WO2002016571A1 - Particules magnetiques ayant une temperature de solution critique de limite inferieure - Google Patents
Particules magnetiques ayant une temperature de solution critique de limite inferieure Download PDFInfo
- Publication number
- WO2002016571A1 WO2002016571A1 PCT/JP2001/007119 JP0107119W WO0216571A1 WO 2002016571 A1 WO2002016571 A1 WO 2002016571A1 JP 0107119 W JP0107119 W JP 0107119W WO 0216571 A1 WO0216571 A1 WO 0216571A1
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- WIPO (PCT)
- Prior art keywords
- fine particles
- magnetic fine
- biotin
- critical solution
- solution temperature
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
- C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
- C07D495/04—Ortho-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
- C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
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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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
- G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/255—Salmonella (G)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
- G01N2446/86—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids the coating being pre-functionalised for attaching immunoreagents, e.g. aminodextran
Definitions
- the present invention relates to a magnetic fine particle having a lower critical solution temperature, a method for converting a substance using the same, a method for separating or concentrating a microorganism, a method for modifying a denatured protein, a method for detecting a nucleic acid, a separating agent, and a biological material. And a method for separating the same.
- the present inventors have conducted intensive studies in view of the above-mentioned problems of the related art. As a result, if magnetic particles having a lower critical solution temperature and at least one selected from biotin and avidin are immobilized, enzymes and antibodies are immobilized on the polymer on the magnetic particles for each purpose of use. The present inventors have found that there is no need to perform the process, and it is no longer necessary to directly bind nucleic acids to proteins and the like, and based on this finding, have completed the present invention.
- an object of the present invention is to provide a magnetic fine particle having a lower critical solution temperature that is highly versatile in applications such as a separating agent, a method for converting a substance using the same, and a method for separating microorganisms.
- Another object of the present invention is to provide a concentration method, a modified protein modification method, a nucleic acid detection method, and a biological substance separation method.
- the present invention has the following configuration.
- Magnetic fine particles having a lower critical solution temperature in which at least one selected from biotin and avidin is immobilized.
- a method for modifying a denatured protein comprising using the magnetic fine particles having a lower critical solution temperature according to any one of the above items 1 to 8.
- a method for purifying, detecting, or concentrating a nucleic acid comprising using the magnetic fine particles having a lower critical solution temperature according to any one of the above items 1 to 8.
- a method for detecting a nucleic acid comprising amplifying a nucleic acid obtained by the method for purifying or concentrating a nucleic acid according to the above (12).
- a separating agent containing the magnetic fine particles according to any one of (1) to (8).
- biotin means biotin or iminobiotin
- avidin means avidin or streptavidin
- the magnetic fine particles having LCST used in the present invention refer to a solvent containing magnetic fine particles when the temperature of the solvent containing the magnetic fine particles falls below a specific temperature. When the temperature of the magnetic fine particles becomes higher than the specific temperature, the magnetic fine particles are precipitated and aggregated in the solvent.
- LCST is the specific temperature.
- the phenomenon in which the magnetic fine particles dissolve and precipitate at the boundary of the LCST is called LCST characteristics.
- the above-mentioned solvent is not particularly limited, and specific examples thereof include water and a liquid containing 50% by weight or more of water. Further, specific examples of the liquid containing 50% by weight or more of water include physiological saline and buffer.
- the solvent may be a mixture of an organic solvent such as acetone and water, as long as the solvent exhibits the LCST characteristic in a state containing the polymer.
- composition and structure of the LCST magnetic fine particles used in the present invention are not particularly limited, but it is preferable that the polymer having LCST is immobilized on the surface of the magnetic fine particles.
- Examples of the polymer having an LCST include poly N-substituted acrylamide derivatives and copolymers thereof, poly N-substituted methacrylamide derivatives and copolymers thereof, polyvinyl methyl ether, polypropylene oxide, and polyethylene oxide. Examples include xide, poly (N-vinylalkylamide), and poly (N-isopropylacrylamide).
