JP2003516165A - Methods and compositions for electrical detection of nucleic acid reactions - Google Patents

Abstract

  (57) [Summary] The present invention relates to the detection of molecular interactions between biological molecules. In particular, the invention relates to the electrical detection of interactions, such as nucleic acid or peptide antigen-antibody hybridization, using arrays of peptides or oligonucleotides. In particular, the invention relates to devices and methods for detecting nucleic acid hybridization or peptide binding using electrical methods including AC impedance. In some embodiments, no electrochemical or other labels are used. In other words, an electrochemically active label is used to detect a reaction on the hydrogel array, including a genotyping reaction such as a single base extension reaction.

Description

Detailed Description of the Invention

The present invention is directed to US patent application Ser. No. 09/458, filed Dec. 9, 1999,
501; U.S. Patent Application No. 09/459, filed December 13, 1999,
No. 685 and US patent application Ser. No. 09/458, filed Dec. 9, 1999
, 533, which are expressly incorporated by reference herein.

TECHNICAL FIELD OF THE INVENTION The present invention relates to the detection of molecular interactions between biological molecules. In particular, the present invention relates to the electrical detection of interactions such as nucleic acid or peptide antigen-antibody hybridization using arrays of peptides or oligonucleotides. In particular, the invention relates to devices and methods for detecting nucleic acid hybridization or peptide binding using electrical methods including AC impedance. In some embodiments, no electrochemical label or other label is used. In other words, an electrochemically active label (or label) to detect reactions on the hydrogel array, including genotyping reactions such as single base extension reactions.
To use.

BACKGROUND OF THE INVENTION Numerous commonly used biological applications, including, for example, the diagnosis of genetic disorders, the analysis of sequence polymorphisms and the study of receptor / ligand interactions, are between probe and target molecules. Rely on the ability of analytical techniques to quickly detect events related to the interaction of These molecular detection techniques have conventionally utilized radioactive isotopes or fluorescent substances to monitor the interaction between the probe and the target molecule, but the electrical detection method of the intermolecular interaction is the radioactive label or fluorescent label. It provides an attractive alternative to detection technology that relies on.

Electrical and electrochemical detection techniques are based on detecting changes in the electrical properties of the electrode that result from the interaction between probe molecules on the electrode surface and target molecules in the reaction mixture. Electrical or electrochemical detection eliminates many of the disadvantages inherent in using radioactive or fluorescent labels to identify interactions between molecules. For example, this process provides a detection technique that is safe, inexpensive, sensitive, and does not suffer from the need for crackdown due to complex and cumbersome rules.

Despite these advantages, however, there are numerous obstacles to using electrical or electrochemical detection techniques to analyze intermolecular interactions.
Among some methods, one such obstacle is the requirement to incorporate an electrochemical label into the target molecule. For example, labeled target molecules have been used to increase the signal generated during hybridization tests (or assays) based on the formation of nucleic acid duplexes.

For example, Meade et al. (US Pat. Nos. 5,591,578, 5,705,3
48, 5,770,369, 5,780,234 and 5,824.
, 473) propose a method of selectively covalently modifying nucleic acids using redox activators such as transition metal complexes. Meade et al. Further disclose nucleic acid hybridization tests using such covalently modified nucleic acid molecules. Further, a similar study was conducted in US Pat. No. 6,090,933, International Publication W.
O98 / 20162, International Publication WO98 / 12430, International Publication WO00 /
16089, International Publication WO99 / 57317, International Publication WO99 / 6742
5, International Publication WO99 / 37819, International Publication WO00 / 38836, International Application PCTUS00 / 19889 and International Publication WO99 / 57319.

Heller et al. (US Pat. Nos. 5,605,662 and 5,632,95
No. 7) proposes a method for controlling molecular biology reactions in a microscope format that utilizes self-addressable, self-assembling microelectronic devices. Furthermore, Heller et al. Used a fluorescent dye to label (
(Or labeled) target molecule is transferred to a specific test site, where the target molecule reacts with a specific probe that is thereby enriched and bound to the test site. . Unbound or non-specifically interacting target molecules are then removed by reversing the electrical polarity of the test site. The interaction between probe and target molecule is then assayed using optical means.

Certain alternative methods have been disclosed in the prior art that do not use labeled target nucleic acids. For example, Hollis et al. (US Pat. Nos. 5,653,939 and 5
, 846, 708) propose a method and apparatus for identifying molecular structures within a sample material using a monolithic array of test sites formed on a substrate on which the sample material is used. In the method of Hollis et al., The change in the electromagnetic or acoustic properties of the test site following the addition of the sample substance-eg the change in resonance frequency-determines which probe interacted with the target molecule in the sample substance. For being detected.

Further, Eggers et al. (US Pat. Nos. 5,532,128, 5,670,
322 and 5,891,630) propose methods and apparatus for identifying molecular structures within sample material. In the method of Eggers et al., Multiple test sites with attached probes are exposed to the sample material and an electrical signal is then applied to the test sites. The dielectric properties of the test site are subsequently detected to determine which probe interacted with the target molecule in the sample material.

Another obstacle in developing simple and cost-effective electrical or electrochemical detection devices for detecting molecular interactions is to probe molecules onto the substrate of microelectrodes or microarrays. Including being attached (or attached). For example, the prior art proposes a microarray that uses a polyacrylamide pad to attach an oligonucleotide probe to a solid support, whereas the prior art proposes a pad associated with an electrical or electrochemical detection device. There wasn't.

Guschin et al. (Anal. Biochem. Vol. 250: 203-1
1 (1997)) describes a technique for detecting molecular interactions between a target molecule in a biological sample (or biological sample) solution and a probe immobilized on a glass substrate by polyacrylamide gel. . In the technique disclosed by Guschin et al., The molecular interaction between probe and target molecule is detected using an optical reporter. The Guschin et al. Reference does not teach or suggest any electrical or electrochemical detection techniques for detecting hybridization between a target molecule and an immobilized probe.

Guschin et al. (Appl. Environ. Microbiol. Vol.
． 63: 2397-402 (1997)) also describes assembling a microarray via immobilization of oligonucleotide probes on a polyacrylamide gel pad placed in contact with a glass substrate. In this technique disclosed by Guschin et al., Parallel hybridization between target nucleic acid and immobilized probe is detected using an optical reporter moiety. This Gusc
The Hin et al. reference also does not teach or suggest any electrical or electrochemical detection technique in combination with immobilization of the probe on a polyacrylamide gel pad.

Furthermore, Yang et al. (Anal. Chim. Acta., Vol. 346: 2).
59-75 (1997)) assembled a microarray on a polyacrylamide gel pad via immobilization of a nucleic acid probe, and then the molecular interaction between the probe and the target molecule was determined by an optical reporter moiety. It describes that it detects using. Yang et al. Further describe an alternative technique in which the molecular interaction between the nucleic acid probe attached directly to the solid electrode and the labeled target molecule is detected using electrical or electrochemical means. However, Yang et al.
It does not suggest the use of electrical or electrochemical detection techniques in combination with immobilization of the probe on a polyacrylamide gel pad.

Detection of single base mutations and genetic polymorphisms in nucleic acids is an important tool (or tool) in modern diagnostic and biological research. In addition, nucleic acid-base assays also play an important role in identifying infectious organisms such as bacteria and viruses, as well as testing levels of normal and deleted gene expression, and oncogenes. Plays an important role in detecting and identifying mutants associated with diseases such as. Therefore, improving the speed, efficiency, economy and specificity of such assays is an important requirement in medical technology.

Ideally, these assays are sensitive, specific, and easily automated. Efforts to improve the sensitivity of nucleic acids in assays are known in the prior art. For example, the polymerase chain reaction (Mullis, US Pat.
83,195, issued July 28, 1987, produced an effective amount of specific nucleic acid (about 1 microgram) in a sample with essentially low amount of original specific nucleic acid (about 1 picogram). Presented the ability to do. However, this prior art has not been very successful in improving the specificity of nucleic acid hybridization tests.

The specificity of a nucleic acid assay is determined by the degree of molecular complementarity of hybridization between the probe and target sequence. Although it is theoretically possible to distinguish complementary targets from one or two mismatched targets under tightly defined conditions, the dependence of hybridization on target / probe concentration and hybridization conditions Limits the extent to which hybridization mismatches can be used to reliably detect mutations and genetic polymorphisms, among others.

Detection of single base extensions has been used in the prior art for mutations and genetic polymorphisms.

US Pat. No. 5,925,520 describes at least two types of dideoxy chain terminating nucleotide triphosphates, each chain terminating nucleotide labeled with a detectable and distinguishable fluorescent labeling group. Disclosed is a method of using triphosphates to detect genetic polymorphisms using capture groups and single base extensions on oligonucleotide probes.

US Pat. No. 5,710,028, each labeled with a detectable and distinguishable fluorescent labeling group, uses detectably labeled chain-terminating nucleotides to detect the nucleic acid of interest. Disclosed is a method for identifying a nucleotide base at a specific position in (or a method for determining the identity of nucleotide bases).

US Pat. No. 5,547,839 discloses a method of identifying a nucleotide base at a specific position in a nucleic acid of interest using a chain-terminating nucleotide having an optically removable protecting group. Is disclosed.

US Pat. No. 5,534,424 describes a target nucleic acid, such as 4 which is annealed to an extension primer and extends with one of the four chain terminating species and further with all four chain extending nucleotides. Disclosed is a method of identifying nucleotide bases at specific positions in a nucleic acid of interest using one of the methods whereby the identification (or identity) of nucleotides at the position of interest is more than a single base. This is done due to insufficient primer being extended.

US Pat. No. 4,988,617 describes the annealing of two adjacent nucleotide primers to a target nucleic acid, as well as a linking agent (or crosslinker) such as a ligase that covalently links two oligonucleotides. The two oligonucleotides are in the 3'extent of the first oligonucleotide and 5'of the second oligonucleotide only in the context of a perfect match to the nucleic acid. Disclosed a method for identifying a nucleotide base at a specific position in a nucleic acid of interest by providing a linking agent that results in a linked oligonucleotide of

US Pat. No. 4,656,127 uses primer extension with chain terminating nucleotides or other nucleotides having anti-exonucleose linkages,
Furthermore, it disclosed a method for identifying nucleotide bases at specific positions in a nucleic acid of interest by subjecting multiple extension products to exonucleases to detect resistant species.

One common feature of this prior art is that single base extension has been detected by incorporating a fluorescent label in the extended nucleic acid species.

A significant disadvantage of single-base extension methods based on fluorescent label detection is the need for expensive and technically complex optics for detecting fluorescent labels. Although the fluorescent probes used in such methods provide a sufficient level of discrimination between extended and unextended positions in the oligonucleotide array, these methods typically use four different fluorescent labels. Therefore, each label requires detection down to a label that has a unique excitation frequency and fluorescence emission frequency. As a result of these features, such assay systems are capable of generating and distinguishing all of these different excitation and emission frequencies of light, producing and operating the devices in which they are actually used. It must add significant cost and complexity.

Another method for detecting a target nucleic acid molecule is to combine electrochemical means such as cyclic voltammetry, some of which are described above,
The use of an electrochemical tag (or tag) such as a redox (or redox) moiety.

Further, a disclosure of similar methods for detecting biological molecules such as DNA and proteins is provided in “Ihara et al., Nucleic Acids Research 24: 4273-4280 (1996); Li.
vache et al., Synthetic Metals 71: 2143-214.
6 (1995); Hashimoto, Supramolecular Chem. , 2
Volume: 265-270 (1993), Millan et al., Anal. Chem. ,
65: 2317-2323 (1993) ".

However, many of the methods known in the prior art that rely on electrochemical tags include
It requires probe / target hybridization in the presence of a redox intercalating agent. Electrochemical detection based on redox intercalating agents is generally not reproducible for electrochemical attachment to covalently attach moieties. This redox intercalator method does not diminish the actual signal but relies notably on washing conditions to remove excess label. As a result, false positives are often obtained using these methods. The specificity of the redox intercalator method is often less than that achievable with covalently linked redox sticks.

The current technology provides a simple method for detecting single base extension products in nucleic acid assays for detecting mutations and gene polymorphisms in a biological sample (or biological sample) containing a nucleic acid of interest. There is a need for economical and efficient methods.

Similarly, current technology requires the development of alternatives to amperometric detection methods used to detect interactions between biological molecules, especially nucleic acids and peptides.
In particular, current technology requires the development of electrical or electrochemical methods for detecting interactions between biological molecules that do not require modification of target or probe molecules bearing reporter (or reporter) labels. There is. The development of such methods is widely applied to medical technology, genetic technology and molecular biology technology. Furthermore, current technology requires the development of alternative techniques for attaching such biological molecules to the microelectrodes or substrates of electrical (or device) or electrochemical devices.

SUMMARY OF THE INVENTION The present invention provides an apparatus and method for detecting the nature and extent of molecular interactions between a probe and a target molecule using cations in an electrolyte solution. The most preferable aspect of the method of the present invention is to utilize the AC impedance for the detection. The devices and methods of the present invention have the advantage of providing an electrical detection method that does not require the target molecule to be further labeled with a reporter (reporter or reporter) molecule.

In a preferred embodiment of the invention, the apparatus and method comprises an ordered array (or
Molecular interactions such as nucleic acid hybridization (or hybridization) between an oligonucleotide probe molecule bound to a defined (or specific) region of a dered array and a nucleic acid target molecule capable of interacting with the probe molecule. Useful for detecting effects. In another aspect of the invention, the devices and methods are useful for detecting peptide-to-peptide (eg, receptor-ligand binding or antibody recognition of antigen) interactions.

In a further preferred aspect of the invention, the device of the invention comprises a support substrate (support substrate or support substrate), an array of microelectrodes (or microelectrodes) in contact with a support substrate to which probes are immobilized ( array), at least one counter electrode (or counter-electrode) in electrochemical contact with the support substrate, means for generating electrical impedance in each microelectrode, each in the presence or absence of a target molecule. It comprises means for detecting impedance changes at the microelectrodes, and an electrolyte solution in contact with the plurality of microelectrodes.

In another preferred embodiment, the device of the present invention comprises a support substrate, a microelectrode in contact with the support substrate, a plurality of polyacrylamide gels in contact with the microelectrode and having probes immobilized thereon. A pad, at least one counter electrode in electrochemical contact with a support substrate, means for generating electrical impedance at each microelectrode, means for detecting impedance changes at each microelectrode in the presence or absence of target molecules, and It comprises an electrolyte solution in contact with a plurality of microelectrodes. Alternatively, multiple electrodes can be defined on the substrate, which can be covered with a continuous unpatterned layer of polyacrylamide or other polymer.

In a further preferred embodiment of the invention, the microelectrodes include, but are not limited to, dense or porous film metals such as gold, platinum, titanium or copper, metal oxides, metal nitrides. Prepared from metal, metal carbide or carbon.

In a further preferred embodiment of the invention, the electrolyte solution comprises a metal cation or a polymeric cation which is ionically conductive and capable of reacting with the probe or probe-target complex. In a more preferred embodiment, the electrolyte solution comprises a salt of a lithium cation, and most preferably lithium perchlorate.

The device of the present invention further comprises at least one reference electrode. In another aspect of the invention, the device of the invention further comprises a plurality of depressions (or wells), each of which is interrogare responsive to at least one microelectrode and the entire array. Sufficient for at least one counter electrode.

In a preferred method of the present invention, the above-mentioned electrolyte solution is preferably placed in contact with a plurality of microelectrodes on which nucleic acid probes having a neutral polypyrrole layer are immobilized. The AC impedance of the microelectrode is first measured in the absence of added target nucleic acid. The microelectrode is then added to the biological sample containing the target nucleic acid molecule (or, most preferably, by adding the sample to the electrolyte solution, or replacing the electrolyte solution with a sample contained or diluted in the electrolyte solution). (Biological sample) Contact with material. The probe and target molecule are allowed to interact, preferably by hybridization, after which the AC impedance is measured.

In another aspect of the method of the present invention, the above-mentioned electrolyte solution is placed in contact with a polyacrylamide gel pad having a plurality of microelectrodes and nucleic acid probes immobilized thereon. The AC impedance of the microelectrode is first measured in the absence of added target nucleic acid. A microelectrode is then added to the biological sample material containing the target nucleic acid molecule, most preferably by adding the sample to the electrolyte solution or by replacing the electrolyte solution with a sample contained or diluted in the electrolyte solution. Contact. The probe and target molecule are allowed to interact, preferably by hybridization, after which the AC impedance is measured.

