Reagent Layer and Sensor

Abstract

A polymer 36 contained in a reagent layer 32 of a sensor for the measurement of an analyte in a sample includes: a first binding site 46 that forms a covalent bond with a protein 38 that acts on an analyte; and a second binding site 48 that binds with the protein 38 through electrostatic interaction.

Description

[Technical field]

[0001] The present disclosure relates to polymers, reagent layers and sensors. [Background technology]

[0002] Conventionally, sensors that measure analytes in a sample by reacting proteins with the analytes are known. One such sensor is an electrochemical sensor that uses an enzyme. A representative example of an electrochemical sensor that uses an enzyme is an electrochemical glucose biosensor that is used for self-measurement of blood glucose. The electrochemical glucose biosensor uses glucose oxidoreductase.

[0003] In recent years, implantable electrochemical sensors have been developed that continuously measure analytes in living organisms. Such implantable electrochemical sensors are generally implanted in living organisms for long periods of time, such as several days to several weeks. Therefore, it is necessary to prevent the oxidoreductase from leaking out of the sensor. In response to this, a technique is known in which a reagent layer containing the oxidoreductase is covered with a protective film to prevent the oxidoreductase from leaking out of the sensor (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2019 / 146788 Summary of the Invention [Problem to be solved by the invention]

[0005] By suppressing the outflow of oxidoreductases using a protective film, it is possible to suppress the decrease in durability and measurement sensitivity of the electrochemical sensor and the negative effects on living organisms. On the other hand, there is always a demand to further suppress the decrease in durability and measurement sensitivity of electrochemical sensors and to further suppress the negative effects on living organisms. Furthermore, even in sensors that are not implanted or sensors that use proteins other than oxidoreductases, it is naturally required to maintain durability and measurement sensitivity. Therefore, it is desirable to further suppress the outflow of proteins to the outside of the sensor.

[0006] The present disclosure has been made in light of these circumstances, and one of its objectives is to provide a technique for suppressing the outflow of proteins to the outside of a sensor. [Means for solving the problem]

[0007] In order to solve the above problems, one aspect of the present disclosure is a polymer contained in a reagent layer of a sensor for measuring an analyte in a sample, the polymer having a first binding site that covalently binds to a protein that acts on the analyte, and a second binding site that binds to the protein through electrostatic interaction.

[0008] Another aspect of the present disclosure is a reagent layer of a sensor for measuring an analyte in a sample, the reagent layer comprising the polymer of the above aspect and a protein that binds to the polymer and acts on the analyte.

[0009] Another aspect of the present disclosure is a sensor. The sensor includes an electrode portion including a working electrode and a counter electrode, and the reagent layer of the above aspect that is disposed so as to be in contact with the working electrode.

[0010] Any combination of the components described above, and any conversion of the expressions of the present disclosure between methods, devices, systems, etc. are also valid as aspects of the present disclosure. Effect of the Invention

[0011] According to the present disclosure, it is possible to suppress the outflow of proteins to the outside of the sensor. [Brief description of the drawings]

[0012] [Figure 1] Fig. 1(A) is a diagram showing the front side of a sensor according to an embodiment, and Fig. 1(B) is a diagram showing the back side of a sensor according to an embodiment. [Diagram 2] 2(A) to 2(D) are diagrams showing the manufacturing process of the sensor. [Diagram 3] 3(A) to 3(D) are diagrams showing the manufacturing process of the sensor. [Figure 4] 4(A) to 4(C) are diagrams showing the manufacturing process of the sensor. [Diagram 5] FIG. 2 is a cross-sectional view taken along the line AA shown in FIG. [Figure 6] Fig. 6(A) is a cross-sectional view taken along line BB in Fig. 5. Fig. 6(B) is a cross-sectional view taken along line CC in Fig. 5. [Figure 7] FIG. 2 is a schematic diagram showing the state of a polymer, a protein, and a redox mediator in a reagent layer. [Figure 8] FIG. 1 shows combinations of reactive groups capable of covalent bonding. [Figure 9] FIG. 2 is a schematic diagram for explaining the measurement principle of a sensor. [Figure 10] FIG. 2 is a diagram showing the polymerization reaction of polymer A. [Figure 11] FIG. 2 is a diagram showing the polymerization reaction of polymer B. [Figure 12] FIG. 2 is a diagram showing the polymerization reaction of polymer C. [Figure 13] FIG. 1 shows the absorption spectra of each mediator-bound polymer. [Figure 14] FIG. 13 is a diagram showing the results of durability evaluation of a sensor. [Figure 15] FIG. 2 is a diagram showing the polymerization reaction of polymer E. [Figure 16] FIG. 2 is a diagram showing the polymerization reaction of polymer F. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present disclosure will be described below with reference to the drawings based on preferred embodiments. The embodiments are illustrative and do not limit the present disclosure, and all features and combinations thereof described in the embodiments are not necessarily essential to the present disclosure. The same or equivalent components, members, and processes shown in each drawing are given the same reference numerals, and duplicated descriptions are omitted as appropriate. In addition, the scale and shape of each part shown in each drawing are set for convenience to facilitate explanation, and are not to be interpreted as being limiting unless otherwise specified. In addition, when terms such as "first" and "second" are used in this specification or claims, unless otherwise specified, these terms do not represent any order or importance, but are intended to distinguish one configuration from another. In addition, some of the members that are not important in explaining the embodiment in each drawing are omitted.

