WO2006003991A1 - 遺伝子検出方法、及び挿入剤 - Google Patents
遺伝子検出方法、及び挿入剤 Download PDFInfo
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- WO2006003991A1 WO2006003991A1 PCT/JP2005/012080 JP2005012080W WO2006003991A1 WO 2006003991 A1 WO2006003991 A1 WO 2006003991A1 JP 2005012080 W JP2005012080 W JP 2005012080W WO 2006003991 A1 WO2006003991 A1 WO 2006003991A1
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- stranded nucleic
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/66—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light electrically excited, e.g. electroluminescence
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
Definitions
- the present invention relates to a gene detection method for detecting a specific gene sequence present in a sample with high sensitivity, and particularly to a technique for electrochemically detecting a gene with an intercalating agent.
- a conventional DNA chip for electrochemically detecting a specific gene sequence has a single-stranded nucleic acid probe having a base sequence complementary to the target gene to be detected immobilized on the electrode surface. After hybridizing the nucleic acid probe and the target gene sample denatured into a single strand, the nucleic acid probe and the target gene sample are inserted into the double-stranded nucleic acid specifically and electrochemically active. An agent is added to the reaction system of the nucleic acid probe and the gene sample, and the intercalating agent bound to the double-stranded nucleic acid is detected by electrochemical measurement via the electrode. The presence of the target gene is confirmed by detecting the nucleic acid probe hybridized with (see, for example, Patent Document 1 and Patent Document 2).
- the intercalating agent refers to a substance that recognizes the double-stranded nucleic acid and specifically binds to the double-stranded nucleic acid.
- Each of the intercalating agents has a plate-like insertion group such as a phenyl group in the molecule, and the insertion group intervenes between the base pair of the double-stranded nucleic acid, thereby Join.
- the binding between the intercalating agent and the double-stranded nucleic acid is binding by electrostatic interaction or hydrophobic interaction, and the intercalation of the intercalating agent between the base strand of the double-stranded nucleic acid and its base It is a bond by equilibrium reaction in which separation from the pair is repeated at a constant rate.
- the intercalating agent specifically binds only to the double-stranded nucleic acid, and the amount of intercalating agent bound to the double-stranded nucleic acid is accurately detected. It was important.
- intercalating agents used for gene detection have a single-stranded nucleic acid probe or the like due to a chemical bond such as a coordination bond or a covalent bond, or an electrostatic interaction or a hydrophobic interaction.
- a chemical bond such as a coordination bond or a covalent bond, or an electrostatic interaction or a hydrophobic interaction.
- This non-specifically adsorbed intercalating agent becomes knock ground noise when detecting the amount of intercalating agent bound to the double-stranded nucleic acid, causing a decrease in detection sensitivity.
- Patent Document 1 Japanese Patent No. 2573443
- Patent Document 2 Japanese Patent No. 3233851
- the intercalating agent and the double-stranded nucleic acid are bound by an electrostatic interaction or a hydrophobic interaction! As a result, the binding force is weak! For this reason, the single-stranded nucleic acid probe and the double-stranded nucleic acid are bound to the double-stranded nucleic acid during the washing step of removing the nonspecifically adsorbed intercalating agent from the surface of the electrode on which the nucleic acid probe is immobilized. There is a problem that the intercalating agent is also dissociated, and conversely, the detection sensitivity is lowered.
- the single-stranded nucleic acid probe and the electrode surface that cause knock ground noise when considering that the intercalating agent bound to the double-stranded nucleic acid does not dissociate.
- the detection sensitivity is lowered because the intercalating agent adsorbed nonspecifically cannot be sufficiently removed.
- the binding reaction between the intercalating agent and the double-stranded nucleic acid is an equilibrium reaction, the ratio of the intercalating agent inserted between the base pairs of the double-stranded nucleic acid is low, and the detection sensitivity is low.
