JP2011007785A - Target molecular interaction substance-immobilized carrier - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a target molecular interaction substance immobilized carrier with a large binding-amount of target molecular interaction substances and a totally uniform structure even under a neutrality condition, capable of acting each other effectively.SOLUTION: The target molecular interaction substance immobilized carrier includes a solid phase carrier, and a structure containing a target molecular interaction substance and water-soluble polymer that are immobilized to the solid phase carrier and bound each other. A method for manufacturing includes the steps of (1) copolymerizing a predetermined ethylenically unsaturated polymerizable monomer with a phosphorylcholine similar group and a predetermined ethylenically unsaturated polymerizable monomer with a functional group bonded thereto via a spacer to bind covalently to a target molecular interaction substance to synthesize water-soluble polymer, (2) mixing the water-soluble polymer with molecules acting with target molecules each other, and (3) supplying the mixture to the surface of a solid phase with a functional group for binding covalently to the functional group or target molecular interaction substance to manufacture the target molecular interaction substance immobilized carrier.

Description

  The present invention relates to a carrier on which a target molecule interaction substance is immobilized and a method for producing the same. Specifically, the present invention relates to a biochip on which a target molecule interaction substance such as a sensor chip or a target molecule separation / purification chip is immobilized.

  In the fields of medical / diagnosis, genetic analysis, proteomics, microelectronics, membrane separation, etc., diagnosis by detecting the target molecule using the interaction between the target molecule and a substance that specifically interacts with the target molecule, Inspection, analysis, purification, etc. are performed. In order to appropriately detect such target molecules, various techniques have been developed for immobilization carriers for substances that interact with the target molecules.

  In particular, the amount of the target molecule interacting substance (hereinafter sometimes simply referred to as “interacting substance”) supported, and the minute space containing the interacting substance (that is, the interaction between the interacting substance and the target molecule) (Patent Document 1) has been studied, and Patent Document 1 discloses an interactive substance (protein) prepared by supplying and concentrating a mixed solution of an interactive substance and a polymer on a substrate. And a chip in which polymer aggregates are fixed in a laminated state is disclosed. In this chip, the amount of the interaction substance immobilized is increased as compared with the conventional method, and as a result, the interaction between the target molecule and the interaction substance can be detected with high sensitivity. However, in the above method, when the mixed solution of the interacting substance and the polymer is supplied and concentrated on the substrate, a thick laminate is produced around the supplied droplet, and conversely, the laminate is laminated inside the spot. There was a problem of non-uniformity that the structure was thin. This phenomenon was particularly remarkable when a chip was produced under neutral conditions. In order to solve this problem, a method for producing the chip using an alkaline solution has been proposed. However, when an alkaline and easily denatured protein is used as an interaction substance, protein inactivation becomes a problem. It was.

International Publication WO2006 / 038456 Pamphlet

  In the present invention, the amount of binding of the interacting substance is large, and the microspace containing the interacting substance is a space in which the interaction between the interacting substance and the target molecule can be effectively performed under neutral conditions. However, an object is to provide a carrier on which a structure having a uniform structure as a whole is fixed.

  As a result of intensive studies to solve the above problems, the inventors of the present invention include a solid phase carrier, a target molecule interacting substance immobilized on the solid phase carrier and bound to each other, and a water-soluble polymer. The water-soluble polymer is a water-soluble polymer of a compound containing a spacer and a polymerizable group, and the target molecule interacting substance is bound to the water-soluble polymer through the spacer. The present inventors have found that a target molecule interaction substance-immobilized carrier can solve the above problems. More specifically, (1) a predetermined unsaturated polymerizable monomer having a hydrophilic or amphiphilic functional group and a functional group for covalently binding the interacting substance are bonded via a spacer. (2) mixing the water-soluble polymer and the interaction substance, and (3) mixing the mixture with the interaction substance or the water-soluble polymer. It has been found that a target molecule-interacting substance-immobilized carrier that solves the above-mentioned problems can be produced by supplying a solid-phase surface having a functional group for covalently bonding a compound. The present invention is based on these findings.

（式中Ｒ₇は水素原子又はメチル基を示し、Ｗは標的分子相互作用物質を共有結合するための官能基を示す。）
［６］前記水溶性重合体が、スペーサーと重合性基を含む化合物と、親水性又は両親媒性の官能基を有する不飽和重合性モノマーとの共重合体である、［１］〜［５］のいずれかに記載の標的分子相互作用物質固定化担体。
［７］下記（１）〜（３）に示す工程を少なくとも含む方法により製造されることを特徴とする、［６］に記載の標的分子相互作用物質固定化担体；
（１）親水性又は両親媒性の官能基を有する不飽和重合性モノマーと、前記一般式（II）で表されるエチレン系不飽和重合性モノマーとを共重合させて水溶性重合体を合成する工程
（２）（１）の工程で得られた水溶性重合体と、標的分子相互作用物質とを混合する工程、
（３）（２）の工程で得られた混合物を、前記官能基Wを共有結合するための官能基又は標的分子相互作用物質を共有結合するための官能基を有する固相表面に供給する工程。
［８］親水性又は両親媒性の官能基を有する不飽和重合性モノマーが下記式（I）で表わされるホスホリルコリン類似基を有するエチレン系不飽和重合性モノマーである、［６］または［７］に記載の標的分子相互作用物質固定化担体。

（式中、Ｒ₁は炭素数２〜１０の２価の炭化水素基を示し、Ｒ₂は炭素数１〜４の２価の炭化水素基を示し、Ｒ₃、Ｒ₄及びＲ₅はそれぞれ独立して水素原子又は炭素数１〜６の炭化水素基を示し、Ｒ₆は水素原子又はメチル基を示す。）
［９］水溶性重合体全体に対する、前記一般式（I）で表わされるホスホリルコリン類似基を有するエチレン系不飽和重合性モノマーに由来する構成単位の割合が、３０モル％以上であることを特徴とする、［８］に記載の標的分子相互作用物質固定化担体。
［１０］前記水溶性重合体が、さらに疎水性ユニットを有する不飽和重合性モノマーに由来する構成単位を含む、［６］〜［９］のいずれかに記載の標的分子相互作用物質固定化担体。
［１１］前記疎水性ユニットを有する不飽和重合性モノマーがｎ−ブチルメタクリレートである、［１０］に記載の標的分子相互作用物質固定化担体。
［１２］下記（１）〜（３）に示す工程を少なくとも含む、［６］〜［１１］のいずれかに記載の標的分子相互作用物質固定化担体の製造方法；
（１）親水性又は両親媒性の官能基を有する不飽和重合性モノマーと、前記一般式（II）で表わされるエチレン系不飽和重合性モノマーとを共重合させて水溶性重合体を合成する工程、
（２）（１）の工程で得られた水溶性重合体と、標的分子相互作用物質とを混合する工程、
（３）（２）の工程で得られた混合物を、前記官能基Ｗを共有結合するための官能基又は標的分子相互作用物質を共有結合するための官能基を有する固相表面に供給する工程。
［１３］ 下記（１）〜（３）に示す工程を少なくとも含む方法により製造されることを特徴とする、標的分子を捕捉・検出するための、標的分子相互作用物質の標的分子相互作用物質固定化担体；
（１）下記一般式（I）で表わされるホスホリルコリン類似基を有するエチレン系不飽和重合性モノマーと、下記一般式（II）で表わされるエチレン系不飽和重合性モノマーとを共重合させて水溶性重合体を合成する工程、

（式中Ｒ₁は炭素数２〜１０の２価の炭化水素基を示し、Ｒ₂は炭素数１〜４の２価の炭化水素基を示し、Ｒ₃、Ｒ₄及びＲ₅はそれぞれ独立して水素原子又は炭素数１〜６の炭化水素基を示し、Ｒ₆は水素原子又はメチル基を示す。）

（式中Ｒ₇は水素原子又はメチル基を示し、Ｙは炭素数１〜１０のアルキレンオキシ基又はアルキレン基を示し、Ｗは標的分子相互作用物質を共有結合するための官能基を示し、ｑは１〜２０の整数を示す。ｑが２以上２０以下の整数である場合、繰り返されるＹは、それぞれ同一であっても異なっていてもよい。）
（２）（１）の工程で得られた水溶性重合体と、標的分子相互作用物質とを混合する工程、
（３）（２）の工程で得られた混合物を、前記官能基Ｗを共有結合するための官能基又は標的分子相互作用物質を共有結合するための官能基を有する固相表面に供給する工程。
［１４］ 前記（３）の工程により固相表面に供給された混合物を乾燥させる工程をさらに含む方法により製造される、［１３］に記載の標的分子相互作用物質固定化担体。
［１５］ 前記（１）の工程において、前記一般式（I）で表わされるホスホリルコリン類似基を有するエチレン系不飽和重合性モノマーが、共重合に用いられる全モノマーの３０モル％以上であることを特徴とする［１３］または［１４］に記載の標的分子相互作用物質固定化担体。
［１６］前記（１）の工程において、さらに疎水性ユニットを有するエチレン系不飽和重合性モノマーを共重合させることにより製造される、［１３］〜［１５］のいずれかに記載の標的分子相互作用物質固定化担体。
［１７］前記疎水性ユニットを有するエチレン系不飽和重合性モノマーがｎ−ブチルメタクリレートである［１６］に記載の標的分子相互作用物質固定化担体。
［１８］下記（１）〜（３）に示す工程を少なくとも含む、［１３］〜［１７］のいずれかに記載の標的分子相互作用物質固定化担体の製造方法；
（１）下記一般式（I）で表わされるホスホリルコリン類似基を有するエチレン系不飽和重合性モノマーと、下記一般式（II）で表わされるエチレン系不飽和重合性モノマーとを共重合させて水溶性重合体を合成する工程、

（式中Ｒ₁は炭素数２〜１０の２価の炭化水素基を示し、Ｒ₂は炭素数１〜４の２価の炭化水素基を示し、Ｒ₃、Ｒ₄及びＲ₅はそれぞれ独立して水素原子又は炭素数１〜６の炭化水素基を示し、Ｒ₆は水素原子又はメチル基を示す。）

（式中Ｒ₇は水素原子又はメチル基を示し、Ｙは炭素数１〜１０のアルキレンオキシ基又はアルキレン基を示し、Ｗは標的分子相互作用物質を共有結合するための官能基を示し、ｑは１〜２０の整数を示す。ｑが２以上２０以下の整数である場合、繰り返されるＹは、それぞれ同一であっても異なっていてもよい。）
（２）（１）の工程で得られた水溶性重合体と、標的分子相互作用物質とを混合する工程、
（３）（２）の工程で得られた混合物を、前記官能基Ｗを共有結合するための官能基又は標的分子相互作用物質を共有結合するための官能基を有する固相表面に供給する工程。
［１９］標的分子が、標的分子相互作用物質を介して結合した、［１］〜［１１］および［１３］〜［１７］のいずれかに記載の標的分子相互作用物質固定化担体。
［２０］標的分子が蛋白質、ペプチド、核酸および低分子化合物からなる群より選ばれる、［１９］に記載の標的分子相互作用物質固定化担体。
［２１］［１９］または［２０］に記載の標的分子相互作用物質固定化担体を含むバイオチップ。 That is, the present invention is as follows.
[1] a solid phase carrier;
A structure comprising a target molecule interacting substance and a water-soluble polymer which are bonded to each other and fixed to the solid phase carrier;
A target molecule interaction substance-immobilized carrier comprising:
The water-soluble polymer is a water-soluble polymer of a compound containing a spacer and a polymerizable group, and the target molecule-interacting substance captures the target molecule bonded to the water-soluble polymer through the spacer. A target molecule interaction substance-immobilized carrier for detection.
[2] The water-soluble polymer is a water-soluble polymer of a compound in which a functional group for covalently binding a target molecule interacting substance is bonded to a polymerizable group via a spacer, and the target molecule interacting substance Is a target molecule-interacting substance-immobilized carrier according to [1], which is bound to a water-soluble polymer through the functional group.
[3] The target molecule interaction substance-immobilized carrier according to [1] or [2], wherein the spacer is represented by the following formula.
-(Y) _q-
Here, Y represents an alkyleneoxy group having 1 to 10 carbon atoms or an alkylene group, and q represents an integer of 1 to 20, but when q is an integer of 2 or more and 20 or less, the repeated Ys are the same. Or different.
[4] The target molecule-interacting substance-immobilized carrier according to any one of [1] to [3], wherein the polymerizable group is an acryl group or a methacryl group.
[5] The target molecule-interacting substance-immobilized carrier according to [4], wherein the compound is an ethylenically unsaturated polymerizable monomer represented by the following general formula (II).

