JP4818056B2 - Structure having a binding functional group on a substrate for binding a capture molecule for capturing a target substance - Google Patents

Description

  The present invention relates to a structure including a binding functional group on a substrate for binding non-specific adsorption of a biomolecule and binding a capture molecule for capturing a target substance.

  Conventionally, interaction between molecules has been used to detect a target substance in a specimen. This interaction includes an interaction between two molecules where one molecule captures the other molecule. As such interaction, for example, antigen-antibody reaction, binding reaction between a molecule and its receptor (receptor), hybridization reaction between complementary nucleic acids, enzyme substrate binding reaction and the like are known. Using such an interaction, the target substance in the specimen can be detected. For example, a capture molecule capable of capturing a target substance is immobilized on the substrate surface, a reaction is performed by bringing a specimen containing the target substance into contact therewith, and the target substance is captured by the capture molecule on the substrate surface. In general, a method for detecting a target substance in a specimen by detecting s.

  When quantitatively measuring the target substance that interacts with the capture molecule immobilized on the substrate, depending on the nature of the substrate surface or the immobilization method, non-specific adsorption to the substrate surface is possible in addition to the target substance that interacts with the capture molecule. There is also a risk that the detected biomolecule may be detected at the same time. This causes a decrease in the minimum detection sensitivity in a sensor that requires a small amount of detection. Therefore, a method for detecting only the target substance while suppressing non-specific adsorption is required.

  Anal. Chem. 1996, 68, 490-497 discloses that a self-assembled (SAM) film is formed on a gold substrate surface with an alkanethiol whose end is modified with oligoethylene glycol. Furthermore, this document discloses that only a ligand molecule as a target substance can be detected while activating a part of the ethylene glycol end on the SAM membrane to immobilize a receptor molecule while preventing nonspecific adsorption. ing.

  However, since the SAM film is formed by the van der Waals force between molecules, if there is a curvature or unevenness of several nanometers on the substrate, the film may not be uniform or the SAM may not be formed. In addition, in the case of a substrate manufactured at a low cost, as shown in FIG. 1, as shown in FIG. There is a risk of falling into a state. In FIG. 1, 10 is a base material, 12 is an alkanethiol group, 14 is a target substance capture molecule, 16 is a target substance, and 18 is a non-specifically adsorbed substance (biomolecule). 29 is oligoethylene glycol.

  Japanese Patent Application Laid-Open No. 2004-264027 also discloses a structure in which a SAM film is formed on the surface of a gold substrate. In this publication, a heterobifunctional type polyethylene glycol (PEG) having a functional group that reacts with the end of the SAM film and a functional group for immobilizing the capture molecule at both ends is reacted with the SAM film, It is disclosed to immobilize capture molecules via

  However, if a functional molecule is to be immobilized on a SAM film composed of alkanethiol using a previously synthesized heterobifunctional PEG as a spacer, the steric hindrance of the polymer will occur at the stage of reacting PEG on the SAM film. This creates a gap. Therefore, as shown in FIG. 2, there is a possibility that small molecules (biomolecules) contained in the specimen may be adsorbed on the SAM film or the substrate. In FIG. 2, 10 is a substrate, 12 is an alkanethiol group, 14 is a capture molecule, 16 is a target substance, and 18 is a non-specific adsorption substance. 20 is a heterobifunctional polyethylene glycol (PEG).

  On the other hand, as a technique for effectively preventing non-specific adsorption to the substrate surface, a technique for preventing non-protein specific adsorption and cell adhesion by forming a brush-like polymer at a high density on the surface of a silicon substrate is known. This technique is disclosed in Biomacromolecules 2004, 5, 2308-2314. This document describes that a brush-like polymer having 2-methacryloyloxyethyl phosphorylcholine (hereinafter referred to as MPC) as a monomer is formed on a silicon surface with high density by atom transfer radical polymerization. However, this document does not devise fixing a functional molecule at the polymer molecule tip.

  Another method for suppressing non-specific adsorption is disclosed in JP-T-2003-516519. Japanese Patent Publication No. 2003-516519 discloses a technique in which a functionalized polymer grafted on a substrate is applied and a target substance capturing molecule is partially arranged on the functionalized polymer to suppress nonspecific adsorption by the grafted region. Is disclosed. However, it has not been disclosed to arrange a functional group that suppresses nonspecific adsorption on the side chain of the grafted region, and there is room for improvement to suppress nonspecific adsorption more fully.

As described above, there is a room for improvement in various methods for preventing nonspecific adsorption of biomolecules and capturing a target substance in a specific region on a substrate. The present invention provides a technique capable of detecting a target substance with high sensitivity by efficiently preventing non-specific adsorption and reliably capturing the target substance in a specific region on the substrate.
JP 2004-264027 A Special table 2003-516519 gazette Anal.Chem. 1996,68,490-497 Biomacromolecules 2004,5,2308-2314

A structure having a binding functional group on a substrate for binding a capture molecule for capturing the target substance from a sample containing the biomolecule and the target substance provided by the present invention,
A target substance- capturing polymer in which the base is bonded to one end of the main chain and the bonding functional group is bonded to the other end via a sulfur atom;
A suppressing polymer in which the base is bonded to one end of the main chain, and a suppressing functional group that suppresses adsorption of the biomolecule to the structure via a sulfur atom is bonded to the other end;
Have
A suppressive functional group that suppresses adsorption of the biomolecule to the structure is bound to both side chains of the target substance capturing polymer and the suppressor polymer ,
The density of the polymer on the substrate is in the range of 0.1 molecule / nm ² or more and 1.0 molecule / nm ² or less .

Further, a method for producing a structure provided with a binding functional group on a substrate for binding a capture molecule for capturing the target substance from a sample containing the biomolecule and the target substance provided by the present invention,
A polymer having a main chain in which one end is bonded to the surface of the substrate by polymerizing an unsaturated monomer having a side chain having a suppression functional group that suppresses the adsorption of the biomolecule to the structure from the base surface. A growing process;
Chain transfer containing a chain transfer agent containing the bonding functional group and a sulfur atom at the growth end of the main chain of the polymer obtained by the polymerization, and a chain transfer agent containing the suppression functional group and a sulfur atom React with the agent,
A target substance- capturing polymer in which the base is bonded to one end of the main chain and the bonding functional group is bonded to the other end via a sulfur atom;
A suppressing polymer in which the base is bonded to one end of the main chain, and a suppressing functional group that suppresses adsorption of the biomolecule to the structure via a sulfur atom is bonded to the other end;
Obtaining
I have a,
The density of the polymer on the substrate is in the range of 0.1 molecule / nm ² or more and 1.0 molecule / nm ² or less .

