JP4985916B2 - Method for producing carboxyl group-containing particles - Google Patents

Description

  The present invention uses a carboxyl group and can be chemically bonded to a biological substance such as an antibody or an antigen, and contains a carboxyl group with little non-specific adsorption of biological protein or nucleic acid. The present invention relates to a method for producing particles.

  Organic polymer particles and magnetic particles are used as a reaction solid phase of a diagnostic agent utilizing antigen-antibody reaction in order to detect test substances such as infectious diseases, cancer markers, and hormones. In such a diagnostic agent, an inspection probe (primary probe) such as an antibody or an antigen is immobilized on the particle. The substance to be inspected in the sample is captured on the particle via the primary probe and then reacts with the second inspection probe. The second inspection probe (secondary probe) is labeled with a fluorescent substance or an enzyme, and is detected by fluorescence or an enzyme reaction. In recent years, for the purpose of early detection of diseases, etc., it has been required to increase the sensitivity of tests, and improving the sensitivity of diagnostic agents has become a major issue. Also in diagnostic agents using magnetic particles, in order to improve sensitivity, a method using enzyme color development as a detection method is being switched to a method using fluorescence or chemiluminescence that can obtain higher sensitivity.

  With the development of these detection techniques, it is theoretically said that it has reached a level at which even the presence of a single molecule of a test substance can be detected, but in reality, sufficient sensitivity has not been obtained. The cause is that non-specific adsorption of secondary probes and contaminants to the particle surface. For example, even with an inspection technique that can theoretically detect a single molecule of a test target substance, single molecule detection is impossible when several molecules of secondary probes are adsorbed nonspecifically on the particle surface. For this reason, there is a strong demand to reduce the nonspecific adsorption of substances used for testing on the particle surface.

  Conventionally, a method called blocking has been performed as a method for reducing such non-specific adsorption. Blocking is a method in which the primary probe is immobilized on the particle surface, and then the particle surface is coated with a blocking agent such as albumin or skim milk that hardly adsorbs secondary probes or contaminants. However, the covering effect of the blocking agent may not be sufficiently obtained, and the quality stability of the blocking agent which is a biological material may be low. On the other hand, even when blocking is sufficiently performed, nonspecific adsorption may occur due to changes in the action over time due to alteration of the blocking agent, etc., and sufficient nonspecific adsorption reduction effect has not been obtained It was.

  As a method for solving the problem of non-specific adsorption, a method of introducing a hydrophilic polymer to the surface of an immunoassay substrate typified by a 96-well plate has been proposed (Patent Documents 1 to 3). However, in the immunoassay substrate using such a flat surface, the area for immobilizing the primary probe is limited, and the reaction between the primary probe and the test substance is a solid-liquid reaction. There are disadvantages such as poor efficiency and long inspection time.

  Furthermore, as countermeasures for reducing non-specific adsorption, microspheres (Patent Documents 4, 5, and 6) in which a physiologically active substance is bound to organic polymer particles made of a styrene-glycidyl methacrylate copolymer or the like via a spacer, Organic polymer particles (Patent Documents 7 and 8) in which hydrophilic spacers are introduced on the particle surface have been proposed. However, none of these has a sufficient effect of reducing non-specific adsorption, and the sensitivity is insufficient for immunoassay.

As hydrophilic monomers, the present inventors have hydroxyalkyl (meth) acrylate, alkoxyalkyl (meth) acrylate, polyoxyalkylene (C2-C4) group-containing (meth) acrylate, epoxy group-containing (meth) acrylate, and phosphorylcholine analog. A magnetic particle for immunoassay with little non-specific adsorption, in which a group-containing monomer or the like is copolymerized on the particle surface, has been proposed (Patent Document 9), but further highly sensitive expression is desired.
Japanese Patent Laid-Open No. 11-174057 JP 2000-304749 A JP 2001-272406 A Japanese Patent Laid-Open No. 10-195099 JP 2000-300823 A International Publication No. 04/025297 Pamphlet JP 2004-331953 A International Publication No. 04/040305 Publication Pamphlet JP 2005-69926 A

  An object of the present invention is to provide a method for producing carboxyl group-containing particles, which can obtain a highly sensitive and low noise carboxyl group-containing particle with a small amount of non-specific adsorption of a biological substance.

  In order to achieve the above object, the present inventors have conducted extensive research and obtained carboxyl group-containing particles obtained by reacting the hydroxyl group of the organic polymer particles having a hydroxyl group with a carboxylic acid anhydride to form an ester bond. However, the present inventors have found that nonspecific adsorption of biologically related substances such as proteins and nucleic acids is extremely small, and that high sensitivity that is special in the fields of biochemistry and pharmaceuticals is developed.

The method for producing a carboxyl group-containing particle according to the first aspect of the present invention includes:
A step of reacting the hydroxyl group of the organic polymer particles having a hydroxyl group with a carboxylic acid anhydride to form an ester bond.

The method for producing carboxyl group-containing particles according to the second aspect of the present invention comprises:
A step of reacting a hydroxyl group derived from the 2,3-dihydroxypropyl group of the organic polymer particles having a 2,3-dihydroxypropyl group with a carboxylic acid anhydride to form an ester bond.

