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Description  |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a voltammetric sequence-selective sensor
for target polynucleotide sequences, and more particularly to a
polynucleotide probe having one end immobilized on an amperometric
electrode and a target binding region.
2. Description of Prior Art
The application of recombinant DNA techniques is emerging as a powerful
tool in the area of molecular diagnostic medicine. For example, the
development of DNA and RNA molecular probes for the detection of viral and
bacterial genomes and genetic defects in mammalian chromosomes may replace
current immunochemical approaches.
Polynucleotide hybridization assays are used as research tools for the
detection and identification of unique or specific polynucleotide
sequences in samples of complete, fragmented, or mixed nucleic acids.
Various hybridization diagnostic techniques have been developed.
The southern blot technique is based on a polynucleotide hybridization
technique employing radiolabeled nucleic acid probes. This procedure
permits autoradiographic detection of probe/analyte hybrids and
identification of the polynucleotide sequence of the analyte. However, the
Southern procedure, as well as the other diagnostic procedures employing
radiolabeled nucleic acid probes, are very complex, time consuming, and
have the additional problems and expenses generally associated with
radioactive materials such as disposal and personnel monitoring. Thus,
such assays have remained a tool of basic research and are not generally
employed in applied or commercial areas such as clinical diagnosis.
Most of the existing methods used to attach a polynucleotide probe to a
solid support are non-specific and the number of attachment sites per
nucleic acid is difficult to control. It has been found that multiple
attachment reduces the degree of freedom of the immobilized nucleic acid.
The physical adsorption of single stranded DNA, covalent attachment via
diazo-linkage, epoxidation, cyanogen bromide activation and photochemical
reactions are associated with the complication of non-specific linkage
between the nucleic acids and the solid support.
Canadian Patent 1,223,222, issued on Jun. 23, 1987, discloses an
immobilized nucleic acid-containing probe coupled to a solid support in a
manner which is site specific, which does not interfere with the ability
of the nucleic acid to hybridize and which involves preferably a single
chemical covalent linkage per nucleic acid to the solid support.
Specifically, the nucleotide is coupled to the nucleic acid employing an
enzyme and the nucleotide is chemically modified.
Canadian Patent 1,236,410, issued on May 10, 1988, discloses methods and
reagents for determining the presence of specific DNA and RNA base
sequences on single stranded target polynucleotides. The method involves
the preparation of a specific single stranded ribonucleic acid or
deoxyribonucleic acid molecule into which a bifunctional crosslinking
molecule has been covalently incorporated. The incorporation is such that
the crosslinking molecule retains the capacity to undergo a second
addition to the nucleic acid of the bacterial, viral, or mammalian
chromosome which is the target for the probe. The single stranded DNA or
RNA probe is designed so that its nucleic acid base sequence is
complementary to a unique region of the bacterial, viral, or mammalian
chromosome target sequence. The nucleic acid, for example, from a blood,
tissue, or cell sample is reacted with the probe under conditions where
hybridization of the probe with the target will occur. Following
hybridization, the sample is subjected to a photochemical or chemical
procedure which causes crosslinking of the probe to the target
complementary sequence. If no target genomic sequence is present, then no
crosslinking of the probe will occur. In some cases hybridization of the
probe to the target will precede both reactions of the bifunctional
crosslinking reagent.
Canadian Patent 1,293,937 issued on Jan. 7, 1992, discloses polynucleotide
probe compositions, diagnostic kits, and nonradiometric hybridization
assays useful in the detection and identification of at least one target
polynucleotide analyte in a physiological sample. There is provided a
first polynucleotide probe having a catalyst attached thereto and which is
substantially complementary to a first single-stranded region of the
analyte; and a second polynucleotide probe having an apoluminescer
attached thereto and which is substantially complementary to a second
single-stranded region of the analyte. The second region is substantially
mutually exclusive from the first region, such that upon hybridization of
the first and second probes with the analyte, the catalyst and the
apoluminescer are close enough to each other to permit the catalyst to act
on a substrate to release a transformation radical to convert the
apoluminescer to a luminescer.
U.S. Pat. No. 4,882,013 issued on Nov. 21, 1989, discloses the use of
tetrathiafulvalene (TTF) and its derivatives as mediator molecules in the
transfer of electrons between redox systems and electrodes in
bioelectrochemical processes. There is also disclosed an assay procedure
for assaying a substance based on this redox system and using an
electrode. This assay does not include the detection of hybridized
polynucleotide probes with a target polynucleotide since no redox system
is involved in this hybridization.