- the problem Mame since the binding reaction between the intercalating agent and the double-stranded nucleic acid is an equilibrium reaction, the ratio of the intercalating agent inserted between the base pairs of the double-stranded nucleic acid is low, and the detection sensitivity is low.
- An object of the present invention is to provide a gene detection method and an insertion agent capable of detecting a gene to be detected in a specimen sample with high sensitivity. To do.
- the gene detection method of the present invention is a gene detection method for detecting a gene having a specific sequence, wherein a gene to be detected is denatured into a single strand and a gene sample
- a gene sample preparation step for preparing a DNA sample a fixation step for fixing a single-stranded nucleic acid probe having a base sequence complementary to the base sequence of the gene sample to be detected
- an intercalating agent having a substituent consisting of Lb—Ib, one Lc Fb, V, or one or a combination of both is added to each site of the following general formula (1). Insert And adding step,
- Fa and Fb are electrochemically active sites having electrochemical activity
- la and lb are specifically inserted into a double-stranded nucleic acid, and form a covalent bond with the double-stranded nucleic acid upon irradiation with light.
- Single-stranded nucleic acid binding sites, La, Lb, and Lc are linking sites that link the double-stranded nucleic acid binding site and the electrochemically active site
- the intercalator molecule Also present in the intercalator molecule The number of double-stranded nucleic acid binding sites and electrochemically active sites increases, and as a result, the insertion rate of double-stranded nucleic acid between base pairs increases, and the electric power of the intercalating agent bound to the double-stranded nucleic acid increases. Since the gas chemical output signal also becomes large, the gene sample can be detected with high sensitivity.
- Fa and Fb, la and lb are the same compound.
- a voltage is applied to the electrode, and the amount of electrochemiluminescence by the intercalating agent covalently bonded to the double-stranded nucleic acid is measured. .
- Ia and lb are compounds having photosensitivity.
- the intercalating agent inserted into the double-stranded nucleic acid binds strongly and irreversibly to the double-stranded nucleic acid, so that the intercalating agent is removed from the double-stranded nucleic acid even after a strong washing treatment.
- gene samples can be detected with high sensitivity.
- the photosensitive compound is a furocoumarin derivative.
- the gene detection method of the present invention is the furocoumarin derivative-powered psoralen derivative.
- the Fa and Fb are compounds having oxidation-reduction properties.
- the presence of the gene to be detected can be detected by measuring the redox current generated during the redox reaction.
- the compound having redox properties is a compound that exhibits electrochemiluminescence.
- the compound exhibiting electrochemiluminescence is a metal complex having a heterocyclic compound as a ligand, rubrene, anthracene, coronene, pyrene, fluoranthene, taricene, phenanthrene, Perylene, binaphthyl, or octatetraene.
- the metal complex having a heterocyclic compound as a ligand is a metal complex having a pyridine moiety as a ligand.
- the gene detection method of the present invention is any one of a metal complex force metal biviridine complex and a metal phenantorin phosphorus complex having a pyridine moiety in the ligand.
- the gene detection method of the present invention is one in which the central metal force of the metal complex having a heterocyclic compound as the ligand is ruthenium or osmium.
- intercalating agent of the present invention is an intercalating agent represented by the following general formula (1):
- Fa is an electrochemically active site having electrochemical activity
- la is a double-stranded nucleic acid bond that specifically inserts into a double-stranded nucleic acid and forms a covalent bond with the double-stranded nucleic acid upon irradiation with light.
- Site, La is a linking site linking the double-stranded nucleic acid binding site and the electrochemically active site
- lb is specifically inserted into a double-stranded nucleic acid and covalently bonded to a double-stranded nucleic acid by irradiation with light at each site in the general formula (1).
- a double-stranded nucleic acid binding site, Fb is an electrochemically active site having electrochemical activity, and Lb and Lc are linking sites.
- Fa and Fb, la and lb are the same compound.
- Ia and lb are compounds having photosensitivity.