[0014] Fig. 1(A) is a diagram showing the front side of a sensor 1 according to an embodiment. Fig. 1(B) is a diagram showing the back side of a sensor 1 according to an embodiment. In this embodiment, the front side of the sensor 1 is the surface on which a working electrode 26 and a reference electrode 28 are arranged. The back side of the sensor 1 is the surface on which a counter electrode 30 is arranged. The front and back of the sensor 1 are not particularly limited.

[0015] The sensor 1 according to this embodiment is a sensor for measuring an analyte in a sample, for example a biosensor. As an example, the sensor 1 is an electrochemical sensor, and has a sensing unit 2 and a terminal unit 4. The sensing unit 2 is inserted into a living body (for example, a human body). The terminal unit 4 electrically connects the sensing unit 2 to a circuit unit (not shown) that is attached to the surface of the living body. The sensing unit 2 transmits an electrical signal to the circuit unit via the terminal unit 4.

[0016] By maintaining the state where the sensing unit 2 is inserted into the living body, the presence and concentration of an analyte such as glucose in a sample such as blood can be continuously measured. In this embodiment, an implantable electrochemical sensor is described as an example of the sensor 1, but the sensor 1 may be a non-implantable type or may be a measurement method other than the electrochemical type. The type of the sample and the analyte is not particularly limited. The sample may be, for example, a culture medium. Examples of the analyte include hemoglobin, glycated hemoglobin, amino acids such as glutamine and glutamic acid, glycated amino acids such as fructosyl lysine and fructosyl valine, glycated peptides such as fructosyl valyl histidine, glycated albumin, sugars such as glucose, vitamins such as vitamin C, alcohols, cholesterol, lactic acid, pyruvic acid, and ketone bodies (3-hydroxybutyric acid).

[0017] The sensing unit 2 is covered with a protective film 6. The protective film 6 covers at least the reagent layer described later. The protective film 6 suppresses leakage of substances contained in the reagent layer (e.g., oxidoreductase and redox mediator) to the outside of the protective film 6. The protective film 6 also has holes through which the analyte present outside the protective film 6 can penetrate into the protective film 6. In an implantable biosensor, the sensing unit 2 is inserted into a living body. For this reason, the protective film 6 is preferably one that is difficult for proteins and cells to adsorb, and is generally made of a polymer having such properties. Examples of polymers that can constitute the protective film 6 include copolymers of methyl methacrylate and hydroxyethyl methacrylate, copolymers of butyl methacrylate and hydroxyethyl methacrylate, and poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate). It should be noted that (meth)acrylate-based compounds having the same main chain as these exemplified polymers can also be used.

[0018] A reference electrode connecting terminal 8 and a working electrode connecting terminal 10 are provided on the front surface of the terminal portion 4. A counter electrode connecting terminal 12 is provided on the back surface of the terminal portion 4. The portions of the terminal portion 4 other than the reference electrode connecting terminal 8, the working electrode connecting terminal 10, and the counter electrode connecting terminal 12 are covered with an insulating resist film 14.

[0019] 2(A) to 2(D), 3(A) to 3(D), and 4(A) to 4(C) are diagrams showing the manufacturing process of the sensor 1. First, as shown in FIG. 2(A) and FIG. 2(C), an insulating substrate 16 is formed. The insulating substrate 16 is made of a resin material such as polyethylene terephthalate, polyether ketone, polycarbonate, polyimide, polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyoxymethylene, monomer cast nylon, polybutylene terephthalate, methacrylic resin, ABS resin, or a glass material. Preferably, the insulating substrate 16 is made of polyethylene terephthalate.

[0020] Then, as shown in Fig. 2(B), a conductive thin film 18 is formed on the front surface of the insulating substrate 16. Also, as shown in Fig. 2(D), a conductive thin film 18 is formed on the rear surface of the insulating substrate 16. The conductive thin film 18 can be formed by forming a film of a conductive material such as gold (Au), platinum (Pt), palladium (Pd), iridium (Ir), carbon (C), etc. on both sides of the insulating substrate 16 using a method such as sputtering, vapor deposition, ion plating, or screen printing.

[0021] Next, as shown in FIG. 3(A), the conductive thin film 18 on the front side is divided by laser drawing to form a reference electrode conductor 20 and a working electrode conductor 22. Then, as shown in FIG. 3(B), a resist film 14 is formed on the conductive thin film 18 on the front side by screen printing or the like. A part of the reference electrode conductor 20 included in the sensing section 2 and a part of the reference electrode conductor 20 included in the terminal section 4 are exposed without the resist film 14 being laminated thereon. This forms the reference electrode forming region 24 and the reference electrode connecting terminal 8. Also, a part of the working electrode conductor 22 included in the sensing section 2 and a part of the reference electrode conductor 22 included in the terminal section 4 are exposed without the resist film 14 being laminated thereon. This forms the working electrode 26 and the working electrode connecting terminal 10. Next, as shown in FIG. 3(C), a film of silver / silver chloride (Ag / AgCl) or the like is formed in the reference electrode forming region 24 by screen printing, inkjet printing or the like to form a reference electrode 28.