  In the method for producing the carboxyl group-containing particles, the organic polymer particles may contain a magnetic substance.

The carboxyl group-containing particles of the third aspect of the present invention are
It includes a mother particle including a core particle, a magnetic layer of superparamagnetic fine particles provided on the surface of the core particle, and an organic polymer layer made of a crosslinked polymer provided so as to cover the mother particle.

  According to the above-mentioned method for producing carboxyl group-containing particles, the amount of non-specific adsorption of biologically related substances such as proteins and nucleic acids is small, high sensitivity that is outstanding in the biochemistry / pharmaceutical field, and for biochemical examinations. Particles capable of obtaining a high S / N ratio can be obtained.

  When the particles obtained by the above-described method for producing carboxyl group-containing particles are used, for example, as a diagnostic agent utilizing an antigen-antibody reaction, it is possible to express high sensitivity and low noise.

1. Carboxyl group-containing particles and method for producing the same The method for producing carboxyl group-containing particles according to an embodiment of the present invention includes a step of reacting the hydroxyl group of the organic polymer particles having a hydroxyl group with a carboxylic acid anhydride to form an ester bond. including. That is, the carboxyl group-containing particles obtained by the production method have a functional group having an ester bond.

1.1. Method for producing carboxyl group-containing particles 1.1.1. Organic polymer particles having a hydroxyl group The hydroxyl group of the organic polymer particle is preferably an alcoholic hydroxyl group, more preferably a polyhydric alcohol containing α, β-diol, most preferably 2,3-dihydroxy. Propyl group. The organic polymer particles preferably have at least a hydroxyl group on the surface.

  Examples of the organic polymer particles having a hydroxyl group include the following (i) to (iv).

(I) Organic polymer particle having 2,3-dihydroxypropyl group (i) The 2,3-dihydroxypropyl group has two hydroxyl groups in one functional group. (I) The organic polymer particles having a 2,3-dihydroxypropyl group include, for example, homopolymerizing a monomer (A) having a 2,3-dihydroxypropyl group (hereinafter also simply referred to as “monomer (A)”). It may be produced by copolymerization with other monomers, or the monomer (B) having a 2,3-dihydroxypropyl group (hereinafter also simply referred to as “monomer (B)”) by hydrolysis is homopolymerized. Alternatively, it may be produced by copolymerizing with other monomers to obtain an organic polymer and hydrolyzing the organic polymer. Alternatively, organic polymer particles may be produced using both monomer (A) and monomer (B). Since more hydroxyl groups can be introduced, it is more preferable to use the monomer (B) in that lower noise can be expressed and polymerization stability is excellent.

  Here, examples of the monomer (A) having a 2,3-dihydroxypropyl group include radical polymerizable monomers such as glycerol (meth) acrylate and allylglycerol ether.

  Moreover, as a monomer (B) which produces | generates a 2, 3- dihydroxypropyl group by hydrolysis, the monomer which has a functional group which protected the hydroxyl group with the well-known protecting group is mentioned, for example, (B-1) 2,3 -Monomers having an epoxypropyl group, (B-2) a monomer obtained by acetalizing a 2,3-dihydroxypropyl group, (B-3) a monomer obtained by silylated a 2,3-dihydroxypropyl group. Specific examples of the monomer (B-1) having a 2,3-epoxypropyl group include glycidyl (meth) acrylate and allyl glycidyl ether. (B-2) Specific examples of monomers obtained by acetalizing 2,3-dihydroxypropyl groups include 1,3-dioxolan-2-one-4-ylmethyl (meth) acrylate and 1,3-dioxolane-2. , 2-dimethyl-4-ylmethyl (meth) acrylate and the like. (B-3) As a monomer obtained by silylating a 2,3-dihydroxypropyl group, specifically, a di (t-butyl) silylated product of 2,3-dihydroxypropyl (meth) acrylate, 2,3-dihydroxy A di (trimethylsilyl) product of propyl (meth) acrylate can be exemplified.

  The conditions for hydrolysis of the functional group contained in the monomer (B) depend on the type of the monomer (B), but are usually heated with acid, base, or fluoride salt as a catalyst in a state where particles are dispersed in water. The functional group is hydrolyzed by stirring for several hours to several tens of hours under conditions. The hydrolysis of the functional group contained in the monomer (B) does not necessarily require that all the functional groups in the monomer (B) are hydrolyzed as long as storage stability is not hindered. Although the hydrolysis of the functional group contained in the monomer (B) is usually carried out after the polymerization of the monomer part, a part thereof may be hydrolyzed during the polymerization.

  The monomer (A) and the monomer (B) are preferably copolymerized with the crosslinkable monomer (C). The crosslinkable monomer (C) (hereinafter also simply referred to as “monomer (C)”) can be copolymerized with other monomers to be used, and has two or more radically polymerizable unsaturated bonds in one molecule. It is a monomer having.

  Examples of the crosslinkable monomer (C) include ethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, pentaerythritol triacrylate, pentaerythritol trimethacrylate, dipentaerythritol hexaacrylate, Examples thereof include polyfunctional (meth) acrylates such as dipentaerythritol hexamethacrylate, conjugated diolefins such as butadiene and isoprene, divinylbenzene, diallyl phthalate, allyl acrylate, and allyl methacrylate.

