WO2005085853A1 - バイオチップおよびその製造方法、ならびに該チップを用いた化学物質の相互作用検出方法 - Google Patents
バイオチップおよびその製造方法、ならびに該チップを用いた化学物質の相互作用検出方法 Download PDFInfo
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- WO2005085853A1 WO2005085853A1 PCT/JP2005/003963 JP2005003963W WO2005085853A1 WO 2005085853 A1 WO2005085853 A1 WO 2005085853A1 JP 2005003963 W JP2005003963 W JP 2005003963W WO 2005085853 A1 WO2005085853 A1 WO 2005085853A1
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- biochip
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- 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
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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
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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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
-
- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/545—Synthetic resin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/548—Carbohydrates, e.g. dextran
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/552—Glass or silica
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
Definitions
- the present invention relates to a biochip, a method for producing the same, and a method for detecting an interaction of a biological substance using the chip.
- biochip-related technology in functional analysis of proteins such as DNA has been recognized.
- biochips those called protein chips, DNA chips, sugar chain chips, and cell chips are generally widely known.
- a protein chip which is a form of a biochip, is used as a means for immobilizing a protein on a substrate and detecting interacting proteins.
- a glass substrate on which a metal thin film for binding a protein to each of several regions on the substrate is coated is formed.
- the treated sample is adopted as a chip, and first, a different protein-containing liquid sample is circulated for each region described above, and the protein of the protein-containing liquid sample is bound to the chemical substance and antigen-antibody on the substrate and fixed to the substrate.
- a different sample liquid sample is circulated for each region to detect the interaction between the sample and the protein immobilized on the substrate (prior art (ii)).
- the interaction between the protein immobilized on the substrate and the sample is detected by detecting the change in mass such as the binding or dissociation between two molecules, V, and the surface plasmon resonance (SPR) signal.
- the method of detecting is adopted.
- a crystalline protein to be immobilized on a substrate is produced by an insect virus, and a protein called a crystal protein inclusion body is used to attach the protein to the substrate.
- a protein called a crystal protein inclusion body is used to attach the protein to the substrate.
- it is fixed (prior art (iv): see JP-A-2003-155300).
- the present invention focuses on and solves the above-mentioned problems of the prior art. Specifically, the present invention provides the following biochip, a method for producing the same, and a biochip using the biochip.
- An object of the present invention is to provide a method for detecting an interaction between substances.
- a first object of the present invention is to enable a chemical substance to be securely fixed to a substrate in a liquid without losing activity, and to provide one or more chemical substances fixed to the substrate.
- An object of the present invention is to provide a Noochip capable of detecting an interaction with a sample in a liquid.
- a second object of the present invention is to enable a chemical substance to be securely fixed to a substrate in a liquid without losing its activity, and to be combined with one or more chemical substances fixed to the substrate.
- An object of the present invention is to provide a method for producing a Noo chip capable of manufacturing a Noo chip capable of detecting an interaction with a sample in a liquid.
- a third object of the present invention is to provide a method in which a chemical substance is securely fixed to a substrate in a liquid without losing its activity, and one or more chemical substances fixed to the substrate and a specimen are combined with each other.
- a chemical substance interaction detection method capable of avoiding noise detection, detecting an interaction appropriately, and detecting the interaction of a chemical substance with a small amount of a sample when performing interaction detection. Nimble.
- the present invention provides a chemical substance-bound carrier in which a chemical substance is bound to a carrier, in a liquid, It is a biochip fixed to a plate.
- the present invention is the above-described nanochip, wherein two or more kinds of chemical substance-binding carriers each having a different chemical substance bonded thereto are fixed to a substrate in a liquid. According to the present invention, it is possible to detect the interaction between a specimen and two or more chemical substances in a liquid.
- the present invention is the above-mentioned biochip, wherein the maximum length of the carrier is 110 ⁇ m.
- a biochip having a high degree of integration can be obtained.
- the present invention is the above biochip, wherein the carrier has at least one kind of power selected from agarose, cellulose, silica, and acrylic acid resin.
- a carrier for general-purpose liquid chromatography can be used.
