WO2021177201A1 - 共重合体およびその製造方法、測定装置ならびに測定用担体 - Google Patents

共重合体およびその製造方法、測定装置ならびに測定用担体 Download PDF

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WO2021177201A1
WO2021177201A1 PCT/JP2021/007608 JP2021007608W WO2021177201A1 WO 2021177201 A1 WO2021177201 A1 WO 2021177201A1 JP 2021007608 W JP2021007608 W JP 2021007608W WO 2021177201 A1 WO2021177201 A1 WO 2021177201A1
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Prior art keywords
polymer
copolymer
structural unit
substance
formula
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English (en)
French (fr)
Japanese (ja)
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安藤　剛
敦郎 村瀬
秀治 栗岡
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Kyocera Corp
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Kyocera Corp
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Priority to US17/908,834 priority Critical patent/US20230110097A1/en
Priority to EP21764970.6A priority patent/EP4116340A4/en
Priority to CN202180018439.5A priority patent/CN115244094B/zh
Priority to JP2022504338A priority patent/JP7333462B2/ja
Publication of WO2021177201A1 publication Critical patent/WO2021177201A1/ja
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
    • G01N29/02—Analysing fluids
    • G01N29/022—Fluid sensors based on microsensors, e.g. quartz crystal-microbalance [QCM], surface acoustic wave [SAW] devices, tuning forks, cantilevers, flexural plate wave [FPW] devices
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/10—Esters
    • C08F220/34—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate
    • C08F220/36—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate containing oxygen in addition to the carboxy oxygen, e.g. 2-N-morpholinoethyl (meth)acrylate or 2-isocyanatoethyl (meth)acrylate
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/02—Alkylation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/02—Indexing codes associated with the analysed material
    • G01N2291/025—Change of phase or condition
    • G01N2291/0255—(Bio)chemical reactions, e.g. on biosensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/02—Indexing codes associated with the analysed material
    • G01N2291/025—Change of phase or condition
    • G01N2291/0256—Adsorption, desorption, surface mass change, e.g. on biosensors
    • G01N2291/0257—Adsorption, desorption, surface mass change, e.g. on biosensors with a layer containing at least one organic compound

Definitions

  • the present disclosure relates to a copolymer for forming a polymer film used in a measuring device, a method for producing the same, a measuring device provided with the polymer film, and a carrier for measurement.
  • a surface acoustic wave sensor is disclosed in Patent Document 1 as an example of a measuring device for measuring the concentration of a substance to be measured (for example, a biomolecule).
  • a measuring device for measuring the concentration of a substance to be measured (for example, a biomolecule).
  • a measuring device includes a detection unit in which a substance (for example, an antibody) that interacts with a substance to be measured contained in a sample is fixed.
  • a polymer film is often formed on the detection unit in order to fix a substance that interacts with the substance to be measured contained in the sample.
  • the polymer film formed in the detection part of the measuring device is required to be able to fix the above-mentioned interacting substances and to have high chemical stability.
  • the copolymer according to one embodiment is a copolymer containing a first structural unit represented by the following formula (1) and a second structural unit represented by the following formula (2).
  • R 1 is a hydrogen atom or a methyl group.
  • R 2 and R 3 are independently hydrogen atoms or alkyl groups having 1 to 4 carbon atoms.
  • x1 is an integer of 1 to 4.
  • R 4 is a hydrogen atom or a methyl group.
  • R 5 and R 6 are independently hydrogen atoms or alkyl groups having 1 to 4 carbon atoms.
  • x2 is an integer of 1 to 4, and y is an integer of 3 to 7.
  • the method for producing a copolymer according to an embodiment is a method for producing a copolymer containing a first structural unit represented by the following formula (1) and a second structural unit represented by the following formula (2).
  • R 1 is a hydrogen atom or a methyl group.
  • R 2 and R 3 are independently hydrogen atoms or alkyl groups having 1 to 4 carbon atoms.
  • x1 is an integer of 1 to 4.
  • R 4 is a hydrogen atom or a methyl group.
  • R 5 and R 6 are independently hydrogen atoms or alkyl groups having 1 to 4 carbon atoms.
  • x2 is an integer of 1 to 4, and y is an integer of 3 to 7.
  • FIG. 1 shows an outline of the sensor 2 of the measuring device 100 according to the present embodiment.
