JPWO2016140216A1 - Solid phase carrier, ligand-bound solid phase carrier, target substance detection or separation method, and method for producing the solid phase carrier - Google Patents

Abstract

To provide a solid phase carrier that can be produced by a simple method, hardly causes non-specific adsorption, and exhibits excellent target substance capturing performance when a ligand is bound thereto. A solid phase carrier, wherein a copolymer comprising the following structural units (A) to (C) forms at least a solid surface. (A) Structural unit derived from a monomer having a zwitterionic structure: 1 to 35% by mass relative to all structural units (B) Structural unit derived from a monomer having a functional group capable of chemically bonding to a ligand: all structural units 70% by mass or less based on (C) a structural unit derived from a poorly water-soluble monomer having no functional group capable of chemically bonding to a ligand

Description

  The present invention relates to a solid phase carrier, a ligand-bound solid phase carrier, a method for detecting or separating a target substance, and a method for producing the solid phase carrier.

  Solid phase carriers are used for the purpose of detecting and separating target substances such as proteins, nucleic acids and cells from samples such as blood. As a detection / separation method using a solid phase carrier, a method is generally used in which a ligand is bound to a solid phase carrier and a sample is brought into contact therewith to cause a target substance to react with the ligand. In some cases, target substances and contaminants in the sample are adsorbed nonspecifically on the surface of the solid phase carrier, not the ligand, and this causes noise.

  For this reason, a copolymer using a highly hydrophilic monomer such as a monomer having a functional group capable of chemically bonding to a ligand and a zwitterionic structure or a monomer having a polyethylene glycol chain of a certain length or more is used as a solid phase. Attempts have been made to suppress non-specific adsorption by increasing the hydrophilicity of the surface by forming it on the surface of the carrier (Patent Documents 1 to 3).

JP 2012-181181 A International Publication No. 2011-125618 JP 2012-177691 A JP 2008-191128 A

However, simply increasing the hydrophilicity of the solid phase carrier surface using a highly hydrophilic monomer as described above does not sufficiently suppress non-specific adsorption. There was also room for improvement in the capture amount of the target substance.
In Patent Documents 2 and 3, a copolymer is bonded to the surface of a solid support using a radiation-induced graft polymerization method or a silane coupling method, but a special technique such as grafting is used. It is desired to develop a solid support that can be easily produced without using it. An emulsion polymerization method is known as a simple technique for forming a copolymer on the surface of a solid phase carrier (Patent Document 4). A polymer containing a structural unit derived from a highly hydrophilic monomer is solidified by an emulsion polymerization method. It is difficult to form it on the surface of the phase carrier, and it has been difficult to satisfy both the fact that it can be produced by a simple technique such as an emulsion polymerization method and sufficiently suppressing nonspecific adsorption.

  Therefore, the problem to be solved by the present invention is to provide a solid phase carrier that can be produced by a simple method, hardly causes non-specific adsorption, and exhibits excellent target substance capturing performance when a ligand is bound thereto. .

  Therefore, as a result of intensive studies, the present inventors have found that (A) a structural unit derived from a monomer having a zwitterionic structure: 1 to 35% by mass with respect to all structural units, and (B) a chemical bond with a ligand. A structural unit derived from a monomer having a functional group: a copolymer comprising 70% by mass or less with respect to all structural units, and (C) a structural unit derived from a poorly water-soluble monomer having no functional group capable of chemically bonding to a ligand. A solid-phase carrier characterized in that the polymer forms at least a solid-phase surface can be easily produced using an emulsion polymerization method, non-specific adsorption hardly occurs, and an excellent target when a ligand is bound. The present invention was completed by finding that it exhibits a substance-trapping performance.

  That is, the present invention provides the following <1> to <4>.

<1> A solid phase carrier, wherein a copolymer containing the following structural units (A) to (C) forms at least a solid surface.
(A) Structural unit derived from a monomer having a zwitterionic structure: 1 to 35% by mass relative to all structural units
(B) Structural unit derived from a monomer having a functional group capable of chemically binding to a ligand: 70% by mass or less based on the total structural unit (C) Derived from a poorly water-soluble monomer having no functional group capable of chemically binding to a ligand Structural unit

  <2> A ligand-bound solid phase carrier obtained by binding a ligand to the solid phase carrier of <1> above.

  <3> A method for detecting or separating a target substance in a sample, comprising using the ligand-bound solid phase carrier according to <2>.

  <4> Monomer having a zwitterionic structure: 1 to 35% by mass with respect to all monomers, and a monomer having a functional group capable of chemically bonding with a ligand: 70% by mass or less with respect to all monomers, and chemical bonding with the ligand A method for producing a solid phase carrier, comprising a step of radical polymerization of a poorly water-soluble monomer having no functional group in the presence of a raw material carrier.

The solid phase carrier of the present invention can be produced by a simple method. In addition, non-specific adsorption is unlikely to occur, and excellent target substance capture performance is exhibited when a ligand is bound. Therefore, the target substance is bound to the solid support of the present invention with high sensitivity and low noise. Can be detected.
In addition, according to the production method of the present invention, a non-specific adsorption is unlikely to occur, and a solid phase carrier that exhibits excellent target substance capturing performance when a ligand is bound can be easily produced.

[Solid support]
The solid phase carrier of the present invention is characterized in that a copolymer containing the following structural units (A) to (C) (hereinafter also referred to as copolymer (I)) forms at least a solid surface. Is. Here, in this specification, “form the surface of the solid phase” means that the solid phase (base material) itself contains the copolymer (I) or the copolymer (I) is a solid phase (group). It is said that the copolymer (I) forms a solid phase surface by, for example, being physically coated with the material), and that the copolymer (I) is a graft chain formed from the solid surface. It is a concept to exclude.
(A) Structural unit derived from a monomer having a zwitterionic structure: 1 to 35% by mass relative to all structural units
(B) Structural unit derived from a monomer having a functional group capable of chemically binding to a ligand: 70% by mass or less based on the total structural unit (C) Derived from a poorly water-soluble monomer having no functional group capable of chemically binding to a ligand Structural unit

<Structural unit (A)>
Copolymer (I) contains 1 to 35% by mass of structural units derived from a monomer having a zwitterionic structure based on the total structural units. Due to the zwitterionic structure contained in such a structural unit, the hydrophilicity of the solid support surface can be increased, and nonspecific adsorption is suppressed. The structural unit (A) has an alkali metal ion such as sodium ion and potassium ion, an alkaline earth metal ion such as calcium ion and magnesium ion, a counter ion such as ammonium ion, hydrogen ion and hydroxide ion. It may be. The same applies to any of the following embodiments.

Examples of the structural unit derived from a monomer having a zwitterionic structure include a structural unit derived from a (meth) acrylate monomer having a zwitterionic structure and a (meth) acrylamide monomer having a zwitterionic structure. The structural unit derived from the structural unit and the styrene-type monomer which has a zwitterionic structure is mentioned, Among these, 1 type may be included and 2 or more types may be included.
Among these, from the viewpoint of suppressing non-specific adsorption, a structural unit derived from a (meth) acrylate monomer having a zwitterionic structure, a structural unit derived from a (meth) acrylamide monomer having a zwitterionic structure is preferable, A structural unit derived from a (meth) acrylate monomer having a zwitterionic structure is more preferable.

The zwitterionic structure includes a quaternary ammonium salt type cationic functional group, — (C═O) O ⁻ , —SO ₃ ^— and —O— (O═PO) from the viewpoint of suppressing nonspecific adsorption. ^- ) Having a monovalent or divalent anionic functional group selected from -O- is preferred, and those represented by the following formula (1) or (2) are particularly preferred.

