JP2010260877A - Organic polymer particle and probe-bonded particle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain particles that extremely hardly cause unspecific adsorption of a biochemical substance and separates a target biochemical substance at a very high purity in a high yield. <P>SOLUTION: The organic polymer particles for separating a biochemical substance from a biological sample utilizing an interaction with a probe includes an active functional group for bonding to the probe, has a surface area giving a value of 20-5,000 Å<SP>2</SP>when divided by the amount of the active functional group, and bears a nonionic hydrophilic group. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to organic polymer particles for separating biochemical substances.

In recent years, research on biochemical substances such as cells, proteins, and nucleic acids has been actively conducted in the fields of biochemistry and pharmaceuticals. By conducting detailed research on the structure, function, abundance, etc. of these biochemical substances, it is possible, for example, to scientifically elucidate unknown life phenomena or to create new drugs with excellent efficacy. It is expected to be possible.
In order to carry out these studies, specific biochemical substances contained in biological samples (cell suspension, cell disruption fluid, blood, saliva, etc.) are contained in countless contaminants. It is a technology that separates from.

Conventional biochemical substance separation techniques can be roughly classified into the following two types.
(A) A method of separation using the physicochemical properties (size, shape, specific gravity, isoelectric point, polarity, etc.) of the target substance. For example, a filter, a filter membrane, a centrifuge, an electrophoresis device, a separation method using an adsorption carrier, and the like can be given.
(B) A technique for separating using the biochemical properties of the target substance (affinity to proteins, DNA, compounds, etc.). For example, a gel carrier in which a probe is bonded to the surface, a particle carrier, or a separation method using column chromatography packed with these carriers can be used.

Since the separation technique (A) uses the physicochemical properties of the target substance, it is not suitable for use in separating two or more biochemical substances having similar physicochemical properties. Usually, these separation techniques are often used for concentration and crude purification of biochemical substances.
On the other hand, since the separation technique (B) uses the affinity of the target substance with the probe, it is possible in principle to separate a specific biochemical substance. However, there are some problems in the prior art.

The problem with the conventional separation technique (B) is that, for example, when the surface of the carrier used has a property of non-specifically adsorbing biochemical substances, the purity of the target substance to be separated is low. There was a problem. In addition, when the probe cannot maintain its activity on the carrier, there is a problem that the yield of the target substance to be separated is lowered. Furthermore, when the probe is not bonded to the carrier through a chemical bond, the probe is also collected when the target substance is separated, so that there is a problem that the purity of the separated target substance is lowered. .
If the purity of the biochemical substance to be separated is low and impurities are mixed, it may be difficult to identify the target biochemical substance or perform functional analysis, or may lead to incorrect analysis results. There is. In addition, if the yield of the separated biochemical substance is low, it will be below the detection limit of the equipment used in the analysis, and it will be difficult to identify and analyze the biochemical substance, or the measurer will show the separation fact itself. You may miss it.
Therefore, it is necessary to improve the capture amount of the target substance and reduce the amount of nonspecific adsorption.

Problems in the conventional separation technique (B) will be described more specifically.
Examples of the carrier used in the separation technique (B) include gel carriers based on sugar chains such as agarose and dextran, organic polymer particles such as polystyrene and acrylic, and organic-inorganic composite carriers containing inorganic substances such as silica and magnetic substances. Can be mentioned.
Among these, in the gel carrier, since the carrier surface is hydrophilic, there is an advantage that non-specific adsorption of the biochemical substance is relatively small, but the activity of the probe cannot be maintained on the carrier, There was a problem that the yield of the target substance was reduced. Moreover, since these gel carriers have low mechanical strength, they have a problem that they cannot be used under pressurized conditions and a problem that solvent resistance is poor.

On the other hand, organic polymer particles and organic-inorganic composite carriers have the advantage that the mechanical strength of the carriers is high, but there is a problem that biochemical substances are adsorbed non-specifically on the hydrophobic carrier surface. . Moreover, there is a problem that the yield of the target substance is reduced because the activity of the probe cannot be maintained on the carrier.

In addition, in particles used for conventional diagnostic agents, albumin, casein, skim milk, etc. could be used as a blocking agent for suppressing nonspecific adsorption of biochemical substances. There is a problem that this method cannot be applied to particles for the purpose of separation or analysis of biochemical substances because the purity of the separated substances is lowered.

As a technique for suppressing non-specific adsorption, for example, Japanese Patent Application Laid-Open No. 2006-131771 proposes a method in which particles are dispersed in styrene and glycidyl methacrylate and finely dispersed by ultrasonic treatment and then polymerized. However, since the coating of the magnetic material is insufficient, the reduction of non-specific adsorption is insufficient, and the average particle size is 200 nm or less and the particle size is small, so that the magnetic separation property is also insufficient. In addition, since the magnetic substance is exposed, there is a problem that the particles collapse in a solvent such as an acid. Furthermore, this method has room for improvement in the yield of the target substance.

  Furthermore, Japanese Patent Application Publication No. 2006-511935 proposes that a magnetic substance is deposited on the surface and inside of a particle and further coated with a polymer having a glycidyl group. However, in this method, when the magnetic particles are deposited to the surface of the particles, the coating with the polymer having a glycidyl group alone is insufficient to coat the magnetic particles, and the nonspecific adsorption property cannot be reduced.

In Japanese Patent Publication No. 10-505118, a reaction for introducing an amino group into a magnetic particle into which a glycidyl group is introduced is described. This magnetic particle is a porous particle for the purpose of ion exchange. Non-specific adsorption is remarkable.
The present applicant, in Japanese Patent No. 3738847, is capable of easily and efficiently producing diagnostic pharmaceutical particles having a uniform particle size without dropping off of a magnetic material and elution of a substance derived from a magnetic material component such as iron ions. For this purpose, a method for producing magnetic particles including a step of providing a two-layer polymer coat is disclosed, and further, in JP 2005-83904 A and JP 2005-83905 A, magnetic particles with reduced non-specific adsorption are disclosed. In recent years, it has been desired to further reduce non-specific adsorption.

As described above, although there are many techniques for conventional biochemical substance separation techniques, specific biochemical substances (cells, proteins, nucleic acids, etc.) contained in biological samples are contained in a myriad of contaminants. Until now, there has been no technique applicable to the use for separation with extremely high purity and good yield.

JP 2006-131771 A JP-T 2006-511935 Japanese National Patent Publication No. 10-505118 Japanese Patent No. 3738847 JP 2005-83904 A JP 2005-83905 A

  An object of the present invention is to obtain particles that have very little non-specific adsorption of biochemical substances and that separate target biochemical substances with extremely high purity and high yield.

In order to achieve the above-mentioned object, the present inventors have conducted intensive research. As a result, the present invention has an active functional group for binding a probe and a nonionic hydrophilic group, and the surface area of the particle is determined based on the active functional group. divided by the amount (parking area) is relative to satisfy organic polymer particles 20～5000A ^2, by using a carrier obtained by coupling via a chemical bond to the probe, the biological sample From the inside, it was found that biochemical substances could be separated with extremely high purity and high yield, and the present invention was completed.
According to the present invention, it is possible to provide an organic polymer particle for binding a probe, and a probe binding particle in which a probe is bonded to the organic polymer particle via a chemical bond.

The aspect of the organic polymer particles in the present invention is:
A value (parking area) having an active functional group for immobilizing the probe on the particle and a nonionic hydrophilic group and dividing the surface area of the particle by the amount of the active functional group is 20 to 5000 Å. An organic polymer particle characterized by satisfying the condition of ² .

  The organic polymer particles can contain a magnetic material.

  The organic polymer particle may have a carboxyl group, an amino group, or a tosyl group as an active functional group.

  The organic polymer particles can have 2,3-dihydroxypropyl groups on the particles as nonionic hydrophilic groups.

  The organic polymer particles include a mother particle having a superparamagnetic fine particle layer on the surface, an organic polymer layer having an active functional group for binding a probe and a nonionic hydrophilic group provided so as to cover the mother particle, Can have a structure consisting of

The aspect of the probe-binding particle in the present invention is:
A probe-bonded particle in which a probe is bonded to the organic polymer particle through an active functional group on the particle through a chemical bond.

The probe-binding particles can have antibodies, antigens, nucleic acids, compounds, affinity tag capture substances and the like as probes.

The organic polymer particles of the present invention have very little non-specific adsorption of biochemical substances such as proteins, nucleic acids, and cells, and bind the probe to the particle surface through chemical bonds while maintaining high activity. Can be made.
The probe-binding particle of the present invention can separate a specific biochemical substance or a complex of biochemical substances from a biological sample with extremely high purity.

  Hereinafter, organic polymer particles and probe-bonded particles according to an embodiment of the present invention will be described.

1. Organic polymer particle and method for producing the same The organic polymer particle of the present invention is an organic polymer particle for separating a biochemical substance from a biological sample by using an interaction with a probe, and the organic polymer particle have active functional has a group, and the value obtained by dividing the amount of active functional groups the surface area of the particles is 20～5000A ^2, further nonionic hydrophilic groups for binding the probe.
In the present invention, the “biological sample” is a biological test sample, and examples thereof include cells, tissues, blood, bone marrow, body fluid, saliva, semen, urine, and oral swabs.
In the present invention, “biochemical substance” refers to all substances related to the living body. Examples of the biochemical substance include a substance contained in a living body, a substance derived from a substance contained in a living body, and a substance that can be used in a living body.

The “probe” in the present invention refers to a protein such as an antigen / antibody that can bind to a specific biochemical substance in a biological sample, a nucleic acid, a sugar chain, a chemical substance, and the like.
The “active functional group” in the present invention is a functional group that can be bonded to the probe through a chemical reaction, and is a carboxyl group, amino group, tosyl group, epoxy group, carbonyl group, mercapto group, succinimide group, silanol. A group. In view of storage stability of the functional group, ease of introduction of the probe, activity of the probe after introduction, and the like, preferred active functional groups are a carboxyl group, an amino group, and a tosyl group. The method for introducing these functional groups will be described later.