  • the measuring device 100 can detect a specific substance (first substance) as a target from the measurement target (sample).
  • the first substance 5 is, for example, a substance in the living body. Examples of the above-mentioned substances in the living body include proteins, DNAs, substrates for enzymatic reactions, and the like.
  • 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.
  • the sensor 2 may be, for example, a sensor that uses elastic waves, QCM (Quartz Crystal Microbalance), SPR (Surface Plasma Resonance), FET (Field Effect Transistor), or the like. That is, it suffices that the sensor 2 can mutually convert an electric signal, an elastic wave, a QCM, an SPR, a FET, and the like.
  • the sensor 2 according to one embodiment is a sensor that uses elastic waves. That is, the measuring device 100 according to the embodiment can detect the change of the elastic wave due to the presence of the first substance as the change of the electric signal by using the sensor 2.
  • 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, and the like. , Information specific to sensors that utilize elastic waves may be included.
  • the sensor 2 has an external terminal 21.
  • the sensor 2 can be electrically connected to the 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, the first substance based on the electric signal input from the sensor 2.
  • the control device 6 may calculate, for example, 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 terminal 21 may be manufactured by a conventionally known technique. Further, the configuration for electrically connecting the sensor 2 and the control device 6 is not limited to the external terminal 21. For example, if the sensor 2 and the control device 6 can be electrically connected, the sensor 2 and the control device 6 do not have to be physically connected by 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 the measurement becomes unnecessary, and the influence of insufficient cleaning on the measurement result can be eliminated.
  • FIG. 2 shows a plan view of the sensor 2.
  • the sensor 2 includes a substrate 22, a detection unit 23 located on the substrate 22, a reference unit 24, and a pair of first IDT (Inter Digital Transducer) electrodes 25a arranged so as to sandwich the detection unit 23 on the substrate 22. , A pair of second IDT electrodes 25b.
  • 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.
  • the substrate 22 is, for example, a substrate having piezoelectricity.
  • the substrate 22 is, for example, a crystal substrate.
  • the substrate 22 is not limited to a quartz substrate as long as it can propagate elastic waves. That is, the substrate 22 may be made of any material capable of propagating elastic waves.
  • the substrate 22 may be a substrate containing a metal such as gold, silver, copper, platinum and aluminum, and a single crystal having piezoelectricity such as lithium tantalate and quartz. Further, the substrate 22 may be manufactured by a conventionally known method.
  • a substance (second substance) that reacts with the first substance 5 is fixed to the detection unit 23. Therefore, in the detection unit 23, the first substance contained in the sample and the fixed second substance can react with each other.
  • the detection unit 23 changes the propagation characteristics of elastic waves of the substrate 22 by reacting the first substance with the second substance. Specifically, the detection unit 23, for example, reacts the first substance with the second substance to change the weight applied to the substrate 22 or the viscosity of the liquid in contact with the surface of the substrate 22. The magnitude of these changes correlates with the amount of reaction between the first substance and the second substance. Further, the characteristics of elastic waves (for example, phase, amplitude, period, etc.) are changed by propagating 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, the magnitude of the viscosity of the liquid in contact with the surface of the substrate 22, and the like. Therefore, the sensor 2 can detect the first substance based on the change in the characteristics of the elastic wave. Specifically, the measuring device 100 can measure, for example, the concentration of the first substance contained in the sample. The 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.
  • the elastic waves propagating on the surface of the substrate 22 are also referred to as surface acoustic waves (SAW: Surface Acoustic Wave).
  • SAW Surface Acoustic Wave
  • the pair of first IDT electrodes 25a may be positioned on the substrate 22 so as to sandwich the detection unit 23.
  • an electric signal is input to one of the pair of first IDT electrodes 25a.
  • the input electric signal is converted into an elastic wave propagating toward the detection unit 23 and transmitted from one of the first IDT electrodes 25a.
  • the transmitted elastic wave passes through the detection unit 23.
  • the other first IDT electrode 25a can receive elastic waves that have passed through the detection unit 23.
  • the received elastic wave is converted into an electric signal.
  • the pair of first IDT electrodes 25a may be made of 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 multi-layer electrode made of a plurality of materials.
  • the sensor 2 may have two or more combinations of the detection unit 23 and the pair of first IDT electrodes 25a.
  • the measuring device 100 may detect different types of target substances for each combination, for example.
  • the measuring device 100 may detect, for example, a plurality of combinations of target substances of the same type and compare the detection results of each.
