WO2002052266A1 - Procede permettant de fixer un acide nucleique sur un diamant, conjugue diamant-mononucleoside et conjugue diamant-acide nucleique - Google Patents

Procede permettant de fixer un acide nucleique sur un diamant, conjugue diamant-mononucleoside et conjugue diamant-acide nucleique Download PDF

Info

Publication number
WO2002052266A1
WO2002052266A1 PCT/JP2001/011101 JP0111101W WO02052266A1 WO 2002052266 A1 WO2002052266 A1 WO 2002052266A1 JP 0111101 W JP0111101 W JP 0111101W WO 02052266 A1 WO02052266 A1 WO 02052266A1
Authority
WO
WIPO (PCT)
Prior art keywords
diamond
nucleic acid
mononucleoside
conjugate
binding
Prior art date
Application number
PCT/JP2001/011101
Other languages
English (en)
Japanese (ja)
Inventor
Toshihiro Ando
Koichi Ushizawa
Yoichiro Sato
Original Assignee
Japan Science And Technology Corporation
National Institute For Materials Science
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Japan Science And Technology Corporation, National Institute For Materials Science filed Critical Japan Science And Technology Corporation
Publication of WO2002052266A1 publication Critical patent/WO2002052266A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • the present invention relates to a method for binding a nucleic acid to a diamond, a diamond-mononucleoside conjugate used in the method, and a diamond-nucleic acid conjugate obtained by binding a nucleic acid to the diamond-mononucleoside conjugate.
  • diamond is expected to be useful in many other fields.
  • a diamond-mononucleoside conjugate used in this method and a diamond-nucleic acid conjugate obtained by bonding a nucleic acid to the diamond-mononucleoside conjugate.
  • the technology for fixing DNA includes the technology for fixing DNA using a glass slide as a fixing substrate, and is well known as a DNA chip.
  • the DNA chip is used, for example, as a DNA sensor.
  • a DNA having a nucleotide sequence complementary to the DNA sequence to be detected is bound to the glass surface, the nucleic acid of the DNA sequence to be detected is present. And are selectively extracted. As a result, it is possible to confirm the presence or absence of a DNA nucleic acid as a detection target, and it is also possible to perform a physicochemical property test, and thus conduct a drug effect test and the like.
  • nucleic acid base sequence testing identification
  • DNA microarrays Arrayed DNA chips
  • the DNA is immobilized via a conjugated compound (intermediate compound) different from the base material, the bond with glass is indirect, and the bond between the base material and the intermediate layer and the DNA between the intermediate layer and the intermediate layer are indirect. Both are based on Van der Waals forces or weak electrostatic coupling.
  • the bond between the substrate and the DNA is not due to a strong chemical bond, but is due to van der Waals force or the like. It depends on weak electrostatic force. Due to the weak binding force, there are inconveniences such as separation from the DNA force base material and mixing into other spots for various operations for the assay, and the exact characteristics of the DNA to be tested There was also an inconvenience that there was a case where it could not be grasped. In addition, there was also an inconvenience that the degradation of DNA may be caused due to the low biocompatibility of the base material.
  • the present invention has a strong chemical binding property to nucleic acid, excellent stability, and a chemical reaction in an organic solvent, acid or alkaline solution.
  • a nucleic acid / diamond binding method, and a diamond / mononucleoside conjugate, which is a nucleic acid chip, is capable of realizing a nucleic acid chip which is excellent in stability, has a high binding density of nucleic acid, and has excellent biocompatibility and biocompatibility.
  • An object of the present invention is to provide a diamond-nucleic acid conjugate in which a nucleic acid is bonded to a diamond-mononucleoside conjugate.
  • Still another object of the present invention is to provide a nucleic acid sensor that uses a diamond-nucleic acid conjugate to detect a state change of a nucleic acid as an electric signal. Disclosure of the invention
  • a method for binding a nucleic acid to diamond according to the present invention is characterized in that nucleic acid is bound to diamond.
  • the surface carbon atom Ningling bonds can be used for binding to nucleic acids, and binding points to nucleic acids can be dense and uniform.
  • the chemical bond between diamond and dangling bonds is large in binding energy, the bond between dangling bonds and nucleic acids can be strengthened via nucleosides.
  • the carbon atom of diamond is chemically inert, it has high biocompatibility and does not induce nucleic acid alteration.
  • the present invention is characterized in that a diamond / mononucleoside conjugate in which a specific dinucleoside is bonded to the surface of a diamond and a nucleic acid having a specific base at a terminal are complementarily bonded.
  • the mononucleoside specified above is either thymidine, adenosine, guanosine, cytosine, or peridine.
  • the target nucleic acid can be bonded by complementary base binding by binding mononucleosides corresponding to the type of the target nucleic acid to the diamond surface.
  • the above-mentioned diamond mononucleoside conjugate forms a carboxyl group at a carbon atom on the surface of the diamond, halogenates the carboxy group, and binds the hapoxylated lipoxyl group to a specific mononucleoside. It can be formed.
  • the carboxyl group is preferably formed by oxidizing the surface of diamond with an acid and then hydrolyzing it with an alkali.
  • the acid is a mixture of nitric and sulfuric acids and the alkali is caustic soda.
  • a carboxyl group can be formed at a carbon atom on the diamond surface.
  • Halogenated carboxyl groups can be formed by the hydroxylation of the carboxylic acid group.
  • the halogenating agent is preferably composed of any one of chloridion thionyl, phosphorus trichloride, and pentachloride, or a combination thereof.
  • the carboxy group is halogenated, and can be esterified with the mononucleoside.
  • Itoyoshii of a halogenated carboxyl group and a specific mononucleoside is an organic compound containing a catalyst of a halogenated carboxyl group and a specific mononucleoside. It can be formed by an ester reaction in a solvent.