[0022] 3(D), a resist film 14 is formed on the conductive thin film 18 on the back surface side by screen printing or the like. A portion of the conductive thin film 18 on the back surface side that is included in the sensing section 2 and a portion of the conductive thin film 18 that is included in the terminal section 4 are exposed without being covered with the resist film 14. In this way, the counter electrode 30 and the counter electrode connecting terminal 12 are formed.

[0023] Next, as shown in FIG. 4(A), a reagent solution is applied onto the working electrode 26 and dried to form a reagent layer 32. Therefore, the reagent layer 32 is disposed so as to contact the working electrode 26. The composition of the reagent layer 32 will be described in detail later. Thereafter, as shown in FIG. 4(B) and FIG. 4(C), the portion corresponding to the sensing unit 2 is immersed in a polymer solution for a protective film and dried to form a protective film 6. As a result, the reagent layer 32 is covered with the protective film 6. The protective film 6 in this embodiment covers the entire sensing unit 2.

[0024] Through the above steps, a sensor 1 is obtained, which has a sensing portion 2 covered with a protective film 6, and a terminal portion 4 which is not covered with the protective film 6 and in which a reference electrode connecting terminal 8, a working electrode connecting terminal 10, and a counter electrode connecting terminal 12 are exposed.

[0025] Fig. 5 is a cross-sectional view taken along line AA in Fig. 1(A). Fig. 5 shows only the sensing unit 2. Fig. 6(A) is a cross-sectional view taken along line BB in Fig. 5. Fig. 6(B) is a cross-sectional view taken along line CC in Fig. 5.

[0026] 5 and 6(A), an insulating substrate 16 is disposed in the center of the tip region of the sensing unit 2. A working electrode 26 and a reagent layer 32 are laminated in this order on the front surface side of the insulating substrate 16. A counter electrode 30 is laminated on the back surface side of the insulating substrate 16. The insulating substrate 16, the working electrode 26, the reagent layer 32, and the counter electrode 30 are covered with a protective film 6.

[0027] As shown in Fig. 5 and Fig. 6(B), an insulating substrate 16 is disposed in the center of a region closer to the terminal section 4 than the tip region of the sensing section 2. A reference electrode conductor 20 and a working electrode conductor 22 are laminated on the front surface side of the insulating substrate 16, and a resist film 14 is laminated thereon. A reference electrode 28 is laminated on the front surface side of the reference electrode conductor 20. A conductive thin film 18 and a resist film 14 are laminated in this order on the rear surface side of the insulating substrate 16. The insulating substrate 16, the reference electrode conductor 20, the working electrode conductor 22, the resist film 14 on the front surface side, the reference electrode 28, the conductive thin film 18, and the resist film 14 on the rear surface side are covered with a protective film 6.

[0028] The working electrode 26, the reference electrode 28, and the counter electrode 30 constitute an electrode section 34. Therefore, the electrode section 34 of the present embodiment has a three-electrode electrode structure. Note that the electrode section 34 may be of a two-electrode type having only the working electrode 26 and the counter electrode 30, as long as it includes the working electrode 26 and the counter electrode 30.

[0029] Next, the reagent layer 32 will be described in detail. FIG. 7 is a schematic diagram showing the state of the polymer 36, the protein 38, and the redox mediator 40 in the reagent layer 32. As shown in FIG. 7, the reagent layer 32 includes the polymer 36 and the protein 38. Examples of the protein 38 include an oxidoreductase and an antibody (immunoglobulin). In this embodiment, the protein 38 is an oxidoreductase. The reagent layer 32 of this embodiment includes a redox mediator 40 having an electron transfer ability. Furthermore, the reagent layer 32 includes a first crosslinker 42, a second crosslinker 44, and a third crosslinker 54. The reagent layer 32 may also include other components such as conductive particles such as carbon particles, and buffer solution components.

[0030] The polymer 36 has a first binding site 46 that is covalently bonded to the protein 38, and a second binding site 48 that is bonded to the protein 38 by electrostatic interaction. The polymer 36 and the protein 38 are bonded to each other by covalent bonds and electrostatic interactions, so that a high molecular weight complex in which the polymer 36 and the protein 38 are firmly bonded can be formed. This makes it possible to prevent the protein 38 from permeating the protective film 6 and flowing out of the sensor 1. Note that, although the second binding site 48 has a positive charge and the protein 38 has a negative charge in FIG. 7, the opposite may be true.

[0031] In this embodiment, the first crosslinker 42 is interposed between the protein 38 and the first binding site 46, binding them together. This allows the complex to have a higher molecular weight, and therefore the outflow of the protein 38 can be further suppressed. The protein 38 and the first binding site 46 may be directly covalently bound without the first crosslinker 42.

[0032] Furthermore, the polymer 36 has a third binding site 50 that is covalently bonded to the redox mediator 40. The polymer 36 and the redox mediator 40 are bonded to each other by a covalent bond, thereby forming a high molecular weight complex in which the polymer 36 and the redox mediator 40 are bonded to each other. This makes it possible to prevent the redox mediator 40 from permeating the protective film 6 and flowing out of the sensor 1.