- the intercalating agent inserted into the double-stranded nucleic acid binds strongly and irreversibly to the double-stranded nucleic acid, so that the intercalating agent is removed from the double-stranded nucleic acid even after a strong washing treatment.
- gene samples can be detected with high sensitivity.
- the compound having photosensitivity is a furocoumarin derivative.
- the intercalating agent of the present invention is the furocoumarin derivative-powered psoralen derivative.
- the Fa and Fb are compounds having oxidation-reduction properties.
- the presence of the gene to be detected can be detected by measuring the redox current generated during the redox reaction.
- the compound having redox properties is a compound showing electrochemiluminescence.
- the presence of the gene to be detected can be detected by measuring the electrochemiluminescence.
- the intercalating agent of the present invention is such that the compound exhibiting electrochemiluminescence is a metal complex having a heterocyclic compound as a ligand, rubrene, anthracene, coronene, pyrene, fluoranthene, taricene, phenanthrene, perylene. , Binaphthyl, or octatetraene.
- the intercalating agent of the present invention is such that the metal complex having a heterocyclic compound in the ligand is a metal complex having a pyridine moiety in the ligand.
- the metal complex having a pyridine moiety in the ligand is any of a metal biviridine complex and a metal phenantorin complex.
- the intercalating agent of the present invention is one in which the central metal of the metal complex having a heterocyclic compound as the ligand is either ruthenium or osmium.
- the invention's effect when detecting a gene having a specific sequence, an insertion agent that is electrochemically active and covalently binds to a double-stranded nucleic acid by light irradiation is used.
- the double-stranded nucleic acid and the intercalating agent can be covalently bound to each other by light irradiation, so that the double-stranded nucleic acid and the intercalating agent can be bound irreversibly and firmly.
- the intercalating agent bound to the double-stranded nucleic acid is not dissociated.
- the sample can be detected with high sensitivity.
- the intercalating agent since the intercalating agent has a plurality of electrochemically active sites or a plurality of double-stranded nucleic acid binding sites, the intercalating agent double strand Since the insertion ratio between nucleic acid base pairs increases and the electrochemical output signal of the intercalating agent force bound to the double-stranded nucleic acid also increases, it is possible to detect the gene sample to be detected with higher sensitivity. Can do.
- the intercalating agent and the double-stranded nucleic acid are firmly and irreversibly provided with a double-stranded binding site that is covalently bound to the double-stranded nucleic acid by light irradiation. It is possible to provide an intercalating agent that does not dissociate from the double-stranded nucleic acid even by washing treatment.
- the intercalating agent of the present invention since it has a plurality of electrochemically active sites or a plurality of double-stranded nucleic acid binding sites, insertion of the intercalating agent between base pairs of double-stranded nucleic acids. It is possible to provide an intercalating agent that can increase the ratio and increase the electrochemical output signal from the intercalating agent bound to the double-stranded nucleic acid.
- FIG. 1 shows an electrode x obtained by Example 1 of the present invention on which a double-stranded nucleic acid is formed and an electrode y on which a double-stranded nucleic acid is formed. It is a figure which shows the maximum amount of electrochemical luminescence detected.
- the gene sample in the following embodiments refers to, for example, blood, white blood cells, serum, urine, feces, semen, saliva, cultured cells, tissue cells such as various organ cells, and any other gene containing genes. From the sample, cells in the sample are destroyed to release double-stranded nucleic acids, which are dissociated into single-stranded nucleic acids by heat treatment or alkali treatment.
- the gene sample in this embodiment may be a nucleic acid fragment that has been cut with a restriction enzyme and purified by electrophoresis separation or the like.
- a gene sample to be examined is created. As described above, this gene sample is denatured into a single strand by heat treatment or alkali treatment by destroying cells in an arbitrary sample to release double-stranded nucleic acid.
- the cells in the sample can be destroyed by a conventional method, for example, by applying a physical action such as shaking or ultrasonic waves from the outside.