  In addition, the detection apparatus for detecting a target substance in a specimen provided by the present invention contacts a light source, a detection apparatus for detecting light, and a capture molecule for capturing the target substance in the specimen. And a detection device for detecting a target substance in a specimen by an optical method, wherein a capture molecule for capturing the target substance is bound to a binding functional group in the reaction area. The structure is arranged.

  The present inventors have found a structure suitable as a substrate for capturing a target substance having a high-density polymer layer having the ability to prevent nonspecific adsorption of biomolecules on the substrate.

  In this structure, a binding functional group for binding a capture molecule that captures a target substance is partially fixed to the other end of the polymer layer having one end fixed to the substrate, that is, the sample side end, and the binding functional group is fixed. A functional group for suppressing non-specific adsorption of biomolecules is fixed to a terminal that is not formed. Furthermore, in the structure of the present invention, a functional group for suppressing nonspecific adsorption is also bonded to the side chain of the polymer. By adopting this structure, as shown in FIG. 3, nonspecific adsorption molecules (biological molecules) can be obtained by both the nonspecific adsorption inhibiting functional group 26 fixed to the polymer end and the nonspecific adsorption inhibiting functional group 24 bonded to the polymer side chain. (Molecule) adsorption is effectively suppressed. Then, the target substance 16 can be effectively captured by the target substance capturing molecule 14 fixed to the binding functional group 28 fixed to the polymer terminal. As a result, as shown in FIG. 3, even when a substrate having a surface with a curvature or unevenness of several nanometers is used, the target substance can be captured with high efficiency while suppressing nonspecific adsorption of biomolecules. . When this structure is used for sensing a target substance, the target substance can be detected with high sensitivity.

  In FIG. 3, 10 is a substrate, 12 is an alkanethiol, and 22 is a polymer main chain. In the present invention, one end of the polymer is bonded to the substrate, not only when one end of the polymer is directly bonded to the surface of the substrate, but also another molecule or film is formed on the substrate, and one end of the polymer is bonded on the other. This includes cases where

  The present invention includes the following aspects.

  The structure of the present invention is a structure provided on a substrate with a binding functional group for binding a capture molecule for capturing a target substance, and one end of a polymer is bound to the substrate, The end has a binding functional group bound to it and a binding functional group that suppresses the adsorption of biomolecules to the structure, and the side chain of the polymer also suppresses the suppression. The functional group for use is bonded. As the functional group for suppression, hydroxyl group, methoxy group, ethoxy group, propoxy group, isopropoxy group, 2-hydroxyethyl group, 2-hydroxypropyl group, 2-hydroxyisopropyl group, 2-hydroxybutyl group, sulfonyl group, Phosphonyl group, amino group, methylamino group, ethylamino group, isopropylamino group, amide group, methylamide group, ethylamide group, isopropylamide group, pyrrolidone group, ethylene glycol group and polymers thereof, choline group, phosphatidylcholine group Can be used. As the bonding functional group, any of a carboxyl group, an aldehyde group, a succinimide group, a maleimide group, a glycidyl group, and an amino group can be used.

  The structure of the present invention can contain sulfur atoms between the other end of the polymer and the bonding functional group, and between the other end of the polymer and the suppressing functional group. As the polymer, a vinyl polymer compound can be used. The vinyl polymer compound has a hydroxyl group, methoxy group, ethoxy group, propoxy group, isopropoxy group, 2-hydroxyethyl group, 2-hydroxypropyl group, 2-hydroxyisopropyl group, 2-hydroxyl group on the side chain. Hydroxybutyl group, sulfonyl group, phosphonyl group, amino group, methylamino group, ethylamino group, isopropylamino group, amide group, methylamide group, ethylamide group, isopropylamide group, pyrrolidone group, ethylene glycol group and its polymer, choline Group or a phosphatidylcholine group-containing monomer polymer.

In the present invention, the density of the polymer on the substrate can be in the range of 0.1 molecule / nm ² or more and 1.0 molecule / nm ² or less. The average molecular weight of the polymer can be in the range of 500 or more and 100,000 or less.

  The present invention also includes a structure in which a capture molecule is bound to a binding functional group.

  The method for producing a structure of the present invention is a method for producing a structure having a binding functional group for binding a capture molecule that captures a target substance on a substrate, and is based on a predetermined position on the surface of the substrate. A step of polymerizing an unsaturated monomer having a suppression functional group in a side chain that suppresses adsorption of a biomolecule to the structure; and as a terminal of the polymerization, the binding functional group and the suppression functional group And a step of adding.

  In the present invention, a chain transfer agent containing a sulfur atom can be used as a terminal of polymerization when a bonding functional group and a suppression functional group are added.

  First, the structure of a structure having a binding functional group for binding a capture molecule for capturing a target substance of the present invention on a substrate will be described. In the present invention, non-specific adsorption refers to adsorption of a biomolecule that interacts with a capture molecule that captures a target substance on the structure of the present invention including the substrate surface and the polymer.

(Substrate)
The substrate in the present invention is a material capable of forming a polymer in a brush shape at a high density on the surface of the substrate. It should be noted that some molecule or film may be formed on the surface of the substrate, and a polymer may be formed thereon. The material of the substrate in the present invention may be any material as long as it can form the structure of the present invention. Preferably, a metal such as gold, silver, copper, platinum or aluminum to which an amino group or thiol group can be bonded, a semiconductor such as CdS or ZnS, a metal oxide such as titanium oxide or aluminum oxide, or a silanol group is bonded. Possible glass, silicon, titanium oxide, ceramic, ceramic capable of binding a carboxyl group, carbon and the like. Moreover, the plastic which can show a carboxyl group by oxidizing the surface by oxygen plasma treatment, UV treatment, etc. may be used. The above reason is deeply related to the method of forming the polymer on the substrate surface. The method for forming the polymer will be described later. Further, the shape of the substrate in the present invention may be any shape such as a flat plate, a particle, or a microstructure.

(polymer)
In the structure of the present invention, one end of the polymer molecule is fixed to the substrate surface. The other end of the polymer coexists with a binding functional group for binding a capture molecule and a binding functional group for suppressing nonspecific adsorption of a biomolecule. And the side chain of each molecule | numerator of a polymer has the functional group for suppression which suppresses nonspecific adsorption | suction of a biomolecule. By fixing these polymers to the substrate, a non-specific adsorption preventing layer made of these polymers is formed. Examples of the polymer that can be used in the present invention include vinyl polymer compounds. The vinyl polymer compound has a hydroxyl group, methoxy group, ethoxy group, propoxy group, isopropoxy group, 2-hydroxyethyl group, 2-hydroxypropyl group, 2-hydroxyisopropyl group, 2-hydroxy group on the side chain. Butyl group, sulfonyl group, phosphonyl group, amino group, methylamino group, ethylamino group, isopropylamino group, amide group, methylamide group, ethylamide group, isopropylamide group, pyrrolidone group, ethylene glycol group and its polymer, choline group A polymer of a monomer having any of phosphatidylcholine groups can be employed.