[0033] In this embodiment, the second crosslinker 44 is interposed between the redox mediator 40 and the third binding site 50, binding them together. This allows the complex to have a higher molecular weight, and therefore the outflow of the redox mediator 40 can be further suppressed. The redox mediator 40 and the third binding site 50 may be directly covalently bonded to each other without the second crosslinker 44 being interposed therebetween.

[0034] Furthermore, by binding the oxidoreductase as the protein 38 and the redox mediator 40 to the polymer 36, the distance between the oxidoreductase and the redox mediator 40 can be reduced. This can improve the responsiveness of the reagent layer 32. Furthermore, the molecular weight of the complex can be increased compared to when the polymer 36 binds only one of the protein 38 and the redox mediator 40. This can further suppress the outflow of the protein 38 and the redox mediator 40.

[0035] The reagent layer 32 also contains a plurality of polymers 36. The plurality of polymers 36 contained in the reagent layer 32 may be of only one type, or may be a combination of a plurality of types. Each polymer 36 has a fourth binding site 52 that is covalently bonded to the other polymers 36. The polymers 36 can be bonded to each other by covalently bonding the fourth binding sites 52 to each other. In this embodiment, the third crosslinker 54 is interposed between the two fourth binding sites 52 and bonds them together. The polymers 36 are bonded to each other, so that the complex can have a higher molecular weight. The third crosslinker 54 is interposed between the polymers 36 and bonds them together, so that the complex can have a higher molecular weight. This makes it possible to further suppress the outflow of the protein 38 and the redox mediator 40. The fourth binding sites 52 may be directly covalently bonded to each other without the third crosslinker 54.

[0036] Each component contained in the reagent layer 32 will now be described in detail.

[0037] (Polymer 36) Polymer 36 of this embodiment teeth It contains, as constituent units, a first monomer represented by the following formula (1) and a second monomer represented by the following formula (2).

[0038] [ka] [In formula (1), A 1 is a hydrocarbon group which may have a substituent and may contain a heteroatom in the main chain, R 1 is a hydrocarbon group which may have a substituent, an acyl group which may have a substituent, an oxyacyl group which may have a substituent, or a phenylene group which may have a substituent, X is a functional group capable of covalently bonding to a protein 38, and when there are a plurality of first monomers, each A 1 , R 1 and X may be the same or different.

[0039] [ka] [In formula (2), A 2 is a hydrocarbon group which may have a substituent and may contain a heteroatom in the main chain, R 2 is a hydrocarbon group which may have a substituent, an acyl group which may have a substituent, an oxyacyl group which may have a substituent, or a phenylene group which may have a substituent; Y is a functional group capable of binding to the protein 38 through electrostatic interaction; and when there are a plurality of second monomers, each A 2 , R 2 and Y may be the same or different.

[0040] A 1 and A 2 The heteroatoms that may be contained in the main chain are, independently of one another, oxygen (O), nitrogen (N), and silicon (Si). 1 As an example, -CH 2 -CHR 1 -, -CH 2 -C(CH 3 )R 1 - and so on are examples. A 2 As an example, -CH 2 -CHR 2 -, -CH 2 -C(CH 3 )R 2 - etc. are examples. R 1 and R 2 As for each independently, -CH 2 -, -C(=O)O-CH 2 -CH 2 -, -C 6 H 5 -CH 2 - etc. are examples. 6 H 5 - is a benzene ring.

[0041] The functional group X is preferably a group derived from at least one selected from the group consisting of amines, alcohols, phenols, thiols, carboxylic acids, hydrazines, alkoxyamines, hydroxyamines, imidazoles, pyridines, acyl azides, acyl halides, alkyl halides, acid anhydrides, aldehydes, ketones, alkyl sulfonates, maleimides, epoxides, aziridines, isocyanates, isothiocyanates, imide esters, sulfonyl halides, and activated esters. Examples of activated esters include succinimidyl esters, benzotriazole esters, or aryl esters substituted with electron-withdrawing groups including sulfo, nitro, or halo groups, or carboxylic esters having a carbodiimide group.

[0042] At least a part of the functional group X constitutes the first binding site 46. In this case, the type of the functional group X can be appropriately selected according to the type of the protein 38 to be bound. FIG. 8 is a diagram showing a combination of reactive groups capable of covalent bonding. The first binding site 46 and the protein 38 can be covalently bonded to each other if one of them has the first reactive group shown in FIG. 8 and the other has the second reactive group shown in FIG. 8. In addition, when the first crosslinker 42 is interposed between the first binding site 46 and the protein 38, one of the first crosslinker 42 and the first binding site 46 may have the first reactive group and the other may have the second reactive group, and one of the first crosslinker 42 and the protein 38 may have the first reactive group and the other may have the second reactive group.

