WO2001042508A2 - Procedes et compositions se rapportant a la detection electrique des reactions d'acides nucleiques - Google Patents

Procedes et compositions se rapportant a la detection electrique des reactions d'acides nucleiques Download PDF

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Publication number
WO2001042508A2
WO2001042508A2 PCT/US2000/033497 US0033497W WO0142508A2 WO 2001042508 A2 WO2001042508 A2 WO 2001042508A2 US 0033497 W US0033497 W US 0033497W WO 0142508 A2 WO0142508 A2 WO 0142508A2
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Prior art keywords
microelectrodes
electrode
contact
nucleic acid
target molecule
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PCT/US2000/033497
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English (en)
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WO2001042508A3 (fr
WO2001042508A9 (fr
Inventor
Vi-En Choong
Sean Gallagher
Mike Gaskin
Changming Li
George Maracas
Song Shi
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Motorola, Inc.
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Priority claimed from US09/458,533 external-priority patent/US20020051975A1/en
Priority claimed from US09/459,685 external-priority patent/US6518024B2/en
Application filed by Motorola, Inc. filed Critical Motorola, Inc.
Priority to EP00993326A priority Critical patent/EP1238114A2/fr
Priority to AU29072/01A priority patent/AU2907201A/en
Priority to JP2001544379A priority patent/JP2003516165A/ja
Priority to CA002393733A priority patent/CA2393733A1/fr
Priority to US10/149,319 priority patent/US20030209432A1/en
Publication of WO2001042508A2 publication Critical patent/WO2001042508A2/fr
Publication of WO2001042508A3 publication Critical patent/WO2001042508A3/fr
Publication of WO2001042508A9 publication Critical patent/WO2001042508A9/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors

Definitions

  • 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 ohgonucleotides. 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, electrochemicaily active labels are used to detect reactions on hydrogel arrays, including genotyping reactions such as the single base extension reaction.
  • 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 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 surface of the electrode and target molecules in the reaction mixture. Electrical or electrochemical 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.
  • 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.
  • labeled target molecules have been used to increase the signal produced upon the formation of nucleic acid duplexes during hybridization assays.
  • Heller ef 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 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
  • Hollis ef al. in U S. Patent Nos. 5,653,939 and 5,846,708, provide a meth ⁇ "d 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
  • changes in the electromagnetic or acoustic properties - for example, 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.
  • 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
  • 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 dielect ⁇ cal properties of the test sites are subsequently detected to determine which probes have interacted with target molecules in the sample substance
  • 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
  • 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 Mtcrobiol 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
  • 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 electrochemical detection techniques in combination with the immobilization of probes on polyacrylamide gel pads
  • 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 speed, efficiency, economy and specificity of such assays are thus significant needs in the medical arts
  • nucleic acid assays should be sensitive, specific and easily amenable to automation
  • Efforts to improve sensitivity in nucleic acid assays are known in the prior art
  • 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 ⁇ g) of a specific nucleic acid in a sample in which the original amount of the specific nucleic acid is substantially smaller (about 1 pg)
  • the prior art has been much less successful in improving specificity of nucleic acid hybridization assays
  • nucleic acid 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
  • 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 t ⁇ phosphates, each labeled with a detectable and distinguishable 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 specific 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 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
  • 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 covalentiy links the two ohgonucleotides to produce a third, combined oligonucleotide only under circumstances wherein the two ohgonucleotides are perfectly matched to the target nu
  • 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
  • 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
  • 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
  • assay systems must be capable of producing and distinguishing light at all of these different 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
  • 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 reporter molecule
  • 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
  • the apparatus and methods are useful for detecting interactions between peptides (e g , receptor- ligand binding or antibody recognition of antigens)
  • 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 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
  • the apparatus of the present invention comprises a 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
  • multiple electrodes can be defined on a substrate and covered with a continuous, unpattemed layer of polyacrylamide or other polymer
  • 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
  • the electrolyte solution comprises metal cations or polymerized cations that are ion conductive and capable of reacting with probes or probe-target complexes
  • the electrolyte solution comprises a salt of a lithium cation, most preferably L ⁇ CI0 4
  • the apparatus of the present invention may further comprise at least one reference electrode
  • 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
  • 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 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
  • an electrolyte solution as 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 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
  • 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
  • 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 ohgonucleotides 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
  • 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
  • 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
  • the apparatus and methods are useful for detecting interactions between peptides (e g , receptor-ligand binding or antibody recognition of antigens)
  • 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, 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 electrical 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
  • multiple electrodes can be defined on a substrate and covered with a continuous, unpattemed layer of polymeric hydrogel
  • 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
  • the probes are oligonucleotide probes having a sequence comprising from about 10 to about 100 nucleotide residues, and said probes are attached to the polyacrylamide gel pads using techniques known to those with skill in the art
  • the probes are peptides, such as receptors, gands, 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