- the present invention is the above-mentioned biochip, wherein the chemical substance is a biological substance.
- the present invention is the above biochip, wherein the biological substance is at least one selected from the group consisting of proteins, peptides, DNA, RNA, sugar chains, and cells.
- the present invention is a method for producing the above-mentioned biochip, comprising a step of fixing a chemical substance-binding carrier to a substrate in a liquid. According to the present invention, a biochip can be produced without losing activity.
- the present invention is also a method for producing the above-mentioned biochip, comprising a step of disposing a chemical substance-binding carrier on a substrate in a liquid. According to the present invention, it is possible to arrange a carrier at a desired position on a substrate.
- the present invention is a method for producing the above-mentioned biochip, comprising the steps of: disposing a chemical substance-binding carrier on a substrate in a liquid; and immobilizing the chemical substance-binding carrier on the substrate in the liquid. Performing the method.
- the present invention it is possible to arrange a carrier at a desired position on a substrate and produce a biochip without losing activity.
- the present invention is the above-mentioned method, wherein the step of disposing the chemical substance-bound carrier on the substrate is performed by laser masation.
- the present invention is a method for detecting an interaction between a sample and a chemical substance in a liquid using the above-described biochip, wherein the method comprises the steps of:
- the method includes a step of contacting, and a step of detecting an interaction only in a predetermined range of the substrate.
- the interaction is detected only in a predetermined range of the substrate, so that efficient detection can be performed.
- the present invention is the above-mentioned method, characterized in that fluorescence excited by using a process force SNOM probe for detecting an interaction only in a predetermined range is detected.
- one or more chemical substance-bound carriers obtained by binding a chemical substance to a carrier in a liquid can be immobilized on a substrate.
- a biochip that can reliably fix chemical substances to a substrate without losing activity and that can detect the interaction of one or more chemical substances fixed to the substrate with a sample be able to.
- biochip manufacturing method of the present invention in manufacturing a biochip, one or more kinds obtained by binding a chemical substance to a carrier in a liquid.
- the chemical substance binding carrier is fixed to the substrate so that the chemical substance can be securely fixed to the substrate without losing the activity, and one or more chemical substances fixed to the substrate can be fixed.
- a biochip capable of detecting an interaction between a substance and a sample can be manufactured.
- the chemical substance of a chemical substance binding carrier immobilized on a substrate of a biochip and the chemical substance of a desired sample can be used.
- the interaction should be detected only within a predetermined range on a substrate on which one or more chemical substance-bound carriers in which a chemical substance is bound to a carrier.
- Chemicals without losing activity When detecting the interaction between one or more chemical substances fixed to the substrate and the chemical substance of the sample, which is firmly fixed to the substrate, avoid noise detection and detect the interaction appropriately.
- FIG. 1 is a conceptual diagram showing an example of a configuration of a biochip (protein chip) of the present invention.
- (a) is a plan view showing the entire appearance of the biochip
- (b) is a plan view showing an enlarged portion indicated by b in (a)
- (c) is a plan view showing the portion shown in (b). It is a schematic front view which expands and shows a part.
- FIG. 2 is a flowchart showing a method for producing a biochip of the present invention.
- FIG. 3 is a flowchart showing a method for detecting an interaction of a chemical substance using the biochip of the present invention.
- FIG. 4 is a schematic front view showing a state where a specimen is dropped in the present invention.
- FIG. 5 is a plan view showing a fluorescence state in a Noo chip according to the present invention. Here, (a) and (b) are obtained by dropping different specimens.
- FIG. 1 is a conceptual diagram showing an example of the biochip of the present invention.
- the biochip 100 of the present embodiment has a plurality of carriers (beads) 121, 131, 141... In a liquid (not shown).
- examples of the chemical substances A, B, C ′ include biomolecules such as proteins, peptides, DNAs, sugar chains, and cells. Therefore, when a protein is immobilized on a substrate, it constitutes a protein chip, and when DNA is immobilized on a substrate, it constitutes a DNA chip, and any chemical substance according to the purpose is used. be able to.