  Examples of the crosslinkable monomer (C) further include hydrophilic monomers such as polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, and poly (meth) acrylic ester of polyvinyl alcohol.

  The ratio of the crosslinkable monomer (C) is preferably 0 to 30% by weight and more preferably 5 to 20% by weight in 100% by weight of the copolymer. If the ratio of the monomer (C) in the copolymer exceeds 30% by weight, the particles may become porous and increase nonspecific adsorption.

  The monomer (A) and the monomer (B) may further be copolymerized with another monomer (D). Other monomers (D) include (meth) acrylates having a hydrophilic functional group such as 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, methoxyethyl acrylate, methoxyethyl methacrylate, polyethylene glycol monoacrylate, and polyethylene glycol monomethacrylate. Hydrophilic monomers such as acrylamide, methacrylamide, N-methylol acrylamide, N-methylol methacrylamide, diacetone acrylamide, and aromatic vinyl monomers such as styrene, α-methylstyrene, halogenated styrene, vinyl acetate, Vinyl esters such as vinyl propionate, unsaturated nitriles such as acrylonitrile, methyl acrylate, methyl methacrylate, ethyl acrylate Ethylenic unsaturation such as ethyl methacrylate, butyl acrylate, butyl methacrylate, 2-ethylhexyl acrylate, 2-ethylhexyl methacrylate, lauryl acrylate, lauryl methacrylate, stearyl acrylate, stearyl methacrylate, cyclohexyl acrylate, cyclohexyl methacrylate, isobornyl acrylate, isobornyl methacrylate Carboxylic acid alkyl esters can be exemplified. As the other monomer (D), a monomer having a carboxyl group such as acrylic acid, methacrylic acid, maleic acid, and itaconic acid may be used as long as the effects of the present invention are not hindered. Preferably no monomer is used.

  The most preferred monomer combination is 80-95 parts of monomer (B) and 5-20 parts of monomer (C).

  The organic polymer particles having a hydroxyl group obtained by polymerization of the above monomer can be produced by using a conventional method such as emulsion polymerization, soap-free polymerization, suspension polymerization or the like. More specifically, a two-stage swelling polymerization method using seed particles (mother particles) described in JP-B-57-24369, Journal of Polymer Science, Polymer Letter Edition, 937, Vol. 21, 1963 (J. Polymer. Sci., Polymer Letter Ed. 21,937 (1963)), JP-A Nos. 61-215602, 61-215603, and 61-215604. It can be produced by the method described in the publication. Among these methods, the two-stage swelling polymerization method using seed particles (mother particles) is preferable in that the coefficient of variation in particle size can be reduced. As the seed particles (mother particles), polystyrene or a styrene copolymer can be suitably used. And the polymer part added by a two-stage swelling polymerization method consists of a copolymer of the above-mentioned monomer. In these polymerizations, known emulsifiers, dispersants, initiators and the like can be used.

  As described above, radical polymer particles have been described as organic polymer particles having 2,3-dihydroxypropyl groups, but polyurethanes end-capped with glycerin or the like as condensation polymer particles having 2,3-dihydroxypropyl groups have been described. Examples thereof include particles, polyamide particles, and polyester particles.

  (Ii) Organic polymer particles having a polyhydric alcohol containing α, β-diol other than the above (i) include, for example, polyurethane particles, polyamide particles, polyester particles and the like end-capped with sugar or reducing sugar; Examples thereof include condensation polymer particles end-capped with reducing sugars; crystalline polysaccharides such as chitin, chitosan, starch, and cellulose; and crosslinked particles of natural or synthetic polysaccharides such as crosslinked dextrin, crosslinked cyclodextrin, and crosslinked pullulan.

  (Iii) Examples of the organic polymer particles containing an alcoholic hydroxyl group other than the above (i) and (ii) include monomers having a hydroxyalkyl group such as hydroxyethyl (meth) acrylate and hydroxybutyl (meth) acrylate. ) Polymer particles; Partial hydrolysis products of polyvinyl acetate particles; Cross-linked polyvinyl alcohol particles.

  (Iv) Examples of organic polymer particles having a hydroxyl group other than the above (i) to (iii) include, for example, silanol-modified organic polymer particles; composite polymer particles of metal oxides such as silica, alumina, and titania and organic polymer particles Polysilsesquioxane particles and the like.

  In the organic polymer particles having a hydroxyl group used in the method for producing a carboxy group-containing particle according to an embodiment of the present invention, the hydroxyl group is a functional group capable of reacting with a carboxylic acid anhydride to form an ester bond. In addition, it is a factor expressing low nonspecific adsorption and high sensitivity. The amount of the hydroxyl group is preferably 10 μmol / g or more, more preferably 50 μmol / g or more, and most preferably 100 μmol / g or more based on the solid content of the organic polymer particles having a hydroxyl group. When the amount of the hydroxyl group is less than 10 μmol / g, nonspecific adsorption may increase.