- nucleic acid can be released using a nucleic acid extraction solution (for example, a solution containing a surfactant such as SDS, Triton-X, Tween-20, or a solution containing saponin, EDTA, or protease). You can also.
- the nucleic acid probe may be a nucleic acid obtained by cleaving a nucleic acid extracted from a biological sample with a restriction enzyme and purified by separation by electrophoresis or the like, or a single-stranded nucleic acid obtained by chemical synthesis.
- nucleic acid extracted from a biological sample it is preferably dissociated into single-stranded nucleic acid by heat treatment or alkali treatment.
- the nucleic acid probe obtained as described above is fixed to the electrode.
- the electrode used in the present invention may be any electrode as long as it can be used as an electrode.
- a noble metal electrode such as gold, platinum, platinum black, palladium, rhodium, graphite
- Carbon electrodes such as glassy carbon, pyrolytic graphite, carbon paste, carbon fiber, oxide electrodes such as titanium oxide, tin oxide, manganese oxide, lead oxide, Si, Ge, ZnO, CdS, TiO And semiconductor electrodes such as GaAs.
- These electrodes may be coated with a conductive polymer. By coating in this way, a more stable probe fixing electrode can be prepared.
- As a method of immobilizing the nucleic acid probe to the electrode a known method is used.
- a thiol group is introduced at the 5′—or 3′—end (preferably the 5′—end) of the nucleic acid probe to be immobilized, and the covalent bond between gold and iow.
- the nucleic acid probe is fixed to the gold electrode via Methods for introducing thiol groups into this nucleic acid probe are described in the literature (M. Maeda et al., Chem. Lett., 1805-1808 (1994) and BA Connolly, Nucleic Ac
- a nucleic acid probe having a thiol group obtained by the above method is dropped on a gold electrode and left at low temperature for several hours, whereby the nucleic acid probe is fixed to the electrode and a nucleic acid probe is produced.
- the electrode is glassy carbon
- the carbonic acid is first oxidized with potassium permanganate to introduce carboxylic acid groups on the electrode surface.
- the nucleic acid probe is fixed to the glassy carbon electrode surface by an amide bond.
- the actual immobilization method for immobilizing a nucleic acid probe to this glassy carbon electrode is described in detail in the literature (KM Millan et al., Analytical Chemistry, 65, 2317-2323 (1993))! .
- an intercalating agent is added to the electrode on which the double-stranded nucleic acid is formed, and the intercalating agent is inserted into the double-stranded nucleic acid.
- the insertion agent of this insertion agent may be added to the specimen sample before forming the double-stranded nucleic acid, that is, before the hybridization reaction.
- the double-stranded nucleic acid to which the intercalating agent is added is irradiated with light to form a covalent bond between the double-stranded nucleic acid and the intercalating agent.
- the intercalating agent inserted into the double-stranded nucleic acid will be described.
- a substance that is specifically inserted into the double-stranded nucleic acid and has a characteristic of being covalently bonded to the double-stranded nucleic acid by light irradiation is used.
- the intercalating agent is strongly and irreversibly bound to the double-stranded nucleic acid, so that the intercalating agent bound to the double-stranded nucleic acid is converted into the double-stranded nucleic acid cassette in the subsequent washing step. In the washing step where dissociation does not occur, unreacted intercalating agent can be removed by binding to the double-stranded nucleic acid.
- the intercalating agent of the present invention uses a substance having the characteristic of being electrochemically active. Accordingly, the presence of the double-stranded nucleic acid, that is, the gene sample can be detected with high sensitivity by detecting an electrochemical signal derived from the intercalating agent specifically bound to the double-stranded nucleic acid.
- the intercalating agent that satisfies the above-mentioned two characteristics specifically inserts into the double-stranded nucleic acid and binds to the double-stranded nucleic acid covalently upon irradiation with light, and has an electrochemical activity. And a linking site La for linking the double-stranded nucleic acid binding site la and the electrochemically active site Fa.
- such an intercalating agent can be represented by the following general formula (1).