The density of the polymer on the substrate is in the range of 0.1 molecule / nm ^{2 to} 1.0 molecule / nm ² . To fix the polymer molecule at one terminal to the substrate, a method in which a polymer already synthesized is brought into contact with the substrate surface and fixed may be used. Preferably, a method of polymerizing the polymer on the substrate is used. preferable. Living radical polymerization is particularly preferred as the polymerization method on the substrate. Living radical polymerization will be described later. The density of the polymer formed by such a polymerization method varies depending on the side chain, but is generally about 0.5 molecule / nm ^{2 in} the case of a polymer composed of a small molecule monomer, but it is much higher. May be. Moreover, the average molecular weight of a preferable polymer is 500 or more and 100,000 or less, More preferably, it is 1000 or more and 10,000 or less. The film thickness of the resulting polymer film is 5 nm or more, more preferably 10 nm or more and 100 nm or less.

  The polymer of the present invention comprises a step of polymerizing an unsaturated monomer having a side chain with a functional group for suppression that suppresses adsorption of biomolecules to the structure based on a predetermined position on the surface of the substrate; And a step of adding a functional group for bonding and a functional group for suppression.

  The polymer of the structure of the present invention can be formed on a substrate by polymerization to form a polymer layer so as to fill a gap according to the unevenness even when there are unevenness of about several nm on the flat plate. Is possible. In the case where the shape of the substrate is a particle or a microstructure, it is difficult to fix the functional molecule while forming a thin polymer layer in a minute space on the surface of the substrate by the conventional method. However, in the present invention, it is possible to form a high-density polymer layer along the surface shape having minute changes in the substrate.

  The polymer of the present invention includes a polymer having a binding functional group for binding a capture molecule to the liquid contact end (analyte side end) of the polymer molecule, and a functional group having a nonspecific adsorption inhibiting ability at the liquid contact end. And a polymer to which is bound. That is, the polymer is formed so that both a polymer molecule in which the functional group for binding is introduced at the end on the wetted side and a polymer molecule in which a functional group having nonspecific adsorption inhibiting ability is introduced at the end on the wetted side are obtained. Is done. In order to fix two or more kinds of binding functional groups or to introduce two or more kinds of functional groups having nonspecific adsorption inhibiting ability, two or three kinds of polymers are present on the substrate. You may do it. When two or more types of capture molecules are immobilized, functional groups for immobilizing separate capture molecules may be introduced, or different capture molecules may be immobilized using the same functional group. Further, these two or more types of polymers may be polymers each composed of a plurality of monomers. A method for immobilizing a capture molecule and a method for introducing a functional group having nonspecific adsorption inhibiting ability will be described later.

(Living radical polymerization)
Generally, according to living radical polymerization, the molecular weight distribution of a polymer to be synthesized is small, and a polymer layer can be grafted at a high density on a substrate. Therefore, if living radical polymerization is performed on an unsaturated monomer having a functional group having nonspecific adsorption inhibiting ability in the side chain, a high density nonspecific adsorption inhibiting layer can be provided on the substrate. In addition, after the polymerization main chain is grown from the substrate surface as a starting point to obtain a predetermined length (molecular weight), the functional group is introduced into the liquid contact end, and the active group is subjected to steric hindrance of the polymer. It is possible to fix without. Examples of the living radical polymerization method include a method using any of the following polymerizations.
(1) Atom transfer radical polymerization (ATRP) using an organic halide as an initiator and a transition metal complex as a catalyst.
(2) Nitroxide-mediated polymerization (NMP) using a radical scavenger such as a nitroxide compound.
(3) Photoinitiator polymerization using a radical scavenger such as dithiocarbamate.
In the present invention, the functional structure may be produced by any method, but it is preferable to utilize atom transfer radical polymerization for ease of control.

(Atom transfer radical polymerization)
When the living radical polymerization is atom transfer radical polymerization, an organic halide as shown in Chemical Formulas 1 to 3 or a sulfonyl halide compound as shown in Chemical Formula 4 can be used as a polymerization initiator.

  After adding the substrate into which the atom transfer radical polymerization initiator has been introduced to the reaction solvent, the unsaturated monomer and transition metal complex to be used as a non-specific adsorption prevention layer are added, and the reaction system is replaced with an inert gas to replace the atom transfer radical. Polymerize. This allows the polymerization to proceed while keeping the graft density constant. That is, polymerization can proceed like a living room, and all non-specific adsorption preventing layers can be grown almost uniformly on the substrate.

  The reaction solvent is not particularly limited. Isopropyl cellosolve, butyl cellosolve, acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, ethyl acetate, butyl acetate, ethyl propionate, trioxane, tetrahydrofuran and the like can be used. These may be used alone or in combination of two or more.

  Nitrogen gas or argon gas can be used as the inert gas.

  The transition metal complex used consists of a metal halide and a ligand. The metal species of the metal halide is preferably a transition metal from Ti of atomic number 22 to Zn of 30, and more preferably Fe, Co, Ni, and Cu. Among these, cuprous chloride and cuprous bromide are preferable.

  The ligand is not particularly limited as long as it can be coordinated to a metal halide. For example, 2,2′-bipyridyl, 4,4′-di- (n-heptyl) -2,2′-bipyridyl, 2- (N-pentyliminomethyl) pyridine, (-)-sparteine, tris (2-dimethylaminoethyl) amine, ethylenediamine, dimethylglyoxime, 1,4,8,11-tetramethyl-1,4,8,11 -Tetraazacyclotetradecane, 1,10-phenanthroline, N, N, N ′, N ″, N ″ -pentamethyldiethylenetriamine, hexamethyl (2-aminoethyl) amine and the like can be used.

  The addition amount of the transition metal complex is 0.001 to 10% by weight, preferably 0.05 to 5% by weight, based on the unsaturated monomer serving as the nonspecific adsorption preventing layer.

  The polymerization temperature is in the range of 40 ° C to 100 ° C, preferably in the range of 50 ° C to 80 ° C.

  Moreover, when performing polymerization, it is preferable to add a free polymerization initiator not fixed to the substrate to the reaction system. The free polymer produced from the free polymerization initiator can be used as an indicator of the molecular weight and molecular weight distribution of the non-specific adsorption preventing layer grafted to the substrate.