- Examples of the liquid used in the present invention include a buffer solution and the like. Since the chemical substance-bound carriers 120, 130, 140 are fixed to the substrate 110 in this liquid, the chemical substances A, B, As for C ',', the activity can be prevented from being lost, and the activity can be appropriately maintained.
- Examples of the material of the substrate 110 include a nitrocellulose film, a PVDE film, a metal, and glass.
- the substrate 110 may be in the form of particles. If the chemical substance bonded to the carrier can be easily fixed to the substrate, the following effect can be obtained.
- Examples of the material of the carriers 121, 131, 141 ⁇ ⁇ ⁇ include agarose, cellulose, silica, acrylic acid resin, various fillers for liquid chromatography, and the like.
- the shapes of the carriers 121, 131, 141 ⁇ ⁇ ⁇ are not particularly limited.
- the size of these carriers 121, 131, 141 ⁇ ⁇ ⁇ is not particularly limited, but those having a maximum length of 1 ⁇ m to 10 m are preferably used. If this is the case, the chip itself can be significantly reduced in size, and the effect of enabling detection and analysis with a small amount of sample can be obtained.
- the maximum length refers to the length of the largest portion of the carrier, and if it is in the form of beads, it means its diameter.
- Means for binding the chemical substances A, B, C. to the carriers 121, 131, 141 ⁇ ⁇ ⁇ include, for example, means for amino bonding, means for physical adsorption, and means for thiol bonding. And means for causing an antigen-antibody reaction.
- a chemical treatment a means for amino bonding, a means for physically adsorbing, a thiol, Binding means, antigen-antibody reaction, etc.
- biological modification anti- (A body, a receptor, etc.)
- a means for fixing the substrate by changing the light with irradiation light a means for fixing the substrate by changing the light with irradiation light.
- the biochip 100 two types of chemicals A, B, and C obtained by binding different chemicals A, B, and C to the plurality of carriers 121, 131, 141 in a liquid are described.
- the chemical substance binding carriers 120, 130, and 140 are fixed to the substrate 110, and the chemical substances A, B, and C can be reliably fixed to the substrate 110 without losing activity. It is possible to detect the interaction between two or more chemical substances A, B, and C immobilized on the sample and the chemical substance X in the sample.
- FIG. 2 is a flowchart showing the method for producing a biochip of the present invention.
- a liquid not shown
- two or more types of chemical substances A, B, C ' The procedure of fixing a plurality of chemical substance binding carriers 120, 130, 140,... To the substrate 110 is adopted.
- the desired chemical substances A, B, C When binding the chemical substances A, B, C 'to the carriers 121, 131, 141, as described above, for example, a means for binding to an amino, a means for physically adsorbing, or a means for binding to thiol And means for reacting the antigen and antibody.
- the chemical substance binding carriers 120, 130, 140,... are arranged on the substrate 110, they can be performed by laser manipulation such as optical tweezers.
- a chemical treatment a means for amino bonding or a physical adsorption Means, thiol binding, antigen-antibody reaction, etc.
- biological modification an antibody, receptor, etc.
- a plurality of carriers 121, 131, 141 ⁇ ⁇ ⁇ At least two types of chemical substance binding carriers 120, 130, 140 obtained by combining different chemical substances A, B, C are fixed to the substrate 110, and the activity is not lost.
- the dangling substances A, B, and C can be reliably fixed to the substrate 110 via the carrier, and the two or more chemical substances A, B, and C fixed to the substrate 110 via the carrier and the chemical substance of the sample
- the biochip 110 capable of detecting the interaction with X can be manufactured.
- FIG. 3 is a flowchart showing a method for detecting an interaction of a chemical substance using the biochip of the present invention.
- the interaction detection for example, when the chemical substance of the chemical substance binding carrier is a protein, a means for fluorescently labeling the chemical substance as a specimen and detecting the fluorescence, Means for detecting a change in mass due to binding or dissociation between the two molecules as a surface plasmon resonance signal, means for electrochemically measuring a sample labeled with an electrochemically active substance, and the like can be employed.
- the detection analysis of the chemical substance X of the sample interacting with the chemical substances A, B, C, ... of the chemical substance-bound carriers 120, 130, 140 ⁇ ⁇ ⁇ By dropping the chemical substance X of the fluorescently labeled sample onto the biochip 100, the sample interacting with the chemical substances A, B, C ' The fluorescence of chemical substance X can be detected.