[0043] Preferably, the functional group X is a group derived from at least one selected from the group consisting of amine, thiol, carboxylic acid, imidazole, and pyridine. This allows a more readily available crosslinking agent to be selected as the first crosslinking agent 42, and therefore makes it easier to achieve covalent bonding between the polymer 36 and the protein 38 via the first crosslinking agent 42. Furthermore, in the crosslinking reaction between the polymer 36 and the protein 38 with or without the first crosslinking agent 42, the reaction conditions, such as temperature and pH, can be made relatively mild. This makes it possible to suppress inactivation of the protein 38.

[0044] Moreover, at least a part of the functional group X constitutes the third binding site 50. In this case, the type of the functional group X can be appropriately selected according to the type of the redox mediator 40 to be bound. That is, the third binding site 50 and the redox mediator 40 can be covalently bound to each other as long as one of them has a first reactive group and the other has a second reactive group. Furthermore, when the second crosslinker 44 is interposed between the third binding site 50 and the redox mediator 40, one of the second crosslinker 44 and the third binding site 50 may have a first reactive group and the other may have a second reactive group, and one of the second crosslinker 44 and the redox mediator 40 may have a first reactive group and the other may have a second reactive group.

[0045] The first binding site 46 and the third binding site 50 preferably have the same functional group. In other words, it is preferable that the same type of functional group X constitutes both the first binding site 46 and the third binding site 50. This allows one type of first monomer to achieve covalent bonds with the protein 38 and the redox mediator 40. Therefore, the types of monomers constituting the polymer 36 can be reduced, and the method for synthesizing the polymer 36 can be simplified.

[0046] Furthermore, at least a part of the functional groups X constitutes the fourth binding site 52. In this case, the type of the functional group X can be appropriately selected depending on the type of the fourth binding site 52 of the other polymer 36 that is the binding partner. In other words, the fourth binding sites 52 of two polymers 36 can be covalently bonded to each other if one of them has a first reactive group and the other has a second reactive group. Furthermore, when the third crosslinker 54 is interposed between the two fourth binding sites 52, it is only necessary that the combination condition of the first reactive group and the second reactive group is satisfied between the third crosslinker 54 and each fourth binding site 52.

[0047] At least two of the first binding site 46, the third binding site 50, and the fourth binding site 52 preferably have the same functional group, and more preferably, three of them have the same functional group. In other words, it is preferable that the same type of functional group X constitutes at least two of the first binding site 46, the third binding site 50, and the fourth binding site 52. This makes it possible to realize covalent bonds with the protein 38, the redox mediator 40, and the other polymers 36 with fewer types of first monomers. Therefore, the types of monomers constituting the polymer 36 can be reduced, and the method for synthesizing the polymer 36 can be simplified.

[0048] The functional group Y is preferably a cationic group derived from at least one selected from the group consisting of quaternary ammonium, quaternary phosphonium, tertiary sulfonium, and aromatic cations, or an anionic group derived from at least one selected from the group consisting of carboxylic acid, phosphonic acid, and sulfonic acid.

[0049] Quaternary ammonium has four organic substituents R on the nitrogen atom. 2 , R 3 , R 4 , R 5 A quaternary phosphonium has four organic substituents R bonded to the phosphorus atom. 2 , R 3 , R 4 , R 5 Tertiary sulfonium has a structure in which three organic substituents R are bonded to the sulfur atom. 2 , R 3 , R 4 It has a structure in which R 2 is as mentioned above. R 3 ~R 5 is, for example, C 1-6 a linear or branched, saturated or unsaturated hydrocarbon group, an acyl group which may have a substituent, an alkoxy group which may have a substituent, or a phenyl group which may have a substituent; 3 ~R 5 may be the same or different.

[0050] Examples of aromatic cations include imidazolium, oxazolium, pyrazinium, pyrazolium, pyridazinium, pyrrolidinium, pyridinium, piperidinium, thiazolium and triazolium.

[0051] The functional group Y constitutes the second binding site 48. The type of the functional group Y can be appropriately selected depending on the type of the protein 38 to be bound. If the second binding site 48 and the protein 38 have opposite charges, they can bind to each other through electrostatic interaction. As an example, the functional group Y is a group different from the functional group X. Also, the functional group Y is preferably a cationic group. When the protein 38 is an oxidoreductase, many general oxidoreductases have a negative charge under near-neutral conditions in which the sensor 1 is mainly used. For this reason, by making the functional group Y a cationic group, it is possible to more easily realize binding between the polymer 36 and the protein 38 through electrostatic interaction.

[0052] Specific examples of the polymer 36 include those shown in the following formulae (3) to (7). m and n in formulae (3) to (6) and l, m and n in formula (7) indicate the molar ratio of each monomer. The molar ratio of each monomer can be appropriately set. In addition, in formulae (3) to (7), Cl is used as an example of a counter ion. - This shows that.