- the magnetic fine particles to which the polymer having LCST is immobilized are not particularly limited, but the material may be an organic substance or an inorganic substance as long as it is a substance exhibiting magnetism at room temperature. Specific examples include nickel oxide particles, ferrite particles, magnetite particles, cobalt iron oxide, barium fluoride, carbon steel, tungsten steel, KS steel, fine particles of rare earth cobalt magnets, and hematite particles. Can be done.
- the particle size of the magnetic fine particles is not particularly limited in the present invention, even if the magnetic force is applied to the solvent in which the magnetic fine particles are dispersed, as long as the magnetic fine particles do not adsorb the magnetic fine particles. Although not limited, it is preferably in the range of 1 nm to 1 m, more preferably 1 ⁇ ! 1100 nm.
- the method for preparing the magnetic fine particles used in the present invention will be described by taking a case where magnetite is used as an example. Magnetite is formed into double micelles using sodium oleate and sodium dodecylbenzenesulfonate, and by dispersing the micelles in an aqueous solution, magnetic magnetite fine particles having a particle size of several tens of nm are obtained. Can be. This method is described in Biocatalysis 1991, Vol. 5, pp. 61-69.
- Immobilization of the LCST polymer on the surface of the magnetic fine particles may be performed by any of the functions. Physical adsorption or chemical bonding such as hydrogen bonding or covalent bonding may be used. Specific examples of the immobilization method include a method of polymerizing an LCST polymer in the presence of magnetic fine particles, a method of bringing an LCST polymer into contact with magnetic fine particles in a solvent, and a method of coupling a magnetic fine particle having a functional group such as an SH group. A method in which a binding agent is bonded and the LCST polymer is graft-polymerized based on the functional group.
- the magnetic fine particles of the present invention obtained by a method of polymerizing an LCST polymer in the presence of the magnetic fine particles are immobilized even when repeated dissolution, precipitation, and aggregation in the liquid containing the same.
- the LCST polymer is preferably used because it is difficult to detach from the magnetic fine particles.
- the method for immobilizing at least one selected from biotin and avidin on the LCST polymer immobilized on the magnetic microparticles is not particularly limited, and the already synthesized LCST polymer may be selected from biotin and avidin.
- Immobilization of one or more of the above hereinafter referred to as the “immobilization method”
- polymerization method or polymerization of a monomer whose structure is composed of biotin or avidin and a monomer that forms an LCST polymer by polymerization.
- polymerization method To introduce one of the pair of substances into the LCST polymer (hereinafter referred to as “polymerization method”).
- the immobilization method described above is preferably a covalent bond, but may be a bond using an ion complex or a charge transfer complex, or a bond using biochemical affinity.
- the polymerization method can be preferably used in the present invention because the polymerization ratio in the polymer is relatively easy to control.
- the method for immobilizing biotin on the UCST polymer immobilized on magnetic fine particles will be described in detail, taking the polymerization method using a monomer having biotin as a part of its structure as an example.
- Examples of monomers that form an LCST polymer by polymerization include N-substituted acrylamide derivatives, N-substituted methacrylamide derivatives, vinyl methyl ether, propylene oxide, ethylene oxide, N-vinyl alkylamide, and N- And isopropylacrylamide (the following general formula (1)).
- Examples of the monomer having piotin as a part of its structure include (meth) acrylami and (meth) acrylate derivatives using a terminal carboxyl group of piotin, but are not limited to these in the present invention. Not something.
- Examples of the monomer which forms the LCS polymer which can be preferably used in the polymerization method include the above-mentioned isopropyl acrylamide, and as a monomer having biotin as a part of its structure. May include a polymerizable biotin derivative represented by the following general formula (2).
- R 2 represents a hydrogen atom or an alkyl group.
- R 3 and R 4 each independently represent a hydrogen atom, an alkyl group or an aryl group.
- W represents a single bond, a carbonyl group, a thiocarbonyl group or an alkylene group having 1 to 5 carbon atoms.