In a preferred embodiment of the method of the present invention, the AC impedance is measured at various frequencies in order to improve the sensitivity of the method. Probe-target interactions are detected by the difference in AC impedance signal at individual microelectrodes before and after such interactions. Most preferably, hybridization between the oligonucleotide probe immobilized on the microelectrode and the complementary (or complementary) target nucleic acid ("complete" hybridization) and the target nucleic acid mismatched with the immobilized oligonucleotide. This method is used to distinguish differences between ("mismatch" hybridizations) and hybridizations.
Nucleotide sequence of oligonucleotides immobilized on each microelectrode (nucleoti
The information about de sequence) is used in conjunction with the “complete” or “mismatch” hybridization detected by the method of the invention to determine the presence or absence of a particular target nucleic acid in a sample. It

In another aspect, the invention electrically determines the nature and extent of molecular interactions between a probe molecule and an electrochemically active reporter-labeled target molecule. Alternatively, it provides an apparatus and method for electrochemical detection. In a preferred embodiment of the invention, the device and method provide an oligonucleotide probe molecule bound to a defined (or specific) region of an ordered array and an electrochemically active molecule capable of interacting with the probe molecule. It is useful for detecting molecular interactions such as nucleic acid hybridization between reporter-labeled nucleic acid target molecules. In another aspect of the invention, the devices and methods are useful for detecting peptide-to-peptide (eg, receptor-ligand binding or antigen antibody recognition) interactions.

In a further preferred aspect of the invention, the device of the invention comprises a support substrate, a microelectrode array in contact with the support substrate, a plurality of polymer contacts in contact with the microelectrodes and having probes immobilized thereon. Hydrogel pad, at least one counter electrode in electrochemical contact with a supporting substrate, means for generating an electrical signal at each microelectrode, in the presence of an electrochemically active reporter-labeled target molecule Alternatively, it comprises means for detecting changes in the electrical signal at each microelectrode in the absence and a plurality of hydrogel porous microelectrodes and an electrolyte solution in contact with the counter electrode. Alternatively, multiple electrodes can be defined on the substrate, which can be covered with a continuous layer of unpatterned polymer hydrogel.

In a preferred embodiment of the invention, the microelectrodes are prepared from a dense or porous film of metal such as gold, platinum, titanium or copper, metal oxides, metal nitrides, metal carbides or graphitic carbon. Has been done.

In some aspects of the invention, the probe is an oligonucleotide probe having a sequence of about 10 to about 100 nucleotide residues, and the probe uses techniques known to those of skill in the art. Applied to a polyacrylamide gel pad. In another aspect, the probe is a receptor, ligand, antibody, antigen, or peptide such as a synthetic peptide, and the probe is attached to the polymer hydrogel pad using techniques known to those of skill in the art. .

The device of the present invention further comprises at least one reference electrode. In another aspect of the invention, the device further comprises a plurality of depressions, each of which is adapted to send (or interrogate) a response command signal to at least one hydrogel porous microelectrode and the entire array. Sufficient at least one counter electrode.

In the method of the present invention, the molecular interaction between the probe molecule and the electrochemically active reporter-labeled target molecule is determined by conventional electrical or electrochemical reactions such as AC impedance. It is detected using a static detection method. In one embodiment, AC of a plurality of hydrogel porous microelectrodes with immobilized nucleic acid probes
Impedance is first measured in the absence of electrochemically labeled target nucleic acid. The hydrogel porous microelectrode is then contacted with a biological sample material containing an electrochemically active reporter-labeled target molecule. The probe and target molecule are allowed to interact, preferably by hybridization, after which the AC impedance is measured.

In a preferred embodiment of the method of the present invention, the AC impedance is measured at various frequencies to improve the sensitivity of the method. Interactions between probe molecules and electrochemically labeled target molecules are detected by the difference in AC impedance signals at the individual hydrogel porous microelectrodes prior to such interactions. Most preferably, hybridization between the immobilized oligonucleotide probe on the hydrogel porous microelectrode and the complementary target nucleic acid ("complete" hybridization) and target nucleic acid mismatched with the immobilized oligonucleotide ( This method is used to distinguish between hybridization and non-matching hybridization. Information about the nucleotide sequence of the oligonucleotides immobilized on each hydrogel porous microelectrode is detected by the method of the invention to determine the presence or absence of a particular target nucleic acid in the sample. Used in conjunction with "consisting" or "mismatch" hybridization.

Other aspects of the invention include, but are not limited to, cyclic voltammetry, stripping voltammetry, pulse voltammetry, square wave voltammetry, AC voltammetry, hydrodynamic modulation voltammetry. , Electropotentiation, potentiometry, amperometry, amperometry, and combinations thereof, and other electrical and / or electrochemical methods include probe molecules-electrochemically labeled target molecules. Used to detect interactions between.

The present invention further comprises methods and devices 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 devices of the invention is accomplished by sequence-specific incorporation of a chemically-terminated chain-terminating nucleotide species with an electrochemical species. In a preferred embodiment, single base extension is performed using hybridization to an oligonucleotide array, and most preferably an addressable array,
There, the sequence of each oligonucleotide in the array is known and associated with a particular address in the array. In a more preferred embodiment, single base extensions are detected using extension products labeled with an electrochemical reporter group, wherein the electrochemical reporter group comprises a transition metal complex, and most Preferably,
Contains transition metal ions which are ruthenium, cobalt, iron or osmium.

In practicing the methods of the invention, the invention provides an array of oligonucleotide probes immobilized to a surface defining a first electrode. Preferably, each oligonucleotide sequence at each of the particular identified positions (or "addresses") in the array is understood and at least one such oligonucleotide is to be tested (""Target" or "target nucleic acid") is complementary to a sequence in a nucleic acid contained in a biological sample. In one preferred embodiment, the sequence of at least one of said oligonucleotides hybridizes to a position immediately adjacent to a nucleotide position in the sample nucleic acid that should send a response command signal (ie for mutations or genetic polymorphisms). Selected to soybean.
The term "contiguous" in the present specification is intended to include a position 1 nucleotide base upstream of the base to which a response command signal is to be sent, that is, a position 3'with respect to the template chromatid of the target DNA. The hybridization of the oligonucleotides in the array to the nucleic acids in the sample is carried out in the reaction chamber and in the hybridization buffer at a temperature that causes hybridization between the nucleic acids in the sample and their complementary oligonucleotides in the array. Then, it will be held for a certain time. Single base extension is performed with a polymerase, and most preferably with a thermostable polymerase in the presence of a chain terminating primer extension unit covalently attached to an electrochemical label. Be seen. In a preferred embodiment, each chain terminating nucleotide species (eg dideoxy ddATP, ddGTP, ddCTP, and d.
dTTP) is labeled with various electrochemical labels, and most preferably with various, distinct and specifically detectable oxidation / reduction potentials. Single base extensions are detected by applying conventional electrochemical detection methods such as cyclic voltammetry or stripping voltammetry. Other electrical and / or electrochemical methods that may be used include, but are not limited to, AC impedance, pulse voltammetry, square wave voltammetry, AC voltammetry (ACV), hydrodynamically modulated voltammetry. , Potential step method, potentiometric measurement, amperometric measurement, current step method, and combinations thereof.

In another embodiment, the 3'residue of the oligonucleotide hybridizes to a nucleotide position in the sample nucleic acid that should (or interrogate) for a mutation or genetic polymorphism. At position, at least one sequence of said oligonucleotide is selected to hybridize to the target nucleic acid. In this array, oligonucleotides having sequence identity to the oligonucleotide that hybridizes to the target nucleic acid at the 3'residue will also hybridize to the target, but the 3'residue of such oligonucleotide will Results in a "mismatch" and does not hybridize at the 3'residue. Single base extension is performed using a polymerase that does not recognize mismatches and only oligonucleotides that hybridize to the target including the 3'residue are extended. In these aspects of the invention, if the nucleotide sequence of each oligonucleotide in the array is understood and is correctly associated with a position in the array, a single electrochemical species-labeled single Only chain terminating species can be used, or the same electrochemical species can be used for all four chain terminating species. Therefore, detecting an electrochemical signal from a redox species at a particular position in the array using conventional electrochemical detection methods such as cyclic voltammetry identifies the 3'residue of the probe. And will therefore identify (or provide the identity of complementary nucleotides) with complementary nucleotides at corresponding positions in the target nucleic acid.

In practicing the preferred embodiments of the method of the present invention and using the device of the present invention, an electric current is applied to each specific chain-terminating nucleotide species labeled with an electrochemically active reporter. Recorded as a function of sweep voltage for one electrode. In a preferred embodiment, the current at each specific potential is detected at each address in the array where a single base extension occurs with the corresponding chain terminating nucleotide species labeled with a particular electrochemical reporter group. Detection of the electrical signal at a particular position in the array, where the nucleotide sequence of the oligonucleotide occupying that position is known, allows the identification (or identity) of the extended nucleotide to be measured, It thus allows the identification of the mutation or gene polymorphism to be measured.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to various electrical and electrochemical techniques used to detect the presence of target analytes, particularly nucleic acids, in a sample. Generally, this method relies on molecular interactions such as nucleic acid hybridization or protein-protein binding reactions performed on a solid support with an array of capture binding ligands. As a result of such interactions, some electrical properties of the system change and detection is achieved.

This may be done in various ways. In a preferred embodiment, the method of the present invention utilizes AC impedance for detection. In some embodiments, the devices and methods of the invention have the advantage of providing an electrical detection method that does not require the target molecule to be further labeled with a reporter molecule. That is, the electrical impedance of the system changes as a result of the specific binding of the target analyte to the corresponding capture binding ligand (herein referred to as the capture probe when the analyte is a nucleic acid).

Alternatively, the use of an electrochemically active label allows the detection of specific interactions in a manner similar to known fluorescence systems. In this aspect, for example, when the target is a nucleic acid, it can be labeled with an electrochemically active label (ECA) during an amplification reaction such as PCR, or a second labeling system (or labeling).・ System) can be used for labeling.

In a preferred embodiment, when the target is a nucleic acid, these ECA labels have been developed to allow identification of specific bases within the target sequence, generally as outlined below.

The methods and compositions of the invention are used to detect target analytes in a sample. The molecule, compound or particle to be detected here means the “target analyte” or “analyte”. As outlined below, the target analyte preferably binds to the binding ligand fully described above. As will be appreciated by those in the art, numerous analytes may be detected using current methods; basically, as described herein, the target analyte for which the binding ligand is made is It will be detected using the method of the invention.

Suitable analytes include organic and inorganic molecules, including biomolecules. In a preferred embodiment, analytes include environmental contaminants (including fungicides, insecticides, toxins, etc.); chemicals (including solvents, polymers, organic substances, etc.); therapeutic molecules (therapeutic agents, drugs of abuse, antibiotics, etc.). Biomolecules (including hormones, cytokines, proteins, lipids, carbohydrates, cell membrane antigens, and receptors (neutral, hormonal, nutrients and cell surface receptors) or their ligands); whole cells (
Prokaryotic cells (including pathogenic bacteria) and eukaryotic cells, including mammalian tumor cells); viruses (including retroviruses, herpesviruses, adenoviruses, lentiviruses, etc.); spores and the like.

Particularly preferred analytes are environmental pollutants; nucleic acids; proteins (including enzymes, antibodies, antigens, growth factors, cytokines, etc.); therapeutic agents and drugs of abuse; cells; viruses.

In a preferred embodiment, the target analyte is a nucleic acid. "Nucleic acid" or "oligonucleotide" or grammatical equivalents (or substantially the same as them) means at least two nucleotides covalently linked together. Nucleic acids of the invention generally contain phosphodiester bonds, but, as outlined below, in some cases have alternating backbones and include, for example, phosphoramide (Beauca).
ge et al. Tetrahedron 49 (10): 1925 (1993) and references therein; Letsinger, J. et al. Org. Chem. 35:38
00 (1970); Sprinzl et al., Eur. J. Biochem. 81: 5
79 (1977); Letsinger et al., Nucl. Acids Res. 1
4: 3487 (1986); Sawai et al., Chem. Lett. 805 (19
84); Letsinger et al. Am. Chem. Soc. 110: 447
0 (1988); and Pauwels et al., Chemica Scripta.
26: 1419 (1986)), phosphorothioates (Mag et al., Nucle.
ic Acids Res. 19: 1437 (1991), and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem.
． Soc. 111: 2321 (1989)), O-methyl phosphoramidite linkage (Eckstein, oligonucleotides and analogs: a practical approach:
Oxford Press), and peptide nucleic acid backbones and bonds (Eghorm)
Et al., J. Am. Chem. Soc. 114: 1895 (1992); Meier.
Chem. Int. Ed. Engl. 31: 1008 (1992); Nie
lsen, Nature 365: 566 (1993); Carlsson et al.
Nature 380: 207 (1996), all of which are incorporated by reference herein, for nucleic acid analogs.
log) is included. Other analog nucleic acids are positive (positive).
) Having a main chain (Denpcy et al., Proc. Natl. Acad. Sc)
i. USA 92: 6097 (1995); nonionic backbone (US Pat. No. 538).
6023, 5637684, 560240, 5216141 and 446.
9863); Kiedrowshi et al., Angew. Chem. Intl. Ed
． English 30: 423 (1991); Letsinger et al., J. Am. A
m. Chem. Soc. 110: 4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13: 1597 (199).
4); ACS Symposium, Series 580, Chapters 2 and 3, "Modification of Carbohydrates in Antisense Studies," Ed. Y. S. Sanghui and P.M. Dan
Cook, Mesmaeker et al., Bioorganic & Medici.
nal Chem. Lett. 4: 395 (1994); Jeffs et al.
Biomolecular NMR 34:17 (1994); Tetrahhe.
dron Lett. 37: 743 (1996)) and US Pat. No. 5235.
033 and 5034506 and ACS Symposium, Series 580, Chapters 6 and 7 "Modification of Carbohydrates in Antisense Studies" (Ed.YS.S.
anghui and P. Non-ribose backbone, including Dan Cook). Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (Jenkins et al., Chem. Soc.
． Rev. (1995) 169-176). Some nucleic acid analogs are RAW
Is, C & E News, June 2, 1997, p. 35. Nucleic acid analogs also include "locked nucleic acids". All of these references are expressly incorporated by reference. Such modification of the ribose-phosphate backbone may be done to facilitate the addition of electronic transition moieties, or to increase the stability and half-life of the molecule in a physiological environment. May be done.

As appreciated by those of skill in the art, these nucleic acid analogs are also used in the present invention. Furthermore, a mixture of a naturally occurring nucleic acid and an analog can be prepared, for example, at a site of a conductive oligomer or an accessory site of an electronic transition part, and an analog structure can also be used. Alternatively, mixtures of different nucleic acid analogs as well as mixtures of naturally occurring nucleic acids and analogs can be prepared.

As outlined herein, a nucleic acid may have a specific (or specific) single-stranded structure, a double-stranded structure, or a double-stranded structure and a single-stranded structure. It may include both parts of the double-stranded structure sequence. Nucleic acid includes DNA, genome and cDNA
, RNA, or hybrids, in which the nucleic acid is a base including deoxyribonucleotides and ribonucleotides, and uracil, adenine, thymine, cytosine, guanine, inosine, xanthanine hypoxasanine, isocytosine, isoguanine, and the like. May be a combination of. As used herein, the term "nucleoside" includes modified nucleosides such as nucleotides, nucleosides and nucleoside analogs, amino modified nucleosides and the like. In addition, "nucleoside" includes non-naturally occurring analog structures. Thus, for example, individual units of peptide nucleic acid, each containing a base.
Is here a nucleoside.

In a preferred aspect, the present invention provides a method for detecting a target nucleic acid. By "target nucleic acid" or "target sequence" or grammatical equivalents (or substantially equivalents) is meant a nucleic acid sequence in the single-stranded structure of a nucleic acid. Target sequences include genes, regulatory sequences (or regulatory sequences), genomic DNA, cDNA, mRNA and rRNA.
It may be RNA containing or other part. Nucleic acids can be of any length, with the understanding that longer sequences are more specific. It may be desirable to cleave the sample nucleic acid into fragments of 100-10,000 base pairs, and in some preferred embodiments cleave or split into nucleic acids with fragments of approximately 500 base pairs. As will be appreciated by those in the art, complementary target sequences may take many forms. For example, it may be comprised within a larger nucleic acid sequence, ie in particular in a part of a gene or mRNA, a restriction fragment of a plasmid or genomic DNA.

As will be more fully described below, probes (including primers) are hybridized to a target sequence to determine (or measure) the presence or absence of the target sequence in a sample. Generally speaking, this term is well understood by those skilled in the art.