[In Formula (1),
R ¹ and R ² each independently represent a single bond or a divalent organic group having 1 to 10 carbon atoms,
R ³ represents — (C═O) O 2 ^— or —SO ₃ ^— ,
R ⁴ and R ⁵ each independently represents a methyl group or an ethyl group. ]

[In Formula (2),
R ⁶ and R ⁷ each independently represents a single bond or a divalent organic group having 1 to 10 carbon atoms,
R ⁸ , R ⁹ and R ¹⁰ each independently represent a methyl group or an ethyl group. ]

R ¹ and R ² in formula (1) and R ⁶ and R ^{7 in} formula (2) each independently represent a single bond or a divalent organic group having 1 to 10 carbon atoms. From the viewpoint of suppressing adsorption, a divalent organic group having 1 to 10 carbon atoms is preferable. The organic group is preferably a divalent hydrocarbon group or a group having at least one selected from an ether bond, an amide bond and an ester bond between carbon-carbon atoms of a divalent hydrocarbon group having 2 or more carbon atoms. A divalent hydrocarbon group is more preferred.

  When the divalent organic group is a divalent hydrocarbon group, the carbon number is preferably 1 to 8, more preferably 1 to 6, still more preferably 1 to 4, and particularly preferably 1 to 3. On the other hand, when the divalent organic group is a group having one or more selected from an ether bond, an amide bond and an ester bond between carbon-carbon atoms of a divalent hydrocarbon group having 2 or more carbon atoms, the total carbon As a number, 2-8 are preferable, 2-6 are more preferable, 2-4 are still more preferable, and 2 or 3 is especially preferable.

The “divalent hydrocarbon group” in R ¹ , R ² , R ⁶ and R ⁷ is preferably a divalent aliphatic hydrocarbon group. The divalent aliphatic hydrocarbon group may be linear or branched.
The divalent aliphatic hydrocarbon group is preferably an alkanediyl group, specifically, a methane-1,1-diyl group, an ethane-1,1-diyl group, an ethane-1,2-diyl group, Propane-1,1-diyl group, propane-1,2-diyl group, propane-1,3-diyl group, propane-2,2-diyl group, butane-1,2-diyl group, butane-1,3 -Diyl group, butane-1,4-diyl group, pentane-1,4-diyl group, pentane-1,5-diyl group, hexane-1,5-diyl group, hexane-1,6-diyl group, etc. Can be mentioned.

R ³ in the formula (1) is preferably — (C═O) O 2 ^— .
As R < ⁴ > and R < ⁵ > in Formula (1) and R <^8> , R < ⁹ > and R < ¹⁰ > in Formula (2), a methyl group is preferable.

  Preferable specific examples of the structural unit (A) include a structural unit represented by the following formula (3).

[In Formula (3),
R ¹¹ represents a hydrogen atom or a methyl group,
R ¹² represents — (C═O) —O— *, — (C═O) —NR ¹⁴ — * (R ¹⁴ represents a hydrogen atom or a methyl group, and * represents R ^{13 in} formula (3). Or a phenylene group,
R ¹³ represents a zwitterionic structure. ]

In formula (3), R ¹² is preferably — (C═O) —O— * or — (C═O) —NR ¹⁴ — * from the viewpoint of suppressing nonspecific adsorption, and — (C═O). -O- * is more preferred.
Further, zwitterionic structure represented by R ¹³ are as defined above zwitterion structure, from the viewpoint of inhibiting nonspecific adsorption, and the quaternary ammonium salt type cationic functional group, - (C = O) O - , —SO ₃ ^— and —O— (O═PO ^— ) ^— O— are preferred, and those having a monovalent or divalent anionic functional group are represented by the formula (1) or (2). Are particularly preferred.
R ¹⁴ is preferably a hydrogen atom.

  Examples of the monomer having a zwitterionic structure include [2-((meth) acryloyloxy) ethyl] (carboxylatomethyl) dimethylaminium, [2-((meth) acryloyloxy) ethyl] (carboxylatoethyl). Dimethylaminium, [2-((meth) acryloyloxy) ethyl] (carboxylatopropyl) dimethylaminium, [2-((meth) acryloyloxy) ethyl] dimethyl- (3-sulfomethyl) ammonium hydroxide, [2 -((Meth) acryloyloxy) ethyl] dimethyl- (3-sulfoethyl) ammonium hydroxide, [2-((meth) acryloyloxy) ethyl] dimethyl- (3-sulfopropyl) ammonium hydroxide, O- [2- ((Meth) acryloyloxy) ethoxy ( Kishirato) phosphinyl] choline and the like, may be using one of these may be used in combination of two or more.

Content of a structural unit (A) is 1-35 mass% with respect to all the structural units in copolymer (I). Since the solid phase carrier of the present invention has a low content of the structural unit (A) in the copolymer (I), it can be easily produced using an emulsion polymerization method. On the other hand, the solid phase carrier of the present invention is less likely to cause non-specific adsorption despite the low content of the structural unit (A) in the copolymer (I) as described above, and binds the ligand. When used, the target substance can be detected with low noise.
The content of the structural unit (A) is preferably 3% by mass or more with respect to all the structural units, and preferably 30% by mass or less from the viewpoint of nonspecific adsorption suppression, target substance capturing performance, and the like. Preferably it is 25 mass% or less, More preferably, it is 20 mass% or less, More preferably, it is 15 mass% or less, Most preferably, it is 10 mass% or less.
The content of each structural unit in the copolymer (I) may be measured using, for example, X-ray photoelectron spectroscopy or elemental analysis.

<Structural unit (B)>
Copolymer (I) contains 70% by mass or less of structural units derived from monomers having a functional group capable of chemically bonding to a ligand, based on the total structural units. The structural unit (B) means a unit other than the structural unit (A) among structural units derived from a monomer having a functional group capable of chemically bonding to a ligand.
Examples of the structural unit derived from a monomer having a functional group capable of chemically bonding to a ligand include, for example, a structural unit derived from (meth) acrylic acid or a salt thereof, and a (meth) acrylate system having a functional group capable of chemically bonding to a ligand. Examples include structural units derived from monomers, structural units derived from (meth) acrylamide monomers having functional groups capable of chemically bonding to ligands, and structural units derived from styrene monomers having functional groups capable of chemically bonding to ligands. One of these may be included, or two or more may be included.
Among these, (meth) acrylates having a functional unit capable of chemically binding to a structural unit derived from (meth) acrylic acid or a salt thereof, from the viewpoint of being able to be produced by a simple method and from the viewpoint of suppressing nonspecific adsorption A structural unit derived from a monomer or a structural unit derived from a (meth) acrylamide monomer having a functional group capable of chemically bonding to a ligand is preferred, and derived from a (meth) acrylate monomer having a functional group capable of chemically bonding to a ligand. A structural unit is more preferable.

  Examples of the functional group that can be chemically bonded to the ligand include a carboxy group, a tosyl group, an amino group, an epoxy group, and an azide group. Among these, from the viewpoint of target substance capturing performance, and when using biomolecules such as proteins and nucleic acids as ligands, it is possible to bind to a solid phase carrier using a functional group that the ligand originally has. Group, tosyl group, amino group and epoxy group are preferred, and carboxy group is more preferred.

Further, the content of the functional group capable of chemically binding to the ligand is preferably 1 μmol or more, more preferably 10 μmol or more, more preferably 10 μmol or more, per 1 g of the solid content of the solid phase carrier, from the viewpoint of non-specific adsorption suppression, target substance capture performance, etc. The amount is preferably 25 μmol or more, particularly preferably 50 μmol or more, and is preferably 800 μmol or less, more preferably 650 μmol or less, and still more preferably 500 μmol or less, from the viewpoint of nonspecific adsorption suppression, target substance capture performance, and the like.
The content of the functional group that can be chemically bonded to the ligand can be measured by, for example, an electrical conductivity measurement method when the functional group that can be chemically bonded to the ligand is a carboxy group. According to the method described in 1. In addition, when the functional group that can be chemically bonded to the ligand is a tosyl group, it can be determined by measuring the ultraviolet-visible light absorption of the tosyl group, and when the functional group that can be chemically bonded to the ligand is an amino group, It can be determined by reacting N-succinimidyl-3- (2-pyridyldithio) propionate with an amino group, followed by reduction and measuring the absorbance of the free thiopyridyl group.

  Preferable specific examples of the structural unit (B) include a structural unit represented by the following formula (4).