(Wherein R ₇ represents a hydrogen atom or a methyl group, and W represents a functional group for covalently bonding the target molecule interacting substance.)
[6] The water-soluble polymer is a copolymer of a compound containing a spacer and a polymerizable group and an unsaturated polymerizable monomer having a hydrophilic or amphiphilic functional group, [1] to [5] ] The target molecule interaction substance immobilization support according to any one of the above.
[7] The target molecule-interacting substance-immobilized carrier according to [6], which is produced by a method including at least the following steps (1) to (3):
(1) A water-soluble polymer is synthesized by copolymerizing an unsaturated polymerizable monomer having a hydrophilic or amphiphilic functional group and the ethylenically unsaturated polymerizable monomer represented by the general formula (II). The step (2) the step of mixing the water-soluble polymer obtained in the step (1) and the target molecule interacting substance,
(3) A step of supplying the mixture obtained in the step (2) to a solid phase surface having a functional group for covalently binding the functional group W or a functional group for covalently binding a target molecule interacting substance. .
[8] The unsaturated polymerizable monomer having a hydrophilic or amphiphilic functional group is an ethylenically unsaturated polymerizable monomer having a phosphorylcholine-like group represented by the following formula (I): [6] or [7] 2. A target molecule interaction substance-immobilized carrier according to 1.

(In the formula, R ₁ represents a divalent hydrocarbon group having 2 to 10 carbon atoms, R ₂ represents a divalent hydrocarbon group having 1 to 4 carbon atoms, and R ₃ , R ₄ and R ₅ each represents Independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, and R ₆ represents a hydrogen atom or a methyl group.)
[9] The ratio of the structural unit derived from the ethylenically unsaturated polymerizable monomer having the phosphorylcholine-like group represented by the general formula (I) to the entire water-soluble polymer is 30 mol% or more. The target molecule interaction substance-immobilized carrier according to [8].
[10] The target molecule-interacting substance-immobilized carrier according to any one of [6] to [9], wherein the water-soluble polymer further includes a structural unit derived from an unsaturated polymerizable monomer having a hydrophobic unit. .
[11] The target molecule-interacting substance-immobilized carrier according to [10], wherein the unsaturated polymerizable monomer having a hydrophobic unit is n-butyl methacrylate.
[12] The method for producing a target molecule-interacting substance-immobilized carrier according to any one of [6] to [11], comprising at least the steps shown in the following (1) to (3);
(1) A water-soluble polymer is synthesized by copolymerizing an unsaturated polymerizable monomer having a hydrophilic or amphiphilic functional group and the ethylenically unsaturated polymerizable monomer represented by the general formula (II). Process,
(2) A step of mixing the water-soluble polymer obtained in the step (1) and the target molecule interacting substance,
(3) A step of supplying the mixture obtained in the step (2) to a solid phase surface having a functional group for covalently binding the functional group W or a functional group for covalently binding a target molecule interacting substance. .
[13] Immobilization of a target molecule-interacting substance for target molecule-interacting substance for capturing / detecting the target molecule, which is produced by a method including at least the steps shown in the following (1) to (3) Carrier;
(1) Water-soluble by copolymerizing an ethylenically unsaturated polymerizable monomer having a phosphorylcholine-like group represented by the following general formula (I) and an ethylenically unsaturated polymerizable monomer represented by the following general formula (II) A step of synthesizing a polymer;

(Wherein R ₁ represents a divalent hydrocarbon group having 2 to 10 carbon atoms, R ₂ represents a divalent hydrocarbon group having 1 to 4 carbon atoms, and R ₃ , R ₄ and R ₅ are each independently selected. And represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, and R ₆ represents a hydrogen atom or a methyl group.)

(Wherein R ₇ represents a hydrogen atom or a methyl group, Y represents an alkyleneoxy group or alkylene group having 1 to 10 carbon atoms, W represents a functional group for covalently bonding the target molecule interacting substance, q Represents an integer of 1 to 20. When q is an integer of 2 or more and 20 or less, the repeated Ys may be the same or different.
(2) A step of mixing the water-soluble polymer obtained in the step (1) and the target molecule interacting substance,
(3) A step of supplying the mixture obtained in the step (2) to a solid phase surface having a functional group for covalently binding the functional group W or a functional group for covalently binding a target molecule interacting substance. .
[14] The target molecule-interacting substance-immobilized carrier according to [13], which is produced by a method further comprising a step of drying the mixture supplied to the solid surface by the step (3).
[15] In the step (1), the ethylenically unsaturated polymerizable monomer having a phosphorylcholine-like group represented by the general formula (I) is 30 mol% or more of the total monomers used for copolymerization. The target molecule interaction substance-immobilized carrier according to [13] or [14], which is characterized.
[16] The target molecule interaction according to any one of [13] to [15], which is produced by copolymerizing an ethylenically unsaturated polymerizable monomer having a hydrophobic unit in the step (1). Active substance immobilization carrier.
[17] The target molecule-interacting substance-immobilized carrier according to [16], wherein the ethylenically unsaturated polymerizable monomer having a hydrophobic unit is n-butyl methacrylate.
[18] The method for producing a target molecule interaction substance-immobilized carrier according to any one of [13] to [17], comprising at least the steps shown in the following (1) to (3);
(1) Water-soluble by copolymerizing an ethylenically unsaturated polymerizable monomer having a phosphorylcholine-like group represented by the following general formula (I) and an ethylenically unsaturated polymerizable monomer represented by the following general formula (II) A step of synthesizing a polymer;

(Wherein R ₁ represents a divalent hydrocarbon group having 2 to 10 carbon atoms, R ₂ represents a divalent hydrocarbon group having 1 to 4 carbon atoms, and R ₃ , R ₄ and R ₅ are each independently selected. And represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, and R ₆ represents a hydrogen atom or a methyl group.)

(Wherein R ₇ represents a hydrogen atom or a methyl group, Y represents an alkyleneoxy group or alkylene group having 1 to 10 carbon atoms, W represents a functional group for covalently bonding the target molecule interacting substance, q Represents an integer of 1 to 20. When q is an integer of 2 or more and 20 or less, the repeated Ys may be the same or different.
(2) A step of mixing the water-soluble polymer obtained in the step (1) and the target molecule interacting substance,
(3) A step of supplying the mixture obtained in the step (2) to a solid phase surface having a functional group for covalently binding the functional group W or a functional group for covalently binding a target molecule interacting substance. .
[19] The target molecule interaction substance-immobilized carrier according to any one of [1] to [11] and [13] to [17], in which the target molecule is bound via a target molecule interaction substance.
[20] The target molecule-interacting substance-immobilized carrier according to [19], wherein the target molecule is selected from the group consisting of proteins, peptides, nucleic acids, and low molecular compounds.
[21] A biochip comprising the target molecule interaction substance-immobilized carrier according to [19] or [20].

  The target molecule-interacting substance-immobilized carrier according to the present invention has a larger amount of the interacting substance supported than that produced by the conventional production method. In the target molecule-interacting substance-immobilized carrier of the present invention, the microspace containing the interacting substance is a space in which the interaction between the interacting substance and the target molecule can be effectively performed. Since the target molecule-interacting substance-immobilized carrier according to the present invention has a structure having a uniform structure even under neutral conditions, the activity is maintained particularly when a protein or the like is used as an interacting substance. It can be supported. By using the target molecule-interacting substance-immobilized carrier according to the present invention, in the diagnosis, inspection, analysis, and purification using proteins and the like as the interacting substance, the target molecule is markedly compared with the conventional target molecule-interacting substance-immobilized carrier. The contact efficiency is high, and there is an effect that a uniform and highly reproducible signal can be obtained in diagnosis, inspection and analysis.

(A)-(c) is a schematic diagram which expands and shows the surface vicinity of an example of the target molecule interaction material immobilization support | carrier of this invention. A well in which an interacting substance structure in which polymer A and mouse IgG are bound is immobilized, a well in which an interacting substance structure in which polymer B and mouse IgG are bound is immobilized, and polymer C and mouse IgG are bound. It is a figure which shows the comparison data of the capture amount of protein A in the well in which the interacting substance structure was immobilized, and the well in which only mouse IgG was immobilized. A gold substrate spotted with an interacting substance structure bound to polymer A and mouse IgG, a gold substrate spotted with an interacting substance structure bound to polymer B and mouse IgG, and an interaction bound to polymer C and mouse IgG It is a figure which shows the comparison data of the imaging image of a spot in the gold substrate which spotted the substance structure, and the gold substrate which spotted only mouse IgG. A gold substrate spotted with an interacting substance structure bound to polymer A and mouse IgG, a gold substrate spotted with an interacting substance structure bound to polymer B and mouse IgG, and an interaction bound to polymer C and mouse IgG It is a figure which shows the comparison data of the fixed amount of the interaction substance structure or mouse | mouth IgG in the gold substrate which spotted the substance structure, and the gold substrate which spotted only mouse | mouth IgG. A gold substrate spotted with an interacting substance structure bound to polymer A and mouse IgG, a gold substrate spotted with an interacting substance structure bound to polymer B and mouse IgG, and an interaction bound to polymer C and mouse IgG It is a figure which shows the comparison data of the capture amount of protein A in the gold substrate which spotted the substance structure, and the gold substrate which spotted only mouse IgG. A gold substrate spotted with an interacting substance structure bound to polymer B and mouse IgG, a gold substrate spotted with an interacting substance structure bound to polymer C and mouse IgG, and a gold substrate spotted with only mouse IgG (2D ) And comparative data of the captured amount of protein A. Fluorescence of Cy5 derived from the bound target molecule in the spot of the interactor structure in which polymer B and mouse IgG are bound, the spot of the interactor structure in which polymer C and mouse IgG are bound, and the spot of only mouse IgG It is a figure which shows an image (photograph). Comparison data of the capture amount of protein A between the gold substrate spotted with the interacting substance structure bound to polymer D and mouse IgG and the gold substrate spotted with the interacting substance structure bound to polymer E and mouse IgG It is a figure (photograph) which shows the fluorescence image of Cy5 derived from the target molecule couple | bonded with the figure to show.

[I Target molecule interaction substance immobilization support of the present invention]
The target molecule-interacting substance-immobilized carrier of the present invention is a solid-phase carrier and a target molecule-interacting substance that is bonded to the solid-phase carrier (hereinafter also simply referred to as an interacting substance). And a structure comprising a water-soluble polymer, a target molecule interaction substance-immobilized carrier, wherein the water-soluble polymer is a water-soluble polymer of a compound containing a spacer and a polymerizable group, and the target The molecular interaction substance is a target molecule interaction substance immobilization carrier for capturing and detecting a target molecule, which is bound to the water-soluble polymer via the spacer.
Here, the water-soluble polymer is a water-soluble polymer of a compound in which a functional group for covalently bonding the target molecule interacting substance is bonded to the polymerizable group via a spacer, and the target molecule interacting substance is It is preferably bonded to the water-soluble polymer via a functional group.
More preferably, the water-soluble polymer is a copolymer of a compound in which a target molecule interaction substance is bonded to a polymerizable group via a spacer and an unsaturated polymerizable monomer having a hydrophilic or amphiphilic functional group. It is a coalescence.
The target molecule-interacting substance-immobilized carrier of the present invention preferably has an unsaturated polymerizable monomer having a hydrophilic or amphiphilic functional group and a functional group for covalently binding the interacting substance via a spacer. A step of synthesizing a water-soluble polymer by copolymerizing with a predetermined unsaturated polymerizable monomer, a step of coexisting the water-soluble polymer and an interacting substance in a predetermined medium (hereinafter, as appropriate, It is produced by a method including at least a step of supplying a mixture obtained in the mixing step to a solid phase surface having a functional group for covalently binding an interacting substance.
In the target molecule interaction substance-immobilized carrier of the present invention, more preferably, a predetermined ethylenically unsaturated polymerizable monomer having a phosphorylcholine-like group and a functional group for covalently bonding the interaction substance are bonded via a spacer. A step of synthesizing a water-soluble polymer by copolymerizing with a predetermined ethylenically unsaturated polymerizable monomer, a step of mixing the water-soluble polymer and an interacting substance in a predetermined medium, and a mixing step The mixture obtained in (1) is produced by a method including at least a step of supplying the mixture to a solid surface having a functional group for covalently binding an interacting substance.

