A reagent layer and a biocom equipped with a reagent layer

Abstract

An embodiment of the present invention provides a reagent layer containing: a polymer that contains a proton acceptor group as a repeating unit and has a pH-buffering capacity; and an oxidoreductase that oxidizes or dehydrogenates an analyte.

Description

Technical Field

[0001] The present invention relates to a biosensor including a reagent layer.

Background Art

[0002] In recent years, in various fields such as the medical field, a biosensor can be used to measure an analyte in a sample (such as a cell culture solution). As a method for measuring an analyte, for example, an electrochemical measurement method can be used.

[0003] As a biosensor in an electrochemical measurement method, there is one including an electrode (working electrode, counter electrode, reference electrode) and an enzyme film provided on the electrode (see Patent Document 1). The biosensor including the electrode and the enzyme film can be disposed in a weighing unit having a structure that enables continuous supply of a buffer solution having a pH buffering ability and injection of a sample into the buffer solution. By appropriately selecting an enzyme in which an analyte to be measured acts as a substrate, various analytes can be measured.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Here, the inventors of the present application newly found that there are matters that can be improved in the configuration of a conventional biosensor. Specifically, a conventional biosensor can be disposed in a structure that enables continuous supply of a buffer solution and injection of a sample into the buffer solution. However, such a conventional biosensor is difficult to cope with a method of continuously and directly monitoring a sample without continuously supplying the buffer solution. Therefore, it is desirable that a reagent layer (corresponding to the enzyme film of Patent Document 1) as a component of the biosensor has a property of pH buffering ability.

[0006] This invention has been made in view of the above problems. That is, the object of this invention is to provide a reagent layer having pH buffering capacity and a biosensor comprising the reagent layer. [Means for solving the problem]

[0007] To achieve the above objective, in one embodiment of the present invention, A reagent layer is provided, comprising a polymer having a proton-receptor group in its repeating units and pH buffering capacity, and an oxidoreductase that oxidizes or dehydrogenates the analyte.

[0008] One embodiment of the present invention provides a biosensor comprising the above-mentioned reagent layer. [Effects of the Invention]

[0009] According to one embodiment of the present invention, the reagent layer itself can have pH buffering properties. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 is a schematic cross-sectional view showing the configuration of a reagent layer containing a reagent according to one embodiment of the present invention. [Figure 2] Figure 2 is a schematic cross-sectional view showing the main components of a biosensor according to one embodiment of the present invention. [Figure 3] Figure 3 is a schematic cross-sectional view illustrating the mechanism of action provided by a biosensor based on the main components of one embodiment of the present invention. [Figure 4] Figure 4 is a schematic cross-sectional view illustrating the mechanism of action provided based on the main configuration of a biosensor according to another embodiment of the present invention. [Figure 5] Figure 5 is a graph showing the relationship between the measurement time and the current response value in Example 1A. [Figure 6] Figure 6 is a graph showing the relationship between the measurement time and the current response value in Comparative Example 1A. [Figure 7]FIG. 7 is a graph showing the relationship between the measurement elapsed time and the current response value in Example 2A. [Figure 8] FIG. 8 is a graph showing the relationship between the measurement elapsed time and the current response value in Comparative Example 2A.

BEST MODE FOR CARRYING OUT THE INVENTION

[0011] Hereinafter, embodiments of the present invention (Invention A) will be specifically described. First, the overall configuration of the biosensor will be described. Then, the characteristic parts of the present invention will be described.

[0012] [Overall Configuration of Biosensor] Hereinafter, the overall configuration of the biosensor in one embodiment of the present invention will be described. FIG. 1 is a cross-sectional view schematically showing the configuration of a reagent layer according to one embodiment of the present invention. FIG. 2 is a cross-sectional view schematically showing the main configuration of a biosensor according to one embodiment of the present invention.

[0013] A biosensor 100 according to one embodiment of the present invention includes a working electrode 30 located on the surface of a substrate, a reagent layer 10 located on the surface of the working electrode, and a protective film 20 located on the surface of the reagent layer 10 (see FIGS. 1 and 2). In this embodiment, for the sake of convenience, the working electrode 30 as a component of the electrode part is illustrated, and it is confirmatorily noted that the illustration of the counter electrode and the reference electrode as other components of the electrode part is omitted.

[0014] As used herein, a "biosensor" is a measurement device that utilizes a combination such as an enzyme-substrate, converts a chemical change generated by a molecular recognition reaction between a substrate (corresponding to an analyte) and an enzyme (corresponding to a receptor) into an electrical signal, and measures the metabolic rate, concentration, etc. of the analyte according to the strength of the obtained electrical signal.

[0015] As the substrate, an insulating substrate can be used. Although not particularly limited, the substrate can be composed of materials such as polyethylene terephthalate, polyamide, polyimide, etc. with a thickness of several hundred μm. In one embodiment, the electrode portion of the biosensor 100 can be inserted into a liquid sample (such as a cell culture solution). Further, after the insertion of the biosensor 100, a voltage can be applied to the electrode portion.

[0016] As specifically described below, the reagent layer 10 can contain at least enzyme B (see FIG. 1). As enzyme B, one in which the analyte to be measured acts as a substrate can be selected. By such a selection, measurement of various analytes becomes possible. Enzyme B can be a redox enzyme that oxidizes or dehydrogenates the analyte.

[0017] For example, as the redox enzyme, glucose oxidase, lactate oxidase, cholesterol oxidase, bilirubin oxidase, glucose dehydrogenase, lactate dehydrogenase, amino acid oxidase, amino acid dehydrogenase, glutamate oxidase, glutamate dehydrogenase, fructosyl amino acid oxidase, fructosyl peptide oxidase, 3-hydroxybutyrate dehydrogenase, alcohol oxidase, and / or alcohol dehydrogenase, etc. can be mentioned.

[0018] In addition, the above reagent layer can further contain a mediator and / or conductive particles. Examples of the conductive sexual grain particles include carbon black and carbon nanotubes. As used herein, the "mediator" generally means a redox substance that mediates electron transfer, and specifically refers to a substance that is responsible for the electron transfer caused by the redox reaction of the analyte in the following biosensor.

[0019] While not particularly limited, mediators include metal complexes (e.g., osmium complexes, ruthenium complexes, iron complexes, etc.), quinone compounds (e.g., benzoquinone, naphthoquinone, phenanthrenequinone, phenanthrolinequinone, anthraquinone, and their derivatives, etc.), phenazine compounds, viologen compounds, phenothiazine compounds, and phenol compounds.

[0020] More specifically, one or more substances selected from the group consisting of potassium ferricyanide, hexaammineruthenium, ferrocene, poly(1-vinylimidazole)-bis(bipyridine)chloroosmium, hydroquinone, 2-methyl-1,4-benzoquinone, 1,2-naphthoquinone-4-sulfonate, 9,10-phenanthrenequinone-2-sulfonate, 9,10-phenanthrenequinone-2,7-disulfonate, 1,10-phenanthroline-5,6-dione, anthraquinone-2-sulfonate, phenazine derivatives such as 1-methoxy-5-methylphenadinium methyl sulfate and 1-methoxy-5-ethylphenadinium ethyl sulfate, methyl viologen, benzyl viologen, methylene blue, methylene green, 2-aminophenol, 2-amino-4-methylphenol, and 2,4-diaminophenol are used as mediators.

[0021] The protective membrane 20 may be configured to cover the reagent layer 10 and the electrode portion. The protective membrane 20 is positioned to control the permeation rate of analytes (such as lactic acid) from cells in the culture medium, while allowing the analytes to penetrate toward the working electrode. Furthermore, the protective membrane 20 is positioned to suppress the outflow of components contained in the reagent layer 10 on the working electrode to the outside of the protective membrane 20. In other words, the "protective membrane" as used herein is a membrane that contributes to suppressing the leakage of substances contained in the reagent layer to the outside of the protective membrane, and has pores that allow analytes present outside the protective membrane to move toward the reagent layer through the protective membrane.

[0022] The protective film preferably contains a biocompatible polymer. The protective film can be obtained, for example, by dissolving the polymer and crosslinking agent in an alcohol solvent, such as a buffer-alcohol-containing solvent, to form a film solution, and then applying this film solution to the reagent layer, drying it, or by immersing the electrode and reagent layer laminate in the film solution, removing it, and drying it.

[0023] The polymer included in the protective film may have a biocompatible phosphorylcholine group and a polymerizable methacryloyl group or acryloyl group. For example, 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer may be included.

[0024] Furthermore, the polymer included in the protective film may include t-butyl acrylate having a heterocyclic nitrogen group. As the heterocyclic nitrogen group, for example, a pyridyl group can be selected. As an example, random copolymers of styrene-2-vinylpyridine-ter.butyl methacrylate (S2VPtBuMA), random copolymers of tripropylene glycol methyl ether methacrylate-styrene-4-vinylpyridine (TGMAS4VP), and / or poly(ter.butyl methacrylate-b-4-vinylpyridine (tBuMA4VP)) can be selected.

[0025] As a crosslinking agent, one having a reactive group that can react with the heterocyclic nitrogen group mentioned above can be used. One example is poly(ethylene glycol) diglycidyl ether.

[0026] The mechanism of reaction provided by the biosensor 100 having the above-described components will be explained below. Figure 3 is a schematic cross-sectional view showing the mechanism of action provided by the main configuration of a biosensor according to one embodiment of the present invention. Figure 4 is a schematic cross-sectional view showing the mechanism of action provided by the main configuration of a biosensor according to another embodiment of the present invention.

[0027] In one example of the present invention, with the electrode immersed in the culture medium, the analyte, which moves from the culture medium to the reagent layer 10 via the protective film 20, is oxidized in the reagent layer 10 using enzyme B as a catalyst and dissolved oxygen. By electrically detecting the hydrogen peroxide produced at that time, the concentration of the analyte can be measured.