[In Formula (4),
R ¹⁵ represents a hydrogen atom or a methyl group,
R ¹⁶ represents — (C═O) —O— *, — (C═O) —NR ¹⁸ — * (R ¹⁸ represents a hydrogen atom or a methyl group, and * represents R ^{17 in} formula (4). Or a phenylene group,
When R ¹⁶ is — (C═O) —O— *, R ¹⁷ represents a hydrogen atom or an organic group having a functional group capable of chemically bonding to a ligand, and R ¹⁶ represents — (C═O) —. NR ¹⁸ - If a * or a phenylene group, R ¹⁷ represents an organic group having a ligand capable of chemically bonding functional groups. ]

In the formula (5), R ¹⁶ is preferably — (C═O) —O— * or — (C═O) —NR ¹⁸ — * from the viewpoint of suppressing nonspecific adsorption, and — (C═O). -O- * is more preferred. R ¹⁸ is preferably a hydrogen atom.

R ¹⁷ is preferably an organic group having a functional group that can be chemically bonded to a ligand from the viewpoint of being able to be produced by a simple method and from the viewpoint of suppressing nonspecific adsorption. As the organic group having a functional group capable of chemically bonding to the ligand, an organic group represented by the following formula (5) is preferable. The functional group capable of being chemically bonded to the ligand in R ¹⁷ and Y is the same as the functional group capable of being chemically bonded to the ligand, preferably a carboxy group, a tosyl group, an amino group, or an epoxy group, and more preferably a carboxy group. .

[In Formula (5),
R ¹⁹ represents a divalent organic group,
Y represents a functional group that can be chemically bonded to a ligand. ]

The divalent organic group represented by R ¹⁹ includes a divalent hydrocarbon group, an ether bond, an imino group, an amide bond and an ester bond between carbon-carbon atoms of a divalent hydrocarbon group having 2 or more carbon atoms. Groups having one or more selected groups, and groups having at least one selected from an ether bond, an imino group, an amide bond and an ester bond between carbon-carbon atoms of a divalent hydrocarbon group having 2 or more carbon atoms. Is preferred.

When the divalent organic group is a divalent hydrocarbon group, the carbon number is preferably 1-20, more preferably 1-10, still more preferably 1-8, and particularly preferably 1-6.
In the case where the divalent organic group is a divalent hydrocarbon group, the divalent hydrocarbon group includes a divalent aliphatic hydrocarbon group, a divalent alicyclic hydrocarbon group, and a divalent hydrocarbon group. The concept includes an aromatic hydrocarbon group, but a divalent aliphatic hydrocarbon group is preferable. The divalent aliphatic hydrocarbon group may be linear or branched.
The divalent aliphatic hydrocarbon group is preferably an alkanediyl group, specifically, a methane-1,1-diyl group, an ethane-1,1-diyl group, an ethane-1,2-diyl group, Propane-1,1-diyl group, propane-1,2-diyl group, propane-1,3-diyl group, propane-2,2-diyl group, butane-1,2-diyl group, butane-1,3 -Diyl group, butane-1,4-diyl group, pentane-1,4-diyl group, pentane-1,5-diyl group, hexane-1,5-diyl group, hexane-1,6-diyl group, etc. Can be mentioned.

On the other hand, when the divalent organic group is a group having one or more selected from an ether bond, an imino group, an amide bond and an ester bond between carbon-carbon atoms of a divalent hydrocarbon group having 2 or more carbon atoms, As the total carbon number, 2-20 are preferable, 2-16 are more preferable, 2-12 are still more preferable, and 2-8 are especially preferable.
In addition, the group having one or more selected from an ether bond, an imino group, an amide bond and an ester bond between carbon-carbon atoms of a divalent hydrocarbon group having 2 or more carbon atoms is represented by the following formula (6). Preferred is a divalent group.

[In Formula (6),
R ²⁰ and R ²¹ each independently represent a divalent hydrocarbon group,
X represents an ether bond, an imino group, an amide bond or an ester bond,
* Indicates a bonding position with Y in the formula (5). ]

X is preferably an ester bond.
The divalent hydrocarbon group represented by R ²⁰ and R ²¹ is a concept including a divalent aliphatic hydrocarbon group, a divalent alicyclic hydrocarbon group, and a divalent aromatic hydrocarbon group.
As carbon number of a bivalent aliphatic hydrocarbon group, 1-10 are preferable, 1-8 are more preferable, and 1-6 are especially preferable. The divalent aliphatic hydrocarbon group may be linear or branched.
The divalent aliphatic hydrocarbon group is preferably an alkanediyl group, specifically, a methane-1,1-diyl group, an ethane-1,1-diyl group, an ethane-1,2-diyl group, Propane-1,1-diyl group, propane-1,2-diyl group, propane-1,3-diyl group, propane-2,2-diyl group, butane-1,2-diyl group, butane-1,3 -Diyl group, butane-1,4-diyl group, pentane-1,4-diyl group, pentane-1,5-diyl group, hexane-1,5-diyl group, hexane-1,6-diyl group, etc. Can be mentioned.
As carbon number of a bivalent alicyclic hydrocarbon group, 5-10 are preferable and 5-7 are more preferable. The divalent alicyclic hydrocarbon group is preferably a cycloalkanediyl group, and specific examples include a cyclohexanediyl group and a cycloheptanediyl group.
As carbon number of a bivalent aromatic hydrocarbon group, 6-18 are preferable and 6-12 are more preferable. The divalent aromatic hydrocarbon group is preferably an arylene group, and specific examples include a phenylene group and a naphthylene group.

As a combination of R ²⁰ and R ²¹ , R ²⁰ is a divalent aliphatic hydrocarbon group, and R ²¹ is a divalent aliphatic hydrocarbon group, a divalent alicyclic hydrocarbon group, or 2 A combination that is a divalent aromatic hydrocarbon group is preferable, and a combination that R ²⁰ is a divalent aliphatic hydrocarbon group and R ²¹ is a divalent aliphatic hydrocarbon group is more preferable.

  Examples of the monomer having a functional group capable of chemically bonding with a ligand include hydrogen 1-((meth) acryloyloxy) ethyl succinate, 1- (1- (meth) acryloyloxyethyl) phthalate, and 1,2-cyclohexane. Hydrogen dicarboxylate 1- [1-((meth) acryloyloxy) ethyl], hydrogen 1-((meth) acryloyloxy) propyl succinate, hydrogen 1- [1-((meth) acryloyl) 1,2-cyclohexanedicarboxylate Oxy) propyl], succinic acid 1- (2- (meth) acryloyloxyethyl), phthalic acid 1- (2- (meth) acryloyloxyethyl), hydrogen hexahydrophthalate 1- (2- (meth) acryloyloxy) Ethyl), hydrogen succinate 2-((meth) acryloyloxy) propyl, hydrogen phthalate 1- (2- (methyl ) Acryloyloxypropyl), hydrogen hexahydrophthalate 1- (2- (meth) acryloyloxypropyl), hydrogen succinate 3-((meth) acryloyloxy) propyl, hydrogen phthalate 1- (3- (meth) acryloyl) Oxypropyl), hydrogen 1,2-cyclohexanedicarboxylate 1- [3-((meth) acryloyloxy) propyl] and the like, and one of these may be used, or two or more may be used in combination. Good.

Content of a structural unit (B) is 70 mass% or less with respect to all the structural units in a copolymer. With such a configuration, nonspecific adsorption is less likely to occur, and the target substance capturing performance is improved.
The content of the structural unit (B) is preferably 1% by mass or more, more preferably 10% by mass or more, and particularly preferably 20% from the viewpoints of nonspecific adsorption suppression, target substance capturing performance, and the like with respect to all structural units. In addition, from the viewpoint of nonspecific adsorption suppression and target substance capturing performance, it is preferably 60% by mass or less, more preferably 50% by mass or less, and particularly preferably 40% by mass or less.

  Moreover, as mass ratio [(A) :( B)] of content of a structural unit (A) and a structural unit (B), it is 5: 95-60 from viewpoints of nonspecific adsorption | suction suppression, target substance capture | acquisition performance, etc. : 40 is preferable, 10:90 to 50:50 is more preferable, 15:85 to 40:60 is more preferable, 17.5: 82.5 to 35:65 is still more preferable, and 17.5: 82.5 to 30:70 is more preferable, and 17.5: 82.5 to 25:75 is particularly preferable.