The target sequence may be composed of different target domains, which may be contiguous (ie, in contact) or separated. For example, ligation chain reaction (LCR, ligation chain reaction)
Method), a first primer may hybridize to a first target domain and a second primer may hybridize to a second target domain. Domains may be contiguous or separated by one or more nucleotides in combination with the use of polymerase and dNTPs, as more fully outlined below.

The terms “first” and “second” refer to the 5′-3 ′ orientation of a target sequence.
It does not mean to give (or to give) the orientation of the array. For example,
Given the 5'-3 'orientation of the complementary target sequence, the first target domain may be located 5'to the second domain or 3'to the second domain. good.

In a preferred embodiment, the target analyte is a protein. As will be appreciated by those in the art, there are numerous target analytes for proteins that may be detected using the present invention. As used herein, “protein” or grammatical equivalents (or substantially equivalent thereto) means proteins, oligopeptides and peptides, including non-naturally occurring amino acids and amino acid analogs and derivatives containing the structure of peptides and Means an analog. This side chain may have an (R) or (S) structure. In a preferred embodiment, the amino acid is (S) or L-structure (or body). As described below, the use of proteins as binding ligands slows down degradation by sample contaminants, so it is preferable to utilize protein analogs.

Suitable protein target analytes include, but are not limited to, (1) immunoglobulins, particularly IgEs, IgGs, and IgMs, and more particularly therapeutically or diagnostically relevant antibodies, including, but not limited to: However, for example, antibodies against human albumin, apolipoproteins (including apolipoprotein E), human chorionic gonadotropin, cortisol, α-fetoprotein, thyroxine, thyroid stimulating hormone (TSH), antithrombin, antibodies against drugs ( Anti-erosion drugs (phenitone, primidone, carbaryzepine, ethosuximide, valproic acid, and phenobarbitol), cardioactive drugs (digoxin, lidocaine, procainamide, and disopyramide), bronchodilators (theophylline), antibiotics (chloramphenicol, Sur Lonamide), antidepressants, immunosuppressants, drugs of abuse (amphetamines, methamphetamines, cannabinoids, cocaine and opium drugs) and antibodies against numerous viruses (including orthomyxoviruses (eg influenza virus)), paramyxoviruses (eg, Trachea syncytium virus, mumps virus, measles virus), adenovirus, rhinovirus, coronavirus,
Reovirus, togavirus (for example, rubella virus), parvovirus, poxvirus (for example, smallpox virus, cowpox virus), enteric virus (for example, poliovirus, Kokusakievirus), hepatitis virus (A, B, Type C), herpes virus (eg, herpes simplex virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus), rotavirus, noouk virus, hantavirus, arenavirus, rhabdovirus (eg, Rabies virus), retroviruses (HIV, HTLV-I and HTL)
V-II), papovaviruses (eg papiromavirus), polyomaviruses, and picomaviruses, etc.), and bacteria (bacillus; Vibrio such as cholera; Escherichia such as enterotoxic E. coli, Shigella such as dysentery; Salmonella). Typhus; Mycobacteria such as tuberculosis, leprosy; Clostridium such as Botrynus, tetanus, Difficile, Perfringen; Cornibacterium such as Diphtheria; Streptococcus, Pseudomonas, Mycobacterium tuberculosis; Staphylococcus such as Aureus; Haemophilus such as influenza; Neisseria such as meninges Flame, phospora; Yersinia, eg Lambria, plague, Pseudomonas, eg Elginosa, Putida; Chlamydia, eg Trachoma; Bordetella, eg Pertussis; Treponema, eg pa A wide range of pathogenic or non-pathogenic prokaryotes, including (), (2) enzymes (and other proteins), including but not limited to, indicators and treatments for heart disease Enzymes including creatine kinase, creatine kinase, lactate dehydrogenase, aspartate aminotransferase, troponin T, myoglobin, fibrinogen, cholesterol,
Triglyceride, thrombin, tissue plasminogen activator (tPA)
Pancreatic disease indicators including amylase, lipase, chymotrypsin and trypsin; liver function enzymes and proteins including cholinesterase, bilirubin and alkaline phosphatase: aldolase, prostatic acid phosphatase, terminal deoxynucleotidyl transferase, and bacterial and viral such as HIV protease Sex enzymes; (3) hormones and cytokines, many of which serve as ligands for cellular receptors, erythropoietin (EPO), thrombopoietin (TPO), interleukins (including IL-1 to IL-17) , Insulin, insulin-like growth factor (including IGF-1 and IGF-2), epidermal growth factor (EGF), transforming growth factor (including TGF-α and TGF-β), human growth hormone, trans Ferrin, epidermal growth factor (EGF), low density lipoprotein, high density lipoprotein, leptin, VEGF, PDGF, ciliary neurotrophic factor, prolactin, adrenocorticotropic hormone (ACTH), calcitonin, human chorionic gonadotropin, Koturisol, estradiol, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), progesterone and testosterone; and (4) other proteins (α-fetoprotein, carcinoembryonic antigen CEA, cancer marker, etc.) including).

In addition, biomolecules for which antibodies can be discovered, or biomolecules for which antibodies can likewise be discovered directly; that is, detection of viral or bacterial cells, therapeutic agents, drugs of abuse, etc., can be performed directly. .

Suitable target analytes include, but are not limited to, labels for breast cancer (C
A15-3, CA549, CA27.29), mucin-like carcinoma associated antigen (MCA), ovarian cancer (CA125), pancreatic cancer (DE-PAN-2), prostate cancer (PSA), CEA and colorectal cancer. And pancreatic cancer (CA19, CA50
, CA 242).

Suitable target analytes include, but are not limited to, aluminum, arsenic,
It contains metal ions including cadmium, selenium, cobalt, copper, chromium, lead, silver and nickel, especially heavy metals and / or toxic metals.

These target analytes include, but are not limited to, blood, lymph, saliva, vaginal and anal secretions, body fluids including urine, stool, sweat, tears, and liver, spleen, bone marrow, lungs. There may be many different sample types of solid tissue, including muscle, brain, etc.

Therefore, the present invention provides a device for the detection of target analytes comprising a solid substrate. As described herein, this solid substrate can be made from a variety of materials and can be constructed in a number of ways, as will be apparent to those skilled in the art. In addition, a single device may be composed of more than one substrate, eg, interfaced (or interfaced with,) in its separate “detection” cassette.
There may be "sample processing" cassettes that are terfaced. The source sample is added to the sample processing cassette and manipulated to prepare the detection sample, which (or the detection sample) is removed from the sample processing cassette and added to the detection cassette. There may be cassettes with additional functionality to which this device fits, eg heating elements (or heating elements, heating elements) placed in contact with the sample cassette to effect a reaction such as PCR. ) May be sufficient. In some cases, a portion of the substrate may be removable; for example, the sample cassette has a removable detection cassette so that the entire sample cassette does not contact the detection device. For example, US Pat. No. 5,603,351 and PCT US96 / 17, which are incorporated herein by reference.
See 116.

The composition of the solid substrate depends on the technique used to make the device, the device usage, the composition of the sample, the analytes to be detected, the size of the wells (or wells) and microchannels, the electronic components. component
) Depending on various factors such as the presence or absence of. Also, in general, the device of the present invention needs to be easily sterilized.

In a preferred embodiment, the solid substrate can be made from a wide variety of materials. Preferred embodiments utilize ceramic components as the solid substrate, as will be appreciated by those skilled in the art, although the devices of the present invention include other materials, as outlined more generally below. These include, but are not limited to, silicon wafers, silicon dioxide, silicon nitride, glass and fused silica, gallium arsenide,
Indium phosphide, aluminum, ceramics, polyimide, quartz, plastics, resins and polymethylmethacrylate, acrylics, polyethylene, polyethylene terephthalate, polycarbonate, polystyrene and other styrene copolymers (or styrene copolymers), polypropylene, polytetrafluoroethylene Containing polymer (or polymer), superalloy, zircaloy, steel (or steel), gold, silver, copper, tungsten, molybdenum, tantalum, kovar (KOV)
AR), Kevlar, Kapton, Mylar, brass, sapphire, etc. When light-based technology (or light-related technology) is required in the sample handling process, high quality glass such as high melting point borosilicate or fused silica is preferred for UV transparency of the glass. obtain. Further, as outlined herein, the inner surface of the device is optionally such that a binding ligand can be attached to it to reduce biocompatibility or nonspecific binding to flow resistance. Various coatings may be applied.

In a preferred embodiment, the solid support is US patent application Ser. No. 09/235081;
09/337086; 09/464490; 09/492013; 09/466
325; 09/460281; 09/460283; 09/387691; 09
/ 438600; 09/506178 and 09/458534, which are incorporated herein by reference in their entirety. In this aspect, the device is formed from layers of green sheets that are laminated and sintered together to form a substantially monolithic structure. The green sheet is a composite material containing inorganic particles of glass, glass ceramics, ceramics or mixtures thereof, dispersed in a polymeric binder and may also contain additives such as plasticizers and dispersants. The green sheet is preferably in sheet form with a thickness of 50-250 microns. Ceramic particles are typically metal oxides such as aluminum oxide or zirconium oxide. Examples of such green sheets containing glass-ceramic particles include E.I. I. DuPont de Nemours and Company (EI Du Pont de Nemours and Comp
There is "AX951" sold by Any. An example of a green sheet containing aluminum oxide particles is "Ferro Alumina" sold by Ferro Crop. The composition of the green sheet is customized by the customer to suit the specific application.
It is also possible to do). The greensheet layers are laminated together and fired to form a substantially monolithic multilayer laminate structure. The production, processing and application of ceramic green sheets is carried out by Richard E. Richard.
E. Mistler's “Tape Casting: The Basic Process fo” that meets the needs of the electronics industry.
r Meeting the Needs of Electronics I
ndustry "Ceramic Bulletin (Ceramic Bulletin)
), Volume 69 (No. 6) 1022-26 (1990) and U.S. Pat. No. 399.
1029 (incorporated herein by reference).

The method of manufacturing a device (such as the devices shown in FIGS. 27-30 as devices 100 and 200) begins by providing a sheet of green sheet, preferably 50-250 microns thick. Sheets of this greensheet are typically cut into desired sizes, such as 6 inches by 6 inches, in conventional processing, although smaller or larger devices may be used as desired. Each greensheet layer is biased () to form the desired structure.
Various techniques such as vias, channels (or channels), cavities, etc. may be used to texture the final multilayer structure.

Various techniques may be used to make the green sheets. For example, portions of the greensheet layer are punched (or punched) to form a bias or groove pattern. This operation is carried out by Pacific Trinetics (
Pacific Trends Corp. ) Model APS-8718 Automated Punching System (Model APS-8718 Automated)
It is performed using a conventional multilayer ceramic punch such as the Punch System). Instead of punching out a portion of the material, groove-like and well-like shapes (fe
A negative image of the desired structure (or negative image, n
The surface of the green sheet may be embossed (or may be embossed or embossed) by pressing the green sheet against an embossed plate having an aggressive image. Further, such work (or surface processing, texturing) may be achieved by a laser device (or laser equipment) having a laser by a system such as LVS-3012 manufactured by Pacific Trinetics.

Then, various kinds of materials may be used, preferably in the form of a thick film paste, for each of the crafted (or surface-treated) green sheet layers. For example,
A metal-containing thick film paste is deposited (or deposited, deposi) on the green sheet layer.
t) to provide an electrically conductive path. Thick film pastes may typically include a desired material such as a metal or a dielectric, in the form of a powder that is dispersed in an organic vehicle (or organic vehicle). The paste is constructed to have the proper viscosity for the desired vapor deposition technique such as screen printing. This organic vehicle contains resin, solvent,
It may contain a surfactant and a flow control agent. The thick film paste may also contain a small amount of flux such as glass frit so as to be suitable for sintering. This thick film technology is described in J. D. Provence, “Performance Review of Thick Fi
lm Materials ”Isolation / Circuit (April 1977), and Morton. L. Morton L. Topfer, “Thick Film Microelectronics, Assembly, Design, Applications (Thick Film Microelec)
tronics, February, Design and Appli
"Cations)" (1977) pp. 41-59 (incorporated herein by reference).

By adjusting the amount of organic vehicle present in the thick film paste, the porosity of the thick film produced can be adjusted. In particular, the porosity of the thick film can be increased by increasing the proportion of organic vehicle in the thick film paste. Similarly, increasing the proportion of organic binder can increase the porosity of the greensheet layer. Another way to increase the porosity of thick film and greensheet layers is to disperse them in an organic vehicle, an organic binder, in another organic phase that is not soluble in the organic vehicle. Polymer microparticles (or microspheres, m
Microspheres) can advantageously be used for this purpose.

To add an electrically conductive pathway, the thick film paste is typically silver, platinum,
Includes metal particles such as palladium, gold, copper, tungsten, nickel, tin, or alloys thereof. Silver paste is preferred. An example of a suitable silver paste is E.I. Eye. Silver conductive composition No. 7 sold by DuPont de Nemours and Company. 7025 and 7713.

The thick film paste is preferably used for the green sheet layer by screen printing. In the screen printing process, the thick film paste is pressed through a patterned silk screen to deposit in a pattern corresponding to the greensheet layer. Typically, the silkscreen pattern is photolithographically formed in the mask by exposure. In this way, conductive traces are applied to the surface of the greensheet layer. The bias present in the green sheet layer may be filled with a thick film-filling paste. When filled with a thick film paste containing an electrically conductive material, this bias acts to provide an electrical connection between the layers.

After the desired structure is formed in each layer of the green sheet, it is preferable that the adhesive layer is applied to either surface of the green sheet. Preferably the adhesive is a room temperature adhesive. Since such a room temperature adhesive has a glass transition point below room temperature, that is, below about 20 ° C., it is possible to bond substrates at room temperature. Moreover, rather than undergoing a chemical change, or chemically reacting with a component of the substrate, or dissolving the component of the substrate, such room temperature adhesives penetrate the substrate surface, thereby causing Are joined together. Room temperature adhesives are sometimes also referred to as "pressure sensitive adhesives". Suitable room temperature adhesives are typically supplied as water-based emulsions and are available from Rohm and Haas and Air Products. For example, "Flexcryl" from Air Products
) 1653 "was found to work well.

The room temperature adhesive is applied (or applied) to the green sheet by conventional coating techniques. Depending on the coating technique used and the viscosity and solids of the starting materials, it is often desirable to dilute the supplied pressure sensitive adhesive with water to facilitate coating. After coating, the room temperature adhesive is dried.
The dry thickness of the room temperature adhesive film is preferably 1 to 10 microns and the thickness should be uniform over the entire surface of the greensheet. Film thicknesses above 15 microns are not desirable. With such thick films of adhesive, voids or delaminations occur during combustion due to the large amount of organic material that must be removed. When dried, a film of about 0.5 micron or less is too thin because of poor adhesion between layers.

Among conventional coating techniques, spin coating and spraying are
This is the preferred method. When using spin coating, it is preferable to add 1 g of deionized water to 10 g of "Flexcryl (1653)". When using the spray method, higher dilution levels are preferred for ease of atomization. Furthermore, when spraying the room temperature adhesive, a high temperature, for example 60-70, is used so that the material dries immediately when the adhesive is deposited on the green sheet.
It is preferable to keep the green sheet at ℃. Flash drying results in a more uniform and homogeneous adhesive film.

After applying the room temperature adhesive to the greensheet layer, the films are laminated to form a multilayer greensheet structure. The layers are preferably stacked in an alignment mold (or alignment die) to maintain the desired alignment between the structures of each layer. When using this alignment mold, alignment holes must be added to each greensheet layer. When using room temperature adhesives, typically only the overlay process is sufficient to bond the greensheet layers together. In other words, little or no pressure is required to bond the layers together. However, in order to make the bonding of the layers more secure, it is preferred to stack the layers and then laminate them together.

The laminating process involves applying pressure to the stacked layers. For example, in the conventional laminating process, about 1000-1500 psi 1
Axial pressure is applied to the stacked greensheet layers, then 7
At elevated temperature, such as 0 ° C., an equilibrium pressure (or isobar) of about 3000-5000 psi is applied for about 10-15 minutes. There is no need to use an adhesive to bond the greensheet layers when using the conventional laminating process.