[0028] Taking the detection of lactic acid as an example, lactic acid (corresponding to Lac in Figure 3) that has moved from the culture medium to the reagent layer 10 through the protective membrane 20 is oxidized by an enzymatic reaction with the enzyme in the reagent layer 10 (corresponding to lactate oxidase (LOx) in Figure 3). This oxidation can lead to the formation of pyruvate (corresponding to Pyru in Figure 3), hydrogen peroxide, and ionized protons (H+). By electrically measuring the hydrogen peroxide produced during this formation, it is possible to measure the concentration of lactic acid. In this case, the biosensor 100 can function as a lactic acid sensor.

[0029] Furthermore, as shown in Figure 4, the reagent layer 10 may also contain a mediator. As described above, the mediator is a redox substance that mediates the transfer of electrons generated by the redox reaction of analyte (corresponding to lactic acid) in the biosensor 100. Therefore, the presence of this mediator allows for favorable transfer of electrons.

[0030] [Features of the present invention (reagent layer)] The following describes the features of the present invention. The present invention is characterized by the configuration of the reagent layer 10, which is a component of the biosensor (see Figure 1).

[0031] The inventors of the present invention have diligently studied a configuration for continuously and directly monitoring analytes present in cell culture media, etc., without requiring a continuous supply of a buffer solution with pH buffering capacity, as in conventional biosensors. As a result, the inventors of the present invention have devised a configuration in which the reagent layer 10, a component of the biosensor 100, includes the enzyme B described above, as well as a polymer A that has pH buffering capacity and contains a proton-receiving group in its repeating unit. The "proton-receiving group" here refers to a proton (H) contained in the repeating unit of the polymer.+ This refers to a functional group that can accept ).

[0032] The repeating units of polymer A contain proton-receiving groups, which allows them to accept ionized protons that may be generated when the analyte is oxidized by an enzymatic reaction with enzyme B within the reagent layer 10. From the viewpoint of preventing polymer A from detaching to the outside of the reagent layer 10, it is preferable that the weight-average molecular weight (Mw) of polymer A is 10,000 or more.

[0033] As a result, polymer A remains near enzyme B within the reagent layer 10, preventing the reaction field pH from deviating from the optimal pH for enzyme B. That is, the reaction field pH can be maintained within a certain range. Therefore, deactivation of enzyme B can be avoided, and as a result, the durability of the biosensor 100 can be improved. Specifically, by directly inserting the biosensor 100 containing the reagent layer 10 having the above characteristics into a culture vessel supplied with culture medium, suitable continuous measurement of analytes becomes possible. In this specification, "sensor durability" refers to the maintenance rate of the sensor's current response value during the measurement period.

[0034] A heterocyclic nitrogen group can be used as the proton-accepting group included in the repeating unit of polymer A described above. For example, the heterocyclic nitrogen group can be at least one selected from the group consisting of imidazole group, pyridyl group, indolyl group, quinolyl group, isoquinolyl group, tetrahydroquinolyl group, thiazole group, indolidyl group, imidazopyridyl group, acridinyl group, tetrazole group, triazole group, pyrazyl group, morpholyl group, and piperadyl group.

[0035] An example of polymer A containing heterocyclic nitrogen groups in its repeating units is, for example, the polyvinylimidazole PolyIMZ shown below. [C1] TIFF0007894935000001.tif6069

[0036] Another example of polymer A, which contains heterocyclic nitrogen groups in its repeating units, is poly-L-histidine (PolyH), as shown below. [C2] TIFF0007894935000002.tif8159

[0037] The proton-accepting group included in the repeating unit of polymer A described above can be at least one selected from the group consisting of ionized phosphate groups, sulfo groups, and carboxyl groups. An example of polymer A containing the above functional groups in an ionized state in the repeating unit is sodium polyphosphate, for example, having a degree of polymerization of 700 to 1000 and a weight-average molecular weight (Mw) of 70,000 to 100,000. [C3] TIFF0007894935000003.tif54114

[0038] Furthermore, the polymer A described above may include ionic groups in addition to proton-accepting groups in its repeating units. Here, "ionic groups" refers to functional groups in an ionic state that are capable of providing hydrophilicity and are included in the repeating units of the polymer.

[0039] As an example, polymer A may contain a repeating unit that is derived from 4-vinylpyridine and a repeating unit that is derived from 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate. That is, in the repeating unit of polymer A, the first portion having an ionic group may contain 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate. Also, in the repeating unit of polymer A, the second portion having a proton-accepting group may contain 4-vinylpyridine. For example, polymer A may have the following structure. [C4] TIFF0007894935000004.tif12080

[0040] [Method for fabricating a biosensor] The following describes an example of a method for fabricating the above-mentioned biosensor.

[0041] First, an insulating substrate is prepared. After preparing the insulating substrate, carbon ink or the like is printed onto the insulating substrate to form conductive thin films that constitute the working electrode 30, the counter electrode, and the wiring. In addition, Ag / AgCl ink is printed onto the conductive thin film to form the reference electrode.

[0042] Alternatively, a conductive thin film (corresponding to the working electrode) selected from a metal such as gold, platinum, or palladium can be deposited by sputtering, vapor deposition, or ion plating.

[0043] The surface of this working electrode can be coated with Nafion, which has a fluorocarbon main chain and sulfo groups as side chains. The thickness of the conductive thin film can be 10 nm to several hundred nm. The counter electrode and / or reference electrode may be placed on the periphery of the working electrode, for example, on the back side of the substrate. An insulating resist layer may be formed on areas other than these electrode formation areas.

[0044] Subsequently, the reagents constituting the reagent layer of the present invention are applied to the working electrode. The reagents include polymer A and enzyme B, which contain proton-receiving groups in the repeating units described above. In addition to polymer A and enzyme B, the reagents may further include a suspension of conductive particles such as carbon particles for imparting conductivity and an aqueous solution of a mediator. After applying the reagents, the reagent layer 10 can be formed by drying the reagents applied to the working electrode.

[0045] After the reagent layer 10 is formed, a polymer solution for the protective film 20 is applied to the surface of the reagent layer 10 and then dried at room temperature. Alternatively, the electrode 30 with the reagent layer 10 attached is repeatedly immersed in the polymer solution for the protective film 20, removed, and dried multiple times. This allows the protective film 20 to be formed so as to cover the reagent layer 10 and the electrode. As a result, the biosensor 100 can be fabricated. [Examples]

[0046] The following describes embodiments of the present invention (Invention A).

[0047] Example 1A [Fabrication of biosensors] A biosensor was fabricated through the following steps (1) to (6).

[0048] ●Process (1): Electrode fabrication Using a 250IP screen printing machine (manufactured by Seria Corporation), carbon ink (manufactured by Fujikura Chemical Co., Ltd.) was printed onto an insulating substrate to form a conductive thin film. The conductive thin film constitutes the working electrode and wiring. Subsequently, a first resist film was laminated onto the insulating substrate to restrict the extent of the reagent layer. Furthermore, a water-repellent film was attached to the first resist film to restrict the extent of the cation exchange film and protective film.

[0049] ●Step (2): Preparation of reagent solution The reagents listed below were mixed to the following final concentrations and reacted for approximately 1 hour to prepare the reagent solution. • Sodium phosphate buffer (pH 6.5), final concentration 10 mM • Sodium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) Final concentration 20 mM • Carbon dispersion, final concentration 5 mg / mL • Polymer-bound PNT final concentration 4 (absorbance at 608 nm) • Hydroxypropylcellulose (NISSO HPC-VH, manufactured by Nippon Soda Co., Ltd.) Final concentration 0.05% (w / v) • Polyvinylimidazole, final concentration 6.59 mg / mL • Lactate oxidase (LOX T-47: manufactured by Asahi Kasei Pharma Co., Ltd.) Final concentration 400 U / mL • Glutaraldehyde 25% solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) Final concentration 0.002% (w / v)

[0050] The sodium phosphate buffer solution was prepared using disodium hydrogen phosphate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and sodium dihydrogen phosphate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0051] The carbon dispersion was obtained by mixing Ketjenbrak (EC300J, manufactured by Lion Specialty Chemicals Co., Ltd.) with a 5 mg / mL solution of hydroxypropyl cellulose (NISSO HPCL, manufactured by Nippon Soda Co., Ltd.) to a carbon concentration of 16 mg / mL, and then treating the mixture with an ultrasonic homogenizer for at least 3 minutes.

[0052] The carbon dispersion was treated in an ultrasonic bath for approximately 10 minutes before use. The concentration of polymer-bound PNTs was determined by diluting the polymer-bound PNT solution 25-fold, adding 100 μL to a microplate, and measuring the absorption spectrum with a plate reader. The concentration of PNTs in the solution was then adjusted based on this value. For example, a final concentration of 4 refers to the concentration at which the polymer-bound PNTs exhibit an absorbance of 0.16 when diluted 25-fold.

[0053] Furthermore, the polymer-bound PNTs described above were obtained through the following process. [Synthesis of I.PNT-70] (1) Synthesis of sulfonic acid fragments [5] TIFF0007894935000005.tif30100 The above reaction first synthesized a sulfonic acid fragment.

[0054] (2) Synthesis of carboxylic acid fragments [6] Carboxylic acid fragments were synthesized by a two-step reaction in acetonitrile and tetrahydrofuran (THF) as described above.

[0055] (3) Synthesis of PNT-34 [7] The sulfonic acid fragment and carboxylic acid fragment synthesized above were suspended in MeOH / H2O, and 50% Ag2CO3 / Celite was added in portions over 15 minutes at an internal temperature of approximately 47°C. After addition, the mixture was stirred for 2.5 hours at an internal temperature of approximately 68°C. After cooling to room temperature, Celite filtration was performed, and the filtrate was concentrated. The residue was purified multiple times by silica gel column chromatography to obtain PNT-34.

[0056] (4) Synthesis of the condensate (PNT-68) [8] Under an Ar atmosphere, PNT-34 was dissolved in dichloromethane, and amino-PEG12-t-butyl ester and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl) were added. The mixture was then stirred at room temperature for 3 hours. After concentrating the reaction mixture, the condensate (PNT-68) was obtained by purifying it three times by silica gel column chromatography.