  • 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
  • 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
  • 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
  • the AC impedance is measured at different frequencies in order to increase the sensitivity of the method
  • Interactions 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
  • 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 immobilized oligonucleotide and a mismatched target nucleic acid (“mismatch" hybridization)
  • 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, potentiomet ⁇ c measurements, amperomet ⁇ c measurements, current step method, and combinations thereof
  • 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
  • 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
  • the invention provides an array of oligonucleotide probes immobilized to a surface that defines a first electrode
  • the sequence of each oligonucleotide at each particular identified position (or “address") in the array is known and at least one of said ohgonucleotides is complementary to a sequence in a nucleic acid contained in the biological sample to be assayed (termed the "target” or “target nucleic acid")
  • 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, / e , for mutation or genetic polymorphism
  • the term "adjacent" in this context is intended to encompass positions that are one nucleotide base upstream of base to be interrogated, / e in the 3' direction with respect to the template strand of the target DNA Hybridization of the
  • 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 polymorphism
  • ohgonucleotides 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 ohgonucleotides 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
  • only a single chain-terminating species labeled with an electrochemical species can be employed, or the same electrochemical
  • 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
  • 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 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
  • Figures 1 A and 1 B illustrate a schematic representation of the structure of a hydrogel porous 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 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 w 1 2 ( Figure 4A) and the plot of high frequency resistance versus w ( 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 mm at 37°C, and curve 4 was obtained following washing of hybridized molecules for 30 mm at 38°C),
  • Figure 6 illustrates a plot of low frequency resistance versus w " 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 mm at 37°C, and curve 4 was obtained following washing of hybridized molecules for 30 m 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 L ⁇ CI0 4 ,
  • 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),
  • Figure 9 illustrates a plot of capacitance versus frequency for a polypyrrole microelectrode with an attacKed 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 1 1 illustrates a hydrogel porous microelectrode
  • 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),
  • 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
  • FIG 15 illustrates single base extension using chain-terminating nucleotdie species labeled with an electrochemical reporter group (ECA label)
  • the present invention is directed 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
  • the methods of the invention utilize AC impedance for the 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 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 gand (frequently referred to herein as "capture probes" when the analyte is a nucleic acid)
  • electrochemically active labels allows the detection of specific interactions, in a manner similar to known fluorescent systems
  • the target can be labeled with an electrochemically active (ECA) label, for example during an amplification reaction such as PCR when the target is a nucleic acid, or through the use of secondary labeling systems
  • ECA electrochemically active
  • these ECA labels are exploited to allow the identification of specific bases within a target sequence, as is generally outlined below
  • target analyte or “analyte” or grammatical equivalents herein is meant any molecule, compound or particle to be detected
  • target analytes preferably bind to binding ligands, as is more fully described above
  • a large number of analytes may be detected using the present methods, basically, any target analyte for which a binding hgand, described herein, may be made may be detected using the methods of the invention.
  • Suitable analytes include organic and inorganic molecules, including biomolecules.
  • 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, pids, 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.
  • an environmental pollutant including pesticides, insecticides, toxins, etc.
  • a chemical including solvents, polymers, organic materials, etc.
  • therapeutic molecules including
  • Particularly preferred analytes are environmental pollutants; nucleic acids; proteins (including enzymes, antibodies, antigens, growth factors, cytokines, etc); therapeutic and abused drugs, cells; and viruses.
  • the target analyte is a nucleic acid.
  • nucleic acid or “oligonucleotide” or grammatical equivalents herein means at least two nucleotides covalentiy 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; Letsmger, J. Org. Chem. 35:3800 (1970); Sp ⁇ nzl et al., Eur. J. Biochem. 81 :579 (1977); Letsmger et al., Nucl. Acids Res. 14:3487 (1986),
  • nucleic acids containing one or more carbocychc sugars are also included within the definition of nucleic acids (see Jenkins et al , Chem.
  • nucleic acid analogs may find use in the present invention
  • mixtures of naturally occurring nucleic acids and analogs can be made, for example, at the site of conductive o gomer or electron transfer moiety attachment, an analog structure may be used
  • mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made
  • 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 deoxy ⁇ bo- and ⁇ bo-nucleotides, and any combination of bases, including uracil, adenme, thymme, cytosine, guanine, inosine, xathanme hypoxathanme, isocytosme, isoguanine, etc
  • the term "nucleoside” includes nucleotides and nucleoside and nucleotide analogs, and modified nucleosides such as am o modified nucleosides
  • "nucleoside” includes non- ⁇ aturally occu ⁇ ng analog structures
  • 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 It may be any length, with the understanding that longer sequences are more specific
  • the complementary target sequence may take many forms For example, it may be contained within a larger nucleic acid sequence, i e all or part of a gene or mRNA, a restriction fragment of a plasmid or genomic DNA, among others
  • probes are made to hybridize to target sequences to determine the presence or absence of the target sequence in a sample Generally speaking, this term will be understood by those skilled in the art
  • the target sequence may also be comprised of different target domains, which may be adjacent ( ⁇ e contiguous) or separated
  • 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 polymerase 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.
  • the target analyte is a protein.
  • proteins or grammatical equivalents herein is meant proteins, oligopeptides and peptides, derivatives and analogs, including proteins containing non-naturally occurring ammo acids and ammo acid analogs, and peptidomimetic structures.
  • the side chains may be in either the (R) or the (S) configuration.
  • the ammo acids are in the (S) or L-configuration.