  • a catalyst of a halogenated carboxyl group and a specific mononucleoside can be formed by an ester reaction in a solvent.
  • 3 ⁇ 4 is 4-dimethylaminopyridine
  • the organic solvent is anhydrous pyridine.
  • the complementary bond between the diamond-mononucleoside conjugate and the nucleic acid having a specific base at the end is preferably an enzyme that binds the diamond-mononucleoside conjugate and the nucleic acid in a buffer containing the enzyme.
  • This is a ligation reaction.
  • the enzyme when the terminal of the nucleic acid is adenosine and the mononucleoside of the diamond's mononucleoside conjugate is thymidine, the enzyme is T4 DNA ligase, and the buffer is polyamine and It is a mixture of divalent metal salt and polyethylene glycol.
  • the mononucleoside of the diamond 'mononucleoside conjugate and the base at the end of the nucleic acid are combined with each other to fix the target nucleic acid to the diamond fixing substrate.
  • the present invention is characterized in that a nucleic acid fluorescent dye is added to a diamond / mononucleoside conjugate having a nucleic acid complementarily bonded thereto, the product is irradiated with ultraviolet light to generate fluorescence, and the nucleic acid is detected by the fluorescence.
  • the diamond-mononucleoside conjugate of the present invention is characterized in that it has a mononucleoside bonded to a carbon atom on the surface of the diamond.
  • This diamond 'mononucleoside conjugate has a mononucleoside esterified to a surface atom of the diamond.
  • the diamond is a polycrystalline or single crystal particle having a particle diameter of 10 nm or more, either bulk or thin film. This diamond is an undoped diamond or a doped diamond.
  • the self-doping diamond is an insulator, a conductor, or a semiconductor diamond.
  • the above-mentioned semiconductor diamond is p-type doped with boron or zeolite. Or n-type semiconductor diamond.
  • the target nucleic acid can be firmly fixed on the diamond surface.
  • the diamond-nucleic acid conjugate of the present invention is characterized in that a nucleic acid is complementary-bonded to a mononucleoside of a diamond 'mononucleoside conjugate having a mononucleoside bonded to a carbon atom on the surface of the diamond.
  • the nucleic acid is firmly and densely fixed with good biocompatibility, so that the DNA is not easily exposed to acids, alkalis, organic solvents, or other biomolecules. It is possible to supply a DNA chip capable of performing a physicical dungeon characteristic test.
  • the nucleic acid sensor of the present invention has a feature that it has a conductor diamond, a semiconductor diamond, or a diamond having a pn junction, and detects a state change of a nucleic acid as an electric signal.
  • a change in the state of the nucleic acid can be immediately detected as an electric signal, and the change in the state of the nucleic acid can be performed with high precision, at a high speed, and at a high speed.
  • the present invention has been completed based on the following findings of the inventors.
  • Diamond has unique properties.
  • the present invention effectively applies the unique properties of diamond to the field of genetic engineering.
  • Diamond is composed of strong covalent bonds of carbon atoms.
  • the carbon atoms on the surface can form a strong bond with hydrogen, oxygen, fluorine, etc., that is, a stable chemisorption state.
  • the hydrogen adsorbed by dangaku is stable even when exposed to air up to about 200 ° C. even at room temperature.
  • the surface condition of the diamond does not change.
  • These properties of diamond are in contrast to many other semiconductors and metals. Many semiconductors and metals are easily oxidized by surface forces to form an oxide phase there.
  • the surface of diamond chemically adsorbed with the above-mentioned elements is inert and stable to organic solvents such as ethanol.
  • Diamond can impart conductivity without impairing the above excellent properties.
  • impurities such as boron (p-type), phosphorus (n-type), and zeo (n-type) may be doped.
  • boron p-type
  • phosphorus n-type
  • zeo n-type
  • only the surface can be made conductive by adsorbing hydrogen on the surface.
  • the use of carbon nanotubes enables ultra-fine lines.
  • the present invention utilizes an organic chemical reaction technology and a genetic engineering technology to bond DNA to a diamond surface by chemical bonding. As a result, a strong bond in which the nucleic acid is chemically bonded to the diamond is obtained. Since the bond between the nucleic acid and the diamond is strong and stable as described above, and as described in i) above, the surface of the diamond is stable. However, reliable data can be obtained without breaking the binding force between the nucleic acid and the diamond or generating undesirable side reaction force.
  • a nucleic acid molecule bound to the surface of the immobilized base material is brought into contact with another nucleic acid, amino acid, or a biomolecule such as a protein enzyme.
  • diamond is excellent in biocompatibility, so that a preferable side reaction does not occur and the nucleic acid is not deteriorated, so that reliable data can be obtained.
  • nucleic acid and diamond are in a chemically stable bond state, and as described in iii) above, diamond can add conductivity without sacrificing surface stability and biocompatibility. Can be detected as an electric signal on the diamond side. For example, charge transfer (electrons or holes) between the nucleic acid and the diamond surface due to a change in the state of the nucleic acid, such as when the nucleic acid binds to the diamond surface or when the nucleic acid force that has bound to the diamond surface comes into contact with a living body. Or a change in dipole interaction occurs, which can be detected as an electrical signal on the diamond side Noh.
  • the target nucleic acid can be detected with high density and stability. It is possible to provide a diamond-nucleic acid conjugate which has a strong chemical bond and excellent stability, and can be used in an organic solvent, an acid or a weak alkaline solution.
  • the nucleic acid sensor of the present invention by using the nucleic acid sensor of the present invention, the presence or absence of a nucleic acid or a change in the state of a nucleic acid can be electrically detected.