  • FIG. 3 shows a conceptual diagram showing the polymer film 1 included in the detection unit 23.
  • the polymer film 1 containing the polymer 3 is fixed on the substrate 22. Further, a substance that reacts with the first substance 5 (second substance 4) is fixed on the polymer film 1.
  • the polymer membrane 1 is a membrane adjusted so as to have high specific adsorptivity, and is a membrane adjusted to reduce non-specific adsorption.
  • 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.
  • a reaction is a reaction in which the first substance 5 and the second substance 4 are bonded by, for example, a redox reaction, an enzymatic reaction, an antigen-antibody reaction, a chemical adsorption, an intermolecular interaction, or an ionic interaction. You may.
  • the reaction between the first substance 5 and the second substance 4 may be a reaction for producing a new substance (third substance) by an enzymatic reaction or the like.
  • the second substance 4 fixed to the detection unit 23 may be appropriately selected according to the first substance 5.
  • the first substance 5 is a specific protein, DNA, cell, or the like in a sample
  • the second substance 4 an antibody, a peptide, an aptamer, or the like may be used.
  • an antigen may be used as the second substance 4.
  • an enzyme may be used as the second substance 4.
  • the measuring device 100 may indirectly detect the target first substance 5.
  • 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 using the first substance 5 as an antigen and the first substance 5 may be reacted in advance, and the unreacted antibody may be reacted with the fixed second substance 4.
  • the measuring device 100 can indirectly calculate the amount of the first substance 5 from the amount of the detected antibody, for example, if the amount of the antibody is known.
  • the polymer 3 constitutes the polymer film 1.
  • an example of the polymer 3 of the aspect of the present disclosure hereinafter, abbreviated as “polymer of the present embodiment”.
  • the polymer of the present embodiment is a copolymer containing a first structural unit represented by the following formula (1) and a second structural unit represented by the following formula (2).
  • R 1 is a hydrogen atom or a methyl group.
  • R 2 and R 3 are independently hydrogen atoms or alkyl groups having 1 to 4 carbon atoms.
  • x1 is an integer of 1 to 4.
  • R 4 is a hydrogen atom or a methyl group.
  • R 5 and R 6 are independently hydrogen atoms or alkyl groups having 1 to 4 carbon atoms.
  • x2 is an integer of 1 to 4, and y is an integer of 3 to 7.
  • the first structural unit is derived from, for example, N- (carboxymethyl) -N, N-dimethyl-2-[(2-methyl-1-oxo-2-propen-1-yl) -oxy] etanaminium (CBMA1). It may be a structural unit to be used.
  • the second structural unit may be, for example, a structural unit derived from the compound represented by the following formula (8).
  • R 4, R 5, R 6, x2 and y respectively, the same meanings as R 4, R 5, R 6 , x2 and y in formula (2).
  • the polymer of the present embodiment can improve the chemical stability of the polymer film by having the first structural unit represented by the formula (1), which is chemically stable. Further, the polymer of the present embodiment has a carboxyl group in the second structural unit represented by the formula (2), which has good reactivity with the second substance 4, so that the second substance 4 can be easily fixed. Become. Therefore, the polymer of the present embodiment having these two structural units can easily fix the second substance 4 and produce a chemically stable polymer film. In addition, the polymer of the present embodiment has high thermal stability and hydrolysis resistance.
  • the polymer of the present embodiment When used in a measuring device such as a biosensor, it is contained in the above-mentioned copolymer in that it has high chemical stability and good reactivity with the second substance 4.
  • the total content of the first structural unit and the second structural unit may be 80 mol% or more, 90 mol% or more, or 95 mol% or more.
  • the polymer of the present embodiment has high chemical stability and reduces non-specific adsorption, even if the proportion of the first structural unit contained in the copolymer is 50 mol% or more. It may be 60 mol% or more, and may be 80 mol% or more. Further, the above ratio may be 95 mol% or less, or 90 mol% or less.
  • the molar ratio of the second structural unit to the first structural unit contained in the copolymer is such that the reactivity with the second substance 4 is improved while reducing the non-specific adsorption.
  • (Second structural unit / first structural unit) may be 1 or less, 0.7 or less, or 0.3 or less.
  • the molar ratio may be 0.05 or more, or 0.1 or more, in order to express the reactivity of the substance to be measured contained in the sample and the substance that interacts with the substance.
