligonucleotides
having a particular
nucleotide sequence, or groups of such oligonucleotides having related (e.g.,
overlapping) nucleotide
sequences, are immobilized at each of the plurality of microelectrodes. In
further preferred
embodiments, the nucleotide sequences) ofthe immobilized oligonucleotides at
each microelectrode,
3 0 and the identity and correspondence between a particular microelectrode
and the nucleotide
sequence of the oligonucleotide immobilized thereto, are known.
The primer or probe used in the present invention is preferred to be an
oligonucleotide having a
length, both the upper and lower limits of which are empirically determined.
The lower limit on probe
length is stable hybridization: it is known in the art that probes that are
too short do not form
3 5 thermodynamically-stable duplexes sufficient for single base extension
under the hybridization
34
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/L1S00/33497
conditions of the assay. The upper limit on probe length are probes that
produce a duplex in a region
other than that of the predetermined interrogation target, leading to
artifactual incorporation of primer
extension units) labeled with electrochemically active moieties. Preferred
oligonucleotide primer or
probes used in the present invention have a length of from about 8 to about
50, more preferably from
about 10 to about 40, even more preferably from about 12 to about 30, and most
preferably from
about 15-25 nucleotides. However, longer probes, i.e. longer than 40
nucleotides, may also be used.
In the present invention, the primer or probe is preferably immobilized
directly on the first electrode
surface through an anchoring group. As will be appreciated by those in the
art, advantageous
anchoring groups include, for example, moieties comprising thiols,
carboxylates, hydroxyls, amines,
hydrazines, esters, amides, halides, vinyl groups, vinyl carboxylates,
phosphates, silicon-containing
organic compounds, and their derivatives. For example, an oligonucleotide
which is complementary
to a target DNA is covalently linked to a metallic gold electrode through a
thiol-containing anchoring
group. In a preferred embodiment, the length of these anchoring groups is
chosen such that the
conductivity of these molecules do not hinder electron transfer from the
electrochemical reporter
1S groups, to the electrode, via the hybridized probe and target DNA, and
these anchoring groups in
series. Stated differently, these anchoring groups are preferred to have
higher conductivities than
double-stranded nucleic acid. A conductivity, S, of from between about 10'~ to
about 10'° W-' cm-',
more preferably from about 10-5 to about 10' W-' cm-', corresponds to a length
for the anchoring
groups ranging from about 5 A to about 200 ~.
2 0 Alternatively, the primer or probe can be covalently bound onto an
intermediate support that is placed
on top of the first electrode. The support is preferred to be either a thin
layer of porous inorganic
material such as TiOx, Si02, NOx or a porous organic polymer such as
polyacrylamide, agarose,
nitrocellulose membranes, nylon, and dextran supports. Primers are covalently
bound to the support
through a linker. Preferred linker moieties include, but are not limited to,
thioethers, ethers, esters,
2 5 amides, amines, hydrazines, carboxylates, halides, hydroxyls, vinyls,
vinyl carboxylates, thiols,
phosphates, silicon containing organic compounds, and their derivatives and
other carboxylate
moieties. More preferably, biotin-streptavidin pairs are advantageous arranged
to provide probe
binding onto the intermediate support.
The apparatus of the invention also includes a second electrode and a
reference electrode to permit
3 0 current flow. The second electrode is most preferably comprised of any
conducting material,
including, for example, metals such as gold, silver, platinum, copper, and
alloys; conductive metal
oxides such as indium oxide, indium-tin oxide, zinc oxide; or other conductive
materials such as
carbon black, conductive epoxy; most preferred is a platinum (Pt)-wire
auxiliary electrode. The
reference electrode is preferably a silver wire electrode, a silverlsilver
chloride (Ag/AgCI) reference
3 5 electrode, or a saturated calomel electrode.
The apparatus also comprises one or a multiplicity of reaction chambers, each
reaction chamber
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
being in electrochemical contact with at least one of each of the
aforementioned electrodes, wherein
each of the electrodes are connected to a power source and a means for
controlling said power
source. For the purposes of this invention, the term "in electrochemical
contact" is intended to mean,
interalia, that the components are connected such that current can flow
through the electrodes when
a voltage potential is created between the two electrodes.
Electrochemical contact is advantageously provided using an electrolyte
solution in contact with each
of the electrodes or microelectrode arrays of the invention. Electrolyte
solutions useful in the
apparatus and methods of the invention include any electrolyte solution at
physiologically-relevant
ionic strength (equivalent to about 0.15M NaCI) and neutral pH. Nonlimiting
examples of electrolyte
solutions useful with the apaaratus and methods of the invention include but
are not limited to
phosphate buffered saline, HEPES buffered solutions, and sodium bicarbonate
buffered solutions.
Preferred polymerises for performing single base extensions using the methods
and apparatus of
the invention are polymerises having little or no exonuclease activity. More
preferred are
polymerises that tolerate and are biosynthetically-active at temperatures
greater than physiological
temperatures, for example, at 50°C or 60°C or 70°C or are
tolerant of temperatures of at least 90°C
to about 95°C. Preferred polymerises include Taq polymerise from T.
aquaficus (commercially
available from Perkin-Elmer Cetus, Foster City, CA), Sequenase~ and
ThermoSequenase~
(commercially available from U.S. Biochemical, Cleveland, OH), and Exo(-)Pfu
polymerise
(commercially available from New England Biolabs, Beverley, MA).