Surface area divided by the amount of the active functional groups of the organic polymer particles of the present invention (hereinafter, referred to as "parking area") is 20~5000Å ^2. A more suitable range, reason for limitation, etc. will be described later.

The “nonionic hydrophilic group” in the present invention is a functional group such as a hydroxyl group, an ethylene oxide group or an amide group, preferably an alcoholic hydroxyl group, more preferably a 2,3-dihydroxypropyl group. The nonionic hydrophilic group is an essential component for preventing the contaminants in the biological sample from adsorbing to the particles and separating the target biochemical substance with high purity. In addition, as an index of the introduction amount of nonionic hydrophilic groups, the contact angle between the dry coating film obtained from the aqueous dispersion of organic polymer particles and water is preferably 60 ° or less, more preferably 5 to 50 °, Most preferably, it is 10 to 40 °. When the introduction amount of the nonionic hydrophilic group is insufficient, that is, when the contact angle exceeds 60 °, nonspecific adsorption may increase. About the measuring method of the contact angle of the dry coating film obtained from the aqueous dispersion of organic polymer particles and water, it is as having described in Unexamined-Japanese-Patent No. 2007-127426.
As the internal structure of the organic polymer particle of the present invention, the whole particle may be uniform, or a layer structure in which an organic polymer layer is formed on the mother particle using the mother particle as a nucleus may be used. In the manufacturing method described below, organic polymer particles having a layer structure are described.

1. Organic polymer particles and method for producing the same Organic polymer particles according to an embodiment of the present invention include a mother particle that may contain a magnetic material, and an organic polymer layer provided so as to cover the mother particle. The organic polymer layer has an active functional group for binding the probe and a nonionic hydrophilic group. Further, the value obtained by dividing the surface area of the particle by the amount of the active functional group is in the range of 20 to 5000 ² , more preferably 20 to 1000 ² .
The method for producing organic polymer particles according to an embodiment of the present invention includes a step of forming an organic polymer layer on the mother particle after producing the mother particle which may contain a magnetic material. Get. The active functional group and the hydrophilic group may be introduced in the organic polymer layer production step, or may be introduced after the organic polymer layer production step.

1.1. Mother particles As the shape of the mother particles, for example, (I) particles made of a nonmagnetic material such as an organic polymer, (II) particles in which superparamagnetic fine particles are dispersed in a continuous phase of a nonmagnetic material such as an organic polymer, III) Particles having a secondary aggregate of superparamagnetic fine particles as a core and a nonmagnetic material such as an organic polymer as a shell, and (IV) Particles made of a nonmagnetic material such as an organic polymer as a core, and secondary particles of the superparamagnetic fine particles. And particles having an aggregate layer (magnetic layer) as a shell.
When the particles are used in applications such as a carrier for affinity column chromatography, the mother particles in the form (I) that do not contain a magnetic substance are more preferable. On the other hand, when used in applications such as magnetic particles that can be magnetically separated by a magnet, it has excellent magnetic responsiveness and the particle diameter can be uniformly controlled. Mother particles are more preferred.
The mother particles having the form (IV) can be obtained by providing a secondary aggregate layer (magnetic material layer) of superparamagnetic fine particles on the mother particles having the form (I). Hereinafter, the constitution and production method of the mother particles in the forms (I) and (IV) will be described.

1.1.1. Mother particle (I)
The base particle is basically a non-magnetic substance, and any of an organic substance and an inorganic substance can be used. As a typical example of the organic substance, for example, a polymer can be mentioned. Such a polymer is particularly preferably a vinyl polymer, and most preferably crosslinked polystyrene or crosslinked polymethyl methacrylate. These polymers may have a functional group such as a carboxyl group introduced therein.
The material of the mother particles is preferably an organic substance such as a polymer from the viewpoints of processability and lightness when combined.

  The average particle diameter of the mother particles is preferably 0.1 to 200 μm, more preferably 0.5 to 100 μm, and most preferably 0.8 to 20 μm. If the average particle diameter of the mother particles is less than 0.1 μm, it may be difficult to separate the particles from the dispersion medium. On the other hand, when the average particle size of the mother particles exceeds 200 μm, the particles are significantly settled by gravity, so that the reaction field may be non-uniform when reacting with the sample as probe-bound particles. In addition, since the effective surface area per weight of the particles becomes small, the reaction efficiency may decrease.

  The polymer particles as the base particles having an average particle diameter in the specific range described above can also be obtained, for example, by suspension polymerization of a vinyl monomer or pulverization of a polymer bulk. As a method for producing core particles having a uniform particle size, a swelling polymerization method described in JP-B-57-24369 or a polymerization method previously proposed by the present applicant (Japanese Patent Laid-Open Nos. 61-215602, 61). -215603 and 61-215604).

1.1.2. Mother particle (IV)
The mother particles (IV) have the mother particles (I) as a core and the secondary aggregate layer (magnetic material layer) of superparamagnetic fine particles as a shell.
1.1.2.1. The superparamagnetic particles superparamagnetic particles, a particle size not greater than 20 nm (preferably particle size 5 to 20 nm) are fine particles typically iron _{oxide-based, MFe 2 O 4 (M =} Co, Ni, Mg, Cu, Li _0.5 Fe _0.5 or the like), magnetite represented by Fe ₃ O ₄ , or γ-Fe ₂ O ₃ . In particular, it is preferable to include either γ-Fe ₂ O ₃ or Fe ₃ O ₄ having a strong saturation magnetization and a small residual magnetization.

  The superparamagnetic fine particles preferably have a hydrophobized surface from the viewpoint of the affinity and compatibility between the core and the monomer used in the subsequent step. Examples of the method for hydrophobizing the surface of the magnetic fine particles include the method described in Japanese Patent No. 3738847. Such superparamagnetic fine particles can also be obtained by precipitating and washing particles from a known magnetic fluid with a suitable poor solvent.

1.1.2.2. Mother particle (mother particle (IV)) with a magnetic layer on its surface
As a method of forming a magnetic layer containing superparamagnetic fine particles on the surface of the core (mother particle (I)), the core and superparamagnetic fine particles are mixed and the superparamagnetic fine particles are physically adsorbed on the surface of the core. Thus, a method of forming a magnetic layer is preferred. In the present invention, the “physical adsorption method” refers to an adsorption method and a bonding method that do not involve a chemical reaction. As a method for adsorbing the superparamagnetic fine particles on the surface of the core, for example, a method is used in which the core and the superparamagnetic fine particles are dry blended and both particles are combined by applying a physically strong force from the outside. A method is mentioned. As a method of applying a physically strong force, for example, a mortar, an automatic mortar, a ball mill, a method using a mechanochemical effect such as a blade pressure type powder compression method, a mechanofusion method, a jet mill, a hybridizer The thing using the impact method in high-speed air currents, such as these, is mentioned. It is desirable that the physical adsorption force is strong in order to efficiently and firmly perform the composite. In order to carry out complexing with a strong physical adsorption force, 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 equipped with a stirring blade. It is possible to carry out compounding. When the peripheral speed of the stirring blade is lower than 15 m / sec, it may not be possible to obtain sufficient energy for adsorbing the superparamagnetic fine particles on the surface of the core particles. In addition, although there is no restriction | limiting in particular about the upper limit of the circumferential speed of a stirring blade, It determines automatically from points, such as an apparatus to be used and energy efficiency.

  The mixing ratio (core: superparamagnetic fine particles) when the secondary aggregate layer of superparamagnetic fine particles is provided on the core is preferably 95: 5 to 20:80 by weight. If the amount of superparamagnetic particles is less than this range, the magnetic separation of the resulting magnetic particles may be inferior. On the other hand, if the amount of superparamagnetic fine particles is larger than this range, a part of the superparamagnetic fine particles remain in the system without being complexed. May be non-uniform.

1.2. Organic polymer layer having active functional group and hydrophilic group As the form of the organic polymer layer having an active functional group and a nonionic hydrophilic group for binding a probe formed on the mother particle, for example, (V) mother particle Particles having a polymer layer having an active functional group and a hydrophilic group directly on the surface thereof, (VI) a hydrophobic first polymer layer (hereinafter also simply referred to as “first polymer layer”) on the surface of the mother particle. In addition, particles having a second polymer layer (hereinafter, also simply referred to as “second polymer layer”) having an active functional group and a hydrophilic group on the surface thereof are included.
When using mother particles containing superparamagnetic fine particles, the organic polymer layer of the form (VI) is more preferable in that exposure of superparamagnetic fine particles can be suppressed. When superparamagnetic fine particles are exposed, non-specific adsorption of biochemical substances increases, and the purity of the separated substances decreases. Moreover, superparamagnetic fine particles may be dissolved by the addition of acid or the like.
Hereinafter, the manufacturing method of the organic polymer layer of the form (VI) is demonstrated.

As a production method for forming the organic polymer layer in the form of (VI) on the mother particle, after forming the hydrophobic first polymer layer on the surface of the mother particle, for example, (VII) a monomer having an active functional group, , A method of copolymerizing with a monomer having a hydrophilic group, (VIII) a monomer having an active functional group and / or a monomer having a hydrophilic group is (co) polymerized, and then the active functional group and / or the hydrophilic group is chemically Examples thereof include a method of forming the second polymer layer by a method of introducing by modification. In these, the manufacturing method of (VIII) is more preferable at the point that it is easy to control the quantity of the functional group introduce | transduced into an organic polymer particle.

The organic polymer particle according to an embodiment of the present invention has an active functional group for binding a probe. The active functional group preferably has at least one group selected from the group consisting of a carboxyl group, an amino group, and a tosyl group, for example. Examples of a method for introducing these active functional groups into the organic polymer layer produced by the production method (VIII) by chemical modification include the following methods.
(A) Carboxyl group introduction A carboxylating agent is reacted with a hydroxyl group on the organic polymer layer.
(B) Introduction of amino group An aminating agent is reacted with the epoxy group on the organic polymer layer.
(C) Introduction of tosyl group A tosylating agent is reacted with a hydroxyl group on the organic polymer layer.