1.1.2. Carboxylic acid anhydride and ester bond thereof The carboxylic acid anhydride that can be used in the method for producing a carboxy group-containing particle according to an embodiment of the present invention is a polyvalent carboxylic acid anhydride, and specific examples thereof include itaconic anhydride. Aliphatic dicarboxylic acid anhydrides such as acid, succinic anhydride, citraconic anhydride, dodecenyl succinic anhydride, tricarbanilic anhydride, glutaric anhydride, maleic anhydride, hexahydrophthalic anhydride, methyltetrahydrophthalic anhydride, and hymic anhydride; Alicyclic polycarboxylic dianhydrides such as 1,2,3,4-butanetetracarboxylic dianhydride and cyclopentanetetracarboxylic dianhydride; phthalic anhydride, pyromellitic anhydride, trimellitic anhydride Aromatic polycarboxylic anhydrides such as benzophenone tetracarboxylic anhydride; Examples include ester group-containing acid anhydrides such as water trimellitate and glycerin tris anhydrous trimellitate. Of these, 1,2-dicarboxylic anhydrides such as succinic anhydride, maleic anhydride, and phthalic anhydride are more preferable.

  As a specific method for producing an ester bond by reacting the hydroxyl group of the organic polymer particles having a hydroxyl group with a carboxylic acid anhydride, for example, an organic polymer having a hydroxyl group in an organic solvent in which the carboxylic acid anhydride is dissolved. A method of dispersing a dry powder of particles and stirring at room temperature to 80 ° C. for 1 to 24 hours can be mentioned. The organic solvent used here is not limited, and examples thereof include pyridine, acetone, methyl ethyl ketone, and tetrahydrofuran. As the catalyst, sulfuric acid, p-toluenesulfonic acid, zinc chloride, sodium acetate, pyridine, 4-dimethylaminopyridine, 4-pyrrolidinopyridine, and triethylamine may be used. Of these organic solvents and catalysts, pyridine is suitable as an organic solvent and catalyst.

  In addition, it is not necessary that all the hydroxyl groups on the organic polymer particles react with the carboxylic acid anhydride to be esterified, and it is preferable that a part of the hydroxyl groups remain as hydroxyl groups without being esterified.

1.2. Carboxyl Group-Containing Particles The number average particle diameter (hereinafter simply referred to as “particle diameter”) of the carboxyl group-containing particles according to an embodiment of the present invention is preferably 0.1 to 15 μm, more preferably 0.3. 10 μm, most preferably 1 to 10 μm. The particle size can be measured by a laser diffraction / scattering method. Here, when the particle size is less than 0.1 μm, it takes a long time for separation using centrifugation or the like, and the separation between the washing solvent such as water and the particles becomes insufficient. Removal of biologically related substances such as proteins and nucleic acids may be insufficient, and sufficient purification may not be possible. On the other hand, when the particle size exceeds 15 μm, the specific surface area becomes small, and the amount of physiologically active substance trapped decreases, and as a result, the sensitivity may decrease.

  The carboxyl group-containing particles according to an embodiment of the present invention are obtained by the production method and have a functional group having an ester bond. The functional group having an ester bond can function as a functional group for binding a protein. The functional group having an ester bond can be obtained by reacting a hydroxyl group with a carboxylic acid anhydride in the production method.

  The carboxyl group-containing particles may further have a hydroxyl group. For example, when both of two hydroxyl groups in 2,3-dihydroxypropyl group are esterified by the reaction between 2,3-dihydroxypropyl group in organic polymer particles and carboxylic acid anhydride, the diester structure is changed. The resulting carboxyl group-containing particles are obtained.

  In addition, for example, by reacting a 2,3-dihydroxypropyl group in a 2,3-dihydroxypropyl group with a carboxylic acid anhydride, either one of two hydroxyl groups in the 2,3-dihydroxypropyl group is an ester. When converted, carboxyl group-containing particles having a monoester structure are obtained.

  The carboxyl group-containing particles according to an embodiment of the present invention can further have a carboxyl group. In the carboxyl group-containing particles, the carboxyl group is not only a functional group for binding proteins, but also a factor expressing the dispersibility of the particles. The amount of the carboxyl group is preferably 2 μmol / g or more, more preferably 5 μmol / g or more, and most preferably 10 to 100 μmol / g based on the solid content of the particles. If the amount of the carboxyl group is less than 2 μmol / g, the signal may decrease.

  The contact angle between the dried coating film and the water from the aqueous dispersion of carboxyl group-containing particles according to an embodiment of the present invention is preferably 60 ° or less, more preferably 50 ° or less, and most preferably 10 °. ~ 40 °. The dry coating film from the aqueous dispersion was prepared by applying 0.2 ml of an aqueous dispersion containing 50 mg of particles to a smooth substrate such as a slide glass using an applicator and the like, and having a humidity of 40% and an air temperature of 25 ° C. Obtained by drying for a period of time. The contact angle between the dry coating and water is about 1 μL of water droplets dropped onto the dry coating, and the image from the horizontal direction is immediately captured as data by the camera, and the contour of the water droplet is assumed to be part of the circumference. It can be determined from the angle with the horizontal line of the film. By making the contact angle between the dried coating film and water from the aqueous dispersion of carboxyl group-containing particles according to an embodiment of the present invention within these ranges, it is possible to achieve both low non-specific adsorption and high sensitivity. it can.

