Copolymers, polymer membranes, measuring devices, and measuring carriers

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

[Technical Field]

[0001] This disclosure relates to copolymers used to form polymer films used in measuring devices. Furthermore, this disclosure relates to polymer films obtained using such copolymers, and measuring devices or measuring carriers equipped with such polymer films. [Background technology]

[0002] As an example of a measuring device for measuring the concentration of a target substance (e.g., biomolecules), a surface acoustic wave sensor is disclosed in Patent Document 1. Such a measuring device includes a detection unit on which a substance that interacts with the target substance contained in the sample (e.g., an antibody) is immobilized. A polymer film is often formed on the detection unit in order to immobilize the substance that interacts with the target substance contained in the sample. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2008-286606 [Overview of the Initiative]

[0004] A copolymer according to one embodiment comprises two hydrophilic blocks A, each containing at least one structural unit (a) from among the structural units (a) represented by the following formulas (1) to (6), [ka] [ka] (In the formula, R 1 ~R 7 Each of these is independently either H or CH3, and X 1 ~X 4 Each is independently O or NH, and p 1 ~p 5 Each of these is an independent integer between 1 and 3, and q 1 ~q 2is each independently an integer of 1 or more and 5 or less. Between the two hydrophilic blocks A, a hydrophobic block B containing at least one structural unit (b) represented by the following formulas (7) and (8),

Chemical formula

[0006] [Figure 1] This is a schematic diagram showing an example of a measuring device related to this disclosure. [Figure 2] This is a plan view showing the sensors included in the above measuring device. [Figure 3] Figure 1 is a conceptual diagram showing the polymer contained in the detection unit 23. [Figure 4] This is a plan view showing an example of a measuring carrier related to this disclosure. [Figure 5] This graph shows the relationship between the unit ratio (CBMA1 / BMA) and the amount of non-specific adsorption in the triblock copolymer according to this disclosure. [Figure 6] This graph shows the relationship between the average degree of polymerization of CBMA1 in the triblock copolymer relating to this disclosure and the amount of nonspecific adsorption. [Modes for carrying out the invention]

[0007] [Embodiment 1] The sensors provided in the measuring device according to this disclosure will be described below, with appropriate use of the drawings. Figure 1 shows a schematic of the sensor 2 of the measuring device 100 according to this embodiment.

[0008] The measuring device 100 can detect a specific substance (first substance) from the object to be measured (sample). The first substance 5 is, for example, a biomolecule. Examples of the biomolecule include proteins, DNA, substrates for enzyme reactions, etc. The measuring device 100 includes a sensor 2 capable of detecting the first substance and a control device 6 capable of controlling the measuring device 100.

[0009] Sensor 2 can be any sensor that utilizes, for example, elastic waves, QCM (Quartz Crystal Microbalance), SPR (Surface Plasmon Resonance), or FET (Field Effect Transistor). In other words, sensor 2 only needs to be able to convert between electrical signals and elastic waves, QCM, SPR, FET, etc. Sensor 2 according to one embodiment is a sensor that utilizes elastic waves. That is, the measuring device 100 according to one embodiment can detect changes in elastic waves based on the presence of the first substance as changes in electrical signals by using sensor 2. Sensor 2 can be manufactured by conventionally known methods, except for the polymer film 1 described below. In this case, the inspection information included in the identification information may include information specific to sensors that utilize elastic waves, such as the initial phase of the elastic wave and the orientation of the substrate 22.

[0010] 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 the external terminal 21. That is, the sensor 2 and the control device 6 can input and output electrical signals to and from each other via the external terminal 21. Therefore, the control device 6 can detect, for example, a first substance based on the electrical signal input from the sensor 2. The control device 6 may, for example, calculate the concentration of the first substance contained in the sample. Alternatively, the control device 6 may, for example, identify the first substance. The control device 6 and the external terminal 21 can be manufactured by conventionally known techniques. Furthermore, 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, they do not need to be physically connected by terminals or the like. For example, the sensor 2 and the control device 6 may be electrically connected by electromagnetic induction.

[0011] Sensor 2 may be a disposable cartridge. This eliminates the need to clean Sensor 2 after measurement, thus eliminating the influence of insufficient cleaning on the measurement results.

[0012] Figure 2 shows a plan view of sensor 2. Sensor 2 comprises a substrate 22, a detection unit 23 located on the substrate 22, a reference unit 24, a pair of first IDT (Inter Digital Transducer) electrodes 25a and a pair of second IDT electrodes 25b arranged on the substrate 22 so as to sandwich the detection unit 23. The detection unit 23, the reference unit 24, the pair of first IDT electrodes 25a, and the pair of second IDT electrodes 25b may be located on the substrate 22.