On the solid phase surface supplied with the above mixture, a structure composed of a chain composed of an interacting substance and a water-soluble polymer (hereinafter sometimes referred to as an interacting substance structure) is formed. Yes. The chain is one described in paragraph [0089] of JP-A No. 2006-111050, and is formed by bonding an interacting substance and a water-soluble polymer to each other. An interactive substance is bonded to the water-soluble polymer through a spacer, and a chain-like and / or network-like structure is formed by repeating the structure.
This chain is assembled or bonded, for example, to form a matrix structure (matrix) of the interactive substance structure in the present invention. That is, the interactive substance structure in the present invention is a structure containing an interactive substance and a water-soluble polymer as schematically shown in FIGS. 1 (a) to 1 (c), and its skeleton is It is formed by a chain formed by binding an interacting substance and a water-soluble polymer. Thereby, the interacting substance structure in the present invention is configured as a matrix having a structure in which the chains are linked in a chain and / or a network.
1 (a) to 1 (c) show the surface of an example of the interactive substance structure in the present invention immobilized on a solid phase carrier, in order to explain the matrix structure of the interactive substance structure in the present invention. It is a schematic diagram which expands and shows the vicinity. Moreover, in FIG. 1 (a)-FIG.1 (c), a circular part represents an interactive substance and a linear part represents a water-soluble polymer. Moreover, a dotted line part represents a spacer among linear parts.

Hereinafter, the chain | strand of the interaction substance structure in this invention and the matrix structure comprised from the said chain | strand are demonstrated.
As described above, the interacting substance structure in the present invention is a structure having a chain composed of an interacting substance and a water-soluble polymer. Moreover, the chain | strand of the interaction substance structure in this invention comprises the frame | skeleton of a matrix structure, and, specifically, an interaction substance and a water-soluble polymer couple | bond together. Specifically, an interacting substance is bonded to a water-soluble polymer via a spacer, and a chain-like and / or network-like structure is formed by repeating the structure.
Therefore, the interaction substance structure in the present invention usually has two or more partial structures represented by the following formula (A).
R ¹ -R ² (A)
{In the above formula (A), R ¹ represents an interacting substance, and R ² represents a water-soluble polymer. However, when the interacting substance structure is bound to the solid phase carrier, R ² represents a water-soluble polymer not directly bound to the solid phase carrier. Also, each R ¹ and R ² may be the same or different. }
That is, the interacting substance structure in the present invention is a structure in which a partial structure in which an interacting substance and a water-soluble polymer are bound as in the above formula (A) is bound in a linear and / or network form. Preferably there is. Specifically, R ¹ in the above formula (A) is independently bonded to one or more other R ^{2 s} , and R ² is independently bonded to one or more other R ^{1 s} . It is preferable. However, the interaction substance structure in the present invention may include a partial structure in which, for example, the interaction substances R ¹ or the water-soluble polymers R ² are bonded to each other. Here, the bonds between R ¹ and R ² indicate bonds by physical interaction such as intermolecular attractive force, lyophobic interaction, and electrical interaction.
Therefore, the interaction substance structure in the present invention has at least a bridge structure in which an interaction substance exists between water-soluble polymers and a water-soluble polymer exists between interaction substances. It is preferable to have a part. And it is preferable that the chain | strand of a matrix structure is comprised with both the interaction substance and the water-soluble polymer.

The fact that the interactive substance structure has a chain composed of the interactive substance and the water-soluble polymer can be confirmed, for example, by the following method.
Since the interaction substance structure in the present invention has a chain composed of the interaction substance and the water-soluble polymer as described above, the structure of the interaction substance structure can be obtained by decomposing the binding of the interaction substance as a component. Collapse. If this is utilized to decompose only the interacting substance of the interacting substance structure, a chain composed of the interacting substance and the water-soluble polymer can be confirmed. For example, when the interacting substance is decomposed while preventing the water-soluble polymer from being decomposed, the interacting substance structure in the present invention greatly collapses. Further, by investigating the collapsed compounds, it is possible to specify the compounds constituting the interacting substance structure other than the interacting substance constituent elements. Further, when the interacting substance is decomposed while preventing the water-soluble polymer from being decomposed, the interacting substance structure in the present invention is fixed to the solid phase carrier via the interacting substance in the water-soluble polymer. The part which has been converted is released from the solid phase carrier.

  Therefore, specifically, the interaction substance is decomposed by an enzyme or other chemicals that are not decomposed by the water-soluble polymer and only the interaction substance is decomposed, and the substance detached from the solid support by this treatment is examined. Alternatively, by examining the substance remaining on the surface of the solid phase carrier, the compound constituting the interacting substance structure other than the interacting substance constituent element can be specified. If the interacting substance structure has a chain composed of the interacting substance and the water-soluble polymer, the water-soluble polymer is detected in the detached substance or the substance remaining on the surface of the solid support. The

Any enzyme or chemical for decomposing the interaction substance used in the above method may be used appropriately depending on the type of the interaction substance or water-soluble polymer used. When the specific example is given, when an interaction substance is a nucleic acid, nucleolytic enzymes, such as ribonuclease and deoxyribonuclease, etc. are mentioned, for example.
When the interacting substance is a protein, for example, proteolytic enzymes such as microbial protease, trypsin, chymotrypsin, papain, rennet, and V8 protease, cyanogen bromide, 2-nitro-5-thiocyanobenzoic acid, hydrochloric acid, sulfuric acid And chemical substances having protein resolution such as sodium hydroxide.
Furthermore, when the interaction substance is a lipid, for example, lipolytic enzymes such as lipase and phospholipase A2 can be mentioned.
In addition, when the interacting substance is a sugar, examples thereof include glycolytic enzymes such as α-amylase, β-amylase, glucoamylase, pullulanase, and cellulase.
In addition, the enzyme and chemical | medical agent for decomposing | disassembling an interaction substance may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and a ratio.
However, among the exemplified substances, those that may decompose not only the interacting substance but also the water-soluble polymer should be avoided because the chain cannot be confirmed accurately.

Moreover, when decomposing the interaction substance and confirming the substance remaining on the solid support after the decomposition, the specific confirmation method is arbitrary. For example, surface plasmon resonance (SPR), crystal resonator This can be confirmed by measurement using a microbalance (QCM), electron microscope, ellipsometry, or the like.
Furthermore, when analyzing a substance detached from a solid phase carrier after decomposition of an interactive substance, the specific analysis method is arbitrary. For example, liquid chromatography, gas chromatography, mass spectrometry (MS), red Examples include external spectroscopy, nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC), gel electrophoresis, capillary electrophoresis, absorbance measurement, fluorescence measurement, elemental analysis, amino acid quantification and the like. In the analysis, each measurement method may be used alone, or two or more kinds may be arbitrarily combined.

[I-1 Water-soluble polymer]
The water-soluble polymer in the present invention is a water-soluble polymer of a compound containing a spacer and a polymerizable group, and preferably a functional group for covalently binding the target molecule interacting substance is bonded to the polymerizable group via the spacer. Is a water-soluble polymer obtained by carrying out a polymerization reaction using the resulting compound as a monomer (hereinafter sometimes referred to as “a monomer with a spacer” or “first monomer”). However, when the interacting substance reacts with the functional group and binds to the spacer, the functional group is no longer a constituent of the water-soluble polymer.
Here, examples of the spacer include a structure represented by the following formula.
-(Y) _q-

  Here, Y represents an alkyleneoxy group having 1 to 10 carbon atoms or an alkylene group. Although q shows the integer of 1-20, the more preferable target molecule capture effect is acquired and the range which can synthesize | combine a water-soluble polymer more efficiently is 2-8. When q is an integer of 2 or more and 20 or less, the repeated Ys may be the same or a chain of different alkyleneoxy groups. A preferred structure of the spacer includes polyoxyethylene. In particular, when a water-soluble polymer is obtained by polymerizing the first monomer alone, the spacer needs to be a polyalkyleneoxy group.

  The polymerizable group may be a group that can be polymerized by being reacted alone or with another polymerizable group, but is preferably an addition polymerizable group, more preferably an acryl group, a methacryl group, or a group containing these.

ここで、Ｒ₇は水素原子又はメチル基を示す。 Specifically, as a compound in which a functional group for covalently binding a target molecule interaction substance is bonded to a polymerizable group via a spacer, an ethylenically unsaturated polymerizable monomer represented by the following general formula (II) is Can be mentioned.

Here, R ₇ represents a hydrogen atom or a methyl group.

The definition and preferred examples _of- (Y) _q- in formula (II) are as described above. However, if there is no spacer (when q is 0), a monomer in which an active ester is directly bonded to the main chain ( For example, even if a water-soluble polymer is synthesized using N-acryloyloxysuccinimide), neutral p
In the medium in the H region, the interaction substance cannot be efficiently bound.
This is because the hydrophobic succinimide group is buried inside the water-soluble polymer, making it difficult to contact and bind to the interacting substance. In particular, in the case of a water-soluble polymer using a phosphorylcholine-like group as the second monomer described later, this tendency is further strong because the phosphorylcholine-like group is relatively bulky.

  W represents a functional group that can be covalently bonded to the target molecule interacting substance. The functional group is not particularly limited as long as it can be covalently bonded to the above-described interacting substance, and any functional group can be used. Usually, it is possible to select an appropriate one according to the type of the interacting substance and the use of the water-soluble polymer to which the interacting substance is bound in the present invention (hereinafter sometimes referred to as the interacting substance structure). preferable. In addition, a functional group may be used individually by 1 type, and may be used 2 or more types by arbitrary combinations and a ratio.

  A preferable functional group includes an active ester group. Specific examples of the active ester group include an N-hydroxysuccinimide active ester group, a p-nitrophenyl active ester group, a succinimide active ester group, and a phthalimide active ester group. When the interacting substance is a protein, usually, an amino group present on the surface layer of the protein is bound to the active ester group. When the interacting substance is a nucleic acid, the amino group introduced into the end of the nucleic acid is usually bound to the active ester group. Further, even when the interacting substance is a sugar, the amino group present in the side chain of the sugar is usually bonded to the active ester group.

  The water-soluble polymer in the present invention is a combination of the first monomer and an unsaturated polymerizable monomer having a hydrophilic or amphiphilic functional group (hereinafter sometimes referred to as “second monomer”). A water-soluble polymer obtained by polymerization is preferred.

  The second monomer constituting the water-soluble polymer is not particularly limited as long as it is an unsaturated polymerizable monomer having a hydrophilic functional group or an amphiphilic unsaturated polymerizable monomer. Specific examples of the unsaturated polymerizable monomer having a hydrophilic or amphiphilic functional group include (meth) acrylic acid, itaconic acid, 2-hydroxyethyl (meth) acrylate, and 2-hydroxy (meth) acrylic acid. Propyl, sodium p-styrenesulfonate, (meth) acrylamide, N, N-dimethyl (meth) acrylamide, N-isopropylacrylamide, N-vinylformamide, (meth) acrylonitrile, N- (meth) acryloylmorpholine, N-vinyl Examples include pyrrolidone, N-vinylacetamide, N-vinyl-N-acetamide, polyethylene glycol mono- (meth) acrylate, glycidyl (meth) acrylate, N-acryloylmorpholine, 2-methacryloyloxyethyl phosphorylcholine and the like.

Examples of the water-soluble polymer in the present invention include at least an ethylenically unsaturated polymerizable monomer having a phosphorylcholine-like group represented by the following general formula (I) and an ethylenically unsaturated polymerization represented by the following general formula (II): And a copolymer with a functional monomer.

In the formula, R ₁ represents a divalent hydrocarbon group having 2 to 10 carbon atoms, R ₂ represents a divalent hydrocarbon group having 1 to 4 carbon atoms, and R ₃ , R ₄ and R ₅ are each independently selected. Represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, and R ₆ represents a hydrogen atom or a methyl group. Here, specific examples of R ₁ include an ethylene group, a propylene group, and a butylene group, and an ethylene group is preferable. Specific examples of R ₂ include a methylene group, an ethylene group, a propylene group, and a butylene group, with an ethylene group being preferred. Specific examples of R ₃ , R ₄ , and R ₅ include a hydrogen atom, a methyl group, an ethyl group, a propyl group, and a butyl group, with a methyl group being preferred.

  Specific examples of the compound represented by (I) include 2-methacryloyloxyethyl phosphorylcholine, 2-methacryloyloxyethoxyethylphosphorylcholine, 6-methacryloyloxyhexylphosphorylcholine, 10-methacryloyloxydecylphosphorylcholine, 2-methacryloyloxyallyl. Phosphorylcholine, 2-methacryloyloxybutenylphosphorylcholine, 2-methacryloyloxyhexenylphosphorylcholine, 2-methacryloyloxyoctenylphosphorylcholine, 2-methacryloyloxydecenylphosphorylcholine, etc., among which 2-methacryloyloxyethyl Phosphorylcholine is preferably used.

