WO2023210671A1 - Copolymer, polymer film, measuring device, and support for measurement - Google Patents

Abstract

This copolymer comprises: two blocks A including at least one structural unit (a) selected from among structural units (a) represented by formulae (1) to (6); and a block B interposed between the two blocks A, the block B including at least one structural unit (b) selected from between structural units (b) represented by formulae (7) and (8). The block B has a disulfide bond between structural units (b).

Description

Copolymers, polymer membranes, measurement devices and measurement carriers

The present disclosure relates to a copolymer used to form a polymer membrane used in a measuring device. The present disclosure also relates to a polymer membrane obtained using the copolymer, and a measuring device or a measuring carrier provided with the polymer membrane.

A surface acoustic wave sensor is disclosed in Patent Document 1 as an example of a measuring device that measures the concentration of a substance to be measured (for example, a biomolecule). Such a measuring device includes a detection section on which a substance (for example, an antibody) that interacts with a substance to be measured contained in a specimen is immobilized. A polymer film is often formed in the detection section in order to immobilize a substance that interacts with the substance to be measured contained in the sample.

Japanese Patent Application Publication No. 2008-286606

（式中、Ｒ^１～Ｒ^７はそれぞれ独立してＨまたはＣＨ_３であり、Ｘ^１～Ｘ^４はそれぞれ独立してＯまたはＮＨであり、ｐ^１～ｐ^５はそれぞれ独立して１以上３以下の整数であり、ｑ^１～ｑ^２はそれぞれ独立しては１以上５以下の整数である。）
　前記２つの親水性ブロックＡの間に、下記式（７）および（８）で表される構造単位（ｂ）のうち少なくとも１つの構造単位（ｂ）を含む疎水性ブロックＢと、を含み、

（式中、Ｒ^８およびＲ^１０はそれぞれ独立してＨまたはＣＨ_３であり、Ｒ^９、Ｒ^１１およびＲ^１２はそれぞれ独立して炭素数１以上６以下のアルキル基であり、Ｘ^５およびＸ^６はＯまたはＮＨであり、ｒは１以上３以下の整数である。）
　前記疎水性ブロックＢは、前記構造単位（ｂ）の間にジスルフィド結合を有する、共重合体である。 The copolymer according to one embodiment includes two hydrophilic blocks A containing at least one structural unit (a) among the structural units (a) represented by the following formulas (1) to (6),

(In the formula, R ¹ to R ⁷ are each independently H or CH ₃ , X ¹ to X ⁴ are each independently O or NH, and p ¹ to p ⁵ are each independently 1 to 3 The following integers are shown, and q ¹ to q ² are each independently an integer between 1 and 5.)
Between the two hydrophilic blocks A, a hydrophobic block B containing at least one structural unit (b) among the structural units (b) represented by the following formulas (7) and (8),

(In the formula, R ⁸ and R ¹⁰ are each independently H or CH ₃ , R ⁹ , R ¹¹ and R ¹² are each independently an alkyl group having 1 to 6 carbon atoms, and X ⁵ and ⁶ is O or NH, and r is an integer from 1 to 3.)
The hydrophobic block B is a copolymer having disulfide bonds between the structural units (b).

（式中、Ｒ^１～Ｒ^７はそれぞれ独立してＨまたはＣＨ_３であり、Ｘ^１～Ｘ^４はそれぞれ独立してＯまたはＮＨであり、ｐ^１～ｐ^５はそれぞれ独立して１以上３以下の整数であり、ｑ^１～ｑ^２はそれぞれ独立しては１以上５以下の整数である。）
　下記式（７）および（８）で表される構造単位（ｂ）のうち、少なくとも１つの構造単位（ｂ）を含む疎水性ブロックＢと、を含み、

（式中、Ｒ^８およびＲ^１０はそれぞれ独立してＨまたはＣＨ_３であり、Ｒ^９、Ｒ^１１およびＲ^１２はそれぞれ独立して炭素数１以上６以下のアルキル基であり、Ｘ^５およびＸ^６はＯまたはＮＨであり、ｒは１以上３以下の整数である。）
　前記疎水性ブロックＢの前記親水性ブロックＡと結合している側とは反対側の末端が、チオール基またはジチオエステル基を有する共重合体を含む、高分子膜である。 The polymer membrane according to one embodiment includes a hydrophilic block A containing at least one structural unit (a) among structural units (a) represented by the following formulas (1) to (6);

(In the formula, R ¹ to R ⁷ are each independently H or CH ₃ , X ¹ to X ⁴ are each independently O or NH, and p ¹ to p ⁵ are each independently 1 to 3 The following integers are shown, and q ¹ to q ² are each independently an integer between 1 and 5.)
A hydrophobic block B containing at least one structural unit (b) among the structural units (b) represented by the following formulas (7) and (8),

(In the formula, R ⁸ and R ¹⁰ are each independently H or CH ₃ , R ⁹ , R ¹¹ and R ¹² are each independently an alkyl group having 1 to 6 carbon atoms, and X ⁵ and ⁶ is O or NH, and r is an integer from 1 to 3.)
The end of the hydrophobic block B opposite to the side bonded to the hydrophilic block A is a polymer membrane containing a copolymer having a thiol group or a dithioester group.

FIG. 1 is a schematic diagram showing an example of a measuring device according to the present disclosure. It is a top view showing the sensor with which the above-mentioned measuring device is provided. FIG. 2 is a conceptual diagram showing a polymer included in the detection section 23 shown in FIG. 1. FIG. FIG. 2 is a plan view showing an example of a measurement carrier according to the present disclosure. It is a graph showing the relationship between the unit ratio (CBMA1/BMA) in the triblock copolymer and the amount of non-specific adsorption according to the present disclosure. It is a graph showing the relationship between the average degree of polymerization of CBMA1 in the triblock copolymer according to the present disclosure and the amount of non-specific adsorption.

[Embodiment 1]
Hereinafter, a sensor included in a measuring device according to the present disclosure will be described using the drawings as appropriate. FIG. 1 schematically shows a sensor 2 of a measuring device 100 according to this embodiment.

The measurement device 100 can detect a specific substance (first substance) as a target from a measurement target (sample). The first substance 5 is, for example, an in-vivo substance. Examples of the above-mentioned biological substances include proteins, DNA, substrates for enzymatic reactions, and the like. The measuring device 100 includes a sensor 2 that can detect the first substance and a control device 6 that can control the measuring device 100.