[0013] (substrate) The substrate 22 is, for example, a piezoelectric substrate. Specifically, the substrate 22 is, for example, a quartz substrate. The substrate 22 is not limited to a quartz substrate as long as it can propagate elastic waves. That is, the substrate 22 can be made of any material that can propagate elastic waves. For example, the substrate 22 may be a substrate containing a piezoelectric single crystal such as a metal such as gold, silver, copper, platinum, and aluminum, lithium tantalate, or quartz. Furthermore, the substrate 22 may be manufactured by conventionally known methods.

[0014] (Detection unit) A substance (second substance 4) that reacts with the first substance 5 is fixed in the detection unit 23. Therefore, the first substance 5 contained in the sample and the fixed second substance 4 can react in the detection unit 23. By reacting the first substance 5 and the second substance 4 in the detection unit 23, the propagation characteristics of the elastic waves of the substrate 22 change. Specifically, for example, by reacting the first substance 5 and the second substance 4 in the detection unit 23, the weight on the substrate 22 or the viscosity of the liquid in contact with the surface of the substrate 22 changes. The magnitude of these changes correlates with the amount of reaction between the first substance 5 and the second substance 4. In addition, the characteristics of the elastic waves (e.g., phase, amplitude, or period) change as they propagate through the detection unit 23. The magnitude of the change in characteristics correlates with, for example, the weight on the substrate 22 or the viscosity of the liquid in contact with the surface of the substrate 22. Therefore, the sensor 2 can detect the first substance 5 based on the change 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.

[0015] A pair of first IDT electrodes 25a can generate elastic waves between them. Of the generated elastic waves, those that propagate along the surface of the substrate 22 are also called surface acoustic waves (SAWs). The pair of first IDT electrodes 25a only need to be positioned on the substrate 22 so as to sandwich the detection unit 23. 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 unit 23 and is emitted from one of the first IDT electrodes 25a. The emitted elastic wave passes through the detection unit 23. The other first IDT electrode 25a can receive the elastic wave that has passed through the detection unit 23. The received elastic wave is converted into an electrical signal. The pair of first IDT electrodes 25a may be made of a metallic material such as gold, chromium, or titanium. Furthermore, the pair of first IDT electrodes 25a may be single-layer electrodes made of a single material, or multi-layer electrodes made of multiple materials.

[0016] Sensor 2 may have two or more combinations of a detection unit 23 and a pair of first IDT electrodes 25a. In this case, the measuring device 100 may, for example, detect different types of target substances for each combination. Alternatively, the measuring device 100 may, for example, detect the same type of target substance in multiple combinations and compare the detection results of each.

[0017] Figure 3 shows a conceptual diagram of the polymer film 1 provided in the detection unit 23. In the detection unit 23, the polymer film 1 containing polymer 3 is fixed on the substrate 22. Furthermore, a substance (second substance 4) that reacts with the first substance 5 is fixed on the polymer film 1.

[0018] Polymer film 1 is a film that has been adjusted to have high specific adsorption properties, and is a film that has been adjusted to reduce nonspecific adsorption.

[0019] (Substance 1 and Substance 2) The reaction between the first substance 5 and the second substance 4 can be any reaction that causes a change in the output of sensor 2. Such a reaction may be one in which the first substance 5 and the second substance 4 combine through redox reactions, enzymatic reactions, antigen-antibody reactions, chemiadsorption, intermolecular interactions, or intermolecular interactions. Alternatively, the reaction between the first substance 5 and the second substance 4 may be a reaction that generates a new substance (third substance) through an enzymatic reaction or the like.

[0020] The second substance 4, which is immobilized in the detection unit 23, can be appropriately selected according to the first substance 5. For example, if the first substance 5 is a specific protein, DNA, or cell in the sample, the second substance 4 may be an antibody, peptide, or aptamer. Also, for example, if the first substance 5 is an antibody, the second substance 4 may be an antigen. Also, for example, if the first substance 5 is a substrate, the second substance 4 may be an enzyme.

[0021] The measuring device 100 may indirectly detect the target substance 5. 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 that uses the first substance 5 as an antigen may be reacted with the first substance 5 in advance, and the antibody that did not react may be reacted with the fixed second substance 4. In this case, 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 antibody detected.

[0022] (Triblock copolymer) Next, we will describe a triblock copolymer of the present disclosure, which is an example of a copolymer used to form the polymer film 1.

[0023] The triblock copolymer of this disclosure is a copolymer comprising two hydrophilic blocks A and a hydrophobic block B between the two hydrophilic blocks.

[0024] <Hydrophilic Block A> The hydrophilic block A includes at least one structural unit (a) from among the structural units (a) represented by the following formulas (1) to (6). [ka] [ka]

[0025] In the formula, R 1 ~R 7 Each of these is independently either H or CH3. 1 ~X 4 Each is independently O or NH, p 1 ~p 5 Each of these is an independent integer between 1 and 3 (1, 2, or 3). 1 ~q 2 Each of these is an independent integer between 1 and 5 (1, 2, 3, 4, or 5).