<Structural unit (C)>
Copolymer (I) contains, in addition to the structural units (A) and (B), a structural unit derived from a poorly water-soluble monomer having no functional group capable of chemically bonding to a ligand. With such a configuration, nonspecific adsorption is less likely to occur, and the target substance capturing performance is improved.
In the present specification, the poorly water-soluble monomer means a monomer having a solubility of 10 g or less with respect to 100 g of water.

Examples of the structural unit derived from a poorly water-soluble monomer that does not have a functional group that can be chemically bonded to a ligand include, for example, a structural unit derived from a poorly water-soluble (meth) acrylate monomer that does not have a functional group that can be chemically bonded to a ligand. A structural unit derived from a poorly water-soluble (meth) acrylamide monomer that does not have a functional group capable of chemically binding to a ligand, and a structural unit derived from a poorly water-soluble styrene monomer that does not have a functional group capable of chemically binding to a ligand. 1 type may be included among these, and 2 or more types may be included. Further, the poorly water-soluble monomer for deriving these structural units may be a monofunctional monomer or a polyfunctional monomer.
Among these, from the viewpoint of suppressing non-specific adsorption, etc., it has a structural unit derived from a poorly water-soluble (meth) acrylate monomer that does not have a functional group that can be chemically bonded to the ligand, and has a functional group that can be chemically bonded to the ligand. A structural unit derived from a poorly water-soluble (meth) acrylamide monomer is preferred, and a structural unit derived from a poorly water-soluble (meth) acrylate monomer having no functional group capable of chemically bonding to a ligand is more preferred.

  As the structural unit (C), the following structural units (C-1) to (C-3) are preferable. In addition, copolymer (I) may contain 1 type in these, and may contain 2 or more types.

  (C-1) Monoacrylate monomer that retains intermediate water when polymerized as a homopolymer and coexisting with free water, or a structural unit derived from a monomethacrylate monomer having the same side chain structure as the monoacrylate monomer

  (C-2) Structural unit derived from a mono (meth) acrylate monomer represented by the following formula (7)

[In Formula (7),
R ²² represents a hydrogen atom or a methyl group,
R ²³ represents a hydrocarbon group. ]

  (C-3) Structural unit derived from polyfunctional monomer

(Structural unit (C-1))
The structural unit (C-1) is derived from a monoacrylate monomer which is polymerized to form a homopolymer and retains intermediate water when free water coexists, or a monomethacrylate monomer having the same side chain structure as the monoacrylate monomer. A structural unit.

Here, the homopolymer that retains intermediate water when free water coexists means that the water temperature is less than −10 ° C. in the DSC curve obtained by differential scanning calorimetry using a differential scanning calorimeter (DSC) in the temperature rising process. A homopolymer in which an exothermic peak due to crystallization is observed.
Free water refers to water that is observed as a melting point (endothermic peak) near 0 ° C. in the DSC curve obtained by differential scanning calorimetry in the temperature rising process.
The intermediate water refers to water that is observed as an exothermic peak due to crystallization at a temperature of less than −10 ° C. in the DSC curve obtained by differential scanning calorimetry in the temperature rising process.
The temperature raising process refers to a temperature raising process after cooling to −100 ° C. or lower in differential scanning calorimetry.

  As the structural unit (C-1), a structural unit derived from a monomer represented by the following formula (8) or (9) is preferable, and a structural unit derived from a monomer represented by the following formula (9) is more preferable. .

[In Formula (8),
R ²⁴ represents a hydrogen atom or a methyl group,
R ²⁵ represents an alkanediyl group having 1 to 3 carbon atoms;
R ²⁶ represents an alkyl group having 1 to 3 carbon atoms,
n shows the integer of 1-5. ]

[In Formula (9),
R ²⁷ represents a hydrogen atom or a methyl group,
m represents 1 or 2. ]

In formula (8), the carbon number of the alkanediyl group represented by R ²⁵ is preferably 2 or 3, and more preferably 2. The alkanediyl group may be linear or branched. Specific examples thereof include ethane-1,1-diyl group, ethane-1,2-diyl group, propane-1,1-diyl group, propane- 1,2-diyl group, propane-1,3-diyl group, propane-2,2-diyl group and the like can be mentioned.
The number of carbon atoms of the alkyl group represented by R ²⁶ is preferably 1 or 2. The alkyl group may be linear or branched, and specific examples include a methyl group, an ethyl group, an n-propyl group, and an isopropyl group.
n represents an integer of 1 to 5, and an integer of 1 to 3 is preferable.
In formula (9), m represents 1 or 2, but 1 is preferable.

  As the monomer for deriving the structural unit (C-1), methoxyethyl acrylate, methoxyethyl methacrylate, tetrahydrofurfuryl acrylate, tetrahydrofurfuryl methacrylate, di (ethylene glycol) monomethyl ether acrylate, di (ethylene glycol) monomethyl ether methacrylate, Di (ethylene glycol) monoethyl ether acrylate, di (ethylene glycol) monoethyl ether methacrylate, tri (ethylene glycol) monomethyl ether acrylate, tri (ethylene glycol) monomethyl ether methacrylate, tri (ethylene glycol) monoethyl ether acrylate, tri ( Ethylene glycol) monoethyl ether methacrylate, etc. Well with seeds, it may be used in combination of two or more.

(Structural unit (C-2))
The structural unit (C-2) is a structural unit derived from a mono (meth) acrylate monomer represented by the following formula (7).

[In Formula (7),
R ²² represents a hydrogen atom or a methyl group,
R ²³ represents a hydrocarbon group. ]

The hydrocarbon group represented by R ²³ is a concept including an aliphatic hydrocarbon group, an alicyclic hydrocarbon group, and an aromatic hydrocarbon group.
The number of carbon atoms of the aliphatic hydrocarbon group for R ^23, preferably from 1 to 20, more preferably 1 to 12, 1 to 8 is more preferable. The aliphatic hydrocarbon group may be linear or branched. As the aliphatic hydrocarbon group, an alkyl group is preferable, and specifically, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, A pentyl group, a hexyl group, a heptyl group, an octyl group, etc. are mentioned.
The number of carbon atoms of the alicyclic hydrocarbon group in R ^23, 6 to 12 is preferable, 6 to 10 is more preferable. The alicyclic hydrocarbon group is roughly classified into a monocyclic alicyclic hydrocarbon group and a bridged ring hydrocarbon group. Examples of the monocyclic alicyclic hydrocarbon group include cycloalkyl groups such as a cyclopropyl group and a cyclohexyl group. Examples of the bridged ring hydrocarbon group include an isobornyl group.
The number of carbon atoms of the aromatic hydrocarbon group for R ^23, 6 to 12 is preferable. Examples of the aromatic hydrocarbon group include aryl groups such as a phenyl group.

Among these hydrocarbon groups, R ²³ is preferably an alicyclic hydrocarbon group, and more preferably a cycloalkyl group.

  Examples of the monomer for deriving the structural unit (C-2) include methyl (meth) acrylate, ethyl (meth) acrylate, stearyl (meth) acrylate, cyclohexyl (meth) acrylate, isobornyl (meth) acrylate, and the like. Of these, one type may be used, or two or more types may be used in combination.

(Structural unit (C-3))
The structural unit (C-3) is a structural unit derived from a polyfunctional monomer. Examples include polyfunctional (meth) acrylate monomers; polyfunctional aromatic vinyl monomers such as divinylbenzene; conjugated diolefins such as butadiene and isoprene. Among these, a polyfunctional (meth) acrylate monomer is preferable. Multifunctional (meth) acrylate monomers include bifunctional (meth) acrylate monomers such as ethylene glycol di (meth) acrylate and allyl (meth) acrylate; trifunctionality such as trimethylolpropane tri (meth) acrylate (Meth) acrylate monomers; hexafunctional (meth) acrylate monomers such as dipentaerythritol hexa (meth) methacrylate and the like.

  Moreover, as the structural unit (C), from the viewpoint of non-specific adsorption suppression and target substance capturing performance, those containing at least the structural unit (C-1) and those containing at least the structural unit (C-3) are preferable. Those containing at least the structural unit (C-1) are more preferred, and those containing at least the structural units (C-1) and (C-3) are particularly preferred.