- U represents a single bond or —NH— group.
- X represents a single bond, a hydrocarbon bond having 1 to 8 carbon atoms, an oxygen atom or an —NH— group.
- Y represents a single bond or a carbonyl group, a thiocarbonyl group, an —NH group, a 1,2-dioxyethylene group or a 1,2-diaminoethylene group.
- Z represents a single bond or a carbonyl group, a thiocarbonyl group, an alkylene group having 1 to 5 carbon atoms, an oxygen atom or a mono-NH— group.
- V represents a single bond or an alkylene group having 1 to 5 carbon atoms.
- polymerizable biotin derivatives represented by the following general formulas (3) to (5) can be preferably used in the polymerization method.
- R 1 represents a single bond or an alkylene group having 1 to 4 carbon atoms
- R 5 represents an alkylene group having 2 or 3 carbon atoms
- X 1 represents an oxygen atom or a sulfur atom
- X 2 to X 5 each independently represent an oxygen atom or a mono-NH— group.
- T, R 2 , R 3 and R 4 are the same as defined in the above general formula (2).
- the polymerizable biotin derivative represented by the above general formula (3) is generally obtained by appropriately removing the side chain carboxyhydroxyl group of biotin or the biotin derivative represented by the following general formula (6). After conversion into a group, it can be obtained by a condensation reaction with an acrylic derivative represented by the following general formula (7).
- the polymerizable biotin derivative represented by the general formula (4) generally comprises a biotin derivative represented by the following general formula (8), and a suitable acrylate agent (including a methacrylate agent, for example, acrylic acid, acrylic acid chloride).
- a suitable acrylate agent including a methacrylate agent, for example, acrylic acid, acrylic acid chloride.
- Acrylic acid, acrylic acid, acryloxysuccinimide, etc. acrylic acid, methacrylic acid, methacrylic acid chloride, methacrylic anhydride, methacryloxysuccinimide, etc.
- a cryating agent, etc. A cryating agent, etc.
- the polymerizable biotin derivative represented by the above general formula (5) is generally obtained by converting a biotin derivative represented by the following general formula (9) into tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), ether, dimethylformamide (DMF). ), Dichloromethane, chloroform, ethyl acetate, acetone, aliphatic hydrocarbons, benzene, toluene, etc., in an aprotic solvent such as an isocyanate substance represented by the general formula (10). Further, a polymerizable biotin derivative represented by the following general formula (11) can be particularly preferably used in the polymerization method.
- a biotin metaclearamide derivative represented by the general formula (12) and a biotin derivative represented by the general formula (13) can be exemplified.
- the polymerization method will be described more specifically.
- a solution containing 0.01 to 10% by weight of a monomer forming an LCST polymer the above biotin monomer is preferably used in a molar ratio (monomer forming an LCST polymer: iminopiotin monomer) of 10000 :: !! To 5: 1, more preferably 1000: 1 to 10: 1, and magnetic fine particles are further added to 0.01 to 5% by weight.
- a polymerization initiator eg, potassium persulfate
- a polymerization accelerator eg, tetramethylene diamine
- the polymerization may be performed in a nitrogen atmosphere.
- the solution temperature during the polymerization is not particularly limited, but is preferably about 20 to 60 ° C.
- the molecular weight of the obtained polymer is not particularly limited, but is preferably about tens of thousands to one million.
- the LCST magnetic fine particles of the present invention are produced by a method of polymerizing an LCST polymer in the presence of magnetic fine particles, impurities such as unreacted monomers and salts react. Coexists in liquid.
- the removal of the contaminants may be performed by dialysis, or may be performed by setting the temperature of the solution to L CST or higher, aggregating the solution, collecting the aggregate with a magnet, and removing the supernatant.
- immobilization to LCST magnetic microparticles may be at least one selected from biotin and avidin.However, it is difficult to immobilize avidin directly to LCST polymer. Preferably, there is.