  • Suitable protein target analytes include, but are not limited to, (1 ) immunoglobu ns, particularly IgEs, IgGs and IgMs, and particularly therapeutically or diagnostically relevant antibodies, including but not limited to, for example, antibodies to human albumin, apo poproteins (including apohpoprotein E), human chononic gonadotropm, cortisol, ⁇ -fetoprotein, thyroxin, thyroid stimulating hormone (TSH), antithrombm, antibodies to pharmaceuticals (including antieptileptic drugs (phenyto , p ⁇ midone, carbariezepin, ethosuximide, valproic acid, and phenobarbitol), cardioactive drugs (digoxin, lidocaine, procainamide, and disopyramide), bronchodilators ( theophyllme), antibiotics (chloramphenicol, sulfonamides), antidepressants, immunosuppresants, abused drugs (amphetamine, methamphetamine,
  • influenza virus influenza virus
  • paramyxoviruses e.g respiratory syncytiai virus, mumps virus, measles virus
  • adenoviruses e.g. respiratory syncytiai virus
  • mumps virus e.g. mumps virus
  • measles virus e.g. adenoviruses
  • rhmoviruses coronaviruses
  • reoviruses e.g. rubella virus
  • parvoviruses poxviruses (e.g. " variola virus, vaccinia virus), enteroviruses (e.g pohovirus, coxsackievirus), hepatitis viruses (including A, B and C), herpesviruses (e.g.
  • Vibrio e.g. V. cholerae
  • Esche ⁇ chia e.g. Enterotoxigenic E.
  • TSH leutinzmg hormone
  • LH leutinzmg hormone
  • progeterone and testosterone
  • other proteins including ⁇ -fetoprotem, carcmoembryonic antigen CEA, cancer markers, etc.
  • 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., 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-hke 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)
  • 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 nickeT
  • 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, perspiration and tears, and solid tissues, including liver, spleen, bone marrow, lung, muscle, brain, etc.
  • bodily fluids including blood, lymph, saliva, vaginal and anal secretions, urine, feces, perspiration and tears, and solid tissues, including liver, spleen, bone marrow, lung, muscle, brain, etc.
  • 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 the art
  • 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 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
  • 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
  • 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 ste ⁇ lizable as well
  • 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, 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
  • 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, 09/387,691 , 09/438,600, 09/506,178, and 09/458,534, all of which are expressly incorporated by reference in their entirety
  • 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 as plasticizers and dispersants
  • the green-sheet is preferably in the form of sheets that are 50 to
  • the ceramic particles are typically metal oxides, such as aluminum oxide or zirconium oxide
  • metal oxides such as aluminum oxide or zirconium oxide
  • An example of such a green-sheet that includes glass-ceramic particles is "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 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
  • 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 T ⁇ netics Corp Model APS- 8718 Automated Punch System
  • features, such as channels and wells may be embossed into the surface of the green-sheet by pressing the green- 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 T ⁇ netics LVS-3012
  • 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 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," Insulation/Circuits (April, 1977) and in Morton L Topfer, Thick Film Microelectronics, Fabrication Design, and Applications (1977), pp 41-59, which are incorporated here
  • the porosity of the resulting thick-film can be adjusted by adjusting the amount of organic vehicle 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 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
  • 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
  • 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
  • the silk screen pattern is created photographically by exposure to a mask
  • 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
  • the adhesive is a room-temperature adhesive
  • room-temperature adhesives have glass transition temperatures below room temperature, / e , below about 20° C, so that they can bind substrates together at room 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
  • pressure-sensitive adhesives Suitable room-temperature adhesives are typically supplied as water-based emulsions and are available from Rohm and Haas, Inc and from Air Products, Inc For example, a material sold by Air Products, Inc as
  • 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 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 removed Films that are less than about 0 5 microns thick when dried are too thin because they provide insufficient adhesion between the layers 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
  • the layers are stacked together to form a multilayered green-sheet structure
  • the layers are stacked in an alignment die, so as to maintain the desired registration between the structures of each layer
  • alignment holes must be added to each green-sheet layer
  • 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
  • the layers are preferably laminated together after they are stacked
  • the lamination process involves the application of pressure to the stacked layers
  • a uniaxial 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
  • Adhesives do not need to be applied to bind the green-sheet layers together when the conventional lamination process is used
  • 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 lower pressures are more desirable to allow the formation of larger structures, such as cavities and channels
  • pressures less than T000 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 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
  • pressures less than about 100 psi may be applied by hand
  • many devices may be present on each sheet
  • 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
  • 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
  • appropriate sintering temperatures range from about 950 to about 1600° C, depending on the material
  • sintering temperatures between 1400 and 1600° C are typical
  • Other ceramic materials, such as silicon nitride, aluminum nitride, and silicon carbide require higher sintering temperatures, namely 1700 to 2200° C
  • 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
  • metal particles may require sintering temperatures anywhere from 550 to 1700° C, depending on the metal
  • 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
  • polymers are present as a binder in the green-sheet and in the room-temperature adhesive The firing should be of sufficient temperature and duration to decompose these polymers and to allow for their removal from the multilayered structure
  • 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 14 to 17% is typically observed
  • the volume change due to firing 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
  • 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
  • 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
  • silver is the preferred metal for providing electrically conductive pathways
  • some other metal, such as platinum must be used due to the relatively low melting point of silver (961 ° C)
  • 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
  • two "halves" of a device can be glued or fused together
  • reagent mixture such as a hydrogel or biological components that are not stable at high temperature
  • 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
  • the substrates comprise arrays of capture binding ligands
  • any number of different capture binding ligands, or capture probes 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, 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
  • 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
  • the oligonucleotide probes are attached to the microelectrodes through a neutral polypyrrole matrix
  • 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
  • nucleic acid duplex possesses a high negative charge density Following electrical perturbation of the nucleic acid, strong interactions, such as the intercalation or binding of metal ions to the nucleic acid, occur
  • nucleic acid molecules such as a probe
  • the result of this molecular interaction is a change in AC impedance This change is used in the methods and apparatus of the invention to distinguish between "complete” hybridization and incomplete or “mismatch” hybridization between the immobilized oligonucleotide probe and target nucleic acid
  • 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 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
  • 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 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.