2 0 The inventive methods for SNP detection provided by the invention
generally comprise: (1 ) preparing
a sample containing the target nucleic acids) of interest to obtain single-
stranded nucleic acid that
spans the specific position (typically by denaturing the sample); (2)
contacting the single- stranded
target nucleic acid with an oligonucleotide primer of known sequence that
hybridizes with a portion
of the nucleotide sequence in the target nucleic acid immediately adjacent the
nucleotide base to be
2 5 interrogated (thereby forming a duplex between the primer and the target
such that the nucleotide
base to be interrogated is the first unpaired base in the target immediately
5' of the nucleotide base
annealed with the 3'-end of the primer in the duplex; this oligonucleotide is
preferably a specific
oligonucleotide occupying a particular address in an addressable array); (3)
contacting the duplex with
a reagent which includes an aqueous carrier, a polymerise, and at least one
primer extension unit,
3 0 wherein the primer extension unit comprises an extension moiety, an
optional linker, and an
electrochemical detection moiety. The primer extension reaction catalyzed by
the polymerise results
in incorporation of the extension moiety of the primer extension unit at the
3'-end of the primer, and
the extension of the primer by a single base; (4) removing the unincorporated
primer extension unit(s);
and (5) determining the identity of the incorporated primer extension unit in
the extended duplex by
3 5 its unique electrochemical detection moiety.
The extension moiety in the primer extension unit is preferably a chain-
terminating moiety, most
36
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/LTS00/33497
preferably dideoxynucleoside triphosphates (ddNTPs), such as ddATP, ddCTP,
ddGTP, and ddTTP;
however other terminators known to those skilled in the art, such as
nucleotide analogs or arabinoside
triphosphates, are also within the scope of the present invention. These
ddNTPs differ from
conventional deoxynucleoside triphosphates (dNTPs) in that they lack a
hydroxyl group at the 3'
position of the sugar component. This prevents chain extension of incorporated
ddNTPs, and thus
terminates the chain. Unlike conventional detection moieties that have been
either fluorescent dyes
or radioactive labels, the present invention provides primer extension units
labeled with an
electrochemical reporter group that are detected electrochemically, most
preferably by redox
reactions. Any electrochemically-distinctive redox label which does not
interfere with the incorporation
of the ddNTP into a nucleotide chain is preferred.
Optionally, the target DNA in the sample to be investigated can be amplified
by means of in vitro
amplification reactions, such as the polymerise chain reaction (PCR) technique
well known to those
skilled in the art. Enriching the target DNA in a biological sample to be used
in the methods of the
invention provides more rapid and more accurate template-directed synthesis by
the polymerise. The
use of such in vitro amplification methods, such as PCR, is optional in the
methods of the invention,
which feature advantageously distinguishes the instantly-disclosed methods
from prior art detection
techniques, which typically required such amplification in order to generate
sufficient signal to be
detected. Because of the increased sensitivity of the instantly-claimed
methods, the extensive
purification steps required after PCR and other in vitro amplification methods
are unnecessary; this
2 0 simplifies performance of the inventive methods.
Single base extension is performed using a polymerise in the presence of at
least one primer
extension unit in a buffer solution appropriate for the biochemical activity
of the polymerise. A
general formula of a preferred embodiment of the primer extension unit is:
ddNTP-L-R
2 5 where ddNTP represents a dideoxyribonucleotide triphosphate including
ddATP, ddGTP, ddCTP,
ddTTP, L represents an optional linker moiety, and R represents an
electrochemical reporter group,
preferably an electrochemically-active moiety and most preferably a redox
moiety.
In preferred embodiments, each chain-terminating nucleotide species (for
example, dideoxy(dd)ATP,
ddGTP, ddCTP and ddTTP) is labeled with a different electrochemical reporter
group, most preferably
3 0 wherein each different reporter group has a different and
electrochemically-distinguishable
reduction/oxidation (redox) potential. In this regard, it will be appreciated
that nucleotides comprising
a DNA molecule are themselves electrically active; for example, guanine and
adenine can be
electrochemically oxidized around 0.75 V and 1.05 V, respectively. Thus, it is
generally preferable
for the redox potential of the electrochemical reporter group comprising the
primer extension units of
3 5 the invention to be distinguishable from the intrinsic redox potential of
the incorporated nucleotides
themselves. The following electrochemical species are non-limiting examples of
electrochemically-
active moieties provided as electrochemical reporter groups of the present
invention, the oxidation
37
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/LTS00/33497
(+) potential or reduction (-) potential being listed in the parenthesis (in
volt units):
- Redox moieties useful against an aqueous saturated calomel reference
electrode include 1,4-
benzoquinone (-0.54V), ferrocene (+0.307), tetracyanoquinodimethane (+0.127, -
0.291 ),
N,N,N',N'-tetramethyl-p-phenylenediamine (+0.21 ), tetrathiafulvalene (+0.30).