[0026] In terms of further reducing nonspecific adsorption, structural unit (a) may have a betaine structure. Also, in terms of facilitating the immobilization of antibodies, etc., structural unit (a) may have a COO structure. - It may have a base.

[0027] Examples of structural units (a) include the following: [ka] [ka] [ka] [ka]

[0028] The proportion of structural units (a) constituting hydrophilic block A in the triblock copolymer of this disclosure may be 20 mol% or more, 30 mol% or more, or 40 mol% or more, in terms of reducing nonspecific adsorption. Furthermore, the above proportion may be 70 mol% or less, 60 mol% or less, or 50 mol% or less.

[0029] The hydrophilic block A may have structural units other than structural unit (a) to the extent that it does not impair the effects of the present disclosure.

[0030] If hydrophilic block A contains two or more structural units, each structural unit may be included in block A by any of the following methods: random copolymerization, block copolymerization, etc.

[0031] The two hydrophilic blocks A contained in the triblock copolymer of this disclosure may or may not have the same structure. The two hydrophilic blocks A having the same structure means that they have equivalent structural units (a) to the extent that the effects of this disclosure are not impaired. Specifically, equivalent structural units (a) may be, for example, the same structural units (a), and they may have structural units other than structural units (a). Furthermore, equivalent structural units (a) may be, for example, the same structure composed of structural units (a), such that the relative positions and number-average degrees of polymerization of structural units (a) are the same.

[0032] <Hydrophobic Block B> The hydrophobic block B includes at least one structural unit (b) from among the structural units (b) represented by the following formulas (7) and (8). [ka]

[0033] In the formula, R 8 and R 10 Each of these is independently either H or CH3. 9 , R 11 and R 12Each of these is an alkyl group having between 1 and 6 carbon atoms. 5 and X 6 The expression is O or NH. r is an integer between 1 and 3 (1, 2, or 3).

[0034] Examples of alkyl groups having 1 to 6 carbon atoms include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, heptyl, and hexyl groups.

[0035] Examples of structural units (b) include the following: [ka] [ka]

[0036] The proportion of structural units (b) constituting hydrophobic block B in the triblock copolymer of this disclosure may be 30 mol% or more, 40 mol% or more, or 50 mol% or more, in terms of the ease of micelle formation of the polymer. Furthermore, the above proportion may be 80 mol% or less, 60 mol% or less, or 50 mol% or less.

[0037] The hydrophobic block B may have structural units other than structural unit (b) to the extent that it does not impair the effects of the present disclosure.

[0038] If hydrophobic block B contains two or more structural units, each structural unit may be included in block B by any of the following methods: random copolymerization, block copolymerization, etc.

[0039] Hydrophobic block B has disulfide bonds between structural units (b). There may be one or more disulfide bonds.

[0040] At least one end of the triblock copolymer of this disclosure may have a thiol group or a dithioester group, or it may be a thiol group, in terms of ease of immobilizing the polymer film onto the substrate 22 (measurement substrate 12).

[0041] The ratio of moles of structural unit (a) to moles of structural unit (b) in the triblock copolymer of this disclosure may be 0.20 or more, 0.50 or more, or 0.70 or more, in terms of reducing nonspecific adsorption and facilitating micelle formation of the copolymer. Furthermore, the above ratio may be 2.5 or less, 2.0 or less, or 1.5 or less.

[0042] The number-average degree of polymerization of the triblock copolymer of this disclosure may be 100 or more, or 200 or more, in terms of ease of micelle formation and film formation density of the copolymer.

[0043] The total number-average degree of polymerization of the two hydrophilic blocks A in the triblock copolymer of this disclosure may be 30 or more, 60 or more, 100 or more, 200 or less, or 150 or less, in terms of reducing nonspecific adsorption.

[0044] The number-average degree of polymerization of each hydrophobic block B in the triblock copolymer of this disclosure may be 25 or more, 30 or more, 50 or more, or 80 or more, depending on the ease of micelle formation of the copolymer.

[0045] The triblock copolymers of this disclosure may be identified by conventionally known organic analytical methods. For example, the copolymer may be identified by NMR (Nuclear Magnetic Resonance), or by liquid chromatography, or by infrared spectroscopy. In other words, when identifying the copolymer, any apparatus capable of performing these methods should be used. The identification methods and apparatus are not limited to these, as long as they are capable of identifying the copolymer.

[0046] <Method for producing triblock copolymer>

[0047] An example of a method for producing the triblock copolymer of this disclosure includes a synthesis step of hydrophobic block B (Step 1) and a synthesis step of hydrophilic block A (Step 2). Each step is described below.

[0048] <Step 1: Synthesis process of hydrophobic block B> In this process, hydrophobic block B is synthesized by polymerizing hydrophobic monomer compounds. Examples of hydrophobic monomer compounds include the following compounds. Specific examples of hydrophobic monomer compounds include butyl methacrylic acid (BMA). Hydrophobic monomer compounds may be used individually or in combination of two or more types.