  The total content of the structural unit (C) is preferably 10% by mass or more, more preferably 30% by mass or more, and still more preferably from the viewpoints of non-specific adsorption suppression and target substance capturing performance, etc. It is 40% by mass or more, more preferably 50% by mass or more, particularly preferably 55% by mass or more, and preferably 90% by mass or less, more preferably 80% from the viewpoint of non-specific adsorption suppression and target substance capturing performance. It is at most 70% by mass, particularly preferably at most 70% by mass.

Moreover, as mass ratio [(A) :( C)] of content of a structural unit (A) and a structural unit (C), it is 1: 99-60 from viewpoints of nonspecific adsorption | suction suppression, target substance capture | acquisition performance, etc. : 40 is preferable, 5:95 to 50:50 is more preferable, 10:90 to 40:60 is more preferable, 10:90 to 30:70 is still more preferable, and 10:90 to 20:80 is particularly preferable.
Moreover, as mass ratio [(B) :( C)] of content of a structural unit (B) and a structural unit (C), it is 1: 99-99 from viewpoints of nonspecific adsorption | suction suppression, target substance capture | acquisition performance, etc. : 1 is preferable, 10:90 to 60:40 is more preferable, 20:80 to 45:55 is further preferable, and 20:80 to 40:60 is particularly preferable.

  Moreover, when copolymer (I) has a structural unit (C-1), content of a structural unit (C-1) is nonspecific adsorption | suction suppression, target substance capture performance, etc. with respect to all the structural units. From the viewpoint, it is preferably 10% by mass or more, more preferably 20% by mass or more, still more preferably 30% by mass or more, still more preferably 40% by mass or more, and particularly preferably 50% by mass or more. And 90% by mass or less, more preferably 80% by mass or less, still more preferably 70% by mass or less, and particularly preferably 60% by mass or less from the viewpoints of target substance capturing performance and the like.

  Moreover, when copolymer (I) has a structural unit (C-2), content of a structural unit (C-2) becomes like this. Preferably it is 10 mass% or more with respect to all the structural units, More preferably, it is 20 It is at least 30% by mass, particularly preferably at least 30% by mass, preferably at most 90% by mass, more preferably at most 80% by mass, particularly preferably at most 70% by mass.

  Moreover, when copolymer (I) has a structural unit (C-3), content of a structural unit (C-3) is nonspecific adsorption | suction suppression, target substance capture performance, etc. with respect to all the structural units. From the viewpoint, it is preferably 1% by mass or more, more preferably 3% by mass or more, and from the viewpoints of non-specific adsorption suppression and target substance capturing performance, etc., preferably 70% by mass or less, more preferably 30% by mass or less. Especially preferably, it is 15 mass% or less.

The copolymer (I) may have a structural unit other than the structural units (A) to (C) (hereinafter also referred to as a structural unit (D)).
Examples of the structural unit (D) include structural units derived from hydrophilic monomers other than monomers having a zwitterionic structure. More specifically, structural units derived from methoxypolyethylene glycol # 400 methacrylate (Shin Nakamura Chemical Co., Ltd.) and the like.

Moreover, when copolymer (I) does not have a crosslinked structure, as a number average molecular weight (Mn), 5,000-1,000,000 are preferable and 10,000-500,000 are more preferable.
Moreover, when copolymer (I) does not have a crosslinked structure, as a weight average molecular weight (Mw), 5,000-1,000,000 are preferable and 10,000-500,000 are more preferable.
Moreover, when copolymer (I) does not have a crosslinked structure, as molecular weight distribution (Mw / Mn), 1.0-3.0 are preferable.
In addition, a number average molecular weight and a weight average molecular weight mean the average molecular weight of polyethylene glycol conversion measured by gel permeation chromatography.

  In addition, the arrangement of the structural units in the copolymer (I) is not particularly limited and may be any of a random copolymer, a block copolymer, and an alternating copolymer. Random copolymers are preferred from the standpoints that can be made.

The solid phase carrier of the present invention is not limited so long as the copolymer (I) forms at least the surface of the solid phase, even if the solid phase (base material) itself contains the copolymer (I). The solid phase (base material) may be physically coated with the combined body (I).
Further, the solid phase substrate (raw material carrier) may be an organic material or an inorganic material such as a metal or a metal oxide, and is not particularly limited, but preferably includes a resin. The resin may be a natural polymer composed of a polysaccharide such as agarose, dextran, or cellulose, or may be a synthetic polymer.
Further, the form of the solid phase carrier of the present invention is not particularly limited and may be any of particles, monoliths, membranes, fibers, chips, etc., but particles are preferable, and magnetic particles are more preferable. When the solid phase carrier of the present invention is in the form of particles, particularly magnetic particles, excellent redispersibility is exhibited, and operations such as detection and separation can be greatly simplified. In the present specification, the “magnetic particle” means a particle having a magnetic material.

The magnetic material may be ferromagnetic, paramagnetic, or superparamagnetic, but is preferably superparamagnetic from the viewpoint of facilitating separation by a magnetic field and redispersion after removing the magnetic field. Examples of the magnetic material include metals such as ferrite, iron oxide, iron, manganese oxide, manganese, nickel oxide, nickel, cobalt oxide, and cobalt, or alloys.
Specific examples of the magnetic particles include those in which the copolymer (I) forms at least the surface of any of the following particles (i) to (iv). Porous or non-porous magnetic polymer particles are preferred.

(I) Particles in which magnetic fine particles are dispersed in a continuous phase containing a non-magnetic material such as a resin (ii) Particles having a secondary aggregate of magnetic fine particles as a core and a non-magnetic material such as a resin as a shell (Iii) Core particles having core particles composed of a non-magnetic material such as a resin and a magnetic layer (secondary aggregate layer) containing magnetic fine particles provided on the surface of the core particles are used as a core. Particles in which a non-magnetic layer such as resin is provided as a shell on the outermost layer of the mother particle (iv) A resin or silica in which a non-magnetic layer such as resin is provided as a shell on the outermost layer of the particle Particles in which magnetic fine particles are dispersed in pores of porous particles made of, etc. The number average particle diameter of the particles (i) to (iv) is preferably 0.05 to 250 μm, more preferably It is 0.1-50 micrometers, Most preferably, it is 0.1-25 micrometers. The particles (i) to (iv) are all known and can be produced according to a conventional method.

Although the resin in said (i)-(iv) is not specifically limited, For example, resin derived from 1 type, or 2 or more types chosen from a monofunctional monomer and a polyfunctional monomer is mentioned.
Examples of the monofunctional monomer include monofunctional aromatic vinyl monomers such as styrene, α-methylstyrene, and halogenated styrene; methyl (meth) acrylate, ethyl (meth) acrylate, stearyl (meth) acrylate, cyclohexyl ( Monofunctional (meth) acrylates such as (meth) acrylate, isobornyl (meth) acrylate, glycidyl (meth) acrylate, allyl glycidyl ether, 2-aminoethyl (meth) acrylate, 1,4-cyclohexanedimethanol mono (meth) acrylate Based monomers. Examples of the polyfunctional monomer include polyfunctional aromatic vinyl monomers such as divinylbenzene; ethylene glycol di (meth) acrylate, trimethylolpropane tri (meth) acrylate, dipentaerythritol hexa (meth) methacrylate, allyl ( Examples thereof include polyfunctional (meth) acrylate monomers such as (meth) acrylate; conjugated diolefins such as butadiene and isoprene.

The average particle diameter (volume average particle diameter) when the solid phase carrier of the present invention is a particle is preferably 0.02 to 500 μm, more preferably 0.1 to 250 μm, and still more preferably 0.8. It is 2-100 micrometers, More preferably, it is 0.3-50 micrometers. By setting it as such a range, when a solid-phase support | carrier is a magnetic particle, a magnetic flux collection speed becomes quick and handling property is improved. Moreover, even when the solid phase carrier of the present invention is a relatively small particle having an average particle size of about 0.05 to 0.3 μm, good redispersibility can be obtained.
Further, the coefficient of variation of the average particle diameter may be about 20% or less.
Moreover, the specific surface area should just be about 1.0-2.0 m < ² > / g.
The average particle size and specific surface area can be measured by laser diffraction / scattered particle size distribution measurement or the like.