  The carboxyl group-containing particles according to an embodiment of the present invention are usually used after being dispersed in an appropriate dispersion medium. As a dispersion medium that can be used, a dispersion medium that does not dissolve organic polymer particles or swell organic polymer particles is preferable. As a preferable dispersion medium, for example, an aqueous medium can be used. Here, the aqueous medium refers to water or a mixture of water and an organic solvent miscible with water (for example, alcohols, alkylene glycol derivatives, etc.).

1.3. Method for Producing Carboxyl Group-Containing Particles Containing Magnetic Material The carboxyl group-containing particles according to an embodiment of the present invention may contain a magnetic material. Hereinafter, the carboxyl group-containing particles containing a magnetic substance are also referred to as “magnetic substance-containing carboxyl group-containing particles”.

  Since the magnetic substance-containing carboxyl group-containing particles can be separated using a magnet without using, for example, a centrifuge, it is useful in that the process of separating particles from a specimen can be simplified or automated. It is.

  The magnetic substance-containing carboxyl group-containing particles include, for example, (I) particles in which magnetic fine particles are dispersed in a non-magnetic continuous phase such as an organic polymer, and (II) secondary aggregates of magnetic fine particles. , Particles having a non-magnetic material such as an organic polymer as a shell, (III) a core particle made of a non-magnetic material such as an organic polymer, and a secondary aggregate layer of magnetic fine particles provided on the surface of the core particle (magnetic And particles having a core as a core and an organic polymer layer as an outermost layer of the mother particle as a shell. Among these, (III) particles having a core particle including a secondary aggregate layer of the magnetic fine particles as a core and an organic polymer layer as a shell are preferable. The organic polymer in the magnetic substance-containing carboxyl group-containing particles having the structures (I) to (III) must have a hydroxyl group on the outermost surface of the particle except for the core portion of the core-shell type particle. It is.

  The most preferred magnetic substance-containing carboxyl group-containing particles are a core particle including a core particle, a magnetic layer of superparamagnetic fine particles provided on the surface of the core particle, and a cross-link provided to cover the host particle. And an organic polymer layer made of a polymer. That is, in the magnetic substance-containing carboxyl group-containing particles, the base particles are the core and the crosslinked polymer is the shell. Here, the preferred crosslinked polymer is more specifically 40 to 95 parts by weight of monomer (B), 5 to 30 parts by weight of crosslinkable monomer (C), and 0 to 55 parts by weight of other monomer (D). The monomer part consisting of can be polymerized to obtain a copolymer, and this copolymer can be obtained by hydrolysis.

  In the production of particles having the structure of (III) above, as a method for producing mother particles in which a magnetic layer of superparamagnetic fine particles is formed on the surface of the core particles, for example, nonmagnetic organic polymer particles and superparamagnetic fine particles are used. It can be produced by dry blending and combining both particles by applying a physically strong force from the outside. As a method of applying a physically strong force, for example, a mortar, an automatic mortar, a ball mill, a blade pressurizing powder compression method, a method using a mechanochemical effect such as a mechanofusion method, a jet mill, a hybridizer, etc. And those using the high-speed air-flow impact method. It is desirable that the physical adsorption force is strong in order to efficiently and firmly perform the composite. As the method, it is mentioned that the peripheral speed of the stirring blade is preferably 15 m / second or more, more preferably 30 m / second or more, and further preferably 40 to 150 m / second in a vessel with a stirring blade. When the peripheral speed of the stirring blade is lower than 15 m / sec, it may not be possible to obtain sufficient energy to adsorb the superparamagnetic fine particles on the surface of the nonmagnetic organic polymer particles. In addition, although there is no restriction | limiting in particular about the upper limit of the peripheral speed of a stirring blade, It determines automatically from points, such as an apparatus to be used and energy efficiency. As the superparamagnetic fine particles used in the present invention, for example, ferrite and / or magnetite fine particles having a particle diameter of about 5 to 20 nm can be suitably used.

  The polymer part (shell) is obtained by polymerizing a monomer part composed of 40 to 95 parts by weight of the monomer (B), 5 to 30 parts by weight of the crosslinkable monomer (C), and 0 to 55 parts by weight of the other monomer (D). A polymer can be obtained and formed by hydrolyzing the copolymer. Each monomer component is as described above. A more specific polymerization method is as disclosed in JP-A-2004-205481 and the like. The conditions for the hydrolysis treatment after polymerization are also as described above.

  In order to prevent dissolution of the superparamagnetic fine particles on the mother particles, the mother particles having a superparamagnetic fine particle magnetic layer formed on the surface of the core particles are mixed with 0 to 30 parts by weight of the crosslinkable monomer (C) and other parts. After another monomer part consisting of 70 to 100 parts by weight of monomer (D) is polymerized to form a coating layer, 40 to 95 parts by weight of monomer (B) and a crosslinkable monomer with the mother particles containing this coating layer as the core (C) By copolymerizing a monomer part consisting of 5 to 30 parts by weight and another monomer (D) 0 to 55 parts by weight, particles forming a polymer part (shell) are obtained, and the particles are hydrolyzed. Thus, magnetic substance-containing carboxyl group-containing particles may be formed. In the case of magnetic substance-containing carboxyl group-containing particles in which such superparamagnetic fine particles are coated with a coating layer, hydrolysis conditions having a wide range of strong acidity to strong basicity can be selected in the above-described hydrolysis treatment.