Here, it is a known technique that a hydroxyl group is obtained by hydrolyzing an epoxy group. Therefore, in the production method of (VIII), after forming the hydrophobic first polymer layer and the second polymer layer having an epoxy group, hydrolysis is appropriately performed, and further, the above (a) to (c) The organic polymer particles according to one embodiment of the present invention can be obtained by introducing an active functional group by the method (hereinafter, this production method is referred to as production method (IX)). The production method (IX) is not only easy to control the amount of functional groups introduced into the organic polymer particles, but also easily converted from a single base particle to particles having a plurality of types of active functional groups. It is preferable.
In addition, the epoxy group introduce | transduced on a 2nd polymer layer can also be used as an active functional group for couple | bonding a probe.
Hereafter, the manufacturing method of said (IX) is demonstrated in detail.

1.2.1. Formation of Hydrophobic First Polymer Layer The monomer for forming the hydrophobic first polymer layer (hereinafter also referred to as “first monomer part”) contains 80% by weight or more of a hydrophobic monomer, preferably It contains 95 wt% or more of hydrophobic monomer, more preferably 98 wt% or more of hydrophobic monomer. If the hydrophobic monomer in the first monomer part is less than 80% by weight, non-specific adsorption may deteriorate. Here, the hydrophobic monomer is a simple substance or a mixture of polymerizable monomers having a solubility in water at 25 ° C. of 2.5% by weight or less. The hydrophobic monomer may be either a monofunctional (non-crosslinkable) monomer or a crosslinkable monomer, or may be a mixture of a monofunctional monomer and a crosslinkable monomer.

  In the presence of the mother particles, the auxiliary raw material polymerization initiator, emulsifier, dispersing agent, electrolyte, cross-linking agent, molecular weight regulator, etc. are added as necessary in the liquid, and the main raw material is 80% by weight or more. A first polymer layer can be formed by polymerizing the first monomer portion containing a hydrophobic monomer. In particular, when the mother particles contain a magnetic material, the magnetic material can be effectively coated by forming the first polymer layer by polymerization, and non-specific adsorption can be effectively performed. Can be reduced.

  Among the hydrophobic monomers that can be used in the first monomer portion, monofunctional monomers include aromatic vinyl monomers such as styrene, α-methylstyrene, and halogenated styrene, methyl acrylate, methyl methacrylate, ethyl acrylate, and ethyl methacrylate. And ethylenically unsaturated carboxylic acid alkyl esters such as stearyl acrylate, stearyl methacrylate, cyclohexyl acrylate, cyclohexyl methacrylate, isobornyl acrylate and isobornyl methacrylate. Among the hydrophobic monomers, crosslinkable monomers include ethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, pentaerythritol triacrylate, pentaerythritol trimethacrylate, dipentaerythritol hexaacrylate. And polyfunctional (meth) acrylates such as dipentaerythritol hexamethacrylate, conjugated diolefins such as butadiene and isoprene, divinylbenzene, diallyl phthalate, allyl acrylate, and allyl methacrylate.

  The first monomer part may contain less than 20% by weight of a non-hydrophobic monomer (hydrophilic monomer). Among non-hydrophobic monomers, monofunctional monomers include monomers having a carboxyl group such as acrylic acid, methacrylic acid, maleic acid, itaconic acid, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, glycerol acrylate, glycerol methacrylate , Methoxyethyl acrylate, methoxyethyl methacrylate, polyethylene glycol acrylate, polyethylene glycol methacrylate, polypropylene glycol acrylate, polypropylene glycol methacrylate, polyethylene glycol-polypropylene glycol (meth) acrylate, methoxypolyethylene glycol mono (meth) acrylate, 2-dimethylaminoethyl ( (Meth) acrylate, 2-diethylaminoethyl (Meth) acrylate having a hydrophilic functional group (for example, hydroxyl group, amino group, alkoxy group, etc.) such as meth) acrylate, 2-dimethylaminopropyl (meth) acrylate, 3-dimethylaminopropyl (meth) acrylate, acrylamide, Methacrylamide, N-methylolacrylamide, N-methylolmethacrylamide, diacetoneacrylamide, N- (2-diethylaminoethyl) (meth) acrylamide, N- (2-dimethylaminopropyl) (meth) acrylamide, N- (3- Examples thereof include dimethylaminopropyl) (meth) acrylamide. Among the non-hydrophobic monomers, hydrophilic monomers such as polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, and poly (meth) acrylic ester of polyvinyl alcohol can be exemplified as the crosslinkable monomer. The non-hydrophobic monomer contained in the first monomer part is less than 20% by weight, preferably less than 5% by weight, and more preferably less than 2% by weight.

  The ratio of the crosslinkable monomer (including the hydrophobic monomer and the non-hydrophobic monomer) in the first monomer part is preferably 1 to 40% by weight in 100% by weight of the monomer part constituting the first polymer layer, Preferably, it is 5 to 20% by weight. When the ratio of the crosslinkable monomer in the first monomer part exceeds 40% by weight, the particles may become porous and increase nonspecific adsorption.

  The polymerization initiator is preferably an oil-soluble polymerization initiator from the viewpoint of solubility in water. When a water-soluble polymerization initiator is used, the polymerization of only the hydrophobic monomer is prioritized over the polymerization on the surface of the mother particles, and a large amount of new particles tend to be generated.

  Examples of oil-soluble polymerization initiators include benzoyl peroxide, lauroyl peroxide, tertiary butyl peroxy 2-ethylhexanate, 3,5,5-trimethylhexanoyl peroxide, peroxide compounds such as azobisisobutyronitrile, azo compounds, etc. Can be mentioned.

Water-soluble initiators include potassium persulfate, ammonium persulfate, persulfates such as sodium persulfate, hydrogen peroxide, 2-2 azobis (2-aminopropane) mineral acid salt, azobiscyanovaleric acid and its alkali metal salts, and Examples thereof include ammonium salts, and redox initiators in which persulfates, hydrogen peroxide salts and sodium bisulfite, sodium thiosulfate, ferrous chloride, etc. are combined. Among them, persulfates are preferably used. .
The ratio of these polymerization initiators to the whole monomer is preferably in the range of 0.01 to 8% by weight.

  As the emulsifier, a commonly used anionic surfactant or nonionic surfactant can be used alone or in combination. For example, anionic surfactants include alkali metal salts of higher alcohol sulfates, alkali metal salts of alkylbenzene sulfonic acids, alkali metal salts of dialkyl ester succinic acids, alkali metal salts of alkyl diphenyl ether disulfonic acids, polyoxyethylene In addition to anionic surfactants such as sulfuric acid ester salts of alkyl (or alkylphenyl) ethers, phosphoric acid ester salts of polyoxyethylene alkyl (or alkylphenyl) ethers, formalin condensates of sodium naphthalene sulfonate, Eleminol JS- 2, JS-5 [product name, manufactured by Sanyo Chemical Co., Ltd.], Latemul S-120, S-180A, S-180, PD-104 [product name, manufactured by Kao Corporation], Aqualon HS-10, HS -20, KH-10 [Product , Dai-ichi Kogyo Seiyaku Co., Ltd.], ADEKAREASOAP SE-10N, SR-10 [product name, and the like can be mentioned reactive emulsifier such as Asahi Denka Kogyo Co., Ltd.].

  Examples of the nonionic surfactant include polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, Aqualon RS-20 (manufactured by Daiichi Kogyo Seiyaku Co., Ltd.), Adekari Soap NE-20 ( Reactive nonionic surfactants such as Asahi Denka Kogyo Co., Ltd.).

  The method for adding the monomer to the polymerization system in the formation of the first polymer layer is not particularly limited, and may be any of a batch method, a division method, or a continuous addition method. The polymerization temperature varies depending on the polymerization initiator, but is usually 10 to 90 ° C, preferably 30 to 85 ° C, and the time required for the polymerization is usually about 1 to 30 hours.

  Moreover, the thickness of the first polymer layer is preferably 0.005 to 20 μm, and more preferably 0.01 to 5 μm. Further, when the mother particle contains a magnetic substance, it is preferable that the first polymer layer completely covers the magnetic substance.

1.2.2. Formation of Second Polymer Layer Having Epoxy Group The monomer for forming the second polymer layer (hereinafter also referred to as “second monomer part”) contains 20% by weight or more of an epoxy group-containing monomer, preferably 40 The epoxy group-containing monomer is included in an amount of not less than wt%, and more preferably not less than 80 wt%. As the other monomer constituting the second polymer layer, the hydrophobic monomer and / or the non-hydrophobic (hydrophilic) monomer exemplified as the monomer used in the first monomer portion can be used.
By polymerizing the second monomer part containing a predetermined amount of the epoxy group-containing monomer, a second polymer layer having an epoxy group can be obtained.

  The second polymer layer can be formed by a method basically similar to the method for forming the first polymer layer. That is, in the presence of particles in which the first polymer layer is formed, in a liquid in which a polymerization initiator, an emulsifier, a dispersant, an electrolyte, a crosslinking agent, a molecular weight modifier, and the like, which are auxiliary materials, are added as necessary, A second polymer layer can be formed by polymerizing the second monomer part which is the main raw material.

  Here, examples of the copolymerizable monomer containing an epoxy group include glycidyl acrylate, glycidyl methacrylate, and allyl glycidyl ether.

  The second monomer part preferably further contains a crosslinkable monomer, and the ratio thereof is preferably 1 to 40% by weight, more preferably 5 to 20% by weight in 100% by weight of the second monomer part. is there. If the ratio of the crosslinkable monomer in the second monomer part exceeds 40% by weight, the particles may become porous and increase nonspecific adsorption.

  The method for adding the monomer to the polymerization system in the formation of the second polymer layer is not particularly limited, and may be any of a batch method, a division method, or a continuous addition method. The polymerization temperature varies depending on the polymerization initiator, but is usually 10 to 90 ° C, preferably 30 to 85 ° C, and the time required for the polymerization is usually about 1 to 30 hours.

  The thickness of the second polymer layer can be made thinner than that of the first polymer layer, and is preferably 0.005 to 5 μm, more preferably 0.005 to 1 μm.