Current methods for the diagnosis of inherited diseases employ digestion of
a prepared DNA sample with restriction enzymes to form short,
double-stranded segments, gel electrophoresis to separate these segments
according to size, transfer of the separated segments to a thin membrane
material, such as nylon, hybridization of the segments of interest with a
labeled oligonucleotide (of complementary sequence to the known disease
sequence), and detection of the label. The complete procedure requires
about 24 hours, is labor-intensive, and is not readily automated.
Furthermore, these methods usually employ radioactive labels, with their
inherent safety and disposal problems. None of the above-mentioned
diagnostic systems discloses a probe which can be treated to be reusable
for hybridization. Thus, these systems are for a unique usage.
None of the above-mentioned diagnostic systems are highly sequence specific
to enable the diagnosis of point mutation inherited diseases.
It would be highly desirable to be provided with a sequence-selective
system for target polynucleotide sequences that uses a nonradiometric
label in a system that is simple to use, highly specific and sensitive and
reusable.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide a sensor capable of
precisely detecting target polynucleotide sequences which enables the
diagnosis of inherited diseases such as sickle-cell anemia,
.beta.-thalassemia and cystic fibrosis.
Another aim of the present invention is to provide a system with a solid
support having a polynucleotide probe immobilized thereon in a system that
can be easily reset pursuant to an assay and may be continuously reused.
In accordance with the present invention, there is provided a voltammetric
sequence-selective sensor for target polynucleotide sequences, which
comprises:
a) an amperometric electrode;
b) an immobilized polynucleotide probe having one end covalently bound onto
the electrode and the immobilized probe having a target binding region
capable of hybridizing to the target polynucleotide sequences to form an
immobilized heteroduplex having at least a hybridized region; and
c) means for voltammetrically detecting the immobilized heteroduplex.
In accordance with the present invention, there is also provided a method
for detecting the presence of a target polynucleotide analyte in a
physiological sample, which comprises the steps of:
a) incubating the physiological sample with the voltammetric
sequence-selective sensor in accordance with the present invention;
b) voltammetrically detecting the immobilized heteroduplex; and
c) comparing the resulting voltammogram with a control voltammogram.
In accordance with the present invention, there is also provided a kit for
detecting the presence of a target polynucleotide analyte in a
physiological sample, which comprises:
a) an amperometric electrode;
b) an immobilized polynucleotide probe having one end covalently bound onto
the electrode and the immobilized probe having a target binding region
capable of hybridizing to the target polynucleotide sequences to form an
immobilized heteroduplex having at least a hybridized region;
c) an electroactive complex which reversibly binds with heteroduplexes; and
d) control polynucleotide sequences.
The voltammetric sequence-selective sensor in accordance with the present
invention may be used for the detection of base pair mutation-associated
diseases such as sickle-cell anemia, muscular dystrophy, osteodystrophy,
phenylketonuria, hemophilia, hypercholesterolemia, thalassemia and cystic
fibrosis.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a scheme of the activation of glassy carbon electrodes and the
covalent binding of the probe onto its surface;
FIG. 2 is the calibration curves of voltammetric peak current for the
reduction of Co(bpy).sub.3.sup.3+ to Co(bpy).sub.3.sup.2+ against
Co(bpy).sub.3.sup.3+ concentration; and
FIG. 3 is the cyclic voltammogram of repeated regeneration and
hybridization with denatured poly (dA)poly(dT).
DETAILED DESCRIPTION OF THE INVENTION
The sequence-selective DNA sensor is used to determine whether a particular
DNA sequence is present in a sample containing DNA of unknown sequence.
Applications of this sensor include the diagnosis of human or animal
genetic disorders, such as cystic fibrosis or sickle-cell anemia, where
the particular alterations in the normal DNA sequence that produce the
disease are known. Among other applications, the sensor may also be used
for the identification of certain viruses, for which components of the
viral DNA sequence are known.
A sensor capable of detecting a precisely defined sequence of
deoxyribonucleic acid (DNA) would have tremendous clinical utility in the
diagnosis of inherited diseases such as sickle-cell anemia,
.beta.-thalassemia and cystic fibrosis. The DNA sensor of the present
invention consists of single-stranded DNA (ssDNA) covalently bound to an
electrode surface. Selective recognition of the complementary sequence
results in double-strand formation. The double-stranded DNA (dsDNA) is
then detected during or after exposure to a solution of a redox-active,
DNA-binding complex that preconcentrates at the surface.
The sequence-selective sensor of the present invention is based on
hybridization indicators and can replace existing procedures for the
detection of known sequences of DNA in an unknown DNA sample.