[0057] (5) Synthesis of the deprotector (PNT-69) [9] The condensate (PNT-68) obtained in TIFF0007894935000009.tif56130(4) was dissolved in dichloromethane, trifluoroacetic acid (TFA) was added, and the mixture was stirred at room temperature for 3 hours. After concentrating the reaction mixture, the deprotected product (PNT-69) was obtained by azeotropic reaction several times with toluene.

[0058] (6) Synthesis of PNT-69NHS compound (PNT-70) [C10] The deprotected compound (PNT-69) was dissolved in dichloromethane, and N-hydroxysuccinimide (NHS) and EDCI·HCl were added. The mixture was then stirred at room temperature for 4 hours. The reaction mixture was purified by silica gel column chromatography to obtain PNT-69NHS compound (PNT-70), in which the carboxyl groups at the end of the PEG chain (PEG12) were activated (NHS esterified).

[0059] [II. Synthesis of Polymer C] [C11] First, 2-aminoethyl methacrylate hydrochloride, (4-vinylphenyl)methaneamine, and methacloylcholinchloride were prepared. Then, 0.97 mmol of 2-aminoethyl methacrylate hydrochloride, 0.97 mmol of (4-vinylphenyl)methaneamine, 11.97 mmol of methacloylcholinchloride, 0.08 mmol of V-50, and 10.49 g of ethanol were placed in a four-necked flask. Polymer C corresponds to a polymer with a molar ratio of n:m:l = 21.2:8.1:70.7, calculated from the results of 1H-NMR. Furthermore, as measured by gel permeation chromatography (GPC), the number-average molecular weight Mn of the polymer was 37774, and the weight-average molecular weight Mw was 214367.

[0060] [Synthesis of polymer-bound PNTs] PNT-69NHS (PNT-70) was dissolved in MilliQ water to a concentration of 21.05 mg / mL (Solution (1)). As a high molecular weight polymer, polymer C was dissolved in MilliQ water to a concentration of 15 mg / mL (Solution (2)). Next, WSC (DOJINDO W001) was dissolved in MilliQ water to a concentration of 20 mg / mL (Solution (3)). 40 μL of Solution (1), 140.3 μL of Solution (2), and 383.4 μL of Solution (3) were mixed with 96 μL of separately prepared 250 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.0), and the total volume was adjusted with MilliQ water to 1200 μL. The mixture was then allowed to react at room temperature for approximately 20 hours with stirring. Subsequently, the liquid was collected after several ultrafiltrations using a centrifugal ultrafiltration filter (Amicon Ultra-4 50k; Merck Millipore) to remove low molecular weight particles. This process yielded polymer-bound PNTs.

[0061] ●Step (3): Application of reagent solution 0.9 μL of the reagent prepared in step (2) was spread onto the electrode fabricated in step (1), and dried overnight. This formed a reagent layer on the working electrode.

[0062] ●Step (4): Preparation of polymer solution for protective film 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer (Lipidure-CM5206, NOF Corporation) was dissolved in ethanol at a concentration of 4% (w / w). In addition, styrene-2-vinylpyridine-ter-butyl methacrylate random copolymer (hereinafter referred to as "S2VPtBuMA") was dissolved in 2-propanol at a concentration of 7% (w / w).

[0063] ●Step (5): Application of protective film solution In step (3), 1 μL of Lipidure-CM5206 solution prepared in step (4) was applied to the electrode and dried at room temperature. Then, 1 μL of S2VPtBuMA solution was applied and dried at room temperature. This formed a protective film on the reagent layer.

[0064] ●Process (6): Formation of electrode section The biosensor electrode prepared in step (5) was used as the working electrode, and a gold electrode was used as the counter electrode, and an Ag / AgCl electrode (saturated KCl) (manufactured by B.A.S. Co., Ltd.) was used as the reference electrode to create a three-electrode electrode section.

[0065] Comparative Example 1A Comparative Example 1A differs from Example 1A in that polyvinylimidazole is not added in step (2): preparation of the reagent solution. Other aspects are the same as in Example 1A, and therefore, to avoid duplication, the explanation is omitted.

[0066] [Electrochemical measurements using biosensors, etc.] Next, electrochemical measurements were performed using the biosensors prepared in Comparative Example 1A and Example 1A. Furthermore, the pH of the measurement solution was measured when lactic acid was added at each concentration in Comparative Example 1A.

[0067] For the electrochemical measurements using the biosensor described above, a potentiostat (manufactured by BAS Corporation) was used, and the change in current value was measured by adding lactic acid after a predetermined time had elapsed in RPMI medium heated to approximately 37°C using the amperometric method.

[0068] For the RPMI medium, RPMI-1640 Medium (Sigma-Aldrich, R1383) was used. To simulate the buffering capacity in a CO2 incubator, MES (2-Morpholinoethanesulfonic acid monohydrate) (Dojin Chemical Co., Ltd.) and MOPS (3-Morpholinopropanesulfonic acid) (Dojin Chemical Co., Ltd.) were added as buffer components to a final concentration of 25 mM each, and the pH was adjusted to 7.4.

[0069] Specifically, the current response values ​​of the sensor were continuously measured when lactic acid solution was added to the culture medium at 500-second intervals starting 1000 seconds after the start of measurement, so that the final concentrations were 10 mM, 20 mM, 30 mM, and 40 mM. The lactic acid solution used was L-lactic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) diluted to 0.5 M with 0.5 M MOPS solution.

[0070] After the measurement was completed, the sensor was immersed in RPMI culture medium and stored at 37°C. Then, to evaluate the durability of the lactate sensor, the current response values ​​described above were measured before storage, 7 days after storage, and 13 days after storage, respectively.

[0071] [Measurement results] ● pH values ​​of the measurement solution when lactic acid is added at each concentration in Comparative Example 1A The pH of the measurement solution when lactic acid was added at each concentration in Comparative Example 1A was as follows: From the results in Table 1 below, it was found that as the lactic acid concentration in the measurement solution increases, the pH of the measurement solution may decrease and deviate from the optimal pH (6-8). (Table 1) TIFF0007894935000012.tif19117

[0072] ● Relationship between measurement time and current response value in Example 1A and Comparative Example 1A Figure 5 shows the relationship between the measurement time and the current response value in Example 1A, and Figure 6 shows the relationship between the measurement time and the current response value in Comparative Example 1A.

[0073] [Sensor responsiveness] As shown in Figures 5 and 6, in Example 1A, in which polyvinylimidazole was added to the sensor reagent solution, the sensor's current response value to lactic acid at each concentration (10 mM to 40 mM) increased stepwise compared to Comparative Example 1A, in which polyvinylimidazole was not added, and the current value after lactic acid addition remained constant.

[0074] On the other hand, in Comparative Example 1A, it was found that the current response value of the sensor decreased significantly after the addition of lactic acid, especially at high concentrations (40 mM).

[0075] The reason for the decrease in the current response value of this sensor can be understood as follows: as described above, when the lactic acid concentration was high, the pH of the measurement solution deviated from the optimal pH for the enzyme (see Table 1), and the pH of the reaction site decreased due to the protons generated during the enzymatic reaction, resulting in the enzyme being inactivated and not in a suitable active state.

[0076] In contrast, as described above, in Example 1A (a configuration in which polyvinylimidazole was added to the sensor reagent solution), the sensor's current response values ​​to lactic acid at each concentration (10 mM to 40 mM) were more stable compared to Comparative Example 1A (a configuration in which polyvinylimidazole was not added to the sensor reagent solution). This suggests that polyvinylimidazole played a role in pH buffering, effectively suppressing the pH near the enzyme from falling outside the optimal pH range for the enzyme.

[0077] In other words, in Example 1A, it is thought that the pH buffering capacity of polyvinylimidazole suppressed the inactivation of lactate oxidase.

[0078] [Durability] Furthermore, in Comparative Example 1A, it was found that the sensor's responsiveness to lactic acid at each concentration (10 mM to 40 mM) decreased with the passage of the storage period (before storage of the sensor ⇒ 7 days after storage ⇒ 13 days after storage).

[0079] In contrast, in Example 1A, it was found that the sensor's responsiveness to lactic acid at each concentration (10 mM to 40 mM) was maintained even after the storage period (before storage of the sensor ⇒ 7 days after storage ⇒ 13 days after storage).

[0080] From the above findings, it was found that adding polyvinylimidazole to the sensor reagent solution can improve sensor durability.

[0081] Example 2A [Fabrication of biosensors] A biosensor was fabricated through the following steps (1) to (6).

[0082] ●Process (1): Electrode fabrication Electrodes fabricated by sputtering platinum onto an insulating substrate were immersed in Nafion solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), removed, and dried. This process was repeated multiple times to coat the surface of the platinum electrodes with Nafion.

[0083] ●Step (2): Preparation of reagent solution The reagents listed below were mixed to the following final concentrations and reacted for approximately 1 hour to prepare the reagent solution. • Lactate oxidase (LOX T-47: manufactured by Asahi Kasei Pharma Co., Ltd.) Final concentration 40 U / mL • Bovine serum albumin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) Final concentration 12.5 mg / mL • Poly-L-histidine hydrochloride (Sigma-Aldrich), final concentration 13.7 mg / mL • Glutaraldehyde 25% solution (Fujifilm Wako Pure Chemical Industries, Ltd.), final concentration 0.03125% (w / v)

[0084] ●Step (3): Application of reagent solution One μL of the reagent prepared in step (2) was spread onto the electrode fabricated in step (1), and dried overnight. This formed a reagent layer on the working electrode.