The sensor 2 may be any sensor that uses, for example, elastic waves, QCM (Quartz Crystal Microbalance), SPR (Surface Plasmon Resonance), or FET (Field Effect Transistor). That is, the sensor 2 only needs to be able to mutually convert electrical signals and elastic waves, QCM, SPR, FET, etc. The sensor 2 according to one embodiment is a sensor that uses elastic waves. That is, by using the sensor 2, the measuring device 100 according to one embodiment can detect a change in the elastic wave based on the presence of the first substance as a change in the electrical signal. The sensor 2 may be manufactured by a conventionally known method, except for the polymer film 1 described below. In this case, the inspection information included in the identification information includes the initial phase of the elastic wave, the orientation of the substrate 22, etc. , information specific to a sensor that uses elastic waves may be included.

In the measuring device 100 according to this embodiment, the sensor 2 has an external terminal 21. The sensor 2 can be electrically connected to a control device 6 that controls the measuring device 100 via an external terminal 21 . That is, the sensor 2 and the control device 6 can input and output electrical signals to each other via the external terminal 21. Therefore, the control device 6 can detect, for example, the first substance based on the electrical signal input from the sensor 2. For example, the control device 6 may calculate the concentration of the first substance contained in the sample. Alternatively, the control device 6 may identify the first substance, for example. The control device 6 and the external terminals 21 may be manufactured using conventionally known techniques. Further, the configuration for electrically connecting the sensor 2 and the control device 6 is not limited to the external terminal 21. For example, as long as the sensor 2 and the control device 6 can be electrically connected, the sensor 2 and the control device 6 do not need to be physically connected through a terminal or the like. For example, the sensor 2 and the control device 6 may be electrically connected by electromagnetic induction.

The sensor 2 may be a disposable cartridge. According to this, the step of cleaning the sensor 2 after measurement is unnecessary, and the influence of insufficient cleaning on the measurement results can be eliminated.

FIG. 2 shows a plan view of the sensor 2. The sensor 2 includes a substrate 22, a detection section 23 located on the substrate 22, a reference section 24, and a pair of first IDT (Inter Digital Transducer) electrodes 25a arranged on the substrate 22 so as to sandwich the detection section 23 therebetween. , a pair of second IDT electrodes 25b. The detection section 23, the reference section 24, the pair of first IDT electrodes 25a, and the pair of second IDT electrodes 25b may be located on the substrate 22.

(substrate)
The substrate 22 is, for example, a piezoelectric substrate. Specifically, the substrate 22 is, for example, a crystal substrate. The substrate 22 is not limited to a crystal substrate as long as it can propagate elastic waves. That is, the substrate 22 may be made of any material that can propagate elastic waves. For example, the substrate 22 may be a substrate containing metals such as gold, silver, copper, platinum, and aluminum, lithium tantalate, and a piezoelectric single crystal such as quartz. Further, the substrate 22 may be manufactured by a conventionally known method.

(Detection unit)
A substance (second substance 4) that reacts with the first substance 5 is fixed to the detection unit 23. Therefore, in the detection unit 23, the first substance 5 contained in the specimen and the fixed second substance 4 can react. The detection unit 23 changes the propagation characteristics of the elastic waves of the substrate 22 by causing the first substance 5 and the second substance 4 to react. Specifically, the detection unit 23 changes the weight applied to the substrate 22 or the viscosity of the liquid that contacts the surface of the substrate 22 by causing the first substance 5 and the second substance 4 to react, for example. The magnitude of these changes correlates with the amount of reaction between the first substance 5 and the second substance 4. Further, the characteristics of the elastic wave (for example, phase, amplitude, period, etc.) change as it propagates through the detection unit 23. The magnitude of the change in characteristics correlates with, for example, the magnitude of the weight applied to the substrate 22 or the magnitude of the viscosity of the liquid that contacts the surface of the substrate 22. Therefore, the sensor 2 can detect the first substance 5 based on changes in the characteristics of the elastic waves. Specifically, the measuring device 100 can measure, for example, the concentration of the first substance 5 contained in the sample. Details of the detection unit 23 will be described later.

The pair of first IDT electrodes 25a can generate elastic waves between the pair of first IDT electrodes 25a. Among the generated elastic waves, the elastic waves that propagate on the surface of the substrate 22 are also referred to as surface acoustic waves (SAW). The pair of first IDT electrodes 25a may be located on the substrate 22 so as to sandwich the detection section 23 therebetween. In the measuring device 100 according to one embodiment, an electrical signal is input to one of the pair of first IDT electrodes 25a. The input electrical signal is converted into an elastic wave that propagates toward the detection section 23 and is emitted from one first IDT electrode 25a. The emitted elastic waves pass through the detection section 23. The other first IDT electrode 25a can receive the elastic wave that has passed through the detection section 23. The received elastic waves are converted into electrical signals. The pair of first IDT electrodes 25a may be made of, for example, a metal material such as gold, chromium, or titanium. Further, the pair of first IDT electrodes 25a may be a single layer electrode made of a single material, or a multilayer electrode made of a plurality of materials.

The sensor 2 may have two or more combinations of the detection section 23 and the pair of first IDT electrodes 25a. In this case, the measuring device 100 may detect different types of target substances for each combination, for example. Alternatively, the measuring device 100 may, for example, detect the same type of target substance in multiple combinations and compare the respective detection results.

FIG. 3 shows a conceptual diagram showing the polymer membrane 1 included in the detection section 23. In the detection unit 23, a polymer film 1 containing a polymer 3 is fixed on a substrate 22. Furthermore, a substance (second substance 4) that reacts with the first substance 5 is fixed on the polymer film 1.

The polymer membrane 1 is a membrane that has been adjusted to have high specific adsorption and is a membrane that has been adjusted to reduce non-specific adsorption.

(first substance and second substance)
The reaction between the first substance 5 and the second substance 4 may be any reaction that causes a change in the output of the sensor 2. Such a reaction may be, for example, a reaction in which the first substance 5 and the second substance 4 are bonded together through a redox reaction, an enzyme reaction, an antigen-antibody reaction, chemisorption, intermolecular interaction, or ionic interaction. It's okay. Alternatively, the reaction between the first substance 5 and the second substance 4 may be a reaction in which a new substance (third substance) is produced by an enzymatic reaction or the like.