  • the apparatus is a microarray containing at least 5 microelectrodes on a single substrate to which oligonucleotide probes have been attached.
  • arrayed ohgonucleotides are attached to polyacrylamide gel pads that are in contact with the microelectrodes of the apparatus of the present invention.
  • ohgonucleotides having a particular nucleotide sequence, or groups of such ohgonucleotides having related (e.g., overlapping) nucleotide sequences are immobilized at each of the plurality of microelectrodes.
  • nucleotide sequence(s) of the immobilized ohgonucleotides at each microelectrode and the identity and correspondence between a particular microelectrode and the nucleotide sequence of the oligonucleotide immobilized thereto, are known.
  • the probes are ohgonucleotides 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.
  • 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
  • the immobilized probes are peptides comprising from about 5 to about 500 ammo acid residues
  • the substrate is composed of silicon
  • 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
  • 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
  • probes are attached to conjugated polymers or copolymers including, but not limited to, polypyrrole, polythiphene, polyanihne, polyfuran, polypy ⁇ dine, polycarbazole, polyphenylene, poly(phenylenv ⁇ nylene), polyfluorene, polyindole, their derivatives, their copolymers, and combinations thereof.
  • 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 mm 2 and 5 cm 2 containing between 1 and 1 x 10 8 microelectrodes
  • the substrate has a surface area of 100 mm 2 and contains 10 4 microelectrodes, each microelectrode having an oligonucleotide having a particular sequence immobilized thereto
  • the substrate has a surface area of 100 mm 2 and contains 10 4 microelectrodes, each microelectrode in contact with a polyacrylamide gel pad 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 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
  • 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.
  • 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.
  • the microelectrodes comprise a gold conductor and glass insulator.
  • 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).
  • 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 mm 2 to 0.5 cm 2 In the preferred embodiment, the exposed conductive material is between 100 to 10,000 mm 2 .
  • 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
  • polyacrylamide gel material 7 is packed into a recess etched into the exposed surface of the platinum wire 2
  • oligonucleotide probes are immobilized on the microelectrodes of the apparatus of the present invention using a neutral layer between the ohgonucleotides and the micro lectrodes.
  • this layer comprises neutral polypyrrole.
  • this layer comprises such substances as polythiphene, polyanihne, polyfuran, polypy ⁇ dine, polycarbazole, polyphenylene, poly(phenylenv ⁇ nylene), polyfluorene, 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.
  • oligonucleotide probes are immobilized on polyacrylamide gel pads in contact with the microelectrodes of the apparatus of the present invention
  • 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 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
  • the counter-electrode comprises a conductive material, with an exposed surface that is significantly larger than that of the individual microelectrodes
  • the counter electrode comprises platinum
  • the counter electrode comprises solid or porous films of silver, gold, platinum, titanium, copper, or metal oxides, metal nitrides, metal carbides, or carbon
  • the apparatus comprises at least one reference electrode
  • the reference electrode is used in assays where the further quantification of target molecules is desired
  • the reference electrode comprises a silver/ silver chloride electrode
  • 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 IO ⁇ - conductive and can react with biological molecules, most preferably nucleic acids or peptides
  • 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
  • the electrolyte solution used for the detection of nucleic acid hybridization contains 0 1 M L ⁇ CI0 4 This electrolyte is preferred since CI0 4 is not specifically adsorbed on the electrode surface and thus generates a low background noise
  • ⁇ * is preferred since its small size facilitates intercalation of the Lf cations into the nucleic acid duplex and has less diffusion resistance
  • the AC impedance is measured in hybridization buffers such as 1X SSC following molecular interactions between probe and target molecules
  • 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 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
  • the apparatus further comprises a plurality of wells each of which encompasses at least one microelectrode and at least one counter- electrode
  • 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 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
  • the apparatus and methods of the present invention can be adapted further to be used with arrays of any substance that can participate in a molecular interaction that can be interrogated with cations, most preferably lithium cations
  • Such interactions include gand-receptor interactions, enzyme-inhibitor interactions, and antigen-antibody interactions
  • the apparatus and methods of the present invention are not dependent on labeling the target molecule By removing the labeling step, the cost of the assay is 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 convert the RNA into cDNA The methods of the present invention may also enable new types of assays to be developed 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 nu
  • 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 hydrophihc polymeric materials including but not limited to polyacrylamide, agarose gel, polyethylene glycol, cellular, and sol gels
  • the apparatus is a microarray containing at least 10 3 hydrogel porous microelectrodes to which oligonucleotide probes have been attached.
  • ohgonucleotides having a particular nucleotide sequence, or groups of such ohgonucleotides having related (e g , overlapping) nucleotide sequences are immobilized at each of the plurality of hydrogel porous microelectrodes.
  • the nucleotide sequence(s) of the immobilized ohgonucleotides 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
  • the probes are ohgonucleotides 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
  • the probes are nucleic acids comprising from about 10 to about 5000T)asepa ⁇ rs, more preferably from about 100 to about 1000 basepairs, and most preferably from about 200 to about 500 basepairs
  • the immobilized probes are peptides comprising from about 5 to about 500 ammo acid residues
  • the substrate is composed of silicon
  • 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
  • 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.
  • 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 2 and 5 cm 2 containing between 1 and 1 x 10 8 hydrogel porous microelectrodes.