Next, a method for producing the solid phase carrier of the present invention will be described.
The solid phase carrier of the present invention is, for example, a monomer having a zwitterionic structure: 1 to 35% by mass with respect to the total monomer, and a monomer having a functional group capable of chemically bonding to the ligand: 70% by mass with respect to the total monomer. The following can be produced by a method including radical polymerization of a poorly water-soluble monomer having no functional group capable of chemically bonding to a ligand in the presence of a raw material carrier (base material).

  Radical polymerization may be carried out according to a conventional method with reference to JP-A-2008-191128, etc., but emulsion polymerization, suspension polymerization and the like are preferable, and emulsion polymerization is more preferable from the viewpoint of simplicity and ease, Oil-in-water (O / W) emulsion polymerization is particularly preferred. Specifically, a method may be mentioned in which a pre-emulsion containing the above monomer is dropped into an aqueous medium (water or the like) in which a raw material carrier is dispersed and polymerized.

In addition, radical polymerization is performed by polymerization initiators such as azo initiators, peroxide initiators, and redox initiators; interfaces such as alkyl sulfate esters, alkylaryl sulfate esters, alkyl phosphate esters, and fatty acid salts. It is preferable to use an activator or the like.
Moreover, superposition | polymerization temperature is 40-100 degreeC normally, Preferably it is 50-90 degreeC. The polymerization time is usually 1 to 48 hours.

The solid phase carrier of the present invention can be produced by a simple method, is less likely to cause non-specific adsorption, and exhibits excellent target substance capture performance when bound to a ligand. By binding and using a ligand, a target substance can be detected with high sensitivity and low noise. In addition, the separation of the target substance can be highly purified.
Therefore, by using the solid phase carrier of the present invention as an affinity carrier, immunoassay utilizing antigen-antibody reaction such as enzyme immunoassay, radioimmunoassay, chemiluminescence immunoassay; detection of protein, nucleic acid, etc .; cell, protein, nucleic acid It can be widely used for in-vitro diagnosis and research in the field of biochemistry such as bioseparation of bio-related substances such as; The solid phase carriers of the present invention are particularly suitable for use for immunoassays or for nucleic acid detection.

[Ligand-bound solid support]
The ligand-bound solid phase carrier of the present invention is obtained by binding a ligand to the solid phase carrier of the present invention.
The ligand may be a molecule that binds to a target substance. For example, antibody; antigen; nucleic acid such as DNA and RNA; nucleotide; nucleoside; protein such as protein A, protein G, (strept) avidin, enzyme, and lectin Peptides such as insulin; amino acids; sugars or polysaccharides such as heparin; lipids; vitamins such as biotin; drugs; substrates; hormones;
Among these, antibodies and antigens are preferable from the viewpoint of forming a ligand-bound solid phase carrier suitable for diagnostic drugs and the like. The antibody and the antigen may be any substance that binds to a target substance. For example, anti-antiplasmin antibody, anti-D dimer antibody, anti-FDP antibody, anti-tPA antibody, anti-thrombin / antithrombin complex antibody, anti-FPA antibody, etc. Anticoagulation / fibrinolysis-related antibody or antigen thereto; anti-BFP antibody, anti-CEA antibody, anti-AFP antibody, anti-TSH antibody, anti-ferritin antibody, anti-CA19-9 antibody or other anti-tumor-related antibody or antigen thereto; Serum protein-related testing antibodies such as apolipoprotein antibodies, anti-β2-microbrovulin antibodies, anti-α1-microglobulin antibodies, anti-immunoglobulin antibodies, anti-CRP antibodies, and antigens thereto; endocrine function testing antibodies such as anti-HCG antibodies Or an antigen thereto; an antibody for drug analysis such as an anti-digoxin antibody or an anti-lidocaine antibody or an antigen thereto; HB s antigens, HCV antigens, HIV-1 antigens, HIV-2 antigens, HTLV-1 antigens, mycoplasma antigens, toxoplasma antigens, streptridine O antigens and other infectious disease-related test antigens or antibodies thereto; DNA antigens, heat-denatured humans Examples include antigens for autoimmunity-related examinations such as IgG or antibodies to the same. The antibody may be a polyclonal antibody or a monoclonal antibody.

The amount of ligand binding is preferably 1 to 200 μg, more preferably 1 to 100 μg, still more preferably 1 to 50 μg, and particularly preferably 1 to 30 μg per 1 mg of the solid content of the ligand-bound solid phase carrier.
The amount of ligand binding can be measured according to the method described in Examples described later.

  Ligand binding may be performed according to a conventional method, but is preferably performed by a covalent bond method. For example, when the functional group that can be chemically bonded to the ligand is a carboxy group and the ligand has an amino group, the ligand may be bonded using a dehydrating condensing agent.

  The ligand-bound solid phase carrier of the present invention can be widely used for in vitro diagnosis and research in the field of biochemistry. The ligand-bound solid support of the present invention is particularly suitable for use for immunoassays or for nucleic acid detection.

[Target substance detection or separation method]
A method for detecting or separating a target substance in a sample of the present invention is characterized by using the ligand-bound solid phase carrier of the present invention.

The target substance may be any substance that binds to a ligand. Specifically, antigens; antibodies such as monoclonal antibodies and polyclonal antibodies; cells (normal cells, colon cancer cells, circulating cancer cells in the blood, etc.) Cells); nucleic acids such as DNA and RNA; bio-related substances such as proteins, peptides, amino acids, sugars, polysaccharides, lipids, and vitamins. Drugs that are drug targets, and small molecule compounds such as biotin. The target substance may be labeled with a fluorescent substance or the like.
The sample only needs to contain the target substance or may contain the target substance, and specifically, blood, plasma, serum, a buffer solution containing the target substance, and the like.

  The detection or separation method of the present invention may be performed according to a conventional method except that the ligand-bound solid phase carrier of the present invention is used. For example, the step of bringing the sample containing the ligand-binding solid phase carrier of the present invention and the target substance into contact with each other (contacting step), and the ligand-binding solid phase carrier capturing the target substance using a magnet or the like The method including the process (separation process) which isolate | separates from is mentioned. Note that, after the separation step, a step of detecting the target substance or a step of dissociating the ligand and the target substance may be included.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples.

<Content of functional group (reactive functional group) capable of chemically bonding with ligand>
By measuring the content of the reactive functional group (carboxy group) contained in the chain polymer released from the particles using an electrical conductivity measurement method (Metrohm, 794 Basic Titrino), The content of the reactive functional group was determined.

<Volume average particle diameter>
The volume average particle size of each particle was measured with a laser diffraction / scattered particle size distribution measuring apparatus (Beckman Coulter LS13 320).

[Synthesis Example 1]
Ferrite magnetic material having a hydrophobized surface by adding acetone to oily magnetic fluid ("EXP series, EMG", manufactured by Ferrotec Co., Ltd.) to precipitate particles and drying them. Body fine particles (average primary particle size: 0.01 μm) were obtained.
Next, 30 g of polystyrene particles (number average particle diameter: 0.77 μm) and 15 g of the above-mentioned hydrophobized magnetic fine particles were mixed well with a mixer, and this mixture was mixed with a hybridization system NHS-0 type (Nara Machinery Co., Ltd.). For 5 minutes at a peripheral speed of 100 m / sec (16,200 rpm) of a blade (stirring blade), and a mother particle (number average particle diameter: 1) having a magnetic layer made of magnetic fine particles on the surface. 0.0 μm) was obtained.