The second substance 4 to be fixed to the detection unit 23 may be appropriately selected depending on the first substance 5. For example, when the first substance 5 is a specific protein, DNA, or cell in the specimen, the second substance 4 may be an antibody, a peptide, an aptamer, or the like. Further, for example, when the first substance 5 is an antibody, the second substance 4 may be an antigen. Furthermore, for example, when the first substance 5 is a substrate, the second substance 4 may be an enzyme.

The measuring device 100 may indirectly detect the first substance 5 that is the target. For example, a substance similar to the first substance 5 may be fixed to the detection unit 23 as the second substance 4. That is, for example, an antibody whose antigen is the first substance 5 may be reacted with the first substance 5 in advance, and the unreacted antibody may be reacted with the immobilized second substance 4. In this case, for example, if the amount of antibody is known, the measuring device 100 can indirectly calculate the amount of the first substance 5 from the amount of detected antibody.

(Triblock copolymer)
Next, a triblock copolymer of the present disclosure, which is an example of a copolymer used to form the polymer membrane 1, will be described.

The triblock copolymer of the present disclosure is a copolymer containing two hydrophilic blocks A and a hydrophobic block B between the two hydrophilic blocks.

<Hydrophilic block A>
Hydrophilic block A includes at least one structural unit (a) among the structural units (a) represented by the following formulas (1) to (6).

In the formula, R ¹ to R ⁷ are each independently H or CH ₃ . X ¹ to X ⁴ are each independently O or NH, and p ¹ to p ⁵ are each independently an integer of 1 or more and 3 or less (1, 2 or 3). q ¹ to q ² are each independently an integer from 1 to 5 (1, 2, 3, 4, or 5).

In order to further reduce non-specific adsorption, the structural unit (a) may have a betaine structure. In addition, the structural unit (a) may have a COO 2 ^- group in terms of making it easier to immobilize antibodies and the like.

Examples of the structural unit (a) include the following structural units.

The proportion of the structural unit (a) constituting the hydrophilic block A contained in the triblock copolymer of the present disclosure may be 20 mol% or more, and may be 30 mol% or more in terms of reducing nonspecific adsorption. The amount may be 40 mol% or more. Further, the above ratio may be 70 mol% or less, 60 mol% or less, or 50 mol% or less.

The hydrophilic block A may have a structural unit other than the structural unit (a) within a range that does not impair the effects of the present disclosure.

When the hydrophilic block A contains two or more types of structural units, each structural unit may be contained in the block A by any mode such as random copolymerization or block copolymerization.

The two hydrophilic blocks A contained in the triblock copolymer of the present disclosure may have the same or different structures. In the case where two hydrophilic blocks A have the same structure, it is sufficient that they have the same structural unit (a) within a range that does not impair the effects of the present disclosure. Specifically, the equivalent structural unit (a) may have the same structural unit (a), and may have a structural unit other than the structural unit (a). In addition, the equivalent structural unit (a) may have the same structure as the structural unit (a), for example, the relative position and number average degree of polymerization of the structural unit (a) may be the same. .

<Hydrophobic block B>
Hydrophobic block B includes at least one structural unit (b) among the structural units (b) represented by the following formulas (7) and (8).

In the formula, R ⁸ and R ¹⁰ are each independently H or CH ₃ . R ⁹ , R ¹¹ and R ¹² are each independently an alkyl group having 1 or more and 6 or less carbon atoms. X ⁵ and X ⁶ are O or NH. r is an integer (1, 2 or 3) of 1 or more and 3 or less.

Examples of the alkyl group having 1 to 6 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, heptyl group, hexyl group, etc. .

Examples of the structural unit (b) include the following structural units.

The proportion of the structural unit (b) constituting the hydrophobic block B contained in the triblock copolymer of the present disclosure may be 30 mol% or more in terms of ease of forming micelles of the polymer. , may be 40 mol% or more, or may be 50 mol% or more. Further, the above ratio may be 80 mol% or less, 60 mol% or less, or 50 mol% or less.

The hydrophobic block B may have a structural unit other than the structural unit (b) as long as the effects of the present disclosure are not impaired.

When the hydrophobic block B contains two or more types of structural units, each structural unit may be contained in the block B by any mode such as random copolymerization or block copolymerization.

Hydrophobic block B has a disulfide bond between structural units (b). The number of disulfide bonds may be one or more.

At least one end of the triblock copolymer of the present disclosure may have a thiol group or a dithioester group from the viewpoint of ease of fixing the polymer membrane to the substrate 22 (measurement substrate 12). , may be a thiol group.

The ratio of the number of moles of the structural unit (a) to the number of moles of the structural unit (b) contained in the triblock copolymer of the present disclosure is determined to reduce nonspecific adsorption and form micelles of the copolymer. In terms of ease, it may be 0.20 or more, 0.50 or more, or 0.70 or more. Further, the above ratio may be 2.5 or less, 2.0 or less, or 1.5 or less.

The number average degree of polymerization of the triblock copolymer of the present disclosure may be 100 or more, or even 200 or more, in terms of ease of forming micelles of the copolymer, film formation density, etc. good.

The total number average degree of polymerization of the two hydrophilic blocks A in the triblock copolymer of the present disclosure may be 30 or more, 60 or more, 100 or more in terms of reducing nonspecific adsorption. It may be 200 or less, or it may be 150 or less.

The number average degree of polymerization of the hydrophobic blocks B in the triblock copolymer of the present disclosure may independently be 25 or more, and 30 It may be more than 50, it may be more than 80.

The triblock copolymer of the present disclosure may be identified by conventionally known organic analysis techniques. For example, the copolymer may be identified by NMR (Nuclear Magnetic Resonance). Alternatively, it may be identified, for example, by liquid chromatography. Alternatively, it may be identified, for example, by infrared spectroscopy. That is, when identifying the copolymer, an apparatus capable of implementing these techniques may be used. As long as the copolymer can be identified, the identification method and device are not limited to these methods and devices.

<Method for producing triblock copolymer>

An example of the method for producing a triblock copolymer of the present disclosure includes a step of synthesizing a hydrophobic block B (step 1) and a step of synthesizing a hydrophilic block A (step 2). Each step will be explained below.