  As the compounds represented by the above formulas (I) and (II), commercially available compounds may be used, or those prepared by methods well known to those skilled in the art may be used.

  By using an ethylenically unsaturated polymerizable monomer having a spacer represented by the general formula (II), a succinimide group that is more hydrophobic than a hydrophilic group or an amphiphilic group is exposed in water, and an interactive substance (protein The steric hindrance of the hydrophilic group or the amphiphilic group can be avoided, such as being easily contacted with the like, and the interaction substance can be bound efficiently even in the neutral state. This tendency is particularly remarkable when a phosphorylcholine-like group is used as the second monomer.

  The water-soluble polymer preferably further has a hydrophobic unit (hereinafter sometimes referred to as “third monomer”). The hydrophobic unit means a structural unit derived from an unsaturated polymerizable monomer having a hydrophobic unit, and specifically, methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) having a hydrophobic group. Aromatic rings such as linear or branched alkyl (meth) acrylates such as acrylate, 2-ethylhexyl (meth) acrylate, lauryl (meth) acrylate, stearyl (meth) acrylate, cyclohexyl (meth) acrylate, phenoxyethyl (meth) acrylate, etc. Polyalkylene glycol (meth) acrylates such as (meth) acrylate and polypropylene glycol (meth) acrylate, styrene monomers such as styrene, methylstyrene and chloromethylstyrene, vinyl such as methyl vinyl ether and butyl vinyl ether Examples include ether monomers, vinyl ester monomers such as vinyl acetate and vinyl propionate. These may be used alone or as a mixture of two or more. Of these, n-butyl methacrylate is preferably used. Hydrophobic units impart film-forming properties, and more hydrophobic units are introduced into the polymer chain than hydrophobic active ester groups, making active esters more easily exposed to water. Therefore, it is preferable.

  Moreover, it is also preferable to prevent applying excessive heat or irradiating strong ultraviolet rays so that the monomers do not bond with each other.

  The water-soluble polymer in the present invention can be produced by polymerizing the above monomer by a commonly used method known per se. Specifically, for example, when the monomer is radically polymerized, the polymerization is usually started by mixing a radical polymerization initiator. Any radical polymerization initiator may be used as long as the effects of the present invention are not significantly impaired. Examples of the radical polymerization initiator that can be used include 2,2′-azobisisobutyronitrile (AIBN), 2,2′-azobis- (2-methylpropanenitrile), 2,2′-azobis- ( 2,4-dimethylpentanenitrile), 2,2′-azobis- (2-methylbutanenitrile), 1,1′-azobis- (cyclohexanecarbonitrile), 2,2′-azobis- (2,4-dimethyl) Of azo (azobisnitrile) type, such as -4-methoxyvaleronitrile), 2,2'-azobis- (2,4-dimethylvaleronitrile), 2,2'-azobis- (2-amidinopropane) hydrochloride Initiator, benzoyl peroxide, cumene hydroperoxide, hydrogen peroxide, acetyl peroxide, lauroyl peroxide, persulfate (eg ammonium persulfate), perester For example t- butyl peroxide octoate, etc. α- cumyl peroxypivalate and t- butyl per octoate) peroxide type initiators and the like. The concentration of the radical polymerization initiator added is preferably 1 to 100 mM.

  Furthermore, polymerization may be initiated by mixing a redox initiator. Any redox initiator can be used as long as the effects of the present invention are not significantly impaired. Examples thereof include ascorbic acid / iron (II) sulfate / sodium peroxydisulfate, tert-butyl hydroperoxide / dioxide. Examples include sodium sulfite, tert-butyl hydroperoxide / Na hydroxymethanesulfinic acid. In addition, each component, for example, a reducing component, may be a mixture, for example, a mixture of sodium salt of hydroxymethanesulfinic acid and sodium disulfite.

  The water-soluble polymer in the present invention refers to a water-soluble polymer among the polymers obtained by polymerizing the first monomer alone, the first and second monomers, or the first to third monomers. Here, water-soluble means that at least a part of the polymer may be dissolved in water, and the rest may be dispersed in water. This is because water is used as a medium such as a solvent or a dispersion medium, although the medium used for producing the target molecule interaction substance-immobilized carrier is arbitrary.

In order to make the polymer water-soluble, specifically, for example, the second monomer is prepared in an amount of 30 mol% or more, preferably 40 mol% or more, more preferably 50 mol% or more of the total amount of monomers to be polymerized. Thus, a method for carrying out the copolymerization is used. As the solvent for copolymerization, for example, an alcohol solvent such as ethanol is used, and the reaction conditions can be appropriately selected depending on the type of polymerization initiator or monomer used. As the amount ratio of the monomers to be subjected to the copolymerization reaction, when the water-soluble polymer is prepared by copolymerizing the first monomer and the second monomer, the second monomer is 30 to 99 mol%, preferably 40 to 40%. 99 mol%, more preferably 50-99 mol%, most preferably 60-99 mol%, the first monomer is 1-70 mol%, preferably 1-60 mol%, more preferably 1-50 mol% Most preferably, it is 1 to 40 mol% (provided that the total of the first monomer and the second monomer is 100 mol%).
Furthermore, in the case of preparing a water-soluble polymer by copolymerizing the first monomer, the second monomer, and the third monomer, the second monomer is 30 to 90 mol%, preferably 40 to 90 mol%, more preferably 60 to 90 mol%, the first monomer is 1 to 60 mol%, preferably 1 to 40 mol%, more preferably 1 to 20 mol%, and the third monomer is 10 to 50 mol%, preferably 20 ˜35 mol% (provided that the total of the first monomer, the second monomer and the third monomer is 100 mol%). Specifically, 2-methacryloyloxyethyl phosphorylcholine (hereinafter sometimes referred to as “MPC”) is used as the second monomer, and Methacryl-PEG-NHS (hereinafter referred to as “MA-PEG-NHS”) as the first monomer. And n-butyl methacrylate (hereinafter sometimes referred to as “BMA”) is used as the third monomer, for example, MPC is 30 to 90 mol%, preferably 40 to 90. Mol%, more preferably 60-90 mol%, MA-PEG-NHS 1-60 mol%, preferably 1-40 mol%, more preferably 1-20 mol%, BMA 10-50 mol%, preferably 20-35 mol% (however, the sum of MPC, MA-PEG-NHS and BMA is 100 mol%). That.

  These monomers are suspended in a suitable solvent, added with a polymerization initiator, and then allowed to stand at 50 to 80 ° C. for 2 to 6 hours to carry out a copolymerization reaction. Any solvent can be used without particular limitation as long as it dissolves a monomer, an initiator, and a polymer obtained by polymerization. Specifically, for example, an alcohol solvent such as ethanol is preferably used. The amount of the solvent can be appropriately selected so that the monomer can be sufficiently copolymerized. Specifically, it is preferably about 1 to 20 times the total amount of the monomer.

  The water-soluble polymer thus obtained is preferably purified by a commonly used method known per se. Specifically, reprecipitation is performed with a chloroform-ether mixed solvent (about 2: 8), and the precipitate is collected and dried under reduced pressure.

  The water-soluble polymer is preferably uncharged. If the water-soluble polymer has the same charge (charge having the same sign) as that of the interacting substance, the binding between the water-soluble polymer and the interacting substance may be hindered by electrostatic repulsion. On the other hand, if the water-soluble polymer has a charge opposite to that of the interacting substance (reverse sign charge), the interacting substance and the charged part in the water-soluble polymer are combined by electrostatic attraction. Therefore, there is a possibility that the interactive substance is effectively bonded to the active ester group of the water-soluble polymer. In addition, when the target molecule-interacting substance-immobilized carrier of the present invention is used for separation and purification, the binding between the water-soluble polymer and the interacting substance is an additive such as pH and salt of the solution used at the time of use. Therefore, it is expected that the bond can be easily broken.

  It should be noted that the water-soluble polymer is uncharged means that at least the structural formula is nonionic or zwitterionic (has both positive and negative charges, and the charges mutually cancel each other. ), The water-soluble polymer is uncharged. However, even if the water-soluble polymer has a charge due to hydrolysis of the active ester group or the like in the production process of the interaction substance structure in the present invention, such water-solubility can be used as long as the effect of the present invention is not impaired. A polymer can be used suitably.

  The molecular weight and structure of the water-soluble polymer are not particularly limited and are arbitrary. Therefore, for example, a low molecular weight polymer (oligomer) may be used as the water-soluble polymer, but in that case, it is crosslinked only within one interacting substance to be immobilized, thereby efficiently forming a chain. It may not be possible to form the interacting substance structure in the present invention. In addition, when the cross-linking is performed in the interacting substance, the activity of the interacting substance may not be retained. From the viewpoint of preventing this, the weight average molecular weight of the water-soluble polymer is usually 1,000 or more, preferably 10,000 or more, and usually 1,000,000 or less, preferably 500,000 or less, more preferably 250,000 or less. Various methods can be used for measuring the molecular weight. In addition, the molecular weight in this specification means the molecular weight measured by SEC (size exclusion chromatography) by the method as described in an Example.

  Moreover, when a part of water-soluble polymer is disperse | distributing in a medium, there is no restriction | limiting in the magnitude | size of a water-soluble polymer, and it is arbitrary unless the effect of this invention is impaired remarkably. However, in order to effectively bind the interacting substance and the water-soluble polymer, the diameter of the water-soluble polymer is usually in the state of being mixed in a liquid such as a solvent or a dispersion medium (here, the medium). It is desirable that the thickness is 1 nm or more, preferably 2 nm or more, more preferably 3 nm or more. Moreover, although there is no restriction | limiting in particular in an upper limit, Usually, it is 1 micrometer or less.

  Various methods can be used to measure the size of these water-soluble polymers, but they can be examined by a static light scattering measurement method or the like.

  Further, the amount of the active ester group possessed by the water-soluble polymer is not particularly limited, and cannot be defined unconditionally depending on the type of the water-soluble polymer. For example, a polymer having a molecular weight of 1000 or more was used as the water-soluble polymer. In this case, in order to allow the water-soluble polymer to efficiently bind to the interacting substance and to be miscible with a solvent or a dispersion medium, the water-soluble polymer is 0.05%, preferably 0.05%, based on the water-soluble polymer. It is 0.1%, more preferably 0.5% or more, further preferably 1% or more, and 40% or less, preferably 35% or less, more preferably 30% or less.

[I-2 Target molecule interaction substance]
As the target molecule interaction substance (interaction substance), any substance can be used depending on the purpose as long as the effects of the present invention are not significantly impaired. For example, when the target molecule-interacting substance-immobilized carrier of the present invention is used for purposes such as target molecule purification, a substance that binds to the target molecule is used, and the target molecule is separated and purified using the binding action. To do.

  The target molecule may be any target molecule that is captured and detected by the target molecule interaction substance-immobilized carrier of the present invention. Specific examples include proteins, peptides, nucleic acids, enzymes, antibodies, and low molecular compounds. Here, the “interaction” is not particularly limited, but is usually based on a covalent bond, ionic bond, chelate bond, coordination bond, hydrophobic bond, hydrogen bond, van der Waals bond, and electrostatic force. The effect | action by the force which acts between the substances which arise from at least 1 of a coupling | bonding is shown. In addition, electrostatic coupling includes electric repulsion in addition to electrostatic coupling. In addition, a binding reaction, a synthesis reaction, and a decomposition reaction resulting from the above action are also included in the interaction. The target molecule may be known or unknown. In the case where the target molecule is unknown, for example, a drug compound or drug candidate compound, a receptor whose substrate is not specified is supported as a substance that interacts with the target molecule, and the target molecule-interacting substance-immobilized carrier of the present invention is used. It can be used for identification (screening) of target molecules.

  Combinations of target molecules and interacting substances include enzyme and substrate, antibody and antigen molecule (epitope), lectin and sugar, receptor and ligand, protein A, protein G or protein A / G and Fc, avidin and streptavidin, etc. Biotin-binding protein and biotin, glutathione-S-transferase and glutathione, albumin and albumin-binding compound, maltose-binding protein and maltose, G protein and guanine nucleotide, polyhistidine peptide and metal ions such as nickel or cobalt, DNA-binding protein and DNA , Antibodies, calmodulin and calmodulin-binding peptides, ATP-binding protein and ATP, or various receptor proteins such as estradiol receptor protein and estradiol And its ligand, cells and cell-binding agent, apoenzyme and coenzyme, nucleic acid having a sequence complementary to the nucleic acid and the nucleic acid, and the like.