[0085] ●Step (4): Preparation of polymer solution for protective film The following reagents were mixed to achieve the final concentrations shown below to prepare a polymer solution for the protective film. • Poly(ter-butyl methacrylate-b-4-vinylpyridine) (manufactured by Polymer Source, hereinafter referred to as "tBuMA4VP") Final concentration 7.11% (w / v) • Random copolymer of tripropylene glycol methyl ether methacrylate-styrene-4-vinylpyridine (manufactured by NARD, hereinafter referred to as "TGMAS4VP"), final concentration 0.89% (w / v) • Poly(ethylene glycol) diglycidyl ether (manufactured by Sigma-Aldrich, hereinafter referred to as "PEGDGE") Final concentration 0.98% (w / v) ·HEPES buffer (pH8.0) final concentration 5mM

[0086] tBuMA4VP, TGMAS4VP, and PEGDGE were used after being dissolved in ethanol. HEPES buffer was prepared by dissolving 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (manufactured by Dojin Chemical Co., Ltd.) in Milli-Q water and adjusting the pH with sodium hydroxide.

[0087] ●Step (5): Application of protective film solution In step (3), the electrodes prepared were immersed in a polymer solution for the protective film, removed, and dried. This process was repeated multiple times to form a protective film on each electrode. This resulted in the formation of a protective film on the reagent layer.

[0088] ●Process (6): Formation of electrode section The sensor electrode prepared in step (5) was used as the working electrode, and a palladium electrode was used as the counter electrode, along with an Ag / AgCl electrode as the reference electrode to create a three-electrode electrode section.

[0089] Comparative example 2A Comparative Example 2A differs from Example 2A in that poly-L-histidine hydrochloride is not added in step (2): preparation of the reagent solution. Other aspects are the same as in Example 2A, so to avoid duplication, the explanation is omitted.

[0090] [Electrochemical measurements using biosensors] Next, electrochemical measurements were performed using the biosensors prepared in Comparative Example 2A and Example 2A.

[0091] For the electrochemical measurements using the biosensor described above, the current response values ​​were continuously measured in RPMI medium pre-treated with a 20 mM lactic acid solution, using an amperometric method with a potentiostat in a 37°C constant temperature bath.

[0092] Specifically, the sensor's current response to lactate in RPMI medium, which had lactate pre-added, was continuously measured until 150 hours had elapsed since the start of the measurement.

[0093] For the RPMI medium, RPMI-1640 Medium (Sigma-Aldrich, R1383) was used. To this medium, 20 mM L-lactic acid (Tokyo Chemical Industries, Ltd.), 10% inactivated fetal bovine serum (Thermo Fisher, Inc.), and 1% penicillin-streptomycin-amphotericin B suspension (Fujifilm Wako Pure Chemical Industries, Ltd.) were added. MES (2-Morpholinoethanesulfonic acid monohydrate) (Dojin Chemical Co., Ltd.) and MOPS (3-Morpholinopropanesulfonic acid) (Dojin Chemical Co., Ltd.) were added as buffer components to a final concentration of 25 mM each, and the pH was adjusted to 7.4 and 6.5.

[0094] [Measurement results] ● Relationship between measurement time and current response value in Example 2A and Comparative Example 2A Figure 7 shows the relationship between the measurement time and the current response value in Example 2A, and Figure 8 shows the relationship between the measurement time and the current response value in Comparative Example 2A.

[0095] [Sensor responsiveness] As shown in Figures 7 and 8, in Comparative Example 2A (a configuration in which poly-L-histidine was not added to the sensor reagent solution), the current response value of the sensor differed depending on the pH of the measurement solution, indicating that the sensor's responsiveness to lactic acid was affected by pH.

[0096] On the other hand, in Example 2A (a configuration in which poly-L-histidine is added to the sensor reagent solution), it was found that the sensor's current response value to lactic acid was approximately the same even when there was a difference in the pH of the measurement solution.

[0097] These measurement results suggest that poly-L-histidine played a role in pH buffering, suppressing the pH dependence of the lactate sensor's responsiveness. In other words, in Example 2A, the pH buffering capacity of poly-L-histidine suppressed the pH change near the reagent, preventing a change in lactate oxidase activity due to the decrease in pH.

[0098] [Durability] Furthermore, in Comparative Example 2A, it was found that the sensor's current response value decreased as the measurement time progressed. On the other hand, in Example 2A, it was found that the sensor's current response value was maintained even as the measurement time progressed.

[0099] These measurement results revealed that poly-L-histidine, which plays a role in pH buffering capacity as described above, can also contribute to maintaining the activity of lactate oxidase.

[0100] From the above findings, it was found that adding poly-L-histidine to the sensor reagent solution suppresses the pH dependence of the sensor current response value and improves its durability.

[0101] In summary, the measurement results from Examples 1A, 2A and Comparative Examples 1A, 2A revealed that polyvinylimidazole and poly-L-histidine added to the sensor reagent solution commonly possess heterocyclic nitrogen groups, i.e., proton-accepting groups.

[0102] From the above, it was found that if the reagent layer, which is a component of the final biosensor, has a structure that includes a polymer containing a proton-receiving group in its repeating units, in addition to the enzyme, then ionized protons that may be generated when lactic acid (corresponding to analyte) is oxidized by an enzymatic reaction within the reagent layer can be suitably received.

[0103] In other words, it was found that the reaction site's pH could be suppressed from deviating from the enzyme's optimal pH, and the pH of the reaction site could be maintained within a certain range. Therefore, it was found that enzyme inactivation could be avoided even when performing continuous measurements of analytes, suppressing the pH dependence of the biosensor's responsiveness and improving its durability.

[0104] It should be noted that the present invention is not limited to the embodiments described herein, and various improvements and design modifications are possible without departing from the spirit of the invention.

[0105] Furthermore, the above-described embodiment of the present invention (Invention A) includes the following preferred embodiments. <1A> A reagent layer comprising a polymer containing a proton-receptor group in its repeating units and an oxidoreductase that oxidizes or dehydrogenates an analyte. <2A> The reagent layer according to <1A>, wherein the polymer has pH buffering capacity. <3A> The reagent layer according to <1A> or <2A>, wherein the polymer has buffering capacity within a pH range in which the oxidoreductase can maintain its activity. <4A> The reagent layer according to any one of <1A> to <3A>, wherein the proton-accepting group is a heterocyclic nitrogen group. <5A> The reagent layer according to any one of <1A> to <3A>, wherein the proton-receiving group is at least one selected from the group consisting of an ionized phosphate group, a sulfo group, and a carboxyl group. <6A> The reagent layer according to <4A>, wherein the heterocyclic nitrogen group is at least one selected from the group consisting of an imidazole group, a pyridyl group, an indolyl group, a quinolyl group, an isoquinolyl group, a tetrahydroquinolyl group, a thiazole group, an indolyl group, an imidazopyridyl group, an acridinyl group, a tetrazole group, a triazole group, a pyrazyl group, a morpholyl group, and a piperadyl group. <7A> The reagent layer according to any one of <1A> to <4A>, wherein the polymer is polyvinylimidazole. <8A> The reagent layer according to any one of <1A> to <4A>, wherein the polymer is poly-L-histidine. <9A> The reagent layer according to any one of <1A> to <3A>, or <5A>, wherein the polymer is sodium polyphosphate. <10A> The reagent layer according to any one of <1A> to <9A>, wherein the weight-average molecular weight of the polymer is 10,000 or more. <11A> A biosensor comprising a reagent layer as described in any of <1A> to <10A>. <12A> A lactic acid sensor, as described in <11A>, is a biosensor. [Industrial applicability]

[0106] A biosensor equipped with a reagent layer according to one embodiment of the present invention can be used for suitable measurement of analytes. [Explanation of symbols]

[0107] 10 Reagent layer 20 Protective film 30 Working electrode 100 Biosensors A Polymer B Enzyme

[0108] An embodiment of the present invention (Invention B) will be described below.

[0109] [Title of Invention] Polymer, Reagent containing polymer, Biosensor with a reagent layer containing polymer, and Method for synthesizing polymer [Technical field]

[0110] The present invention relates to a polymer, a reagent containing the polymer, a biosensor comprising a reagent layer containing the polymer, and a method for synthesizing a polymer. [Background technology]

[0111] In recent years, biosensors have been used in various fields, including medicine, to measure analytes (substances to be detected) within cells. One method for measuring analytes is electrochemical measurement. A biosensor in electrochemical measurement comprises a reagent layer containing an enzyme placed on an electrode (corresponding to the working electrode) and a protective membrane covering the reagent layer. By appropriately selecting an enzyme that acts on the analyte to be measured as a substrate, it becomes possible to measure various analytes. For example, lactate oxidase (LO) can be used as the enzyme. X By selecting this option, it becomes possible to measure the concentration of lactic acid as an analyte. [Prior art document] [Patent]

[0112] [Patent Document 2] Japanese Patent Publication No. 2010-517054 [Summary of the Invention] [Problems to be Solved by the Invention]

[0113] Here, the inventors of the present invention have newly discovered that there are areas for improvement in conventional biosensors. Specifically, when analytes in cells undergo a chemical reaction by enzymes, protons (H) are released. + This releases a substance, which can cause the pH of the biosensor's reagent layer to decrease. To suppress this pH decrease, one possible approach is to configure the reagent layer to include a phosphate buffer in addition to the enzyme.

[0114] However, because the molecular weight of the buffering agent mentioned above is relatively small, there is a risk that it may not remain in the reagent layer. Therefore, it may be difficult to secure a predetermined amount of buffering agent in the reagent layer, and thus difficult to suppress the decrease in pH. In biosensors, a decrease in pH can lead to the degradation of enzymes contained in the reagent layer, which may make it difficult to measure analytes properly. For these reasons, a polymer that can suppress the decrease in pH and provide water solubility during reagent formation is desired.

[0115] Therefore, the present invention aims to provide a polymer capable of suppressing pH reduction and providing water solubility, a reagent containing the polymer, a biosensor equipped with a reagent layer containing the polymer, and a method for synthesizing the polymer. [Means for solving the problem]

[0116] To achieve the above objective, in one embodiment of the present invention, A polymer is provided that has a proton-accepting group and an ionic group as repeating units.

[0117] One embodiment of the present invention provides a reagent comprising an oxidoreductase that oxidizes or dehydrogenates the polymer and analyte.

[0118] One embodiment of the present invention provides a biosensor comprising a reagent layer containing the above-mentioned reagent.