- Fa is an electrochemically active electrochemically active site
- la is a double-stranded nucleic acid binding site having a site that is cross-linked with a double-stranded nucleic acid by light irradiation
- La is la and Fa. Represents the connecting part that connects
- the substance that can be used as the double-stranded nucleic acid binding site la shown in the general formula (1) is specifically inserted into the double-stranded nucleic acid and doubled by light irradiation.
- Examples include compounds having photosensitivity that are covalently bonded to a strand nucleic acid.
- Examples of such photosensitive compounds include furocoumarin derivatives, and psoralen derivatives are particularly preferable.
- this psoralen derivative When this psoralen derivative is inserted into a double-stranded nucleic acid, it causes a non-covalent interaction with the double-stranded nucleic acid.
- this psoralen derivative When this psoralen derivative is further irradiated with long-wavelength ultraviolet light (300 to 400 nm), it is inserted into the double-stranded nucleic acid. Since the psoralen derivative part forms a stable covalent bond, the intercalating agent and the double-stranded nucleic acid can be strongly and irreversibly bound as a result.
- psoralen derivatives include psoralen, methoxypsoralen, trimethylenosoralen and the like.
- the substance that can be used as the electrochemically active site Fa shown in the general formula (1) is not particularly limited as long as it is a substance that can be detected electrochemically.
- a compound having redox properties capable of detecting a substance by measuring a redox current generated during a reversible redox reaction can be given.
- the presence of the double-stranded nucleic acid, that is, the gene to be detected can be detected by measuring the redox current generated during the redox reaction.
- Examples of such a compound having acid reductivity include pheucene, catecholamine, metal complexes having a heterocyclic compound as a ligand, rubrene, anthracene, coronene, pyrene, There are funolean lanterns, thalisene, phenanthrene, perylene, binaphthinoles, talented tetratetranes or viologens.
- the above-described metal complex having a heterocyclic compound as a ligand, rubrene, anthracene, coronene, pyrene, funole lanthanum, thalicene, phenanthrene, perylene, binaphthyl, and octatetraene are subjected to oxidation-reduction reaction. Some of them generate electrochemiluminescence. If a substance that generates such electrochemiluminescence is used as an intercalating agent, the presence of the gene sample can be detected by measuring the luminescence.
- the metal complex having a heterocyclic compound in the ligand a heterocyclic compound containing oxygen, nitrogen, etc.
- a metal complex having a pyridine moiety, a pyran moiety, etc. as a ligand for example, a metal complex having a pyridine moiety, a pyran moiety, etc. as a ligand.
- a metal complex having a pyridine moiety as a ligand is preferable.
- the metal complex having the pyridine moiety as a ligand include a metal biviridine complex and a metal phenantorin complex.
- examples of the central metal of the metal complex having a heterocyclic compound as the ligand include ruthenium, osmium, zinc, conoleto, platinum, chromium, molybdenum, and tandastane. And technetium, rhenium, rhodium, iridium, palladium, copper, indium, lanthanum, praseodymium, neodymium, and samarium.
- complexes whose central metal is ruthenium or osmium have good electrochemiluminescence properties.
- Examples of the substance having good electrochemiluminescence properties include a ruthenium biviridine complex, a ruthenium phenantorin complex, an os-umbipyridine complex, and an os-umphenantorin complex.
- any substance that links the electrochemically active site Fa and the double-stranded nucleic acid binding site la can be used.
- the linker sequence of the site is not particularly limited. For example, an alkyl group, an O group, a —CO group, an NH group, or a group having a combination force thereof can be used.
- the intercalating agent of the present invention has the following force -Lb-lb or Lc Fb of the electrochemically active site Fa, the linking site La and the double-stranded nucleic acid binding site la in the general formula (1)
- One of the compounds represented has a substituent which is a combination force of either or both.
- the lb is a double-stranded nucleic acid binding site that specifically inserts into a double-stranded nucleic acid and forms a covalent bond with the double-stranded nucleic acid upon irradiation with light
- the Fb has an electrochemical activity.