  • the substrate has a surface area of 10,000 mm 2 and contains 10 4 hydrogel porous microelectrodes, each of which has an oligonucleotide having a particular sequence immobilized thereto
  • 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.
  • the hydrogel porous microelectrodes are regularly spaced on the solid substrate with a uniform spacing there between
  • 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
  • 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
  • the microelectrodes comprise a gold conductor and glass insulator.
  • 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.
  • 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 mm 2 to 0.5 cm 2 In the preferred embodiment, the exposed conductive material is between 100 to 160,000 mm 2
  • 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
  • polyacrylamide gel material 7 is packed into a recess etched into the exposed surface of the platinum wire 2.
  • the polyacrylamide gel pad is embedded into a recess etched into the surface of the microelectrode.
  • the polyme ⁇ x hydrogel pad is preferably at least about 0 1 to 30 mm thick, more preferably at least about 0.5 to 10 mm thick, and most preferably about 0 5 mm thick
  • oligonucleotide probes are immobilized on the polyacrylamide gel.
  • the apparatus of the present invention comprises at least one counter-electrode.
  • the counter-electrode comprises a conductive material, with an exposed surface that is significantly larger than that of the individual microelectrodes.
  • the counter electrode comprises platinum.
  • the counter electrode comprises solid or porous films of silver, gold, platinum, titanium, or copper, metal oxides, metal nitrides, metal carbides, or carbon
  • the apparatus comprises at least one reference electrode
  • the reference electrode is used in assays where the further quantification of target molecules is desired
  • the reference electrode comprises a silver/ silver chloride electrode
  • the reference electrode comprises solid or porous films of gold, platinum, titanium, or copper, metal oxides, metal nitrides, metal carbides, or carbon
  • the apparatus further comprises a plurality of wells each of which encompasses at least one hydrogel porous microelectrode and at least one counter-electrode
  • 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
  • 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
  • target molecules are labeled with electrochemical reporter groups comprising a transition metal complex, most preferably containing a transition metal ion that is ruthenium, cobalt, iron, or osmium
  • 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-benzoqu ⁇ none, ferrocene, tetracyanoqumodimethane, N,N,N',N'-tetramethyl-p-phenylened ⁇ am ⁇ ne, or tetrathiafulvalene
  • Redox moieities useful against an Ag/AgCI reference electrode include 9-am ⁇ noac ⁇ d ⁇ ne, ac ⁇ dine orange, aclarubicm, daunomycm, doxorubicm, pirarubicin, ethidium bromide, ethidium monoazide, chlortetracycline, tetracyclme, minocycline, Hoechst 33258, Hoechst 33342, 7-am ⁇ noact ⁇ nomyc ⁇ n D, Chromomycm A 3 , mithramycin A, Vinblastme, Rifampicin, Os(b ⁇ py ⁇ d ⁇ ne) 2 (d ⁇ py ⁇ dophenaz ⁇ ne) 2 * , Co(b ⁇ py ⁇ d ⁇ ne) 3 3+ , 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 (CH 2 ) n , 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-S ⁇ -0) 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
  • 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
  • inventive methods disclosed herein are useful for electrochemical detection of molecular interactions between probe molecules bound to defined regions of an ordered array
  • a biochip array (conventionally termed “a biochip array”) and electrochemically-labeled target molecules in a sample which are permitted to interact with the probe molecules
  • a biochip array 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 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 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 detecTmg 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
  • 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, potentiomet ⁇ c measurements, amperomet ⁇ c measurements, current step method, and combinations thereof
  • the electrical signal is current flow in response to an applied voltage at the redox potential of the electrochemical label
  • 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
  • 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, / e , for mutation or genetic polymorphism
  • 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 ohgonucleotides having one of each possible nucleotide at the polymorphic position to ensure hybridization of at least one of the ohgonucleot
  • 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 ohgonucleotides in the array complementary thereto
  • the apparatus of the present invention comprises a supporting substrate, a 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
  • the apparatus of the present invention comprises a supporting substrate, a 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
  • the substrate is composed of silicon
  • the substrate is prepared from substances including, but not 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
  • 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.
  • probes are attached to conjugated polymers or copolymers including, but not limited to, polypyrrole, polythiophene, polyanihne, polyfuran, polypy ⁇ dine, polycarbazole, polyphenylene, poly(phenylenv ⁇ nylene), polyfluorene, polyindole, their derivatives, their copolymers, and combinations thereof.
  • 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 mm 2 and 5 cm 2 containing between 1 and 1 x 10 8 microelectrodes
  • the substrate has a surface area of 100 mm 2 and contains 10" microelectrodes, each microelectrode having an oligonucleotide having a particular sequence immobilized thereto.
  • the substrate has a surface area of 100 mm 2 and contains 10 4 microelectrodes, each microelectrode in contact with a polyacrylamide gel pad 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 between 0 05 mm 2 to 0.5 mm Most preferably, the microelectrodes are regularly spaced on the solid substrate with a uniform spacing there between.