However, for good control over the dimensions (or dimensions) of structures such as internal or external cavities and channels, 2500
Pressures below psi are preferred. Even lower pressures are more preferred to allow the formation of larger structures such as cavities and channels. For example, using a lamination pressure of 2500 psi limits the size of well-formed internal cavities and channels to approximately 20 microns or less. Therefore, 1
Pressures less than 000 psi are more preferred as they allow some dimensional control to form structures with sizes greater than about 100 microns. Three
Pressures below 00 psi are even more preferred as they allow some dimensional control to form structures with sizes greater than 250 microns.
A pressure of less than 100 psi, referred to herein as a "near zero pressure", is most preferred, as there is little constraint on such internal and external cavities and channel sizes that can be formed in the multilayer structure at such pressures. .

In the laminating process, pressure is preferably applied by uniaxial pressing.

Alternatively, a pressure below 100 psi may be applied manually. Many devices may be present on each sheet, such as semiconductor device assembly.

Thus, after lamination, the multi-layer structure may be diced using conventional green sheet dicing or cutting equipment to separate the individual devices. Due to the high level of peeling resistance and shear resistance imparted by the room temperature adhesive, delamination from the edges rarely occurs during the dicing process. If, after dicing, several layers separate near the edge, they can be easily re-laminated by manually applying pressure to the affected edge without affecting the rest of the device.

The final processing step is firing (or burning) to convert the laminated multi-layer greensheet structure from the “green state” to form the completed substantially monolithic multi-layer laminated structure. That's right). This firing process occurs in two important stages as the temperature increases. The first important step is 250-5
A binder burnout (or burnout) stage that occurs in the temperature range of 00 ° C.,
During this time, the binder in the greensheet layer and other organic materials such as organic components in the applied thick film paste are removed from the structure.

In the next important step, the sintering step that occurs at higher temperatures, the inorganic particles are sintered together so that the multilayer structure becomes compact and substantially monolithic. The sintering temperature used depends on the nature of the inorganic particles present in the greensheet. For many types of ceramics, the appropriate sintering temperature depends on the material, but 9
It is in the range of 50 to 1600 ° C. For example, a sintering temperature of 1400 to 1600 ° C. is typical for a green sheet containing aluminum oxide. Other ceramic materials such as silicon nitride, aluminum nitride and silicon carbide,
Further higher sintering temperatures are required, i.e. 1700-2200 ° C. For green sheets with glass-ceramic particles, sintering temperatures of 750-950 ° C are common. Glass particles generally require sintering temperatures in the range of only about 350-700 ° C. Finally, the metal particles, depending on the metal, generally range from 550 to 1
A sintering temperature in the range of 700 ° C. is required.

Typically, the device is fired for about 4 hours to about 12 hours, depending on the material. Generally, firing should be continued for a sufficient period of time to remove the organic material from the structure and to completely sinter the inorganic particles. In particular, the polymer
It is present as a binder in the green sheet and in the room temperature adhesive. Sufficient temperature and time should be used to decompose these polymers and to remove them from the multilayer structure.

Typically, the multilayer structure will undergo a volume reduction during the firing process. During the binder burn-out stage, a volume loss of about 0.5% to 1.5% is usually observed. At higher temperatures, further volume reduction of about 14% to 17% is generally observed during the sintering stage.

On the other hand, the volume change due to firing can be controlled. In particular, (1) particle size; (2) organic components (eg, binders) removed during the combustion process to match volume changes in two materials, such as green sheets and thick film pastes. The percentages (or proportions) of should be combined. In addition, the volume changes need not be precisely matched, but any mismatch will result in internal stress in the device.
However, the symmetrical processing of placing the same material or structure on both sides of the device can absorb the material shrinkage mismatch to some extent. Excessive misalignment in either sintering temperature or volume change can lead to defects or damage to some or all devices. For example, the device may separate into its individual layers, or may bend or twist.

As described above, the different materials added to the green sheet layer are preferably co-fired with the green sheet layer. Such dissimilar materials may be added as a thick film paste, or as another greensheet layer, or later in the post-sintering assembly process. The advantage of co-firing is that the added material is sintered into the greensheet layer and is integrated into a substantially monolithic microfluidic device (Mi
crofluidic device). However, to be co-fireable, the added material should have a sintering temperature and volume change due to firing that is consistent with the greensheet layer. The sintering temperature is mainly material dependent,
Therefore, matching the sintering temperatures simply requires proper selection of materials. For example, silver is the preferred metal for providing an electrically conductive pathway, but if the greensheet layer contains alumina particles that require sintering temperatures in the range of 1400-1600 ° C, silver Due to its relatively low melting point (961 ° C.), other metals such as platinum must be used.

Alternatively, the addition of another substrate or the joining of the two post-sintered pieces can be done using various bonding techniques. For example, two "halves" of the device can be glued or fused together. For example, specific detection platforms and reagent mixtures such as high temperature labile hydrogels or biological components may be combined with two "
You can even sandwich between "half" ones. Alternatively, a ceramic device containing open channels or depressions (or wells) may be made and additional substrates or materials placed in the device after which they are sealed with other materials.

The substrate consists of an array of capture binding ligands. As will be appreciated by those in the art, any number of different capture binding ligands or probes (when the target analyte is a nucleic acid) can be used and can be used in a wide variety of formats. Preferred embodiments utilize microelectrode arrays or hydrogel arrays. These are known techniques, for example, U.S. patent application Ser. Nos. 09 / 458,553; 09/458501; 09.
/ 572187; 09/495992; 09/344217; WO00 / 311
48; 09/439889; 09/438209; 09/344620; PCT
US00 / 17422; 09/478727, all of which
Explicitly incorporated by reference in its entirety.

In some aspects of the invention, the probe is an oligonucleotide probe having a sequence comprising from about 10 to about 30 nucleotide residues, wherein the probe is a conjugated polymer in contact with a microelectrode. Alternatively, it is attached to the copolymer. Conjugated polymers or copolymers used for probe attachment include polypyrrole, polythiophene, polyaniline, polyfuran, polypyridine, polycarbazole, polyphenylene, poly (phenylene vinylene), polyfluorene, polyindole, their derivatives, and their copolymers. , And combinations thereof, but are not limited thereto. In a preferred embodiment,
The oligonucleotide probe is attached to the microelectrode through a neutral polypyrrole matrix.

In another aspect of the invention, the probe is an oligonucleotide probe having a sequence comprising from about 10 to about 30 nucleotide residues, the probe comprising a polyacrylamide gel contacting a microelectrode. It is attached to the pad.

As is known in the art, samples are prepared by various methods and applied to the devices of the invention. The preferred embodiment is directed to the use of a system that does not require the use of ECA labeling and is described below with reference to Figures 1-14 and Examples 1-6.

In this aspect, the device and method of the present invention provides for hybridization (or hybridization) between an oligonucleotide probe, now immobilized on a microelectrode, and a target nucleic acid molecule contained in a biological sample. It demonstrates using. The phosphate groups of nucleic acids are negatively charged at all biologically relevant pH values.
In this way, the nucleic acid duplex has a high negative charge density. Following electrical perturbation of the nucleic acid, strong interactions such as intercalation or binding of metal ions to the nucleic acid occur. These interactions depend on the structure and charge density of the nucleic acid. The result of this molecular interaction is a change in AC impedance, since the structure and electrical properties of nucleic acid molecules (such as probes) change as the probe hybridizes to the appropriate target molecule. This change is used in the devices and methods of the invention to discriminate between "perfect" and imperfect or "mismatch" hybridizations between immobilized oligonucleotide probes and target nucleic acid molecules. .

In one aspect, the device of the invention comprises a support substrate, a plurality of microelectrodes having a probe immobilized thereon, in contact with the support substrate, at least one in contact with the support substrate.
One counter electrode, an electrical impedance generating means at each microelectrode, a means for detecting impedance change at each microelectrode in the presence or absence of a target molecule, and an electrolyte solution in contact with the plurality of microelectrodes.

In another aspect, the device of the invention comprises a support substrate, a plurality of microelectrodes in contact with the support substrate, a plurality of polyacrylamide gel pads in contact with the microelectrodes and having probes immobilized thereon. , At least one counter electrode in contact with the supporting substrate, electric impedance generating means in each microelectrode, means for detecting impedance change in each microelectrode in the presence or absence of a target molecule, and the plurality of microelectrodes With an electrolyte solution in contact with.

In one embodiment, the device is a microarray (or array) comprising at least five microelectrodes having oligonucleotide probes attached to them on a single substrate. Alternatively, the arrayed oligonucleotide probes are attached to a polyacrylamide gel pad in contact with the microelectrodes of the device of the invention. Most preferably, an oligonucleotide having a particular nucleotide sequence,
Alternatively, groups of oligonucleotides with related (eg, overlapping) nucleotide sequences are immobilized on each of the plurality of microelectrodes. In a further preferred embodiment, the nucleotide sequence of the oligonucleotide immobilized on each microelectrode and the identity and correspondence between the particular microelectrode and the nucleotide sequence of the oligonucleotide immobilized on it are known.

In a preferred embodiment, the probe is an oligonucleotide containing from about 10 to about 100 nucleotide residues, more preferably an oligonucleotide containing from about 10 to about 50 nucleotide residues, and most preferably about 15 nucleotides. Is an oligonucleotide containing about 30 nucleotide residues. In another aspect, the probe is about 1
A nucleic acid containing 0 to about 5000 base pairs, more preferably about 00 to about 1000.
Is a nucleic acid containing about 200 to about 500 base pairs. In a more preferred embodiment, the immobilized probe is a peptide containing residues of about 5 to about 500 amino acids.

In a preferred embodiment of the device of the present invention, the substrate consists of silicon. In another aspect, the substrate is made from materials including, but not limited to, glass, plastic, rubber, fibers, or ceramics. The microelectrode is embedded in the substrate or arranged so as to be in contact with the substrate.

In a preferred embodiment, the microelectrode is a metal such as, but not limited to, gold, silver, platinum, titanium or copper, in solid or porous form, preferably a foil or film. It is made of a material containing metal oxide, metal oxide, metal nitride, metal carbide or carbon. In certain preferred embodiments, the probe includes, but is not limited to, polypyrrole, polythiophene, polyaniline, polyfuran, polypyridine, polycarbazole, polyphenylene, poly (phenylene vinylene), polyfluorene, polyindole, their derivatives, and their derivatives. It is attached to conjugated polymers or copolymers, including copolymers, and combinations thereof. In another aspect, the probe is attached to a polyacrylamide gel pad in contact with the microelectrode.

The substrate of the present invention has a surface area of 0.01 mm ^{2 to 5} cm ² and contains 1 to 1 × 10 ⁸ microelectrodes. In one aspect, the substrate has a surface area of 100 mm ² and comprises 10 ⁴ microelectrodes, each microelectrode having an oligonucleotide having a particular sequence immobilized thereon. In another aspect, the substrate has a surface area of 100 mm ² and comprises 10 ⁴ microelectrodes, each microelectrode having a polyacrylamide gel gel having oligonucleotides having a specific sequence immobilized thereon. It is in contact with the pad. In a preferred embodiment, the microelectrodes are arranged on the substrate so as to be separated by a distance of 0.05 mm to 0.5 mm. Most preferably, the microelectrodes are regularly spaced on the solid substrate so that the spacing between the electrodes is constant.

In some aspects of the invention, the microelectrodes project from the surface of the substrate, the projections extending from the substrate surface by 1 × 10 ⁻⁸ to 1 × 10 ⁻⁵ cm.
In another aspect, the microelectrodes consist of a flat disk of conductive material embedded in the substrate and exposed at the substrate surface, the substrate acting as an insulator in the spaces between the microelectrodes.

In a preferred embodiment of the invention, the microelectrode comprises a gold conductor and a glass insulator. In other embodiments, the microelectrode comprises a solid or porous film of silver, platinum, titanium, copper, or a conductive substrate such as metal oxide, metal nitride, metal carbide or carbon (graphite). In another aspect, the microelectrode comprises a substrate and / or an insulating substrate such as glass, silicon, plastic, rubber, fabric (or textile), or ceramic. The microelectrode of the present invention has an exposed conductive surface of 0.01 mm ^{2 to 5} cm ² . In a preferred embodiment, the exposed conductive material is 100-10000 mm ² . One embodiment of the present invention is shown in FIG. 1A, in which the microelectrode comprises a glass capillary tube 1 and an ultrafine platinum wire 2 in which a transition (or communication) wire 3 is located. It has been soldered 6. The transition wire 3 is then soldered 6 to the hook-up wire 4, the wire 4 protruding from the epoxy plug 5 which seals the capillary tube. In one aspect of the invention, polyacrylamide gel material 7 is filled into recesses (or recesses) formed in the exposed surface of platinum wire 2 by etching.

In some embodiments, the oligonucleotide probe is immobilized on the microelectrode of the device of the invention using a neutral layer between the oligonucleotide and the microelectrode. In a preferred embodiment, this layer consists of neutral polypyrrole. In another embodiment, this layer is made of polythiophene, polyaniline, polyfuran, polypyridine, polycarbazole, polyphenylene, poly (phenylene vinylene), polyfluorene, polyindole, their derivatives, their copolymers, and combinations thereof. Including various substances. This layer is preferably at least about 0.
． 001 to 50 mm thick, more preferably at least about 0.01 to 10 mm.
mm thickness, and most preferably at least about 0.5 mm thickness.

In another aspect, the oligonucleotide probe is immobilized on a polyacrylamide gel pad that is in contact with the microelectrodes of the device of the invention. In a preferred embodiment, the polyacrylamide gel pad is embedded in a recess etched in the microelectrode surface. The polyacrylamide gel pad is preferably at least about 0.1-30 mm thick, more preferably at least about 0.5-10 mm thick, and most preferably about 0.5 mm thick.

The device of the present invention comprises at least one counter electrode. In a preferred embodiment of the invention, the counter electrode comprises a conductive material and has an exposed surface that is significantly larger than the surface of the individual microelectrodes. In a preferred embodiment, the counter electrode comprises platinum. In another aspect, the counter electrode is a solid or porous membrane of silver, gold, platinum, titanium, copper, or a metal oxide,
Includes metal nitride, metal carbide or carbon.

In another aspect of the invention, the device comprises at least one reference electrode. This reference electrode is used in the assay if further quantification of the target molecule is desired.
In a preferred embodiment, the reference electrode comprises a silver / silver chloride electrode. In another aspect, the reference electrode comprises a solid or porous film of gold, platinum, titanium or copper, or a metal oxide, metal nitride, metal carbide or carbon. The electrolyte solution that constitutes the device of the present invention is an electrolyte solution that is ionically conductive and contains at least one salt containing polymerized cations or metal cations that is capable of reacting with biomolecules, most preferably nucleic acids or peptides. Most preferably, the salt further comprises an anion that exhibits low specific adsorption to the surface of the microelectrode, thereby reducing noise during detection of molecular interactions between probe and target molecule.

In a preferred aspect of the invention, the electrolyte solution used to detect nucleic acid hybridization comprises 0.1 M LiClO ₄ . This electrolyte is Cl
O ₄ ⁻ is not specifically adsorbed on the surface of the electrode, and thus generates low background noise, which is preferable. In addition, Li ⁺ has the
It is preferred because it promotes the intercalation of Li ⁺ cations into the heavy chain and has a smaller diffusion resistance. However, in other embodiments, the AC impedance is measured in a hybridization buffer such as 1 × SSC,
Track the molecular interactions between the probe and the target molecule.

In the device of the present invention, the means for generating electrical impedance at each microelectrode was a 1260 impedance / gain-phase analyzer with a model 1287 electrochemical interface (Solartron, Houston, Tex.). Can be done using. Other means of measuring electrical impedance include, but are not limited to, a transient method using AC signal perturbation superimposed on a DC potential applied to an electrochemical cell such as an AC bridge and AC voltammetry.
thod) is included. Measurements can be made at certain frequencies determined by scanning the frequencies to ascertain the frequency that produces the maximum signal. Means for detecting impedance changes at each microelectrode in the presence or absence of target molecule are:
It can be done using one of the devices described above.

In yet another aspect of the invention, the device further comprises a plurality of wells, each well comprising at least one microelectrode and at least one counter electrode. The term "recess" is used herein in the conventional sense and is used to describe the portion of the substrate that contains the microelectrode and at least one counter electrode in a defined volume.

The present invention provides devices 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 includes any detection method that does not use or require the reporter labeled moiety to obtain a measurable signal. The impedance is sensitive enough to allow mismatched hybridization between the oligonucleotide probe and the target molecule to be detected. The "discriminating property" of the hybridization reaction.
It is measured at various frequencies to obtain a (signature). The methods of the invention disclosed herein provide for probe molecules bound to defined regions of an ordered array (usually referred to as a "biochip") and samples that are allowed to interact with the probe molecules. It is useful for electrically detecting molecular interactions with target molecules therein. By arraying the microelectrodes with attached individual probe molecules on the biochip, parallel measurements of many probes can be performed in a single assay.