<Step 1: Synthesis step of hydrophobic block B>
In this step, hydrophobic block B is synthesized by polymerizing a hydrophobic monomer compound. Examples of hydrophobic monomer compounds include the following compounds. Moreover, butyl methacrylic acid (BMA) etc. are mentioned as a specific example of a hydrophobic monomer compound. The hydrophobic monomer compounds may be used alone or in combination of two or more.

（式中、Ｒ^８～Ｒ^１２、Ｘ^５およびＸ^６ならびにｒはそれぞれ、式（７）～（８）中のＲ^８～Ｒ^１２、Ｘ^５およびＸ^６と同義である。）

(In the formula, R ⁸ to R ¹² , X ⁵ and X ⁶ and r have the same meanings as R ⁸ to R 12 , ^{X 5 and} X ⁶ in formulas (7) to (8), respectively.)

Polymerization can be produced by known polymerization such as living radical polymerization (LRP). Examples of living radical polymerization include atom transfer radical polymerization (ATRP), single electron transfer polymerization (SET-LRP), reversible chain transfer catalyzed polymerization (RTCP), and RAFT polymerization. (Reversible Addition-Fragmentation chain Transfer Polymerization).

Examples of polymerization initiators include polymerization initiators containing disulfide bonds such as bis[2-(2'-bromoisobutyryloxy)ethyl]disulfide (BiBOEDS)).

（式中、Ｒ^１～Ｒ^７、Ｘ^１～Ｘ^４、ｐ^１～ｐ^５およびｑ^１～ｑ^２はそれぞれ、式（１）～（６）中のＲ^１～Ｒ^７、Ｘ^１～Ｘ^４、ｐ^１～ｐ^５およびｑ^１～ｑ^２と同義である。） <Step 2: Synthesis step of hydrophilic block A>
In this step, hydrophilic blocks A are synthesized at both ends of hydrophobic block B by polymerizing the polymer obtained in step 1 with a hydrophilic monomer compound, thereby obtaining the triblock copolymer of the present disclosure. . Examples of hydrophilic monomer compounds include the following compounds (11) to (16). Further, as a specific example of the hydrophilic monomer compound, N-(carboxymethyl)-N,N-dimethyl-2-[(2-methyl-1-oxo-2-propen-1-yl)-oxy]ethanaminium (CBMA1 ), N,N-dimethylaminoethyl methacrylate (DMAEMA), 2-hydroxypropyl methacrylamide (HPMA), and the like. The hydrophilic monomer compounds may be used alone or in combination of two or more.

(In the formula, R ¹ to R ⁷ , X ¹ to X ⁴ , p ¹ to p ⁵ and q ¹ to q ² are R ¹ to R ⁷ , X ¹ to ⁴ , p ¹ to p ⁵ and q ¹ to q ^2. )

Furthermore, the method for producing a triblock copolymer of the present disclosure may include a quaternary ammonium salt formation step (Step 3) and a deprotection step (Step 4).

<Step 3: Quaternary ammonium formation step of hydrophilic block A>
In this step, the tertiary amine structure of the structural unit (a) of the hydrophilic block A of the polymer obtained in Step 2 is converted into a quaternary ammonium structure (quaternary ammonium formation). For example, quaternary ammonium formation may be performed using a known quaternizing agent such as a halogen compound.

<Step 4: Betainization step of hydrophilic block A>
In this step, a betaine structure is formed in the structural unit (a) of the hydrophilic block A of the polymer obtained in step 3 (betaine formation). For example, betaination may be performed by deprotecting the side chain protecting group (eg, tert-butyl group) of the hydrophilic block A introduced in step 3.

By performing steps 3 and 4, non-specific adsorption can be further reduced.

If necessary, the structure derived from the polymerization initiator in the produced triblock copolymer may be removed. An example of a structure derived from the polymerization initiator is bromine at the terminal of a triblock copolymer when LRP is performed using BiBOEDS as a polymerization initiator.

(polymer membrane)
The polymer membrane of the present disclosure includes a copolymer containing a hydrophilic block A and a hydrophobic block B. The copolymer is a diblock copolymer, and the diblock copolymer is also included in one embodiment of the present disclosure. The diblock copolymer of the present disclosure corresponds to polymer 3 in FIG.

<Diblock copolymer>
In the diblock copolymer of the present disclosure, the end of the hydrophobic block B opposite to the side bonded to the hydrophilic block A has a thiol group or a dithioester group. The structural units of the hydrophilic block A and the hydrophobic block B of the diblock copolymer of the present disclosure and their specific examples are the structural units of the hydrophilic block A and the hydrophobic block B of the triblock copolymer of the present disclosure and their specific examples. This is the same as the specific example.

The ratio of the number of moles of the structural unit (a) to the number of moles of the structural unit (b) contained in the diblock copolymer of the present disclosure is 0.20 in terms of nonspecific adsorption reduction and film formation density. or more, may be 0.50 or more, or may be 0.70 or more. Further, the above ratio may be 2.5 or less, 2.0 or less, or 1.5 or less.

The number average degree of polymerization of the diblock copolymer of the present disclosure may be 25 or more, 50 or more, or 200 or more in terms of film forming density. When the first substance 5 is a substance contained in urine, the diblock copolymer of the present disclosure may have a high molecular weight.

The number average degree of polymerization of the hydrophilic block A in the diblock copolymer of the present disclosure may be 20 or more, 40 or more, or 60 or more in terms of reducing nonspecific adsorption. Generally, it may be 80 or more, 100 or more, or 200 or less.

The number average degree of polymerization of the hydrophobic blocks B in the diblock copolymer of the present disclosure may independently be 25 or more, 30 or more, or 50 or more in terms of film forming density. The number may be 80 or more.

The diblock copolymer of the present disclosure, like the triblock copolymer of the present disclosure, may be identified by conventionally known organic analysis techniques.

The diblock copolymer of the present disclosure can be obtained by cleaving the disulfide bonds of the triblock copolymer of the present disclosure or micelles of the copolymer. A conventionally known reducing agent may be used to cleave the disulfide bond.

<Production method of polymer membrane>
An example of the method for producing a polymer film of the present disclosure includes a step of preparing a coating agent containing micelles of the triblock copolymer of the present disclosure, a step of disposing the coating agent on a substrate, and a step of preparing a coating agent containing micelles of the triblock copolymer of the present disclosure. cleaving disulfide bonds in micelles of the polymer to form a diblock copolymer.