  • the diamond'mononucleoside conjugate of the present embodiment uses diamond as a fixed base material.
  • the diamond bonded to the mononucleoside may be a natural diamond or a human diamond.
  • a natural diamond or a human diamond.
  • Artificial diamonds include Ib, IIa, and lib shapes. In the present embodiment, any of them can be preferably used.
  • Diamond can change physical or chemical properties such as electrical conductivity, surface structure and surface characteristics by doping impurities.
  • diamond doped with boron, phosphorus, zeolite, etc. exhibits p-type and n-type conductivity. Utilizing this conductivity, the charge transfer resulting from the change in the state of the nucleic acid, for example, the charge transfer resulting from the binding between the nucleic acid and diamond may be detected.
  • the p-type layer and the n-type layer may be stacked to form a pn junction, and the charge transfer may be detected using this junction.
  • Doped diamond may exhibit high electrical resistance and may also exhibit other good physicochemical properties.
  • Diamond that has hydrogen adsorbed only on a specific surface has conductivity only on the specific surface.
  • Diamonds with specific sites on the surface connected by conductive polymers or partially connected by carbon nanotubes have only specific lines conductive. diamond • These diamonds can be appropriately selected according to the intended use of the nucleic acid conjugate.
  • the diamond particle size is not a major issue. It can be widely used from a very small particle size to a relatively large surface area of, for example, about ten and several mm 2 .
  • the particle size, surface area and the like may be appropriately selected according to the application.
  • the shape does not matter. For example, it may be plate-shaped. Generally speaking when referring to the particle size, for example 2-5 / m, more preferably 1-2m is most desirable when it is most desirable. If the particle size is less than 1 / m, the separation of the diamond during the organic chemical reaction will be difficult, and there is a possibility that the L is preferable. When it exceeds 100; m, the surface area per unit mass becomes narrow. This may lead to a decrease in the reaction volume, making it difficult to confirm the presence or absence of the reaction, for example, when confirming it with fluorescence.
  • the difference in crystal plane does not basically pose a major problem.
  • the symmetry of the sequence of the binding sites (binding points of nucleic acids) on the surface and the distance between the sites vary depending on the crystal plane.
  • the maximum number of bindable nucleic acid molecules It depends on the force crystal plane, such as the density, the angle between the bound molecule and the crystal plane, and the degree of freedom for the rotation of the molecule. If necessary, the crystal plane can be selected.
  • bonding at a unique site such as a step or kink on a crystal surface takes an array structure different from that of a flat crystal surface, so that this array structure can be effectively utilized depending on the purpose.
  • diamond can also effectively utilize its polycrystal as a fixed base material.
  • various forms of polycrystalline diamond that is, particulate, film-like (plate-like), needle-like (fibrous), and network diamond can be synthesized.
  • the technique of depositing a polycrystalline film on another substrate is most widely applied, and by using this technique, a substrate having the required shape and structure is first prepared, and the surface is diamond-coated.
  • a diamond substrate having a desired shape and structure can be produced.
  • the shape can be controlled not only by synthesis, but also by thermal etching using a gas such as oxygen, fluorine, or hydrogen, plasma etching using a gas containing these elements, or ion sputtering.
  • Etching is for fine surface morphology, such as microfacet and nano It is effective for forming aggregates of needle-shaped diamonds on the order of 1 to micron. Even if the size of the particles constituting the polycrystal is nanometer or several microns or more, there is basically no big problem. As described above, the polycrystal can be used for constructing a diamond fixed base material having various shapes according to the application.
  • Nucleic acids include DNA and RNA. In the present embodiment, both can be preferably used. Nucleic acids can be of any molecular weight. DNA and RNA having different structural formulas may be mixed and mixed on the diamond surface.However, usually, DNA and RNA having the same structural formula suitable for the intended use are used by uniformly binding them. You. When DNA is bound as a nucleic acid, it is often good to use double-stranded DNA. In the case of double-stranded DNA, only one complementary nucleic acid molecule binds to the diamond surface, and the other nucleic acid molecule complementarily binds to the nucleon bound to the diamond surface. May be indirectly bonded to the diamond. With this configuration, it is possible to convert a double-stranded DNA into a single-stranded DNA.
  • the bond between the diamond and the nucleic acid is complementary to the carbon atom on the diamond surface and the nucleic acid via, for example, an ester-bonded nucleoside.
  • the nucleoside structure at the extreme end of the chain has a complementary bond with a carbon atom on the surface of diamond, for example, a nucleoside that is ester-bonded.
  • the thymidine structure is, for example, an ester bond with a carbon atom on the surface of diamond.
  • a diamond-nucleic acid conjugate in which a nucleic acid is bound to diamond can be produced, for example, by going through three or four steps by the following method.
  • the first step is a step of binding a mononucleoside to the surface of the diamond.
  • the second step is a step of replicating the nucleic acid itself.
  • the third step is a step of binding a mononucleoside-bound daiden, ie, a diamond-mononucleoside conjugate, to a nucleic acid body.
  • a diamond 'nucleic acid conjugate in which the nucleic acid is bonded to diamond is obtained.
  • the fourth step is to use diamond 'mononucleoside conjugate And confirming the binding to the nucleic acid body.