  • the apparatus comprises a microarray containing at least 10 3 microelectrodes on a single substrate to which oligonucleotide probes have been attached
  • arrayed ohgonucleotides are attached to polyacrylamide gel pads that are in contact with the microelectrodes of the * apparatus of the present invention
  • ohgonucleotides having a particular nucleotide sequence, or groups of such ohgonucleotides having related (e.g., overlapping) nucleotide sequences are immobilized at each of the plurality of microelectrodes
  • the nucleotide sequence(s) of the immobilized ohgonucleotides at each microelectrode, 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 thermodynamically-stable duplexes sufficient for single base extension under the hybridization 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 un ⁇ t(s) 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
  • longer probes, / e. longer than 40 nucleotides may also be used
  • the primer or probe is preferably immobilized directly on the first electrode surface through an anchoring group
  • advantageous anchoring groups include, for example, moieties comprising thiols, carboxylates, hydroxyls, amines, hydrazines, esters, amides, ha des, vinyl groups, vinyl carboxylates, phosphates, silicon-containing organic compounds, and their derivatives
  • an oligonucleotide which is complementary to a target DNA is covalentiy linked to a metallic gold electrode through a thiol-containmg anchoring group
  • the length of these anchoring groups is chosen such that the conductivity of these molecules do not hinder electron transfer from the electrochemical reporter groups, to the electrode, via the hybridized probe and target DNA, and these anchoring groups in series
  • these anchoring groups are preferred to have higher conductivities than double-stranded nucleic acid A conductivity, S, of from between about 10 "6 to about
  • the primer or probe can be covalentiy 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, S ⁇ 0 2 , NO x or a porous organic polymer such as polyacrylamide, agarose, nitrocellulose membranes, nylon, and dextran supports
  • Primers are covalentiy bound to the support through a linker Preferred linker moieties include, but are not limited to, thioethers, ethers, esters, amides, amines, hydrazines, carboxylates, hahdes, hydroxyls, vinyls, vinyl carboxylates, thiols, phosphates, silicon containing organic compounds, and their derivatives and other carboxylate moieties More preferably, biotm-streptavidin pairs are advantageous arranged to provide probe bindif g onto the intermediate support
  • the apparatus of the invention also includes a second electrode and a reference electrode to permit 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, ⁇ d ⁇ um-t ⁇ n oxide, zinc oxide, or other conductive materials such as carbon black, conductive epoxy, most preferred is a platinum (Pt)-w ⁇ re auxiliary electrode
  • the reference electrode is preferably a silver wire electrode, a silver/silver chloride (Ag/AgCI) reference electrode, or a saturated calomel electrode
  • the apparatus also comprises one or a multiplicity of reaction chambers, each reaction chamber 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
  • the term "in electrochemical contact” is intended to mean, inter alia, 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 apparatus and methods of the invention include but are not limited to phosphate buffered saline, HEPES buffered solutions, and sodium bicarbonate buffered solutions
  • Preferred polymerases for performing single base extensions using the methods and apparatus of the invention are polymerases having little or no exonuclease activity More preferred are polymerases 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 polymerases include Taq polymerase from T aquaticus (commercially available from Perkin-Elmer Cetus, Foster City, CA), Sequenase® and ThermoSequenase® (commercially available from U S Biochemical, Cleveland, OH), and Exo(-)Pfu polymerase (commercially available from New England Biolabs, Beverley, MA)
  • the inventive methods for SNP detection generally comprise (1 ) preparing a sample containing the target nucleic ac ⁇ d(s) 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 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,
  • the extension moiety in the primer extension unit is preferably a chain-terminating moiety, most preferably dideoxy ⁇ ucleoside t ⁇ phosphates (ddNTPs), such as ddATP, ddCTP, ddGTP, and ddTTP, however other terminators known to those skilled in the art, such as nucleotide analogs or arabmoside t ⁇ phosphates, are also within the scope of the present invention
  • ddNTPs differ from conventional deoxynucleoside t ⁇ phosphates (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
  • 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 d
  • the target DNA in the sample to be investigated can be amplified by means of in vitro amplification reactions, such as the polymerase chain reaction (PCR) technique well known to those skilled in the art
  • in vitro amplification reactions such as the polymerase chain reaction (PCR) technique well known to those skilled in the art
  • PCR polymerase chain reaction
  • 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 polymerase
  • in vitro amplification methods such as PCR
  • 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 simplifies performance of the inventive methods
  • a general formula of a preferred embodiment of the primer extension unit is ddNTP-L-R where ddNTP represents a dideoxy ⁇ bonucleotide t ⁇ phosphate 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
  • each chain-terminating nucleotide species (for example, d ⁇ deoxy(dd)ATP, ddGTP, ddCTP and ddTTP) is labeled with a different electrochemical reporter group, most preferably wherein each different reporter group has a different and electrochemically-distmguishable reduction/oxidation (redox) potential
  • redox reduction/oxidation
  • 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
  • the redox potential of the electrochemical reporter group comprising the primer extension units of the invention it is generally preferable for the redox potential of the electrochemical reporter group comprising the primer extension units of 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
  • Redox moieties useful against an aqueous saturated calomel reference electrode include 1 ,4- benzoqumone (-0 54V), ferrocene (+0 307), tetracyanoqumodimethane (+0 127, -0 291 ), N,N,N',N'-tetramethyl-p-phenylened ⁇ am ⁇ ne (+0 21 ), tetrathiafulvalene (+0 30) - Redox moieties useful against a Ag/AgCI reference electrode include 9-am ⁇ noac ⁇ d ⁇ ne
  • 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 about 10 to about 20 Angstroms
  • the linker can be an organic moiety such as a hydrocarbon chain
  • linker 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- mediated chain extension