[0053] [ka]

[0054] [ka]

[0055] [ka]

[0056] [ka]

[0057] [ka]

[0058] (Protein 38) The protein 38 binds to the first binding site 46 and the second binding site 48 of the polymer 36 and acts on the analyte. As described above, examples of the protein 38 include oxidoreductases and antibodies (immunoglobulins). In the present embodiment, the oxidoreductase refers to an enzyme that can oxidize or dehydrogenate an analyte. Examples of the oxidoreductase include glucose oxidase, lactate oxidase, pyruvate oxidase, cholesterol oxidase, amino acid oxidase, glutamate oxidase, fructosyl amino acid oxidase, alcohol oxidase, ascorbate oxidase, fructosyl peptide oxidase, glucose dehydrogenase, lactate dehydrogenase, pyruvate dehydrogenase, amino acid dehydrogenase, glutamate dehydrogenase, 3-hydroxybutyrate dehydrogenase, and alcohol dehydrogenase. A plurality of types of oxidoreductases may be combined as necessary.

[0059] When the protein 38 is an oxidoreductase, typically, an amino group of the oxidoreductase is covalently bonded to the first binding site 46 or the first crosslinker 42. A thiol group, a carboxyl group, or the like of the oxidoreductase may also be covalently bonded to the first binding site 46 or the first crosslinker 42. The amount of the oxidoreductase to be blended is, for example, preferably 0.01 to 100 U (units), more preferably 0.05 to 50 U, and even more preferably 0.1 to 10 U per sensor or per measurement.

[0060] In addition, when the binding site of the protein 38 to the second binding site 48 has a positive charge, the isoelectric point of the protein 38 is preferably equal to or lower than the pH of the usage environment. In addition, when the binding site of the protein 38 to the second binding site 48 has a negative charge, the isoelectric point of the protein 38 is preferably equal to or higher than the pH of the usage environment. The usage environment of the protein 38 refers to at least the reagent solution. Preferably, the usage environment of the protein 38 refers to both the reagent solution and the measurement target sample. As an example, when the binding site to the second binding site 48 has a positive charge, the isoelectric point of the protein 38 is preferably equal to or lower than pH 9, more preferably equal to or lower than pH 7.4, and even more preferably equal to or lower than pH 5.5. In addition, when the binding site to the second binding site 48 has a negative charge, the isoelectric point of the protein 38 is preferably equal to or higher than pH 5.5, more preferably equal to or higher than pH 7.4, and even more preferably equal to or higher than pH 9.

[0061] The oxidoreductase as the protein 38 is preferably a coenzyme-bound enzyme. Examples of the coenzyme include pyrroloquinoline quinone (PQQ), nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), thiamine pyrophosphate (TPP), coenzyme A (CoA), and the like.

[0062] For example, when the analyte is glucose, the oxidoreductase is glucose oxidase or glucose dehydrogenase (GDH). Examples of coenzyme-bound glucose dehydrogenase include pyrroloquinoline quinone (PQQ)-bound GDH and flavin adenine nucleotide (FAD)-bound GDH. Considering the effect of maltose in the living body, the enzyme activity of GDH on maltose is preferably 5% or less, more preferably 3% or less, when the enzyme activity on glucose is 100%. An example of an enzyme having such enzyme activity is FAD-bound GDH. Examples of such FAD-bound GDH include those derived from the genus Aspergillus (oryzae and Terreus) and the genus Mucor.

[0063] (Redox Mediator 40) The redox mediator 40 binds to the third binding site 50 of the polymer 36 and can reversibly become an oxidized form or a reduced form, mediating the transfer of electrons between substances. Examples of the redox mediator 40 include metal complexes such as osmium complexes, ruthenium complexes, and iron complexes; quinone compounds such as benzoquinone, naphthoquinone, phenanthrenequinone, phenanthrolinequinone, anthraquinone, and derivatives thereof; phenazine compounds; viologen compounds; phenothiazine compounds; and phenol compounds. A plurality of types of redox mediators 40 may be combined as necessary.

[0064] Specific examples of redox mediator 40 include derivatives of one or more compounds selected from the group consisting of potassium ferricyanide; hexammineruthenium; ferrocene; 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-methylphenazinium methyl sulfate and 1-methoxy-5-ethylphenazinium ethyl sulfate; methyl viologen; benzyl viologen; methylene green; 2-aminophenol; 2-amino-4-methylphenol; and 2,4-diaminophenol, which have a functional group (the first reactive group or the second reactive group shown in FIG. 8) that can be bonded to polymer 36. Examples of the above salts (sulfonate, disulfonate) include sodium salts, potassium salts, calcium salts, magnesium salts, lithium salts, and the like.

[0065] The amount of the redox mediator 40 blended per sensor or per measurement is, for example, preferably 0.1 pmol to 1000 μmol, more preferably 5 pmol to 250 μmol, and further preferably 100 pmol to 50 μmol.

[0066] The redox mediator 40 has a third binding site 50 or a binding site with the second crosslinker 44. As described above, the binding site of the redox mediator 40 has a reactive group, of the first reactive group and the second reactive group, that is a binding target for the reactive group of the third binding site 50 or the second crosslinker 44.