  When albumin is used as the interacting substance, nonspecific adsorption to the carrier can be suppressed. Further, when avidin is used as the interaction substance, a carrier on which another biotinylated interaction substance or other compound is easily immobilized in a large amount can be constituted. In addition, if protein A is used as the interacting substance, a carrier on which an antibody is easily immobilized in a large amount can be constructed.

[I-3 Mixing step]
In the mixing step, the interacting substance and the water-soluble polymer are allowed to coexist in a predetermined medium. The interacting substance is usually prepared as a solution or dispersion in which the interacting substance is dissolved or dispersed in some solvent. In this case, the solvent or dispersion medium for diluting the interactive substance is preferably prepared in consideration of the activity of the interactive substance, the stability of the structure, and the like. In the mixing step, the interacting substance and the water-soluble polymer are allowed to coexist in a predetermined medium, so that the interacting substance can be bonded via the functional group of the water-soluble polymer. An active substance structure can be obtained. Note that all the functional groups of the water-soluble polymer need not react with the interacting substance, and may partially remain in the interacting substance structure. However, since it is desirable that the target molecule does not remain when interacting with the target molecule, the functional group may be converted to a group that does not react with the target molecule before the target molecule interacts if it remains partially. preferable. In the present specification, a substance that forms a field when an interacting substance and a water-soluble polymer are combined is broadly called a “medium”.

  In the mixing step, a non-binding substance may be allowed to coexist in order to efficiently form a minute space in the interactive substance structure. By forming the minute space, it becomes possible to efficiently perform the interaction between the target molecule and the interacting substance in the minute space.

  The non-binding substance preferably does not bind to the interactive substance and the water-soluble polymer, and it is preferable to use a substance that can form a microspace having a diameter of 300 nm to 100 μm in the interactive substance structure. The non-binding substance is preferably a water-miscible substance, preferably a water-soluble substance, and particularly preferably a water-soluble polymer compound. The non-binding substance is preferably uncharged. It should be noted that the non-binding substance is non-charged at least in terms of the structural formula, non-ionic or zwitterionic (having both positive and negative charges, and the mutual charges substantially cancel each other) If so, the unbound material is uncharged. The non-binding substance may be a synthetic polymer compound or a natural polymer compound. Examples of the non-binding substance include polyethylene glycol. Non-binding substances are described in detail in JP-A-2007-279028.

  After the mixing step, a concentration step for removing the medium and a drying step can be performed, and a step for removing unbound substances can also be performed.

  In the production process of the target molecule-interacting substance-immobilized carrier of the present invention, the effects of the present invention are remarkably applied to the interacting substance, the water-soluble polymer, the non-binding substance and the medium, and the mixture thereof. As long as it does not prevent, arbitrary additives may coexist. Examples of additives include the above-mentioned salts, moisturizers such as acids, bases, buffers, glycerin, polyethylene glycol, saccharides, metal ions such as zinc as stabilizers for biological materials, antifoaming agents, denaturing agents, Examples thereof include preservatives such as sodium azide.

  As the medium, any medium can be used as long as the target molecule-interacting substance-immobilized carrier of the present invention can be produced. Usually, a medium in which the interacting substance and the water-soluble polymer are miscible is used. It is desirable. Furthermore, when a non-binding substance or additive is used, it is desirable to use a substance that can also mix the non-binding substance or additive. At this time, the mixing state of the interacting substance, the water-soluble polymer, the non-binding substance, and the additive is arbitrary, and it may be in a dissolved state or a dispersed state. In order to stably bind these, the interacting substance and the water-soluble polymer are preferably present in a dissolved state in the medium.

  A liquid is usually used as the medium. At this time, the medium forms a field where the interacting substance and the water-soluble polymer are bonded, and may affect the activity and the stability of the structure of the interacting substance and the water-soluble polymer. Therefore, it is preferable to select in consideration of the influence. Usually, the medium includes water and various buffer solutions such as carbonate buffer, phosphate buffer, and acetate buffer. Further, a liquid other than water may be used as the medium, and for example, an organic solvent can be used. Furthermore, among the organic solvents, an amphiphilic solvent, that is, an organic solvent miscible with water is preferable. Specific examples of media other than water include THF (tetrahydrofuran), DMF (N, N-dimethylformamide), NMP (N-methylpyrrolidone), DMSO, in addition to alcohol solvents such as methanol, ethanol, and 1-butanol. (Dimethyl sulfoxide), dioxane, acetonitrile, pyridine, acetone, glycerin and the like.

  Further, when liquid is used as these media, salt may be added to the media. The kind of the salt is arbitrary as long as the effects of the present invention are not significantly impaired. Specific examples include sodium chloride, potassium chloride, sodium phosphate, sodium acetate, calcium chloride, sodium hydrogen carbonate, ammonium carbonate and the like. Moreover, there is no restriction | limiting in the quantity of the salt to be used, Arbitrary quantity salt can be used according to a use. In addition, a medium may be used individually by 1 type and may use 2 or more types together by arbitrary combinations and a ratio.

  The mixing operation in the mixing step is arbitrary, and the order of mixing them is also arbitrary. Furthermore, it is also possible to prepare this mixture on a solid phase carrier for the purpose of immobilizing a water-soluble polymer in which an interacting substance is bound to the solid phase carrier described later.

  The mixing ratio of the interaction substance, water-soluble polymer, non-binding substance, medium, additive, etc. used in the mixing step is arbitrary as long as the target molecule interaction substance-immobilized carrier of the present invention can be obtained. However, the value of the mixing ratio represented by “(weight of interacting substance) / {(weight of interacting substance) + (weight of water-soluble polymer)}” is usually 0.1 or more, preferably 0.8. 2 or more, more preferably 0.4 or more, still more preferably 0.5 or more, and particularly preferably 0.6 or more. Moreover, although there is no restriction | limiting in particular in an upper limit, Usually, it is 0.999 or less.

  Further, the ratio (concentration) of the interacting substance and the water-soluble polymer in the medium is arbitrary as long as the target molecule interacting substance-immobilized carrier of the present invention can be obtained. The total concentration is usually 0.1 g / L or more, preferably 1 g / L or more, more preferably 10 g / L or more.

  Furthermore, the value of the mixing ratio represented by “(weight of non-binding substance) / {(weight of interacting substance) + (weight of water-soluble polymer) + (weight of non-binding substance)}” is usually 0. 0.001 or more, preferably 0.01 or more, more preferably 0.1 or more. Moreover, although there is no restriction | limiting in particular in an upper limit, Usually, 0.95 or less, Preferably it is 0.7 or less, More preferably, it is 0.5 or less.

[I-4 Solid phase carrier]
The solid phase carrier serves as a substrate on which the interacting substance structure is bound to the surface. There is no restriction | limiting in the solid-phase carrier used by this invention, The thing of arbitrary materials, a shape, and a dimension can be used if an interacting substance structure can couple | bond.

  Examples of the material of the solid phase carrier include various resin materials such as polyolefin, polystyrene, polyethylene, polycarbonate, polyamide, and acrylic resin, gels such as agar, carrageenan, gelatin, carboxymethylcellulose, glue, alginic acid, polyvinyl alcohol, and dextran. And inorganic materials such as glass carriers, glass, alumina, carbon, and metals. In addition, the material of the solid phase carrier may be a single material used alone, or a combination of two or more materials in any combination and ratio. In addition, the shape of the solid phase carrier can be appropriately selected depending on the use of the target molecule interaction substance-immobilized carrier of the present invention.

  Further, the solid phase carrier may be used as it is, or the surface of the solid support may be coated with a coating material such as metal or metal oxide, and then the interacting substance structure may be bound. Further, in order to bind the solid phase carrier and the interactive substance structure, as a surface treatment, a solid phase carrier having a functional group W of the general formula (II) or a functional group for covalently binding the interactive substance is used. Use. Examples of the functional group include a hydroxyl group, a carboxyl group, a thiol group, an aldehyde group, a hydrazide group, a carbonyl group, an epoxy group, a vinyl group, an amino group, a succinimide group, and a p-nitrophenyl group. Examples thereof include a functional group capable of binding the carrier and the interacting substance structure.

The binding between the interacting substance structure and the solid surface may be due to the reaction between the interacting substance and the functional group on the solid surface, or the functional group of the water-soluble polymer that has not reacted with the interacting substance. This may be due to the reaction of the functional group on the surface of the solid phase, or both of them.
For example, when the functional group W of the general formula (II) is a succinimide group and the functional group on the solid phase surface is an amino group, the interactive substance structure includes the succinimide group and the amino group on the solid phase surface. Can be bound to the solid surface via a covalent bond between In addition, when the interacting substance is a substance having an amino group and the functional group on the solid phase surface is a succinimide group, the interacting substance structure includes the amino group of the interacting substance and the succinimide group on the solid phase surface. Can be bound to the solid surface via a covalent bond between

  As a method for supplying the interaction substance structure to the solid phase carrier, any method may be used as long as the mixture in the mixing step can be brought into contact with the solid phase carrier. For example, a mixture of constituent components of the interacting substance structure may be prepared in advance, and the mixture may be brought into contact with the solid support, or each component may be prepared separately and mixed on the solid support. While preparing the mixture, the mixture may be brought into contact with the solid phase carrier. A preferable method for contacting the solid phase carrier and the mixture is a method in which the constituent components are separately added or a mixture of the constituent components is dropped onto the solid surface. The amount of dripping can be appropriately selected according to the use of the target molecule-interacting substance-immobilized carrier of the present invention and the conditions at the time of production. Specifically, about 1 pL to 10 μL is preferable.

  The conditions for binding the interacting substance structure to the solid support are arbitrary, but from the viewpoint of avoiding denaturation of the interacting substance, etc., the temperature condition is usually 0 to 3 ° C. or more and 10 to 25 ° C. or less. Furthermore, in order to fix the interacting substance structure to the solid phase carrier, it is desirable that the solid phase carrier is allowed to stand for a predetermined time after the mixture is supplied. Although the standing time is arbitrary, it is usually preferably 1 minute to 1 hour to 12 to 24 hours.

  In addition, the interaction substance structure supplied to the solid phase carrier may be appropriately subjected to a concentration step or a drying step. When drying and concentrating the mixture after immobilization on the solid phase carrier, the pressure condition at that time is also arbitrary, but usually, normal pressure or lower is desirable.

  Furthermore, when a non-binding substance is added in the mixing step, the non-binding substance can be removed from the interactive substance structure by performing a removal step after supplying the mixture to the solid phase carrier.

  There is no limitation on the method for removing the unbound substance, and for example, the unbound substance may be removed by washing the interacting substance structure containing the unbound substance with a washing solvent. The washing solvent is preferably one that does not alter the interactive substance structure. Specific examples of the washing solvent include water-soluble organic solvents such as water, ethanol, 2-propanol, DMSO, and DMF. Of these, water is preferred. Further, the cleaning solvent may contain substances such as a buffer substance and a surfactant.

  The solid-phase carrier to which the thus produced interacting substance structure is bound can be used for detecting the target molecule, but also blocks unreacted functional groups such as active ester groups on the solid-phase surface. For the purpose, it is preferable to contact ethanolamine-HCl aqueous solution or glycine. The contact time is preferably 1 minute to 5 hours. Thereafter, it is preferably washed well with demineralized water or the like for the purpose of removing ethanolamine and the like, and dried at room temperature to obtain the target molecule-interacting substance-immobilized carrier of the present invention.

[II Use of target molecule interaction substance-immobilized carrier of the present invention]
The target molecule interaction substance-immobilized carrier of the present invention can be suitably used, for example, as a biosensor for detecting a target substance. The biosensor described above is, for example, a so-called DNA array or DNA chip, or a sensor chip (biochip) on which DNA or protein or the like is called a protein array or protein chip or the like is used to analyze the interaction. It is. For example, the target molecule interaction substance-immobilized carrier of the present invention can be applied to this sensor chip. That is, a sensor chip can be manufactured by setting the sensor chip body as a solid phase and forming an interactive substance structure on the surface by the above-described method.

  Such sensor chips include fluorescence method, ELISA method, chemiluminescence method, RI method, SPR (surface plasmon resonance) method, QCM (quartz crystal microbalance) method, piezo method cantilever method, laser method cantilever method, mass spectrometry The present invention can be used in a method, an electrochemical method, an electrode method, a field effect transistor (FET) method, a FET using a carbon nanotube, and / or a single electron transistor method.

  Furthermore, since the target molecule interacting substance-immobilized carrier of the present invention can impart antithrombogenicity, cell adhesion, sustained release of proteins such as cell growth factors, etc., and therefore for DDS (drug delivery system) It can be applied to the surface treatment of drugs, the surface treatment of regenerative medical carriers, the surface treatment of artificial organs, the surface treatment of catheters and the like.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples, and may be arbitrarily modified and implemented without departing from the gist of the present invention. Can do.