Given an adequate quantity of analyte DNA (currently about 25 .mu.g of
complementary DNA), the sequence-selective sensor of the present invention
can perform this determination in about 30 minutes. The sensor is
reusable, and may be incorporated into an automated
hybridization/rinse/measure/denature/rinse/measure instrument. The sensor
of the present invention does not employ radioactive chemicals, and no
special precautions are required for its use.
To prepare a physiological sample, DNA is isolated (e.g. from the white
blood cells) and the region of interest is amplified using the polymerase
chain reaction.
The voltammetric sequence-selective sensor of the present invention
essentially comprises three components: 1) an amperometric electrode, 2) a
sequence-selective polynucleotide probe and 3) a hybridization indicator.
1) Amperometric Electrode
The amperometric electrode is a device that monitors local changes in a
physical property that occurs at the surface of the sensor, and converts
this physical property into a measurable electronic signal. For the
purposes of the sensor of the present invention, the amperometric
electrode, measures the local concentration of a redox-active chemical
species.
There may be used as an amperometric electrode in accordance with the
present invention, glassy carbon electrode, graphite electrode, carbon
fiber electrode, carbon paste electrode, reticulated vitreous carbon
electrode, or metal electrodes onto which bifunctional reagents have been
adsorbed, such as gold or platinum with adsorbed thiol- or
disulfide-containing carboxylic acids.
2) Sequence-Selective Polynucleotide Probe
This is a chemical species having two components. The first consists of an
oligodeoxynucleotide, approximately twenty base residues in length, that
has a base sequence complementary to the base sequence in the target DNA.
For example, the oligodeoxynucleotide sequence may be prepared to be
complementary to a known human genetic disease sequence, or to a known
viral DNA sequence. The second component is covalently bound to one end of
this oligodeoxynucleotide, and consists of a chemically reactive group or
series of reactive groups that allow covalent immobilization of the
sequence recognition agent onto the electrode surface.
3) Hybridization Indicator
The hybridization indicator is employed after the
electrode-sequence-selective probe sensor has been exposed to the target
DNA. At this stage, sequence complementarity between the immobilized
polynucleotide and the target DNA will result in hybridization, the
formation of double-stranded DNA, at the electrode surface. The
hybridization indicator is employed to indicate whether or not
double-strand formation, and therefore sequence recognition, has occurred.
The hybridization indicator is a chemical species that interacts
reversibly, in a measurably different way, with single-stranded DNA
compared with double-stranded DNA. This interaction is measured by the
electrode. An amperometric electrode requires a redox-active hybridization
indicator.
There may be used as an hybridization indicator,
tris(2,2'-bipyridyl)cobalt(III) perchlorate, or
tris(1,10-phenanthroline)cobalt(III)perchlorate or any other electroactive
compounds that bind to double-stranded DNA reversibly but not to
single-stranded DNA, such as daunomycin or adriamycin or intercalating
metallo-porphyrins.
Direct Detection of the Local Hybridization Indicator Concentration at the
Electrode Surface
Tris(2,2'-bipyridyl)cobalt(III) perchlorate may be used as a hybridization
indicator. It exhibits reversible redox activity and associates strongly
with double-stranded DNA. Following exposure to target DNA, an electrode
covalently modified with the sequence-selective polynucleotide probe is
placed in a solution containing the cobalt complex. The complex binds to
the double-stranded region of the DNA, so that its local concentration
near the electrode surface is much higher than it is in the bulk of the
solution. The local concentration of the cobalt complex is measured using
standard voltammetric methods in a three-electrode cell, with the
amperometric working electrode used in conjunction with standard reference
and counter electrodes. The applied potential is scanned through the
half-wave potential for the bound complex, and the magnitude of the peak
current for the reduction of tris(2,2'-bipyridyl)cobalt(III) to
tris(2,2'-bipyridyl)cobalt(II) is directly proportional to its local
concentration at the electrode surface. If double-stranded DNA is not
present on the electrode surface, this preconcentration of the cobalt
complex does not occur, and the magnitude of the observed peak current is
significantly lower.
Amplified Detection of the Local Hybridization Indicator Concentration
In addition to redox activity and reversible association with
double-stranded DNA, the hybridization indicator employed may be capable
of acting as a mediator in an enzymatic reaction. This is the case with
tris(2,2'-bipyridyl)cobalt(II), since it can replace one of the two
substrates, molecular oxygen, in the reaction catalyzed by the enzyme
glucose oxidase (GOX):
##STR1##
where (bpy) is 2,2'-bipyridyl. This enzymatic reaction can be employed to
amplify the anodic response current obtained with the sensor. In addition
to the complex itself, the detection solution now contains the enzyme GOX
and its primary substrate, glucose, at a saturating concentration. The
enzymatic reaction converts all Co(bpy).sub.3.sup.3+ generated
electrochemically at the sensor surface to Co(bpy).sub.3.sup.2+ ;
reoxidation of the +2 form to the +3 form of the complex completes the
catalytic cycle, generating an amplified current response.