- Lb and Lc are linking sites that link the double-stranded nucleic acid binding site and the electrochemically active site.
- the introduction position of the above-described substituent represented by -Lb-lb, Lc Fb into the general formula (1) is not particularly limited, and it may be introduced into any part of Fa, La, la.
- the number of the substituents introduced into the general formula (1) is not particularly limited.
- the structure of the substituent may be -Lb-lb, or Lc-Fb, each of which may be used alone.
- the structure may be a combination of forces Lb-lb and Lc-Fb.
- a series structure in which only Lb—lb or only Lc—Fb are connected in series or a series structure in which —Lb—lb and —Lc—Fb are combined and connected in series
- the substance used as the double-stranded nucleic acid binding site lb is a substance that specifically inserts into the double-stranded nucleic acid and covalently binds to the double-stranded nucleic acid by light irradiation.
- a photosensitive compound such as a furocoumarin derivative, and a psoralen derivative are particularly preferable.
- Specific examples of the psoralen derivative include psoralen, methoxypsoralen, trimethylpsoralen, and the like.
- the double-stranded nucleic acid binding sites la and lb may be any substance that can be specifically inserted into the double-stranded nucleic acid and can be covalently bound to the double-stranded nucleic acid by light irradiation.
- La and lb may be different substances, such as la in methoxypsoralen and lb in the substituent being trimethylpsoralen.
- la and lb are different from each other, since la and lb have different ability to insert into double-stranded nucleic acid, considering quantitative detection, for example, both la and lb are trimethyl. For psoralen, it is preferable that la and lb are the same substance as /!
- the substance used as the electrochemically active site Fb is not limited as long as it is an electrochemically detectable substance.
- the substance can be oxidized and reduced.
- a compound having a heterocyclic compound for example, a metal complex having a heterocyclic compound as a ligand.
- examples of the metal complex having a heterocyclic compound in the ligand include heterocyclic compounds containing oxygen, nitrogen and the like, for example, a metal complex having a pyridine moiety, a pyran moiety and the like in the ligand.
- metal complexes having a pyridine moiety as a ligand are preferred.
- examples of the metal complex having the pyridine moiety as a ligand include a metal biviridine complex and a metal phenantorin phosphorus complex.
- the central metal of the metal complex having a heterocyclic compound as the ligand is, for example, ruthenium, osmium, zinc, conoleto, platinum, chromium, molybdenum, tungsten.
- os-um complexes have good electrochemiluminescence properties.
- the substance having good electrochemiluminescence properties include a ruthenium biviridine complex, a ruthenium phenant complex, a os-umbipyridine complex, and a os-umphenant complex.
- the electrochemically active sites Fa and Fb may be both electrochemically detectable substances.
- Fa in the general formula (1) is a ruthenium biviridine complex
- Fa and Fb may be different substances.
- Fa and Fb are different substances, the intensity of the electrochemical detection signal differs between Fa and Fb. Therefore, considering quantitative detection, for example, both Fa and Fb are luteyu. In the case of a mubipyridine complex, it is preferable that Fa and Fb are the same substance as described above.
- the substance used as the linking site Lb, Lc is a substance that links each of Fa, La, la in the general formula (1) and lb or Fb.
- the linker sequence is not particularly limited.
- an alkyl group, an O group, a C 2 O group, a —NH group, a phosphoric acid group, or a group having a combination force thereof can be used.
- intercalating agent as described above is added before or after the gene sample and the nucleic acid probe immobilized on the electrode are hybridized.
- the single-stranded nucleic acid probe not forming the double-stranded nucleic acid immobilized on the electrode surface and the intercalator adsorbed nonspecifically on the electrode surface are removed, and as a result, the hybridized two Only the insert that is specifically covalently bound to the strand nucleic acid remains, and the presence of double-stranded nucleic acid, that is, the gene sample, is detected with high sensitivity by measuring the electrochemical signal derived from this insert. Can do.