The invention further provides devices and methods for detecting cation interactions associated with peptide binding using AC impedance, but labeled with a reporter to obtain a measurable signal. Not used. This method
To electrically detect molecular interactions between a probe molecule bound to a defined region of an ordered peptide array and a target molecule in a sample that is allowed to interact with the probe molecule Used for. By arraying the microelectrodes with attached individual probe molecules on the biochip, parallel measurements of many probes can be performed in a single assay.

Furthermore, the devices and methods of the present invention can be adapted for use with arrays of substances capable of participating in molecular interactions with cations, most preferably lithium cations, which can be (or can be distinguished). Such interactions include ligand-receptor interactions, enzyme-inhibitor interactions and antigen-antibody interactions.

An important advantage of the devices and methods of the invention is that they do not rely on labeling the target molecule. By eliminating the labeling step, the cost of the assay is simplified and reduced, which facilitates electrical detection and is cost effective to use. Moreover, the target molecule does not need to be labeled, thus expanding the range of assays in which the methods of the invention can be used.
For example, the present invention provides for sensitive and high-resolution measurement of RNA concentration in gene expression studies without the need to label chemically labile RNA or the need to convert RNA into cDNA. It is possible. The method of the present invention may allow new types of assays to be developed.

In another aspect, the invention is based on the use of ECA labels and detection on hydrogel arrays. The device and method of the present invention include an oligonucleotide probe immobilized on a polymer hydrogel placed in contact with a microelectrode (hydrogel porous microelectrode) and an electrochemically labeled target contained in a biological sample. Hybridization between nucleic acid molecules will be used to explain.

In a preferred embodiment, the device of the present invention comprises a support substrate, a plurality of microelectrodes in contact with the support substrate, a plurality of polyacrylamide gels in contact with the microelectrodes and having probes immobilized thereon. A pad, at least one counter electrode in contact with a support substrate, means for generating an electrical signal at each microelectrode, each microelectrode in the presence or absence of a target molecule labeled with an electrochemically active reporter Means for detecting changes in the electrical signal of the electrolyte, and an electrolyte solution in contact with the plurality of microelectrodes, the polymer hydrogel pad, and the counter electrode. The polymer hydrogel is made from a hydrophilic polymeric material including, but not limited to, polyacrylamide, agarose gel, polyethylene glycol, cells and sol-gel.

In one aspect, the device is a microarray comprising at least 10 ³ hydrogel porous microelectrodes having oligonucleotide probes attached thereto. Most preferably, an oligonucleotide having a particular nucleotide sequence, or a group of oligonucleotides having related (eg, overlapping) nucleotide sequences, is immobilized on each of a plurality of hydrogel porous microelectrodes. In a more preferred embodiment, the nucleotide sequence of the oligonucleotide immobilized on each hydrogel porous microelectrode and the identity and correspondence between the particular hydrogel porous microelectrode and the nucleotide sequence of the oligonucleotide immobilized thereon. I understand.

In a preferred embodiment, the probe is from about 10 to about 100 nucleotide residues, more preferably from about 10 to about 50 nucleotide residues, most preferably from about 15 to about 30.
Is an oligonucleotide containing the nucleotide residue of. In another embodiment, the probe is a nucleic acid comprising about 10 to about 5000 base pairs, more preferably about 100 to about 1000 base pairs, and most preferably about 200 to about 500 base pairs. In a more preferred embodiment, the immobilized probe is a peptide containing about 5 to about 500 amino acid residues.

In a preferred embodiment of the device of the present invention, the substrate consists of silicon. In another aspect, the substrate is glass, plastic, rubber, fabric (or textile),
Alternatively, it is made of materials including but not limited to ceramics. The hydrogel porous microelectrode is embedded in the substrate or placed in contact with the substrate.

In a preferred embodiment, the microelectrodes include, but are not limited to, gold, silver, platinum, in solid or porous form, preferably foils or films.
It is made from a material containing a metal such as titanium or copper, a metal oxide, a metal nitride, a metal carbide or carbon. In the device of the invention, the probe is attached to a polyacrylamide gel pad that is placed in contact with the microelectrode.

The substrate of the present invention comprises 1-1 × 10 ⁸ hydrogel porous microelectrodes,
It has a surface area of 0.01 mm ^{2 to 5} cm ² . In one aspect, the substrate is 1
It has a surface area of 0000 mm ² and contains 10 ⁴ hydrogel porous microelectrodes, each microelectrode having an oligonucleotide with a specific sequence immobilized thereon. In a preferred embodiment, the hydrogel porous microelectrode has a density of 0.
They are arranged on the substrate so as to be separated by a distance of 05 mm to 0.5 mm. Most preferably, the hydrogel porous microelectrodes are regularly spaced on the solid substrate such that the spacing between the electrodes is constant.

In some example embodiments of the invention, the hydrogel porous microelectrodes are
It projects from the surface of the substrate, and the projection is 5 × 10 ⁻⁸ to 1 × 1 from the surface of the substrate.
And over a range of 0 ^-5 cm. In another embodiment, the hydrogel porous microelectrode has a flat disk of electrically conductive material embedded in the substrate and exposed at the surface of the substrate, the substrate being insulated in the space between the hydrogel porous microelectrodes. It functions as a body.

[0132]   In a preferred embodiment of the invention, the microelectrodes are gold conductors and glass insulation.
Have a body. In other example embodiments, the microelectrodes are silver, platinum, titanium or
Copper solid film or porous film or metal oxide, metal nitride, metal carbon
It has a conductive substrate such as a compound or carbon. In another example embodiment, a microelectrode
Is made of glass, silicone, plastic, rubber, fibers or ceramics.
It has a limbal material and / or a substrate. The microelectrode of the present invention is 0.01 mm ^Two ~ 0.5 cm^TwoHas an exposed conductive surface in the range of. In a preferred embodiment
The exposed conductive material is 100 to 160000 mm^TwoIs the range.

One exemplary embodiment of the present invention is shown in FIG. 1A, in which the microelectrodes are
It has a glass capillary tube 1 and contains an ultrafine platinum wire 2 to which a transition wire 3 is soldered. The transition wire 3 is then soldered 6 to the hook-up wire 4 and protrudes from the epoxy plug 5 which seals the capillary tube. In one embodiment of the present invention, the polyacrylamide gel material 7 is filled in the recesses formed by etching the exposed surface of the platinum wire 2. In a preferred embodiment, the polyacrylamide gel pad is embedded in a recess formed by etching the surface of the microelectrode. The polymeric hydrogel pad is preferably at least about 0.1-30 mm thick, more preferably at least about 0.5-10 mm thick, and most preferably about 0.5 mm thick. . In one exemplary embodiment of the hydrogel porous microelectrode of the present invention, the oligonucleotide probe is immobilized on a polyacrylamide gel pad.

The device of the present invention has at least one counter electrode. In a preferred embodiment of the present invention, the counter electrode comprises a conductive material having an exposed surface area significantly greater than the surface area of the individual microelectrodes. In a preferred embodiment, the counter electrode has platinum. In another embodiment, the counter electrode is made of silver, gold, platinum, titanium, copper solid film or porous film or metal oxide, metal nitride, metal carbide or carbon.

In another exemplary aspect of the invention, the device has at least one reference electrode. This reference electrode is used in the assay if further quantification of the target molecule is desired. In a preferred embodiment, the reference electrode comprises a silver / silver chloride electrode. In another embodiment, the reference electrode comprises gold, platinum, titanium, copper solid or porous film or metal oxide, metal nitride, metal carbide or carbon.

In yet another exemplary embodiment of the present invention, the device further comprises a plurality of depressions, each depression having at least one hydrogel porous microelectrode and at least one.
It has two counter electrodes. The term "well" is used herein in a general sense and is part of the substrate that houses the microelectrode and at least one counter electrode in a given space. It is used to indicate the part where

The method of the invention detects intermolecular interactions between probe molecules bound to hydrogel porous microelectrodes and electrochemically labeled target molecules. Electrochemically labeled target molecules useful in the methods of the invention are prepared by labeling a suitable target molecule with an electrochemically specific redox reporter that does not interfere with the intermolecular interaction being detected. You may. In a preferred embodiment of the method of the invention, the target molecule is labeled with a group of electrochemical reporters having a transition metal complex, most preferably a transition metal complex containing a transition metal ion such as ruthenium, cobalt, iron or osmium. To be done.

In another exemplary embodiment of the method of the present invention, the target molecule may be labeled with, but not limited to, an electrochemically active moiety as follows.

Redox moieties useful for aqueous saturated calomel reference electrodes include transition metal complexes, 1,4-benzoquinone, ferrocene, tetracyanoquinodimethane, N, N, N
Examples include, but are not limited to, ', N'-tetramethyl-p-phenylenediamine or tetrathiafulvalene.

Redox moieties useful for Ag / AgCl reference electrodes include 9-aminoacridine, acridine orange, aclarubicin, daunomycin, doxorubicin, pirarubicin, ethidium bromide, ethidium monoazide, chlorotetracycline, tetracycline, minocycline, Hoechst 33258. , Hoechst 33342, 7-aminoactinomycin D, chromomycin A ₃ , mithramycin A, vinblastine, rifampicin, Os (bipyridine) ₂ (dipyridophenazine) ₂ ⁺ , Co (bipyridine) ₃ ³⁺ , or Fe-. Includes bleomycin.

The electrochemically active portion of the targeting molecule labeled with the electrochemically active reporter of the method of the present invention is a targeting molecule, optionally via a linker having a length of about 10 to about 20 angstroms. It is preferably bound to a molecule. The linker may be an organic moiety such as a hydrocarbon chain (CH ₂ ) _n , or may be an ether, ester, carboxamide, or thioether moiety or a combination thereof. This linker is a siloxane (O-Si-O
) Etc. may be an inorganic part. The length of the linker is chosen so that the electrochemically active moiety does not interfere with the intermolecular interactions that are detected.

Electrochemical contact is advantageously made with an electrolyte solution in contact with each of the hydrogel porous microelectrodes of the present invention. Electrolyte solutions useful in the devices and methods of the invention include electrolyte solutions at either physiologically relevant ionic strength (corresponding to about 0.15 M NaCl) and neutral pH. Examples of electrolyte solutions useful in the devices and methods of the present invention include, but are not limited to, phosphate buffered saline, HEPES buffered saline, and sodium bicarbonate buffered saline.

In an exemplary embodiment, the invention provides an apparatus and method for detecting intermolecular interactions by detecting changes in AC impedance. Impedance is measured at various frequencies to provide a "signature" of the hybridization reaction that is sufficiently sensitive to allow mismatch hybridization between the target molecule to be detected and the oligonucleotide probe. The methods of the invention disclosed herein include probe molecules that bind to defined regions of an ordered array (usually referred to as a "biochip array") array;
It is useful for electrochemically detecting intermolecular interactions between a probe molecule and an electrochemically labeled target molecule in a sample that is allowed to interact. By arranging the microelectrodes in which individual probe molecules are attached on the biochip, it is possible to perform parallel measurement of many probes in a single assay.

In one exemplary embodiment of the device of the present invention, the means for generating electrical impedance at each microelectrode is 1260 impedance / gain-equipped with a model 1287 electrochemical interface (Soratron, Houston, TX). This is done using a phase analyzer. Another means of measuring electrical impedance is A superposed on a DC potential applied to an electrochemical cell such as an AC bridge and AC voltammetry.
Includes, but is not limited to, transient methods using C signal perturbation.
Measurements can be made at a particular frequency determined by scanning the frequency to identify the frequency that produces the maximum signal. The means for detecting the impedance change at each microelectrode in the presence or absence of the electrochemically labeled target molecule is performed using any one of the above-mentioned devices.

In another example embodiment of the present invention, without limitation, cyclic voltammetry, stripping voltammetry, pulse voltammetry, square wave voltammetry, AC voltammetry, hydrodynamic modulation voltammetry. , Probe molecules and electrochemically labeled target molecules using other electrical and / or electrochemical methods including The intermolecular interaction between and can be detected. In these example embodiments, the electrical signal is the flow of current corresponding to the applied voltage at the redox potential of the electrochemical label.

The present invention provides for single nucleotide polymorphisms (S
An apparatus and method for detecting NP) is also provided.

The device of the present invention is particularly useful for analyzing target nucleic acids for the diagnosis of infectious and genetic disorders. The target nucleic acid is generally the part of the gene that has a known nucleotide sequence that is associated with infectious disease and genetic disorders. More specifically, the disease is caused by a single nucleotide mutation (or point mutation). The device includes a nucleic acid oligonucleotide sequence specific for the target gene, as well as a specific one in the target sequence adjacent to the hybridization site of at least one probe in the oligonucleotide sequence (referred to as the “3 ′ offset method”). To detect and measure the identity of two bases, or ((3
Means for including the 3'residue of at least one oligonucleotide probe in the sequence (referred to as the "inclusion method") is incorporated.

The present invention provides a sequence of oligonucleotide primers or probes immobilized on the surface defining the first electrode. Each oligonucleotide sequence at each address in the sequence is known and at least one oligonucleotide in the oligonucleotide sequence is complementary to a portion of the sequence in the nucleic acid of the sample to be assayed. preferable. The sequence of at least one oligonucleotide is
Most preferably, it is chosen to extend to a position immediately adjacent to the nucleotide position in the sample nucleic acid to be investigated (eg to be examined for mutations or genetic polymorphisms). Alternatively, the oligonucleotides are selected to contain sites of mutations or genetic polymorphisms; this latter example embodiment ensures that at least one oligonucleotide in the sequence hybridizes to the nucleic acid in the sample. In order to make it possible, it is generally preferred to provide a multiplicity of oligonucleotides which comprises one of the possible nucleotides at the polymorphic position. The hybridization and extension reactions are carried out in the reaction chamber and in the hybridization buffer for a period of time at a temperature that causes hybridization between the nucleic acid in the sample and the oligonucleotide in the sequence that is complementary thereto.

In one embodiment, the device of the present invention comprises a support substrate, a plurality of first electrodes (or an array of microelectrodes) in contact with the support substrate to which the probe is immobilized, at least one counter electrode and a case. By means of a reference electrode and a plurality of microelectrodes, an counter electrode and an electrolyte solution in contact with the reference electrode.

In another exemplary embodiment, the device of the present invention comprises a support substrate, a plurality of first electrodes (or an array of microelectrodes) in contact with the support substrate, in contact with the microelectrodes and having probes fixed thereto. It has a plurality of polyacrylamide gel pads, at least one counter electrode and optionally a reference electrode, and a plurality of microelectrodes, an electrolyte solution in contact with the counter electrode and the reference electrode. In a preferred embodiment of the device of the present invention, the substrate is composed of silicon. In other example embodiments, the substrate is prepared from materials including, but not limited to, glass, plastic, rubber, fibers, or ceramics.

The electrode having the first surface to which the oligonucleotide or its sequence is attached is formed from at least one of the following materials; metals such as silver, gold, platinum, copper, etc., and electricity of those metals. Conductive alloys; conductive metal oxides such as indium oxide, indium tin oxide, zinc oxide; and other conductive materials such as carbon black and conductive epoxy.

In a preferred embodiment, the microelectrodes include, but are not limited to, metals, metals such as gold, silver, platinum, titanium or copper in solid or porous form, more preferably in the form of foils or films. It is formed from a substrate containing oxide, metal nitride, metal carbide or carbon. In certain preferred embodiments, the probe includes, but is not limited to, polypyrrole, polythiophene, polyaniline, polyfuran, polypyridine, polycarbazole, polyphenylene, poly (phenylenevinylene), polyfluorene, polyindole, their derivatives, copolyenes. Attached to conjugated polymers or copolymers, including coalesces, and combinations thereof. In another example embodiment, the probe is attached to a polyacrylamide gel pad in contact with the microelectrode.

The substrate of the present invention has a surface area of 0.01 mm ^{2 to 5} cm ² and has 1 to 1 × 10 ⁸ microelectrodes. In one exemplary embodiment, the substrate has a surface area of 100 mm ² and has 10 ⁴ microelectrodes, each microelectrode having oligonucleotides having a particular sequence immobilized thereto. In another exemplary embodiment, the substrate has a surface area of 100 mm ² and has 10 ⁴ microelectrodes, each microelectrode being on a polyacrylamide gel pad immobilized with an oligonucleotide having a particular sequence. Are in contact. In a preferred embodiment, the microelectrodes are arranged on the base material so as to be separated by an interval of 0.05 mm to 0.5 mm.
Most preferably, the microelectrodes are regularly spaced apart from one another on a solid substrate.