Diagnostic Applications
The sequence-selective DNA sensor of the present invention represents a new
concept and a new class of sensing device. Applications in the prenatal
diagnostics area are possible since the DNA sequence abnormalities
associated with many inherited diseases are now known.
Examples include cystic fibrosis, muscular dystrophy, osteodystrophy,
phenylketonuria, hemophilia, sickle-cell anemia, thalassemia and
hypercholesterolemia.
With progress continually being made in the sequencing of the human genome,
many more genetically-linked disorders will be diagnosable with the DNA
sensor.
Several alterations in the normal human DNA sequence occur in individuals
having cystic fibrosis. One of these sequence alterations involves a
three-base deletion, called the delta F.sub.504 deletion. Both the normal
and disease DNA sequences are known at this site in the human genome.
Synthetic oligonucleotides complementary to these sequences will be
prepared, incorporated into a sequence recognition agent, and used in a
DNA sensor to detect and diagnose cystic fibrosis from human DNA samples.
EXAMPLE I
Polynucleotide Probe Covalently Bound to an Amperometric Electrode
Activation of the Electrode Surface
Carboxylic acid groups were electrochemically generated on a polished (0.3
.mu.m diamond paste) and rinsed glassy carbon electrode surface (GCE sold
by Bioanalytical Systems, Inc., 0.075 cm.sup.2 area) by oxidation at +1.5
V vs. Ag/AgCl for 15 seconds in aqueous 2.5% potassium dichromate with 10%
nitric acid (1 of FIG. 1). The electrodes were then rinsed with deionized
water and inverted. A 50 .mu.L drop of a reagent solution containing 5 mM
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (Aldrich) and 8 mM sodium
N-hydroxysulfosuccinimide (supplied by Pierce*) in 0.020M potassium
phosphate buffer, pH 6.9, was evaporated to dryness on the surface. Excess
reagents were removed by rinsing with phosphate buffer, leaving
N-hydroxysulfosuccinimide esters of the carboxylic acid groups on the
electrode surface (2 of FIG. 1).
2) Preparation of the Sequence-Selective Polynucleotide Probe
Catalytic elongation of 225 .mu.g oligo(dT).sub.20 (Sigma) in the presence
of a 1000-fold molar excess of deoxyguanoxine triphosphate (dGTP,
Boehringer) was accomplished with 250 units of terminal deoxynucletidyl
transferase (E.C. 2.7.7.31, Boehringer) at 37.degree. C. for 24 hours,
according to the procedure outlined by Boehringer. Products were purified
by phenol-chloroform extraction, reconstituted to 1 mg/mL in 0.020M
phosphate buffer, pH 6.9, and characterized by polyacrylamide gel
electrophoresis (BioRad).
3) Immobilization of the Sequence-Selective Polynucleotide Probe
Covalent immobilization occurred during the evaporation to dryness of 50
.mu.L of a 1 mg/mL solution of the sequence-selective probe in 0.020M
phosphate buffer, pH 6.9, from the activated GCE surface (3 of FIG. 1).
Excess DNA was removed by extensive rinses with phosphate buffer.
Electrodes thus modified were incubated for four hours prior to use in 5
mM tris(hydroxymethyl)aminomethane buffer, pH 7.1, with 20 mM sodium
chloride and stored in this buffer at 4.degree. C.
EXAMPLE II
Detection of Target Polynucleotide Sequence
1) Hybridization with Target DNA
GCEs modified with oligo(dT).sub.20 (dG).sub.110 as described in Example I
were exposed to a 1 mg/mL solution of analyte DNA, either oligo(dA).sub.20
(prepared in the Department of Biology, Concordia University, by standard
phosphoramidite coupling chemistry) or poly(dA) (from denatured
poly(dA)poly(dT), Boehringer), in 5 mM tris(hydroxymethyl)aminomethane
buffer, pH 7.1, with 20 mM sodium chloride, for 15 minutes, to allow
hybridization. The modified electrode was then rinsed with, and
equilibrated in, the same buffer containing no DNA.