- a method for immobilizing avidin on LCST magnetic fine particles a method for immobilizing avidin on UCST polymer via biotin is preferable. That is, avidin is preferably immobilized on the LCST polymer in the form of avidin-linked biotin.
- biotin is immobilized on the magnetic fine particles having LCST, it becomes possible to selectively separate and recover only the target substance on which avidin is immobilized. If immobilized, it is possible to selectively separate and recover only the target substance immobilized with biotin.
- magnetic particles obtained by immobilizing a substance having a specific action on the target substance and avidin immobilized on the LCST magnetic microparticles of the present invention on which biotin is immobilized by a biotin-avidin bond The present invention in which avidin is immobilized by biotin-avidin binding to a substance having a specific action and a biotin immobilized on the LCST magnetic fine particles of the present invention in which avidin is immobilized.
- Magnetic particles obtained by immobilizing on LCST magnetic particles of high efficiency can convert, separate, concentrate and recover substrates with high efficiency.
- a substance having a specific action and a biotin-immobilized substance, which has a specific action mutually, is applied to the LCST magnetic fine particles of the present invention having avidin immobilized thereon.
- magnetic particles obtained by immobilizing the avidin-immobilized LCST magnetic particles of the present invention can keep up to three sites open from the four sites of avidin-biotin binding. Therefore, the conversion, separation, concentration, and recovery of the substrate of the biotinylated target substance can be performed with higher efficiency.
- the substance having a specific action mutually with the target substance is not particularly limited, and specific examples include enzymes, proteins, nucleic acids, peptides, molecular chaperones, heat shock proteins, and antibodies. be able to.
- an enzyme when used as a substance having a specific action with respect to a target substance, it is possible to convert a substrate at a faster rate than a conventional enzyme reaction using an immobilized enzyme.
- the enzyme used at this time is not particularly limited, and examples thereof include an oxidoreductase, a transferase, a hydrolase, a degradase, an isomerase, and a synthase.
- the antibody used in this case may be a monoclonal antibody or a polyclonal antibody.
- the method for separating and concentrating the microorganisms in the solution is not particularly limited. Specifically, the magnetic fine particles are added to a solution containing the microorganism, and the microorganisms and the magnetic fine particles are sufficiently contacted. Thereafter, a method of raising the temperature of the solution to precipitate only the magnetic fine particles on which the microorganisms are adsorbed and removing only the precipitated magnetic fine particles by using a magnetic force can be mentioned. With this method, microorganisms in the solution can be easily separated and concentrated.
- the antibody used is a Salmonella antibody
- only the Salmonella bacteria in the food suspension can be easily separated and concentrated, so that the magnetic fine particles of the present invention having any antibody immobilized thereon can be used.
- an appropriate detection reagent it is also possible to make high-grade microbial test kits or diagnostic agents.
- the LCST magnetic microparticles of the present invention using molecular chaperones or heat shock proteins as substances that have a specific action with the target substance can be repeatedly released because the stability of enzymes and antibodies in the solution is increased. It can support protein production and substance production at the industrial level. Usually, proteins such as molecular chaperones are expensive and difficult to use on an industrial level.
- the LCST magnetic fine particles of the present invention using nucleic acid as a substance having a mutual specific action with the target substance are added to a solution containing nucleic acid, and the magnetic fine particles and nucleic acid are sufficiently contacted.
- a method of precipitating only the magnetic fine particles to which the nucleic acid is adsorbed and removing only the precipitated magnetic fine particles by using a magnetic force can be mentioned.
- the nucleic acid in the solution can be easily separated and concentrated.
- the method for separating and concentrating the nucleic acid can be applied to purification, concentration, and detection of a specific gene.
- the nucleic acid is aggregated and recovered by raising the temperature of the mixed solution, and then the temperature is increased again. By lowering it, it is possible to easily purify, detect, and concentrate any nucleic acid.
- the target DNA or mRNA can be concentrated and purified.