[0119] To achieve the above objective, in one embodiment of the present invention, The first step involves preparing a first solution containing a proton-accepting group, The second step involves preparing a second solution containing ionic groups, A third step involves adding the second solution to the first solution and mixing them, A fourth step involves carrying out a polymerization reaction using a mixture containing the first solution and the second solution in the presence of a polymerization initiator. A method for synthesizing polymers is provided, including [the specified element]. [Effects of the invention]

[0120] According to one embodiment of the present invention, it is possible to provide a polymer capable of suppressing pH reduction and providing water solubility, a reagent containing the polymer, and a biosensor comprising a reagent layer containing the polymer. [Brief description of the drawing]

[0121] [Figure 9] Figure 9 is a schematic cross-sectional view showing the configuration of a reagent layer containing a reagent according to one embodiment of the present invention. [Figure 10] Figure 10 is a schematic cross-sectional view showing the main components of a biosensor 100 according to one embodiment of the present invention. [Figure 11] Figure 11 is a schematic cross-sectional view showing the mechanism of action provided based on the main configuration of the biosensor 100 according to one embodiment of the present invention. [Figure 12] Figure 12 is a graph showing the relationship between the amount of HCl added dropwise and the solution pH in Example 2B and Comparative Example 2B. [Modes for carrying out the invention]

[0122] The embodiments of the present invention (Invention B) will be described in detail below.

[0123] [Polymer of the present invention] First, let me describe the polymer according to one embodiment of the present invention.

[0124] The inventors of this application have diligently studied a novel polymer structure that can suppress pH reduction and provide water solubility. As a result, they have newly discovered that when the repeating units of the polymer possess the following two characteristic groups, it may be possible to suppress pH reduction and provide water solubility.

[0125] Specifically, a polymer according to one embodiment of the present invention is characterized by comprising a "proton-accepting group" and an "ionic group" as repeating units. In this specification, a "proton-accepting group" is a proton (H) contained in the repeating unit of the polymer. + This refers to a functional group that can accept ions. In this specification, "ionic group" refers to a functional group in an ionic state that is contained in the repeating unit of a polymer and is capable of providing hydrophilicity.

[0126] In one embodiment of the present invention, the polymer has the above characteristics, and the presence of a proton-receiving group in the repeating unit allows a proton (H + This enables the acceptance of protons. In addition, the presence of ionic groups in the repeating units allows for suitable hydrophilicity, thereby enabling the provision of water solubility. Since the polymer is composed of a large number of repeating units, it is possible to effectively utilize the proton-accepting function and water-solubility-providing function of these repeating units.

[0127] As described later, when using a biosensor comprising a reagent layer containing the above polymer and enzyme, the effective proton-receiving function of the polymer allows protons (H) that may be released when analytes (e.g., lactate) in cells undergo a chemical reaction with the enzyme to be released. + This allows for the appropriate acceptance of the reagent. This suppresses the decrease in pH in the reagent layer and inhibits the degradation of the enzyme contained in the reagent layer. As a result, appropriate measurement of the analyte becomes possible. Furthermore, due to the effective water-soluble function of the polymer, it becomes possible to appropriately dissolve the polymer in the enzyme-containing solution, thereby allowing for the appropriate formation of the reagent layer as a whole.

[0128] In one embodiment of the present invention, from the viewpoint of effective proton acceptance and water solubility provision, the weight-average molecular weight (Mw) of the polymer can be 10,000 or more, 20,000 or more, 30,000 or more, 40,000 or more, 50,000 or more, 60,000 or more, 70,000 or more, 80,000 or more, 90,000 or more, 100,000 or more, or 110,000 or more, preferably 90,000 or more, for example, 98,000. The upper limit of the weight-average molecular weight (Mw) of the polymer can be 1,000,000 or less from the viewpoint of water solubility and viscosity.

[0129] In a preferred embodiment, the above-mentioned ionic group may be of zwitterionic type. As used herein, "zwitterionic type" refers to a polymer whose repeating unit has both positively charged (+) and negatively charged (-) ionic groups. The zwitterionic nature of the ionic group allows for at least double the number of ionic groups contained in the repeating unit, thereby providing a more favorable hydrophilicity. This can facilitate water solubility. For example, the zwitterionic ionic group may include a quaternary ammonium group in a cationic state and a sulfo or carboxyl group in an anionic state.

[0130] In one preferred embodiment, the above-mentioned proton-accepting group may be a heterocyclic nitrogen group containing an atom with a lone pair of electrons. In this specification, "heterocyclic nitrogen group" means having a ring structure containing nitrogen. Nitrogen has high electronegativity and high electron-withdrawing properties, making it readily acceptor of protons. Furthermore, from the viewpoint of suitably providing a buffering function through proton acceptance, considering the pH range of the reagent layer containing the polymer of the present invention, the pKa of the heterocyclic nitrogen group may be 4 to 8.

[0131] For example, a heterocyclic nitrogen group can be a pyridyl group. [C12] TIFF0007894935000013.tif4254

[0132] In another example, the heterocyclic nitrogen group can be an imidazole group. [C13] TIFF0007894935000014.tif4677

[0133] In another example, the heterocyclic nitrogen group can be a benzimidazole group. [C14] TIFF0007894935000015.tif6497

[0134] In another example, the heterocyclic nitrogen group can be an isoquinolyl group. [C15] TIFF0007894935000016.tif2973

[0135] In one embodiment, the heterocyclic nitrogen group may be at least one selected from the group consisting of the pyridyl group, imidazole group, benzimidazole group, and isoquinolyl group. That is, the heterocyclic nitrogen group may consist of two or more selected from these.

[0136] To give a specific example, the polymer according to one embodiment of the present invention can have the following structure. [C16] TIFF0007894935000017.tif12080

[0137] In the repeating unit of the above polymer, the first portion having an ionic group may contain 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate. Also, in the repeating unit of the above polymer, the second portion having a proton-accepting group may contain 4-vinylpyridine. That is, the repeating unit of the above polymer may contain a portion derived from 4-vinylpyridine and a portion derived from 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate. The portion derived from 4-vinylpyridine and the portion derived from 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate are continuous in a state of covalent bonding to each other, as shown in Chemical Formula 5 above.

[0138] [Method for synthesizing the polymer of the present invention] The following describes a method for synthesizing a polymer according to one embodiment of the present invention.

[0139] A polymer synthesis method according to one embodiment of the present invention is: The first step involves preparing a first solution containing a proton-accepting group, The second step involves preparing a second solution containing ionic groups, A third step involves adding the second solution to the first solution and mixing them, A fourth step involves carrying out a polymerization reaction using a mixture containing the first solution and the second solution in the presence of a polymerization initiator. Includes.

[0140] (1st step) In the first step, a first solution containing a proton-accepting group is prepared as described above. As an example, a first solution containing a heterocyclic nitrogen group as the proton-accepting group is prepared. Furthermore, from the viewpoint of suitably providing a buffering function by proton acceptance, the pKa of the heterocyclic nitrogen group can be 4 to 8, taking into account the pH range of the reagent layer to be formed later. As an example, this heterocyclic nitrogen group can be at least one selected from the group consisting of a pyridyl group, an imidazole group, a benzimidazole group, and an isoquinolyl group. For example, the first solution can be obtained by mixing a non-protic polar solvent (e.g., dimethyl sulfoxide (DMSO)) with the following 4-vinylpyridine (which may be called 4VP) containing a proton-accepting group. [C17] TIFF0007894935000018.tif4040

[0141] (2nd process) In the second step, a second solution containing ionic groups is prepared as described above. This substance containing ionic groups is dissolved in water as a solvent for a few seconds to prepare the second solution. The ionic groups contained in this second solution are preferably of the zwitterionic type from the viewpoint of ensuring good water solubility. As the zwitterionic ionic groups, those containing a quaternary ammonium group in a cationic state and a sulfo group or carboxyl group in an anionic state can be selected.

[0142] As an example, 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate (may be referred to as MAS), which contains an ionic group, is added to water as a solvent and dissolved in an ultrasonic cleaner for 3 to 7 seconds, for example, 5 seconds. This prepares the second solution described above. [C18] TIFF0007894935000019.tif38114

[0143] (3rd step) In the third step, the second solution is added to the first solution and mixed. Specifically, in the third step, the second solution prepared in the second step is quickly added to the first solution prepared in the first step, for example, within 0.5 seconds to 4 seconds, and uniform mixing is performed.

[0144] (4th step) In the fourth step, a polymerization reaction is carried out using a mixture containing the first and second solutions in the presence of a polymerization initiator. As the polymerization initiator, for example, powdered azobisisobutyronitrile (also known as AIBN) can be used as an initiator for radical reactions. [C19] TIFF0007894935000020.tif2359

[0145] Specifically, an initiator is added to the above mixture in a three-necked flask, and then the inside of the flask is replaced with a nitrogen environment to start the polymerization reaction. After starting the polymerization reaction at a predetermined reaction temperature and stirring rate, the polymerization reaction is terminated after a predetermined reaction time has elapsed. After the polymerization reaction is complete, Milli-Q water is added to dissolve the polymer product. Then, in order to purify the polymer product, the solution is added dropwise to hexane to form a precipitate. After that, the precipitate is collected, evaporated, and finally dried under reduced pressure. By doing so, a polymer according to one embodiment of the present invention can be obtained.

[0146] [Reagents containing the polymer of the present invention] The following describes a reagent containing a polymer according to one embodiment of the present invention. Figure 9 is a schematic cross-sectional view showing the structure of a reagent layer containing a reagent according to one embodiment of the present invention.

[0147] The polymer according to one embodiment of the present invention can be used in a reagent containing an enzyme. That is, in one embodiment of the present invention, the reagent may contain at least the polymer A and enzyme B. Reagent layer 10 is a layer containing this reagent (see Figure 9). Enzyme B may be an oxidoreductase that oxidizes or dehydrogenates the analyte. As used herein, "oxidoreductase" means a biochemical substance capable of specifically catalyzing the oxidation or reduction of the analyte.