- Redox moieties useful against a Ag/AgCI reference electrode include 9-
aminoacridine
(+0.85V), acridine orange (+0.830), aclarubicin (+0.774), daunomycin (+0.446),
doxorubicin
(+0.440), pirarubicin (+0.446), ethidium bromide (+0.678), ethidium monoazide
(+0.563),
chlortetracycline (+0.650), tetracycline (+0.674), minocycline (+0.385),
Hoechst 33258
(+0.586), Hoechst 33342 (+0.571 ), 7-aminoactinomycin D (+0.651 ), Chromomycin
A3
(+0.550), mithramycin A (+0.510), Vinblastine (+0.522), Rifampicin (+0.103),
Os(bipyridine)2(dipyridophenazine)2' (+0.72), Co(bipyridine)33'(+0.11 ), Fe-
bleomycin (-0.08)
(The redox data are from Bard & Faulkner, 1980, ELECTROCHEMICAL METHODS,
JohnWiley & Sons,
Inc. and Hshimoto et al., 1994, Anaiytica Chimica Acta. 286: 219-224).
The choice of the electrochemically-active moiety comprising the
electrochemical reporter groups of
the invention is optimized for detection of the moiety to the exclusion of
other redox moieties present
in the solution, as well as to prevent interference of the label with
hybridization between an
oligonucleotide contained in an array and a nucleic acid comprising a
biological sample.
The electrochemically-active moiety comprising the chain-terminating
nucleotides of the invention is
optionally linked to the extension nucleotide through a linker (L), preferably
having a length of from
2 0 about 10 to about 20 Angstroms. The linker can be an organic moiety such
as a hydrocarbon chain
(CHZ)~, or can comprise an ether, ester, carboxyamide, or thioether moiety, or
a combination thereof.
The linker can also be an inorganic moiety such as siloxane (O-Si-O). The
length of the linker is
selected so that R, the electrochemically-active moiety, does not interfere
with either nucleic acid
hybridization between the bound oligonucleotide primer and target nucleic
acid, or with polymerase-
2 5 mediated chain extension.
In preferred embodiments, single base extension is detected by standard
electrochemical means such
as cyclic voltammetry (CV) or stripping voltammetry. In a non-limiting
example, electric current is
recorded as a function of sweeping voltage to the first electrode specific for
each particular labeled
primer extension unit. The incorporation and extension of a specific base is
identified by the unique
3 0 oxidation or reduction peak of the primer extension unit detected as
current flow in the electrode at
the appropriate redox potential.
In additional embodiments, other electric or/and electrochemical methods
useful in the practice of the
methods and apparatus of the invention include, but are not limited to, AC
impedance, pulse
voltammetry, square wave voltammetry, AC voltammetry (ACV), hydrodynamic
modulation
3 5 voltammetry, potential step method, potentiometric measurements,
amperometric measurements,
current step method, and combinations thereof. In all these methods, electric
current is recorded as
38
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
a function of sweeping voltage to the first electrode specific for each
particular labeled primer
extension unit. The difference is the type of input/probe signal and/or shape
of input/probe signal
used to sweep the voltage range. For example, in cyclic voltammetry, a DC
voltage sweep is done.
In ACV, an AC signal is superimposed on to the voltage sweep. In square wave
voltammetry, a
square wave is superimposed on to the voltage sweep. Most preferably, the
signal is recorded from
each position ("address") in the oligonucleotide array, so that the identity
of the extended species can
be determined. The identity of the nucleotide comprising the extension unit is
determined from the
redox potential at which current flow is detected.
In the use of the apparatus of the invention to perform a single base
extension reaction, a reaction
mixture is prepared containing at least one chain-terminating nucleotide
labeled with an
electrochemical label (such as a redox-labeled ddNTP), a hybridization buffer
compatible with the
polymerise and having a salt concentration sufficient to permit hybridization
between the target
nucleic acid and primer oligonucleotides under the conditions of the assay,
and a DNA polymerise
such as Taq DNA polymerise or ThermoSequenase. Single stranded target nucleic
acid, for
example, having been denatured by incubation at a temperature >90°C, is
diluted to a concentration
appropriate for hybridization in deionized water and added to the reaction
mixture. The resulting
hybridization mixture is sealed in a reaction chamber of the apparatus of the
invention containing a
first electrode, wherein the electrode comprises a multiplicity of primers
having known sequence
linked thereto. At least one of the primers has a nucleotide sequence capable
of hybridizing with a
2 0 portion of the nucleotide sequence of the target immediately adjacent the
nucleotide base to be
interrogated under the hybridization conditions employed in the assay.
A duplex between the primer and the target is formed wherein the nucleotide
base to be interrogated
is the first unpaired base in the target immediately 5' of the nucleotide base
that is annealed with the
3'-end of the primer in the duplex. Single base extension of the 3' end of the
annealed primer is
2 S achieved by incorporation of the chain-terminating nucleotide, labeled
with an electrochemically active
moiety, into the primer. The primer sequence and labeled chain-terminating
nucleotide are chosen
so that incorporation of the nucleotide is informative of the identity (i.e.,
mutant, wildtype or
polymorphism) of the interrogated nucleotide in the target.
Alternatively, the probe comprises a 3' terminal residue that corresponds to
and hybridizes with the
3 0 interrogated base. In these embodiments, oligonucleotides having a
"mismatch" at the 3' terminal
residue will hybridize but will not be extended by the polymerise. Detection
of incorporation of the
primer extension unit by interrogating the redox label is then informative of
the identity of the
interrogated nucleotide base, provided that the sequence of the
oligonucleotide probe is known at
each position in the array.