1.2.3. Reaction for introducing a carboxyl group onto the second polymer layer Examples of the reaction for introducing a carboxyl group include (i) a carboxylating agent (for example, a dicarboxylic acid, an aminocarboxylic acid, or an epoxy group contained in the second polymer layer). A reaction that causes an organic compound having three or more carboxyl groups in the molecule to act; (ii) a carboxylating agent (for example, a carboxylic acid) to a hydroxyl group obtained by hydrolyzing an epoxy group contained in the second polymer layer (Iii) reaction of reacting an acid anhydride or carboxylic acid chloride), and (iii) in the presence of a suitable dehydration catalyst, hydroxyl groups obtained by hydrolyzing an epoxy group contained in the second polymer layer, An organic compound having at least two carboxyl groups (for example, dicarboxylic acid or an organic compound having at least three carboxyl groups in the molecule) Reactions to be acted on are listed. By this reaction, polymer particles having a carboxyl group introduced therein are obtained. The carboxyl group-introduced particles can be suitably used for probe binding.

  The reaction of (ii) above is preferable because of easy control of the amount of carboxyl groups introduced, and (ii) a hydroxyl group obtained by hydrolyzing an epoxy group contained in the second polymer layer is used as a carboxylating agent. A reaction in which a carboxylic anhydride is allowed to act is particularly preferred. Here, the carboxylic acid anhydride is a polyvalent carboxylic acid anhydride, and specific examples thereof include itaconic anhydride, succinic anhydride, citraconic anhydride, dodecenyl succinic anhydride, tricarbanilic anhydride, glutaric anhydride, maleic anhydride. Aliphatic dicarboxylic anhydrides such as acid, hexahydrophthalic anhydride, methyltetrahydrophthalic anhydride, and hymic anhydride; 1,2,3,4-butanetetracarboxylic dianhydride, cyclopentanetetracarboxylic dianhydride And alicyclic polycarboxylic acid dianhydrides such as phthalic anhydride, pyromellitic anhydride, trimellitic anhydride, and benzophenone tetracarboxylic anhydride. Of these, 1,2-dicarboxylic anhydrides such as succinic anhydride, maleic anhydride, and phthalic anhydride are more preferable.

The hydrolysis of the epoxy group proceeds, for example, with an appropriate acid catalyst or base catalyst in an aqueous solvent. Preferably, the epoxy group is hydrolyzed in an aqueous solvent using an acid catalyst such as sulfuric acid. Thereby, hydrolysis can be performed promptly and reliably. In this case, the preferable temperature of hydrolysis is 4 to 100 ° C., more preferably 20 to 80 ° C., and the preferable time of hydrolysis is 5 minutes to 24 hours, more preferably 30 minutes to 12 hours.
Hydrolysis of the epoxy group generates a hydroxyl group that is a nonionic hydrophilic group on the particles. In particular, when the epoxy group on the organic polymer particle is derived from a glycidyl group, 2,3-dihydroxypropyl group, which is a nonionic hydrophilic group, is generated on the particle by hydrolysis.
Non-specific adsorption can be reduced by the hydroxyl groups on the particles.

  As a specific method for allowing a carboxylic acid anhydride to act as a carboxylating agent on a hydroxyl group obtained by hydrolyzing an epoxy group contained in the second polymer layer, for example, an organic solvent in which the carboxylic acid anhydride is dissolved And a method of dispersing a dried powder of hydrolyzed particles and stirring at room temperature to 80 ° C. for 1 to 24 hours. Examples of the organic solvent used here include, but are not limited to, pyridine, acetone, methyl ethyl ketone, 1,3-dioxolane, tetrahydrofuran, dimethylformamide, dichloroform, chloroform, toluene and the like. 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 preferable because it can be used as an organic solvent and catalyst.

  In the reaction for introducing a carboxyl group, not all hydroxyl groups need to be esterified. It is preferable that a part of the hydroxyl group is not esterified and remains as a nonionic hydrophilic group on the organic polymer particle as it is. The optimum range of the carboxyl group introduction amount into the polymer particles will be described later.

  In the case of the above (i) to (iii), before the reaction for introducing a carboxyl group, two or more epoxy groups are hydrolyzed or simultaneously with the reaction for introducing a carboxyl group. It is preferred to hydrolyze part of the group. In the case of (i) above, all or part of the remaining epoxy group may be hydrolyzed after the reaction for introducing a carboxyl group.

1.2.4. Reaction for introducing an amino group onto the second polymer layer As a reaction for introducing an amino group, for example, an aminating agent is allowed to act on particles on which a polymer layer having an epoxy group is formed, whereby an amino group is introduced into the polymer layer. The reaction to introduce | transduce is mentioned. By this reaction, polymer particles into which amino groups have been introduced are obtained. The amino group-introduced particles can be suitably used for probe binding.

  Examples of the aminating agent include ammonia, an organic compound having two amino groups in the molecule (diamine), or an organic compound having three or more amino groups in the molecule. An organic compound having two or more amino groups is more preferable.

Here, examples of the diamine include primary diamines such as ethylenediamine, propylenediamine, and o-phenylenediamine. Examples of organic compounds having three or more amino groups in the molecule include 1,2,3-triaminopropane, tetra (aminomethyl) methane, 1,3,5-triaminobenzene, 1,2,3. , 4-tetraaminobenzene and the like.

The reaction for introducing an amino group may be carried out by adding an aminating agent to polymer particles having an epoxy group dispersed in an aqueous solvent, or after drying the polymer particles having an epoxy group, You may implement by dispersing a dry particle in an aminating agent. The aqueous solvent is a mixed solvent of a water-soluble organic solvent and water, or water. Here, examples of the water-soluble organic solvent include methanol, ethanol, acetone, dimethylformamide and the like. The preferred temperature and time for the reaction for introducing an amino group vary depending on the amount of the epoxy group in the polymer layer, the presence or absence of a solvent, the type of solvent, etc., but usually 4 to 100 ° C., preferably 20 to 80 ° C. Usually, 10 minutes to 48 hours, preferably 1 to 24 hours.
In the reaction for introducing an amino group, not all epoxy groups need to be aminated. The optimum range of the amino group introduction amount into the polymer particles will be described later.

  Prior to the reaction for introducing an amino group, a part of two or more epoxy groups may be hydrolyzed. Moreover, you may hydrolyze a part of two or more epoxy groups simultaneously with reaction which introduce | transduces an amino group. Moreover, you may hydrolyze a part or all of the epoxy group which remains after amino group introduction | transduction.

The hydrolysis of the epoxy group proceeds, for example, with an appropriate acid catalyst or base catalyst in an aqueous solvent. Preferably, before or after the reaction for introducing the amino group, the epoxy group is hydrolyzed in an aqueous solvent using an acid catalyst such as sulfuric acid. Thereby, hydrolysis can be performed promptly and reliably. In this case, the preferable temperature of hydrolysis is 4 to 100 ° C., more preferably 20 to 80 ° C., and the preferable time of hydrolysis is 5 minutes to 24 hours, more preferably 30 minutes to 12 hours.
Hydrolysis of the epoxy group generates a hydroxyl group that is a nonionic hydrophilic group on the particles. In particular, when the epoxy group on the organic polymer particle is derived from a glycidyl group, 2,3-dihydroxypropyl group, which is a nonionic hydrophilic group, is generated on the particle by hydrolysis.
Non-specific adsorption can be reduced by the hydroxyl groups on the particles.

1.2.5. Reaction for introducing a tosyl group onto the second polymer layer As a reaction for introducing a tosyl group, for example, polymer particles having an epoxy group are hydrolyzed to obtain polymer particles having a hydroxyl group, followed by drying, an organic solvent Among them, there is a method of introducing a hydroxyl group on the particle by acting with a tosylating agent.
The obtained tosyl group-introduced particles can be suitably used for probe binding.

The conditions for hydrolysis of the epoxy group are as described in the reaction for introducing the carboxyl group. Hydrolysis of the epoxy group generates a hydroxyl group that is a nonionic hydrophilic group on the particles. In particular, when the epoxy group on the organic polymer particle is derived from a glycidyl group, 2,3-dihydroxypropyl group, which is a nonionic hydrophilic group, is generated on the particle by hydrolysis.
Examples of means for drying the polymer particles include vacuum drying and freeze drying.
Examples of the tosylating agent include p-toluenesulfonic acid chloride and p-toluenesulfonic acid. Preferable organic solvents include pyridine.

The reaction for introducing a tosyl group is, for example, by drying polymer particles having a hydroxyl group by vacuum drying, dispersing the dried particles in an organic solvent such as pyridine, adding p-toluenesulfonic acid chloride, and then at room temperature. The reaction is carried out for 1 to 6 hours. Alternatively, the tosylation may be performed by dehydrating condensation of a hydroxyl group of the polymer particles and p-toluenesulfonic acid.
In the reaction for introducing a tosyl group, not all hydroxyl groups need to be tosylated. It is preferable that a part of the hydroxyl group is not tosylated and remains as a nonionic hydrophilic group on the organic polymer particle as it is. Non-specific adsorption can be reduced by the hydroxyl groups on the particles.
The optimum range of the amount of tosyl group introduced into the polymer particles will be described later.

1.2.6. Post-treatment and storage The organic polymer particles according to the present embodiment are stored as an aqueous dispersion or a dry powder. The aqueous dispersion is usually obtained by washing with an aqueous solvent such as distilled water after the step of introducing the active functional group, and adding and dispersing the aqueous solvent. The dry powder is obtained by heat drying, vacuum drying, spray drying, freeze drying or the like of the aqueous dispersion.

1.3. Particle size The average particle size of the organic polymer particles according to this embodiment is preferably 0.1 to 200 µm, more preferably 0.5 to 100 µm, and most preferably 0.8 to 20 µm. If the average particle size is less than 0.1 μm, it may be difficult to separate the particles from the dispersion medium. On the other hand, if the average particle diameter exceeds 200 μm, the gravity sedimentation of the particles becomes significant, and therefore the reaction field may become non-uniform when reacting with the sample as probe-bound particles. In addition, since the effective surface area per weight of the particles becomes small, the reaction efficiency may decrease.