[0148] For example, examples of oxidoreductases include glucose oxidase, lactate oxidase, cholesterol oxidase, bilirubin oxidase, glucose dehydrogenase, lactate dehydrogenase, amino acid oxidase, amino acid dehydrogenase, glutamate oxidase, glutamate dehydrogenase, fructosyl amino acid oxidase, fructosyl peptide oxidase, 3-hydroxybutyrate dehydrogenase, alcohol oxidase, and / or alcohol dehydrogenase.

[0149] The above-mentioned oxidoreductases can be used to detect glucose, lactic acid, cholesterol, bilirubin, amino acids such as glutamine and glutamic acid, glycated amino acids or glycated peptides, ketone bodies (3-hydroxybutyric acid), alcohol, etc. The amount of oxidoreductase is, for example, 0.01 U to 100 U (μmol / min) per biosensor or per measurement, preferably 0.05 U to 10 U, and more preferably 0.1 U to 5 U.

[0150] Furthermore, the above reagent may further contain a mediator and / or conductive particles in addition to the polymer and enzyme. sexual grain Examples of its offspring include carbon black and carbon nanotubes.

[0151] In this specification, "mediator" broadly refers to a redox substance that mediates electron transfer, and narrowly refers to a substance that facilitates electron transfer resulting from the redox reaction of analytes in the biosensors described below.

[0152] While not particularly limited, mediators include metal complexes (e.g., osmium complexes, ruthenium complexes, iron complexes, etc.), quinone compounds (e.g., benzoquinone, naphthoquinone, phenanthrenequinone, phenanthrolinequinone, anthraquinone, and their derivatives, etc.), phenazine compounds, viologen compounds, phenothiazine compounds, and phenol compounds.

[0153] More specifically, one or more substances selected from the group consisting of potassium ferricyanide, hexaammineruthenium, ferrocene, poly(1-vinylimidazole)-bis(bipyridine)chloroosmium, hydroquinone, 2-methyl-1,4-benzoquinone, 1,2-naphthoquinone-4-sulfonate, 9,10-phenanthrenequinone-2-sulfonate, 9,10-phenanthrenequinone-2,7-disulfonate, 1,10-phenanthroline-5,6-dione, anthraquinone-2-sulfonate, phenazine derivatives such as 1-methoxy-5-methylphenadinium methyl sulfate and 1-methoxy-5-ethylphenadinium ethyl sulfate, methyl viologen, benzyl viologen, methylene blue, methylene green, 2-aminophenol, 2-amino-4-methylphenol, and 2,4-diaminophenol are used as mediators.

[0154] The above-mentioned salts are not limited to sodium salts, potassium salts, calcium salts, magnesium salts, lithium salts, etc. The amount of mediator to be added is not particularly limited, and is, for example, 0.1 pmol to 1000 μmol per measurement or per biosensor as described below, preferably 10 pmol to 500 μmol, and more preferably 500 pmol to 100 μmol.

[0155] [Biosensor] The following describes a biosensor according to one embodiment of the present invention. Figure 10 is a schematic cross-sectional view showing the main components of the biosensor 100 according to one embodiment of the present invention.

[0156] As used herein, a "biosensor" is a measuring device that uses combinations such as enzyme-substrate to convert the chemical changes that occur due to molecular recognition reactions between a substrate (corresponding to an analyte) and an enzyme (corresponding to a receptor) into electrical signals, and measures the metabolic rate, concentration, etc. of the analyte according to the strength of the resulting electrical signal.

[0157] A biosensor 100 according to one embodiment of the present invention comprises a reagent layer 10 and a protective film 20 disposed on an electrode portion formed on the surface of a substrate (see Figure 10). For convenience, in this embodiment, only the reagent layer 10 and protective film 20 of the biosensor 100 are shown, while other electrode portions and the like are omitted from the illustration.

[0158] An insulating substrate can be used as the substrate. While not particularly limited, the substrate may be composed of materials such as polyethylene terephthalate, polyamide, or polyimide, with a thickness of several hundred μm. In one embodiment, the electrode portion of the biosensor 100 can be inserted into a liquid culture medium (liquid sample) provided in a culture tank. Furthermore, after inserting the biosensor 100, a voltage can be applied to the electrode portion.

[0159] The electrode portion described above comprises a working electrode and a counter electrode and / or a reference electrode. A reagent layer 10 containing the polymer and enzyme of the present invention may be disposed on the surface of the working electrode.

[0160] The protective film 20 may be configured to cover the reagent layer 10 and the electrode portion. The protective film 20 is configured to control the permeation rate of specific components (such as lactic acid) of cells in the culture medium, while allowing those specific components to penetrate towards the working electrode. Furthermore, the protective film 20 is configured to suppress the outflow of components (such as the polymer and enzyme of the present invention) contained in the reagent layer 10 on the working electrode to the outside of the protective film 20. That is, the "protective film" as used herein is a film that contributes to suppressing the leakage of substances contained in the reagent layer to the outside of the protective film, and is a film that has pores through which analytes present outside the protective film can move towards the reagent layer.

[0161] The protective film may be a biocompatible polymer film. The polymer constituting this protective film may be t-butyl acrylate having heterocyclic nitrogen groups. Examples of heterocyclic nitrogen groups include pyridyl groups and imidazole groups. Furthermore, the polymer constituting this protective film may also include Nafion, which has a fluorocarbon as its main chain and sulfo and carboxyl groups as side chains.

[0162] While not particularly limited, examples of culture media include RPMI-1640 medium, D-MEM medium, F12, and MEM medium. Note that in this invention, the culture media do not contain enzymes.

[0163] Under the above configuration, with the electrode immersed in the culture medium, the analyte, which moves from the culture medium to the reagent layer 10 via the protective film 20, is oxidized in the reagent layer 10 using an enzyme as a catalyst and dissolved oxygen. By electrically detecting the hydrogen peroxide produced at that time, it becomes possible to measure the concentration of the analyte.

[0164] Figure 11 is a schematic cross-sectional view illustrating the mechanism of action provided by a biosensor 100 according to one embodiment of the present invention, based on its main configuration. Taking the detection of lactic acid as an example, lactic acid (corresponding to Lac in Figure 11) that has moved from the culture medium to the reagent layer 10 through the protective membrane 20 is oxidized by an enzymatic reaction with the enzyme in the reagent layer 10 (corresponding to LOx in Figure 11), potentially forming pyruvate (corresponding to Pyru in Figure 11), hydrogen peroxide, and ionized protons (H+). By electrically measuring the hydrogen peroxide produced during this formation, it becomes possible to measure the metabolic rate of lactic acid, etc. In this case, the biosensor 100 can function as a lactic acid sensor.

[0165] As described above, the polymer of the present invention can effectively provide both proton-receiving function and water-soluble function. The biosensor 100 comprises a reagent layer 10 containing polymer A and enzyme B. Therefore, due to the effective proton-receiving function of polymer A, the analyte (lactic acid in Figure 11) that has moved into the reagent layer 10 can release protons (H) when it undergoes a chemical reaction with enzyme B. + This allows for the appropriate acceptance of the reagent layer 10. As a result, the decrease in pH in the reagent layer 10 can be suppressed, and the degradation of enzyme B contained in the reagent layer 10 can be suppressed. Consequently, it becomes possible to directly insert the biosensor 100 into a culture vessel supplied with culture medium and appropriately measure the analyte.

[0166] Furthermore, due to the effective water-soluble function of polymer A, polymer A can be suitably dissolved in the solution containing enzyme B, thereby allowing for the suitable formation of the reagent layer 10 overall. Moreover, compared to conventional low-molecular-weight buffering agents used to suppress pH decrease, polymer A of the present invention has a high molecular weight, which allows it to remain in the reagent layer 10 and suitably suppresses outflow to the culture medium located outside the protective film 20.

[0167] In conventional biosensors, the protective film mentioned above sometimes contains a heterocyclic nitrogen-containing polymer. This protective film merely serves to limit the diffusion of analytes to the working electrode, which is a component of the sensor. On the other hand, in the biosensor 100 of the present invention, the polymer is contained in the reagent layer 10 (not the protective film 20), which is a component of the sensor. Furthermore, this polymer has groups with different properties: a proton-receiving group that contributes to proton acceptance and an ionic group that contributes to providing water solubility. Therefore, compared to the embodiment in which the protective film contains a heterocyclic nitrogen-containing polymer, the polymer of the present invention differs in both its location and function (both proton-receiving and water-solubility functions).

[0168] [Method for fabricating a biosensor] The following describes an example of a method for fabricating the above-mentioned biosensor.

[0169] First, an insulating substrate is prepared. After preparing the insulating substrate, a conductive thin film (corresponding to the working electrode) selected from a metal such as silver, platinum, or palladium is deposited on the insulating substrate by sputtering, vapor deposition, or ion plating. The thickness of the conductive thin film can range from 10 nm to several hundred nm. The counter electrode and / or reference electrode may be placed around the working electrode, for example, on the back side of the substrate. An insulating resist layer may be formed in areas other than these electrode formation areas.

[0170] Subsequently, a reagent containing at least the polymer and enzyme solution of the present invention is applied to the working electrode. In addition to the polymer and enzyme, the reagent may further contain a suspension of conductive particles such as carbon particles for imparting conductivity and an aqueous solution of a mediator. By drying this reagent applied to the working electrode, a reagent layer 10 can be suitably formed. Finally, a protective film 20 is formed to cover the reagent layer 10 and the electrode portion. By doing so, a biosensor 100 can be manufactured. [Examples]

[0171] The following describes embodiments of the present invention (Invention B).

[0172] Example 1B [Synthesis of the polymer of the present invention]

[0173] (Implementation of the first step) In the first step, a first solution was prepared by mixing dimethyl sulfoxide (DMSO) with 4-vinylpyridine (which may be referred to as 4VP) and styrene.