3 5 After the SBE reaction is performed, the electrode is washed at high
stringency (i.e., in a low-salt and
39
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
low dielectric constant solution (such as 0.1X SSC: 0.015M NaCI, l5mM sodium
citrate, pH 7.0),
optionally including a detergent such as sodium dodecyl sulfate at temperature
of between about10-
65°C) for a time and at a temperature wherein the target nucleic acid
is removed. Wash conditions
vary depending on factors such as probe length and probe complexity.
Electrochemical detection is
carried out in an electrolyte solution by conventional cyclic voltammetry.
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 of Polypyrrole Microelectrodes
Polypyrrole microelectrodes were prepared as follows. Ultra-fine platinum wire
having a diameter of
50 mm was inserted into glass capillary tubing having a diameter of 2 mm and
sealed by heating to
form a solid microelectrode structure. The tip of the structure was then
polished with gamma alumina
powder (CH Instruments, Inc., Austin, TX) to expose a flat disk of the
platinum wire. Microelectrodes
were initially polished with 0.3 mm gamma alumina powder, rinsed with
deionized water, and then
polished with 0.005 mm powder. Following polishing, the microelectrodes were
ultrasonically cleaned
2 0 for 2 min. in deionized water, soaked in 1 N HN03 for 20 min., vigorously
washed in deionized water,
immersed in acetone for 10 min., and again washed vigorously in deionized
water. Through the use
of micromanufacturing techniques employed in the fabrication of
semiconductors, modifications ofthis
procedure can be applied to the preparation of microelectrodes of a size
required for the construction
of bioarray chips.
2 5 A neutral polypyrrole matrix was used for attachment of nucleic acid
probes to the exposed platinum
disk ~f the microelectrodes. Electrochemical deposition was performed using a
model 660A
potentiostat (CH Instruments, Inc.), using platinum wire as a counter-
electrode, silver/silver chloride
(Ag/AgCI) as a reference electrode, and cyclic voltammetry (CV). A solution
containing 0.05 M
pyrrole, 2.5 mM 3-acetate-N-hydrodysuccinimido pyrrole, and 0.1 M LiC104/ 95%
acetonitrile was
3 0 prepared as the electrolyte. The potential range for the CV was 0.2 to 1.3
V versus Ag/AgCI for the
first cycle and -0.1 to 1.0 versus Ag/AgCI for 10 additional cycles. The scan
rate was 10 mVlsec. The
electrolyte was purged by nitrogen gas during the entire deposition process.
Alternatively, polypyrrole
film can be formed by oxidation of pyrrole at a constant current of 0.20 to
0.25 mA/cmz in the same
solution described above. This method is more convenient to make array-based
microelectrodes
3 5 since the reference electrode is not required. An electrochemical
oxidation of the pyrrole produced
the polypyrrole shown to the right of the arrow in Figure 2A.
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/ITS00/33497
The polypyrrole electrodes in oxidized form were put into 0.1 M LiCl04 and
cycled over a potential
range of -0.1 to 0.8 for 20 cycles. This procedure stabilizes the polypyrrole
film. To make a
neutralized polypyrrole, the microelectrodes were placed in the electrolyte
again and cycled for 10
cycles over a potential range of -0.2 to 0.3 versus Ag/AgCI, which is the
reduction zone for this
electrochemical system. The neutralization is desired in order to reduce the
background charge of
the probe attachment matrix and thus increase the sensitivity of the
hybridization electrical
measurements. The reaction for neutralizing the polypyrrole film is
illustrated in Figure 2B. Following
neutralization of the polypyrrole film coating the microelectrodes, the
microelectrodes were vigorously
rinsed with deionized water.
EXAMPLE 2
Attachment of Nucleic Acid Probes to PolypYrrole Microelectrodes
To attach nucleic acid probes to the microelectrodes prepared in Example 1,
the microelectrodes were
incubated at room temperature for 4 hours in a solution consisting of 80 mL
dimethylformamide and
mL of 15 nM 5'-amino labeled 15-mer oligonucleotide (5'-C-C-C-T-C-A-A-G-C-A-G-
A-G-G-A-3';
15 SEQ ID NO: 1 ). Following attachment of the probe molecules, the
microelectrodes were washed with
TBE buffer (0.89 M Tris-borate, 0.025 M EDTA), rinsed thoroughly in deionized
water, and allowed
to dry at room temperature.
EXAMPLE 3
Electrical Detection of Nucleic Acid Hybridization
2 0 Using Polypyrrole Microelectrodes
The AC impedance baseline of the microelectrodes prepared according to Example
2 was first
determined in the absence of a complementary target molecule. Microelectrodes
were then exposed
in a sealed conical tube to 35 mL of the complementary target molecule (5'-T-C-
C-T-C-T-G-C-T-T-G-
A-G-G-G-3'; SEQ ID NO: 2) present at concentrations in the micromolar (10-6 M;
mM) to attomolar
2 5 (10-'8 Jvl; aM) range. Hybridization of probe and target molecules was
performed in 1X SSC buffer
(0.15 M NaCI, 0.015 M sodium citrate, pH 7.0) at 37°C for 24 to 48
hours. Following hybridization,
microelectrodes were thoroughly rinsed in an excess volume of 1 X SSC at room
temperature and
then AC impedance was measured.