[Example 1]
Molecular structure that interacts with target molecule on 96-well plate prepared using polymer A or polymer B and mouse IgG (1) Synthesis of water-soluble polymer (polymer A) 2-methacryloyloxyethyl phosphorylcholine (monomer) Hereinafter referred to as “MPC”), 3.7 parts by weight of this, 0.90 parts by weight of n-butyl methacrylate (BMA) (manufactured by Wako Pure Chemical Industries, Ltd.), and synthesized Methacryl- Add 0.67 parts by weight of PEG-NHS (MA-PEG-NHS), 0.049 parts by weight of initiator AIBN (manufactured by Kanto Chemical Co., Inc.), and add ethanol as a solvent to a total volume of 30 ml. went. Then, synthesis was performed by radical polymerization at 60 ° C. for 3 hours. Reprecipitation was performed using a chloroform-ether mixed solvent (2: 8). Polymer A was obtained by collecting the precipitate and drying under reduced pressure.
Here, the synthesis of the MA-PEG-NHS was performed by the following method.
In 300 mL of dehydrated acetone, 8.0121 g of N, N′-disuccinimidyl carbonate (DSC) was added and stirred. Into 100 mL of dehydrated acetone were dissolved 4.4962 g of Polyethylene glycol-monomethacrylate (Blenmer PE-90: manufactured by NOF Corporation) and 6.3411 g of triethylamine (TEA), and the mixture was allowed to react at room temperature for 24 hours. After the reaction, the solvent was removed by evaporation. Thereafter, the resultant was dissolved in 300 mL of chloroform, and washed with 300 mL of diluted hydrochloric acid (pH 4). The chloroform layer was collected, added with magnesium sulfate, and dehydrated overnight. After filtration, the solvent was completely removed by evaporation to obtain a viscous liquid. The yield was 74.7% (6.1442 g).

The obtained polymer A was subjected to SEC (Size Exclusion Chromatography) measurement. As a result, the peak top molecular weight (Mp) of the polymer A was estimated to be about 74470, and the weight average molecular weight (Mw) was estimated to be about 63899.
Here, SEC was measured using Shodex OH pak SB-804HQ (manufactured by Showa Denko), methanol: water = 7: 3 (containing 10 mM LiBr) as a developing solvent. PEG was used as a standard sample for molecular weight calibration.

The molar ratio of MA-PEG-NHS and BMA and MPC contained in the obtained polymer A (MA-PEG-NHS / BMA / MPC) is from ¹ H-NMR measurement MA-PEG-NHS / BMA / MPC = 3.3 / 33/63 was estimated.

(2) Synthesis of water-soluble polymer (Polymer B) 3.7 parts by weight of monomer MPC (manufactured by NOF Corporation), 2.7 parts by weight of synthesized MA-PEG-NHS, initiator AIBN 0.049 parts by weight (manufactured by Kanto Chemical Co., Inc.) and ethanol as a solvent were added so that the total amount was 30 ml, followed by argon substitution. Then, synthesis was performed by radical polymerization at 60 ° C. for 3 hours. Reprecipitation was performed using a chloroform-ether mixed solvent (2: 8). Polymer B was obtained by collect | recovering deposits and drying under reduced pressure.
The obtained polymer B was subjected to SEC (Size Exclusion Chromatography) measurement calibrated with standard polyethylene glycol. As a result, the peak top molecular weight (Mp) of the polymer B was estimated to be about 63508, and the weight average molecular weight (Mw) was estimated to be about 283167. It was lost.

Moreover, the molar ratio (MA-PEG-NHS / MPC) of MA-PEG-NHS and MPC contained in the obtained polymer B is MA-PEG-NAS / MPC = 11/89 from ¹ H-NMR measurement. Estimated.

(3) Formation of molecular structure interacting with target molecule on 96-well plate surface (3-1) Surface treatment of 96-well plate 0.1M N-hydroxysuccinimide (NHS, manufactured by Wako Pure Chemical Industries, Ltd.) aqueous solution 1 mL and 0.4 M 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC, manufactured by Dojindo Laboratories) aqueous solution were mixed with 1 mL, and further diluted with 18 g of demineralized water, 100 μL / well was added to a 96-well plate (Sumilon (registered trademark) ELISA product / carbotype, manufactured by Sumitomo Bakelite Co., Ltd.) into which a group was introduced, and allowed to react for 15 minutes. This is because a succinimide group capable of binding a well and a molecular structure that interacts with a target molecule is introduced into the surface of the well.

(3-2) Preparation and dropping and drying treatment of mixture of interaction substance and water-soluble polymer Next, 10 mg / mL mouse IgG (manufactured by LAMPIRE BIOLOGICAL LABORATORIES) used as an interaction substance was replaced with 10 mM phosphate buffer ( The one exchanged with pH 7.4, pH 8.0) or 10 mM carbonate buffer (pH 9.4) was prepared. A mixture obtained by mixing the buffer containing 10 mg / mL mouse IgG and an aqueous solution of polymer A or polymer B (10 mg / mL) at a weight mixing ratio of 10: 1 (molecule interacting with target molecule: water-soluble polymer), 1 μL spotted into wells of 96 well plate. After drying this at room temperature, for the purpose of blocking unreacted active ester groups, 300 μL of 1M ethanolamine-HCl aqueous solution (pH 8.5) was immersed in 1 well at room temperature for 30 minutes. As a result, a molecular structure that interacts with the target molecule was formed on the well surface of the plate using mouse IgG and polymer A or polymer B as the target molecule interacting substance and water-soluble polymer, respectively.

  Thereafter, the inside of the well was thoroughly washed with demineralized water for the purpose of removing ethanolamine and dried at room temperature to obtain a well carrying a target molecule interacting substance on the surface.

[Comparative Example 1]
Production of carrier for immobilizing target molecule interaction substance on 96-well plate prepared using polymer C and mouse IgG (1) Synthesis of water-soluble polymer (polymer C) N-acryloylmorpholine (NAM, KOHJIN) 2.82 parts by weight of N-acryloyloxysuccinimide (NAS, manufactured by ACROS ORGANICS) and 20.87 parts by weight of dehydrated dioxane (manufactured by Wako Pure Chemical Industries, Ltd.) as a solvent. The mixture was mixed well, poured into a 50 mL four-necked flask, and deaerated with nitrogen at room temperature for 30 minutes to prepare a monomer solution.

  This monomer solution was heated to 70 ° C. in an oil bath, and a solution in which 0.0124 parts by weight of a polymerization initiator azobisisobutyronitrile (AIBN, manufactured by Kishida Chemical Co., Ltd.) was dissolved in 0.50 parts by weight of dehydrated dioxane. The polymerization was started by adding. The polymerization was carried out for 8 hours under a nitrogen atmosphere.

  After the polymerization, the polymer-generated solution was reprecipitated by supplying it to 0.5 L of ethanol (manufactured by Junsei Co., Ltd.), and then pulverized by removing the solvent to obtain a water-soluble polymer polymer C. It was.

The obtained polymer C was subjected to SEC (Size Exclusion Chromatography) measurement calibrated with standard polystyrene. As a result, the weight average molecular weight (Mw) of the polymer C was estimated to be about 59,500.
Here, the measurement of SEC was performed under the following conditions.
Solvent: DMF (with 50 mM LiBr)
Flow rate: 0.5ml / min
Temperature: 80 ° C
In addition, this time, it measured using the following apparatuses, detectors, and columns.
Apparatus: Tosoh HLC-8120GPC
Detector: RI (built-in device)
Column: TOSOH TSKgel SuperHM-M (15 cm × 2)

The molar ratio (NAS / NAM) between NAS and NAM contained in the obtained polymer C was estimated to be NAS / NAM = 23/77 from ¹ H-NMR measurement.

(2) Formation of interacting substance structure on the surface of 96-well plate In the same manner as in “(3-1) Surface treatment of 96-well plate” in Example 1, succinimide groups were formed on the surface in the well of the 96-well plate. Introduced.

  Next, “preparation and dropwise addition of the mixture of the interacting substance and the water-soluble polymer of Example 1 except that the polymer C aqueous solution (10 mg / mL) was used instead of the polymer A aqueous solution or the polymer B aqueous solution. By performing the same operation as “and drying treatment”, an interacting substance structure having mouse IgG and polymer C as a target molecule interacting substance and a water-soluble polymer was formed on the well surface of the plate, respectively.

  In addition, direct fixation (2D) of mouse IgG to the substrate was performed by the same method as described above, except that no polymer was used. Thereafter, the well was thoroughly washed with demineralized water for the purpose of removing ethanolamine and dried at room temperature to obtain a plate carrying only an interacting substance structure or mouse IgG on the surface.

<Evaluation of target molecule-interacting substance-immobilized carrier obtained in Example 1 and Comparative Example 1 by fluorescence intensity method>
The performance of the samples obtained in Example 1 and Comparative Example 1 was analyzed by the fluorescence intensity method. The interactive substance structure plate obtained in Example 1 and Comparative Example 1 was blocked with 200 μl / well of PBS (−) containing 5 wt% BSA at room temperature for 1 hour. After removing the solution, the plate was washed with water, and 100 μl of PBS (−) containing 50 μg / mL FITC Conjugated Protein A (manufactured by EY LABORATORIES, INC.) Was added to the well and incubated at room temperature for 1 hour. After removing the solution, washing was sufficiently performed using PBS (-) containing 0.05% Tween 20 for the purpose of removing excess analyte that was not involved in the reaction. After further washing with water, 100 μL of PBS was added per well, and FITC fluorescence intensity measurement of the whole part of the interactive substance structure was carried out using an MOLECULAR IMAGER (manufactured by BIO-RAD) at an excitation wavelength of 488 nm and a fluorescence wavelength of 530 nm. I did it.

  The results are shown in FIG. In FIG. 2, the present example showed higher fluorescence intensity (Intensity) in the neutral region (Buffer pH 8.0 and pH 7.4) as compared with Comparative Example 1. From this, it was shown that more analytes can be captured by using polymer A or polymer B.

[Example 2] Structure of interacting substance on gold chip using mouse IgG (1) Surface treatment of gold chip A chip coated with gold was treated with 10 mM 16-mercaptohexadecanoic acid (16-MERCAPTOEXADEANOIC ACID: ALDRICH). Manufactured) It was immersed in an ethanol solution and reacted at room temperature for 12 hours for surface treatment. After completion of the reaction, the gold chip was washed with ethanol. This surface treatment is a treatment for introducing a carboxyl group into the surface of the gold chip via a gold-sulfur bond.

  Next, 1 mL of 0.1 M N-hydroxysuccinimide (NHS, manufactured by Wako Pure Chemical Industries, Ltd.) aqueous solution and 0.4 M 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC, Dojindo) The substrate (gold chip) into which the above carboxyl group was introduced was immersed in a solution diluted with 1 mL of an aqueous solution and further diluted with 18 g of demineralized water, and allowed to react for 15 minutes. This is because a succinimide group capable of bonding the substrate and the interactive substance structure is introduced to the substrate surface.

(2) Formation of interaction substance structure Next, an aqueous solution containing 10 mg / mL mouse IgG (phosphate buffer 10 mM, pH 7.4) and an aqueous solution of polymer A or polymer B (10 mg / mL) were mixed at a weight mixing ratio of 10 A mixture of 1 (interactive substance: water-soluble polymer) was spotted on the surface of a gold chip at 1 μL × 4 points. After drying this at room temperature, it was immersed in 3 mL of 1M ethanolamine aqueous solution (pH 8.5) for 15 minutes for the purpose of blocking unreacted active ester groups. Thereby, the interaction substance structure which uses mouse IgG and polymer A or polymer B as an interaction substance and a water-soluble polymer, respectively, was formed on the surface of the gold chip.

  Then, it was washed thoroughly with demineralized water and dried at room temperature to obtain an interactive substance structure gold chip carrying an interactive substance structure on its surface.

[Comparative Example 2] Interactive substance structure on gold chip using mouse IgG (1) Surface treatment of gold chip Introduction of succinimide group into the gold chip was carried out in accordance with “(1) Surface treatment of gold chip of Example 2. ".

(2) Formation of interacting substance structure Next, “(2) interacting substance structure of Example 2 except that polymer A aqueous solution (10 mg / mL) was used instead of polymer A aqueous solution or polymer B aqueous solution. By performing the same operation as “formation”, an interacting substance structure having mouse IgG and polymer C as a target molecule interacting substance and a water-soluble polymer, respectively, was formed on the surface of the gold chip. In addition, direct fixation (2D) of mouse IgG to the substrate was performed by the same method as described above, except that no polymer was used.