[0174] The usage amount, concentration, and product number are as follows. [Amount used] • DMSO: 24.8 mL • 4VP: 3.539mL • Styrene: 0.077 mL [4VP concentration] 12.45 v / v% [Styrene concentration] 0.27 v / v% [Product number] • DMSO: 045-28335 (Manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) • 4VP: V0150 (manufactured by Tokyo Chemical Industry Co., Ltd.)

[0175] (Implementation of the second step) In the second step, 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate (which may be referred to as MAS) was added to water as a solvent and dissolved in an ultrasonic cleaner for 5 seconds. This prepared the second solution.

[0176] The usage amount, concentration, and product number are as follows. [Amount used] MAS: 9.36g MilliQ Water: 5.0 mL [MAS concentration] 65.2 (w / w%) [Part Number] MAS: M1971 (Manufactured by Tokyo Chemical Industry Co., Ltd.)

[0177] (Implementation of the third step) In the third step, the second solution was added to the first solution and mixed. Specifically, the second solution prepared in the second step was added to the first solution prepared in the first step within 1 second and mixed uniformly. The mixing ratio (molar ratio) in this mixing was styrene:4VP:MAS = 1:49:50.

[0178] (4th step) In the fourth step, a polymerization reaction was carried out using a mixture containing the first and second solutions in the presence of a polymerization initiator. Specifically, the initiator (azobisisobutyronitrile (also known as AIBN)) was added to the mixture in a three-necked flask. The solution temperature at the time of AIBN addition was 24°C. Subsequently, the flask was replaced with a nitrogen environment, and the polymerization reaction was started.

[0179] The amount, concentration, and product code of the initiator to be used are as follows. [Amount used] AIBN: 0.064g [Concentration of AIBN] Add the AIBN powder to a three-necked flask. AIBN concentration: 100% [Product number] AIBN: 019-04932 (Manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

[0180] Subsequently, the polymerization reaction was started at a reaction temperature of 80°C and a stirring speed of 500 rpm, and terminated after a reaction time of 42.5 hours. After the polymerization reaction was complete, Milli-Q water was added to dissolve the polymer product. Then, the solution was added dropwise to hexane to form a precipitate, which was collected, evaporated, and finally dried under reduced pressure. Through these steps, the polymer of the present invention was synthesized.

[0181] Example 2B [Characterization of the polymer of the present invention] The properties of the polymer of the present invention synthesized as described above were evaluated by the following method.

[0182] First, 1 mL of the polymer of the present invention (250 mg / mL) was added to 50 mL of distilled water (DW) and dissolved. HCl at a concentration of 0.1 mol / L was then added sequentially to the resulting solution. To ensure reproducibility, the same procedure was repeated. Afterward, the pH of each solution was evaluated using a pH meter.

[0183] As a comparative example that can be compared to Example 2B, HCl at a concentration of 0.1 mol / L was sequentially added to 50 mL of DW (distilled water). To ensure reproducibility, the same procedure was repeated. After that, the pH of each sample was evaluated using a pH meter.

[0184] Evaluation result 1 is shown in Figure 12.

[0185] As shown in Figure 12, in a comparative example where HCl at a concentration of 0.1 mol / L was sequentially added to DW without using the polymer of the present invention, the pH, which was around 5.5 at the start of HCl addition, immediately decreased to around 4. Specifically, the pH became around 4 when the concentration of added HCl was around 0.14 mmol / L.

[0186] On the other hand, in Example 2B, where HCl at a concentration of (0.1 mol / L) was sequentially added to a solution containing the polymer of the present invention in DW (distilled water), the pH, which was around 7 at the start of HCl addition, finally dropped to around 4 when the concentration of added HCl reached approximately 3.4 mmol / L. Furthermore, in Example 2B, 1 mL of the polymer of the present invention (250 mg / mL) could be suitably added to 50 mL of DW (distilled water). From the above, it was found that the polymer of the present invention has both a pH decrease suppression function and a water solubility providing function.

[0187] Furthermore, the weight-average molecular weight (Mw) and number-average molecular weight (Mn) of the obtained polymer of the present invention were measured using GPC (Gel Permeation Chromatography). The measurement results for each average molecular weight were as follows. ·Weight average molecular weight (Mw): 98,000 ·Number average molecular weight (Mn): 13,000

[0188] Example 3B The following steps were followed: [1. Polymer synthesis], [2. Fabrication of a sensor containing the synthesized polymer], and [3. Sensor immersion experiment]. Note that, to avoid duplication with the description in Example 1B, the relevant sections are omitted.

[0189] [1. Polymer Synthesis] (Implementation of the first step) In the first step, dimethyl sulfoxide (DMSO) was mixed with 4-vinylpyridine and styrene to prepare the first solution. The preparation conditions for the first solution in this first step were the same as in Example 1B.

[0190] The usage amount, concentration, and product number are as follows. [Amount used] • DMSO: 24.8 mL • 4VP: 3.539 mL (105.14 / mol) Styrene: 0.077 mL (104.15 / mol) [4VP concentration] 12.45 v / v% [Styrene concentration] 0.27 v / v% [Product number] • DMSO: 045-28335 (Manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) • 4VP: V0150 (manufactured by Tokyo Chemical Industry Co., Ltd.)

[0191] (Implementation of the second step) In the second step, 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate was added to water as the solvent to prepare the second solution. The preparation conditions for the second solution in this second step were the same as in Example 1B.

[0192] The usage amount, concentration, and product number are as follows. [Amount used] MAS: 9.36 g (279.35 / mol) Millipore water: 5.0 mL [MAS concentration] 65.2 (w / w%) [Product number] MAS: M1971 (manufactured by Tokyo Chemical Industry Co., Ltd.)

[0193] (Implementation of the third step) In the third step, the above second solution was added to the first solution and mixed. Regarding the mixing ratio (molar ratio), styrene: 4VP: MAS = 1: 49: 50. The addition and mixing conditions of the first solution and the second solution in this third step are the same as those in Example 1B.

[0194] (Fourth step) In the fourth step, a polymerization reaction was carried out using the mixture containing the above first solution and second solution in the presence of a polymerization initiator. The polymerization reaction conditions in this fourth step are the same as those in Example 1B.

[0195] After the polymerization reaction was completed, Millipore water was added to dissolve the polymerization reaction product. Then, the dissolved solution was dropped into hexane to form and collect precipitates, evaporation was performed, and finally vacuum drying was carried out. Thus, the polymer of the present invention could be synthesized. In the obtained polymer, the molecular weight of the polymer basic structural unit composed of 4VP and MAS was 191.63.

[0196] After polymer synthesis, 250.0 mg of the same polymer was weighed, 1 mL of DW (distilled water) was added, and a polymer solution was prepared. Thereby, the concentration of the same polymer solution was set to 250 mg / mL (0.25 mg / μL, 1304.6 mmol / L).

[0197] [2. Fabrication of a sensor containing the synthesized polymer] After polymer synthesis, a sensor was fabricated through the following steps.

[0198] ●(1): Fabrication of the working electrode Using a screen printing machine (manufactured by Celia Corporation), carbon ink XC-3180 (manufactured by Fujikura Kasei Co., Ltd.) was printed on an insulating substrate to form a conductive thin film. As the insulating substrate, PET W400J (manufactured by Mitsubishi) was used.

[0199] ●(2): Preparation of the water-repellent resist layer Using a screen printing machine (manufactured by Celia Corporation), resist ink XB-3342 (manufactured by Fujikura Kasei Co., Ltd.) was printed on the working electrode to form a water-repellent resist.

[0200] ●(3) Corona discharge treatment The surface of the working electrode was treated using a corona discharge surface modification device (manufactured by Shinko Electric Instrument Co., Ltd.).

[0201] ●(4): Preparation of the water-repellent film layer A fluorine-based water-repellent film SS4A (manufactured by Nippa) was attached onto the above water-repellent resist layer to fabricate a water-repellent film layer.

[0202] ●(5): Coating of the polymer solution On the working electrode fabricated in step (1), 1 μL of the 250 mg / mL polymer solution formed in [1. Polymer synthesis] was coated and dried at room temperature to form a polymer film.

[0203] ●(6): Preparation and coating of the solution for the protective film After the above drying, 0.6 mg of a neutralized Nafion solution for film coating at 16.125 wt% was coated on the polymer film and dried at room temperature to form a Nafion film. Subsequently, after the above drying, 0.6 mg of a 4VP-tBuMA solution for film coating was coated on the Nafion film and dried at room temperature to form a 4VP-tBuMA film. Thus, a protective film was formed on the polymer film.

[0204] (Preparation of the neutralized Nafion solution for film coating) <(被膜用中和ナフィオン溶液の調製)>0000925The above-mentioned neutralized Nafion solution for coating was prepared through the following steps. Specifically, 26250.7 mg of 20 wt% Nafion dispersion (Catalog No. 663492, manufactured by Sigma-Aldrich) was added to a bottle. Next, 7355.2 mg of ethanol (Catalog No. 057-00451, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the bottle. Then, 1394.08 mg of 5 mol / L sodium hydroxide aqueous solution (Catalog No. 196-05375, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the bottle. Finally, the precipitate was completely dissolved by repeatedly stirring for 60 seconds and sonicating for 5 seconds using VORTEX-GENIE 2. The neutralized Nafion solution for coating was thus prepared.

[0205] (Preparation of 4VP-tBuMA solution for coating) Furthermore, the 4VP-tBuMA solution for coating was prepared through the following steps. Specifically, prior to preparing the 4VP-tBuMA solution for coating, 4.0 g of 4VP-tBuMA (product code P42487, [molecular weight ratio] 4VP:tBuMA = 120,000:270,000, manufactured by Polymer Source) was added to a bottle, followed by 36 g of ethanol (product code 057-00451, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and the mixture was stirred using a ROTATOR VMR-3R. This prepared a 10 wt% 4VP-tBuMA stock solution. Separately, 1.0 g of PEGDGE1000 (product code 805505, manufactured by Sigma-Aldrich) was added to a bottle, followed by 7.83 g of ethanol, and the mixture was stirred using a ROTATOR VMR-3R. This prepared an 11.32 wt% PEGDGE1000 stock solution.