2) Detection Using the Hybridization Indicator
Prior to and following the hybridization step (1), the GCE covalently
modified with the sequence recognition agent was placed in a
water-jacketed (25.degree. C.) three-electrode electrochemical cell
containing a platinum wire auxiliary electrode, an Ag/AgCl reference
electrode, and 10.00 mL of 5 mM Tris buffer, pH 7.1, with 20 mM NaCl.
Cyclic voltammetry was performed using a Bioanalytical Systems 100A
potentiostat, between +500 mV and -200 mV, at a scan rate of 0.050 V/s.
Aliquots of a stock solution containing 5 mM
tris(2,2'-bipyridyl)cobalt(III) in the same buffer were added, with cyclic
voltammetry being performed after each addition, to construct calibration
curves of peak current vs. concentration.
Calibration curves of voltammetric peak current for the reduction of
Co(bpy).sub.3.sup.3+ to Co(bpy).sub.3.sup.2+ against Co(bpy).sub.3.sup.3+
concentration are given in FIG. 2. Five curves are shown, corresponding to
(a) the signals observed at an unmodified GCE, (b) those seen at the GCE
following modification with the sequence recognition agent, (c) peak
currents observed after hybridization to oligo(dA).sub.20, (d) those seen
after hybridization to a denatured solution of poly(dA)poly(dT), and (e)
those seen after attempted hybridization to a model interferent,
poly(dAdT).
The results given in FIG. 2 indicate (a) that the currents measured at the
sensor depend on whether single- or double stranded DNA is present on the
electrode surface, (b) that the hybridization indicator used,
Co(bpy).sub.3.sup.3+, interacts with both single- and double-stranded DNA,
but that binding to double-stranded DNA occurs to a much greater extent,
(c) that covalent immobilization does not interfere with the ability of
the oligo(dT).sub.20 segment to recognize and hybridize with a
complementary sequence, and (d) that a potential nonspecific interferent,
such as poly(dAdT), will not give rise to increased signals, or "false
positive" results.
Control immobilizations of the sequence recognition agent were performed in
the absence of the carbodiimide and N-hydroxysulfosuccinimide coupling
reagents to ensure that covalent immobilization, as opposed to adsorption,
was occurring. These control electrodes yielded responses identical to
those observed at the unmodified GCE.
Immobilizations were attempted with a variety of species of DNA in order to
determine that deoxyguanosine residues were selectively bound to the
activated GCE surface. Repeated attempts to immobilize denatured
poly(dA)poly(dT) and native and denatured calf thymus DNA (42% GC content)
were unsuccessful, while denatured poly(dG)poly(dC) yielded large response
currents for Co(bpy).sub.3.sup.3+.
Separate immobilizations were attempted with the synthetic
oligodeoxynucleotides oligo(dG).sub.20 and oligo (dC).sub.20, followed by
hybridizations with their complements; the results of these experiments
indicated that increased current responses were only obtained for the
oligo(dG).sub.20 immobilization. Taken together, these experiments show
that consecutive deoxyguanosine residues are required for covalent
immobilization using the carbodiimide/N-hydroxysulfosuccinimide reaction
described above.
EXAMPLE III
Regeneration of the Sequence-Selective Sensor
Regeneration of single-stranded oligo(dT).sub.20 on the GCE surface was
achieved by rinsing the modified electrode with hot, deionized water.
Repeated regeneration and hybridization with denatured poly(dA)poly(dT)
yielded consistent signal decreases and increases, respectively. FIG. 3
shows cyclic voltammograms obtained over such a cycle of reuse. No
significant signal deteriorations were observed over 10
regeneration/hybridization cycles, and average responses of 2.7.+-.0.2
.mu.A and 1.5.+-.0.2 .mu.A were obtained with 0.12 mM Co(bpy).sub.3.sup.3+
after hybridization and regeneration, respectively. These results show
that the DNA sensor of the present invention is reusable over multiple
determinations.
FIG. 3 shows cyclic voltammograms of 0.12 mM Co(bpy).sub.3.sup.3+ in 5 mM
tris, pH 7.1, with 20 mM NaCl, at a GCE modified with oligo(dT).sub.20
(dG).sub.110, after (A) hybridization to poly(dA), (B) denaturation by
rinsing with hot, deionized water, and (C) rehybridization to poly(dA).
While the invention has been described in connection with specific
embodiments thereof, it will be understood that it is capable of further
modifications and this application is intended to cover any variations,
uses, or adaptations of the invention following, in general, the
principles of the invention and including such departures from the present
disclosure as come within known or customary practice within the art to
which the invention pertains and as may be applied to the essential
features hereinbefore set forth, and as follows in the scope of the
appended claims.
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