  • the second step can be omitted if the necessary amount of the nucleic acid is sufficiently secured in advance.
  • the first step may be performed, for example, as follows.
  • the surface is oxidized by heating the diamond with a mixed acid solution of concentrated nitric acid and sulfuric acid.
  • the mixed acid concentration ratio is preferably 1: 8 to 9 by the mass ratio of concentrated nitric acid to concentrated sulfuric acid.
  • the mixing ratio between diamond and the mixed acid is preferably 500 to 900 parts by mass with respect to 100 parts by mass of the diamond.
  • the reaction temperature is preferably 150 to 200 ° C, and the reaction time is preferably several (about 7 to 8) hours to 3 days. During the reaction, the reaction solution may be refluxed.
  • the diamond whose surface has been exposed to the mixed acid may then be hydrolyzed with an alcohol. Prior to hydrolysis, it is preferably washed thoroughly with pure water.
  • Na2OH aqueous solution or the like is preferably used for the decomposition of carohydrate.
  • the concentration is preferably 0.1 to 0.2 mol Z liter.
  • the amount used may be from 12 to 20 parts by mass per 100 parts by mass of diamond.
  • the reaction temperature is preferably 80 to 90 ° C, and the reaction time is preferably several (about 7 to 8) hours to 3 days.
  • hydrochloric acid having a concentration of 0.1 to 0.2 mol / liter is preferable. In this way, many carboxylic groups are formed on the diamond surface.
  • carboxyl groups can be formed with higher reproducibility by smoothing the diamond surface by exposing it to microwave hydrogen plasma.
  • the carbon atoms on the diamond surface are hydrogenated to form CH, CH 2 , CH 3 groups and the like, and carboxyl groups can be formed with higher reproducibility.
  • the carboxyl-bonded diamond-carboxylic acid then halogenates the hydroxyl groups in the carboxylic acid. Thereby, a diamond acid halide is obtained.
  • a halogenating agent for example, thionyl chloride, phosphorus trichloride, phosphorus pentachloride or the like may be used alone or in combination.
  • the obtained diamond ⁇ -halogenide and mononucleoside are then The reaction is carried out by introducing the compound into a solvent and synthesizes a diamond-mononucleoside conjugate.
  • the mononucleoside to be subjected to the reaction may be L of thymidine, adenosine, guanosine, cytosine, or lysine, or may be different. These similar structures may be used.
  • thymidine may be selected as the mononucleoside to be used for the reaction.
  • the bond between the acid halide and the mononucleoside is preferably an ester reaction, for example.
  • Preferred examples of the organic solvent include anhydrous pyridine.
  • As a reaction catalyst 4-dimethylaminopyridine is preferred.
  • the reaction temperature is preferably room temperature, and the reaction time is preferably (3-4) hours to 3 days.
  • reaction formula is shown below based on an example where the carbon atom on the diamond crystal surface constitutes a methyl group. It is not limited to the methyl group, CH, CH 2 or other alkyl groups derconnection may.
  • [C] of [C] -CH 3 indicates a diamond crystal, and 1 C indicates a surface carbon atom of the diamond crystal. .
  • the second step may be performed, for example, as follows.
  • the PCR method is used.
  • the gene containing information of the nucleic acid that binds to diamond is amplified by involving a primer that transcribes the relevant range.
  • ⁇ 3 ⁇ 4 may be automatically performed using a thermal cycler device or the like.
  • a primer having a phosphate group at the 5 'end.
  • the nucleic acid is strongly bonded to the 5 'end of the amplified nucleic acid.
  • Many of the PCR-amplified DNAs have adenosine bound to the 3 'end, and when adenosine is bound, ligation (enzymatic reaction) with thymidine complementary thereto proceeds smoothly.
  • the amplification method is not limited to the PCR method.
  • the best method can be selected by comprehensively determining the labor, time, recovery rate, etc.
  • the third step may be performed, for example, as follows.
  • nucleic acid main body molecule a chain reaction between the nucleic acid main body molecule and the diamond-mononucleoside conjugate is caused to occur.
  • Mononucleosides and chain-like nucleic acids bound to diamond The endmost nucleoside structure of the main molecule may not be complementary.
  • a nucleoside complementary to the mononucleoside bound to the diamond may be further bound to the extreme end of the chain nucleic acid body molecule and extended.
  • the nucleic acid body molecule may be shortened until a mononucleoside in a complementary relationship appears at the extreme end.
  • the nucleic acid having adenosine bonded to the 3 ′ end and the diamond-thymidine conjugate having thymidine force binding may be bound in the presence of, for example, T4 DNA ligase.
  • the binding reaction is usually performed in a buffer.
  • a buffer a mixed solution of a polyamine, a divalent metal salt, and polyethylene glycol is often used.
  • the divalent metal salt for example, Mg 2 + salt is used. Kit products are sold by Takara Shuzo (supplier).
  • the reaction temperature is preferably about 16 ° C.
  • the reaction time is 90 to 120 hours. Usually, it may be performed using a heat block device. "TaKa Ra PCR Thermal Cycler 480" (trade name) is known.
  • An example of the reaction formula is shown below as an example of a binding reaction between a diamond-thymidine conjugate and a double-stranded DNA having a terminal adenosine.
  • COT indicates thymidine ester. The thymidine structure and the adenosine structure bind complementarily.
  • a shi-) ⁇ After the elapse of the DNA reaction time, remove the unreacted DNA from the diamond that is presumed to have nucleic acid binding. For example, water washing is performed. The number of washings should be 5 to 7 times.
  • the fourth step is performed, for example, as follows.
  • nucleic acid fluorescent stain examples include the chemical name EtBr (ethidiumpromide).
  • EtBr ethidiumpromide
  • Gelster solution trade name “GelSter Nucleic Acid Stain> manufactured by BMA.