1.4. Amount of active functional group introduced (parking area)
The organic polymer particle which concerns on one Embodiment of this invention has at least 1 group chosen from the group which consists of a carboxyl group, an amino group, and a tosyl group as an active functional group, for example.
The amount of active functional groups is expressed in the parking area. The parking area means an average area occupied by one active functional group molecule on the particle surface. When the number of functional groups per unit area (functional group density) of the particles increases, the parking area decreases. Conversely, when the functional group density per unit area decreases, the parking area increases.

Regarding the “particle surface area” used for calculating the parking area, a measured value of an effective surface area obtained from a gas adsorption method or the like is used instead of a calculated value predicted from an average particle diameter and specific gravity. Thus, even if the particle surface has an uneven shape, the effective surface area can be accurately estimated, and the actual functional group density on the particle surface can be expressed more accurately. In addition, there is an advantage that the functional group density can be expressed in a form independent of the specific gravity of the particles.
The parking area of the active functional group introduced into the organic polymer particles according to this embodiment is preferably 20 to 5000 cm ² , more preferably 20 to 1000 cm ² . When the parking area of the active functional group is less than 20 ² , when the probe is bound to the polymer particle, the activity of the probe is impaired, so that the specific capture amount of the biochemical substance may be reduced. In addition, since many active functional groups that are not used for binding to the probe remain on the particle, the amount of nonspecific adsorption of the biochemical substance may increase. On the other hand, when the parking area of the active functional groups is more than 5000 Å ^2, because fewer bonds of the probe to the polymer particles, there are cases where the amount of specific capture biochemicals is reduced.

・・・式（１）
１．４．１．１．粒子の比表面積
粒子の比表面積は、粒子１ｇ当たりの表面積である。粒子を水で洗浄後、乾燥させた後、ガス吸着量測定装置により測定し、この乾燥粒子の比表面積を「粒子の比表面積」とした。 1.4.1. Parking Area Measurement Method The parking area in the present invention is calculated using the following equation (1). A method for measuring the specific surface area and the amount of the active functional group necessary for the calculation will be described later.

... Formula (1)
1.4.1.1. Specific surface area of particles The specific surface area of particles is the surface area per 1 g of particles. The particles were washed with water and dried, and then measured with a gas adsorption amount measuring device. The specific surface area of the dry particles was defined as “specific surface area of particles”.

1.4.1.2. Active Functional Group Amount on Particles As the amount of active functional group in the present invention, 1.4.1.2.1. Of carboxyl group, amino group and tosyl group. Determination of the amount of carboxyl groups Using an aqueous dispersion containing 1 g of particles (solid content), the apparent surface charge (X) of these particles was determined by conductivity titration described in JP-A-10-270233, Further, after obtaining the background charge amount (Y) by the same measurement using only the dispersion medium (water), the difference (XY) between these charge amounts is calculated as the amount of carboxyl groups per 1 g of particles. did.

1.4.1.2.2. After taking 5 mg (solid content) of amino group quantitative particles in a tube and removing the supernatant, 1 ml of a 100 mM methanol solution of N-succinimidyl 3- (2pyridyldithio) propionate (manufactured by Dojindo) was added, For 60 minutes. After washing the particles twice with methanol, a dithiothreitol solution (20 mM, 1 mL) dissolved in a 1: 1 mixed solution of carbonate buffer solution (50 mM, pH 9.4) and methanol was added, and 30 minutes at room temperature. Reduce reaction. Thereafter, the amount of pyridine 2-thione released by this reduction reaction is quantified using an ultraviolet-visible spectrophotometer, and the amount of this released pyridine 2-thione corresponds to the amount of amino groups on the particles. did. Converted to the amount per 1 g of particles, this was the amino group amount. In the determination of pyridine 2-thione, the absorbance at a wavelength of 343 nm is measured, and 8080 is used as the molar extinction coefficient ε.

1.4.1.2.3. Quantification of the amount of tosyl group (p-toluenesulfonyl group) Take 5 mg of particles (solid content) in a tube, remove the supernatant, add 1 ml of 10% tris aqueous solution, and react at 70 ° C. for 16 hours under stirring conditions. . After the reaction, centrifugation or magnetic separation is performed, and the amount of p-toluenesulfonic acid released in the supernatant is quantified using an ultraviolet-visible spectrophotometer, and the amount of the released p-toluenesulfonic acid is measured on the particles. The amount of tosyl group in The amount was converted to the amount per 1 g of particles, and this was used as the tosyl group amount. In the determination of p-toluenesulfonic acid, the absorbance at a wavelength of 261 nm is measured, and 380 is used as the molar extinction coefficient ε.

1.5. Use Of the organic polymer particles according to the present embodiment, according to particles into which carboxyl groups or amino groups are introduced, in actual use, after mixing the probe and the particles, a known water-soluble carbodiimide or the like The probe can be chemically bound to the surface of the particle by activating the carboxyl group on the particle or the probe with the activator.

  Among the organic polymer particles according to the present embodiment, according to the tosyl group-introduced particles, in actual use, the probe can be chemically bonded to the surface of the particle simply by mixing the probe and the particle. it can.

After binding the probe to the surface of the particle, excess probe is washed and unreacted active groups are inactivated as necessary. As the inactivating agent, it is preferable to use an inactivating agent containing a hydroxyl group such as ethanolamine or tris (hydroxymethyl) aminomethane (hereinafter referred to as “Tris”). Thereafter, the process may be shifted to a normal process using particles.
The organic polymer particles according to this embodiment can be suitably used as a particle carrier for probe binding.

2. Probe-binding particle and method for producing the same The probe-binding particle manufacturing method according to an embodiment of the present invention includes, for example, the organic having at least one group consisting of a carboxyl group, an amino group, and a tosyl group as an active functional group. Introducing a probe into the surface of the polymer particle via a chemical bond.

2.1. Introduction of probe into organic polymer particle having carboxyl group or amino group As a reaction for introducing the probe into organic polymer particle having carboxyl group or amino group through a chemical bond, for example, a probe is placed on the organic polymer particle. A method of forming a chemical bond between a particle and a probe with an appropriate activator after physical adsorption is exemplified. Suitable activators include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide met-p-toluenesulfonate. Is mentioned.

  As a specific method for introducing the probe, for example, after adding the probe to an aqueous dispersion of organic polymer particles having a carboxyl group or an amino group, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride The method of adding salt and stirring at 2-80 degreeC for 1 to 24 hours is mentioned.

2.2. Introduction of probe into organic polymer particle having tosyl group As a reaction for introducing a probe into organic polymer particle having tosyl group via a chemical bond, for example, a probe is added to an aqueous dispersion of organic polymer particle having tosyl group. And a method of stirring at 2 to 80 ° C. for 1 to 72 hours.

2.3. Post-treatment and storage The probe-binding particles according to this embodiment are usually washed after the probe introduction step, after washing the excess probe and inactivating unreacted active groups as necessary, and then adding an aqueous solvent. It is obtained by dispersing. As the inactivating agent, it is preferable to use an inactivating agent containing a hydroxyl group such as ethanolamine or tris (hydroxymethyl) aminomethane.
When the probe binding particles are used for biochemical substance separation, it is not necessary to block the surface of the particles with proteins such as albumin, skim milk, and casein.
The probe binding particles according to this embodiment are usually stored as an aqueous dispersion.

2.4. Applications The probe-binding particles according to this embodiment can be suitably used as an affinity carrier for the concentration, purification, and separation of biochemical substances in the biochemical field and the pharmaceutical field, and can be used as a target for living with extremely high purity. A chemical substance or a complex containing this biochemical substance can be concentrated, purified and separated. The separation substance may be either a known substance or an unknown substance.
Concentration, purification, and separation of biochemical substances using the probe-bound particles according to this embodiment can be performed in a container such as a microtube, and can also be packed in column chromatography and used as an affinity column. it can.
Among the probe-binding particles according to the present embodiment, those containing a magnetic substance can be magnetically separated using a magnet, so that biochemical substances can be concentrated, purified, and separated by a simple method. In addition, this process can be automated or semi-automated.
The probe-binding particles according to the present embodiment are used for separating specific cells from biological samples, for separating specific proteins, for separating specific nucleic acids, protein-protein complexes, DNA- It can be suitably used for applications such as separating a transcription factor complex. In addition, these separated substances can be used for functional analysis of biochemical substances.
In addition, the probe-binding particles according to the present embodiment can be suitably used for immunological test applications.

  In the probe binding particle according to the present embodiment, the substances to be captured by the probe are biochemical substances, chemical substances, organisms, and complexes thereof contained in the biological sample. The probe captures them so that they can be separated or examined.

  The biochemical substance to be separated or examined is not particularly limited. For example, a protein (for example, an enzyme, an antibody, an aptamer, a receptor, etc.), a peptide (for example, glutathione), a nucleic acid (for example, DNA or RNA), a sugar Quality, lipids, hormones (eg, luteinizing hormone, human chorionic gonadotropin, thyroid stimulating hormone, insulin, glucagon, growth hormone, etc.) and other cells or substances (eg, various blood cells such as platelets, red blood cells, white blood cells, etc.) Examples include various blood-derived substances including cells, various floating cells, and proteins and nucleic acids that are constituent elements of viruses, bacteria, fungi, protozoa, parasites, and the like. More specifically, examples of proteins include biological proteins, proteins serving as markers for various cancers such as prostate-specific markers and bladder cancer markers, and transcription factors.

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

  The organism to be isolated or 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. ), Bacteria (eg, Neisseria gonorrhoeae, MRSA, E. coli, etc.), fungi (eg, Candida, ringworm, cryptocox, alpergillus, etc.), protozoa / parasites (eg, Toxoplasma, malaria, etc.), and the like.

  The probe that can be carried on the probe binding particle according to the present embodiment is not particularly limited as long as it has a functional group capable of reacting with at least one of the functional groups of the probe binding particle. , Antigens, nucleic acids, peptide nucleic acids, hormones, proteins with a molecular weight of 500 to 1,000,000, sugar chains, polysaccharides, cells, aptamers, viruses, enzymes, various tag capture substances for affinity, coenzymes such as biotin, specific physiological activities A chemical substance having an action (or possibly having a specific physiological activity) can be used.