[0206] Next, 49.98 mg of ethanol was added to the bottle, followed by 106.02 mg of PEGDGE1000 solution (0.6 wt%) prepared above, and then 1144 mg of tBuMA-4VP solution (5.72 wt%). The mixture was then gently stirred using a VORTEX-GENIE 2. After stirring, the opening of the bottle was sealed with Parafilm and polyimide tape, and the mixture was rotated and stirred in a 53°C constant temperature bath for 16 hours. After that, the bottle was removed from the constant temperature bath, and the removed bottle was gently rotated and stirred in a tube rotator until just before application. The 4VP-tBuMA solution for coating was thus prepared.

[0207] [3. Sensor immersion experiment] Subsequently, immersion experiments were conducted on the sensors obtained in [2. Fabrication of sensors containing the synthesized polymer].

[0208] Specifically, the obtained sensors were immersed in 1.5 mL of pure water, and the immersed sensors were left in a 37°C constant temperature bath for 22 hours. After that, the eluate from the sensors was collected, and the MAS concentration in the collected eluate was measured. This MAS concentration was measured using a UV-Vis spectrophotometer (wavelength: 256 nm) (JASCO Corporation, model V-650).

[0209] Comparative Example 3B (An example for comparison with Example 3B) Comparative Example 3B differs from Example 3B in that, during sensor fabrication, phosphate buffer was applied to the working electrode instead of a polymer. The applied phosphate buffer was dried at room temperature after application.

[0210] In Comparative Example 3B, the concentrations of the polymer solution and phosphate buffer applied to the working electrode were set to the same (1304.6 mmol / L), and the application amount was also the same (1 μL). Furthermore, the type and concentration of the constituent materials of the protective film, which is a component of the sensor, were also the same.

[0211] In Comparative Example 3B, the sensor immersion experiment after sensor fabrication was carried out using the same procedure as in Example 3B. Specifically, the obtained sensor was immersed in 1.5 mL of pure water, and the sensor immersed in pure water was left in a constant temperature bath at 37 degrees Celsius for 22 hours.

[0212] Thereafter, the eluate from the sensor was collected, and the phosphoric acid concentration in the collected eluate was measured. This phosphoric acid concentration was measured using ion chromatography (manufactured by Thermo Scientific, model ICS 5000+). From the perspective of avoiding duplication with the content described in Example 3B, the description of the overlapping content is omitted.

[0213] [Measurement Results] In Comparative Example 3B, the phosphoric acid concentration in the eluate was 1.12 μmol, and the elution rate was 85.81%. In contrast, in Example 3B, the MAS concentration in the eluate was 0.042 mg, and the elution rate was 16.61%.

[0214] [Evaluation] From the above measurement results, it was found that in Example 3B, 84% of the MAS constituting the polymer film could remain in the sensor covered with the protective film. On the other hand, in Comparative Example 3B, it was found that only 15% of the phosphoric acid constituting the phosphoric acid buffer film could remain in the sensor covered with the protective film.

[0215] That is, it was found that the elution of MAS to the outside through the protective film in Example 3B was suppressed by about 5.2 times compared to the elution of phosphoric acid to the outside through the protective film in Comparative Example 3B.

[0216] From the above, it was found that compared with the case of using a low-molecular-weight phosphoric acid buffer such as Comparative Example 3B, the polymer in Example 3B tends to remain in the polymer film (corresponding to the reagent layer in the present invention), and it is possible to preferably suppress the outflow to the medium side located outside the protective film.

[0217] Note that the present invention is not limited to the exemplified embodiments, and various improvements and design changes are possible without departing from the gist of the present invention.

[0218] Furthermore, the above-described embodiment of the present invention (Invention B) includes the following preferred embodiments. <1B> A polymer having proton-receiving groups and ionic groups as repeating units. <2B> <1B> A polymer that is water-soluble. <3B> A polymer having a weight-average molecular weight of 10,000 or more in <1B> or <2B>. <4B> A polymer in which the ionic group is of the zwitterionic type in any of <1B> to <3B>. <5B> A polymer in which, in any of <1B> to <4B>, the ionic group comprises a quaternary ammonium group in a cationic state and a sulfo group or carboxyl group in an anionic state. <6B> A polymer in which, in any of <1B> to <5B>, the proton-accepting group is a heterocyclic nitrogen group. <7B> In <6B>, a polymer in which the pKa of the heterocyclic nitrogen group is 4 to 8. <8B> A polymer in <6B> or <7B> in which the heterocyclic nitrogen group is at least one selected from the group consisting of a pyridyl group, an imidazole group, a benzimidazole group, and an isoquinolyl group. <9B> A polymer in which any of <1B> to <8B> comprises 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate in the first portion having the ionic group. <10B> A polymer in which, in any of <1B> to <9B>, the second portion having the proton-receiving group contains 4-vinylpyridine. <11B> A polymer in which, in any of <1B> to <10B>, the repeating unit comprises a portion derived from 4-vinylpyridine and a portion derived from 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate. <12B> A reagent containing an oxidoreductase that oxidizes or dehydrogenates any of the polymers and analytes from <1B> to <11B>. <13B> A biosensor comprising a reagent layer containing reagent <12B>. <14B> <13B> is a biosensor, which is a lactic acid sensor. <15B> The first step involves preparing a first solution containing a proton-accepting group, The second step involves preparing a second solution containing ionic groups, A third step involves adding the second solution to the first solution and mixing them, A fourth step involves carrying out a polymerization reaction using a mixture containing the first solution and the second solution in the presence of a polymerization initiator. A method for synthesizing polymers, including [the specified element]. <16B> A method for synthesizing a polymer, wherein in the first step, a first solution containing a heterocyclic nitrogen group as the proton-accepting group is prepared in <15B>. <17B> A method for synthesizing a polymer in which the pKa of the heterocyclic nitrogen group is 4 to 8 in <15B> or <16B>. <18B> A method for synthesizing a polymer, wherein in <16B> or <17B>, the heterocyclic nitrogen group is at least one selected from the group consisting of a pyridyl group, an imidazole group, a benzimidazole group, and an isoquinolyl group. <19B> A method for synthesizing a polymer, wherein in any of <15B> to <18B>, the first solution is a solution containing 4-vinylpyridine. <20B> A method for synthesizing a polymer, wherein in any of <15B> to <19B>, the second step involves preparing the second solution in which the ionic group is of the zwitterionic type. <21B> A method for synthesizing a polymer, wherein in any of <15B> to <20B>, the ionic group of the second solution contains a quaternary ammonium group in a cationic state and a sulfo group or carboxyl group in an anionic state. <22B> A method for synthesizing a polymer, wherein in any of <15B> to <21B>, the second solution is a solution containing 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate. <23B> A method for synthesizing a polymer, wherein in any of <15B> to <22B>, in the second step, the substance containing the ionic group is dissolved in water as a solvent for 3 seconds or more and 7 seconds or less to prepare the second solution. <24B> A method for synthesizing a polymer, wherein in any of <15B> to <23B>, in the third step, the second solution prepared in the second step is added to the first solution within 0.5 seconds to 4 seconds. [Industrial applicability]

[0219] A biosensor comprising a reagent layer containing a polymer according to one embodiment of the present invention enables suitable measurement of analytes. [Explanation of symbols]

[0220] 10 Reagent layer 20 Protective film 100 Biosensors A Polymer B Enzyme

Claims

1. A reagent layer for an electrochemical sensor comprising a polymer having a weight-average molecular weight of 10,000 to 1,000,000, which contains proton-accepting groups in its repeating units and has buffering capacity in the pH range of 6 to 8; an oxidoreductase that can maintain its activity in the aforementioned pH range for oxidizing or dehydrogenating the analyte; and conductive particles.

2. The reagent layer for an electrochemical sensor according to claim 1, wherein the proton-accepting group is a heterocyclic nitrogen group.

3. The reagent layer for an electrochemical sensor according to claim 1, wherein the proton-receiving group is at least one selected from the group consisting of an ionized phosphate group, a sulfo group, and a carboxyl group.

4. The reagent layer for an electrochemical sensor according to claim 2, wherein the heterocyclic nitrogen group is at least one selected from the group consisting of imidazole group, pyridyl group, indolyl group, quinolyl group, isoquinolyl group, tetrahydroquinolyl group, thiazole group, indolidyl group, imidazopyridyl group, acridinyl group, tetrazole group, triazole group, pyrazyl group, morpholyl group, and piperadyl group.

5. The reagent layer for an electrochemical sensor according to claim 1, wherein the polymer is polyvinylimidazole.

6. The reagent layer for an electrochemical sensor according to claim 1, wherein the polymer is poly-L-histidine.

7. The reagent layer for an electrochemical sensor according to claim 1, wherein the polymer is sodium polyphosphate.

8. The reagent layer for an electrochemical sensor according to claim 1, wherein the polymer comprises a portion of the repeating unit derived from 4-vinylpyridine and a portion derived from 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate.

9. An electrochemical sensor for detecting or quantifying analytes, An electrochemical sensor having a working electrode, a counter electrode, and a reagent layer according to claim 1 disposed on the working electrode, and having a protective film, wherein the polymer does not leak out.

10. The electrochemical sensor according to claim 9, which is a lactic acid sensor.

11. A polymer having a weight-average molecular weight of 10,000 to 1,000,000 and containing a proton-accepting group in its repeating units and having buffering capacity in the pH range of 6 to 8, and an oxidoreductase that can maintain activity in the pH range for oxidizing or dehydrogenating an analyte, A reagent layer for an electrochemical sensor, wherein the polymer is poly-L-histidine.

12. A polymer having a weight-average molecular weight of 10,000 to 1,000,000 and containing a proton-accepting group in its repeating units and having buffering capacity in the pH range of 6 to 8, and an oxidoreductase that can maintain activity in the pH range for oxidizing or dehydrogenating an analyte, A reagent layer for an electrochemical sensor, wherein the polymer is sodium polyphosphate.

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