<Evaluation by SPR measurement of interacting substance structure on gold chip using mouse IgG obtained in Example 2 and Comparative Example 2>
The interaction substance structure gold chip obtained in Example 2 and Comparative Example 2 was measured with an imaging SPR device.

A gold chip on which an interacting substance structure composed of polymer A and IgG or polymer B and IgG is immobilized is set in an imaging SPR device FLEX CHIP ^™ Kinetic Analysis System (HTS Biosystems Inc), and after sending a buffer, spot The imaging image of was observed. In addition, as a comparative example, an interacting substance structure prepared using the polymer C shown in comparative example 2 and a spot (2D) in which mouse IgG was directly immobilized on a gold substrate were used.
The results are shown in FIG. In the interactive substance structure produced using the polymer A, the entire spot was uniform. In the interacting substance structure produced using the polymer B, it was confirmed that the interacting substance structure was formed inside the spot although spots were visible in some places. However, the interaction substance structure (Comparative Example 2) produced using the polymer C has no interaction substance structure formed inside the spot, and the interaction substance structure is formed only around the spot in a ring shape. Was formed. Furthermore, in the case where mouse IgG was directly immobilized on a gold substrate (2D), the amount of immobilization was small and it was difficult to confirm the spot.

In Fig. 3-2, the immobilized amount of the interacting substance structure or mouse IgG immobilized on the circled portion in the spot of the imaging image (Fig. 3-1) was measured by SPR (Surface Plasmon Resonance). Results are shown.
Since the SPR phenomenon depends on the refractive index in the vicinity of the metal thin film, the resonance angle changes when protein or the like is bonded to the surface of the metal thin film. Since the amount of binding coincides with the amount of change in resonance angle, the amount of protein immobilized, the amount of protein-protein interaction, etc. can be examined by measuring the amount of change in resonance angle.
The amount of immobilization decreased in the order of an interactive substance structure using the polymer A, an interactive substance structure using the polymer B, an interactive substance structure using the polymer C, and 2D, which coincided with the imaging image.

  Next, 1 μg / mL protein A (SIGMA) was used as an analyte (specimen), and the interaction between the interacting substance structure and protein A was detected by SPR measurement. The amount of analyte interacting with each interacting substance structure is shown in Figure 3-3. The interacted analyte is in the order of the interaction substance structure using the polymer A, the interaction substance structure using the polymer B, the interaction substance structure using the polymer C, and 2D, in the same manner as the immobilized amount. Diminished.

[Example 3] Target molecule-interacting substance-immobilized carrier prepared using polymer B and mouse IgG (1) Synthesis of water-soluble polymer (polymer B) Synthesis of polymer B, which is a water-soluble polymer, was carried out. In the same manner as in Example 1, “(2) Synthesis of water-soluble polymer (polymer B)”.

(2) Surface treatment of gold chip A chip coated with gold (gold chip) was used as a solid phase. These were immersed in a 10 mM aqueous solution of 2-aminotanthiol hydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.) and reacted at room temperature for 12 hours for surface treatment. After completion of the reaction, the gold chip was washed with demineralized water. This surface treatment is a treatment for introducing an amino group onto the surface of the gold chip via a gold-sulfur bond.
In this way, a solid surface having an amino group as a functional group for covalently bonding the succinimide group contained in the water-soluble polymer polymer B was obtained.

(3) Formation of interacting substance structure on solid-phase surface having amino group Next, the buffer of 10 mg / mL mouse IgG solution (manufactured by LAMPIRE BIOLOGICAL LABORATORIES) was replaced with 10 mM phosphate buffer (pH 7.4). I prepared something. This aqueous solution containing 10 mg / mL mouse IgG (phosphate buffer 10 mM, pH 7.4) and the aqueous solution of polymer B (10 mg / mL) were mixed at a weight mixing ratio of 10: 1 (interacting substance: water-soluble polymer). 1 μL of the obtained mixture was supplied to the surface of the gold chip having amino groups prepared as described above. After drying this at room temperature, for the purpose of blocking unreacted succinimide groups, 3 mL of 1M ethanolamine aqueous solution (pH 8.5) was dropped so as to cover the entire surface of the gold chip, and left for 30 minutes. As a result, an interacting substance structure having mouse IgG and polymer B on the surface of the gold chip as an interacting substance and a water-soluble polymer was produced.

  Thereafter, it was thoroughly washed with demineralized water and dried at room temperature to obtain an interactive substance structure on the gold chip surface.

[Comparative Example 3] Synthesis of interacting substance structure (1) water-soluble polymer (polymer C) on a gold chip prepared using polymer C and mouse IgG "(1) Water-soluble polymer (1) of Comparative Example 1" Same as “Synthesis of polymer C)”.

(2) Surface treatment of gold chip Introduction of amino groups into the gold chip was carried out in the same manner as “(2) Surface treatment of gold chip” in Example 3.

(3) Formation of interacting substance structure “(3) Formation of interacting substance structure on solid surface having amino group” in Example 3 except that polymer C was used as the water-soluble polymer. And so on. As a result, an interacting substance structure having mouse IgG and polymer C on the surface of the gold chip as an interacting substance and a water-soluble polymer was obtained.

  In addition, the direct fixation (2D) of mouse IgG on a gold chip having an amino group was carried out in accordance with “(3) Mutual immobilization on a solid phase surface having an amino group” in Example 3 except that no polymer was used. "Formation of active substance structure".

<Capture and detection of target molecule by interacting substance structure obtained in Example 3 and Comparative Example 3>
The performance of the gold chip having the interacting substance structure obtained in Example 3 and Comparative Example 3 on its surface was analyzed by the fluorescence intensity method. 2 ml of PBS (−) containing 3 wt% BSA was dropped onto a gold chip having the above-mentioned interacting substance structure on the surface, and blocked for 1 hour at room temperature with the gold chip surface completely covered. After removing the solution, it was washed with water, and 1 ml of PBS (−) containing 50 μg / mL Cy5-labeled Protein A as a target molecule was added onto the gold chip on which the interacting substance structure was formed, and incubated at room temperature for 1 hour. After removing the solution, washing was sufficiently performed using PBS (−) containing 0.05% Tween 20 for the purpose of removing excess target molecules not involved in the reaction. After further washing with water, the sample was dried with compressed air, and the fluorescence intensity of Cy5 near the center of the interactive substance structure was measured at an excitation wavelength of 635 nm using an InnoScan700 Microarray Scanner (manufactured by INNOPSYS). Note that MAPIX (manufactured by INNOPSYS) was used as analysis software.
Here, the production of Cy5-labeled Protein A was performed by the following method. The protein aqueous solution (RepliGen) buffer was replaced with a 100 mM carbonate buffer (pH 9.4) to prepare a Protein A concentration of 1 mg / ml. 1 ml of this 1 mg / ml Protein A solution was added to one Cy5 Mono-Reactive Dye Pack (manufactured by GE Healthcare) and mixed. This mixed solution was incubated at room temperature for 30 minutes. Thereafter, PD-10 Columns (manufactured by GE Healthcare) was used, PBS (−) was used as a developing solvent, and unreacted Cy5 was removed from the mixed solution to obtain Cy5-labeled Protein A.

  The results are shown in FIG. In FIG. 4, this example shows higher fluorescence intensity (Intensity) in the neutral region (Buffer pH 7.4) compared to the interacting substance structure (Comparative Example 3) prepared using Polymer C. It was. From this, it was shown that more target molecules can be captured by using the polymer B.

  FIG. 5 shows a fluorescence image of Cy5 derived from the target molecule captured by the interacting substance structure. The interaction substance structure produced using the polymer B was confirmed to have Cy5 fluorescence inside the spot (hereinafter referred to as a region to which a mixture of the interaction substance and the water-soluble polymer was supplied). It can be seen that the structure was formed uniformly. However, the interaction substance structure (Comparative Example 3) produced using polymer C does not have an interaction substance structure formed inside the spot, and Cy5 fluorescence is confirmed only around the spot in a ring shape. It can be seen that the interacting substance structure was formed unevenly.

[Example 4] Structure of interaction substance on gold chip prepared using polymer D and mouse IgG (1) Synthesis of water-soluble polymer (polymer D) N-acryloylmorpholine (NAM, manufactured by KOHJIN) as a monomer ) 0.894 parts by weight, 0.106 parts by weight of MA-PEG-NHS and 4.17 parts by weight of dehydrated dioxane (manufactured by Wako Pure Chemical Industries, Ltd.) as a solvent are mixed well, and 30 mL The solution was poured into a neck flask and deaerated with nitrogen at room temperature for 30 minutes to prepare a monomer solution.

  This monomer solution was heated to 80 ° C. in an oil bath, and a solution obtained by dissolving 0.003 part by weight of a polymerization initiator azobisisobutyronitrile (AIBN, manufactured by Kishida Chemical Co., Ltd.) in 1.5 parts by weight of dehydrated dioxane. The polymerization was started by adding. The polymerization was performed for 6 hours under a nitrogen atmosphere.

  After the polymerization, the solution in which the polymer was formed was reprecipitated by supplying it to 150 mL of a chloroform-diethyl ether mixed solvent [chloroform / diethyl ether = 30/120 (v / v)], and then the solvent was removed. Powdered to obtain water-soluble polymer polymer D.

The obtained polymer D was subjected to SEC (Size Exclusion Chromatography) measurement calibrated with standard polystyrene, and as a result, the weight average molecular weight (Mw) of the polymer D was estimated to be 238954.
Here, the measurement of SEC was performed under the following conditions.
Solvent: DMF (with 50 mM LiBr)
Flow rate: 1.0ml / min
Temperature: 50 ° C
In addition, this time, it measured using the following apparatuses, detectors, and columns.
Apparatus: Waters 2695 made by Nippon Waters Co., Ltd.
Detector: Waters 2410 manufactured by Nippon Waters
Column: Shodex AD-80M / S manufactured by Showa Denko KK

Moreover, the molar ratio (M-PEG-NAS / NAM) of MA-PEG-NHS and NAM contained in the obtained polymer D is MA-PEG-NHS / NAM = 7/93 from ¹ H-NMR measurement. Estimated.

(2) Surface treatment of gold chip A chip coated with gold was used as a solid phase. This was immersed in a 5 mM ethanol solution of 16-mercaptohexadecanoic acid (16-MELCCAPOHEXZDECANIC ACID: manufactured by ALDRICH), and reacted at room temperature for 12 hours for surface treatment. After completion of the reaction, the gold chip was washed with ethanol. This surface treatment is a treatment for introducing a succinimide group to the surface of the gold chip via a gold-sulfur bond.
In this way, a solid phase surface having a succinimide group as a functional group for covalently binding an amino group in mouse IgG as an interacting substance was obtained.

(3) Formation of interacting substance structure on solid surface having succinimide group Next, the buffer of 10 mg / mL mouse IgG solution (manufactured by LAMPIRE BIOLOGICAL LABORATORIES) was exchanged with 10 mM phosphate buffer (pH 7.4). I prepared something. The aqueous solution containing 10 mg / mL mouse IgG (phosphate buffer 10 mM, pH 7.4) and the aqueous solution of polymer D (10 mg / mL) were mixed at a weight mixing ratio of 10: 2 (interacting substance: water-soluble polymer). 1 μL of the prepared mixture was supplied to the surface of the succinimide group-introduced gold chip produced as described above. After drying this at room temperature, for the purpose of blocking unreacted succinimide groups, 3 mL of 1M ethanolamine aqueous solution (pH 8.5) was dropped so as to cover the entire surface of the gold chip and left for 30 minutes. Thereby, the interaction substance structure which uses mouse IgG and polymer D as an interaction substance and a water-soluble polymer, respectively, was formed on the surface of the gold chip.

  Thereafter, it was thoroughly washed with demineralized water and dried at room temperature to obtain an interactive substance structure on the gold chip surface.

[Comparative Example 4] Interaction substance structure on gold chip prepared using polymer E and mouse IgG (1) Synthesis of water-soluble polymer (polymer E) N-acryloylmorpholine (NAM, manufactured by KOHJIN) as a monomer ) And 5.64 parts by weight of N-acryloyloxysuccinimide (NAS, manufactured by ACROS ORGANICS) and 30 parts by weight of dehydrated dioxane (manufactured by Wako Pure Chemical Industries, Ltd.) as a solvent are mixed well. The mixture was poured into a 50 ml four-necked flask and deaerated with nitrogen at room temperature for 30 minutes to prepare a monomer solution.