  • Fluorescence detection is performed as follows. For example, dilute TBE buffer (Tris-Borate-EDTA buffer) 0.5 times. Gelster solution is added to the TBE buffer diluent, and the Gelster solution is diluted 5000 to 10,000 times with TBE buffer diluent. For comparison with the TBE buffer diluent for fluorescence detection, prepare approximately the same amount of pure water separately from the TBE buffer diluent as a reference. It is advisable to prepare a TBE buffer of normal concentration before diluting 0.5-fold by dissolving trisaminomethane, boric acid and EDTA in pure water in advance. The normal concentration of TBE buffer is usually 10.8 g of TBE buffer, 5.5 g of boric acid, 0.5 g of boric acid. Introduce about 4 ml of EDTA solution / liter concentration.
  • a diamond that is presumed to have nucleic acid bound thereto is introduced into a TBE buffer diluent obtained by diluting the Gelster solution.
  • a result usually appears in about 20 minutes.
  • a part of the diamond, which is presumed to be bound to the nucleic acid is also introduced into pure water prepared as a reference.
  • the wavelength of the ultraviolet light is preferably from 302 to 312 nm. It is good to use a transilluminator as a confirmation device. For example, there is a 3UV transilluminator manufactured by Funakoshi, model NLMS-20. Gender equivalent It does not matter what model it has. Fluorescence is not confirmed in the pure water of the comparison standard, but fluorescence is confirmed in the TBE buffer diluent obtained by diluting the Gelster solution, thereby confirming that the nucleic acid is bound to the diamond.
  • the diamond-nucleic acid conjugate of the present embodiment obtained in this manner is driologically stable and stable in many organic solvents, many acidic solutions, and many alkaline solutions.
  • organic solvent include ethyl acetate, phenol, chloroform, pyridine, benzene, tetrachloride, dimethyl sulfoxide (DMSC), and dimethylformamide (DMF).
  • DMSC dimethyl sulfoxide
  • DMF dimethylformamide
  • There are many other alcohols such as ethanol. These organic solvents may be used alone or in combination according to physicochemical properties.
  • the acidic liquid examples include acetic acid, benzoic acid solution, boric acid water, carbonated water, and the like.o In addition, 0.1 mol concentration of hydrogen fluoride water, 0.1 mol concentration of hydrogen chloride water, etc. Can also be mentioned. These acidic liquids may be used alone or in combination according to physicochemical properties.
  • the alkaline solution, bicarbonate Natoriumu salt solution, primary phosphate Natoriumu salt (N a H 2 P0 3) an aqueous solution, or the like can be mentioned. These may be used alone or in combination according to physicochemical properties.
  • a DNA chip using a slide glass as a DNA chip.
  • the present embodiment can provide a more stable and highly reliable chip with a higher degree of integration.
  • the nucleoside which is a structural unit of DNA, is firmly fixed to the substrate by a chemical bond, and the fixing strength is stable.
  • Diamond as a substrate Do itself is chemically stable.
  • the diamond-nucleic acid conjugate of the present embodiment has high durability and can stably maintain the original chemical structure for a long period of time.
  • the reliability of the analysis data is also high. It is also possible to use various organic solvents
  • the DNA can be arranged in a high density plane. Since diamond itself is a biocompatible material, it is unlikely to induce destruction or alteration of biomolecules.
  • the reaction in which the DNA acts is extracted as an electrical signal, and the obtained electrical signal is appropriately processed and processed by a logic circuit in the element to identify the element, thereby realizing a specific function.
  • conductive diamond for the substrate, such an intelligent device becomes possible, and broad application fields can be expected.
  • the DNA sensor It is necessary for the DNA sensor to take out a signal generated by contact with another molecule with a certain signal with high sensitivity. Any new state change in the DNA causes a change in electrical energy transfer, charge transfer, or dipole interaction between the DNA and the diamond. If a conductive diamond is used, it is possible to take out these electrical changes and to image the detection data.
  • Nucleic acid molecules in a solution are usually in a free state where they can swim, Does not occur, and it is currently difficult to effectively suppress the effects. If DNA is immobilized on diamond and DNA and, for example, protein are allowed to act in a solution, it is thought that highly accurate data with few errors can be obtained. For example, it is possible to easily investigate which site force acts on a protein molecule and what kind of interaction it is.
  • proteins with large molecular weights that are difficult to analyze by NMR are crystallized and the structure is determined by X-ray structural analysis. Therefore, its usefulness has been recognized, and it is difficult to crystallize the protein while its structural analysis is in a hurry, and the number of proteins that have not been analyzed is increasing.
  • a combination technique of an organic chemistry method using a specific organic reaction or vibrational spectrum
  • an atom probe such as AFM is used.
  • AFM atom probe
  • the present invention is a basic technology in such a case.
  • the present invention is a basic technology for fixing a DNA to a substrate at one or more locations.
  • Diamond powder (classified to a particle size of 1-211) was treated with a mixed solution of sulfuric acid and nitric acid to remove impurities such as metals that could be mixed. After washing with pure water and drying, the surface of the diamond was smoothed and hydrogenated by microwave plasma CVD equipment at about 800 ° C for 1 hour with hydrogen plasma at a pressure of 4 OTorr.
  • the diamond reacted with the mixed acid was washed with pure water.
  • 0.03 liters of an aqueous solution of sodium hydroxide having a concentration of 0.1 MZ liter was prepared, and all the diamonds washed with water were immersed and allowed to stand in a thermostat at 90 ° C. for several days. Then, after washing thoroughly with pure water, it was allowed to stand at 90 ° C. in 0.1 MZ hydrochloric acid for several days. As a result, a large number of carboxyl groups were bonded to the diamond surface.
  • thionyl chloride was reacted with the diamond having the carboxy group bonded thereto as described above to obtain diamond / acid halide.