The antigen or antibody carried as a probe is not particularly limited as long as it reacts with components generally contained in biological samples. For example, antigens or antibodies for biochemical research, immunodiagnostics, etc. Antigens or antibodies. As the antibody, either a polyclonal antibody or a monoclonal antibody may be used.
Examples of antibodies for biochemical research include anti-proteasome antibodies, anti-Akt-1 antibodies, anti-AUF1 antibodies, anti-Caspase3 antibodies, anti-Caspase6 antibodies, anti-cdk2 antibodies, anti-CREB antibodies, anti-Cyclin B1 antibodies, anti-D4- GDI antibody, anti-FAK antibody, anti-FRA-1 antibody, anti-GSK3β antibody, anti-HDAC1 antibody, anti-Jak2 antibody, anti-MEK1 antibody, anti-Mib1 antibody, anti-NKG2D antibody, anti-Nrf antibody, anti-Paxillin antibody, anti-Phospho-Tyrosine antibody Examples include, but are not limited to, anti-PLC-γ1 antibody and anti-PP2A antibody.

  Examples of antigens or antibodies for immunodiagnosis include 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 for TAT test = Anticoagulation-related test antigens or antibodies such as antithrombin complex antibody, anti-FPA antibody for FPA test; anti-BFP antibody for BFP test, anti-CEA antibody for CEA test, anti-AFP antibody for AFP test, anti-ferritin for ferritin test Antibodies, antigens or antibodies for tumor-related testing such as anti-CA19-9 antibody for CA19-9 testing; anti-apolipoprotein antibodies for testing apolipoprotein, anti-β2-microbrovulin antibody for testing β2-microbrovulin, α1-microglobulin Anti-α1-microglobulin antibody for testing, anti-immunoglob for immunoglobulin testing Antigen or antibody for serum protein-related test such as anti-CRP antibody for CRP test, anti-CRP test for serum protein; Anti-HBs antibody for test for HBs antigen, HBs antigen for test for HBs antigen test HCV antigen for HCV antibody test, HIV-1 antigen for HIV-1 antibody, HIV-2 antigen for HIV-2 antibody test, HTLV-1 antigen for HTLV-1 test, mycoplasma antigen for mycoplasmosis test, Toxoplasma for toxoplasma test Antigens or antibodies for infectious diseases such as antigens, streptolysin O antigen for ASO testing; DNA antigens for testing anti-DNA antibodies, antigens or antibodies for autoimmune testing such as heat-modified human IgG for RF testing; anti-digoxins for testing digoxin Antibody, antigen for drug analysis such as anti-lidocaine antibody for lidocaine test Examples include, but are not limited to, bodies.

  Examples of probes that can be carried on the probe-binding particles according to this embodiment include proteins such as enzymes and hormones, nucleic acids such as DNA and RNA, lipids, and physiologically active sugar chain compounds. It can be bonded to the surface by a chemical bonding method and used as an affinity carrier. Furthermore, a chemical substance to be analyzed (analyzed chemical substance; corresponding to a ligand molecule) is immobilized on the probe binding particle according to the present embodiment by chemical bonding, and a protein having a specific interaction with the analyzed chemical substance Etc. (corresponding to the target molecule) can be separated and purified.

3. Examples Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. In each example and comparative example, the evaluation was performed by the following method.

3.1. Evaluation method 3.1.1. Particle diameter The number average particle diameter of the particles was measured with a laser diffraction particle size distribution analyzer (manufactured by Shimadzu Corporation) SALD-200V, and this was used as the particle diameter of the particles.

3.1.2. The surface area of the particles After washing the particles with water and drying, the specific surface area of the dry particles was measured with a gas adsorption amount measuring device (manufactured by Yuasa Ionics Co., Ltd.) AUTOSORB-1-MP. The surface area per gram of particles was taken.

3.1.3. Amount of active functional group on particle 3.1.3.1. Determination of the amount of carboxyl groups Using an aqueous dispersion containing 1 g of particles (solid content), the apparent surface charge (X) of these particles was determined by conductivity titration described in JP-A-10-270233, Further, after obtaining the background charge amount (Y) by the same measurement using only the dispersion medium (water), the difference (XY) between these charge amounts is calculated as the amount of carboxyl groups per 1 g of particles. did.

3.1.3.2. After taking 5 mg (solid content) of amino group quantitative particles in a tube and removing the supernatant, 1 ml of a 100 mM methanol solution of N-succinimidyl 3- (2pyridyldithio) propionate (manufactured by Dojindo) was added, For 60 minutes. After washing the particles twice with methanol, a dithiothreitol solution (20 mM, 1 mL) dissolved in a 1: 1 mixed solution of carbonate buffer solution (50 mM, pH 9.4) and methanol was added, and 30 minutes at room temperature. A reduction reaction was performed. Thereafter, the amount of pyridine 2-thione liberated by this reduction reaction was quantified using an ultraviolet-visible spectrophotometer. The amount of free pyridine 2-thione was assumed to correspond to the amount of amino groups present on the particles. Converted to the amount per 1 g of particles, this was the amino group amount. In the determination of pyridine 2-thione, the absorbance at a wavelength of 343 nm was measured, and 8080 was used as the molar extinction coefficient ε.

3.1.3.3. Quantitative determination of p-toluenesulfonyl group (tosyl group) Take 5 mg of particles (solid content) in a tube, remove the supernatant, add 1 ml of 10% aqueous Tris solution, and react at 70 ° C. for 16 hours under stirring conditions. It was. After the reaction, centrifugation or magnetic separation was performed, and the amount of p-toluenesulfonic acid released in the supernatant was quantified using an ultraviolet-visible spectrophotometer. The amount of p-toluenesulfonic acid thus liberated corresponds to the amount of tosyl groups present on the particles. The amount was converted to the amount per 1 g of particles, and this was used as the tosyl group amount. In the determination of p-toluenesulfonic acid, the absorbance at a wavelength of 261 nm was measured, and 380 was used as the molar extinction coefficient ε.

・・・式（１） 3.1.4. Parking area The parking area was calculated using the following equation (1). Regarding the specific surface area and the amount of active functional groups required for the calculation, the values obtained by the above measurement method were used.

... Formula (1)

3.1.5. Performance of probe-bound particles The degree of specific capture of probe-bound particles and the degree of non-specific adsorption were evaluated by the methods described below. This evaluation is a model experiment targeting the 20S proteasome contained in the cell lysate.

3.1.5.1. Degree of specific capture 50 μL of a 1% by weight dispersion of particles on which the probe was immobilized was placed in a tube, and the supernatant was removed by centrifugation or magnetic separation. Pour 20 μl of Jurkat cell lysate, which has been confirmed to contain the target protein (20S proteasome), and further shake the mixture with a touch mixer to disperse the particles, followed by shaking at room temperature for 2 hours. I let you. Subsequently, the supernatant was removed by centrifugation or magnetic separation, and 1 ml of 10 mM HEPES containing 0.05% nonionic surfactant NP40 was poured to disperse the particles with a touch mixer. After the same treatment was repeated twice more, the contents were transferred to another unused tube, and the supernatant was removed by centrifugation or magnetic separation. Subsequently, 25 μl of 0.5% sodium dodecyl sulfate aqueous solution was poured into this tube as an extract, and the particles were dispersed by applying very light vibration with a touch mixer. After standing for 10 minutes, centrifugation or magnetic separation was performed, and 20 μl of supernatant was collected (extraction step). 2-mercaptoethanol was dissolved so that the concentration in the premix sample buffer manufactured by Bio-Rad was 2 wt%, and 20 μl of this was collected in a tube. This was mixed with 20 μl of the supernatant collected in the extraction step, and heated with a tube heater at 95 ° C. for 5 minutes. Gel 1 using Biorad vertical electrophoresis system “Mini Protian 3”, BioRad precast polyacrylamide gel “Lady Gel J (12.5%)”, and BioRad premix electrophoresis buffer 10 μl per lane was applied and electrophoresis was performed. Staining was performed by a standard method using a Bio stain silver stain plus kit. The stained gel was scanned and imaged with a densitometer “GS-700” manufactured by Bio-Rad.
In the stained gel image, the case where several bands around the molecular weight of 31k corresponding to the protein constituting the subunit of the 20S proteasome are clearly confirmed is “good”, and the case where it is confirmed that the band is unclear is confirmed. “Slightly good”, otherwise “bad”.

3.1.5.2. Degree of non-specific adsorption In the gel image obtained in the above-described evaluation of specific capture, “good” indicates that no band is confirmed except in the vicinity of a molecular weight of 31 k. “Slightly good” and other than “bad”.

3.2. Synthesis example 3.2.1. Synthesis Example 1 Production of Organic Polymer Particles 3.2.1.1. Preparation of mother particles 75% di (3,5,5-trimethylhexanoyl) peroxide solution (“Parroyl 355-75 (S)” manufactured by NOF Corporation, hereinafter referred to as “Perroyl”) 2 parts by mass of 1% dodecyl It mixed with 20 mass parts of sodium sulfate aqueous solution, and finely emulsified with the ultrasonic disperser. This was put into 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 at 25 ° C. for 12 hours. In a separate container, 95 parts by mass of methyl methacrylate (hereinafter referred to as “MMA”) and 5 parts by mass of trimethylolpropane trimethacrylate (hereinafter referred to as “TMP”) are emulsified in 400 parts by mass of a 0.1% sodium dodecyl sodium sulfate aqueous solution. The mixture was placed in the reactor and stirred at 40 ° C. for 2 hours, and 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. This is designated as mother particle A-1. The particle diameter was 1.5 μm.

3.2.1.2. Formation of polymer layer having glycidyl group 375 g of 0.5 wt% aqueous solution of sodium dodecylbenzenesulfonate was charged into a 1 L separable flask, and then 22 g of mother particle A-1 was charged and dispersed with a homogenizer. Heated. A pre-emulsion prepared by dispersing 18 g of MMA, 2 g of TMP, and 0.4 g of parroyl in 100 g of a 0.5% by weight aqueous solution of sodium dodecylbenzenesulfonate in a separate container was added to the 1 L separable flask controlled at 60 ° C. The solution was added dropwise over 1 hour 30 minutes.