  The monomer solution was heated to 80 ° C. in an oil bath, and a solution prepared by dissolving 0.018 parts by weight of a polymerization initiator azobisisobutyronitrile (AIBN, manufactured by Kishida Chemical Co., Ltd.) in 4 parts by weight of dehydrated dioxane was added. The polymerization was started. The polymerization was carried out for 6 hours under a nitrogen atmosphere.

After the polymerization, the solution in which the polymer was formed was reprecipitated by supplying it to 150 ml of a chloroform-diethyl ether mixed solvent [chloroform / diethyl ether = 30/120 (v / v)], and then the solvent was removed. Powdered to obtain water-soluble polymer polymer D.
Polymer E was obtained.

The obtained polymer E was subjected to SEC (Size Exclusion Chromatography) measurement calibrated with standard polystyrene. As a result, the weight average molecular weight (Mw) of the polymer was estimated to be 170320.
Here, the SEC was measured in the same manner as in the method described in “(1) Synthesis of water-soluble polymer (polymer D)” in Example 4.

The molar ratio (NAS / NAM) between NAS and NAM contained in the obtained polymer E was estimated to be NAS / NAM = 8.4 / 91.6 from ¹ H-NMR measurement.

(2) Surface treatment of gold chip A gold chip was used as a solid phase. The introduction of the succinimide group of the gold chip was carried out in the same manner as in “(2) Surface treatment of gold chip” in Example 4.

(3) Formation of interacting substance structure using polymer E Except that polymer E was used as the water-soluble polymer, “(3) interacting substance on solid surface having succinimide group” in Example 4 Same as “Formation of structure”. As a result, an interacting substance structure having mouse IgG and polymer E as the interacting substance and water-soluble polymer on the surface of the gold chip was obtained.

<Capture and detection of target molecule by interacting substance structure obtained in Example 4 and Comparative Example 4>
As described above, the performance of the gold chip having the interacting substance structure produced in Example 4 and Comparative Example 4 on its surface was analyzed by the fluorescence intensity method. 2 ml of PBS (−) containing 3 wt% BSA was dropped onto the above interacting substance structure gold chip so as to cover the surface, and blocked at room temperature for 1 hour. After removing the solution, it was washed with water, and 1 ml of PBS (−) containing 50 μg / mL of Cy5-labeled Protein A as a target molecule was added so as to cover the surface of the gold chip on which the interacting substance structure was formed. Incubated for 1 hour. After removing the solution, washing was sufficiently performed using PBS (−) containing 0.05% Tween 20 for the purpose of removing excess target molecules not involved in the reaction. After further washing with water, the sample was dried with compressed air, and the fluorescence intensity of Cy5 near the center of the interactive substance structure was measured at an excitation wavelength of 635 nm using an InnoScan700 Microarray Scanner (manufactured by INNOPSYS). Note that MAPIX (manufactured by INNOPSYS) was used as analysis software.
Here, the production of Cy5-labeled Protein A was performed by the following method. The protein aqueous solution (RepliGen) buffer was replaced with a 100 mM carbonate buffer (pH 9.4) to prepare a Protein A concentration of 1 mg / ml. 1 ml of this 1 mg / ml Protein A solution was added to one Cy5 Mono-Reactive Dye Pack (manufactured by GE Healthcare) and mixed. This mixed solution was incubated at room temperature for 30 minutes. Thereafter, PD-10 Columns (manufactured by GE Healthcare) was used, PBS (−) was used as a developing solvent, and unreacted Cy5 was removed from the mixed solution to obtain Cy5-labeled Protein A.

  The results are shown in FIG. In FIG. 6, this example showed higher fluorescence intensity (Intensity) in the neutral region (Buffer pH 7.4) compared to Comparative Example 4. From this, it was shown that more target molecules can be captured by using the polymer D.

  Further, FIG. 6 shows a fluorescence image of Cy5 derived from the target molecule captured by the target molecule interaction substance-immobilized carrier. The interaction substance structure produced using the polymer D was confirmed to have Cy5 fluorescence inside the spot (hereinafter referred to as a region to which a mixture of the interaction substance and the water-soluble polymer was supplied). It can be seen that the structure was formed uniformly. However, the interaction substance structure (Comparative Example 4) produced using the polymer E has a very low fluorescence intensity inside the spot, and Cy5 fluorescence is strongly confirmed only around the spot in a ring shape. It can be seen that is formed unevenly.

  The target molecule-interacting substance-immobilized carrier of the present invention can be suitably used in the fields of medicine / diagnosis, gene analysis, proteomics, microelectronics, membrane separation and the like.

Claims (21)

A solid support;
A structure comprising a target molecule interacting substance and a water-soluble polymer which are bonded to each other and fixed to the solid phase carrier;
A target molecule interaction substance-immobilized carrier comprising:
The water-soluble polymer is a water-soluble polymer of a compound containing a spacer and a polymerizable group, and the target molecule-interacting substance captures the target molecule bonded to the water-soluble polymer through the spacer. A target molecule interaction substance-immobilized carrier for detection.   The water-soluble polymer is a water-soluble polymer of a compound in which a functional group for covalently bonding a target molecule interacting substance is bonded to a polymerizable group via a spacer, and the target molecule interacting substance is the functional group. The target molecule-interacting substance-immobilized carrier according to claim 1, which is bound to a water-soluble polymer via a group. The target molecule interaction substance immobilization support according to claim 1 or 2, wherein the spacer is represented by the following formula.
-(Y) _q-
Here, Y represents an alkyleneoxy group having 1 to 10 carbon atoms or an alkylene group, and q represents an integer of 1 to 20, but when q is an integer of 2 or more and 20 or less, the repeated Ys are the same. Or different. The target molecule interaction substance immobilization support according to any one of claims 1 to 3, wherein the polymerizable group is an acrylic group or a methacryl group. The target molecule-interacting substance-immobilized carrier according to claim 4, wherein the compound is an ethylenically unsaturated polymerizable monomer represented by the following general formula (II).

(Wherein R ₇ represents a hydrogen atom or a methyl group, and W represents a functional group for covalently bonding the target molecule interacting substance.) The water-soluble polymer is a copolymer of a compound containing a spacer and a polymerizable group and an unsaturated polymerizable monomer having a hydrophilic or amphiphilic functional group. Item 6. A target molecule interaction substance-immobilized carrier according to Item. The target molecule-interacting substance-immobilized carrier according to claim 6, which is produced by a method including at least the steps shown in the following (1) to (3);
(1) A water-soluble polymer is synthesized by copolymerizing an unsaturated polymerizable monomer having a hydrophilic or amphiphilic functional group and the ethylenically unsaturated polymerizable monomer represented by the general formula (II). The step (2) the step of mixing the water-soluble polymer obtained in the step (1) and the target molecule interacting substance,
(3) A step of supplying the mixture obtained in the step (2) to a solid phase surface having a functional group for covalently binding the functional group W or a functional group for covalently binding a target molecule interacting substance. . The target molecule according to claim 6 or 7, wherein the unsaturated polymerizable monomer having a hydrophilic or amphiphilic functional group is an ethylenically unsaturated polymerizable monomer having a phosphorylcholine-like group represented by the following formula (I): Interactive substance-immobilized carrier.

(In the formula, R ₁ represents a divalent hydrocarbon group having 2 to 10 carbon atoms, R ₂ represents a divalent hydrocarbon group having 1 to 4 carbon atoms, and R ₃ , R ₄ and R ₅ each represents Independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, and R ₆ represents a hydrogen atom or a methyl group.)   The proportion of structural units derived from an ethylenically unsaturated polymerizable monomer having a phosphorylcholine-like group represented by the general formula (I) with respect to the entire water-soluble polymer is 30 mol% or more, Item 9. The target molecule interaction substance-immobilized carrier according to Item 8.   The target molecule-interacting substance-immobilized carrier according to any one of claims 6 to 9, wherein the water-soluble polymer further includes a structural unit derived from an unsaturated polymerizable monomer having a hydrophobic unit.   The target molecule interaction substance immobilization support according to claim 10, wherein the unsaturated polymerizable monomer having a hydrophobic unit is n-butyl methacrylate. The method for producing a target molecule-interacting substance-immobilized carrier according to any one of claims 6 to 11, comprising at least the steps shown in (1) to (3) below:
(1) A water-soluble polymer is synthesized by copolymerizing an unsaturated polymerizable monomer having a hydrophilic or amphiphilic functional group and the ethylenically unsaturated polymerizable monomer represented by the general formula (II). Process,
(2) A step of mixing the water-soluble polymer obtained in the step (1) and the target molecule interacting substance,
(3) A step of supplying the mixture obtained in the step (2) to a solid phase surface having a functional group for covalently binding the functional group W or a functional group for covalently binding a target molecule interacting substance. . A target molecule interaction substance-immobilized carrier for capturing and detecting a target molecule, which is produced by a method including at least the steps shown in the following (1) to (3);
(1) Water-soluble by copolymerizing an ethylenically unsaturated polymerizable monomer having a phosphorylcholine-like group represented by the following general formula (I) and an ethylenically unsaturated polymerizable monomer represented by the following general formula (II) A step of synthesizing a polymer;

(Wherein R ₁ represents a divalent hydrocarbon group having 2 to 10 carbon atoms, R ₂ represents a divalent hydrocarbon group having 1 to 4 carbon atoms, and R ₃ , R ₄ and R ₅ are each independently selected. And represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, and R ₆ represents a hydrogen atom or a methyl group.)

(Wherein R ₇ represents a hydrogen atom or a methyl group, Y represents an alkyleneoxy group or alkylene group having 1 to 10 carbon atoms, W represents a functional group for covalently bonding the target molecule interacting substance, q Represents an integer of 1 to 20. When q is an integer of 2 or more and 20 or less, the repeated Ys may be the same or different.
(2) A step of mixing the water-soluble polymer obtained in the step (1) and the target molecule interacting substance,
(3) A step of supplying the mixture obtained in the step (2) to a solid phase surface having a functional group for covalently binding the functional group W or a functional group for covalently binding a target molecule interacting substance. .   The target molecule-interacting substance-immobilized carrier according to claim 13, which is produced by a method further comprising a step of drying the mixture supplied to the solid surface by the step (3).   In the step (1), the ethylenically unsaturated polymerizable monomer having a phosphorylcholine-like group represented by the general formula (I) is 30 mol% or more of the total monomers used for copolymerization. The target molecule interaction substance immobilization support according to claim 13 or 14.   The target molecule interaction substance immobilization according to any one of claims 13 to 15, which is produced by copolymerizing an ethylenically unsaturated polymerizable monomer further having a hydrophobic unit in the step (1). Carrier.   The target molecule-interacting substance-immobilized carrier according to claim 16, wherein the ethylenically unsaturated polymerizable monomer having a hydrophobic unit is n-butyl methacrylate. The method for producing a target molecule-interacting substance-immobilized carrier according to any one of claims 13 to 17, comprising at least the steps shown in the following (1) to (3);
(1) Water-soluble by copolymerizing an ethylenically unsaturated polymerizable monomer having a phosphorylcholine-like group represented by the following general formula (I) and an ethylenically unsaturated polymerizable monomer represented by the following general formula (II) A step of synthesizing a polymer;

(Wherein R ₁ represents a divalent hydrocarbon group having 2 to 10 carbon atoms, R ₂ represents a divalent hydrocarbon group having 1 to 4 carbon atoms, and R ₃ , R ₄ and R ₅ are each independently selected. And represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, and R ₆ represents a hydrogen atom or a methyl group.)

(Wherein R ₇ represents a hydrogen atom or a methyl group, Y represents an alkyleneoxy group or alkylene group having 1 to 10 carbon atoms, W represents a functional group for covalently bonding the target molecule interacting substance, q Represents an integer of 1 to 20. When q is an integer of 2 or more and 20 or less, the repeated Ys may be the same or different.
(2) A step of mixing the water-soluble polymer obtained in the step (1) and the target molecule interacting substance,
(3) A step of supplying the mixture obtained in the step (2) to a solid phase surface having a functional group for covalently binding the functional group W or a functional group for covalently binding a target molecule interacting substance. . The target molecule interaction substance immobilization carrier according to any one of claims 1 to 11 and 13 to 17, wherein the target molecule is bound via a target molecule interaction substance. The target molecule-interacting substance-immobilized carrier according to claim 19, wherein the target molecule is selected from the group consisting of proteins, peptides, nucleic acids, and low molecular weight compounds. A biochip comprising the target molecule-interacting substance-immobilized carrier according to claim 19 or 20.

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