  • the reaction amount was 0.04 mol of 1 g of diamond to thionyl chloride.
  • the reaction temperature was maintained at normal temperature or higher and 50 ° C or lower. Using an oil bath, the mixture was stirred at Magnetics Yuichira.
  • the resulting diamond acid halide and thymidine were then introduced into an organic solvent, anhydrous pyridine, and reacted. 1 g of diamond acid nodogenide, 0.058 of thymidine, and 0.03 liter of organic solvent were used.
  • As a reaction catalyst 4-dimethylaminopyridine was used. The reaction temperature was room temperature of 18 to 25 ° C, and the reaction time was 3 hours to 3 days.
  • thermocycler which is a method amplifier
  • a part of the ansipirin resistance gene was expanded by 123 base pairs.
  • a primer having a phosphate group at the 5 'end of the primer was used, and a phosphate group was added to the 5' end of the amplified DNA.
  • the total amount of the diamond 'thymidine conjugate was 1 g, and 0.001 g of the conjugate was complementarily bound to the nucleic acid, and the rest was stored as a comparative sample.
  • the nucleic acid amplified by the PCR method amplifier and the diamond-thymidine conjugate were complementarily bound as follows.
  • the reaction was carried out in a TBE buffer using T4DNALigase as a ligase and a DNA Ligation kit Ver 2> manufactured by Takara Shuzo Co., Ltd.
  • a heat block was used as the reactor, the reaction temperature was 16 ° C, and the reaction time was 2 hours. After the reaction time had elapsed, the diamond presumed to have the nucleic acid bound thereto was washed several times with water.
  • a commercially available diamond powder of the same type as in Example 1 (classified to a particle size of 1 to 2 / im) was similarly treated with a mixed solution of sulfuric acid and nitric acid to remove impurities. This is washed with pure water, dried, and then subjected to microwave plasma CVD with 0.5 V o 1% of methane gas (CH 4 ), 5 O vol ppm of diborane gas (B 2 H 6 ), and the remaining amount of hydrogen gas (approx. (95 V 0 1%) mixed gas as raw material, sample temperature 850. Grow for 2 hours at C I got it.
  • CH 4 methane gas
  • B 2 H 6 diborane gas
  • the original diamond is covered with an epitaxial layer of boron-doped ⁇ -type diamond with an average thickness of about 0.5 ⁇ , and a semiconductor diamond with a grain size of 2-3 m is obtained.
  • a starting material thymidine binding, amplification of DNA and treatment of ligase were carried out in the same manner as in Example 1, and finally, DNA binding was confirmed by a fluorescence detection test.
  • Example 1 The diamond-nucleic acid conjugate obtained in Example 1 was immersed in 0.02 liter of hydrogen fluoride water, and the liquid was stirred at 200 rpm for 2 hours. The concentration of aqueous hydrogen fluoride was adjusted to 0.1 mol Z liter.
  • the diamond / nucleic acid conjugate was subjected to spectral inspection with an extended Fourier transform infrared spectrometer (commonly known as DRIFT). No dangling or physical changes were observed in the nucleic acid.
  • DRIFT extended Fourier transform infrared spectrometer
  • Example 1 The diamond-nucleic acid conjugate obtained in Example 1 was treated in the same manner as in Example 2 except that a 5% by weight aqueous solution of sodium hydrogen carbonate was used instead of the aqueous hydrogen fluoride solution. No chemical or physical changes were observed in the nucleic acids.
  • Example 1 The diamond'nucleic acid conjugate obtained in Example 1 was treated in the same manner as in Example 2 except that a pyridine solution was used instead of the hydrogen fluoride water. No chemical or physical changes were observed in the nucleic acids.
  • Example 1 The diamond-nucleic acid conjugate obtained in Example 1 was immersed in 0.02 liter of sodium methylate solution and stirred at 200 rpm for 2 hours. DRIFT showed that the nucleic acid had completely disappeared from the diamond. When the mass was measured, the difference between before and after immersion in the sodium methylate solution clearly showed that 10 ng of DNA force was bound per 1 cm 2 of the diamond surface.
  • Example 2 Poly L one lysine surface responsibility 1 cm 2 in coated ground glass, the DNA by attaching to a glass slide, a nucleic acid conjugate of DNA and identical chemical structure as used in Example 1 Preparation Then, the same experiment as in Example 2, Example 3, or Example 4 was performed.
  • a slide glass having a chemical structure identical to that of the diamond nucleic acid conjugate and the nucleic acid portion of Example 1 and a DNA conjugate were prepared, and 0.2 liter of hydrogen fluoride water (concentration 0.1 mol / liter) was prepared. ) And the solution was stirred at 200 rpm for 2 hours. Examination by DRIFT showed that the nucleic acid had completely disappeared from the slide glass. The mass was measured and compared with the mass before immersion. As a result, it was found that 0.1 ng of DNA per 1 cm 2 of the surface glass was bound to the slide glass' nucleic acid conjugate.
  • the chemical binding with the nucleic acid is strong and the chemical stability is excellent, and the method can be used in an organic solvent, an acid or an alkaline solution.
  • a nucleic acid chip with excellent chemical stability, high nucleic acid binding density, and excellent biocompatibility and biocompatibility can be realized.
  • the chemical bond with nucleic acid is strong and the chemical stability is excellent, and the chemical stability is excellent in an organic solvent, an acid or an alkaline solution.
  • the chemical bond with nucleic acid is strong and excellent in chemical stability, the chemical stability in an organic solvent, acid or alkaline solution is excellent, and It can be used as a DNA specimen with high binding density and excellent biocompatibility and biocompatibility. Then, the presence or absence of a nucleic acid can be detected by using the method for detecting the presence or absence of a nucleic acid using the diamond'mononucleoside conjugate of the present invention.
  • nucleic acid sensor that uses the diamond-nucleic acid conjugate of the present invention and detects a state change of a nucleic acid as an electric signal is used, the state change of a nucleic acid can be detected as an electric signal.