Micelles of the triblock copolymer of the present disclosure are also included in one embodiment of the present disclosure. The micelles are flower-shaped micelles.

Further, an example of the method for producing a polymer membrane of the present disclosure includes a step of preparing a coating agent containing micelles of a diblock copolymer obtained by cleaving the disulfide bonds of the triblock copolymer of the present disclosure; arranging the coating agent on the substrate.

Micelles of diblock copolymers obtained by cleaving the disulfide bonds of the triblock copolymers of the present disclosure are also included in one embodiment of the present disclosure. The micelles are W/O type micelles.

A polymer membrane formed by a diblock copolymer obtained by cleaving disulfide bonds after forming micelles of the triblock copolymer of the present disclosure has a high density of the diblock copolymer. Furthermore, a high density polymer membrane can also be achieved by using micelles of a diblock copolymer obtained by cleaving the disulfide bonds of the triblock copolymer of the present disclosure. Furthermore, the polymer membrane formed by the diblock copolymer obtained from the triblock copolymer of the present disclosure has a reduced amount of nonspecific adsorption.

Methods for immobilizing the diblock copolymer of the present disclosure on the substrate 22 include, for example, a method in which a polymer solution obtained by dissolving polymer 3 in a solvent is applied to the substrate 22 and dried, graft polymerization using radiation or ultraviolet rays, Examples include a chemical reaction with a functional group of the substrate 22. By these methods, the polymer film 1 made of the polymer 3 is formed on the substrate 22.

An example of a method for fixing the second substance 4 onto the polymer 3 (polymer membrane 1) is a method of covalently bonding the second substance 4 to a carboxyl group that the polymer 3 has. For example, polymer 3 is reacted with N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (NHS/EDC activation). Then, the carboxyl group that polymer 3 has is substituted with an NHS ester group. The second substance 4 is fixed on the polymer 3 (polymer membrane 1) by reacting the activated NHS ester group with the amino group of the second substance 4. NHS/EDC activation of the polymer 3 may be performed before immobilizing on the substrate 22 or after immobilizing the substrate 22. Since the polymer of this embodiment has high chemical stability, it is unlikely to be decomposed by NHS/EDC activation. Therefore, non-specific adsorption caused by decomposition products accompanying NHS/EDC activation can be reduced.

[Embodiment 2]
Hereinafter, a measurement carrier according to one embodiment will be described using the drawings as appropriate. For convenience of explanation, members having the same functions as those described in the above embodiments are denoted by the same reference numerals, and the description thereof will not be repeated. FIG. 4 shows a schematic configuration of the measurement carrier 11 according to this embodiment. An example of the measurement carrier 11 is a plate for ELISA (enzyme-linked immunosorbent).

The measurement carrier 11 has a detection region 31 for specifically capturing the target substance (first substance 5) contained in the sample and a non-detection region 32 for non-selectively adsorbing a blocking agent etc. on the surface of the measurement substrate 12. We are preparing for The polymer film 1 is fixed to the detection region 31 .

A second substance 4 that reacts with the first substance 5 is fixed on the polymer membrane 1, similar to the polymer membrane 1 described in the first embodiment. A membrane adjusted to increase non-specific adsorption may be fixed to the non-detection region 32.

(Example of use of measurement carrier)
First, a desired second substance 4 is fixed on the polymer film 1 in the detection region 31 . Further, the blocking agent is non-selectively adsorbed to the non-detection region 32 . Thereafter, by bringing the specimen into contact with the detection region 31, the first substance 5, which is a target substance contained in the specimen, reacts with the second substance 4, and the reaction is detected by the detection reagent. Examples of detection reagents include redox substances, fluorescent substances, enzymes, and dye compounds.

(Measurement board)
The measurement substrate 12 may be made of, for example, metals such as gold, silver, copper, platinum, and aluminum; plastics such as polyethylene and polypropylene; and inorganic materials such as titanium oxide, silica, glass, and ceramics. The measurement substrate 12 is not limited to these examples.

The shape of the measurement substrate 12 may be, for example, a plate, a particle, a microstructure, a microtiter plate, or the like. The shape of the measurement substrate 12 is not limited to these examples.

(Measurement kit)
A measurement kit including a measurement substrate 12 on which a polymer film 1 is fixed, a second substance 4, and a detection reagent is also included within the scope of the present disclosure. The second substance 4 may be fixed to the polymer membrane 1 in advance during product manufacture, or may be fixed by the user before measurement.

The measurement kit according to this embodiment may include other reagents and instruments. For example, components other than the second substance 4 and the detection reagent described above may be included. Further, a buffer or the like may be provided. Furthermore, the measurement kit according to the present embodiment may include a plurality of different reagents mixed in appropriate volumes and/or forms, or may be provided in separate containers. Further, the measurement kit according to the present embodiment may include an instruction sheet describing the procedure for detecting the reaction between the first substance 5 and the second substance 4. It may be written or printed on paper or other media, or it may be attached to an electronic medium such as a magnetic tape, a computer readable disk, or a CD-ROM or the like.

（式中、Ｒ^１～Ｒ^７はそれぞれ独立してＨまたはＣＨ_３であり、Ｘ^１～Ｘ^４はそれぞれ独立してＯまたはＮＨであり、ｐ^１～ｐ^５はそれぞれ独立して１以上３以下の整数であり、ｑ^１～ｑ^２はそれぞれ独立しては１以上５以下の整数である。）
　前記２つの親水性ブロックＡの間に、下記式（７）および（８）で表される構造単位（ｂ）のうち少なくとも１つの構造単位（ｂ）を含む疎水性ブロックＢと、を含み、

（式中、Ｒ^８およびＲ^１０はそれぞれ独立してＨまたはＣＨ_３であり、Ｒ^９、Ｒ^１１およびＲ^１２はそれぞれ独立して炭素数１以上６以下のアルキル基であり、Ｘ^５およびＸ^６はＯまたはＮＨであり、ｒは１以上３以下の整数である。）
　前記疎水性ブロックＢは、前記構造単位（ｂ）の間にジスルフィド結合を有する。 〔summary〕
The copolymer according to aspect 1 of the present disclosure comprises two hydrophilic blocks A containing at least one structural unit (a) among the structural units (a) represented by the following formulas (1) to (6). ,

(In the formula, R ¹ to R ⁷ are each independently H or CH ₃ , X ¹ to X ⁴ are each independently O or NH, and p ¹ to p ⁵ are each independently 1 to 3 The following integers are shown, and q ¹ to q ² are each independently an integer between 1 and 5.)
Between the two hydrophilic blocks A, a hydrophobic block B containing at least one structural unit (b) among the structural units (b) represented by the following formulas (7) and (8),

(In the formula, R ⁸ and R ¹⁰ are each independently H or CH ₃ , R ⁹ , R ¹¹ and R ¹² are each independently an alkyl group having 1 to 6 carbon atoms, and X ⁵ and ⁶ is O or NH, and r is an integer from 1 to 3.)
The hydrophobic block B has a disulfide bond between the structural units (b).