  • single base extension is detected by standard electrochemical means such as cyclic voltammetry (CV) or stripping voltammetry
  • CV cyclic voltammetry
  • stripping voltammetry 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 oxidation or reduction peak of the primer extension unit detected as current flow in the electrode at the appropriate redox potential
  • 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 voltammetry, potential step method, potentiomet ⁇ c measurements, amperomet ⁇ c measurements, current step method, and combinations thereof
  • electric current is recorded as 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
  • a DC voltage sweep is done In ACV, an AC signal is superimposed on to the voltage sweep
  • square wave voltammetry a square wave is superimposed on to the voltage sweep
  • the signal is recorded from each position ("address") in the oligonucleotide array, so that the identity of the extended species can be determined
  • 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 polymerase and having a salt concentration sufficient to permit hybridization between the target nucleic acid and primer ohgonucleotides under the conditions of the assay, and a DNA polymerase such as Tag DNA polymerase 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
  • 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 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 (/ e , mutant, wildtype or polymorphism) of the interrogated nucleotide in the target
  • the probe comprises a 3' terminal residue that corresponds to and hybridizes with the interrogated base
  • ohgonucleotides having a "mismatch" at the 3' terminal residue will hybridize but will not be extended by the polymerase 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
  • the electrode is washed at high stringency (i e , in a low-salt and low dielectric constant solution (such as 0 1X SSC 0 015M NaCI, 15mM sodium citrate, pH 7 0), optionally including a detergent such as sodium dodecyl sulfate at temperature of between aboutl 0- 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
  • a neutral polypyrrole matrix was used for attachment of nucleic acid probes to the exposed platinum disk ⁇ jf 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-hydrodysucc ⁇ n ⁇ m ⁇ do pyrrole, and 0 1 M LiCIOJ 95% acetonit ⁇ le was 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 mV/sec
  • the electrolyte was purged by nitrogen gas during the entire deposition
  • the microelectrodes were incubated at room temperature for 4 hours in a solution consisting of 80 mL dimethylformamide and 20 mL of 15 nM 5'-am ⁇ no 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 microelectrodes were washed with
  • TBE buffer (0 89 M T ⁇ s-borate, 0 025 M EDTA), rinsed thoroughly in deionized water, and allowed to dry at room temperature
  • 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 (10 18 JVI, aM) range Hybridization of probe and target molecules was performed in 1X SSC buffer
  • Figure 3B illustrates the frequency complex curves, as seen in Figure 3A, for the high frequency zone (where Z' ⁇ 5 x 10 4 ) 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 (/ 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 L ⁇ +
  • 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 signals were obtained, demonstrating that methods of the present invention can be also used to quantify the amount of target hybridized onto the electrode-immobilized probe.
  • this method can be used in conjunction with appropriate reference electrodes to measure the absolute quantities of nucleic acid in a test sample.
  • 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.
  • 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', SEQ ID NO: 2) and four other microelectrodes were incubated in a solution containing 2 /M of a 15-mer target molecule containing three mismatched bases relative to the attached probe (5'- C-C-C-T-C-A-A-G-C-A-G-A-G-G-G-A-3'; SEQ ID NO: 1 ) using the conditions described in Example
  • Microelectrodes 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
  • the behavior of the microelectrode is treated as an "ideal" polarization electrode under conditions of an electrolyte solution comprising 0 1 M L ⁇ Cl0 4 with purging N 2 and before hybridization to a suitable target molecule is reasonable since there is no electrochemically active species and no specific adsorption
  • 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, / e , electrochemical reaction resistance and R ⁇ 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
  • Microelectrodes were prepared as described in Example 1 ( Figure 1 A) The exposed flat disk of platinum was then etched in hot aqua regia to form a recess (/ 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 11 ). A hydrogel porous microelectrode having a diameter of 258 mm was used in the following Examples.
  • hydrogel porous microelectrodes Prior to attachment of probe molecules, hydrogel porous microelectrodes were activated by incubation for 10 mm. in 2% t ⁇ fluoroacetic acid, and rinsed for 2 mm. in deionized water. Microelectrodes were then incubated for 15 mm. in 0.1 M sodium pe ⁇ odate, and rinsed for 2 mm. in deionized water. Following this treatment, microelectrodes were thoroughly washed by incubation in deionized water for 15 mm., and then air-dried. Microelectrodes were subsequently incubated for 10 mm. in 2% dimethyl dichlorosilane solution and 2% octamethylcyclotetrasiloxane, washed in ethanol, rinsed in deionized water, and air-dried.
  • 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'-am ⁇ no-3'fluoresce ⁇ n 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 microelectrodes were washed with TBE buffer, rinsed thoroughly in deionized water, and allowed to dry at room temperature.
  • 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 sealed conical tube to either 35 mL of a complementary target molecule (5'-T-C-C-T-C-T-G-C-T-T-)
  • Hybridization of the probe with either target molecule was performed in 1X SSC buffer at room temperature for 1 hour. Following hybridization, microelectrodes were thoroughly rinsed for 20 mm at room temperature in an excess volume of 1X 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 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
  • OCV open circuit voltage
  • 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 for 2 mm in deionized water, soaked in 1 N HN0 3 for 20 mm , vigorously washed in deionized water, immersed in acetone for 10 mm , and again washed vigorously in deionized water Through the use of micromanufactu ⁇ ng techniques employed in the fabrication of semiconductors, modifications of this procedure can be applied to the preparation of microelect
  • Hydrogel porous microelectrodes were prepared from the above-described microelectrodes as 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)
  • Figure 2 A hydrogel porous microelectrode having a diameter of 258 mm was used in the Example 2.