In one example embodiment, the device comprises a microarray having at least 10 ³ microelectrodes on a single substrate to which oligonucleotide probes are attached. Alternatively, the arrayed oligonucleotide probes are attached to a polyacrylamide gel pad that contacts the microelectrodes of the device of the invention. Most preferably, an oligonucleotide having a particular nucleotide sequence, or a group of oligonucleotides having related (eg, overlapping) nucleotide sequences, is immobilized on each of the plurality of microelectrodes. In a further preferred embodiment, the nucleotide sequence of the oligonucleotides immobilized on each microelectrode and the correspondence and correspondence between the particular microelectrode and the nucleotide sequence of the oligonucleotides immobilized thereon is found. Has been issued.

The primer or probe used in the present invention is preferably an oligonucleotide having a predetermined length whose upper limit length and lower limit length are empirically determined. The lower limit of probe length is stable hybridization; probes that are too short may not form thermodynamically stable duplexes sufficient for single base extension under the hybridization conditions of the assay. , Known to those skilled in the art. The upper limit of probe length is a probe that forms a duplex in a region other than the predetermined response target region that results in artificial incorporation of the primer extension unit labeled with an electrically active moiety. Preferred oligonucleotide primers or probes used in the present invention are about 8 to about 50, more preferably about 1
0 to about 40, more preferably about 12 to about 30, most preferably about 15 to about 25.
Has a nucleotide length. However, longer probes may be used, i.e. probes 40 nucleotides or more in length.

In the present invention, the primer or probe is attached to the first group via the anchoring group.
It is preferably fixed directly on the surface of the electrode. As will be appreciated by those skilled in the art, advantageous anchoring groups include, for example, thiols, carboxylates, hydroxyls, amines, hydrazines, esters, amides, halides,
Included are groups or moieties that include vinyl groups, vinyl carboxylates, phosphates, silicon-containing organic compounds and their derivatives. For example, an oligonucleotide complementary to the target DNA is covalently attached to a metal gold electrode via a thiol-containing anchoring group. In a preferred embodiment, the length of these anchoring groups is such that the conductivity of these molecules is such that the hybridized probe and target D
It is chosen so as not to interfere with the transfer of electrons from the electrochemical reporter group to the electrode via NA and these anchoring groups in series. In other words, these anchoring groups preferably have higher conductivity than double-stranded nucleic acid. The conductivity S is about 10 ⁻⁶ to 10 ⁻⁴ W ⁻¹ cm ⁻¹ , more preferably about 10 ⁻⁵ to 1.
0 ³ W ^-1 cm ^-1 , which corresponds to an anchoring group with a length of about 5 Å to about 200 Å.

Alternatively, the primer or probe can be covalently bound to an intermediate support located on top of the first electrode. This support is made of TiOx, Si
It is preferably a thin layer of a porous inorganic material such as O ₂ or NOx, or a thin layer of a porous organic polymer such as polyacrylamide, agarose, nitrocellulose membrane, nylon, and dextran support. The primer is covalently attached to the support via a linker. Preferred linker moieties include, but are not limited to, thioethers, ethers, esters, amides, amines, hydrazines, carboxylates, halides, hydroxyls, vinyls, vinylcarboxylates, thiols. , Phosphates, silicon-containing organic compounds and their derivatives, and other carboxylate moieties. More preferably, the biotin-streptavidin pair is placed free to provide probe binding to the intermediate support.

The device of the present invention has a second electrode carrying a current and a reference electrode. The second electrode is
For example, metals such as gold, silver, platinum, copper and alloys thereof; conductive metal oxides such as indium oxide, indium tin oxide, zinc oxide; or other conductive materials such as carbon black and conductive epoxy. It is most preferred to have a conductive material. The most preferable of these is a platinum (Pt) wire auxiliary electrode. The reference electrode is preferably a silver wire electrode, a silver / silver chloride (Ag / AgCl) reference electrode, or a saturated calomel electrode.

This device consists of one or more reaction chambers, each reaction chamber being in electrochemical contact with at least one of the above-mentioned electrodes, each electrode being connected to a power supply and a power supply control means. There is. For the purposes of the present invention, the term “electrochemically contacting” means connecting components so that a current flows through the electrodes, especially when an electrical potential develops between the two electrodes.

Electrochemical contact is advantageously provided using an electrolyte solution in contact with each of the electrode arrays or microelectrode arrays of the present invention. Electrolyte solutions useful in the devices and methods of the present invention include electrolyte solutions of either physiologically relevant ionic strength (corresponding to about 0.15 M NaCl) and neutral pH. Examples of electrolyte solutions useful in the devices and methods of the present invention include, but are not limited to, phosphate buffered saline, HEPES buffered saline, and sodium bicarbonate buffered saline.

Preferred polymerases for performing single base extensions using the devices and methods of the present invention are those that exhibit little or no exonuclease activity. Higher than physiological temperature, ie 50 ° C or 60
More preferred are polymerases that withstand temperatures of 70 ° C or 70 ° C and are biosynthetically active, or polymerases that withstand temperatures of at least 90 ° C up to about 95 ° C.
Preferred polymerases include T. Taq obtained from aquaticus (commercially available from Perkin Elmer, Inc., Foster City, CA)
Polymerase, Sequenase (R) and Thermo Sequenase (R) (commercially available from US Biochemicals, Inc., Cleveland, Ohio), Exo-Pfu Polymerase (New England By Labo, Burberry, Mass.) Commercially available from).

The method of SNP detection provided by the present invention comprises: (1) preparing a sample containing a target nucleic acid of interest (typically Denaturing); (2) contacting the single-stranded target nucleic acid with an oligonucleotide primer of known sequence that hybridizes to the portion of the nucleotide sequence in the target nucleic acid that flanks the responding nucleotide bases (and thereby The primer and target of the target should be such that the nucleotide base to be responded to is the first unpaired base in the target adjacent to the 5'end of the nucleotide base that anneals at the 3'end of the double-stranded primer. It forms a double strand in between; this oligonucleotide is preferably a particular oligonucleotide that occupies a particular address in the addressable sequence. (3) A double-stranded aqueous carrier, a polymerase, and at least one primer extension unit comprising an extension moiety, an optional linker, and an electrochemical detection moiety. Contact with a reagent containing
In this case, by the primer extension reaction catalyzed by polymerase,
(The extension part of the primer extension unit is incorporated at the 3'end of the primer, resulting in extension of the primer by a single base); (4) Excluding unincorporated primer extension unit; and (5) Extended double strand Measuring the identity of the incorporated primer extension unit in the specific electrochemical detection moiety.

The extension portion in the primer extension unit is preferably a chain termination portion,
Most preferred are dideoxynucleotide triphosphates (ddNTPs) such as ddATP, ddCTP, ddGTP, and ddTTP; Within the range of. These ddNTPs are
They differ from normal deoxynucleotide triphosphates (dNTPs) in that they lack a hydroxyl group at the 3'position of the sugar moiety. This prevents chain extension of the incorporated ddNTP and causes chain termination. Unlike conventional detection moieties that were fluorescent dyes or radioactive labels, the present invention provides extension units labeled with an electrochemical reporter group that is detected electrochemically, most preferably by a redox reaction. . Electrochemically discriminating redox labeling that does not interfere with the incorporation of ddNTPs into the nucleotide chain is preferred.

Optionally, the target DNA in the sample to be investigated can be amplified by an in vitro amplification reaction such as the polymerase chain reaction technique (PCR) known to those skilled in the art. By increasing the target DNA in the biological sample used in the method of the present invention, template-directed synthesis by the polymerase is performed faster and more accurately. In vit such as PCR
The use of the ro amplification method is optional in the method of the present invention, a feature of which is that, compared to conventional detection techniques that generally require such amplification to generate sufficient signal to detect, Advantageously characterizes the method disclosed in the present invention. Due to the high sensitivity of the method of the invention, the purification steps and other in vitro amplification methods required after PCR are unnecessary, which simplifies the implementation of the method of the invention.

Single base extension is performed by using the polymerase in the presence of at least one primer extension unit in a buffer suitable for the biochemical activity of the polymerase. The general formula of a preferred embodiment of the primer extension unit is: ddNTP-LR where ddNTP is ddATP, ddGTP, ddCTP, and ddTT.
Represents a dideoxyribonucleotide triphosphate containing P, L represents any linker moiety, and R represents an electrochemical reporter group, preferably an electrochemically active moiety, most preferably a redox moiety. ]

In a preferred embodiment, each chain terminating nucleotide species (eg dideoxy (dd))
ATP, ddGTP, ddCTP, and ddTTP) are labeled with different electrochemical reporter groups, in which case it is likely that each reporter group will have a different electrochemically distinguishable different redox potential. preferable. In this regard, it is understood that nucleotides containing DNA molecules are themselves electrically active; for example, guanine and adenine are electrochemically oxidized at about 0.75V and 1.05V, respectively. Will Therefore, it is generally preferred that the reduction / oxidation potential of the electrochemical reporter group bearing the primer extension unit of the invention be distinguishable from the intrinsic reduction / oxidation potential of the nucleotide itself to be incorporated. The following electrochemical species are examples of, but not limited to, the electrochemically active moieties provided as the electrochemical reporter group of the present invention (
Oxidation (+) or reduction (-) potential (in volts) is shown in parentheses).

Redox moieties useful for aqueous saturated calomel reference electrodes include 1,4-benzoquinone (-0.54V), ferrocene (+0.307), tetracyanoquinodimethane (
+ 0.127V, -0.291V), N, N, N ', N'-tetramethyl-p-
It includes phenylenediamine (+0.21) and tetrathiafulvalene (+ 0.30V). Useful redox moieties for Ag / AgCl reference electrodes include 9-aminoacridine (
+ 0.85V), acridine orange (+ 0.830V), aclarubicin (+
0.774V), daunomycin (+ 0.446V), doxorubicin (+0.
440V), pirarubicin (+ 0.446V), ethidium bromide (+0.
678V), ethidium monoazide (+ 0.563V), chlorotetracycline (+ 0.650V), tetracycline (+ 0.674V), minocycline (+ 0.385V), Hoechst 33258 (+ 0.586V), Hoechst 333.
42 (+ 0.571V), 7-aminoactinomycin D (+ 0.651V),
Chromomycin A ₃ (+ 0.550V), Mitramycin A (+ 0.510V)
, Vinblastine (+ 0.552V), Rifampicin (+ 0.103V),
Os (bipyridine) ₂ (dipyridophenazine) ₂ (+ 0.72V), Co (bipyridine) ₃ (+ 0.11V), or Fe-bleomycin (-0.08V)
Is included. (This redox data is from Bird & Falkner, Electroche
medical Method (1980), John Wiley & Sons, and Hashimoto et al., Analyticala Chimica Acta 286 : 2.
(Reproduced from 19-224 (1994))

The selection of electrochemically active moieties having an electrochemical reporter group of the present invention is for detecting moieties other than other redox moieties present in solution as well as included in nucleic acids and arrays of biological samples. Optimized to avoid interference with labeling due to hybridization with the oligonucleotide.

The electrochemically active moiety comprising a chain-terminating nucleotide of the present invention optionally comprises a linker, preferably a linker (L) having a length of about 10 to about 20 angstroms.
) To the extended nucleotide. This linker may be an organic moiety such as a hydrocarbon chain (CH ₂ ) _n , or may have an ether, ester, carboxamide, or thioether moiety or a combination thereof. The linker may be an inorganic moiety such as siloxane (O-Si-O). The length of the linker is chosen such that the electrochemically active moiety R does not interfere with nucleic acid hybridization between the bound oligonucleotide primer and the target nucleic acid or chain extension by the polymerase.

In a preferred embodiment, the single base extension is cyclic voltammetry (CV).
, Or by standard electrochemical means such as stripping voltammetry. For example, the current is recorded as a function of the sweep voltage for the first electrode specific for each particular labeled primer extension unit. Specific base incorporation and extension is identified by the oxidation or reduction peaks characteristic of the primer extension unit, which is detected as the amount of current in the electrode at the appropriate redox potential.

In further exemplary embodiments, other electrical and / or electrochemical methods useful in practicing the methods and devices 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, potentiometric, amperometric, current step method, and combinations thereof. In all of these methods, the current is recorded as a function of the sweep voltage for the first electrode specific for each particular labeled primer extension unit. The difference is the type of input / probe signal and / or the shape of the input / probe signal used to sweep the voltage range. For example, in cyclic voltammetry, DC voltage sweeping is performed. In ACV, the AC signal is superimposed on the voltage sweep. In square wave voltammetry, a square wave is superimposed on a voltage sweep. Most preferably, the signal is recorded from each position ("address") in the oligonucleotide sequence so that the identity of the extended species can be measured. The identity of the nucleotides containing the extension unit is determined from the redox potential at which the current is detected.

In using the apparatus of the present invention to perform a single base extension reaction, at least one chain-terminating nucleotide labeled with an electrochemical label (such as redox-labeled ddNTP) is used in combination with a polymerase. A hybridization buffer that is soluble and has a salt concentration sufficient to allow hybridization between the target nucleic acid and the primer oligonucleotide under test conditions, and Taq
A reaction mixture containing a DNA polymerase or a DNA polymerase such as Thermo Sequenase is prepared. For example, 1 denatured by culturing at a temperature of 90 ° C or higher
The double-stranded target nucleic acid is diluted with deionized water to a concentration suitable for hybridization and added to the reaction mixture. The resulting hybridization mixture is enclosed in the reaction chamber of the device of the invention having a first electrode having a diversity of primers having a known sequence to which the electrode is linked. At least one of the primers
One has a nucleotide sequence capable of hybridizing with the portion of the target nucleotide sequence immediately adjacent to the responding nucleotide base under the hybridization conditions used in the test.

A double strand is formed between the primer and the target, in which case the nucleotide base to respond to is the 5'side of the 5'nucleotide base that is annealed at the 3'end of the primer in the double strand. Is the first unpaired base in the target adjacent to. Single base extension at the 3'end of the annealed primer is achieved by incorporating into the primer a terminating nucleotide labeled with an electrochemically active moiety. The primer sequences and labeled chain-terminating nucleotides allow the incorporation of nucleotides to provide information about the identity (ie, mutant, mutant, wild-type, or polymorphic) of the responding nucleotides in the target. To be selected.

Alternatively, the probe has a 3'terminal residue that corresponds and hybridizes to the responsive base. In this example embodiment, oligonucleotides with a "mismatch" at the 3'terminal residue hybridize but do not extend by the polymerase. Detecting incorporation of a primer extension unit responsive to a redox label, provided that the sequence of the oligonucleotide probe is known at each position in the sequence, provides information about the identity of the responsive nucleotide bases. It

After performing the SBE reaction, the electrode was placed in a solution of strict (ie low salt and low dielectric constant (eg 0.1 × SSC: 0.015M NaCl, 15 mM sodium citrate, pH 7.0)). If necessary, wash with a detergent such as sodium dodecyl sulfate, and wash for a predetermined time at a temperature of about 10 to 65 ° C. and at a temperature at which the target nucleic acid is removed. It depends on factors such as: Electrochemical detection is performed in electrolyte solution by conventional cyclic voltammetry.

The following examples provide a more complete description of the methods of using the invention described above and illustrate the best mode contemplated for practicing the various aspects of the invention. It should be understood that these examples are provided for purposes of illustration and are not intended to limit the scope of the invention in any way. All references cited are incorporated herein by reference.

[0177]

【Example】

Example 1 Production of Polypyrrole Micro Electrode A polypyrrole micro electrode was produced as follows. An ultrafine platinum wire with a diameter of 50 mm was inserted into a glass capillary tube with a diameter of 2 mm and sealed by heating to form a solid microelectrode structure. The tip of this structure was polished with gamma-alumina powder (CH Instruments, Inc, Austin, Tex.) To expose a platinum wire platen. The microelectrodes were first polished with 0.3 mm gamma alumina powder, washed with deionized water, and then 0.005 mm powder. After polishing, the microelectrode was ultrasonically cleaned in deionized water for 2 minutes, soaked in 1N HNO ₃ nitric acid for 20 minutes, washed vigorously in deionized water, soaked in acetone for 10 minutes, and again in deionized water. Washed well. Through the use of microfabrication techniques employed in semiconductor fabrication, modifications of this method can be applied to the fabrication of microelectrodes with the size required for bioarray chip construction.