In the copolymer according to Aspect 2 of the present disclosure, in Aspect 1, the proportion of the structural unit (b) constituting the hydrophobic block B contained in the copolymer is 30 mol% or more and 80 mol% or less. It may be.

In the copolymer according to Aspect 3 of the present disclosure, in Aspect 1 or 2, the proportion of the structural unit (a) constituting the hydrophilic block A contained in the copolymer is 20 mol % or more and 70 mol %. % or less.

The copolymer according to Aspect 4 of the present disclosure is provided in any one of Aspects 1 to 3, in which the number of moles of the structural unit (a) is 1 per mole of the structural unit (b) contained in the copolymer. The numerical ratio may be 0.20 or more and 2.5 or less.

In the copolymer according to Aspect 5 of the present disclosure, at least one terminal of the copolymer according to any one of Aspects 1 to 4 may have a thiol group or a dithioester group.

（式中、Ｒ^１～Ｒ^７はそれぞれ独立してＨまたはＣＨ_３であり、Ｘ^１～Ｘ^４はそれぞれ独立してＯまたはＮＨであり、ｐ^１～ｐ^５はそれぞれ独立して１以上３以下の整数であり、ｑ^１～ｑ^２はそれぞれ独立しては１以上５以下の整数である。）
　下記式（７）および（８）で表される構造単位（ｂ）のうち、少なくとも１つの構造単位（ｂ）を含む疎水性ブロックＢと、を含み、

（式中、Ｒ^８およびＲ^１０はそれぞれ独立してＨまたはＣＨ_３であり、Ｒ^９、Ｒ^１１およびＲ^１２はそれぞれ独立して炭素数１以上６以下のアルキル基であり、Ｘ^５およびＸ^６はＯまたはＮＨであり、ｒは１以上３以下の整数である。）
　前記疎水性ブロックＢの前記親水性ブロックＡと結合している側とは反対側の末端が、チオール基またはジチオエステル基を有する共重合体を含む。 The polymer membrane according to aspect 6 of the present disclosure includes a hydrophilic block A containing at least one structural unit (a) among structural units (a) represented by the following formulas (1) to (6);

(In the formula, R ¹ to R ⁷ are each independently H or CH ₃ , X ¹ to X ⁴ are each independently O or NH, and p ¹ to p ⁵ are each independently 1 to 3 The following integers are shown, and q ¹ to q ² are each independently an integer between 1 and 5.)
A hydrophobic block B containing at least one structural unit (b) among the structural units (b) represented by the following formulas (7) and (8),

(In the formula, R ⁸ and R ¹⁰ are each independently H or CH ₃ , R ⁹ , R ¹¹ and R ¹² are each independently an alkyl group having 1 to 6 carbon atoms, and X ⁵ and ⁶ is O or NH, and r is an integer from 1 to 3.)
The end of the hydrophobic block B opposite to the side bonded to the hydrophilic block A contains a copolymer having a thiol group or a dithioester group.

A measuring device according to Aspect 7 of the present invention includes the polymer membrane of Aspect 6 above.

A measurement carrier according to Aspect 8 of the present invention includes the polymer membrane of Aspect 6 above.

The invention according to the present disclosure has been described above based on the drawings and examples. However, the invention according to the present disclosure is not limited to each embodiment described above. That is, the invention according to the present disclosure can be modified in various ways within the scope shown in the present disclosure, and the invention according to the present disclosure also applies to embodiments obtained by appropriately combining technical means disclosed in different embodiments. Included in technical scope. In other words, it should be noted that those skilled in the art can easily make various changes or modifications based on the present disclosure. It should also be noted that these variations or modifications are included within the scope of this disclosure.

In the following examples, unless otherwise specified, % represents mass %.

[Example 1] Production of triblock copolymer <Step 1. Synthesis of polybutyl methacrylate <p(BMA)>
p(BMA) corresponding to hydrophobic block B was synthesized according to the scheme below.

p(BMA) was synthesized by the SET-LRP method. 2.08 mol/L of monomer butyl methacrylic acid (BMA) and 20.0 mol/L of bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide (BiBOEDS) were added to 2-propanol that had been previously subjected to deoxygenation treatment. 8 mmol/L, copper (II) bromide was dissolved at 2.08 mmol/L, and N,N,N',N'',N''-pentamethyldiethylenetriamine (PMDETA) was dissolved at 15.0 mmol/L. . Next, the obtained solution was added to a copper wire that had been reduced with hydrazine, and reacted in nitrogen at 40° C. for 6 hours. The amount of copper wire was such that the concentration would be 305 mmol/L assuming that it was all dissolved. After the reaction, the reaction solution was filtered and poured into a large amount of methanol (15 to 20 times the volume of the polymerization solution). Then, p(BMA) was obtained by filtering and drying the precipitate. The degree of polymerization of the obtained p(BMA) was calculated by nuclear magnetic resonance (NMR) spectroscopy. Specifically, the area of the peak (peak around 2.9 ppm) derived from the methylene group bonded to sulfur of BiBOEDS in ^{the 1} H-NMR spectrum and the side chain (methylene group bonded to oxygen) in the BMA unit of the polymer are determined. ) and the area of the peak (peak around 3.90 ppm to 4.00 ppm). As a result, the degree of polymerization of the obtained p(BMA) was estimated to be 52.

<Step 2. Polymerization of N,N-dimethylaminoethyl methacrylate (DMAEMA)>
DMAEMA was polymerized according to the following scheme.