[0049] [ka] (In the formula, R 8 ~R 12 , X 5 and X 6 Furthermore, r is the R in equations (7) and (8), respectively. 8 ~R 12 , X 5 and X 6 (This is synonymous with...)

[0050] Polymerization can be carried out by known polymerization methods 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 catalytic polymerization (RTCP), and reversible addition-fragmentation chain transfer polymerization (RAFT).

[0051] Examples of polymerization initiators include those containing disulfide bonds, such as bis[2-(2'-bromoisobutyryloxy)ethyl]disulfide (BiBOEDS).

[0052] <Step 2: Synthesis process of hydrophilic block A> In this process, hydrophilic monomer compounds are polymerized onto the polymer obtained in step 1 to synthesize hydrophilic blocks A at each end of hydrophobic block B, thereby obtaining the triblock copolymer of this disclosure. Examples of hydrophilic monomer compounds include the following compounds (11) to (16). Specific examples of hydrophilic monomer compounds include N-(carboxymethyl)-N,N-dimethyl-2-[(2-methyl-1-oxo-2-propen-1-yl)-oxy]ethanaminonium (CBMA1), N,N-dimethylaminoethyl methacrylate (DMAEMA), 2-hydroxypropyl methacrylamide (HPMA), etc. Hydrophilic monomer compounds may be used individually or in combination of two or more. [ka] [ka] (In the formula, R 1~R 7 , X 1 ~X 4 , p 1 ~p 5 and q 1 ~q 2 These are R in equations (1) to (6), respectively. 1 ~R 7 , X 1 ~X 4 , p 1 ~p 5 and q 1 ~q 2 (This is synonymous with...)

[0053] Furthermore, the method for producing the triblock copolymer according to this disclosure may include a step of forming a quaternary ammonium salt (step 3) and a deprotection step (step 4).

[0054] <Step 3: Quaternary ammonium treatment of hydrophilic block A> In this step, the tertiary amine structure of structural unit (a) of hydrophilic block A of the polymer obtained in step 2 is converted to a quaternary ammonium structure (quaternary ammonium conversion). For example, quaternary ammonium conversion may be carried out using a known quaternizing agent such as a halogen compound.

[0055] <Step 4: Betaine formation process of hydrophilic block A> In this step, a betaine structure is formed on the structural unit (a) of the hydrophilic block A of the polymer obtained in step 3 (betaine formation). For example, betaine formation may be carried out by deprotecting the protecting group (e.g., tert-butyl group) of the side chain of the hydrophilic block A introduced in step 3.

[0056] By performing steps 3 and 4, nonspecific adsorption can be further reduced.

[0057] If necessary, structures derived from the polymerization initiator may be removed from the manufactured triblock copolymer. An example of such a structure is the terminal bromine of the triblock copolymer when BiBOEDS is used as the polymerization initiator in LRP.

[0058] (polymer membrane) The polymer film of this disclosure comprises a copolymer containing a hydrophilic block A and a hydrophobic block B. The copolymer is a diblock copolymer, and this diblock copolymer is also included in one embodiment of this disclosure. The diblock copolymer of this disclosure corresponds to polymer 3 in Figure 3.

[0059] <Diblock copolymer> In the diblock copolymer of this disclosure, the end of hydrophobic block B opposite to the side bonded to hydrophilic block A has a thiol group or a dithioester group. The constituent units and specific examples of hydrophilic block A and hydrophobic block B of the diblock copolymer of this disclosure are the same as the constituent units and specific examples of hydrophilic block A and hydrophobic block B of the triblock copolymer of this disclosure.

[0060] The ratio of moles of structural unit (a) to moles of structural unit (b) in the diblock copolymer of this disclosure may be 0.20 or more, 0.50 or more, or 0.70 or more, in terms of reducing nonspecific adsorption and film formation density. Furthermore, the above ratio may be 2.5 or less, 2.0 or less, or 1.5 or less.

[0061] The number-average degree of polymerization of the diblock copolymer of this disclosure may be 25 or more, 50 or more, or 200 or more in terms of film formation density. When the first substance 5 is a substance contained in urine, the diblock copolymer of this disclosure may have a high molecular weight.

[0062] The number-average degree of polymerization of hydrophilic block A in the diblock copolymer of this disclosure may be 20 or more, 40 or more, 60 or more, 80 or more, 100 or more, or 200 or less, in terms of reducing nonspecific adsorption.

[0063] The number-average degree of polymerization of hydrophobic blocks B in the diblock copolymer of this disclosure may be 25 or higher, 30 or higher, 50 or higher, or 80 or higher, in terms of film formation density.

[0064] The diblock copolymers of this disclosure may be identified by conventionally known organic analytical methods, similar to the triblock copolymers of this disclosure.

[0065] The diblock copolymers of this disclosure are obtained by cleaving the disulfide bonds of the triblock copolymer or micelles of the copolymer. Disulfide bond cleavage can be performed using conventionally known reducing agents.