AC impedance was measured using a model 1260 Impedance/Gain-Phase Analyser
with model 1287
3 0 Electrochemical Interface (Solartron Inc., Houston, TX). The counter and
reference electrodes were
platinum and Ag/AgCi, respectively, and the impedance measurements were made
under open circuit
voltage (OCV) conditions in a 1 M LiCl04 solution. The measured complex
impedance (Z) versus
frequency for a polypyrrole microelectrode with attached 15-mer
oligonucleotide before and after
hybridization with a 2 1M solution of the complementary target molecule is
shown in Figure 3A
41
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
(Complex impedance is described by the equation Z = Z' + ~Z", in which Z' is
real part of the
impedance, i is O-1 and Z" is imaginary part of the impedance). A significant
difference was observed
between the microelectrodes before and after hybridization to the target
molecule (Figure 3A). The
large signals produced at low target concentration (i. e., 21M of target is
equivalent to 0.1 amol of target
molecules) indicates the high sensitivity of the methods of the present
invention for detecting
hybridization between oligonucleotide probes and target nucleic acid
molecules. The frequency
increases from 0.1 Hz at large values of Z' to 1 MHz at a Z' of -0.
Figure 3B illustrates the frequency complex curves, as seen in Figure 3A, for
the high frequency zone
(where Z' < 5 x 10'). Frequency dependent semicircle impedance curves were
observed at high
frequencies before and after hybridization. Generally, such curves at high
frequencies indicate the
existence of a Faraday resistance (i.e., electrochemical reaction resistance)
in parallel with a
capacitance. Semicircular curves such as those shown in Figure 3B can be used
to obtain the
electrochemical reaction resistance and the double layer capacitance by
equivalent circuit simulation.
The simulation results obtained using the data shown in Figure 3B is shown in
Table I. These results
indicate that following hybridization of the probe and target molecules, the
high frequency
electrochemical resistance decreases and the capacitance increases, which is
as expected. This
demonstrates that the hybridized DNA has a strong electrochemical interactions
with Li'.
TABLEI
Equivalent Circuit Parameters Obtained from High Frequency Impedance Data
2 0 Nucleic Acid Status Faraday Resistance R (W) Capacitance, C (nF)
Single-stranded 2236 0.1 gg
Double-stranded 922 0.597
The real part of the complex impedance data shown in Figure 3A, i.e., the
resistance (R) versus
the square root of the frequency (W "z), is plotted in Figures 4A and 4B.
Linear regions are
2 5 observed, demonstrating that the Li' diffusion process dominates the
measurements at lower
frequencies. Figures 4A and 4B show that a significant change in the
resistance occurs after
hybridization of the single-stranded probe with the target molecule. The
decrease in high
frequency resistance following hybridization (Figure 4B) can be explained by a
decrease in the
Faraday resistance of the hybridized nucleic acid. At low frequencies, the
large ion diffusion
3 0 resistance dominates the impedance and thus the resistance is higher for
the hybridized probe-
target duplex (Figure 4A). As the frequency increases, the contribution of the
frequency-
dependent diffusion resistance decreases and thus the smaller Faraday
resistance dominates.
The limit of detection in the experiments described above was reached at
approximately 0.1
attomol of target molecule. With increased target molecule concentrations,
higher hybridization
3 5 signals were obtained, demonstrating that methods of the present invention
can be also used to
42
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
quantify the amount of target hybridized onto the electrode-immobilized probe.
Thus, this method
can be used in conjunction with appropriate reference electrodes to measure
the absolute
quantities of nucleic acid in a test sample. For example, the methods of the
present invention
enable one to perform high sensitivity, high resolution measurements of RNA
concentrations in
gene expression studies. Comparative gene expression studies performed using
such a method
permits the direct measurement of the quantity of expressed RNA, rather than
relying on a
determination of the ratio between the RNA of interest and a control RNA.
EXAMPLE 4
Specificity of Electrical Detection Using Polypyrrole Microelectrodes
Microelectrodes with attached oligonucleotide probes were prepared as
described in Examples 1
and 2. Four microelectrodes were incubated in a solution containing 2 pM of a
15-mer target
molecule that was fully complementary to the attached probe (5'-T-C-C-T-C-T-G-
C-T-T-G-A-G-G-
G-3'; SEO ID NO: 2) and four other microelectrodes were incubated in a
solution containing 2 fM
of a 15-mer target molecule containing three mismatched bases relative to the
attached probe (5'-
1 S C-C-C-T-C-A-A-G-C-A-G-A-G-G-A-3 ; SEQ ID NO: 1 ) using the conditions
described in Example
3.
Following hybridization, individual microelectrodes were washed at
successively higher
temperatures to electrically measure the melting of the duplexes. Washing was
performed by
placing the microelectrodes in 1 X SSC for 30 min. at either 37°C or
38°C. AC impedance curves
2 0 for the microelectrodes that were not hybridized, hybridized to the
target, or washed at 37°C or
38°C are shown in Figures 5A (fully complementary target molecule) and
5B (mismatched target
molecule).
AC impedance measurements showed a pronounced difference between the fully
complementary
(perfect) and mismatched hybridized nucleic acid duplexes. The impedance
curves obtained for
25 the fully complementary target molecule remained unchanged following the
washes, indicating that
the melting temperature of the perfect duplex was not exceeded. The impedance
curves obtained
for the mismatched target molecule moved toward the baseline (i.e.,
unhybridized probe) following
washes, indicating that the melting of this duplex was occurring at
temperatures near to that
duplexes melting temperature. The ability to discriminate between matched and
mismatched
3 0 nucleic acid sequences demonstrates the applicability of the methods of
the present invention in
the detection of gene polymorphism. Figure 6 indicates that the resistance in
the mismatched
DNA system continuously decreases with increasing wash temperature going back
to the baseline
of the single-stranded DNA.