Polymerization of DMAEMA was performed by the SET-LRP method using p(BMA) synthesized in Step 1 as a macroinitiator. Add 1.50 mol/L of monomer DMAEMA, 15.0 mmol/L of p(BMA) synthesized in step 1, and 1.50 mmol/L of copper(II) bromide to 2-propanol that has been previously deoxidized. , PMDETA was dissolved to a concentration of 10.8 mmol/L. Next, the obtained solution was added to a copper wire that had been reduced with hydrazine, and reacted in nitrogen at 40° C. for 8 hours. The amount of copper wire was such that the concentration would be 220 mmol/L assuming that all of the copper wire was dissolved. After the reaction, 45°C warm water was added. Then, the precipitate was filtered and dried to obtain p(BMA)-bp(DMAEMA). The degree of polymerization of the obtained p(BMA)-bp(DMAEMA) was calculated by nuclear magnetic resonance (NMR) spectroscopy. Specifically, it was calculated from the ratio of the area of the peak around 1.5 ppm originating from the BMA unit and the area of the peak around 2.3 ppm originating from the DMAEMA unit of the polymer in the ¹ H-NMR spectrum. As a result, the ratio of BMA units to DMAEMA units (BMA:DMAEMA) was estimated to be 52:42.

<Step 3. Quaternary ammonium conversion of DMAEMA unit>
The side chain of the DMAEMA unit was converted into a quaternary ammonium according to the scheme below.

To 2-propanol in which p(BMA)-bp(DMAEMA) synthesized in step 2 was dissolved so that the DMAEMA unit was 500 mmol/L, tert-butyl bromoacetate was added at a concentration of 750 mmol/L. The reaction was allowed to proceed at room temperature for 20 hours. N-hexane was added to the reaction. The precipitate was then filtered and dried to convert the DMAEMA unit side chain into a quaternary ammonium. By nuclear magnetic resonance (NMR) spectroscopy, a peak derived from a tert-butyl group was observed in the ¹ H-NMR spectrum, confirming quaternary ammonium formation of the side chain of the DMAEMA unit.

　ステップ３で得られた、ＤＭＡＥＭＡユニットの側鎖が４級アンモニウム化されたブロック共重合体をトリフルオロ酢酸に溶解させ、室温で２０時間反応させた。反応後、エバポレーションによりトリフルオロ酢酸を留去し、ポリマーの粉末を回収した。ポリマーの粉末をメタノールに溶解させ、ジエチルエーテルを投入して再沈殿させ、沈殿物をろ過して乾燥させることにより、ポリマーの粉末を得た。核磁気共鳴（ＮＭＲ）分光法により、^１Ｈ－ＮＭＲスペクトルにてｔｅｒｔ－ブチル基に由来するピークが消失していた。ＮＭＲ分光法の結果から、ｔｅｒｔ－ブチル基が脱保護され、目的のトリブロック共重合体であるｐ（ＢＭＡ）－ｂ－ｐ（ＣＢＭＡ１）が得られたことを確認した。 <Step 4. Betaining DMAEMA unit>
The side chain of the DMAEMA unit was betaineated according to the scheme below.

The block copolymer obtained in step 3 in which the side chain of the DMAEMA unit was quaternary ammonium was dissolved in trifluoroacetic acid and reacted at room temperature for 20 hours. After the reaction, trifluoroacetic acid was distilled off by evaporation, and polymer powder was recovered. A polymer powder was obtained by dissolving the polymer powder in methanol, adding diethyl ether to cause reprecipitation, and filtering and drying the precipitate. Nuclear magnetic resonance (NMR) spectroscopy revealed that the peak derived from the tert-butyl group had disappeared in the ¹ H-NMR spectrum. From the results of NMR spectroscopy, it was confirmed that the tert-butyl group was deprotected and the desired triblock copolymer p(BMA)-bp(CBMA1) was obtained.

The triblock copolymer obtained by the above synthesis scheme is represented by the following formula.
p(CBMA1)-p(BMA)-SS-p(BMA)-p(CBMA1)
"p(CBMA1") is the hydrophilic block A, and "p(BMA)-SS-p(BMA)" is the hydrophobic block B. "SS" in hydrophobic block B represents a disulfide bond.

The degree of polymerization of each block of the triblock copolymer obtained by the above synthesis scheme was 42-52-SS-52-42. That is, by the above synthesis scheme, an ABA type triblock copolymer containing a hydrophobic block B containing a disulfide bond and having a degree of polymerization of 104 between two hydrophilic blocks A having a degree of polymerization of 42 was obtained. It was done.

A triblock copolymer (p(CBMA1)-p(BMA)-SS-p(BMA)-p(CBMA1)) having the following degree of polymerization was also produced.
・24-27-SS-27-24
・24-31-SS-31-24
・20-84-SS-84-20
・71-31-SS-31-71
・80-84-SS-84-80

Furthermore, a triblock copolymer (p(HPMA)-p(BMA)-SS-p(BMA)-p(HPMA)) was also produced according to the following scheme.

The polymerization of HPMA was carried out by the SET-LRP method using p(BMA) in which the p(BMA) unit was an 86-mer synthesized in the same manner as in Step 1 as a macroinitiator. Add 2.00 mol/L of monomer HPMA, 10.0 mmol/L of p(BMA) synthesized in step 1, and 2.00 mmol/L of copper(II) bromide to 2-propanol that has been previously deoxidized. , Me ₆ TREN (tris[2-(dimethylamino)ethylamine) was dissolved at a concentration of 15.0 mmol/L. Next, the obtained solution was added to a copper wire that had been reduced with hydrazine, and reacted in nitrogen at 40° C. for 91 hours. The amount of copper wire was such that the concentration would be 305 mmol/L assuming that it was all dissolved. After the reaction, water was added to the soluble portion of the solution. Then, the soluble portion was removed by a decanting method, and p(BMA)-bp(HPMA) was obtained by drying. The degree of polymerization of the obtained p(BMA)-bp(HPMA) was calculated by nuclear magnetic resonance (NMR) spectroscopy. Specifically, it was calculated from the ratio of the area of the peak around 4 ppm originating from the BMA unit and the area of the peak around 2.9 ppm originating from the HPMA unit of the polymer in ^{the 1} H-NMR spectrum. As a result, the ratio of BMA units to HPMA units (BMA:HPMA) was estimated to be 86:14.