  • the proportion of the second structural unit contained in the copolymer may be 5 mol% or more, and 10 mol% in terms of improving the reactivity with the second substance 4. It may be more than 20 mol% or more. Further, from the viewpoint of reducing non-specific adsorption, the above ratio may be 50 mol% or less, 45 mol% or less, or 40 mol% or less.
  • the polymer of the present embodiment may have a structural unit other than the first structural unit and the second structural unit as long as the effects of the present disclosure are not impaired.
  • the polymer of the present embodiment may further contain a third structural unit represented by the following formula (3).
  • R 7 is a hydrogen atom or a methyl group.
  • R 8 and R 9 are independently alkyl groups having 1 to 4 carbon atoms.
  • z is an integer of 1 to 4.
  • the third structural unit is derived from, for example, N, N-dimethylaminoethyl (meth) acrylate (DMAEMA), N, N-diethylaminoethyl (meth) acrylate, N, N-diisopropylaminoethyl (meth) acrylate and the like. It may be a structural unit.
  • the proportion of the third structural unit contained in the copolymer may be 20 mol% or less, and 10 mol% or less. It may be present, and may be 5 mol% or less.
  • At least one end of the main chain of the polymer of the present embodiment has a thiol group or a dithioester group in terms of ease of fixation to the polymer film on the substrate 22 (measurement substrate 12). It may be a thiol group.
  • the number average degree of polymerization of the polymer of the present embodiment may be 25 or more, 50 or more, or 200 or more in terms of film formation density.
  • the polymer of the present embodiment may have a high molecular weight.
  • the polymer of the present embodiment may be any of a random copolymer, a block copolymer, and a graft copolymer.
  • the copolymer containing the first structural unit and the second structural unit can be identified by a conventionally known organic analysis method.
  • the copolymer may be identified by NMR (Nuclear Magnetic Resonance).
  • NMR Nuclear Magnetic Resonance
  • it may be identified by a liquid chromatograph.
  • infrared spectroscopy That is, when identifying the copolymer, an apparatus capable of carrying out these methods may be used.
  • the identification method and device are not limited to these methods and devices as long as the copolymer can be identified.
  • the polymer production method of the present embodiment can be produced by a known polymerization method such as radical polymerization.
  • An example of the method for producing the polymer of the present embodiment is shown below.
  • Examples of the polymer production method of the present embodiment include an intermediate synthesis step (steps 1 and 2), an intermediate deprotection step (step 3), and a substitution step of substituting the end of the polymer with a thiol group (SH group). (Step 4) is included. Hereinafter, each step will be described.
  • Steps 1 and 2 Intermediate synthesis step>
  • the polymer containing the third structural unit represented by the above formula (3) (hereinafter, may be abbreviated as “starting polymer”) is represented by the compound represented by the formula (4) and the formula (hereinafter, may be abbreviated as “starting polymer”).
  • An intermediate is synthesized by the step of reacting with the compound represented by 5).
  • the intermediate is a polymer containing a structural unit represented by the formula (6) and a structural unit represented by the formula (7).
  • the starting polymer may be a commercially available product or a synthetic product.
  • the ratio of the third structural unit contained in the starting polymer may be 80 mol% or more, or 90 mol% or more.
  • n is an integer from 25 to 1000.
  • the starting polymer can be synthesized, for example, by RAFT polymerization (reversible addition-fragmentation chain transfer polymerization).
  • the starting polymer can be synthesized by RAFT polymerization using a monomer such as DMAEMA (2- (dimethylamino) ethyl methacrylate) as a starting material and a RAFT agent and a polymerization initiator. Since the polymer obtained by RAFT polymerization has a dithioester group at the terminal, the step of substitution with a thiol group (step 4) described later can be performed easily and in a short time.
  • RAFT agents include dithioesters, trithiocarbonates, dithiocarbamates, dithiocarbonates and the like.
  • polymerization initiator include persulfates, peroxides, azo compounds and the like.
  • reaction solvent, reaction catalyst, reaction temperature, reaction time, etc. used in RAFT polymerization the description of Examples and general conditions of RAFT polymerization can also be referred to.
  • the starting polymer is reacted with the compound represented by the formula (4) and then reacted with the compound represented by the formula (5).
  • the starting polymer may be reacted with the compound represented by the formula (4) and the compound represented by the formula (5) separately.