[Example 1]
300 g of a 0.50 mass% aqueous solution of sodium dodecyl sulfate was put into a 500 mL separable flask, 10 g of the mother particles obtained in Synthesis Example 1 were added, and the mixture was dispersed with a homogenizer, and then heated to 60 ° C. to maintain the temperature.
Next, in another container, 45 g of a 0.50 mass% aqueous solution of sodium dodecyl sulfate, 0.5 g of [2- (methacryloyloxy) ethyl] (carboxylatomethyl) dimethylaminium (hereinafter referred to as “CBMA”), 2- Methacryloyloxyethyl succinic acid (hereinafter referred to as “SA”) 2.0 g, tetrahydrofurfuryl methacrylate (hereinafter referred to as “THFMA”) 3.5 g, trimethylolpropane trimethacrylate (hereinafter referred to as “TMP”) 0 0.5 g, and di (3,5,5-trimethylhexanoyl) peroxide 75% solution (Perroyl 355-75 (S) manufactured by NOF Corporation (hereinafter referred to as “Parroyl 355-75 (S)”) ) 0.15 g was added and dispersed to obtain a pre-emulsion. The entire amount of this pre-emulsion was dropped into the 500 mL separable flask maintained at 60 ° C. over 2 hours. After completion of dropping, the mixture was kept at 60 ° C. and stirred for 2 hours. Next, the particles in the 500 mL separable flask were separated using magnetism, and then repeatedly washed with distilled water. The volume average particle diameter of the obtained particles was 1.2 μm.

[Example 2]
As raw materials for preparing the pre-emulsion, 45 g of sodium dodecyl sulfate 0.50 mass% aqueous solution, CBMA 1.0 g, SA 2.0 g, THFMA 3.0 g, TMP 0.5 g, and Parroyl 355-75 (S) 0.15 g are used. Except that, the same operation as in Example 1 was performed. The volume average particle diameter of the obtained particles was 1.2 μm.

Example 3
As raw materials for preparing the pre-emulsion, 45 g of sodium dodecyl sulfate 0.50 mass% aqueous solution, CBMA 2.0 g, SA 2.0 g, THFMA 2.0 g, TMP 0.5 g, and Parroyl 355-75 (S) 0.15 g are used. Except that, the same operation as in Example 1 was performed. The volume average particle diameter of the obtained particles was 1.2 μm.

Example 4
As raw materials for preparing a pre-emulsion, 45 g of sodium dodecyl sulfate 0.50 mass% aqueous solution, CBMA 0.5 g, SA 2.0 g, 3.5 g of cyclohexyl methacrylate (hereinafter referred to as “CHMA”), TMP 0.5 g, and Parroyl 355 The same operation as in Example 1 was performed except that 0.15 g of -75 (S) was used. The volume average particle diameter of the obtained particles was 1.2 μm.

Example 5
As raw materials for preparing the pre-emulsion, 45 g of sodium dodecyl sulfate 0.50 mass% aqueous solution, CBMA 0.5 g, SA 1.0 g, THFMA 4.5 g, TMP 0.5 g, and Parroyl 355-75 (S) 0.15 g are used. Except that, the same operation as in Example 1 was performed. The volume average particle diameter of the obtained particles was 1.2 μm.

Example 6
As raw materials for preparing pre-emulsion, 45 g of sodium dodecyl sulfate 0.50 mass% aqueous solution, CBMA 0.5 g, SA 3.0 g, THFMA 2.5 g, TMP 0.5 g, and Parroyl 355-75 (S) 0.15 g are used. Except that, the same operation as in Example 1 was performed. The volume average particle diameter of the obtained particles was 1.2 μm.

Example 7
As a raw material for preparing the pre-emulsion, except that 45 g of sodium dodecyl sulfate 0.50 mass% aqueous solution, CBMA 0.5 g, SA 2.0 g, THFMA 4.0 g, and Parroyl 355-75 (S) 0.15 g were used. The same operation as in Example 1 was performed. The volume average particle diameter of the obtained particles was 1.2 μm.

Example 8
As raw materials for preparing the pre-emulsion, 45 g of a 0.50 mass% aqueous solution of sodium dodecyl sulfate, 0.5 g of O- [2- (methacryloyloxy) ethoxy (oxylato) phosphinyl] choline (hereinafter referred to as “MPC”), The same operation as in Example 1 was performed, except that SA 2.0 g, THFMA 3.5 g, TMP 0.5 g, and Parroyl 355-75 (S) 0.15 g were used. The volume average particle diameter of the obtained particles was 1.2 μm.

Example 9
As raw materials for preparing the pre-emulsion, 45 g of sodium dodecyl sulfate 0.50 mass% aqueous solution, CBMA 0.5 g, SA 2.0 g, diethylene glycol monomethyl ether methacrylate (hereinafter referred to as “DEGMA”) 3.5 g, TMP 0.5 g, The same operation as in Example 1 was performed except that 0.15 g of Parroyl 355-75 (S) was used. The volume average particle diameter of the obtained particles was 1.2 μm.

Example 10
300 g of magnetic fluid EMG 6 g, CBMA 0.5 g, SA 2 g, THFMA 3.5 g, TMP 0.5 g and Parroyl 355-75 (S) 0.15 g dispersed in 300 g of 0.50 mass% sodium dodecyl sulfate aqueous solution 10 g in a 500 mL separable flask The mixture was added and dispersed to obtain a pre-emulsion, and then the temperature was raised to 60 ° C. and held for 2 hours. Next, the particles in the 500 mL separable flask were separated using magnetism, and then repeatedly washed with distilled water. The volume average particle diameter of the obtained particles was 0.2 μm.

[Comparative Example 1]
Example 1 with the exception that 45 g of 0.50% by weight sodium dodecyl sulfate aqueous solution, 4.5 g of CBMA, 2.0 g of SA, and 0.15 g of parroyl 355-75 (S) were used as raw materials for preparing the pre-emulsion. The same operation was performed. The volume average particle diameter of the obtained particles was 1.2 μm.

[Comparative Example 2]
As the raw material for preparing the pre-emulsion, except that 45 g of sodium dodecyl sulfate 0.50 mass% aqueous solution, SA 2.0 g, THFMA 4.0 g, TMP 0.5 g, and Parroyl 355-75 (S) 0.15 g were used. The same operation as in Example 1 was performed. The volume average particle diameter of the obtained particles was 1.2 μm.

[Comparative Example 3]
As raw materials for preparing the pre-emulsion, 45 g of sodium dodecyl sulfate 0.50 mass% aqueous solution, CBMA 3.0 g, SA 2.0 g, THFMA 1.0 g, TMP 0.5 g, and Parroyl 355-75 (S) 0.15 g are used. Except that, the same operation as in Example 1 was performed. The volume average particle diameter of the obtained particles was 1.2 μm.

[Comparative Example 4]
As raw materials for preparing the pre-emulsion, 45 g of sodium dodecyl sulfate 0.50 mass% aqueous solution, CBMA 0.5 g, SA 5.0 g, THFMA 0.5 g, TMP 0.5 g, and Parroyl 355-75 (S) 0.15 g are used. Except that, the same operation as in Example 1 was performed. The volume average particle diameter of the obtained particles was 1.2 μm.

[Comparative Example 5]
As raw materials for preparing the pre-emulsion, 300 g of 0.50 mass% aqueous solution of sodium dodecyl sulfate, 6 g of magnetic fluid dispersed in 10 g of heptane, 2.0 g of SA, 4.0 g of THFMA, 0.5 g of TMP, and Parroyl 355-75 (S) The same operation as in Example 10 was performed except that 0.15 g was used. The volume average particle diameter of the obtained particles was 0.2 μm.

[Test Example 1]
1 mg of magnetic particles obtained in each example and comparative example were dispersed in 2 mL of water. This aqueous dispersion was put into an Eppendorf tube, particles were separated using magnetism, and the supernatant was removed. The particles were then dispersed in 990 μL of MES buffer (100 mM, pH 5.0), 10 μL of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (10 mg / mL) was added and incubated for 30 minutes at room temperature. . Particles were separated using magnetism, the supernatant was removed, dispersed in 1 mL of MES buffer (100 mM, pH 5.0), and 20 μg of anti-TSH antibody (Funakoshi) was added. After incubation for 12 hours at room temperature, the particles were separated using magnetism and the supernatant was removed. The antibody-bound particles were obtained by washing 5 times with TBS-T (0.05 mass% Tween 20) buffer. The amount of antibody binding was determined by BCA Assay. The results are shown in Table 2.