A neutral polypyrrole matrix was used to attach the nucleic acid probe to the exposed platinum disk of the microelectrode. Platinum electrode as counter electrode, silver as reference electrode /
Electrochemical deposition was performed using a model 660A potentiostat (CH Instruments, Inc) with silver chloride (Ag / AgCl) and cyclic voltammetry (CV). 0.05M of pyrrole, 2.5 mM of 3-acetate -N- hydroxysuccinimide pyrrole and 0.1M LiClO _4/9,
A solution containing 5% acetonitrile was prepared as an electrolytic solution. The potential range for CV is 0.2-1.3 V vs. Ag / AgCl in the first cycle, and 10
In the additional cycles, the voltage was -0.1 to 1.0 V with respect to Ag / AgCl. The scan speed was 10 mV / sec. The electrolyte was nitrogen purged throughout the electrochemical deposition process. Alternatively, the polypyrrole film is 0.20-0 in the same solution as above.
． It can be formed by oxidizing pyrrole with a constant current of 25 mA / cm ² . This method does not require a reference electrode and is therefore more convenient to make array-based microelectrodes. The polypyrrole shown to the right of the arrow in FIG. 2A was prepared by electrochemical oxidation of pyrrole.

An oxidized polypyrrole electrode was placed in 0.1 M LiClO ₄ to give -0.1
It was cycled 20 times over a potential range of 0.8. This treatment stabilizes the polypyrrole film. In order to make the neutralized polypyrrole, the microelectrode was re-immersed in the electrolyte and 10 over a potential range of -0.2 to 0.3 with respect to the reduction zone Ag / AgCl of this electrochemical system. It was cycled twice. Neutralization is desirable to reduce the background charge (or charge) of the probe attachment matrix and increase the sensitivity of hybridization electrometry. The reaction to neutralize the polypyrrole film is shown in Figure 2B. Following neutralization of the polypyrrole film coating the microelectrodes, the microelectrodes were rinsed vigorously with deionized water.

Example 2 Attachment of Nucleic Acid Probe to Polypyrrole Microelectrode To attach the nucleic acid probe to the polypyrrole microelectrode prepared in Example 1, a microelectrode was attached to 80 mL of dimethylformamide and 20 mL of 15
A 15-mer oligonucleotide (5'-C labeled with nM 5'-amino.
-C-C-T-C-A-A-G-C-A-G-A-G-G-A-3 '; SEQ
Incubated for 4 hours at room temperature in a solution consisting of ID NO: 1). Following attachment of the probe molecule, the microelectrode was placed in TBE buffer (0.89M Tris-
Washed with borate, 0.025M EDTA), rinsed thoroughly in deionized water,
It was dried at room temperature.

Example 3 Electrical Detection of Nucleic Acid Hybridization Using Polypyrrole Microelectrodes First, the AC impedance baseline of the microelectrodes prepared in Example 2 was determined in the absence of complementary target molecules. The microelectrodes were placed in a sealed conical tube with complementary target molecules (5′-T-C-C-T-C-T-G-C-T-T-G-
A-GG-G-3 '; SEQ ID NO: 2) is micromolar (10 ^-6 M)
, MM) to attomole (10 ⁻¹⁸ M; aM), which was exposed to 35 mL present at a concentration range. Hybridization of the probe and target molecule is performed in 1xSSC buffer (
0.15M NaCl, 0.015M sodium citrate, pH 7.0) at 37 ° C. for 24-48 hours. Following hybridization, the microelectrodes were thoroughly rinsed in excess 1 × SSC at room temperature, after which the AC impedance was measured.

The AC impedance is model 1260 impedance / gain-phase analyzer (Solartron Inc., Houston, Houston, with model 1287 electrochemical interface).
(Texas). The counter electrode and the reference electrode were platinum and Ag / AgCl, respectively, and the AC impedance measurement was performed in a 1M LiClO ₄ solution under open circuit voltage (OCV) conditions. The measured complex impedance (Z) vs. frequency for a polypyrrole microelectrode with attached 15-mer oligonucleotides before and after hybridization with a 2 fM solution of complementary target molecules is shown in Figure 3A (complex impedance is Z = Z '+ i
Z ″ is a real part of impedance, i is 0 to 1, and Z ″ is an imaginary part of impedance). A significant difference was observed between the microelectrodes before and after hybridization to the target molecule (Fig. 3A). A large signal generated at low concentrations of the target molecule (ie 2 fM of the target corresponds to 0.1 amol of the target molecule) is a signal for detecting hybridization between the oligonucleotide probe and the target nucleic acid molecule. It shows the high sensitivity of the method of the invention. The frequency is 0.1 Hz (value at large Z ') to 1 MHz (nearly zero (~ 0)
Value at Z ').

FIG. 3B shows the frequency complex curve (seen in FIG. 3A) for the high frequency region (Z ′ <5 × 10 ⁴ ). A frequency-dependent semi-circular impedance curve was observed at high frequencies before and after hybridization. Such a curve at high frequency shows that the Faraday resistance (ie the electrochemical reaction resistance) is in parallel with the capacitance. A semi-circular curve, such as the curve shown in Figure 3B,
It can be used to obtain electrochemical reaction resistance and double layer capacitance by equivalent circuit simulation. Table 1 shows the simulation results obtained using the data shown in FIG. 3B. These results show that, as expected, the high frequency electrochemical resistance decreases and the capacitance increases after hybridization of the probe with the target molecule. This means that hybridized DN
It is revealed that A has a strong electrochemical interaction with Li ⁺ .

[0184]

[Table 1] Equivalent circuit parameters obtained from high frequency impedance data

The real part of the complex impedance data shown in FIG. 3A, that is, the square root of the frequency (W ^−1/2 ) with respect to the resistance (R), is plotted in FIGS. 4A and 4B. A linear region was observed, indicating that at low frequencies the diffusion process of Li ⁺ is dominant for the measurements. 4A and 4B show that a significant change in resistance occurs after hybridization of the single-stranded probe and the target molecule. Decrease in high frequency resistance following hybridization (
FIG. 4B) can be explained by the decrease in Faraday resistance of the hybridized nucleic acid. At low frequencies, a large ionic diffusion resistance dominates the impedance and the resistance is higher for the hybridized probe-target duplex (Fig. 4A). As the frequency increases, the contribution of the frequency-dependent diffused resistance decreases, so that the small Faraday resistance dominates.

The limit of detection in the above experiment reached approximately 0.1 attomole of the target molecule. Higher hybridization signals were obtained as the target molecule concentration increased, indicating that the method of the invention can be used for quantification of target hybridized to electrode-immobilized probes. Thus, the method can be used with a suitable reference electrode to measure the absolute amount of nucleic acid in a test sample.
For example, the method of the present invention enables highly sensitive and highly analytical determination of RNA in gene expression studies. Comparative gene expression studies performed using such methods allow for direct quantification of expressed RNA, rather than by determining the ratio of RNA of interest to control RNA.

Example 4 Specificity of Electrical Detection Using Polypyrrole Microelectrodes Microelectrodes with attached oligonucleotide probes were prepared as described in Examples 1 and 2. Four microelectrodes attached to the probe (
5′-T-C-C-T-C-T-G-C-T-T-G-A-G-G-G-3 ';
SEQ ID NO: 2) was incubated in a solution containing 2 pM of the 15-mer target molecule perfectly complementary to SEQ ID NO: 2), and the other four microelectrodes were used in Example 3
The attached probe (5'-C-C-C-T-C-A) using the conditions described in.
-A-G-C-A-G-A-G-G-A-3 '; SEQ ID NO: 1) 15-mer target molecule containing 3 mismatch bases was incubated in a solution containing 2 fM. .

After hybridization, the individual microelectrodes were washed by successively increasing the temperature in order to electrically measure the melting point of the double strand. 1 microelectrode for cleaning
It was carried out by placing in SSC at 37 ° C or 38 ° C for 30 minutes. The AC impedance curves of the unhybridized, target-hybridized, or washed at 37 ° C or 38 ° C electrodes are shown in Figure 5A (fully complementary target molecule) and Figure 5B (mismatched target molecule). Show.

AC impedance measurements showed clear differences between perfectly complementary (perfect) and mismatched hybridized nucleic acid duplexes. The impedance curves obtained for the perfectly complementary target molecule are unchanged after washing, indicating that the melting point of the perfect duplex is not exceeded. The impedance curve obtained for the mismatched target molecule shifts towards the baseline (ie, non-hybridizing probe) after washing, which causes the melting point of the duplex to be near the melting point of the duplex. It is shown that it occurs in. The ability to discriminate between matched and mismatched nucleic acid sequences indicates that the method of the invention is applicable to the detection of genetic polymorphisms. Figure 6 shows that the mismatched DNA system decreases continuously with increasing wash temperature, returning to the baseline for single-stranded DNA.

Example 5 Polypyrrole Microelectrodes for Molecular Interactions Use of Li ⁺ Reporters in Electrical Detection Microelectrodes were prepared as described in Examples 1 and 2, and also Example 3
Hybridized to the appropriate target molecule as described in. AC impedance before and after nucleic acid hybridization is shown in FIG. Microelectrodes with attached oligonucleotide probes showed ideal polarized electrode properties prior to hybridization with the target molecule. The equivalent circuit in this state is shown in FIG. 8A, and the AC impedance response is shown in FIG. 8B. In this state, the microelectrode
_Formula: it can be described by _{Z = R s -j (wC dl} ) -1. Where Z is impedance, R _s is solution resistance, j is 0 to 1, w is 2, and C _dl is double layer capacitance.

The behavior of the microelectrode is reasonable prior to hybridization to the appropriate target molecule, as it has no electrochemically active species and no specific adsorption, and 0.1 M LiC
Treated as an “ideal” polarized electrode under conditions with a nitrogen purge of the electrolyte solution containing 10 ₄ . However, after hybridization to the appropriate target molecule, a large deviation from the ideal curve was observed in the same electrolyte, indicating a significant increase in impedance. With the AC impedance measured on the electrodes after hybridization, the electrochemical process and equivalent circuit under such conditions is shown in FIG. 8C (where R _t is the Faraday or electrochemical reaction resistance and R _w is the Warburg. It is a resistance). Resistance from both electrochemical reactions and diffusion processes causes the behavior of the electrode after hybridization to deviate from the ideal polarization curve.

Simulations will allow calculation of all parameters of the equivalent circuit for this condition, but the equivalent circuit can also be simplified for R and C. The results of such simplifications are shown in FIGS. 9 and 10, which show that the resistance from both R and C has increased by an order of magnitude. Experimental results described above (particularly Example 4)
(Described in (1) above), Li ⁺ in the electrolyte can act as a reporter, allowing detection of mismatches between the probe and the target molecule. Since the method of the invention relies on the intercalation or binding of cations, more preferably Li ⁺ cations, to allow electrical detection, this method does not require labeling of the target molecule.

Example 6 Preparation of Hydrogel Porous Microelectrode A microelectrode was prepared as described in Example 1 (FIG. 1A). afterwards,
The exposed platinum plate was etched in hot aqua regia to form recesses (ie, micro-hole depressions) of specific depth. The depth of the recess was controlled by the length of time the platinum plate was exposed to the etching material. Thereafter, the recess thus formed was filled with a polyacrylamide gel material (FIG. 1B) to form a hydrogel porous microelectrode (FIG. 11). A hydrogel porous microelectrode with a diameter of 258 mm was used in subsequent examples.

Prior to attachment of probe molecules, hydrogel porous microelectrodes were activated by incubating in 2% trifluoroacetic acid for 10 minutes and rinsed in deionized water for 2 minutes. The microelectrode was then incubated in 0.1 M sodium periodate for 15 minutes and rinsed in deionized water for 2 minutes. Following this treatment, the microelectrodes were thoroughly washed by incubation in deionized water for 15 minutes and then air dried. Subsequently, the microelectrode was treated with 2% dimethyldichlorosilane solution and 2%.
Incubated in octamethylcyclotetrasiloxane for 10 minutes, washed in ethanol, rinsed in deionized water and air dried.

Example 7 Attachment of Nucleic Acid Probe to Hydrogel Porous Microelectrode To attach the nucleic acid probe to the microelectrode prepared in Example 6, the microelectrode was attached to 80 mL of dimethylformamide and 20 mL of 2 pM 5′-amino. 15-mer oligonucleotide labeled with -3'-fluorescein (5
'-C-C-C-T-C-A-A-G-C-A-G-A-G-G-A-3'; S
Incubated for 4 hours at room temperature in a solution consisting of EQ ID NO: 1).
Following attachment of the probe molecule, the microelectrode was washed with TBE buffer, rinsed thoroughly in deionized water and dried at room temperature.

Example 8 Electrical Detection of Nucleic Acid Hybridization Using a Hydrogel Porous Microelectrode First, the baseline AC of the hydrogel porous microelectrode prepared in Example 7 was used.
Impedance was determined in the absence of target molecule. The microelectrodes were placed in a sealed conical tube with complementary target molecules (5′-T-C-C-T-C-T-G-C-T-
T-G-A-G-G-G-3 '; SEQ ID NO: 2) was present at a concentration of 2 pM in 35 mL, or the mismatch target molecule (5'-C-C-C-T-C-A-).
A-G-C-A-G-A-G-G-A-3 '; SEQ ID NO: 1) is 30
Exposed to 35 mL present at a concentration of 0 nM. Hybridization of any of the target molecules with the probe was performed in 1 × SSC buffer for 1 hour at room temperature. Following hybridization, the microelectrodes were thoroughly rinsed in excess 1 × SSC for 20 minutes at room temperature, after which the AC impedance was measured.

AC impedance was measured using a model 1260 impedance / gain-phase analyzer with a model 1287 electrochemical interface. The counter electrode and the reference electrode were platinum and Ag / AgCl, respectively, and the AC impedance measurement was performed in 1 × SSC hybridization solution under open circuit voltage (OCV) conditions. The sample is excited with an amplitude of 50 mV. The measured complex impedance (Z) versus frequency for hydrogel porous microelectrodes with attached 15-mer oligonucleotides after hybridization with complementary or mismatched target molecules is shown in FIG.

The signal generated after hybridization of the probe molecule with the mismatched target molecule was indistinguishable from the signal generated in the absence of the target molecule. As shown in FIG. 12, this result indicates that at low frequencies charge transfer is diffusion controlled. The diffusion impedance is represented as the Warburg element W and has a linear region in both the imaginary and real part plots for W ^1/2 . From the imaginary part and the real part of the complex impedance data shown in FIG. 12, the resistance (R) with respect to W ^−1/2
Plots and capacitance (C) plots for W- ^{1 / 2} were obtained and plotted as shown in Figures 13 and 14. A linear region was observed in these plots, indicating that the diffusion process dominates the electronic measurements. These plots show that both resistance and capacitance show single-stranded DNA probe and target D
It shows that there is a significant change after hybridization with NA. After hybridization, resistance decreases and capacitance increases. These results indicate that hybridization of the target molecule to the probe molecule attached to the polyacrylamide gel can improve the charge transfer process by reducing the resistance. The increased capacitance is due to the increased surface charge that is the result of nucleic acid hybridization. The results obtained with hydrogel porous microelectrodes indicate that such microelectrodes can be used to detect 40 fM target molecules in solution.

Example 9 Preparation of Hydrogel Porous Microelectrode A microelectrode was prepared as follows. An ultrafine platinum wire with a diameter of 50 mm was inserted into a glass capillary tube with a diameter of 2 mm and sealed by heating to form a solid microelectrode structure. Gamma alumina powder (CH Instruments
, Inc, Austin, Tex.) To expose a platinum wire platen. The microelectrodes were first polished with 0.3 mm gamma alumina powder, rinsed with deionized water and then with 0.005 mm powder. After polishing, the microelectrodes were ultrasonically cleaned in deionized water for 2 minutes, soaked in 1N HNO ₃ for 20 minutes, washed vigorously in deionized water, soaked in acetone for 10 minutes, and then again in deionized water. Washed. Through the use of microfabrication techniques employed in semiconductor fabrication, modifications of this method can be applied to the fabrication of microelectrodes with the size required for bioarray chip construction.

A hydrogel porous microelectrode was prepared from the above microelectrode as follows. The exposed platinum plate of each microelectrode was etched in hot aqua regia to form recesses (ie, micro-hole depressions) of a specific depth. The depth of the recess was controlled by the length of time the platinum plate was exposed to the etching material. Then, the recess thus formed was filled with a polyacrylamide gel material (FIG. 1B) to form a hydrogel porous microelectrode (FIG. 2). A hydrogel porous microelectrode with a diameter of 258 mm was used in Example 2.