[0066] <Method for manufacturing polymer films> An example of a method for producing the polymer film of the present disclosure includes the steps of: preparing a coating agent containing micelles of the triblock copolymer of the present disclosure; placing the coating agent on a substrate; and cleaving the disulfide bonds of the micelles of the triblock copolymer of the present disclosure to form a diblock copolymer.

[0067] Micelles of the triblock copolymer of the present disclosure are also included in one aspect of the present disclosure. Such micelles are flower-type micelles.

[0068] Furthermore, an example of a method for producing the polymer film of the present disclosure includes the steps 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, and placing the coating agent on a substrate.

[0069] Micelles of diblock copolymers obtained by cleaving the disulfide bonds of the triblock copolymers of the present disclosure are also included in one aspect of the present disclosure. These micelles are W / O type micelles.

[0070] A polymer film formed by a diblock copolymer obtained by cleaving the disulfide bonds after forming micelles of the triblock copolymer of this disclosure exhibits high density. Furthermore, high density of the polymer film can also be achieved by using micelles of the diblock copolymer obtained by cleaving the disulfide bonds of the triblock copolymer of this disclosure. In addition, a polymer film formed by a diblock copolymer obtained from the triblock copolymer of this disclosure exhibits reduced non-specific adsorption.

[0071] Methods for immobilizing the diblock copolymer of this disclosure onto a substrate 22 include, for example, dissolving the polymer 3 in a solvent to obtain a polymer solution, applying it to the substrate 22 and drying it, graft polymerization by radiation or ultraviolet light, and chemical reactions with functional groups of the substrate 22. By these methods, a polymer film 1 composed of the polymer 3 is formed on the substrate 22.

[0072] One method for immobilizing the second substance 4 onto the polymer 3 (polymer film 1) is to covalently bond the second substance 4 to the carboxyl groups of the polymer 3. For example, the polymer 3 is reacted with N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (NHS / EDC activation). The carboxyl groups of the polymer 3 are then replaced with NHS ester groups. By reacting the amino groups of the second substance 4 with these activated NHS ester groups, the second substance 4 is immobilized onto the polymer 3 (polymer film 1). The NHS / EDC activation of the polymer 3 may be performed before immobilization on the substrate 22 or after immobilization on the substrate 22. Since the polymer of this embodiment has high chemical stability, decomposition is unlikely to occur due to NHS / EDC activation. Therefore, nonspecific adsorption caused by decomposition products associated with NHS / EDC activation can be reduced.

[0073] [Embodiment 2] The following describes a measurement carrier according to one embodiment, with the use of drawings as appropriate. For the sake of explanation, components having the same function as those described in the above embodiment will be denoted by the same reference numerals, and their descriptions will not be repeated. Figure 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 assay).

[0074] 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 blocking agents, etc., on the surface of the measurement substrate 12. A polymer film 1 is fixed to the detection region 31.

[0075] On the polymer film 1, a second substance 4 that reacts with the first substance 5 is immobilized, similar to the polymer film 1 described in Embodiment 1. In the non-detection region 32, a film adjusted to enhance non-specific adsorption may be immobilized.

[0076] (Example of use of measuring carrier) First, the desired second substance 4 is immobilized on the polymer film 1 of the detection region 31. A blocking agent is also non-selectively adsorbed onto the non-detection region 32. Then, by bringing the sample into contact with the detection region 31, the target substance 5 contained in the sample reacts with the second substance 4, and this reaction is detected by a detection reagent. Examples of detection reagents include redox substances, fluorescent substances, enzymes, and dye compounds.

[0077] (Measurement board) The measurement substrate 12 may be, for example, a metal such as gold, silver, copper, platinum, and aluminum; a plastic such as polyethylene and polypropylene; or an inorganic material such as titanium oxide, silica, glass, and ceramic. The measurement substrate 12 is not limited to these examples.

[0078] The shape of the measurement substrate 12 may be, for example, a plate, particles, microstructures, or a microtiter plate. The shape of the measurement substrate 12 is not limited to these examples.

[0079] (Measurement Kit) A measurement kit comprising a measurement substrate 12 on which a polymer film 1 is immobilized, a second substance 4, and a detection reagent is also included in the scope of this disclosure. The second substance 4 may be pre-immobilized on the polymer film 1 during the manufacturing of the product, or it may be immobilized by the user before measurement.

[0080] The measurement kit according to this embodiment may include other reagents and instruments. For example, it may include components other than the second substance 4 and the detection reagent described above. It may also include buffers, etc. Furthermore, the measurement kit according to this embodiment may contain a mixture of several different reagents in appropriate volumes and / or forms, or each may be provided in a separate container. The measurement kit according to this embodiment may also include instructions describing the procedure for obtaining detection of the reaction between the first substance 5 and the second substance 4. These instructions may be written or printed on paper or other media, or attached to electronic media such as magnetic tape, a computer-readable disk, or a CD-ROM.