[0067] (First crosslinker 42, second crosslinker 44 and third crosslinker 54) Examples of the first crosslinking agent 42, the second crosslinking agent 44, and the third crosslinking agent 54 include glutaraldehyde; compounds having NHS esters at both ends, such as disuccinimidyl succinate, bis(3-sulfo-N-succinimidyl) suberate disodium salt, and di(N-succinimidyl) 4,7,10,13,16-pentaoxanonadecanedioate (Bis(NHS)PEG5); polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, and the like. compounds having epoxy groups at both ends, such as 1,2-bis(maleimido)ethane and 1,11-bismaleimido-triethyleneglycol; compounds having amino groups at both ends, such as 1,2-bis(2-aminoethoxy)ethane; compounds having different functional groups at both ends, such as Maleimide-PEG2-NHS Ester and Amino-PEG6-carboxylic Acid; Glutarate, 4-arm-PEG10K-Maleimide, 8-arm-PEG10K-Succinimidyl Succinate, and the like.

[0068] When the reagent layer 32 contains the first crosslinking agent 42 and the second crosslinking agent 44, it is preferable that the first crosslinking agent 42 and the second crosslinking agent 44 are the same type of crosslinking agent. When the reagent layer 32 contains the first crosslinking agent 42 and the third crosslinking agent 54, it is preferable that the first crosslinking agent 42 and the third crosslinking agent 54 are the same type of crosslinking agent. When the reagent layer 32 contains the second crosslinking agent 44 and the third crosslinking agent 54, it is preferable that the second crosslinking agent 44 and the third crosslinking agent 54 are the same type of crosslinking agent. When the reagent layer 32 contains the first crosslinking agent 42, the second crosslinking agent 44, and the third crosslinking agent 54, it is preferable that at least two of the first crosslinking agent 42, the second crosslinking agent 44, and the third crosslinking agent 54 are the same crosslinking agent, and it is more preferable that three of the first crosslinking agent 42, the second crosslinking agent 44, and the third crosslinking agent 54 are the same crosslinking agent. As a result, the number of types of crosslinking agents can be reduced, and the method for preparing the reagent layer 32 can be simplified.

[0069] The reagent layer 32 as an example includes a polymer 36 having a first binding site 46, a second binding site 48, a third binding site 50, and a fourth binding site 52, a protein 38, a redox mediator 40, a first crosslinker 42, and a third crosslinker 54. At least two of the first binding site 46, the third binding site 50, and the fourth binding site 52 have the same functional group. The protein 38 binds to the first binding site 46 via the first crosslinker 42. The protein 38 also binds to the second binding site 48. The redox mediator 40 binds directly to the third binding site 50 without the second crosslinker 44. The fourth binding sites 52 of the two polymers 36 are bonded to each other via the third crosslinker 54. The first crosslinker 42 and the third crosslinker 54 are the same crosslinker.

[0070] Next, a description will be given of the measurement principle of an analyte by the sensor 1. Fig. 9 is a schematic diagram for explaining the measurement principle of the sensor 1. As an example, a case will be described in which the sensor 1 is an electrochemical sensor, the analyte is glucose, and the protein 38 is glucose dehydrogenase (GDH).

[0071] First, glucose in the blood or subcutaneous interstitial fluid permeates the protective film 6 and reaches the reagent layer 32. Next, glucose is oxidized by glucose dehydrogenase to produce gluconolactone. At the same time, the redox mediator 40 is reduced, and the oxidized form (M(Ox)) becomes the reduced form (M(Red)). Then, the reduced form of the redox mediator 40 is oxidized on the surface of the working electrode 26, and the reduced form (M(Red)) becomes the oxidized form (M(Ox)), and electrons are generated at the same time. These electrons are transferred to the working electrode 26 and measured as a current value.

[0072] As described above, the polymer 36 according to the present embodiment binds the protein 38 by a covalent bond at the first binding site 46 and an electrostatic interaction at the second binding site 48. This can prevent the protein 38 from flowing out of the sensor. Therefore, the durability and measurement sensitivity of the sensor 1 can be further prevented from decreasing. In addition, negative effects on the living body can be further prevented. The polymer 36 binds the redox mediator 40 by a covalent bond at the third binding site 50. This can prevent the redox mediator 40 from flowing out of the sensor. Therefore, the durability and measurement sensitivity of the sensor 1 can be further prevented from decreasing, and negative effects on the living body can be further prevented. The polymer 36 binds to another polymer 36 by a covalent bond at the fourth binding site 52. This can prevent the durability and measurement sensitivity of the sensor 1 from decreasing, and negative effects on the living body can be further prevented.

[0073] Furthermore, a first crosslinker 42 is interposed between the protein 38 and the first binding site 46. A second crosslinker 44 is interposed between the redox mediator 40 and the third binding site 50. A third crosslinker 54 is interposed between the two fourth binding sites 52. This allows the complex of the polymer 36, the protein 38, and the redox mediator 40 to have a higher molecular weight. This further prevents the protein 38 and the redox mediator 40 from leaking out of the sensor.

[0074] The above describes the embodiments of the present disclosure in detail. The above-mentioned embodiments merely show specific examples of implementing the present disclosure. The contents of the embodiments do not limit the technical scope of the present disclosure, and many design changes such as changes, additions, and deletions of components are possible within the scope of the invention defined in the claims. A new embodiment with design changes has the effects of the combined embodiments and modifications. In the above-mentioned embodiments, the contents for which such design changes are possible are emphasized by adding notations such as "in this embodiment" and "in this embodiment", but design changes are permitted even in contents without such notations. Any combination of the above components is also valid as an aspect of the present disclosure. The hatching on the cross section of the drawing does not limit the material of the object to which the hatching is added.