  • the reaction temperature may be 40 ° C. or higher, 60 ° C. or higher, and the reflux temperature of the solvent from the viewpoint of promoting the reaction. May be.
  • the reaction solvent may be a polar solvent, and examples of the reaction solvent include DMF (dimethylformamide), acetonitrile, methanol, and ethanol, but ethanol may also be used.
  • the amount of the compound represented by the formula (4) added is the amount obtained by multiplying the amount of amino groups contained in the starting polymer by the desired copolymerization ratio of the second structure. May be good.
  • the compound represented by the formula (4) is obtained by multiplying the amount of amino groups contained in the starting polymer by 0.2. May be added.
  • the reaction time may be 6 hours or more, 12 hours or more, or 24 hours or more. You may.
  • the reaction temperature is 0 ° C. from the viewpoint of promoting the reaction.
  • the temperature may be 10 ° C. or higher, or 20 ° C. or higher.
  • the reaction solvent may be a polar solvent, and examples of the reaction solvent include DMF, acetonitrile, methanol, and ethanol, but ethanol may also be used.
  • the formula ( The amount of the compound represented by 5) added is 1.1 times or more the amount of amino groups contained in the reaction product obtained by reacting the starting polymer with the compound represented by the formula (4). It may be 1.2 times or more, or 1.3 times or more.
  • the amount of the compound represented by the formula (5) added is the amino contained in the reaction product obtained by reacting the starting polymer with the compound represented by the formula (4). It may be 3 times or less or 2 times or less the amount of the group.
  • Step 3 Deprotection process of intermediate>
  • the protecting group tert-butyl group of the intermediate obtained in the above step is removed (deprotection step).
  • the intermediate deprotection step gives the polymer of this embodiment.
  • the protecting group can be removed by allowing a strong acid such as TFA (trifluoroacetic acid) to act.
  • TFA trifluoroacetic acid
  • n is an integer from 25 to 1000.
  • Step 4 Substitution step of polymer terminal with thiol group>
  • the end of the polymer main chain is replaced with a thiol group.
  • the polymer can be easily fixed to the substrate 22 (measurement substrate 12).
  • the polymer containing the third structural unit represented by the formula (3) is a polymer obtained by RAFT polymerization
  • the end of the polymer obtained in step 3 has a dithioester group.
  • the dithioester group is replaced with a thiol group.
  • the reaction solvent, reaction catalyst, reaction temperature, reaction time, etc. used in the step of substituting for a thiol group the description of Examples and general conditions for substituting for a thiol group can also be referred to. can.
  • n is an integer of 25 to 1000.
  • Examples of the method for immobilizing the polymer 3 on the substrate 22 include a method in which a polymer solution obtained by dissolving the polymer 3 in a solvent is applied to the substrate 22 and dried, graft polymerization by radiation or ultraviolet rays, and functional groups of the substrate 22. Chemical reaction and the like. By these methods, a polymer film 1 composed of the polymer 3 is formed on the substrate 22.
  • a method of fixing the second substance 4 on the polymer 3 (polymer film 1) for example, a method of covalently bonding the second substance 4 to the carboxyl group of the polymer 3 can be mentioned.
  • polymer 3 is reacted with N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) (NHS / EDC activation). Then, the carboxyl group of the polymer 3 is replaced with the NHS ester group. By reacting the activated NHS ester group with the amino group of the second substance 4, the second substance 4 is fixed on the polymer 3 (polymer film 1).
  • NHS N-hydroxysuccinimide
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
  • the NHS / EDC activation of the polymer 3 may be carried out before immobilization on the substrate 22 or after immobilization of 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 associated with NHS / EDC activation can be reduced.
  • FIG. 4 shows a schematic configuration of the measurement carrier 11 according to the present embodiment.
  • the measurement carrier 11 include a plate for ELISA (enzyme-linked immunosorbent assay).
  • 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 or the like on the surface of the measurement substrate 12. Be prepared for. The polymer film 1 is fixed in the detection region 31.
  • the second substance 4 that reacts with the first substance 5 is fixed on the polymer film 1.
  • a membrane adjusted so as to enhance non-specific adsorptivity may be fixed to the non-detection region 32.
  • the desired second substance 4 is fixed to the polymer film 1 in the detection region 31.
  • the blocking agent is non-selectively adsorbed on the non-detection region 32.