  After completion of the dropwise addition, the mixture was kept at 60 ° C. and stirred for 1 hour, and then added to 75 g of a 0.5 wt% aqueous solution of sodium dodecylbenzenesulfonate in another container, 10.5 g of glycidyl methacrylate, 1.5 g of TMP, and 0. The pre-emulsion in which 3 g was dispersed was dropped into the 1 L separable flask controlled at 60 ° C. over 1 hour and 30 minutes. Thereafter, the temperature was raised to 75 ° C., and polymerization was further continued for 2 hours to complete the reaction.

  Next, the particles in the separable flask were separated by centrifugation and washed with distilled water. Through the above steps, organic polymer particles A-2 (particle size: 2.2 μm) on which a polymer layer having a glycidyl group was formed were obtained.

3.2.2. Synthesis Example 2 Production of magnetic substance-containing polymer particles 3.2.2.1. Preparation of mother particles (formation of magnetic layer)
After adding acetone to oily magnetic fluid (trade name: “EXP series”, manufactured by Ferrotec Co., Ltd.) to precipitate and precipitate the particles, this is dried to obtain a ferrite-based surface having a hydrophobized surface. Superparamagnetic fine particles (average primary particle size: 0.01 μm) were obtained.

  15 g of mother particle A-1 and 20 g of the above-mentioned hydrophobized superparamagnetic fine particles were mixed well with a mixer, and this mixture was mixed with a blade (stirring) using a hybridization system NHS-0 type (manufactured by Nara Machinery Co., Ltd.). Was performed for 5 minutes at a peripheral speed of 100 m / sec (16200 rpm) to obtain mother particles A-3 (particle diameter: 1.7 μm) having a magnetic layer composed of superparamagnetic fine particles on the surface.

3.2.2.2. Formation of polymer layer having glycidyl group 375 g of 0.5 wt% aqueous solution of sodium dodecylbenzenesulfonate was charged into a 1 L separable flask, and then 15 g of mother particle A-3 was charged and dispersed with a homogenizer. Heated. A pre-emulsion prepared by dispersing 18 g of MMA, 2 g of TMP, and 0.4 g of parroyl in 100 g of a 0.5% by weight aqueous solution of sodium dodecylbenzenesulfonate in a separate container was added to the 1 L separable flask controlled at 60 ° C. The solution was added dropwise over 1 hour 30 minutes.

  After completion of the dropwise addition, the mixture was kept at 60 ° C. and stirred for 1 hour, and then added to 75 g of a 0.5 wt% aqueous solution of sodium dodecylbenzenesulfonate in another container, 10.5 g of glycidyl methacrylate, 1.5 g of TMP, and 0. The pre-emulsion in which 3 g was dispersed was dropped into the 1 L separable flask controlled at 60 ° C. over 1 hour and 30 minutes. Thereafter, the temperature was raised to 75 ° C., and polymerization was further continued for 2 hours to complete the reaction.

  Next, the particles in the separable flask were separated using magnetism and washed with distilled water. Through the steps described above, magnetic particles A-4 (particle diameter: 2.7 μm) on which a polymer layer having a glycidyl group was formed were obtained.

3.2.3. Production of polymer particles having active functional groups 3.2.3.1. Synthesis Example 3 Production of Polymer Particle (P-1) Having Carboxyl Group 1.0 g of particles isolated by centrifugation from an aqueous dispersion of organic polymer particle A-2 on which a second polymer layer having a glycidyl group was formed 10 g of a 1% sulfuric acid aqueous solution was added, indirect ultrasonic waves were irradiated for 20 minutes to disperse, and the mixture was stirred at 60 ° C. for 5 hours (hydrolysis of glycidyl group).

  Subsequently, the operation of isolating the particles by centrifugation, dispersing them in pure water and washing them was repeated 5 times and further dried to obtain polymer particles A-5 having 2,3-dihydroxypropyl groups. After washing 1.5 g of the dried particles A-5 with 10 ml of pyridine, a solution of 40 mg of succinic anhydride dissolved in 10 ml of pyridine was added and stirred at room temperature for 4 hours (introduction of carboxyl group).

After the reaction, the particles are isolated by centrifugation, washed 3 times with acetone and then 4 times with distilled water, and then dispersed in distilled water to contain 1.0 g of polymer particles P-1 having carboxyl groups A 1% dispersion was obtained. The particle diameter was 2.2 μm, the specific surface area was 2.6 m ² / g, the amount of carboxyl groups was 9 μmol / g, and the parking area was 48 ² .

3.2.3.2. Synthesis Example 4 Preparation of Magnetic Particle (C-1) Having Carboxyl Group To 1.0 g of particles isolated by magnetic separation from an aqueous dispersion of magnetic particle A-4 on which a second polymer layer having a glycidyl group was formed 10 g of a 1% sulfuric acid aqueous solution was added, and the mixture was dispersed by irradiation with indirect ultrasonic waves for 20 minutes, and then stirred at 60 ° C. for 5 hours (hydrolysis of glycidyl group).

  Subsequently, the operation of isolating the particles by magnetic separation, dispersing in pure water and washing them was repeated five times, and further dried to obtain magnetic particles A-6 having 2,3-dihydroxypropyl groups. After washing 1.0 g of the dry particles A-6 with 10 ml of pyridine, a solution of 2 mg of succinic anhydride dissolved in 10 ml of pyridine was added and stirred at room temperature for 4 hours (introduction of carboxyl group).

After the reaction, the particles are isolated by magnetic separation, washed 3 times with acetone and then 4 times with distilled water, and then dispersed in distilled water to contain 1.0 g of polymer particles C-1 having carboxyl groups A 1% dispersion was obtained. Particle size of 2.7 .mu.m, a specific surface area of 1.6 m ² / g, amount of carboxyl groups is 0.5 [mu] mol / g, the parking area was 531Å ^2.

3.2.3.3. Synthesis Example 5 Preparation of Magnetic Particle (C-2) Having Carboxyl Group 1.0 g carboxyl group was prepared in the same manner as C-1 preparation method described in Synthesis Example 4 except that the amount of succinic anhydride was 12 mg. A 1% dispersion containing polymer particles C-2 with Particle size of 2.7 .mu.m, a specific surface area of 1.6 m ² / g, a carboxyl group content is 3 [mu] mol / g, the parking area was 89Å ^2.

3.2.3.3. Synthesis Example 6 Preparation of Magnetic Particle (C-3) Having Carboxyl Group 1.0 g carboxyl group was prepared in the same manner as C-1 preparation method described in Synthesis Example 4 except that the amount of succinic anhydride was 25 mg. A 1% dispersion liquid containing polymer particles C-3 having a molecular weight was obtained. The particle diameter was 2.7 μm, the specific surface area was 1.6 m ² / g, the amount of carboxyl groups was 6 μmol / g, and the parking area was 44 ² .

3.2.3.4. Synthesis Example 7 Production of Magnetic Particle (C-4) Having Carboxyl Group 1.0 g carboxyl group was prepared in the same manner as C-1 production method described in Synthesis Example 4 except that the amount of succinic anhydride was 40 mg. A 1% dispersion liquid containing polymer particles C-4 having a water content was obtained. The particle diameter was 2.7 μm, the specific surface area was 1.6 m ² / g, the amount of carboxyl groups was 10 μmol / g, and the parking area was 27 cm ² .

3.2.3.8. Synthesis Example 8 Production of Magnetic Particle (M-1) Having Amino Group Particles isolated by magnetic separation from an aqueous dispersion of magnetic particle A-4 on which a second polymer layer having a glycidyl group was formed were dispersed in acetone. After the magnetic separation and washing operation was repeated 5 times, the particles were dispersed again in acetone, the supernatant was removed by magnetic separation, and the particles were dried. Next, 0.50 g of the particles were placed in a 100 ml flask, 25 g of ethylenediamine was added, and then dispersed by irradiation with indirect ultrasonic waves for 20 minutes, and then heated and stirred at 50 ° C. for 3 hours in a nitrogen atmosphere (amino) Group introduction).

  After cooling, the particles are isolated by magnetic separation, dispersed in distilled water, magnetically separated and washed, and then the supernatant is removed by magnetic separation. Then, 1% sulfuric acid is removed. The particles were added to 5 g of an aqueous solution, dispersed by irradiation with indirect ultrasonic waves for 20 minutes, and then stirred at 60 ° C. for 5 hours (hydrolysis of residual glycidyl groups).

Subsequently, the operation of isolating the particles by magnetic separation, dispersing in pure water, magnetically separating and washing 5 times was repeated, and then dispersed in distilled water to obtain 0.49 g of magnetic particles M- having amino groups. A 1% dispersion containing 1 was obtained. The particle diameter was 2.8 μm, the specific surface area was 1.7 m ² / g, the amino group amount was 6 μmol / g, and the parking area was 47 ² .

3.2.3.9. Synthesis Example 9 Preparation of magnetic particle (T-1) having tosyl group After dispersing 1.0 g of magnetic particle A-6 having 2,3-dihydroxypropyl group in 8 ml of pyridine, 15 mg of p-tosyl chloride was added. And stirred at room temperature for 2 hours. After the reaction, the particles are isolated by magnetic separation, washed 4 times with acetone and 4 times with distilled water, and then dispersed in distilled water to disperse 1% containing polymer particles T-1 having 1.0 g of tosyl group. A liquid was obtained. The particle diameter was 2.7 μm, the specific surface area was 1.7 m ² / g, the tosyl group amount was 5 μmol / g, and the parking area was 56 ² .

3.3. Comparative synthesis example 3.3.1. Production of particles whose active functional group parking area is outside the scope of the present invention 3.3.1.1. Comparative Synthesis Example 1 Production of Magnetic Particles Having a Carboxyl Group (C-5) 1.0 g of carboxyl in the same manner as the production method of C-1 described in Synthesis Example 4 except that the amount of succinic anhydride is 70 mg. A 1% dispersion liquid containing polymer particles C-5 having groups was obtained. Particle size of 2.7 .mu.m, a specific surface area of 1.6 m ² / g, amount of carboxyl groups is 17μmol / g, the parking area was 16 Å ^2.