- the nucleic acid can be detected with high sensitivity by amplifying the obtained nucleic acid by various gene amplification methods.
- the nucleic acid amplification method is not particularly limited, but the PCI3 ⁇ 4RT-PCR method can be preferably used in the present invention.
- the separating agent of the present invention is not particularly limited as long as it contains the LCST magnetic fine particles of the present invention, but the content ratio of the magnetic fine particles to the separating agent is in the range of 1 to 100% by weight. It is preferable that the content be in the range of 2 to 30% by weight.
- separating agent of the present invention microorganisms, nucleic acids, proteins, peptides, antigens, environmental hormones and the like can be easily separated.
- Magnetic fine particles were prepared by the following method.
- LCST magnetic fine particles were prepared by the following method. 4 ml of the magnetic fine particles prepared in Example 1, 0.488 g of N-isopropylacrylamide, 12.7 mg of N-piotinyl-methylchlorotrimethyleneamide and 94 ml of distilled water were added to a 300 ml 1-volume flask. Then, the mixture was stirred well at room temperature. 0.1 g of potassium persulfate was added thereto, and the mixture was stirred at room temperature for 6 hours. The resulting solution was dialyzed all day and night to obtain an LCST magnetic fine particle solution having biotin immobilized thereon.
- Example 4 After well mixing the LCST magnetic fine particle solution 501, 1.0% avidin solution 501, 1.0M sodium phosphate buffer (pH 7.0) 1001, and distilled water 800/1 obtained in Example 2 in a test tube The temperature of the solution was raised to 31 ° C or higher. The aggregate was collected with a neodymagnet (4300 G), the supernatant 1001 was removed, denatured with sodium dodecyl sulfate (SDS), and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It was confirmed that there was no band corresponding to avidin.
- SDS-PAGE SDS-polyacrylamide gel electrophoresis
- the temperature of the solution was raised to 31 ° C or higher.
- the aggregate was collected with a neodymium magnet, 100 l of the supernatant was removed, and after denaturation treatment with SDS, SDS-PAGE confirmed that only the band corresponding to avidin was removed from the supernatant.
- Example 5 [Immobilization of avidin-immobilized enzyme on LCST magnetic fine particles]
- LCST magnetic fine particle solution 100 ⁇ 1 obtained in Example 2 commercially available avidin-immobilized peroxide solution (lmg / ml) 1000/1, l.OM sodium phosphate buffer (pH 7.0) 100/1 And distilled water 7001 were added and mixed well. The resulting solution was heated, the aggregates were collected with a magnet, the supernatant 1900/1 was removed, and a fresh 0.1M phosphate buffer (pH 7.0) 1900 ⁇ 1 was added. An LCST magnetic fine particle solution having immobilized thereon was prepared. This solution dissolved below LCST and aggregated above LCST.
- the temperature of the solution was changed in a thermostat, and the operations of dissolution, aggregation, and recovery with a magnet were performed, and the activity of peroxidase in the supernatant was determined as shown below. Oxidase activity was measured. Incidentally, after the recovery of the magnetic particles of the magnet was removed the supernatant 1 9 00 ⁇ 1 each time, was added freshly 0.1M phosphate buffer (pH 7.0) 1900 1.
- Table 11 shows the results of measurement of the enzyme activity of the supernatant in the case where aggregation and dissolution were repeatedly performed by the above method.
- the enzyme activity was shown as a specific activity when the activity at the time of the first dissolution was defined as loo.
- Example 6 [Immobilization of biotin-immobilized enzyme on avidin-immobilized LCST magnetic microparticles] First, in order to obtain avidin-immobilized LCST magnetic microparticles in a state where three sites of avidin-biotin binding were vacant, the procedure of Example 2 was repeated The resulting LCST magnetic particle solution 50 After mixing 1.0% avidin solution 500 ⁇ 1 and 1.0M sodium phosphate buffer (pH7.0) 100 l distilled water 3501 in a test tube, place in a constant temperature bath at 40 ° C.