  • the first substance 5 which is the target substance contained in the sample reacts with the second substance 4, and the reaction is detected by the detection reagent.
  • detection reagents include redox substances, fluorescent substances, enzymes, dye compounds and the like.
  • the measuring substrate 12 may be, for example, a metal such as gold, silver, copper, platinum and aluminum; a plastic such as polyethylene and polypropylene; an inorganic material such as titanium oxide, silica, glass and ceramic; and the like.
  • the measuring substrate 12 is not limited to these examples.
  • the shape of the measurement substrate 12 may be, for example, a plate shape, particles, a microstructure, a microtiter plate, or the like.
  • the shape of the measuring substrate 12 is not limited to these examples.
  • a measurement kit including a measurement substrate 12 on which the polymer film 1 is fixed, a second substance 4, and a detection reagent is also included in the scope of the present disclosure.
  • the second substance 4 may be fixed in advance to the polymer film 1 at the time of manufacturing the product, or may be fixed by the user before the measurement.
  • the measurement kit according to this embodiment may include other reagents and instruments.
  • it may contain components other than the second substance 4 and the detection reagent described above.
  • a buffer or the like may be provided.
  • the measurement kit according to the present embodiment may be a mixture of a plurality of different reagents in appropriate volumes and / or embodiments, or may be provided in separate containers.
  • the measurement kit according to the present embodiment may include an instruction sheet describing a procedure for obtaining detection of the reaction between the first substance 5 and the second substance 4. It may be written or printed on paper or other medium, or attached to a magnetic tape, a readable disc such as a computer, or an electronic medium such as a CD-ROM.
  • CBMA1 refers to N- (carboxymethyl) -N, N-dimethyl-2-[(2-methyl-1-oxo-2-propen-1-yl) -oxy] etanaminium.
  • Polymer C A polymer of CBMA1 and CBMA3, which is a polymer of CBMA1 and CBMA3.
  • the thermal stability of the copolymer having a ratio of 6: 4 and a degree of polymerization of 50 was evaluated using a thermal weight differential thermal analyzer under the following measurement conditions.
  • Measuring device Rigaku EVO-TG 8120 Measurement conditions: Temperature rise rate: 10 ° C / min Measurement temperature: Room temperature to 700 ° C Atmosphere: Nitrogen Measuring container: Pt Reference: Al 2 O 3 Sample amount: Approximately 4 mg The evaluation results are shown in FIG.
  • the weight reduction of all the polymers until the temperature was raised to 100 ° C. is due to the evaporation of water adsorbed on the polymers. It was found that the copolymers A to C had a constant weight from 100 to 130 ° C. and had high thermal stability. Among these, the copolymers A and B were found to have a constant weight up to 150 ° C. and particularly high thermal stability.
  • the powder of each polymer has a concentration of 10 mg / mL. It was dissolved in a mixed solution of 0.2 mol / L NHS, 0.3 mol / L EDC / HCl, and 37.5 mmol / L HCl and activated for 10 minutes. The amounts of ⁇ N + (CH 3 ) 2- and ⁇ N (CH 3 ) 2 in the polymer before and after NHS / EDC activation were measured. The results are shown in Table 1. % In Table 1 is mol%. Each numerical value in Table 1 is a ratio when the content of ⁇ N + (CH 3 ) 2 ⁇ and ⁇ N (CH 3 ) 2 in the polymer is 100 mol%. NHS / EDC activation was performed 3 times, and the values in Table 1 are the values after 3 times.
  • Example 3 Preparation of polymer (copolymer) having a ratio of CBMA1 to CBMA3 of 8: 2 A copolymer was prepared according to the following synthesis scheme.
  • Step_3 Removal of protecting groups (Step_3)> The solid polymer C1 obtained in Step_2 was dissolved in TFA and reacted at room temperature for 8 hours so that the concentration of the amount of the monomer repeating units in the polymer C1 was 0.75 mol / L. After the reaction, the solvent was distilled off to obtain a dry product of polymer F1.
  • Step_4 Substitution of polymer terminal with SH group
  • the polymer F1 obtained by Step_3 was dissolved in water to a concentration of 50 mg / mL, and 30 times the molar amount of ethanolamine was added to the polymer, and the equimolar amount of tris (2-carboxyethyl) phosphine hydrochloride was added to the polymer. (TCEP) was added and reacted at room temperature for 1 hour. After distilling off the solvent, the obtained solid was dissolved in methanol and charged into tetrahydrofuran, and the obtained precipitate was recovered.