3.3.1.2. Comparative Synthesis Example 2 Production of Magnetic Particles Having a Carboxyl Group (C-6) 1.0 g of carboxyl in the same manner as the production method of C-1 described in Synthesis Example 4 except that the amount of succinic anhydride is 100 mg. A 1% dispersion liquid containing polymer particles C-6 having groups was obtained. The particle diameter was 2.7 μm, the specific surface area was 1.6 m ² / g, the amount of carboxyl groups was 25 μmol / g, and the parking area was 11 ² .

3.3.1.3. Comparative Synthesis Example 3 Production of Magnetic Particle (C-7) Having Carboxyl Group 1.0 g of carboxyl in the same manner as C-1 production method described in Synthesis Example 4 except that the amount of succinic anhydride is 250 mg. A 1% dispersion liquid containing polymer particles C-7 having groups was obtained. The particle diameter was 2.7 μm, the specific surface area was 1.6 m ² / g, the amount of carboxyl groups was 36 μmol / g, and the parking area was 7 ² .

3.3.2. Comparative Synthesis Example 4 Preparation of Magnetic Particles (D-1) Containing No Hydrophilic Functional Group 375 g of a 0.5% by weight aqueous solution of sodium dodecylbenzenesulfonate was charged into a 1 L separable flask, and then a magnetic layer was provided on the surface. 15 g of mother particles A-3 were added, dispersed with a homogenizer, and then heated to 60 ° C. A pre-emulsion prepared by dispersing 15 g of styrene, 4 g of methacrylic acid and 0.8 g of parroyl in 100 g of a 0.5% by weight aqueous solution of sodium dodecylbenzenesulfonate in a separate container was controlled at 60 ° C. The solution was added dropwise to the bull flask over 2 hours.
After completion of the dropwise addition, the temperature was raised to 75 ° C., and the polymerization was further continued for 2 hours to complete the reaction.

Next, the particles in the separable flask were separated using magnetism and washed with distilled water. Thereafter, by dispersing in distilled water, a 1% dispersion containing magnetic particles D-1 containing a carboxyl group as an active functional group and containing no hydrophilic functional group was obtained. The particle diameter was 2.2 μm, the specific surface area was 2.2 m ² / g, the amount of carboxyl groups was 15 μmol / g, and the parking area was 24 cm ² .

3.3. Example 3.3.1. Example 1
As a magnetic particle having a carboxyl group, 50 μl of a 1% dispersion of C-1 particles having a parking area of 531 cm ² was placed in a tube, and the supernatant was removed by magnetic separation. After washing three times with 50 mM MES-NaOH pH 6 (hereinafter referred to as “Buffer-1”), the probe is dispersed in 100 μl of Buffer-1 and specifically captures the target substance 20S proteasome; 5 μl of a solution of the protein (anti-20S proteasome α6 / mouse IgG antibody) (concentration: 5 mg / mL) was added, and 50 μg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide was added and stirred. Stirring was performed for 2 hours. After completion of the reaction, the supernatant was removed by magnetic separation. Next, 100 μl of a Tris buffer solution (hereinafter referred to as “TBS-T”) containing 0.05% nonionic surfactant Tween 20 was added, and the mixture was stirred at room temperature for 30 minutes. Furthermore, after washing 5 times with TBS-T, the particles were dispersed with PBS (−) buffer to obtain a 1% dispersion containing probe-bound particles.
The performance of the probe-bound particles was evaluated by the procedure described in the evaluation method. The resulting electrophoretic stained gel image is as shown in FIG. 1. The degree of specific capture was “good” and the degree of non-specific adsorption was “good”.

3.3.2. Example 2
A probe was bound by the method described in Example 1 except that C-2 particles having a parking area of 89 ² were used as magnetic particles having a carboxyl group, and performance evaluation was performed. The resulting electrophoretic stained gel image is as shown in FIG. 1. The degree of specific capture was “good” and the degree of non-specific adsorption was “good”.

3.3.3. Example 3
The probe was bound by the method described in Example 1 except that C-3 particles having a parking area of 44 ² were used as magnetic particles having a carboxyl group, and performance evaluation was performed. The electrophoretic stained gel images obtained as a result were as shown in FIGS. 1 and 2, and the degree of specific capture was “good” and the degree of non-specific adsorption was “good”.

3.3.4. Example 4
As except that the parking area was used C-4 particles 27 Å ² magnetic particles having a carboxyl group, to bind the probe by the method described in Example 1, and performance evaluation. The electrophoretic stained gel image obtained as a result is as shown in FIG. 1. The degree of specific capture was “slightly good” and the degree of non-specific adsorption was “slightly good”.

3.3.5. Example 5
Except the parking area as the magnetic particles having an amino group with M-1 particles 47A ² is similar to the carboxyl group particles, to bind the probe by the method described in Example 1, and performance evaluation. The resulting electrophoretic stained gel image is as shown in FIG. 2, and the degree of specific capture was “good” and the degree of non-specific adsorption was “good”.

3.3.6. Example 6
As magnetic particles having a tosyl group parking area takes a 1% dispersion 50μl of T-1 particles 56A ² in the tube, after removing the supernatant by magnetic separation, dispersed in borate buffer pH9.5 in 100μl It was. To this was added 5 μg of a protein (anti-20S proteasome α6 / mouse IgG antibody) serving as a probe for specifically capturing the 20S proteasome as a target substance, and the mixture was stirred at 37 ° C. for 16 hours. After completion of the reaction, the supernatant was removed by magnetic separation. Next, after washing three times with TBS, the particles were dispersed with PBS (−) buffer to obtain a 1% dispersion containing probe-bound particles T-1a.
The performance of the probe-bound particles was evaluated by the procedure described in the evaluation method. The resulting electrophoretic stained gel image is as shown in FIG. 2, and the degree of specific capture was “good” and the degree of non-specific adsorption was “good”.

3.3.7. Example 7
For using P-1 particles 48 Å ² as the non-magnetic polymer particles having a carboxyl group, and except for using centrifugation separation of the particles, to bind the probe by the method described in Example 1, performance evaluation did. The resulting electrophoretic stained gel image is as shown in FIG. 2, and the degree of specific capture was “good” and the degree of non-specific adsorption was “good”.

3.4. Comparative Example 3.4.1. Comparative Example 1 Magnetic particles whose parking area is outside the scope of the invention (1)
A probe was bound by the method described in Example 1 except that C-5 particles having a parking area of 16 ² were used as magnetic particles having a carboxyl group, and performance evaluation was performed. The resulting electrophoretic stained gel image is as shown in FIG. 1. The degree of specific capture was “poor” and the degree of non-specific adsorption was “poor”.

3.4.2. Comparative Example 2 Magnetic particles whose parking area is outside the scope of the invention (2)
The probe was bound by the method described in Example 1 except that C-6 particles having a parking area of 11 ² were used as magnetic particles having a carboxyl group, and performance evaluation was performed. The resulting electrophoretic stained gel image is as shown in FIG. 1. The degree of specific capture was “poor” and the degree of non-specific adsorption was “poor”.

3.4.3. Comparative Example 3 Magnetic particles whose parking area is outside the scope of the invention (3)
The probe was bound by the method described in Example 1 except that C-7 particles having a parking area of 7 to ² were used as magnetic particles having a carboxyl group, and performance evaluation was performed. The resulting electrophoretic stained gel image is as shown in FIG. 1. The degree of specific capture was “poor” and the degree of non-specific adsorption was “poor”.

3.4.4. Comparative Example 4 Magnetic particle containing no hydrophilic functional group The method described in Example 1 except that D-1 particles having a parking area having a carboxyl group of 24 ² and not containing a hydrophilic functional group were used. The probe was bound with and the performance was evaluated. The resulting electrophoretic stained gel image is as shown in FIG. 2. The degree of specific capture was “poor” and the degree of non-specific adsorption was “poor”.

3.4.5. Comparative Example 5 Magnetic particles whose parking area is outside the scope of the invention (4)
Commercially available magnetic particles parking area 3 Å ² as the magnetic particles having a carboxyl group (Dynal Inc. Dynabeads M-270 Carboxylic Acid, particle size 2.8 .mu.m, the amount of functional groups 150 [mu] mol / g, a specific surface area of 2.6 m ² / g) The probe was bound by the method described in Example 1 except that was used, and the performance was evaluated. The resulting electrophoretic stained gel image is as shown in FIG. 2. The degree of specific capture was “poor” and the degree of non-specific adsorption was “poor”.

It is an electrophoresis pattern which shows the grade of the specific capture | acquisition of the probe binding particle obtained in Examples 1-4, and the grade of non-specific adsorption | suction. It is an electrophoresis pattern which shows the grade of the specific capture | acquisition of the probe binding particle obtained in Example 3, 5-7, and the grade of non-specific adsorption | suction.

Claims (6)

Organic polymer particles for separating a biochemical substance from a biological sample by using an interaction with a probe, the organic polymer particles having an active functional group for binding the probe An organic polymer particle having a value obtained by dividing the surface area of the particle by the amount of the active functional group is 20 to 5,000 ² and further has a nonionic hydrophilic group.   Furthermore, the organic polymer particle of Claim 1 containing a magnetic body.   The organic polymer particle according to claim 1, wherein the active functional group is at least one group selected from the group consisting of a carboxyl group, an amino group, and a tosyl group.   The organic polymer particle according to any one of claims 1 to 3, wherein the nonionic hydrophilic group has a 2,3-dihydroxypropyl group.   A mother particle having a layer of superparamagnetic fine particles on the surface, and an organic polymer layer having an active functional group and a nonionic hydrophilic group for binding a probe provided so as to cover the mother particle. Organic polymer particles according to 2-4.   6. A probe-bound particle comprising the organic polymer particle according to claim 1 and a probe bound via the active functional group.

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