[Example 2] Measurement of non-specific adsorption amount The non-specific adsorption amount of fetal bovine serum to the polymer membrane was measured under the following conditions.
Measuring device: Biacore X100 manufactured by GE Healthcare
Measurement conditions: Running buffer: HBS-P
Temperature: 25℃
Flow rate: 10μL/min Sample contact time: 9 minutes (sample injection volume 90μL)

Using an SPR chip on which a polymer film consisting of p(CBMA1)-p(BMA)-SS-p(BMA)-p(CBMA1) was formed, running buffer was allowed to flow for 9 minutes after sample injection. The difference between the SPR signal value of and the SPR signal value before sample injection was defined as the non-specific adsorption amount, and 10 RU was converted to 1 ng/cm ² .

The conditions for creating an SPR chip with a polymer film formed are shown below. SIA kit Au (manufactured by GE Healthcare) was washed with piranha solution. Next, it was immersed in a 1 mmol/L ethanol solution of 1-dodecanethiol and left overnight. Next, the SPR chip was washed with ethanol and ultrapure water. Next, it was immersed for 18 hours in a methanol solution in which the triblock copolymer was dissolved at a concentration of 0.3 mg/mL to form a polymer film. Thereafter, the chip was washed with ultrapure water and dried in a nitrogen stream to obtain an SPR chip with a polymer film formed thereon.

The measurement results of the amount of non-specific adsorption are shown in Table 1 and Figures 5 and 6. FIG. 5 is a graph showing the relationship between the unit ratio (CBMA1/BMA) of the triblock copolymer and the amount of non-specific adsorption. FIG. 6 is a graph showing the relationship between the average degree of polymerization of CBMA1 in the triblock copolymer and the amount of non-specific adsorption.

The non-specific adsorption amount of the polymer membrane is preferably 20 ng/cm ² or less, and it is known that the higher the film formation density, the lower the non-specific adsorption amount. From the results in Table 1 and Figures 5 and 6, when the unit ratio (CBMA1/BMA) of the triblock copolymer is 0.20 or more and 2.5 or less, the amount of nonspecific adsorption decreases, and the unit ratio is 1 or less. was found to be more preferable. Further, it was found that the number average degree of polymerization of CBMA1 is preferably up to about 70, and more preferably about 20 to 40. Since CBMA1 has the ability to suppress non-specific adsorption, at first glance it seems preferable to have more CBMA1 units. On the other hand, since it is thought that it is more advantageous to have fewer CBMA units for micelle formation, the results of this example suggest that forming micelles is effective in reducing non-specific adsorption.

The present disclosure can be used in a measurement device and a measurement plate that include a detection section on which a polymer film is formed.

1 Polymer membrane 2 Sensor 3 Polymer 4 Second substance 5 First substance 6 Control device 11 Measurement carrier 12 Measurement substrate 21 External terminal 22 Substrate 23 Detection section 24 Reference section 25a First IDT electrode 25b Second IDT electrode 26 Channel member 27 Supply port 28 Discharge port 31 Detection area 32 Non-detection area 100 Measuring device

Claims (12)

Two hydrophilic blocks A containing at least one structural unit (a) among the structural units (a) represented by the following formulas (1) to (6),

(In the formula, R ¹ to R ⁷ are each independently H or CH ₃ , X ¹ to X ⁴ are each independently O or NH, and p ¹ to p ⁵ are each independently 1 to 3 The following integers are shown, and q ¹ to q ² are each independently an integer between 1 and 5.)
Between the two hydrophilic blocks A, a hydrophobic block B containing at least one structural unit (b) among the structural units (b) represented by the following formulas (7) and (8),

(In the formula, R ⁸ and R ¹⁰ are each independently H or CH ₃ , R ⁹ , R ¹¹ and R ¹² are each independently an alkyl group having 1 to 6 carbon atoms, and X ⁵ and ⁶ is O or NH, and r is an integer from 1 to 3.)
The hydrophobic block B is a copolymer having a disulfide bond between the structural units (b). The copolymer according to claim 1, wherein the proportion of the structural unit (b) constituting the hydrophobic block B contained in the copolymer is 30 mol% or more and 80 mol% or less. The copolymer according to claim 1 or 2, wherein the proportion of the structural unit (a) constituting the hydrophilic block A contained in the copolymer is 20 mol% or more and 70 mol% or less. Claims 1 to 3, wherein the ratio of the number of moles of the structural unit (a) to the number of moles of the structural unit (b) contained in the copolymer is 0.20 or more and 2.5 or less. The copolymer according to any one of the items. The copolymer according to any one of claims 1 to 4, wherein at least one terminal of the copolymer has a thiol group or a dithioester group. The copolymer according to any one of claims 1 to 5, wherein the two hydrophilic blocks A have the same structure. The copolymer according to any one of claims 1 to 6, wherein the two hydrophilic blocks A have a number average degree of polymerization of 100 or more. The copolymer according to any one of claims 1 to 7, wherein the two hydrophilic blocks A have a total number average degree of polymerization of 30 or more. The copolymer according to any one of claims 1 to 8, wherein the hydrophobic block B has a number average degree of polymerization of 25 or more. A hydrophilic block A containing at least one structural unit (a) among the structural units (a) represented by the following formulas (1) to (6),

(In the formula, R ¹ to R ⁷ are each independently H or CH ₃ , X ¹ to X ⁴ are each independently O or NH, and p ¹ to p ⁵ are each independently 1 to 3 The following integers are shown, and q ¹ to q ² are each independently an integer between 1 and 5.)
A hydrophobic block B containing at least one structural unit (b) among the structural units (b) represented by the following formulas (7) and (8),

(In the formula, R ⁸ and R ¹⁰ are each independently H or CH ₃ , R ⁹ , R ¹¹ and R ¹² are each independently an alkyl group having 1 to 6 carbon atoms, and X ⁵ and ⁶ is O or NH, and r is an integer from 1 to 3.)
A polymer membrane, wherein the end of the hydrophobic block B opposite to the side bonded to the hydrophilic block A contains a copolymer having a thiol group or a dithioester group. A measuring device comprising the polymer membrane according to claim 10. A measurement carrier comprising the polymer membrane according to claim 10.

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