43
SU8ST1TUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
EXAMPLE 5
Use of Li' Reporter in Polypyrrole Microelectrode Electrical Detection
of Molecular Interactions
Microeiectrodes were prepared as described in Examples 1 and 2 and were
hybridized to suitable
target molecules as described in Example 3. The AC impedance before and after
nucleic acid
hybridization is shown in Figure 7. The microelectrode with attached
oligonucleotide probe
exhibits the characteristics of an ideal polarization electrode prior to
hybridization with a target
molecule. The equivalent circuit for this state is shown in Figure 8A and the
AC impedance
response is shown in Figure 8B. In this state, the microelectrode can be
described by the
equation: Z = RS - j(wCd,)-', where Z is impedance, RS is solution resistance,
j = O-1, w is 2, and Cd,
is double layer capacitance.
The behavior of the microelectrode is treated as an "ideal" polarization
electrode under conditions
of an electrolyte solution comprising 0.1 M LiCIOa with purging NZ and before
hybridization to a
suitable target molecule is reasonable since there is no electrochemically
active species and no
specific adsorption. However, following hybridization to a suitable target
molecule, a large
deviation from the ideal curve was observed in the same electrolyte,
indicating that the impedance
was significantly increased. The AC impedance measured for the microelectrode
following
hybridization suggests that the electrochemical process and equivalent circuit
under such
conditions is as shown in Figure 8C (where R, is the Faraday resistance, i.e.,
electrochemical
2 0 reaction resistance and RW is Warburg resistance). Resistance from both
electrochemical
reactions and the diffusion process causes the electrode behavior following
hybridization to
deviate from the ideal polarization curve.
While simulation would enable the calculation of all the parameters in the
equivalent circuit for this
state, the equivalent circuit can also be simplified for R and C. The results
of such simplification
are shown in Figures 9 and 10, indicating that the resistance from both R and
C increases as one
order of magnitude. The results of the experiments described above
(particularly those described
in Example 4) indicate that Li' in the electrolyte can serve as a reporter,
permitting the mismatches
between probes and target molecules to be detected. Since a method of the
present invention
relies on the intercalation or binding of cations, more preferably Li'
cations, to enable electrical
3 0 detection, this method does not require that target molecules be labeled.
EXAMPLE 6
Preparation of Hydrogel Porous Microelectrodes
Microelectrodes were prepared as described in Example 1 (Figure 1A). The
exposed flat disk of
platinum was then etched in hot aqua regia to form a recess (i.e., micropore
dent) of a specified
44
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
depth. The depth of the recess was controlled by the length of time that the
platinum disk was
exposed to the etching material. The recess thus formed was then packed with
polyacrylamide
gel material (Figure 1 B) to form a hydrogel porous microelectrode (Figure 11
). A hydrogel porous
microelectrode having a diameter of 258 mm was used in the following Examples.
Prior to attachment of probe molecules, hydrogel porous microelectrodes were
activated by
incubation for 10 min. in 2% trifluoroacetic acid, and rinsed for 2 min. in
deionized water.
Microelectrodes were then incubated for 15 min. in 0.1 M sodium periodate, and
rinsed for 2 min.
in deionized water. Following this treatment, microelectrodes were thoroughly
washed by
incubation in deionized water for 15 min., and then air-dried. Microelectrodes
were subsequently
incubated for 10 min. in 2% dim~thyl dichlorosilane solution and 2%
octamethylcyclotetrasiloxane,
washed in ethanol, rinsed in deionized water, and air-dried.
EXAMPLE 7
Attachment of Nucleic Acid Probes to Hydrogel Porous Microelectrodes
To attach nucleic acid probes to the microelectrodes prepared in Example 6,
the microelectrodes
were incubated at room temperature for 4 hours in a solution consisting of 80
mL
dimethylformamide and 20 mL of 2 pM 5'-amino-3'fluorescein labeled 15-mer
oligonucleotide (5'-
C-C-C-T-C-A-A-G-C-A-G-A-G-G-A-3'; SEO ID NO: 1 ). Following attachment of the
probe
molecules, the microelectrodes were washed with TBE buffer, rinsed thoroughly
in deionized
water, and allowed to dry at room temperature.
2 0 EXAMPLE 8
Electrical Detection of Nucleic Acid Hybridization
Using Hydroqel Porous Microelectrodes
The baseline AC impedance of hydrogel porous microelectrodes prepared
according to Example 7
was first determined in the absence of target molecules. Microelectrodes were
then exposed in a
2 5 sealed conical tube to either 35 mL of a complementary target molecule (5'-
T-C-C-T-C-T-G-C-T-T-
G-A-G-G-G-3'; SEO ID NO: 2) present at a concentration of either 2 pM or 35mL
of a mismatched
target molecule (5'-C-C-C-T-C-A-A-G-C-A-G-A-G-G-A-3'; SEQ ID NO: 1 ) present
at a
concentration of 300 nM. Hybridization of the probe with either target
molecule was performed in
1X SSC buffer at room temperature for 1 hour. Following hybridization,
microelectrodes were
3 0 thoroughly rinsed for 20 min. at room temperature in an excess volume of 1
X SSC and then AC
impedance was measured.