  The dispersion of the low nonspecifically adsorbing particles and the magnetic substance-containing carboxyl group-containing particles after hydrolysis is preferably washed with water by a centrifugal separation method, a magnetic separation method or the like to remove the remaining hydrolysis catalyst.

2. Applications The carboxyl group-containing particles according to an embodiment of the present invention can be used in a wide range of fields such as biochemistry, paints, paper, electrophotography, cosmetics, pharmaceuticals, agricultural chemicals, foods, and catalysts.

  More specifically, the carboxyl group-containing particles according to one embodiment of the present invention are a diagnostic agent carrier, a bacteria separation carrier, a cell separation carrier, a nucleic acid separation purification carrier, a protein separation purification carrier, and an immobilized enzyme. It is useful for applications such as carriers and drug delivery. Among them, it can be used as affinity carriers such as particles for compound carriers in the field of biochemistry and particles for chemical binding carriers for diagnostic agents, especially for binding proteins such as antigens or antibodies. As the probe binding particles (protein binding particles) for immunoassay, it is possible to express high sensitivity and low noise.

  In this probe-binding particle, substances to be examined are biologically relevant substances, chemical substances, and organisms contained in the immunoassay reagent and the sample to be examined. In the present invention, “biologically related substance” refers to all substances related to a living body. Examples of the biological substance include substances contained in the living body, substances derived from the substance contained in the living body, and substances that can be used in the living body.

  The biological substance to be examined is not particularly limited. For example, proteins (eg, enzymes, antibodies, aptamers, receptors, etc.), peptides (eg, glutathione), nucleic acids (eg, DNA or RNA), carbohydrates, Lipids and other cells or substances (for example, various blood-derived substances including various blood cells such as platelets, red blood cells, white blood cells, hormones (for example, luteinizing hormone, thyroid stimulating hormone, etc.), viruses, bacteria, fungi, protozoa, Examples include proteins and nucleic acids that are constituent elements of parasites, etc. More specifically, examples of proteins include proteins that are cancer markers such as biologically derived proteins, prostate-specific markers, and bladder cancer markers.

  The chemical substance to be inspected is not particularly limited, and examples thereof include environmental pollutants such as dioxins and pharmaceuticals (for example, antibiotics, anticancer agents, antiepileptic agents, etc.).

  The organism to be examined is not particularly limited. For example, various cancer cells, various floating cells, viruses (for example, hepatitis B virus, hepatitis C virus, herpes simplex virus, HIV virus, rubella virus, influenza virus, etc.), Examples include bacteria (for example, Neisseria gonorrhoeae, MRSA, E. coli, etc.), fungi (for example, Candida, ringworm, Cryptocox, alpergillus, etc.), protozoa and parasites (for example, Toxoplasma, malaria, etc.) and the like.

  According to the carboxyl group-containing particle according to an embodiment of the present invention, the carboxyl group is activated by a known activator such as a water-soluble carbodiimide in actual use since the carboxyl group is introduced on the surface of the particle. The protein can be chemically bonded to the surface of the particle by mixing the protein and the particle.

  After the protein is bound to the surface of the particle, excess protein is washed and unreacted activated carboxyl sites are inactivated as necessary. Further, after the protein is adsorbed on the surface of the particles, a blocking operation that is usually performed may be performed, and in the inactivation step described above, a blocking agent such as albumin may be used in combination. Thereafter, a normal analysis process using particles may be performed.

  The protein that can be carried on the carboxyl group-containing particles for protein binding according to one embodiment of the present invention is preferably an antigen or an antibody. In this case, the antigen or antibody is not particularly limited as long as it reacts with a component generally contained in a subject. For example, anti-antiplasmin antibody for antiplasmin test, anti-D dimer antibody for D dimer test Anti-FDP antibody for FDP test, Anti-tPA antibody for tPA test, Anti-thrombin = antithrombin complex antibody for TAT test, Anticoagulation-related test antigen or antibody such as anti-FPA antibody for FPA test; Anti-BFP for BFP test Anti-Apolipoprotein for testing apolipoprotein such as antibody, anti-CEA antibody for CEA test, anti-AFP antibody for AFP test, anti-ferritin antibody for ferritin test, anti-CA19-9 antibody for CA19-9 test, etc. Antibody, anti-β2-microblobrin antibody for β2-microblobrin test, α1-microglobulin Anti-α1-microglobulin antibody for Blin test, anti-immunoglobulin antibody for immunoglobulin test, antigen or antibody for serum protein-related test such as anti-CRP antibody for CRP test; antigen for endocrine function test such as anti-HCG antibody for HCG test or Antibody: Anti-HBs antibody for HBs antigen test, HBs antigen for HBs antibody test, HCV antigen for HCV antibody test, HIV-1 antigen for HIV-1 antibody, HIV-2 antigen for HIV-2 antibody test, HTLV-1 test HTLV-1 antigen, Mycoplasma antigen for Mycoplasma test, Toxoplasma antigen for Toxoplasma test, Streptridine O antigen for ASO test, etc. Antigen-related test antigen or antibody; Anti-DNA antibody test DNA antigen, RF test heat-modified human Antigen or antibody for autoimmune related test such as IgG; Examples include, but are not limited to, antigens or antibodies for drug analysis such as anti-lidocaine antibodies for lidocaine testing. As the antibody, either a polyclonal antibody or a monoclonal antibody may be used.