  • hydrogel porous microelectrodes Prior to attachment of probe molecules, hydrogel porous microelectrodes were activated by incubation for 10 mm. in 2% t ⁇ fluoroacetic acid, and rinsed for 2 mm. in deionized water. Microelectrodes were then incubated for 15 mm. in 0.1 M sodium pe ⁇ odate, and rinsed for 2 mm. in deionized water. Following this treatment, microelectrodes were thoroughly washed by incubation in deionized water for 15 mm , and then air-dried. Microelectrodes were subsequently incubated for 10 mm. in 2% dimethyl dichlorosilane solution and 2% octamethylcyclotetrasiloxane, washed in ethanol, rinsed in deionized water, and air-dried.
  • 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'-am ⁇ no-3'fluoresce ⁇ n 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 microelectrodes were washed with TBE buffer, rinsed thoroughly in deionized water, and allowed to dry at room temperature.
  • Nucleic acid hybridization between a probe bound to a hydrogel porous microelectrode and an electrochemically-labeled target molecule is detected as follows.
  • the baseline AC impedance of hydrogel 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 (S'-T-C-C-T-C-T-G-C-T-T-G-A-G-G- G-3'; SEQ ID NO.
  • AC impedance is measured using a model 1260 Impedance/Gain-Phase Analyser with model 1287 Electrochemical Interface
  • the counter and reference electrodes are platinum and Ag/AgCI, respectively, and the impedance measurements are made under open circuit voltage (OCV) conditions in 1X SSC hybridization solution. Samples are excited at an amplitude of 50 mV.
  • OCV open circuit voltage
  • 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 2 and contains 10 4 microelectrodes, each microelectrode in contact with a polyacrylamide gel pad that is about 0.5 ⁇ m 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
  • 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 adenosme (A), guanme (G), cytosme (C) or thymid e (T) or uracil (U) residue.
  • the ohgonucleotides are attached to each of the gold electrodes through the polyacrylamide gel pad using a modification of the oligonucleotide at the 5' residue. This residue comprises a thioester linkage that covalentiy 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 third, reference electrode that is a silver/ silver chloride electrode, each electrode being elect ⁇ cally connected to a voltage source
  • 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 "18 M, aM) range.
  • Hybridization of probe and target molecules is 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 1X 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 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
  • 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 (b ⁇ py ⁇ d ⁇ ne) 3 3t , ddGTP labeled with minocychne, ddCTP labeled with ac ⁇ di ⁇ e orange, and ddTTP labeled with ethidium monoazide
  • the redox labels are covalentiy linked to the chain-terminating nucleotides by a hydrocarbon linker (CH 2 ) 2
  • each microelectrode is interrogated by conventional cyclic voltammetry to detect a redox signal
  • identity of ohgonucleotides containing single base extended species is determined by the redox potential of the signal obtained thereby
  • hybridization and single base extension can be performed in the same buffer solution, provided the polymerase is compatible with the hybridization buffer conditions

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Abstract

L'invention se rapporte à la détection d'interactions moléculaires entre les molécules biologiques. Elle concerne plus particulièrement la détection électrique d'interactions telles que l'hybridation entre différents acides nucléiques ou d'interaction antigène - anticorps de peptides utilisant des réseaux de peptides et d'oligonucléotides. Elle concerne un appareil et des procédés pour détecter la liaison de peptides ou l'hybridation d'acides nucléiques au moyen de procédés électriques, y compris l'impédance du c.a. Dans certains modes de réalisation, on n'utilise aucun groupe fonctionnel électrochimique ou autre groupe fonctionnel d'étiquetage. Dans d'autres, on utilise des étiquettes à activité électrochimique pour détecter des réactions sur des réseaux d'hydrogel, y compris des réactions de génotypage telles que la réaction d'extension de base unique.
PCT/US2000/033497 1999-12-09 2000-12-11 Procedes et compositions se rapportant a la detection electrique des reactions d'acides nucleiques WO2001042508A2 (fr)

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EP00993326A EP1238114A2 (fr) 1999-12-09 2000-12-11 Procedes et compositions se rapportant a la detection electrique des reactions d'acides nucleiques
AU29072/01A AU2907201A (en) 1999-12-09 2000-12-11 Methods and compositions relating to electrical detection of nucleic acid reactions
JP2001544379A JP2003516165A (ja) 1999-12-09 2000-12-11 核酸反応の電気的検出に関する方法および組成物
CA002393733A CA2393733A1 (fr) 1999-12-09 2000-12-11 Procedes et compositions se rapportant a la detection electrique des reactions d'acides nucleiques
US10/149,319 US20030209432A1 (en) 2000-12-11 2000-12-11 Methods and compositions relating to electrical detection of nucleic acid reactions

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US09/458,533 US20020051975A1 (en) 1999-12-09 1999-12-09 Reporterless genosensors using electrical detection methods
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US09/459,685 US6518024B2 (en) 1999-12-13 1999-12-13 Electrochemical detection of single base extension

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WO2004079001A1 (fr) * 2003-03-05 2004-09-16 University Of Saskatchewan Technologies Inc. Detection de mesappariement d'acides nucleiques
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WO2005001122A3 (fr) * 2003-06-27 2005-11-24 Adnavance Technologies Inc Detection electrochimique de liaison d'adn
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Publication number Publication date
EP1238114A2 (fr) 2002-09-11
AU2907201A (en) 2001-06-18
JP2003516165A (ja) 2003-05-13
WO2001042508A3 (fr) 2002-03-14
WO2001042508A9 (fr) 2002-05-30
CA2393733A1 (fr) 2001-06-14

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