[Test Example 2]
5 μL of the antibody-bound particle dispersion obtained in Test Example 1 (corresponding to 50 μg of particles) is placed in a test tube, and 5 μIU / mL TSH antigen solution (Lumipulse TSH manufactured by Fujirebio Inc.) supplemented with 50 μL of fetal calf serum (FCS) -III standard TSH solution) 50 μL was mixed and reacted at 25 ° C. for 10 minutes. After separating the particles using magnetism and removing the supernatant, 40 μL of anti-TSH antibody (attached to Lumipulse TSH-III immunoreaction cartridge manufactured by Fujirebio Inc.) labeled with alkaline phosphatase as a secondary antibody was added, and 25 ° C. For 10 minutes. After separating the particles using magnetism and removing the supernatant, the particles obtained by washing three times with PBS were dispersed in 50 μL of 0.01% Triton X-100 and transferred to a new tube. After adding 100 μL of alkaline phosphatase substrate solution (Lumipulse substrate solution manufactured by Fujirebio Inc.) and reacting at 37 ° C. for 10 minutes, the amount of chemiluminescence was measured as a signal. For the measurement of chemiluminescence, a chemiluminescence measuring apparatus (Lumat LB9507) manufactured by Bertol Japan was used. Further, the chemiluminescence amount was measured as noise in the same manner except that a 0 μIU / mL TSH calibrator was used instead of the 5 μIU / mL TSH antigen solution. Further, a value S / N obtained by dividing the signal (S) by the noise (N) was calculated. The results are shown in Table 2.

[Test Example 3]
1 mg of the magnetic particles obtained in each Example and Comparative Example were dispersed in 2 mL of water, and the dispersion was put into an Eppendorf tube. The tube was attached to a magnetic bead separation stand (Magic Trapper (manufactured by Toyobo Co., Ltd.)) and allowed to stand for about 5 minutes to confirm that the particles were collected in a pellet form. Subsequently, the tube was removed from the stand to release the particles from the magnetism, and the state after 1 minute was confirmed and evaluated according to the following criteria. The evaluation results are shown in Table 2.
<Evaluation criteria>
○: The pellet settles in the lower part of the tube while collapsing, and the redispersibility is good.

  Table 1 shows the composition of the monomers constituting the copolymer on the surface of the magnetic particles obtained in each Example and Comparative Example.

  As shown in Table 2, the magnetic particles of Examples 1 to 10 had a high S / N (signal / noise) ratio, and were able to detect antigens with high sensitivity and low noise. The redispersibility was also good.

Claims (21)

A solid phase carrier, wherein a copolymer comprising the following structural units (A) to (C) forms at least a solid surface.
(A) Structural unit derived from a monomer having a zwitterionic structure: 1 to 35% by mass relative to all structural units
(B) Structural unit derived from a monomer having a functional group capable of chemically binding to a ligand: 70% by mass or less based on the total structural unit (C) Derived from a poorly water-soluble monomer having no functional group capable of chemically binding to a ligand Structural unit The solid phase carrier according to claim 1, wherein the structural unit (A) is a structural unit represented by the following formula (3).

[In Formula (3),
R ¹¹ represents a hydrogen atom or a methyl group,
R ¹² represents — (C═O) —O— *, — (C═O) —NR ¹⁴ — * (R ¹⁴ represents a hydrogen atom or a methyl group, and * represents R ^{13 in} formula (3). Or a phenylene group,
R ¹³ represents a zwitterionic structure. ] The solid phase carrier according to claim 1 or 2, wherein the zwitterionic structure is represented by the following formula (1) or (2).

[In Formula (1),
R ¹ and R ² each independently represent a single bond or a divalent organic group having 1 to 10 carbon atoms,
R ³ represents — (C═O) O 2 ^— or —SO ₃ ^— ,
R ⁴ and R ⁵ each independently represents a methyl group or an ethyl group. ]

[In Formula (2),
R ⁶ and R ⁷ each independently represents a single bond or a divalent organic group having 1 to 10 carbon atoms,
R ⁸ , R ⁹ and R ¹⁰ each independently represent a methyl group or an ethyl group. ]   The solid phase carrier according to any one of claims 1 to 3, wherein the structural unit (B) is a structural unit derived from a monomer having a carboxy group. The solid phase carrier according to any one of claims 1 to 3, wherein the structural unit (B) is a structural unit represented by the following formula (4).

[In Formula (4),
R ¹⁵ represents a hydrogen atom or a methyl group,
R ¹⁶ represents — (C═O) —O— *, — (C═O) —NR ¹⁸ — * (R ¹⁸ represents a hydrogen atom or a methyl group, and * represents R ^{17 in} formula (4). Or a phenylene group,
When R ¹⁶ is — (C═O) —O— *, R ¹⁷ represents a hydrogen atom or an organic group having a functional group capable of chemically bonding to a ligand, and R ¹⁶ represents — (C═O) —. NR ¹⁸ - If a * or a phenylene group, R ¹⁷ represents an organic group having a ligand capable of chemically bonding functional groups. ] The solid support according to any one of claims 1 to 5, wherein the structural unit (C) is one or more selected from the following structural units (C-1) to (C-3). .
(C-1) A structural unit derived from a monoacrylate monomer that retains intermediate water when polymerized into a homopolymer in the presence of free water or a monomethacrylate monomer having the same side chain structure as that of the monoacrylate monomer (C -2) Structural unit derived from a mono (meth) acrylate monomer represented by the following formula (7)

[In Formula (7),
R ²² represents a hydrogen atom or a methyl group,
R ²³ represents a hydrocarbon group. ]
(C-3) Structural unit derived from polyfunctional monomer   The solid phase carrier according to claim 6, comprising at least the structural unit (C-1) as the structural unit (C). The solid phase carrier according to claim 6 or 7, wherein the structural unit (C-1) is a structural unit derived from a monomer represented by the following formula (8) or (9).

[In Formula (8),
R ²⁴ represents a hydrogen atom or a methyl group,
R ²⁵ represents an alkanediyl group having 1 to 3 carbon atoms;
R ²⁶ represents an alkyl group having 1 to 3 carbon atoms,
n shows the integer of 1-5. ]

[In Formula (9),
R ²⁷ represents a hydrogen atom or a methyl group,
m represents 1 or 2. ]   The solid phase carrier according to any one of claims 6 to 8, comprising at least a structural unit (C-3) as the structural unit (C).   The solid phase carrier according to any one of claims 1 to 9, wherein the content of the structural unit (C) is 10 to 90 mass% with respect to all the structural units.   The solid phase carrier according to any one of claims 1 to 10, wherein the content of the functional group capable of chemically binding to the ligand is 1 to 800 µmol per 1 g of solid content of the solid phase carrier.   The solid phase carrier according to any one of claims 1 to 11, which is a particle.   The solid phase carrier according to any one of claims 1 to 12, which is a magnetic particle.   The solid phase carrier according to claim 12 or 13, wherein the average particle diameter is 0.02 to 500 µm.   The solid support according to any one of claims 1 to 14, wherein the copolymer is formed by an emulsion polymerization method.   A ligand-bound solid phase carrier obtained by binding a ligand to the solid phase carrier according to any one of claims 1 to 15.   The ligand-bound solid phase according to claim 16, wherein the ligand is an antibody, antigen, nucleic acid, nucleotide, nucleoside, protein, peptide, amino acid, polysaccharide, sugar, lipid, vitamin, drug, substrate, hormone or neurotransmitter. Carrier.   The solid phase carrier or ligand-bound solid phase carrier according to any one of claims 1 to 17, which is used for immunoassay or nucleic acid detection.   A method for detecting or separating a target substance in a sample, which comprises using the ligand-bound solid phase carrier according to any one of claims 16 to 18. Monomer having zwitterionic structure: 1 to 35% by mass with respect to all monomers,
Monomer having a functional group capable of chemically binding to a ligand: 70% by mass or less based on the total monomer
A poorly water-soluble monomer having no functional group capable of chemically bonding to a ligand,
A method for producing a solid phase carrier, comprising a step of radical polymerization in the presence of a raw material carrier.   The production method according to claim 20, wherein the radical polymerization is emulsion polymerization.