  The carboxyl group-containing particles according to an embodiment of the present invention can be suitably used for biochips using the particles, for example, biochips disclosed in JP-A-2005-148048.

  In addition, the use of the carboxyl group-containing particles according to an embodiment of the present invention is not limited to biochemical carrier use, and can be used, for example, in the above-described fields.

3. Examples and Reference Examples Hereinafter, the present invention will be described in more detail with reference to examples , but the present invention is not limited thereto. In this example, “%” and “parts” are based on weight.

3.1. Evaluation method 3.1.1. Measurement of signal by CLEIA (chemiluminescence enzyme immunoassay) 10 μl of particle dispersion (corresponding to 50 μg of particles) obtained by sensitizing anti-AFP (α-fetoprotein) antibody and obtained in each of Examples and Comparative Examples described later is a test tube. The sample was mixed with 50 μl of a standard specimen of AFP antigen (manufactured by Nippon Biotest) diluted to 100 ng / mL with fetal calf serum (FCS), and reacted at 37 ° C. for 10 minutes. After separating the particles by magnetic separation and removing the supernatant, an anti-AFP antibody labeled with alkaline phosphatase (hereinafter referred to as “ALP”) as a secondary antibody (manufactured by Fujirebio Inc., attached to Lumipulse AFP-N) Using reagent) 40 μl was added and allowed to react at 37 ° C. for 10 minutes. Next, the particles were separated by centrifugation to remove the supernatant, and then the particles obtained by repeated centrifugal washing with PBS three times were dispersed in 50 μl of 0.01% Tween 20 and transferred to a new tube. After adding 100 μl of ALP substrate solution (Lumipulse substrate solution: manufactured by Fujirebio Inc.) and reacting at 37 ° C. for 10 minutes, the amount of chemiluminescence was measured. For the measurement of chemiluminescence, a chemiluminescence measuring device (trade name: Lumat LB9507) manufactured by Bertol Japan KK was used.

3.1.2. Measurement of noise Above 3.1.1. In the signal measurement by CLEIA (chemiluminescence enzyme immunoassay), the amount of chemiluminescence as noise was measured in the same manner except that it was not mixed with the standard sample.

3.1.3. Particle size The number average particle size of the particles and the coefficient of variation thereof were measured with a laser diffraction particle size distribution analyzer (manufactured by Shimadzu Corporation) SALD-200V.

3.2. Synthesis example 1
2 parts by mass of 75% di (3,5,5-trimethylhexanoyl) peroxide solution (“Perroyl 355-75 (S)” manufactured by NOF Corporation) is mixed with 20 parts by mass of 1% aqueous sodium dodecyl sulfate solution, and ultrasonically dispersed. The mixture was finely emulsified with a machine and placed in a reactor containing 13 parts by mass of polystyrene particles having a particle size of 0.77 μm and 41 parts by mass of water and stirred for 12 hours at 25 ° C. In another container, 96 parts by mass of styrene A solution prepared by emulsifying 4 parts by mass of divinylbenzene with 400 parts by mass of a 0.1% aqueous sodium dodecyl sulfate solution was placed in the reactor, stirred at 40 ° C. for 2 hours, then heated to 75 ° C. and polymerized for 8 hours. After cooling to room temperature, the particles taken out by centrifugation were further washed with water, dried and pulverized to obtain core particles having a number average particle diameter of 1.5 μm. there were.

  Next, acetone is added to an oil-based magnetic fluid (trade name: “EXP series”, manufactured by Ferrotec Co., Ltd.) to precipitate and precipitate particles, which are then dried to have a surface that has been hydrophobized. Ferrite-based magnetic fine particles (average primary particle size: 0.01 μm) were obtained.

  Next, 15 g of the core particles and 15 g of the magnetic fine particles are mixed well with a mixer, and this mixture is mixed with the periphery of the blade (stirring blade) using a hybridization system NHS-0 type (manufactured by Nara Machinery Co., Ltd.). Treatment was carried out at a speed of 100 m / sec (16200 rpm) for 5 minutes to obtain mother particles having on the surface a magnetic layer composed of magnetic fine particles having an average number particle diameter of 2.0 μm.