  As the free polymerization initiator, it is preferable to select the same type as the atom transfer radical polymerization initiator fixed to the substrate. That is, it is preferable that the free polymerization initiator is ethyl 2-bromoisobutyrate with respect to the polymerization initiator of Chemical Formula 1 (X = Br). Moreover, it is preferable that a free polymerization initiator is ethyl 2-bromopropionate with respect to the polymerization initiator of Chemical formula 2 (X = Br).

  After completion of the polymerization, the substrate can be separated and purified by an appropriate method such as filtration, decantation, fractionation, centrifugation, etc. to obtain a substrate on which the nonspecific adsorption preventing layer is grafted.

(Nitroxide-mediated polymerization)
When the living radical polymerization is nitroxide-mediated polymerization, a nitroxide compound represented by Chemical Formulas 5 to 7 can be used as a polymerization initiator.

  After adding the substrate into which the nitroxide-mediated polymerization initiator has been introduced to the reaction solvent, an unsaturated monomer serving as a non-specific adsorption suppression layer is added, and the reaction system is replaced with an inert gas to perform nitroxide-mediated polymerization. This allows the polymerization to proceed while keeping the graft density constant. That is, polymerization can proceed like a living room, and all non-specific adsorption suppressing layers can be grown almost uniformly on the substrate.

  Although it does not specifically limit as a reaction solvent, The above-mentioned similar solvent can be used. Moreover, you may use them individually or may use 2 or more types together. Nitrogen gas or argon gas can be used as the inert gas. The polymerization temperature is in the range of 40 ° C to 120 ° C, preferably in the range of 50 ° C to 100 ° C. When the polymerization temperature is less than 40 ° C., the formed non-specific adsorption suppressing layer has a low molecular weight, or polymerization is difficult to proceed, which is not preferable. Moreover, when performing superposition | polymerization, it is preferable to add the free polymerization initiator which is not fixed to the base | substrate. The free polymer produced from the free polymerization initiator can be used as an index of the molecular weight and molecular weight distribution of the nonspecific adsorption suppression layer grafted to the substrate. As the free polymerization initiator, it is preferable to select the same type as the nitroxide-mediated polymerization initiator fixed to the substrate. That is, it is preferable that the free polymerization initiator is a nitroxide compound represented by Chemical Formula 8 with respect to the polymerization initiator represented by Chemical Formula 5.

  After completion of the polymerization, the substrate can be separated and purified by an appropriate method such as filtration, decantation, fractionation, centrifugation, etc. to obtain a substrate on which the nonspecific adsorption preventing layer is grafted.

(Photoinitiator polymerization)
When the living radical polymerization is photoinitiator polymerization, an N, N-dithiocarbamine compound as shown in Chemical Formula 9 can be used as a polymerization initiator.

  After adding the substrate into which the photoinitiator polymerization initiator has been introduced to the reaction solvent, an unsaturated monomer that becomes a non-specific adsorption suppression layer is added, the reaction system is replaced with an inert gas, and light irradiation is performed. Perform ether polymerization. This allows the polymerization to proceed while keeping the graft density constant. That is, polymerization can proceed like a living room, and all non-specific adsorption suppressing layers can be grown almost uniformly on the substrate.

  Although it does not specifically limit as a reaction solvent, The above-mentioned similar solvent can be used. Moreover, you may use them individually or may use 2 or more types together.

  Nitrogen gas or argon gas can be used as the inert gas.

  The wavelength of the irradiated light varies depending on the type of the photoinitiator polymerization initiator used. In the case of grafting a non-specific adsorption suppression layer on the surface of a substrate having a photoinitiator polymerization initiator exemplified in Chemical Formula 9, photoinitiator polymerization is improved by irradiating the reaction system with light having a wavelength of 300 nm to 600 nm. proceed.

  The polymerization temperature is preferably room temperature or lower in order to suppress side reactions. However, the temperature range is not limited within a range in which the same effect can be obtained.

  Moreover, when performing superposition | polymerization, it is preferable to add the free polymerization initiator which is not fixed to the base | substrate. The free polymer produced from the free polymerization initiator can be used as an index of the molecular weight and molecular weight distribution of the nonspecific adsorption suppression layer grafted to the substrate.

  As the free polymerization initiator, it is preferable to select the same type as the photoinitiator polymerization initiator fixed to the substrate. That is, it is preferable that the free polymerization initiator is a dithiocarbamate compound represented by Chemical Formula 10 with respect to the polymerization initiator represented by Chemical Formula 9.

  After completion of the polymerization, the substrate can be separated and purified by an appropriate method such as filtration, decantation, precipitation fractionation, and centrifugal separation to obtain a substrate grafted with the nonspecific adsorption suppressing layer.

  The method for fixing the polymerization initiator to the substrate surface is not particularly limited. If the substrate is a metal, a polymerization initiator containing a thiol compound is bound to the substrate surface, or the substrate is bonded with a thiol compound. A method in which a polymerization initiator is subsequently bonded after pretreatment is preferred.

  If the substrate is a metal having an oxide film, there is a method in which a polymerization initiator containing a silane coupling agent is bonded, or after the substrate is pretreated with a silane coupling agent, the polymerization initiator is subsequently bonded. preferable.

  If the substrate is plastic, the surface is oxidized by oxygen plasma treatment, UV treatment, etc. to express carboxyl groups, and then a polymerization initiator containing an amino compound is bound or pretreated with an amino compound, followed by A method of binding a polymerization initiator is preferred.

(Chain transfer agent)
Each polymer molecule constituting the polymer layer of the structure of the present invention has one end fixed to the substrate, and the main chain end on the liquid contact side opposite to the side fixed to the substrate has a capture molecule. A functional group for binding or a functional group that suppresses non-specific adsorption is introduced. The capturing molecule is introduced into the liquid contact end by a method in which a binding functional group is arranged at the end of the main chain and the capturing molecule is bound to this functional group. On the other hand, the introduction of a functional group that suppresses nonspecific adsorption to the wetted end is performed by a method of stopping the progress by placing a functional group that suppresses nonspecific adsorption at the wetted end of the polymer main chain. be able to. For these introductions, radical polymerization using different types of chain transfer agents is suitable.