Abstract

L'invention concerne un conjugué diamant-acide nucléique qui comprend une fraction acide nucléique liée par une liaison normale haute densité, qui présente une liaison chimique forte avec une base solide, qui est chimiquement stable et peut être utilisé de manière stable dans des solvants organiques etc., et qui ne provoque jamais de détérioration de l'acide nucléique. Le diamant est relié par une liaison covalente à la thymidine de manière à former un conjugué diamant-thymidine. On fait ensuite réagir la structure thymidine de ce conjugué diamant-thymidine avec un acide nucléique de manière à la lier avec ce dernier. Pour lier le diamant avec la thymidine, on expose une poudre de diamant à un acide fort afin de fixer un groupe carboxyle à la surface du diamant puis la thymidine est reliée par l'intermédiaire d'une liaison ester à ce groupe carboxyle. Un acide nucléique est ensuite lié à la thymidine sous la liaison ester. L'acide nucléique utilisé est un acide nucléique comprenant une adénosine reliée à l'extrémité 3'. La réaction d'amplification par PCR provoque normalement la liaison d'une adénosine à l'extrémité 3', permettant ainsi d'utiliser l'adénosine, qui est complémentaire à la thymidine, pour fixer l'acide nucléique.
PCT/JP2001/011101 2000-12-25 2001-12-18 Procede permettant de fixer un acide nucleique sur un diamant, conjugue diamant-mononucleoside et conjugue diamant-acide nucleique WO2002052266A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2000-393331 2000-12-25
JP2000393331A JP4019167B2 (ja) 2000-12-25 2000-12-25 核酸とダイヤモンドの結合方法

Publications (1)

Publication Number Publication Date
WO2002052266A1 true WO2002052266A1 (fr) 2002-07-04

Family

ID=18859150

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2001/011101 WO2002052266A1 (fr) 2000-12-25 2001-12-18 Procede permettant de fixer un acide nucleique sur un diamant, conjugue diamant-mononucleoside et conjugue diamant-acide nucleique