- the electrochemical signal derived from the intercalating agent varies depending on the type of intercalating agent to be added, but when an intercalating agent that generates an acid reduction current is used, a potentiostat, a function generator, etc. can also be used. It can be measured with a measurement system. On the other hand, when an intercalating agent that generates electrochemiluminescence is used, measurement can be performed using a photomultiplier or the like.
- a gold electrode was prepared by forming 200 nm of gold on a glass substrate with titanium lOnm as a base using a sputtering device (ULVAC SH-350) and forming an electrode pattern by a photolithographic process.
- nucleic acid probe from the 5, end of the human Cytochrome P-450 gene sequence A 40-base oligodeoxynucleotide (manufactured by Takara Bio Inc.) modified with a thiol group via a 5′-terminal phosphate group having the CCCCCTGGAT CCAGATATGC AATAATTT TC CCACTATCAT sequence at position 629—668 was used.
- the nucleic acid probe was dissolved in 10 mM PBS (sodium phosphate buffer at pH 7.4) and adjusted to 100 M.
- the prepared nucleic acid probe solution is dropped onto the gold electrode and left at room temperature for 4 hours under saturated humidity to bond the thiol group and gold to fix the nucleic acid probe to the gold electrode. 7
- oligodeoxynucleotide manufactured by Takara Bio Inc. having a sequence of ATGATAGT GG GAAAATTATT GCATATCTGG ATCCAGGGGG from the 5′-end complementary to the nucleic acid probe was used.
- the gene sample oligodeoxynucleotide (manufactured by Takara Bio Inc.) having a sequence of ATGATAGT GG GAAAATTATT GCATATCTGG ATCCAGGGGG from the 5′-end complementary to the nucleic acid probe was used.
- the sample was dissolved in a hybridization solution mixed with 10 mM PBS and 2XSSC, and adjusted to 20 M.
- a gold electrode y on which no double-stranded nucleic acid is formed is created as a comparison target.
- the gold electrode y on which the double-stranded nucleic acid is not formed is formed by using the gene sample having a non-complementary sequence with the nucleic acid probe (hereinafter referred to as “comparison gene sample”). The same treatment as that for obtaining the gold electrode X is performed.
- the comparative gene sample a gene sample having a sequence of 40mer Poly-A (manufactured by Tacarano), AAAAAAAAAAAAAAAAAAAAAAAAA was used.
- a psoralen-modified ruthenium complex represented by the following (Chemical Formula 11) was used as an intercalating agent.
- each of the gold electrodes X and y is irradiated with UV light of 365 nm and 5 mWZcm 2 for 10 minutes using a UV crosslinker (Funakoshi UVPCL1000L type) to covalently bind the psoralen and the double-stranded nucleic acid. It was. After the covalent bonding, the gold electrode X was washed with 10 mM PBS for 10 minutes to remove unreacted Ru complex.
- 0.1 M PBS and 0.1 M triethylamine were mixed with each of the electrode x where the double-stranded nucleic acid was formed and the electrode y where the double-stranded nucleic acid was not formed.
- the electrolyte was added dropwise. Thereafter, a voltage was applied to each of the electrodes X and y, and the electrochemiluminescence derived from the intercalator generated at this time was measured. The voltage was applied by scanning from 0 V to 1.3 V and performing electrochemical measurement for 1 second. The amount of electrochemiluminescence was measured using a photomultiplier tube (H7360-01 manufactured by Hamamatsu Photonicus), and the maximum amount of luminescence during voltage scanning was measured.
- Fig. 1 shows the maximum electrochemiluminescence detected in the electrode x in which the double-stranded nucleic acid was formed and the electrode y in which the double-stranded nucleic acid was formed. It is a quantity. As is clear from Fig. 1, the amount of light emitted from the electrode X on which the double-stranded nucleic acid was formed was double-stranded. When nucleic acid is formed, the amount of luminescence at the electrode y is extremely high compared to the amount of light emitted from the electrode y, and double-stranded nucleic acid can be detected with high sensitivity by using the intercalating agent of this example. I know that there is.