[0081] 〔summary〕 The copolymer according to Embodiment 1 of the present disclosure comprises two hydrophilic blocks A, each containing at least one structural unit (a) from among the structural units (a) represented by the following formulas (1) to (6), [ka] [ka] (In the formula, R 1 ~R 7 Each of these is independently either H or CH3, and X 1 ~X 4 Each is independently O or NH, and p 1 ~p 5 Each of these is an independent integer between 1 and 3, and q 1 ~q 2 Each of these is an independent integer between 1 and 5 (inclusive). Between the two hydrophilic blocks A, there is a hydrophobic block B which contains at least one structural unit (b) from among the structural units (b) represented by the following formulas (7) and (8), [ka] (In the formula, R 8 and R 10 Each of these is independently either H or CH3, and R 9 , R 11 and R 12 Each of these is an alkyl group having 1 to 6 carbon atoms, and X 5 and X 6 (where is O or NH, and r is an integer between 1 and 3, inclusive.) The hydrophobic block B has disulfide bonds between its structural units (b).

[0082] In the copolymer according to embodiment 2 of the present disclosure, the proportion of the structural unit (b) constituting the hydrophobic block B contained in the copolymer may be 30 mol% or more and 80 mol% or less.

[0083] In the copolymer according to embodiment 3 of the present disclosure, the proportion of the structural unit (a) constituting the hydrophilic block A contained in the copolymer may be 20 mol% or more and 70 mol% or less in embodiment 1 or 2.

[0084] In the copolymer according to embodiment 4 of the present disclosure, in any of embodiments 1 to 3, the ratio of moles of structural unit (a) to moles of structural unit (b) contained in the copolymer may be 0.20 or more and 2.5 or less.

[0085] In any of embodiments 1 to 4, the copolymer according to embodiment 5 of the present disclosure may have at least one end of the copolymer having a thiol group or a dithioester group.

[0086] The polymer film according to Aspect 6 of the present disclosure includes a hydrophilic block A containing at least one structural unit (a) among the structural units (a) represented by the following formulas (1) to (6), [Chemical formula] [Chemical formula] (In the formula, R 1 ~R 7 are each independently H or CH3, X 1 ~X 4 are each independently O or NH, p 1 ~p 5 are each independently an integer of 1 or more and 3 or less, q 1 ~q 2 are each independently an integer of 1 or more and 5 or less.) and a hydrophobic block B containing at least one structural unit (b) among the structural units (b) represented by the following formulas (7) and (8). <0> [Chemical formula] (In the formula, R 8 and R 10 are each independently H or CH3, R 9 , R 11 and R 12 are each independently an alkyl group having 1 to 6 carbon atoms, X 5 and X 6 [[ID=]](51) is O or NH, and r is an integer of 1 or more and 3 or less.) The polymer film includes a copolymer in which 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.

[0087] The measuring device according to Aspect 7 of the present invention includes the polymer film of Aspect 6.

[0088] The measuring carrier according to Aspect 8 of the present invention includes the polymer film of Aspect 6.

[0089] As described above, the invention according to the present disclosure has been described based on the drawings and examples. However, the invention according to the present disclosure is not limited to each of the above-described embodiments. That is, the invention according to the present disclosure can be variously modified within the scope shown in the present disclosure, and embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the invention according to the present disclosure. In other words, it should be noted that those skilled in the art can easily make various modifications or corrections based on the present disclosure. Also, it should be noted that these modifications or corrections are included in the scope of the present disclosure.

Example

[0090] In the following examples, unless otherwise specified, % represents mass %.

[0091] 〔Example 1〕Production of triblock copolymer <Step 1. Synthesis of poly(butyl methacrylate) <p(BMA)>> According to the following scheme, p(BMA) corresponding to the hydrophobic block B was synthesized.

Chemical formula

[0092] p(BMA) was synthesized by the SET-LRP method. In 2-propanol that had been deoxygenated beforehand, 2.08 mol / L of the monomer butyl methacrylic acid (BMA), 20.8 mmol / L of bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide (BiBOEDS), 2.08 mmol / L of copper(II) bromide, and 15.0 mmol / L of N,N,N',N'',N''-pentamethyldiethylenetriamine (PMDETA) were dissolved. Next, the resulting solution was added to copper wire that had been reduced with hydrazine, and the reaction was carried out under nitrogen at 40°C for 6 hours. The amount of copper wire was such that, assuming complete dissolution, the concentration would be 305 mmol / L. After the reaction, the reaction solution was filtered and added to a large amount of methanol (15-20 times the amount of the polymerization solution). The precipitate was then filtered and dried to obtain p(BMA). The degree of polymerization of the obtained p(BMA) was calculated by nuclear magnetic resonance (NMR) spectroscopy. Specifically, 1 The degree of polymerization was calculated from the ratio of the area of ​​the peak originating from the methylene group bonded to the sulfur in BiBOEDS (peak around 2.9 ppm) and the area of ​​the peak originating from the oxygen-bonded side chain (methylene group) in the BMA unit of the polymer (peak around 3.90 ppm to 4.00 ppm) in the 1H-NMR spectrum. As a result, the degree of polymerization of the obtained p(BMA) was estimated to be 52.