  A chain transfer agent is a substance that moves an active site of a reaction by a chain transfer reaction in a radical polymerization reaction, and is used when it is desired to convert a polymerization terminal to a desired functional group. In a preferred embodiment of the present invention, two or more chain transfer agents are used for living radical polymerization in order to introduce a functional group for immobilizing a capture molecule and a functional group for suppressing nonspecific adsorption of a biomolecule. Used in the process. The chain transfer agent is preferably a thiol compound. This clarifies that the polymer is formed by radical polymerization in the structure of the present invention. As the thiol compound effective as a chain transfer agent, one having a thiol group at one end of an alkyl chain having two or more carbon chains and a desired functional group at the other end is used. Examples of the functional group for fixing the functional molecule include a carboxyl group, an aldehyde group, a succinimide group, a maleimide group, a glycidyl group, and an amino group.

  On the other hand, examples of the functional group for suppressing nonspecific adsorption include a hydroxyl group, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a 2-hydroxyethyl group, a 2-hydroxypropyl group, and a 2-hydroxyisopropyl group. 2-hydroxybutyl group, sulfonyl group, phosphonyl group, amino group, methylamino group, ethylamino group, isopropylamino group, amide group, methylamide group, ethylamide group, isopropylamide group, pyrrolidone group, ethylene glycol group and their heavy Examples include coalescence, choline group, and phosphatidylcholine group. Alternatively, a hydrophilic molecule such as a sugar may be bound later. Of course, the combination of the functional group for immobilizing the functional molecule and the functional group for preventing non-specific adsorption must be different.

  Here, when only a functional group for immobilizing a capture molecule is introduced to the liquid contact end of all polymer molecules using one type of chain transfer agent, multiple locations within the same capture molecule are attached to multiple polymer ends. In some cases, they are coupled and fixed to each other. Then, the capturing structure may be destroyed and the activity may be reduced. Moreover, when only a functional group for suppressing nonspecific adsorption is introduced into the liquid contact end of all polymer molecules, functions such as sensing of a biological substance as a target substance cannot be imparted to the structure.

According to the present invention, it is possible to introduce fewer functional groups that immobilize the capture molecule with respect to the area occupied by the capture molecule described later. More preferably, there is one functional group for the area occupied by the capture molecule. The terminal functional group around the periphery is a functional group that suppresses non-specific adsorption. As a result, the trapping molecules can exhibit their original activity by binding at a smaller number of sites per molecule, preferably by binding at one point. As described above, the ratio of the functional group for immobilizing the capture molecule necessary for exerting the activity is determined by the ratio of the chain transfer agent to be added. That is, it is preferable to grasp the occupation area of one molecule of polymer (reciprocal of polymer density, αnm ² / molecule) and the occupation area of one molecule of capture molecule (βnm ² / molecule). And it is preferable to add so that the ratio (B%) of the functional group for immobilizing the capture molecule at the liquid contact end of the polymer may be (formula) B = α / β × 100 (%). That is, of the two or more chain transfer agents added at the end of the reaction, the ratio of the functional group that fixes the capture molecule is determined by the above formula. Specifically, the ratio of the chain transfer agent having a functional group for immobilizing the capture molecule is preferably 0.1% to 50% of all chain transfer agents at the time of addition. For example, if the capture molecule is an antibody having a molecular weight of about 150 kD on a molar basis, the ratio is more preferably 0.1% to 10%, and further preferably 0.3% to 3%. Further, when the capture molecule is an antibody fragment having a molecular weight of about 25 kD, the ratio is more preferably 0.5% to 50%, and further preferably 2% to 20%. Furthermore, when the capture molecule is a small molecule (for example, biotin) having a molecular weight of about 400, the ratio is more preferably 1% to 50%, and further preferably 10% to 50%. When the said ratio is lower than said each range, there exists a possibility that the said functional group may not react or a capture molecule may not be fixed. Moreover, when the said ratio is larger than said each range, we are anxious about the nonspecific adsorption | suction suppression effect becoming low.

  In the structure of the present invention, a functional group that suppresses non-specific adsorption at a molecular terminal to which a binding molecule on the liquid contact surface (surface opposite to the substrate surface) of the polymer layer provided on the substrate is not fixed. Are combined. This eliminates the need for blocking to prevent non-specific adsorption after capture molecule fixation.

(Capture molecule)
The capture molecule in the present invention is a molecule that interacts with a target substance and selectively (specifically) captures the target substance. Specific examples of such molecules include nucleic acids, proteins, sugar chains, lipids, and complexes thereof. More specifically, DNA, RNA, aptamer, gene, chromosome, cell membrane, virus, antigen, antibody, antibody fragment, lectin, hapten, hormone, receptor, enzyme, peptide, sphingosaccharide, sphingolipid, etc. It is not limited to these. Preferably, it is an antibody, an antibody fragment, or an enzyme capable of capturing a biological substance or converting its structure and properties.

  With the recent development of bioinformatics, the structure and size of biomolecules such as proteins can be assumed from their amino acid sequences and the like. If the density of the polymer to be graft polymerized is determined, it is possible to easily simulate how many polymers exist for one protein molecule immobilized on the brush-like polymer molecule. Two or more kinds of chain transfer agents mixed based on the calculation result are reacted with the polymer terminal to fix the protein to the active group. The immobilized protein is immobilized only at about one point, and all of the non-binding groups of the polymer located in the periphery, that is, the lower part of the protein, use functional groups having a high non-specific adsorption inhibiting ability.

(Immobilization of capture molecules)
In the present invention, the method for immobilizing the capture molecule is not particularly limited, but is preferably a method capable of taking a covalent bond.

(Functional group capable of suppressing nonspecific adsorption of biomolecules)
In the present invention, the functional group having the ability to suppress nonspecific adsorption of biomolecules is a functional group at the liquid contact end and side chain of the polymer and prevents nonspecific adsorption of biomolecules to the substrate surface and structure. Alternatively, any material may be used as long as it is suppressed. Specifically, hydroxyl group, methoxy group, ethoxy group, propoxy group, isopropoxy group, 2-hydroxyethyl group, 2-hydroxypropyl group, 2-hydroxyisopropyl group, 2-hydroxybutyl group, sulfonyl group, phosphonyl group , Amino groups, methylamino groups, ethylamino groups, isopropylamino groups, amide groups, methylamide groups, ethylamide groups, isopropylamide groups, pyrrolidone groups, ethylene glycol groups and polymers thereof, choline groups, phosphatidylcholine groups are preferred. .

  The following can be considered as one of the causes of nonspecific adsorption of protein when the substrate is brought into contact with an aqueous protein solution. When the substrate and the aqueous protein solution come into contact with each other, the different molecules in the substrate break the hydrogen bonds between the water molecules, so that the protein cannot retain its structure, and the exposed part of the hydrophobic part inside the molecule is adsorbed by the different molecules. To do. On the other hand, PEG and MPC are known as functional groups having a high ability to prevent nonspecific adsorption of proteins. It is also known that when these come into contact with an aqueous protein solution, hydrogen bonds between water molecules are maintained relatively stably, and at the same time, the protein is stabilized. The above-mentioned contents are described in detail in Polymer-Water Interface-Approach from Structural Analysis of Water-, Polymer, Vol. 52, January (2003). From such a viewpoint, the functional group that retains hydrogen bonds between water molecules can be used as a functional group having the ability to suppress nonspecific adsorption of biomolecules in the present invention.