- the gene detection method according to the present invention can detect a gene having a specific sequence with high sensitivity, and can be applied to uses such as gene diagnosis, infectious disease diagnosis, and genome drug discovery.
Abstract
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US11/631,609 US7833406B2 (en) | 2004-07-06 | 2005-06-30 | Gene detection method, and intercalator |
JP2006528805A JP4701176B2 (ja) | 2004-07-06 | 2005-06-30 | 遺伝子検出方法、及び挿入剤 |
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Cited By (1)
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JP2011520123A (ja) * | 2008-05-08 | 2011-07-14 | ボード・オブ・リージェンツ・オブ・ザ・ユニバーシティ・オブ・テキサス・システム | 起電性化学発光で使用する発光ナノ構造化材料 |
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EP1757935A4 (en) * | 2004-04-23 | 2008-01-16 | Matsushita Electric Ind Co Ltd | METHOD AND DEVICE FOR DETECTING GENES |
KR101603354B1 (ko) * | 2008-04-11 | 2016-03-14 | 보드 오브 리전츠 오브 더 유니버시티 오브 텍사스 시스템 | 나노입자 전기발생 화학발광 증폭 방법 및 시스템 |
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JP2002531829A (ja) * | 1998-12-01 | 2002-09-24 | シントリックス バイオチップ, インコーポレイテッド | リガンドアレイを有する多孔性コーティング |
JP2003294700A (ja) * | 2002-03-28 | 2003-10-15 | Toshiba Corp | 試料分離検出用チップ |
JP2003322653A (ja) * | 2002-05-07 | 2003-11-14 | Toshiba Corp | プローブ固定支持体及びプローブ固定担体 |
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US5503721A (en) * | 1991-07-18 | 1996-04-02 | Hri Research, Inc. | Method for photoactivation |
JP2573443B2 (ja) | 1990-09-28 | 1997-01-22 | 株式会社東芝 | 遺伝子検出法 |
US5776672A (en) * | 1990-09-28 | 1998-07-07 | Kabushiki Kaisha Toshiba | Gene detection method |
JP3233851B2 (ja) | 1996-04-24 | 2001-12-04 | 繁織 竹中 | 遺伝子の電気化学的検出法およびその装置 |
US6221586B1 (en) * | 1997-04-09 | 2001-04-24 | California Institute Of Technology | Electrochemical sensor using intercalative, redox-active moieties |
FR2805545B1 (fr) * | 2000-02-24 | 2002-05-17 | Argene Sa | Procede electrochimique de detection d'acides nucleiques |
DK1412487T3 (da) * | 2001-07-30 | 2010-08-30 | Meso Scale Technologies Llc | Assayelektroder der har immobiliserede lipid/proteinlag og fremgangsmåder til at fremstille og anvende disse |
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JP2002531829A (ja) * | 1998-12-01 | 2002-09-24 | シントリックス バイオチップ, インコーポレイテッド | リガンドアレイを有する多孔性コーティング |
JP2003294700A (ja) * | 2002-03-28 | 2003-10-15 | Toshiba Corp | 試料分離検出用チップ |
JP2003322653A (ja) * | 2002-05-07 | 2003-11-14 | Toshiba Corp | プローブ固定支持体及びプローブ固定担体 |
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JP2011520123A (ja) * | 2008-05-08 | 2011-07-14 | ボード・オブ・リージェンツ・オブ・ザ・ユニバーシティ・オブ・テキサス・システム | 起電性化学発光で使用する発光ナノ構造化材料 |
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US20090000962A1 (en) | 2009-01-01 |
JPWO2006003991A1 (ja) | 2008-04-17 |
JP4701176B2 (ja) | 2011-06-15 |
US7833406B2 (en) | 2010-11-16 |
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