- the chemical substance A of the chemical substance binding carriers 120, 130, 140 fixed on the substrate 110 of the biochip 100 When detecting the interaction between B and C and the chemical substance X of the desired sample, a chemical substance different for each of the carriers 121, 131, 141 with respect to a plurality of carriers 121, 131, 141 in the liquid
- a chemical substance different for each of the carriers 121, 131, 141 with respect to a plurality of carriers 121, 131, 141 in the liquid Each of the chemical substances of the immobilized substance binding carriers 120, 130, 140 fixed on the substrate 110 on which the two or more kinds of chemical substance binding carriers 120, 130, 140 to which A, B, C are bound are fixed.
- the interaction is detected only in the area around A, B, and C (predetermined range), and the chemical substances A, B, and C are securely fixed to the substrate 110 via the carrier without losing the activity.
- the interaction between two or more chemical substances A, B, and C, which are fixed to the substrate 110 via the carrier, and the chemical substance X of the sample is detected.
- noise detection after which it is possible to detect the proper interaction, it is possible interaction detection of chemicals in the low volume sample.
- FIGS. 5 (a) and 5 (b) show the respective fluorescence states of the biochip on which different samples are dropped. As described above, in the present embodiment, the interaction pattern can be easily grasped for each of the different specimens.
- sugar chain analysis the interaction between sugar chains and lectin, which is a generic term for proteins that specifically recognize sugar chains, is observed.
- the interaction between a lectin and a sugar chain is known to have a dissociation constant of about 10-6 M, and this value is considered to be the weakest binding in vivo. In order to measure such a weak interaction, it is useful to carry out the measurement in a solution in which both the sugar chain and the lectin maintain their activity, and are free from binding.
- a lectin chip is prepared. Lectin lost Because it is active, fixation in a solution is useful to maintain activity.
- lectin bound to small particles is fixed to the substrate one by one.
- lectin binding carriers particle size: 1 ⁇ m-10 m
- lectin chromatography which are used as packing materials for lectin chromatography, are immobilized one by one in a solution to prepare a lectin chip with the activity maintained. can do.
- the lectin-binding carrier For fixing the lectin-binding carrier, for example, by using optical tweezers, the lectin can be arranged at an arbitrary position without damaging the lectin. In addition, since it can be immobilized even if any biomolecule is bound, not only with a lectin-binding carrier, it can be applied to any biochip such as a DNA chip that can only be used with a lectin chip and other biomolecule chips. It comes out.
- fluorescence of sugar chains interacting with lectins is observed by dropping sugar chains labeled with fluorescence onto the prepared lectin chip.
- This fluorescence observation is performed, for example, by fluorescence observation using a fluorescence microscope, or by using a probe of a scanning near-field optical microscope (SNOM) to excite only the lectin-bound carrier force at around a few tens of nm.
- SNOM scanning near-field optical microscope
- the biochip of the present invention the method for producing the biochip, and the method for detecting the interaction of a capitaous substance using the chip are expected to be used in the biochip-related technology industry in functional analysis of proteins such as DNA. be able to.
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JP2004-064933 | 2004-03-09 | ||
JP2004064933A JP2005257275A (ja) | 2004-03-09 | 2004-03-09 | バイオチップおよびその製造方法、ならびに該チップを用いた化学物質の相互作用検出方法 |
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JP2000249706A (ja) * | 1999-02-26 | 2000-09-14 | Hokuto Kagaku Sangyo Kk | 新規の生物学的チップ及び分析方法 |
WO2002061126A2 (en) * | 2001-01-30 | 2002-08-08 | Solexa Ltd. | The preparation of polynucleotide arrays |
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JP2000249706A (ja) * | 1999-02-26 | 2000-09-14 | Hokuto Kagaku Sangyo Kk | 新規の生物学的チップ及び分析方法 |
WO2002061126A2 (en) * | 2001-01-30 | 2002-08-08 | Solexa Ltd. | The preparation of polynucleotide arrays |
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