  • ADVANCE III HD 400 type Measuring conditions Solvent: Heavy water Temperature: 30 ° C Criteria: H 2 O peak is 4.70 ppm Integration: 300 times
  • Calculation method of ratio of CBMA1 and CBMA3 The peak area of 3.30 ppm to 3.35 ppm derived from the methyl group of the quaternary amine in the CBMA1 unit and 3.20 ppm to 3.25 ppm derived from the methyl group of the quaternary amine in the CBMA3 unit. The ratio of CBMA1 and CBMA3 was calculated from the ratio with the peak area.
  • Example 4 Preparation of a polymer (copolymer) in which the ratio of CBMA1 to CBMA3 is 6: 4.
  • the copolymer (polymer E1) was prepared according to the same synthesis scheme as in Example 3 except that the amount of compound B2 added was changed to 3.5 mmol in “3. Reaction with compound B2 (Step_2)”. bottom.
  • Example 5 Relationship between the degree of polymerization of the copolymer and the amount of non-specific adsorption
  • the relationship between the degree of polymerization of (collectively referred to as coalescence E) and the amount of non-specific adsorption was investigated. Specifically, a polymer membrane made of copolymer E having a different degree of polymerization was brought into contact with urine as a sample, and the amount of non-specific adsorption of a urine component on the polymer membrane was measured under the following conditions.
  • Measuring device GE Healthcare Biacore X100 Measurement conditions: Running buffer: HBS-P Temperature: 25 ° C Flow velocity: 10 uL / min Specimen contact time: 9 minutes (Sample injection volume 90 uL)
  • Running buffer HBS-P Temperature: 25 ° C
  • Flow velocity 10 uL / min
  • Specimen contact time 9 minutes (Sample injection volume 90 uL)
  • the difference between the SPR signal value after running a running buffer for 9 minutes after sample injection and the SPR signal value before sample injection was defined as the non-specific adsorption amount.
  • Example 6 Relationship between the ratio of CBMA1 in the copolymer and the amount of antibody-immobilized or serum non-specific adsorption The ratio of CBMA1 in the copolymer and antibody-immobilization using the following four polymers. The relationship between the amount or the non-specific adsorption amount of the component contained in the serum (serum non-specific adsorption amount) was evaluated. The degree of polymerization of each of the four polymers is 200.
  • the amount of antibody immobilized was measured by an SPR device.
  • the amount of non-specific adsorption of serum was measured by the same procedure as in Example 5 except that the sample was changed from urine to serum.
  • the difference between the value and the SPR signal value before injecting the antibody solution was defined as the antibody-immobilized amount.
  • SIA kit Au manufactured by GE Healthcare
  • a 50% methanol solution in which the polymer was dissolved to a concentration of 5 mg / mL for 18 hours.
  • a polymer film was formed. Then, the chips were washed with milli-Q and dried with a stream of nitrogen.
  • FIG. 7 shows the measurement results of the antibody-immobilized amount of each polymer
  • FIG. 8 shows the measurement results of the serum non-specific adsorption amount.
  • Table 3 is a table summarizing the results of Example 6.
  • ratio of CBMA1 indicates the ratio of CBMA1 contained in the polymer. Criteria were set for each evaluation item as follows.
  • a polymer having a CBMA1 ratio of 60% and a polymer having a CBMA1 ratio of 80% have an overall rating of A as shown in Table 3, and are suitable for use in a polymer film of a measuring device or a measuring carrier. Do you get it.
  • the homopolymer of CBMA1 polymer in which the ratio of CBMA1 is 100%
  • the amount of non-specific adsorption of serum was reduced, while the amount of antibody immobilization was low, and the overall evaluation was B.
  • the homopolymer of CBMA3 (a polymer having a ratio of CBMA1 of 0%) had a high amount of antibody immobilization, while a large amount of non-specific adsorption of serum, and the overall evaluation was B.
  • the copolymer containing the first structural unit represented by the above formula (1) and the second structural unit represented by the above formula (2) has high chemical stability. Therefore, it was shown that a polymer film that is easier to handle and can fix a substance that interacts with the substance to be measured contained in the sample can be produced as compared with the conventional material.
  • the present disclosure can be used for a measuring device and a measuring plate provided with a detection unit on which a polymer film is formed.

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