AC impedance was measured using a model 1260 Impedance/Gain-Phase Analyser
with model
1287 Electrochemical Interface. The counter and reference electrodes were
platinum and
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
Ag/AgCI, respectively, and the impedance measurements were made under open
circuit voltage
(OCV) conditions in 1X SSC hybridization solution. Samples were excited at an
amplitude of 50
mV. The measured complex impedance (Z) versus frequency for the hydrogel
porous
microelectrode with attached 15-mer oligonucleotide following hybridization
with the
complementary target molecule or mismatched target molecule is shown in Figure
12.
The signal generated following hybridization of probe molecules with a
mismatched target
molecule was indistinguishable from the signal generated in the absence of
target molecule. The
results, as shown in Figure 12, indicate that the charge transfer has
diffusion control at lower
frequencies. The diffusion impedance is expressed as the Warburg element, W,
and has a linear
region in plots of both imaginary and real parts vs. wi "2. From the imaginary
and real parts of the
complex impedance data shown in Figure 12, plots of resistance (R) vs. w~"2
and of capacitance
(C) vs. vii "2 were extracted and plotted as shown in Figures 13 and 14.
Linear regions are
observed in these plots, proving that a diffusion process dominates the
electronic measurements.
These plots show that both resistance and capacitance exhibit a significant
change after the
hybridization of the single stranded DNA probe with the target DNA. The
resistance decreases
and the capacitance increases following hybridization. These results indicate
that the
hybridization of target molecules to probe molecules attached to the
polyacrylamide gel can
improve the charge transfer process by decreasing the resistance. The increase
in capacitance is
due to the increase in the surface charge as a result of nucleic acid
hybridization. The results
2 0 obtained with the hydrogel porous microelectrode demonstrate that such
microelectrodes can be
used to detect 40 fmol of target molecule in solution.
EXAMPLE 9
Preparation of Hydrogel Porous Microelectrodes
Microelectrodes were prepared as follows. Ultra-fine platinum wire having a
diameter of 50 mm was
2 5 inserted into glass capillary tubing having a diameter of 2 mm and sealed
by heating to form a solid
micrpelectrode structure. The tip of the structure was then polished with
gamma alumina powder (CH
Instruments, Inc., Austin, TX) to expose a flat disk of the platinum wire.
Microelectrodes were initially
polished with 0.3 mm gamma alumina powder, rinsed with deionized water, and
then polished with
0.005 mm powder. Following polishing, the microelectrodes were ultrasonically
cleaned for 2 min.
3 0 in deionized water, soaked in 1 N HN03 for 20 min., vigorously washed in
deionized water, immersed
in acetone for 10 min., and again washed vigorously in deionized water.
Through the use of
micromanufacturing techniques employed in the fabrication of semiconductors,
modifications of this
procedure can be applied to the preparation of microelectrodes of a size
required for the construction
of bioarray chips.
3 5 Hydrogel porous microelectrodes were prepared from the above-described
microelectrodes as
46
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
follows. The exposed flat disk of platinum of each microelectrode was etched
in hot aqua regia to
form a recess (i.e., micropore dent) of a specified depth. The depth of the
recess was controlled by
the length of time that the platinum disk was exposed to the etching material.
The recess thus formed
was then packed with polyacrylamide gel material (Figure 1 B) to form a
hydrogel porous
microelectrode (Figure 2). A hydrogel porous microelectrode having a diameter
of 258 mm was used
in the Example 2.
Prior to attachment of probe molecules, hydrogel porous microelectrodes were
activated by incubation
for 10 min. in 2% trifluoroacetic acid, and rinsed for 2 min. in deionized
water. Microelectrodes were
then incubated for 15 min. in 0.1 M sodium periodate, and rinsed for 2 min. in
deionized water.
Following this treatment, microelectrodes were thoroughly washed by incubation
in deionized water
for 15 min., and then air-dried. Microelectrodes were subsequently incubated
for 10 min. in 2%
dimethyl dichlorosilane solution and 2% octamethylcyclotetrasiloxane, washed
in ethanol, rinsed in
deionized water, and air-dried.
EXAMPLE 10
1 S Attachment of Nucleic Acid Probes to HVdrodel Porous Microelectrodes
To attach nucleic acid probes to the microelectrodes prepared in Example 1,
the
microelectrodes were incubated at room temperature for 4 hours in a solution
consisting of 80 mL
dimethylformamide and 20 mL of 2 pM 5'-amino-3'fluorescein labeled 15-mer
oligonucleotide (5'-C-C-
C-T-C-A-A-G-C-A-G-A-G-G-A-3'; SEQ ID NO: 1 ). Following attachment of the
probe molecules, the
2 0 microelectrodes were washed with TBE buffer, rinsed thoroughly in
deionized water, and allowed to
dry at room temperature.