  Below, the detection apparatus concerning this invention is demonstrated.

  The detection device of the present invention includes a light source, a detection device for detecting light, and a reaction region for bringing the sample into contact with a capture molecule for capturing a target substance in the sample. A detection apparatus for detecting a target substance therein, wherein the structure of the present invention in which a capture molecule for capturing a target substance is bound to a binding functional group is disposed in the reaction region. .

  The detection apparatus of the present invention is an apparatus in which a structure having a polymer layer with nonspecific adsorption suppressing ability on the surface of a substrate and further having a capture molecule arranged on the surface thereof is arranged in the reaction region.

  Below, the detection method and target substance concerning this invention are demonstrated.

(Detection method)
In the present invention, it is preferable to detect these optically as means for detecting a physical and / or chemical amount change in a target substance in a specimen that reacts with a capture molecule. Examples thereof include a fluorescence method, an electrochemiluminescence method, and a plasmon resonance method. In the case of using the fluorescence method or the electrochemiluminescence method, the target substance concentration can be obtained based on the light amount value, so that a detection mechanism having a quantitative function can be simplified. Further, when the plasmon resonance method is used, a physical change during the reaction can be detected, so that the progress of the reaction process can also be used as a parameter for determining the target substance concentration. Further, when such a physical change is measured, since no label is required, the number of reaction steps in the reaction region is reduced, and the time required for detection can be shortened. As the detection means using the plasmon resonance method, there are a surface plasmon resonance method (SPR) and a localized plasmon resonance method (LPR).

(Target substance)
The target substance of the present invention may be any substance as long as it reacts with the capture molecule. More preferably, it is a biological material. Biological materials include biological materials selected from nucleic acids, proteins, sugar chains, lipids, and complexes thereof, and more specifically, biomolecules selected from nucleic acids, proteins, sugar chains, and lipids. Is. More specifically, a substance selected from any of DNA, RNA, aptamer, gene, chromosome, cell membrane, virus, antigen, antibody, lectin, hapten, hormone, receptor, enzyme, peptide, sphingosaccharide, and sphingolipid. The present invention can be applied as long as it is included. Furthermore, bacteria and cells themselves that produce the above-mentioned “biological substances” can also be target substances as “biological substances” targeted by the present invention.

  The interaction between the target substance and the capture molecule may be any interaction as long as the physical / chemical change before and after the binding can be detected by the element of the present invention. Preferably, “antigen-antibody reaction”, “antigen-aptamer (RNA fragment having a specific structure)” “ligand-receptor interaction”, “DNA hybridization” “DNA-protein (transcription factor etc.) interaction”, “ Lectin-sugar chain interaction ”and the like.

  Samples used when the structure of the present invention is used for detection or quantification of a target substance in a sample include various samples to be analyzed and processed products obtained by pre-processing various samples as necessary. . As the pretreatment, for example, when a biological tissue piece or blood is used as a specimen, and the target substance is contained in the specimen, a solution containing the target substance and capable of reacting with the functional structure is obtained. For the purpose.

Example 1 (SPR detection of antigen-antibody reaction)
After depositing 3 nm of chromium on an SF10 transparent glass substrate (thickness 0.3 mm, 12 mm × 10 mm), gold is evaporated 45 nm. The deposition thickness is monitored with a crystal oscillator. The substrate on which gold is deposited is immersed in a piranha solution (sulfuric acid: nitric acid = 7: 3) to remove impurities on the substrate surface.

The substrate is fixed to a Schlenk tube for reaction, 8-mercapto-1-octanol is added, and the mixture is gently stirred to form SAM. After removing the solution, the substrate is washed with dichloromethane, ethyl 2-isobutyric acid bromide is added as a polymerization initiator, and the atom transfer radical polymerization initiator is fixed to the substrate surface. Further, ethyl 2-bromoisobutyrate is added as a free polymerization initiator, and CuBr, 2,2′-bipyridyl, and methanol are added. After removing oxygen in the Schlenk tube by freeze vacuum degassing, the reaction system is replaced with nitrogen, and HEMA (2-hydroxyethyl methacrylate) monomer is reacted for a predetermined time by atom transfer radical polymerization. By adding a large amount of mercaptoacetic acid: mercaptoethanol = 1: 100 (molar ratio) in the reaction system, a carboxyl group and a hydroxyl group are mixed at the terminal of the graft polymer on the substrate. The film thickness of the substrate produced by the above operation is measured by the spectroscopic ellipsometry method, and is calculated from the weight of the substrate that can be measured with a precision gravimetric meter. As a result, the polymer density on the substrate surface is 0.5 / nm. It turns out that it is ² . Furthermore, it is confirmed that a chain transfer agent has been introduced by detecting S atoms using X-ray photoelectron spectroscopy. Then, using a time-of-flight secondary ion mass spectrometer, it is confirmed that two or more types of polymer terminals are formed by detecting ions derived from mercaptoacetic acid and mercaptoethanol bonded to the polymer terminals. . SPR measurement is performed using a substrate having a PHEMA layer surface.

The substrate surface is reacted with water-soluble carbodiimide (WSC) and N-hydroxysuccinimide (NHS) to convert the carboxyl group at the end of the PHEMA layer into an active ester group. The antibody is immobilized by reacting a phosphate buffer solution in which an anti-bovine serum albumin antibody (hereinafter, Anti-BSA) is dissolved. The area required by the antibody is 17.5 × 11.5 nm according to Journal of Molecular Biology 221 (2): 361-5 (1991). This is equivalent to 12 ng / mm ² , that is, equivalent to 1 molecule / 200 nm ^{2 when the} SPR signal obtained by antibody immobilization is converted to weight.

Subsequently, an antigen-antibody reaction is carried out by reacting a phosphate buffer solution in which bovine serum albumin (molecular weight 66000, hereinafter referred to as BSA) is dissolved, and the SPR signal obtained is equivalent to 5 ng / mm ² when converted to weight, that is, BSA1 molecule / 200 nm ² . In addition, when a chicken egg white lysozyme (molecular weight: 15000, hereinafter HEL) solution is contacted instead of BSA, there is almost no SPR signal, and thus an antigen-antibody reaction can be detected with a very high S / N ratio. Yes.