- the temperature was increased to 31 ° C or higher.
- the aggregates were collected with a neodymium magnet (4300 G) to obtain avidin-immobilized LCST magnetic microparticles in which the biotin-binding site other than the polymer binding site was vacant.
- This avidin-immobilized magnetic fine particle solution 1001 commercially available avidin-immobilized peroxidase solution (lmg / ml) 1000Z1, 100M sodium phosphate buffer (pH 7.0) 100 ⁇ 1 and distilled water 7001 And mixed well. The resulting solution was heated, the coagulated matter was collected with a magnet, the supernatant 1900 11 was removed, and a fresh 0.1 M phosphate buffer (pH 7.0) 19001 was added, and the biotin-immobilized peroxide was added. Avidin-immobilized LCST magnetic microparticles with immobilized enzyme were prepared.
- Example 8 [Method of separating and concentrating microorganisms]
- a commercially available biotin-immobilized Salmonella antibody was immobilized on LCST magnetic microparticles in the same manner as in Example 6. The immobilization was confirmed using SDS-PAGE. Subsequently, the magnetic fine particle solution lm 1 was added to 20 ml of a bacterial suspension prepared so that the number of Salmonella was 1 cell / m 1, and after stirring well, the solution was heated in the same manner as in Example 3, After aggregating the magnetic fine particles, they were collected with a magnet, and the supernatant was removed to obtain an lml solution.
- Distilled water 450 ⁇ 1 Commercially available Piochin of labeled DNA fragment 5 00 ⁇ 1 (50 ⁇ 1000bp), avidin immobilization is empty Piochin binding site other than the binding site to the polymer one prepared in Example 6 After adding 50 zl of LCST magnetic fine particles and mixing well, the solution was heated in the same manner as in Example 3, the magnetic fine particles were aggregated, collected by a magnet, and the DNA fragment of the supernatant was confirmed by Rose gel electrophoresis. It was suggested that both DNA fragments were bound to the magnetic microparticles.
- the magnetic fine particles having the lower critical solution temperature of the present invention in which at least one selected from biotin and avidin is immobilized can eliminate the need to immobilize enzymes, antibodies, etc. to the polymer on the magnetic fine particles for each purpose of use. Furthermore, since there is no need to directly bind nucleic acids to proteins and the like, it is highly versatile in applications such as separation agents.
- the method of the present invention for converting a substance using a magnetic fine particle having a lower critical solution temperature includes: It is possible to achieve the object efficiently.
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JP2002522245A JP4797144B2 (ja) | 2000-08-21 | 2001-08-20 | 下限臨界溶液温度を有する磁性微粒子 |
DE60138195T DE60138195D1 (de) | 2000-08-21 | 2001-08-20 | Magnetische partikel mit kritischer lösungstemperatur am unteren bereich |
EP01956971A EP1312671B1 (en) | 2000-08-21 | 2001-08-20 | Magnetic particles having lower limit critical solution temperature |
US10/362,174 US7695905B2 (en) | 2000-08-21 | 2001-08-20 | Magnetic fine particles having lower critical solution temperature |
US11/030,149 US20050158782A1 (en) | 2000-08-21 | 2005-01-07 | Magnetic fine particles having lower critical solution temperature |
US12/219,266 US8313902B2 (en) | 2000-08-21 | 2008-07-18 | Magnetic fine particles having lower critical solution temperature |
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Also Published As
Publication number | Publication date |
---|---|
US7695905B2 (en) | 2010-04-13 |
EP1312671A4 (en) | 2004-07-28 |
US20030165962A1 (en) | 2003-09-04 |
JP4797144B2 (ja) | 2011-10-19 |
US20050158782A1 (en) | 2005-07-21 |
EP1312671A1 (en) | 2003-05-21 |
EP1312671B1 (en) | 2009-04-01 |
DE60138195D1 (de) | 2009-05-14 |
US20080293118A1 (en) | 2008-11-27 |
US8313902B2 (en) | 2012-11-20 |
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