[0093] <Step 2. Polymerization of N,N-dimethylaminoethyl methacrylate (DMAEMA)> DMAEMA was polymerized according to the following scheme. [ka]

[0094] Polymerization of DMAEMA was carried out by the SET-LRP method using p(BMA) synthesized in Step 1 as a macroinitiator. In 2-propanol that had been deoxygenated beforehand, DMAEMA monomer was dissolved at a concentration of 1.50 mol / L, p(BMA) synthesized in Step 1 at a concentration of 15.0 mmol / L, copper(II) bromide at 1.50 mmol / L, and PMDETA at 10.8 mmol / L. Next, the resulting solution was added to copper wire that had been reduced with hydrazine, and the reaction was carried out under nitrogen at 40°C for 8 hours. The amount of copper wire was such that, assuming complete dissolution, the concentration would be 220 mmol / L. After the reaction, 45°C hot water was added. The precipitate was then 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, 1 The ratio was calculated from the area of ​​the peak around 1.5 ppm originating from the BMA unit in the 1H-NMR spectrum and the area of ​​the peak around 2.3 ppm originating from the DMAEMA unit of the polymer. As a result, the ratio of BMA units to DMAEMA units (BMA:DMAEMA) was estimated to be 52:42.

[0095] <Step 3. Quaternary ammonium conversion of DMAEMA units> The side chains of the DMAEMA unit were quaternarily ammoniumated according to the following scheme. [ka]

[0096] The p(BMA)-bp(DMAEMA) synthesized in Step 2 was dissolved in 2-propanol to a concentration of 500 mmol / L of DMAEMA units. To this solution, tert-butyl bromoacetate was added to a concentration of 750 mmol / L, and the reaction was carried out at room temperature for 20 hours. The reaction was then stopped by adding n-hexane. The precipitate was then filtered and dried to quaternary ammonium formation of the DMAEMA unit side chains. Nuclear magnetic resonance (NMR) spectroscopy revealed the following: 1A peak originating from the tert-butyl group was observed in the 1H-NMR spectrum, confirming the quaternary ammonium formation of the DMAEMA unit side chain.

[0097] <Step 4. Betaineization of DMAEMA units> The side chains of the DMAEMA unit were betained according to the following scheme. [ka] The block copolymer obtained in Step 3, in which the side chains of the DMAEMA units were quaternarily ammoniumlated, was dissolved in trifluoroacetic acid and reacted at room temperature for 20 hours. After the reaction, trifluoroacetic acid was removed by evaporation, and the polymer powder was recovered. The polymer powder was dissolved in methanol, diethyl ether was added to reprecipitate, and the precipitate was filtered and dried to obtain the polymer powder. Nuclear magnetic resonance (NMR) spectroscopy revealed that 1 In the 1H-NMR spectrum, the peak originating from the tert-butyl group had disappeared. NMR spectroscopy confirmed that the tert-butyl group was deprotected, yielding the desired triblock copolymer, p(BMA)-bp(CBMA1).

[0098] 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 hydrophilic block A, and "p(BMA)-SS-p(BMA)" is hydrophobic block B. In hydrophobic block B, "SS" represents a disulfide bond.

[0099] The degree of polymerization of each block in the triblock copolymer obtained by the above synthesis scheme was 42-52-SS-52-42. That is, the above synthesis scheme yielded an ABA-type triblock copolymer containing a hydrophobic block B with a degree of polymerization of 104 and a disulfide bond, between two hydrophilic blocks A with a degree of polymerization of 42.

[0100] In addition, a triblock copolymer (p(CBMA1)-p(BMA)-SS-p(BMA)-p(CBMA1)) with 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

[0101] Furthermore, a triblock copolymer (p(HPMA)-p(BMA)-SS-p(BMA)-p(HPMA)) was also produced according to the following scheme. [ka]

[0102] The polymerization of HPMA was carried out by the SET-LRP method, using p(BMA), which is an 86-mer p(BMA) unit synthesized in the same manner as in Step 1, as a macroinitiator. In 2-propanol that had been deoxygenated beforehand, 2.00 mol / L of the monomer HPMA, 10.0 mmol / L of the p(BMA) synthesized in Step 1, 2.00 mmol / L of copper(II) bromide, and 15.0 mmol / L of Me6TREN (tris[2-(dimethylamino)ethylamine) were dissolved. Next, the resulting solution was added to copper wire that had been reduced with hydrazine, and the reaction was carried out under nitrogen at 40°C for 91 hours. The amount of copper wire was such that, assuming complete dissolution, the concentration would be 305 mmol / L. After the reaction, water was added to the soluble part of the solution. The soluble part was then removed by a gradient 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, 1The ratio was calculated from the area of ​​the peak around 4 ppm originating from the BMA unit in the 1H-NMR spectrum and the area of ​​the peak around 2.9 ppm originating from the HPMA unit of the polymer. As a result, the ratio of BMA units to HPMA units (BMA:HPMA) was estimated to be 86:14.