Comparative Example 1 (SPR detection of antigen-antibody reaction)
After removing impurities on the substrate surface in the same manner as in Example 1, the substrate was fixed to a Schlenk tube for reaction, and 7-carboxy-1-heptanethiol and 8-hydroxy-1-octanethiol were used at a ratio of 1: 100. Mix and add. Further dichloromethane is added and gently stirred to form SAM. After removing the solution, the substrate is washed with dichloromethane, and the substrate surface is reacted with water-soluble carbodiimide (WSC) and N-hydroxysuccinimide (NHS) to convert the carboxyl group on the SAM surface into an active ester group. The antibody is fixed by reacting a phosphate buffer solution in which Anti-BSA is dissolved. Subsequently, an antigen-antibody reaction is performed by reacting the BSA solution, and the obtained SPR signal is equivalent to 2 ng / mm ² when converted to weight, that is, BSA1 molecule / 500 nm ² . Further, when a HEL solution is brought into contact instead of BSA, there is almost no SPR signal.

Comparative Example 2 (SPR detection of antigen-antibody reaction)
After removing impurities on the substrate surface in the same manner as in Example 1, the substrate was fixed to a Schlenk tube for reaction, and 8-amino-1-octanethiol hydrochloride and dichloromethane were added and gently stirred to form SAM. . After removing the solution, it was washed with dichloromethane, and a heterobifunctional polyethylene glycol (NHS-PEG-MAL, manufactured by NOF Corporation) solution having a succinimide (NHS) group and a maleimide (MAL) group at the terminal with a molecular weight of 3,400. React. Since the amino group on the SAM surface reacts with the NHS group of NHS-PEG-MAL and the MAL group remains unreacted, a maleimide group can be introduced to the surface via PEG. The antibody is fixed by reacting a phosphate buffer solution in which Anti-BSA is dissolved. Subsequently, an antigen-antibody reaction is performed by reacting with a BSA solution, and the obtained SPR signal is equivalent to 5 ng / mm ² when converted to weight, that is, BSA1 molecule / 200 nm ² . When the HEL solution is brought into contact instead of BSA, the SPR signal increases. When the increase is converted to weight, it is equivalent to 0.1 ng / mm ² , that is, HEL 1 molecule / 2000 nm ² , and it is confirmed that proteins other than the antigen are non-specifically adsorbed.

Comparative Example 3 (Confirmation of nonspecific adsorption preventing effect in SPR)
After removing impurities on the substrate surface in the same manner as in Example 1, the substrate is fixed to a Schlenk tube for reaction, 8-mercapto-1-octanol and dichloromethane are added, and the mixture is gently stirred to form SAM. After removing the solution, the substrate is washed with dichloromethane, ethyl 2-isobutyric acid bromide is added as a polymerization initiator, and the atom transfer radical polymerization initiator is fixed to the substrate surface. Further, ethyl 2-bromoisobutyrate is added as a free polymerization initiator, and CuBr, 2,2′-bipyridyl, and methanol are added. After removing oxygen in the Schlenk tube by freeze vacuum degassing, the reaction system is replaced with nitrogen, and HEMA (2-hydroxyethyl methacrylate) monomer is reacted for a predetermined time by atom transfer radical polymerization. The HEMA monomer is reacted with ATRP for 3 hours, and a hydroxyl group is presented at the end of the graft polymer on the substrate by adding a large amount of mercaptoethanol. After confirming the composite as in Example 1, the SPR signal obtained by adding Anti-BSA is below the detection limit of the device. Furthermore, the SPR signal obtained by flowing the BSA antigen is similarly below the detection limit of the apparatus.

Comparative Example 4 (SPR detection of antigen-antibody reaction)
The same procedure as in Example 1 is followed until the anti-BSA antibody is fixed. In addition to this, by adding ethanolamine, when an unreacted succinimide group is present on the polymer surface, blocking is performed to inactivate it. Thereafter, an antigen-antibody reaction is performed by adding BSA antigen, and the obtained SPR signal is equivalent to 5 ng / mm ² when converted to weight. As a result of the above, the same results as in Example 1 were obtained, and it is understood that the ethanolamine addition operation for blocking nonspecific adsorption in this comparative example is unnecessary.

Comparative Example 5 (SPR detection of antigen-antibody reaction, use of polymer without side chain)
After removing impurities on the substrate surface in the same manner as in Example 1, the substrate is fixed to a Schlenk tube for reaction, and 8-mercapto-1-octanol and dichloromethane are added and gently stirred to form a SAM. After removing the solution, the substrate is washed with dichloromethane, ethyl 2-isobutyric acid bromide is added as a polymerization initiator, and the atom transfer radical polymerization initiator is fixed to the substrate surface. Further, ethyl 2-bromoisobutyrate is added as a free polymerization initiator, and CuBr, 2,2′-bipyridyl, and methanol are added. After removing oxygen in the Schlenk tube by freeze vacuum degassing, the reaction system is replaced with nitrogen, and the ethylene monomer is reacted for a predetermined time by atom transfer radical polymerization. By adding a large amount of mercaptoacetic acid: mercaptoethanol = 1: 100 (molar ratio) in the reaction system, a carboxyl group and a hydroxyl group are mixed at the terminal of the graft polymer on the substrate.

The substrate surface is reacted with water-soluble carbodiimide (WSC) and N-hydroxysuccinimide (NHS) to convert the carboxyl group at the end of the PHEMA layer into an active ester group. The antibody is fixed by reacting a phosphate buffer solution in which Anti-BSA is dissolved. When the SPR signal obtained by antibody immobilization is converted to weight, it corresponds to approximately 1.2 ng / mm ² , that is, approximately 5 × 10 ³ molecules / μm ² . Subsequently, when the BSA solution is reacted under the same conditions as in Example 1, the SPR signal increases. When the obtained SPR signal is converted to weight, it is equivalent to approximately 0.5 ng / mm ² , that is, approximately BSA 5 × 10 ³ molecules / μm ² . From this, it is confirmed that the antigen-antibody reaction is detected. Further, when a HEL solution is brought into contact instead of BSA, the SPR signal increases. When the increase is converted into weight, it is equivalent to approximately 0.01 ng / mm ² , that is, approximately HEL 4 × 10 ² molecules / μm ² . This confirms that non-antigen proteins are adsorbed nonspecifically.