EXAMPLE 11
Electrochemical Detection of Nucleic Acid Hybridization
Nucleic acid hybridization between a probe bound to a hydrogel porous
microelectrode and
2 5 an electrochemically-labeled target molecule is detected as follows. The
baseline AC impedance of
hydro~el porous microelectrodes prepared according to Example 2 is first
determined in the absence
of the electrochemically-labeled target molecules. Microelectrodes are then
exposed in a sealed
conical tube to either 35 mL of a complementary target molecule (5'-T-C-C-T-C-
T-G-C-T-T-G-A-G-G
G-3'; SEQ ID NO: 2) present at a concentration of either 2 pM or 35mL of a
mismatched target
3 0 molecule (5'-T-C-C-T-C-T-G-C-T-T-G-A-G-G-G-3'; SEQ ID NO: 1; present at a
concentration of 300
nM. Hybridization of the probe with either target molecule is performed in 1X
SSC buffer at room
temperature for 1 hour. Following hybridization, microelectrodes are
thoroughly rinsed for 20 min. at
room temperature in an excess volume of 1X SSC and then AC impedance is
measured.
AC impedance is measured using a model 1260 Impedance/Gain-Phase Analyser with
model 1287
3 5 Electrochemical Interface. The counter and reference electrodes are
platinum and Ag/AgCI,
47
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/US00/33497
respectively, and the impedance measurements are made under open circuit
voltage (OCV)
conditions in 1 X SSC hybridization solution. Samples are excited at an
amplitude of 50 mV. The
complex impedance (Z) versus frequency for the hydrogel porous microelectrode
with attached 15-
mer oligonucleotide following hybridization with the complementary target
molecule or mismatched
target molecule is then determined.
EXAMPLE 12
Single Base Extension
An apparatus of the invention is produced as follows. A glass substrate layer
is prepared
comprising an ordered array of a plurality of gold microelectrodes connected
to a voltage source.
The substrate has a surface area of 100 mm~ and contains 104 microelectrodes,
each
microelectrode in contact with a polyacrylamide gel pad that is about 0.5um
thick to which an
oligonucleotide having a particular sequence has been immobilized thereto. The
microelectrodes
are arranged on the substrate so as to be separated by a distance of about 0.1
mm, and are
regularly spaced on the solid substrate with a uniform spacing there between.
To each of the gold electrodes is affixed an oligonucleotide probe having a
length of 25
nucleotides. The resulting ordered array of probes are arranged in groups of
four, whereby the
probes are identical except for the last (most 3') residue. Each group
contains an oligonucleotide
ending in an adenosine (A), guanine (G), cytosine (C) or thymidine (T) or
uracil (U) residue. The
oligonucleotides are attached to each of the gold electrodes through the
polyacrylamide gel pad
2 0 using a modification of the oligonucleotide at the 5' residue. This
residue comprises a thioester
linkage that covalently attaches the oligonucleotide to the polyacrylamide
polymer.
This ordered microelectrode array is placed in a reaction chamber, having
dimensions sufficient to
contain the array and a volume of from about 10 to 100mL of
hybridization/extension buffer. The
reaction chamber also comprises a second counter electrode comprising platinum
wire and a
2 5 third, reference electrode that is a silver/ silver chloride electrode,
each electrode being electrically
conngcted to a voltage source.
In the use of the apparatus of the invention, a volume of from about 10 to
100mL of hybridization
buffer is added to the reaction chamber. This solution also contains a target
molecule, typically at
concentrations in the micromolar (10-6 M; ~M ) to attomolar (10-'e M; aM)
range. Hybridization of
3 0 probe and target molecules is performed in 1 X SSC buffer (0.15 M NaCI,
0.015 M sodium citrate,
pH 7.0) at 37°C for 24 to 48 hours. Following hybridization,
microetectrodes were thoroughly
rinsed in an excess volume of 1 X SSC at room temperature.
A volume of from about 10 to 100mL of extension buffer containing a polymerase
and a plurality of
each of 4 chain-terminating nucleotide species is then added to the reaction
chamber. Each of the
48
SUBSTITUTE SHEET (RULE 26)
CA 02393733 2002-06-06
WO 01/42508 PCT/LTS00/33497
four chain-terminating nucleotide species is labeled with a chemical species
capable of
participating in a reduction/oxidation (redox) reaction at the surface of the
microelectrode. An
example of such a collection of species is: ddATP labeled with cobalt
(bipyridine)33~; ddGTP
labeled with minocycline; ddCTP labeled with acridine orange; and ddTTP
labeled with ethidium
monoazide. The redox labels are covalently linked to the chain-terminating
nucleotides by a
hydrocarbon linker (CH2)2_e. The extension buffer is chosen to accommodate the
polymerase, such
as Thermosequenase (obtained from U.S. Biochemicals, Cleveland, OH). The
extension reaction
is performed at a temperature appropriate for the polymerase, such as about
65'C, that does not
denature the hybridized duplex between the target and the oligonucleotide
probes, and for a time
sufficient for the extension reaction to go to completion. After the extension
reaction is complete,
the array is washed at high stringency in 0.1 X SSC/ 1 % SDS at a temperature
that does not
denature the hybridized duplex.
After washing, a volume of about 10 to 100mL of an electrolyte solution is
added to the reaction
chamber, and each microelectrode is interrogated by conventional cyclic
voltammetry to detect a
redox signal. The identity of oligonucleotides containing single base extended
species is
determined by the redox potential of the signal obtained thereby.
Equivalently, hybridization and single base extension can be performed in the
same buffer
solution, provided the polymerase is compatible with the hybridization buffer
conditions.
49
SUBSTITUTE SHEET (RULE 26)