Country Status (3)

Country Link
JP (1) JP4019167B2 (fr)
TW (1) TWI318297B (fr)
WO (1) WO2002052266A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006320271A (ja) * 2005-05-20 2006-11-30 Tokyo Institute Of Technology 低分子化合物とタンパク質の結合評価方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999040173A1 (fr) * 1998-02-09 1999-08-12 Toyo Kohan Co., Ltd. Substrats d'immobilisation et d'amplification de l'adn, cristaux de blocage d'adn avec de l'adn bloque sur les substrats, et procede d'amplification de l'adn
WO1999041362A1 (fr) * 1998-02-10 1999-08-19 Toyo Kohan Co., Ltd. Appareil pour preparation de genotheque immobilisee, appareil pour amplification de gene, procede de regulation de temperature et procede de comparaison systematique des genes
WO2000022108A1 (fr) * 1998-10-15 2000-04-20 Toyo Kohan Co., Ltd. Supports utilises pour immobiliser de l'adn ou autre

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999040173A1 (fr) * 1998-02-09 1999-08-12 Toyo Kohan Co., Ltd. Substrats d'immobilisation et d'amplification de l'adn, cristaux de blocage d'adn avec de l'adn bloque sur les substrats, et procede d'amplification de l'adn
WO1999041362A1 (fr) * 1998-02-10 1999-08-19 Toyo Kohan Co., Ltd. Appareil pour preparation de genotheque immobilisee, appareil pour amplification de gene, procede de regulation de temperature et procede de comparaison systematique des genes
WO2000022108A1 (fr) * 1998-10-15 2000-04-20 Toyo Kohan Co., Ltd. Supports utilises pour immobiliser de l'adn ou autre

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TAKAHASHI K. ET AL.: "Preservative type of DNA chips using diamond", BIO INDUSTRY, vol. 17, no. 6, 12 June 2000 (2000-06-12), pages 44 - 51, XP002909343 *

Also Published As

Publication number Publication date
JP2002195996A (ja) 2002-07-10
JP4019167B2 (ja) 2007-12-12
TWI318297B (fr) 2009-12-11

Similar Documents

Publication Publication Date Title
JP5743974B2 (ja) Ｄｎａ検出用ナノチューブセンサー装置
Zagorodko et al. Highly sensitive detection of DNA hybridization on commercialized graphene-coated surface plasmon resonance interfaces
Liu et al. A sandwich-type DNA biosensor based on electrochemical co-reduction synthesis of graphene-three dimensional nanostructure gold nanocomposite films
JP2009509175A (ja) 官能性電極上の選択的結合事象を電気的に読み取ることにより分子及びナノ材料を分析する方法及びｃｍｏｓ型デバイス
Gong et al. A sensitive impedimetric DNA biosensor for the determination of the HIV gene based on electrochemically reduced graphene oxide
TWI519774B (zh) 表面增強拉曼散射用金屬粒子及分子感測、以及其製造方法
JP2002542794A (ja) 標識担持標的を検出するための単層および電極ならびにその使用方法
Peng et al. Silver nanoclusters-assisted triple-amplified biosensor for ultrasensitive methyltransferase activity detection based on AuNPs/ERGO hybrids and hybridization chain reaction
Furst et al. DNA-modified electrodes fabricated using copper-free click chemistry for enhanced protein detection
JP2008525822A (ja) ポリヌクレオチド配列のｄｎａ検出／認識用ナノ電子装置
AU2007333001A2 (en) Nanoplasmonic molecular ruler for nuclease activity and DNA footprinting
US6194148B1 (en) Method for detecting a hybridized nucleic acid molecule
Veerbeek et al. Selective functionalization with PNA of silicon nanowires on silicon oxide substrates
Duman et al. A new approach for immobilization of oligonucleotides onto piezoelectric quartz crystal for preparation of a nucleic acid sensor for following hybridization
Dawood et al. Nanostructured Si-nanowire microarrays for enhanced-performance bio-analytics
Al Dine et al. Self-Limited Grafting of Sub-Monolayers via Diels–Alder Reaction on Glassy Carbon Electrodes: An Electrochemical Insight
Ye et al. Screen-printed electrode-based biosensors modified with functional nucleic acid probes and their applications in this pandemic age: a review
Fei et al. Sensitive and selective DNA detection based on the combination of hairpin-type probe with endonuclease/GNP signal amplification using quartz-crystal-microbalance transduction
Moazampour et al. Development of an electrochemical genosensor for quantitative determination of miR-200a based on the current response of ferrocene-functionalized graphene oxide nanosheets
WO2002052266A1 (fr) Procede permettant de fixer un acide nucleique sur un diamant, conjugue diamant-mononucleoside et conjugue diamant-acide nucleique
JP2003083968A (ja) Ｄｎａチップおよびアッセイ方法
Lai Folding-and dynamics-based electrochemical DNA sensors
JP4706074B2 (ja) 生体分子固定化用の三脚型機能性界面分子とこれを用いた遺伝子検出デバイス
Cha et al. Quantitative analysis of H5N1 DNA hybridization on nanowell array electrode
AU2004202326B2 (en) Method for the detection of nucleic acids

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application