Prior to the attachment of probe molecules, the hydrogel porous microelectrodes were activated by incubating in 2% trifluoroacetic acid for 10 minutes and rinsed in deionized water for 2 minutes. The microelectrode was then incubated in 0.1 M sodium periodate for 15 minutes and rinsed in deionized water for 2 minutes. Following this treatment, the microelectrodes were thoroughly washed by incubation in deionized water for 15 minutes and then air dried. Subsequently, the microelectrode was treated with 2% dimethyldichlorosilane solution and 2%.
Incubated in octamethylcyclotetrasiloxane for 10 minutes, washed in ethanol, rinsed in deionized water and air dried.

Example 10 Attachment of Nucleic Acid Probe to Hydrogel Porous Microelectrode To attach the nucleic acid probe to the microelectrode prepared in Example 1, the microelectrode was attached to 80 mL of dimethylformamide and 20 mL of 2 pM 5′-amino. 15-mer oligonucleotide labeled with -3'-fluorescein (5
'-C-C-C-T-C-A-A-G-C-A-G-A-G-G-A-3'; S
Incubated for 4 hours at room temperature in a solution consisting of EQ ID NO: 1).
Following attachment of the probe molecule, the microelectrode was washed with TBE buffer, rinsed thoroughly in deionized water and dried at room temperature.

Example 11 Electrical Detection of Nucleic Acid Hybridization Nucleic acid hybridization between a probe bound to a hydrogel porous microelectrode and an electrochemically labeled target molecule is detected as follows. First, the baseline AC of the hydrogel porous microelectrode prepared in Example 2
Impedance is determined in the absence of electrochemically labeled target molecule. Thereafter, the microelectrode was placed in a sealed conical tube and the complementary target molecule (5'-
T-C-C-T-C-T-G-C-T-T-G-A-G-G-G-3 '; SEQ
35 mL in which ID NO: 2) is present at a concentration of 2 pM, or the mismatch target molecule (5′-T-C-C-T-C-T-G-C-T-T-G-A-G-G-G-). G-
3 '; SEQ ID NO: 1) exposed to 35 mL present at a concentration of 300 nM. Hybridization of any of the target molecules with the probe is performed in 1 × SSC buffer for 1 hour at room temperature. Following hybridization, the microelectrodes are thoroughly rinsed in excess 1 × SSC for 20 minutes at room temperature, after which the AC impedance is measured.

AC impedance is measured using a model 1260 impedance / gain-phase analyzer with a model 1287 electrochemical interface. The counter electrode and the reference electrode are platinum and Ag / AgCl, respectively, and the impedance measurement is performed in 1 × SSC hybridization solution under open circuit voltage (OCV) conditions. The sample is excited with an amplitude of 50 mV. Determine the measured complex impedance (Z) vs. frequency for hydrogel porous microelectrodes with 15-mer oligonucleotides attached after hybridization with complementary or mismatched target molecules.

Example 12 Single Base Extension A device of the invention is made as follows. A glass substrate is prepared with an ordered array of gold microelectrodes connected to a power supply. The substrate has a surface area of 100 mm ² and contains 10 ⁴ microelectrodes, and each microelectrode is immobilized with an oligonucleotide having a specific sequence,
It is in contact with a 0.5 μm thick polyacrylamide gel pad. The microelectrodes are arranged on the substrate so that they are separated by a distance of about 0.1 mm, and are regularly spaced on the solid substrate with a uniform spacing between them.

An oligonucleotide probe having a length of 25 nucleotides is attached to each of the gold electrodes. The regular array of resulting probes is arranged in four groups, whereby the probes are identical except for the last (mostly 3 ') residue. Each group contains oligonucleotides that terminate at adenosine (A), guanine (G), cytonin (C) or thymidine (T) or uracil (U) residues. The oligonucleotide is attached to each gold electrode through a polyacrylamide gel pad using a modification at the 5'residue of the oligonucleotide. This residue contains a thioester bond that covalently attaches the oligonucleotide to the polyacrylamide polymer.

This regular microelectrode array is placed in a reaction chamber sized sufficiently to accommodate the array and approximately 10-100 mL of hybridization / extension buffer. The reaction chamber also comprises a second counter electrode containing a platinum wire and a third reference electrode which is a silver / silver chloride electrode, each electrode being electrically connected to a power supply.

In using the device of the present invention, approximately 10-100 mL of hybridization buffer is added to the reaction chamber. This solution also contains the target molecule, typically in a concentration ranging from micromolar (10 ⁻⁶ M; μM) to attomole (10 ⁻¹⁸ M; aM). Hybridization of the probe and the target molecule was performed by using 1 × SSC buffer (0.
37 in 15M NaCl, 0.015M sodium citrate, pH 7.0)
Perform at 48 ° C for 24-48 hours. After hybridization, the microelectrodes were thoroughly rinsed in excess 1 × SSC at room temperature.

[0209] Thereafter, about 1 comprising a polymerase and a plurality of each 4 chain terminating nucleotide species.
Add 0-100 mL of extension buffer to the reaction chamber. Each of the four chain terminating nucleotide species is labeled with a species that can participate in reduction / oxidation (redox) reactions at the surface of the microelectrode. Examples of such clusters of species are: ddATP labeled with cobalt (bipyridine) ₃ ³⁺ ; ddGTP labeled with minocycline; ddCTP labeled with acridine orange; and ethidium monoazide. DdTTP attached
There is. Redox label is covalently bound to the chain terminating nucleotide by a hydrocarbon linker _{(CH 2) 2-8.} The expansion buffer is a thermo-sequenase (Thermo
sequenase) (obtained from US Biochemicals, Cleveland, Ohio). The extension reaction is sufficient to complete the extension reaction at a temperature suitable for the polymerase, such as a temperature of about 65 ° C., that does not denature the duplex with which the target and the oligonucleotide probe are hybridized. Do in time. After the extension reaction was completed, the array was replaced with 0.1 × SSC / 1% S.
Wash in DS at high stringency and at a temperature that does not denature the duplex.

After washing, approximately 10-100 mL of electrolyte is added to the reaction chamber and each microelectrode is signaled by conventional cyclic voltammetry to detect the redox signal. The identity of the oligonucleotide containing the single base extended species is determined by the redox potential of the signal obtained thereby.

Similarly, hybridization and single base extension can be performed in the same buffer, as long as the polymerase is compatible with the conditions of the hybridization buffer.

[Brief description of drawings]

1A and 1B show a hydrogel porous microelectrode (FIG. 1A).
Figure 1 illustrates a schematic depiction of the tip structure (or tip structure) of Figure 1 and illustrates a schematic depiction of the tip structure of a hydrogel porous microelectrode (Figure IB).

2A and 2B illustrate the electrochemical oxidation of pyrrole (FIG. 2A) and the neutralization of polypyrrole (FIG. 2B).

3A and 3B illustrate frequency complex curves (FIG. 3A) obtained from a polypyrrole microelectrode before and after hybridization of a 15-mer oligonucleotide probe with a complementary nucleic acid target molecule, and Before and after hybridization of the oligomeric oligonucleotide probe with the complementary target molecule (FIG. 2B).
7) illustrates a frequency complex curve obtained in the high frequency zone from the polypyrrole microelectrode of FIG.

4A and 4B illustrate a plot of low frequency resistance versus W ^−1/2 (FIG. 4A) and a plot of high frequency resistance versus W ^−1/2 (FIG. 4B).

5A and 5B are frequency complex curves (() obtained for hybridization of an oligonucleotide probe with a fully complementary nucleic acid target molecule.
FIG. 5A) and shows the frequency complex curve obtained for the hybridization of the oligonucleotide probe with the nucleic acid target molecule having three mismatches (FIG. 5B; curve 1 shows the hybridization of the target molecule to the probe). Got before;
Curve 2 was obtained by subsequent hybridization of the target molecule with the probe for 48 hours; curve 3 was obtained by subsequent washing of the hybridized molecule for 30 minutes at 37 ° C; curve 4 was hybridized. (Obtained by subsequent washing of the molecule for 30 minutes at 38 ° C.).

Figure 6 is a low-frequency resistance versus W was obtained for hybridization with the nucleic acid target molecule with an oligonucleotide probe and three mismatches ^- illustrates a ^half of the plot. (Curve 1 was obtained prior to hybridization of the target molecule to the probe; Curve 2 was obtained by subsequent hybridization of the target molecule with the probe for 48 hours; Curve 3 is of the hybridized molecule. Obtained by subsequent washing for 30 minutes at 37 ° C .; curve 4 was obtained by subsequent washing for 30 minutes at 38 ° C.)

FIG. 7 is a frequency complex obtained from a polypyrrole microelectrode before and after hybridization of a 15-mer oligonucleotide probe with a complementary nucleic acid target molecule in an electrolyte containing 0.1 M lithium perchlorate. A curve is illustrated.

8A-8C show a schematic depiction of the circuit (FIG. 8A), AC impedance response to a polypyrrole microelectrode with attached single-stranded nucleic acid probe prior to hybridization to the target molecule. Illustrates (FIG. 8B),
FIG. 8C illustrates a schematic depiction of the circuit for a polypyrrole microelectrode with attached single-stranded nucleic acid probe after hybridization to the target molecule (FIG. 8C).
.

FIG. 9 illustrates a plot of capacitance versus frequency for a polypyrrole microelectrode with attached single-stranded nucleic acid probe after hybridization to a target molecule.

FIG. 10 illustrates a plot of resistance versus frequency for a polypyrrole microelectrode with attached single-stranded nucleic acid probe after hybridization to a target molecule.

FIG. 11 illustrates a hydrogel porous microelectrode.

FIG. 12 illustrates a frequency complex curve obtained from a hydrogel porous microelectrode with attached 15-mer oligonucleotide probe in the absence of complementary target molecule (curve 1), A frequency complex curve subsequently incubated with 2 pM of the complementary target molecule is illustrated (curve 2) and subsequently 300
A frequency complex curve incubated with nM mismatch target molecule is illustrated (curve 3).

FIG. 13 illustrates a plot of capacitance versus frequency for a hydrogel porous microelectrode with attached single-stranded nucleic acid probe after hybridization to a target molecule.

FIG. 14 illustrates a plot of resistance versus frequency for a hydrogel porous microelectrode with attached single-stranded nucleic acid probe after hybridization to a target molecule.

FIG. 15 illustrates single base extension using a chain terminating nucleotide species labeled with an electrochemical reporter group (ECA group).

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] March 27, 2002 (2002.3.27)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/483 G01N 33/53 M 4B063 33/53 37/00 102 37/00 102 C12Q 1/68 A / / C12N 15/09 G01N 27/46 336M ZNA C12N 15/00 ZNAF C12Q 1/68 F (31) Priority claim number 09 / 459,685 (32) Priority date December 13, 1999 (December 1999. 13) (33) Priority claiming countries United States (US) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC , NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL , AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD , MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Mike Gaskin Amelie United States 85224 West Morelos Street 1132 (72) Chandler, Arizona Inventor Chang Min Li United States 85048 East Muirwood Drive 3138 (72) Inventor George Maracas United States 85048 Arizona Phenix, East Bighorn Avenue 2613 (72) Inventor Si Son United States 85044 East Gold Poppy Way 4521 F Term, Phoenix, Arizona 2G042 BD19 CB03 FB05 HA02 2G045 AA35 DA13 FB02 FB05 GC20 2G060 AA06 AF06 FA09 KA05 4B024 AA11 AA19 BA80 CA01 CA09 CA11 HA12 4B029 AA07 BB20 CC03 FA12 FA15 4B063 QA01 QA18 QQ42 QQ52 QR08 QR42 QR55 QR62 QR82 QS25 QS34 QS36 QX04 QR82

Claims (7)

[Claims] 1. A device for electrical or electrochemical detection of molecular interaction between an immobilized probe and a target molecule labeled with an electrochemically active reporter, comprising: (a) a supporting substrate. , (B) a plurality of microelectrodes in contact with the support substrate, (c) a plurality of polymer hydrogel pads in contact with the microelectrode and having probes immobilized thereon, (d) in contact with the support substrate At least one counter electrode, (e) means for generating an electrical signal at each microelectrode, (f) means for detecting changes in the electrical signal at each microelectrode, and (g) a plurality of microelectrodes, a plurality of polymers The component between the immobilized probe and the target molecule labeled with the electrochemically active reporter comprises a hydrogel pad and an electrolyte solution in contact with the counter electrode. Interaction, the presence or absence of labeled target molecules with an electrochemically active reporter, device detected by detecting a change in electrical signal. 2. A device for detecting a single base extension of an oligonucleotide comprising an oligonucleotide array, wherein the extension is performed by a polymerase and is directed by a nucleotide sequence of a nucleic acid in a biological sample, The device is a first electrode comprising an array of oligonucleotides on a substrate, the electrode comprising a conducting or semiconducting surface, the conducting metal being in contact with an aqueous electrolyte solution. A second counter electrode comprising, and a third reference electrode in contact with the aqueous electrolyte solution, each electrode being connected to a power source, the device further comprising a hybridization solution containing an electrolyte and A reaction chamber containing a polymerase, wherein each electrode further comprises a reaction chamber in electrochemical contact, the solution further comprising a chain-terminating nucleotide species. Comprising a plurality of primer extension units, each different chain terminating nucleotide species being capable of participating in a reduction / oxidation reaction at the surface of a first electrode under conditions in which a potential is applied to the electrode. Labeled with a distinguishable electrochemical label, each labeled chain-terminating nucleotide species has a specific reduction / oxidation potential and contains nucleic acids that hybridize to the oligonucleotides contained in the oligonucleotide array. Is incubated in a reaction chamber under hybridization conditions of moderate to high stringency, and the nucleotide sequence of the hybridized oligonucleotide is extended by incorporating at least one chain terminating nucleotide. , And at a potential specific to the reduction / oxidation potential of the electrochemical label. When a voltage is applied, the detection device current is generated in the apparatus. 3. The oligonucleotide array is an addressable array, the first electrode comprising a plurality of electrodes corresponding to each address of the addressable array, the electrochemical label reducing / reducing electrode. The addressable array after a single base extension with an electrochemical reporter-labeled chain-terminating nucleotide species and an oligonucleotide at the address of the array when a voltage is applied to the electrodes at a potential specific to the oxidation potential. The device of claim 2 wherein the current is generated at a particular address of. 4. The device of claim 2, wherein the chain-terminating nucleotide species is labeled with a transition metal complex. 5. A device for electrical detection of molecular interaction between a fixed probe and a target molecule, comprising: (a) a supporting substrate; and (b) a probe contacting a supporting substrate on which the probe is immobilized. A plurality of microelectrodes, (c) at least one counter electrode in contact with the supporting substrate, (d) an AC / for generating an electrical impedance at each microelectrode
DC power source, (e) electrical detector for detecting impedance change at each microelectrode in the presence or absence of target molecule, and (f) electrolyte in contact with multiple microelectrodes and counter electrodes A device comprising a solution, wherein molecular interaction between an immobilized probe and a target molecule is detected by detecting changes in electrical impedance in the presence and absence of the target molecule. 6. A device for electrical detection of molecular interaction between a fixed probe and a target molecule, comprising: (a) a supporting substrate, (b) a plurality of microelectrodes in contact with the supporting substrate, (C) a plurality of conjugated polymer films in contact with the microelectrodes and having probes immobilized thereon, (d) at least one counter electrode in contact with the support substrate, (e) an electrical impedance for each microelectrode AC / D to generate
A C power source, (f) an electrical detector for detecting a change in impedance at each microelectrode in the presence or absence of a target molecule, and (g) a plurality of microelectrodes, a plurality of conjugated polymer films, And having an electrolyte solution in contact with the counter electrode, the molecular interaction between the immobilized probe and the target molecule detects changes in electrical impedance in the presence and absence of the target molecule. Device detected by. 7. A device for electrical detection of molecular interaction between a fixed probe and a target molecule, comprising: (a) a supporting substrate, (b) a plurality of microelectrodes in contact with the supporting substrate, (C) a plurality of polymer hydrogel pads in contact with the microelectrodes and having probes immobilized thereon, (d) at least one counter electrode in contact with the support substrate, (e) electrical to each microelectrode. AC / D for generating impedance
C power source, (f) electrical detector for detecting impedance change at each microelectrode in the presence or absence of target molecule, and (g) multiple microelectrodes, multiple polyacrylamide gels · A pad and an electrolyte solution in contact with the counter electrode, and the molecular interaction between the immobilized probe and the target molecule detects changes in electrical impedance in the presence and absence of the target molecule. A device that is detected by doing.