  Next, an aqueous solution containing 0.25% by weight of sodium dodecylbenzenesulfonate and 0.25% by weight of a nonionic emulsifier (trade name: “Emulgen 150”, manufactured by Kao Corporation) (hereinafter referred to as “dispersing agent aqueous solution”) 375 g was put into a 1 L separable flask, then 15 g of mother particles having the magnetic layer were put in, dispersed with a homogenizer, and heated to 60 ° C. 150 g of aqueous dispersion agent, 27 g of methyl methacrylate, 3 g of trimethylolpropane trimethacrylate (hereinafter referred to as “TMP”), and di (3,5,5-trimethylhexanoyl) peroxide (manufactured by NOF Corporation; Parroyl 355) The pre-emulsion in which 0.6 g was dispersed was dropped into the 1 L separable flask controlled at 60 ° C. over 1 hour 30 minutes. After completion of dropping, the mixture was kept at 60 ° C. and stirred for 1 hour, and then 13.5 g of glycidyl methacrylate (hereinafter referred to as “GMA”), 1.5 g of TMP, and di (3,5,5-trimethyl) were added to 75 g of the aqueous dispersion agent. A pre-emulsion in which 0.3 g of hexanoyl) peroxide (manufactured by NOF Corporation; Parroyl 355) was added and dispersed was added dropwise to the 1 L separable flask controlled at 60 ° C. over 1 hour 30 minutes. Thereafter, the temperature was raised to 75 ° C., and then polymerization was continued for another 2 hours to complete the reaction. Subsequently, 60 ml of 1 mol / L sulfuric acid was placed in this 1 L separable flask and stirred at 60 ° C. for 6 hours. Subsequently, the particles in the separable flask were separated using magnetism, and then repeatedly washed with distilled water. Thus, magnetic particles having a 2,3-dihydroxypropyl group were obtained (hereinafter referred to as “A-1 particles”).

  Next, 1.0 g of the dry particles obtained by vacuum drying the A-1 particles was washed with 10 ml of pyridine and dispersed in 5 ml of pyridine, and then 3 g of succinic anhydride was dissolved in 25 ml of pyridine. The solution was added and stirred at 60 ° C. for 2 hours. After the reaction, the particles are separated using magnetism, washed 3 times with acetone, then 3 times with a 0.1 M aqueous sodium hydroxide solution and 4 times with distilled water to obtain carboxyl group-containing particles (hereinafter referred to as “B- 1 particle "). The average number particle diameter of the carboxyl group-containing particles (B-1 particles) was 2.9 μm.

3.3. Synthesis example 2
In Synthesis Example 1, the same procedure was used except that 2-hydroxyethyl methacrylate was used instead of GMA, and the step of adding 1 ml / L sulfuric acid in 1 L separable flask and stirring at 60 ° C. for 6 hours was omitted. , Carboxyl group-containing particles (hereinafter referred to as “B-2 particles”) were obtained. The average number particle diameter of the magnetic particles (B-2 particles) was 2.8 μm.

3.4. Comparative Synthesis Example 1
In Synthesis Example 1, 13.5 g of cyclohexyl methacrylate and 1.5 g of methacrylic acid were used instead of 13.5 g of GMA and 1.5 g of TMP, and 60 ml of 1 mol / L sulfuric acid was placed in a 1 L separable flask and stirred at 60 ° C. for 6 hours. A particle (B-3 particle) corresponding to the A-1 particle was obtained in the same procedure except that the step of performing was omitted. The average number particle diameter of the magnetic particles (B-3 particles) was 2.9 μm.

3.5. Reference Example 0.1 mL of 0.1 mM HCl solution in which 5 mg of 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (manufactured by Dojin Chemical) was dissolved in 1 mL of an aqueous dispersion of B-1 particles having a solid content concentration of 1% And 0.1 g of a 0.1 mM HCl solution in which 100 μg of anti-AFP antibody was dissolved, and the mixture was swirled at room temperature for 8 hours. Repeat the operation of adding a phosphate buffer solution (PBS, 0.1% BSA / PBS, pH = 7.2) containing 1% bovine serum albumin and performing magnetic separation three times to prevent unreacted anti-reaction The AFP antibody was removed to obtain probe-bound particles to which the anti-AFP antibody was bound. This probe-bound particle had a signal of 157705 and noise of 71.

3.6. As a result of measuring similarly about Example B-2 particle | grains, the signal was 114432 and the noise was 74.

3. 7 . Comparative Example 1
Probe-bound particles outside the scope of the present invention were obtained in the same manner as in the Reference Example except that B-3 particles were used. The probe-bound particle signal was 36059 and the noise was 306.

  From the above results, the carboxyl group-containing particles obtained in Synthesis Examples 1 and 2 were produced through a process of generating an ester bond by reacting the hydroxyl group of the organic polymer particles having a hydroxyl group with a carboxylic acid anhydride. In comparison synthesis example 1, it can be understood that the sensitivity is high and the noise is low as compared with the particles produced without going through the above steps.

Claims (3)

  The manufacturing method of carboxyl group-containing particle | grains including the process of making the hydroxyl group derived from this monohydroxyalkyl group of the organic polymer particle | grains which have a monohydroxyalkyl group react with a carboxylic anhydride, and producing | generating an ester bond.   The manufacturing method of carboxyl group-containing particle | grains including the process of making the hydroxyl group derived from this 2-hydroxyethyl group of the organic polymer particle which has 2-hydroxyethyl group react with a carboxylic anhydride, and producing | generating an ester bond. In claim 1 or 2,
The manufacturing method of the carboxyl group-containing particle | grains in which the said organic polymer particle contains a magnetic body.