Example 2 (Preparation of fine particles for LPR detection)
A gold fine particle solution having an average particle diameter of 40 nm, mercaptoethanol, and water are added to a centrifuge tube, and gently stirred to adsorb mercaptoethanol on the surface of the fine particles. The reaction solution is removed by centrifuging with dichloromethane three times. After adding gold fine particles having SAM formed on the surface to the Schlenk tube for reaction, ethyl 2-isobutyric acid bromide is added as a polymerization initiator, and the atom transfer radical polymerization initiator is fixed to the substrate surface. Further, ethyl 2-bromoisobutyrate is added as a free polymerization initiator, and CuBr, 2,2′-bipyridyl, and methanol are added. After removing oxygen in the Schlenk tube by freeze vacuum degassing, the reaction system is replaced with nitrogen, and the MPC monomer is reacted for a predetermined time by atom transfer radical polymerization. A PMPC layer is formed in a core shell shape using gold fine particles as a core. By adding a large amount of mercaptoacetic acid: mercaptoethanol = 1: 20 as a chain transfer agent, a carboxyl group and a hydroxyl group are mixed at the terminal of the graft polymer on the substrate. After removing the solution and washing with methanol, water-soluble carbodiimide (WSC), N-hydroxysuccinimide (NHS), and water are added to form a suspension of fine particles in which the carboxyl group at the end of PMPC is converted to an active ester group.

Example 3 (LPR detection of antigen-antibody reaction)
FIG. 4 shows an apparatus for LPR detection. The slide glass 32 having the aminated surface is added to the suspension of the fine particles for LPR detection (30) obtained in Example 2, and the fine particles 30 are fixed to the surface of the slide glass 32. After washing, it is further reacted with a phosphate buffer solution in which anti-BSA-Fab fragment 34 (estimated to be 6 × 6 nm from the X-ray diffraction data in the protein database) is dissolved. The active ester groups remaining on the surface of the fine particles are inactivated by reacting with ethanolamine. Through the above operation, an LPR detection slide glass in which the anti-BSA-Fab fragment 34 is immobilized is produced. The antigen-antibody reaction is performed by reacting the BSA solution, and the reacted BSA antigen 36 can be quantified using the LPR detection device 38.

  The LPR detection device 38 includes a light source 40, a spectrophotometer 42, a slide glass 32, and a device fixed on the slide glass. The BSA antigen can be detected by irradiating the glass slide 32 with light emitted from the light source 40 and measuring the light absorbed by the glass slide 32 and the glass fixed on the glass slide with a spectrophotometer 42. it can. Further, even when a protein other than BSA is brought into contact, since there is almost no LPR signal, an antigen-antibody reaction can be detected with a very high S / N ratio.

It is a schematic diagram about an example of capture molecule immobilization for capturing a target substance related to the prior art. It is the schematic about an example of the capture molecule fixation which capture | acquires the target substance in connection with a prior art. It is a schematic diagram of the structure provided with the functional group for coupling | bonding concerning this invention on the base | substrate. 1 is a schematic diagram of a localized plasmon resonance (LPR) detection apparatus according to the present invention.

Claims (9)

A structure provided on a substrate with a binding functional group for binding a capture molecule for capturing the target substance from a sample containing a biomolecule and the target substance,
A target substance- capturing polymer in which the base is bonded to one end of the main chain and the bonding functional group is bonded to the other end via a sulfur atom;
A suppressing polymer in which the base is bonded to one end of the main chain, and a suppressing functional group that suppresses adsorption of the biomolecule to the structure via a sulfur atom is bonded to the other end;
Have
A suppressive functional group that suppresses adsorption of the biomolecule to the structure is bound to both side chains of the target substance capturing polymer and the suppressor polymer ,
The binding functional group for binding a capture molecule , wherein the density of the polymer on the substrate is in the range of 0.1 molecule / nm ² or more and 1.0 molecule / nm ² or less. A structure provided with a substrate.   The functional group for suppression includes hydroxyl group, methoxy group, ethoxy group, propoxy group, isopropoxy group, 2-hydroxyethyl group, 2-hydroxypropyl group, 2-hydroxyisopropyl group, 2-hydroxybutyl group, sulfonyl group, Phosphonyl group, amino group, methylamino group, ethylamino group, isopropylamino group, amide group, methylamide group, ethylamide group, isopropylamide group, pyrrolidone group, ethylene glycol group and polymers thereof, choline group, phosphatidylcholine group The structure according to claim 1.   The structure according to claim 1, wherein the bonding functional group is any one of a carboxyl group, an aldehyde group, a succinimide group, a maleimide group, a glycidyl group, and an amino group.   The structure according to claim 1, wherein the polymer is made of a vinyl polymer compound.   The vinyl polymer has a hydroxyl group, methoxy group, ethoxy group, propoxy group, isopropoxy group, 2-hydroxyethyl group, 2-hydroxypropyl group, 2-hydroxyisopropyl group, 2-hydroxy group on the side chain. Butyl group, sulfonyl group, phosphonyl group, amino group, methylamino group, ethylamino group, isopropylamino group, amide group, methylamide group, ethylamide group, isopropylamide group, pyrrolidone group, ethylene glycol group and its polymer, choline group The structure according to claim 4, which is a polymer of a monomer having any of phosphatidylcholine groups. Average molecular weight of the polymer, the structure according to any one of claims 1 to 5 in the range of 500 to 100,000. Structure according to any one of claims 1 to 6 wherein the capture molecules bound to the functional group for binding. A method for producing a structure comprising a binding functional group on a substrate for binding a capture molecule for capturing the target substance from a sample containing a biomolecule and the target substance,
A polymer having a main chain in which one end is bonded to the surface of the substrate by polymerizing an unsaturated monomer having a side chain having a suppression functional group that suppresses the adsorption of the biomolecule to the structure from the base surface. A growing process;
Chain transfer containing a chain transfer agent containing the bonding functional group and a sulfur atom at the growth end of the main chain of the polymer obtained by the polymerization, and a chain transfer agent containing the suppression functional group and a sulfur atom React with the agent,
A target substance- capturing polymer in which the base is bonded to one end of the main chain and the bonding functional group is bonded to the other end via a sulfur atom;
A suppressing polymer in which the base is bonded to one end of the main chain, and a suppressing functional group that suppresses adsorption of the biomolecule to the structure via a sulfur atom is bonded to the other end;
Obtaining
I have a,
The binding functional group for binding a capture molecule , wherein the density of the polymer on the substrate is in the range of 0.1 molecule / nm ² or more and 1.0 molecule / nm ² or less. For manufacturing a structure comprising: A light source, a detection device for detecting light, and a reaction region for bringing a specimen into contact with a capture molecule for capturing a target substance in the specimen, and detecting the target substance in the specimen by an optical method A detection apparatus for detecting a target substance in a specimen, wherein the structure according to claim 7 is arranged in the reaction region.

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