[0103] [Example 2] Measurement of non-specific adsorption amount The amount of nonspecific adsorption of fetal bovine serum onto the polymer membrane was measured under the following conditions. Measurement device: GE Healthcare Biacore X100 Measurement conditions: Running buffer: HBS-P Temperature: 25℃ Flow rate: 10μL / min Sample contact time: 9 minutes (Sample injection volume: 90 μL)

[0104] Using an SPR tip with a polymer membrane consisting of p(CBMA1)-p(BMA)-SS-p(BMA)-p(CBMA1) formed on its surface, the difference between the SPR signal value after running buffer is flowed for 9 minutes after sample injection and the SPR signal value before sample injection is defined as the non-specific adsorption amount, and 10 RU equals 1 ng / cm³. 2 That's how it was converted.

[0105] The conditions for creating SPR tips with polymer films are as follows: 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 tips were washed with ethanol and ultrapure water. Then, the tips were immersed for 18 hours in a methanol solution in which triblock copolymer was dissolved to a concentration of 0.3 mg / mL to form a polymer film. After that, the tips were washed with ultrapure water and dried under a nitrogen stream to obtain SPR tips with polymer films.

[0106] The measurement results for non-specific adsorption amounts are shown in Table 1 and Figures 5-6. Figure 5 is a graph showing the relationship between the unit ratio (CBMA1 / BMA) of the triblock copolymer and the amount of non-specific adsorption. Figure 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.

[0107] [Table 1]

[0108] The non-specific adsorption capacity of the polymer film is 20 ng / cm³. 2 The following conditions are preferred, and it is known that the higher the film formation density, the lower the amount of nonspecific adsorption. From the results in Table 1 and Figures 5-6, it was found that when the unit ratio of the triblock copolymer (CBMA1 / BMA) is 0.20 or more and 2.5 or less, the amount of nonspecific adsorption is reduced, and a unit ratio of 1 or less is even more preferable. Furthermore, it was found that the number-average degree of polymerization of CBMA1 is preferably up to about 70, and even more preferably around 20-40. Since CBMA1 has the ability to suppress nonspecific adsorption, it might seem at first glance that a higher number of CBMA1 units is preferable. On the other hand, it is considered that a lower number of CBMA1 units is advantageous for micelle formation, so the results of this example suggest that micelle formation is effective in reducing nonspecific adsorption. [Industrial applicability]

[0109] This disclosure can be used in measuring devices and measuring plates that include a detection unit on which a polymer film is formed. [Explanation of Symbols]

[0110] 1 Polymer membrane 2 sensors 3 Polymers 4 Second substance 5 First substance 6. Control device 11 Measuring Carrier 12 Measurement board 21 External terminals 22 circuit boards 23 Detection unit 24 Reference section 25a 1st IDT electrode 25b 2nd IDT electrode 26 Flow channel members 27 Supply port 28 Outlet 31 Detection area 32 Non-detection areas 100 measuring devices

Claims

1. A copolymer, Two hydrophilic blocks A, each containing at least one structural unit (a) from among the structural units (a) represented by the following formulas (1) to (6), 【Chemistry 1】 【Chemistry 2】 (In the formula, R １ ~R ７ Each of these is independently H or CH ３ X １ ~X ４ Each is independently O or NH, and p １ ~p ５ Each of these is an independent integer between 1 and 3, and q １ Each of the variables ~q3 is an independent integer between 1 and 5 (inclusive). Between the two hydrophilic blocks A, there is a hydrophobic block B which includes at least one structural unit (b) from among the structural units (b) represented by the following formulas (7) and (8), 【Transformation 3】 (wherein, 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, X ５ and X ６ are O or NH, and r is an integer of 1 or more and 3 or less.) The hydrophobic block B has disulfide bonds between the structural units (b), A copolymer in which the ratio of moles of structural unit (a) to moles of structural unit (b) contained in the copolymer is 0.20 or more and 2.5 or less.

2. 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.

3. 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.

4. The copolymer according to claim 1 or 2, wherein at least one end of the copolymer has a thiol group or a dithioester group.

5. The copolymer according to claim 1 or 2, wherein the two hydrophilic blocks A have the same structure as each other.

6. The copolymer according to claim 1 or 2, wherein the two hydrophilic blocks A have a number-average degree of polymerization of 100 or more.

7. The copolymer according to claim 1 or 2, wherein the two hydrophilic blocks A have a combined number-average degree of polymerization of 30 or more.

8. The copolymer according to claim 1 or 2, wherein the hydrophobic block B has a number-average degree of polymerization of 25 or more.

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