[0075] The embodiment may be specified by the items described below. (Item 1) A polymer (36) contained in a reagent layer (32) of a sensor (1) for measuring an analyte in a sample, a first binding site (46) that is covalently linked to a protein (38) that interacts with the analyte; and a second binding site (48) that binds to the protein (38) through electrostatic interaction. Polymer (36). (Item 2) a first monomer represented by formula (1), and Contains a second monomer represented by the above formula (2) as a constituent unit. The polymer (36) according to item 1. (Item 3) the functional group X is a group derived from at least one selected from the group consisting of amines, alcohols, phenols, thiols, carboxylic acids, hydrazines, alkoxyamines, hydroxyamines, imidazoles, pyridines, acyl azides, acyl halides, alkyl halides, acid anhydrides, aldehydes, ketones, alkyl sulfonates, maleimides, epoxides, aziridines, isocyanates, isothiocyanates, imido esters, sulfonyl halides, and activated esters; The functional group Y is a cationic group derived from at least one selected from the group consisting of quaternary ammonium, quaternary phosphonium, tertiary sulfonium and aromatic cations, or an anionic group derived from at least one selected from the group consisting of carboxylic acid, phosphonic acid and sulfonic acid; The polymer (36) according to item 2. (Item 4) The protein (38) is an oxidoreductase. 4. A polymer (36) according to any one of items 1 to 3. (Item 5) The polymer (36) has a third binding site (50) that is covalently bonded to a redox mediator (40) capable of transferring electrons. Item 4. The polymer (36) according to item 4. (Item 6) the first binding site (46) and the third binding site (50) have the same functional group; Item 5. The polymer (36) according to item 5. (Item 7) A reagent layer (32) of a sensor (1) for measuring an analyte in a sample, comprising: A polymer (36) according to any one of items 1 to 6, a protein (38) that binds to the polymer (36) and acts on the analyte; A reagent layer (32). (Item 8) a first cross-linker (42) that binds a protein (38) and a first binding site (46); Item 7. The reagent layer (32) according to item 7. (Item 9) Protein (38) is an oxidoreductase; the polymer (36) has a third binding site (50) that is covalently bonded to a redox mediator (40) capable of transferring electrons; The reagent layer (32) comprises a redox mediator (40) bound to a polymer (36); Item 9. The reagent layer (32) according to item 7 or 8. (Item 10) a second cross-linker (44) that binds the redox mediator (40) and the third binding site (50); Item 9. The reagent layer (32) according to item 9. (Item 11) a first cross-linker (42) that binds an oxidoreductase (38) and a first binding site (46); The first crosslinker (42) and the second crosslinker (44) are the same crosslinker; Item 11. The reagent layer (32) according to item 10. (Item 12) The reagent layer (32) comprises a plurality of polymers (36); Each polymer (36) has a fourth binding site (52) that is covalently bonded to another polymer (36); The reagent layer (32) includes a third cross-linking agent (54) that bonds the fourth binding sites (52) together. 12. The reagent layer (32) according to any one of items 7 to 11. (Item 13) Protein (38) is an oxidoreductase; the polymer (36) has a third binding site (50) that is covalently bonded to a redox mediator (40) capable of transferring electrons; At least two of the first binding site (46), the third binding site (50) and the fourth binding site (52) have the same functional group; Item 13. The reagent layer (32) according to item 12. (Item 14) Protein (38) is an oxidoreductase; the polymer (36) has a third binding site (50) that is covalently bonded to a redox mediator (40) capable of transferring electrons; the reagent layer (32) comprises a first crosslinker (42) that bonds the oxidoreductase (38) and the first binding site (46), and a second crosslinker (44) that bonds the redox mediator (40) and the third binding site (50); At least two of the first crosslinker (42), the second crosslinker (44), and the third crosslinker (54) are the same crosslinker; Item 14. The reagent layer (32) according to item 12 or 13. (Item 15) an electrode portion (34) including a working electrode (26) and a counter electrode (30); and a reagent layer (32) according to any one of items 7 to 14, which is arranged so as to contact the working electrode (26). Sensor(1). (Item 16) A protective film (6) is provided to cover the reagent layer (32). Item 16. The sensor (1) according to item 15. EXAMPLES

[0076] Examples of the present invention will be described below, however, these examples are merely illustrative for suitably explaining the present invention and are not intended to limit the present invention in any way.

[0077] (Synthesis of Polymer A) 10 is a diagram showing the polymerization reaction of polymer A. First, 2-aminoethyl methacrylate hydrochloride (monomer a), (4-vinylphenyl)methanamine (monomer b), and methacryloylcholine chloride (monomer c) were prepared. Then, 0.97 mmol of monomer a, 0.97 mmol of monomer b, 11.97 mmol of monomer c, 0.08 mmol of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (hereinafter referred to as V-50), and 227 mmol of ethanol were added to a four-neck flask.

[0078] Next, a stirrer tip, a thermometer, and a vacuum / Ar gas line were set in the four-neck flask. While stirring at room temperature, the vacuum / Ar gas line was used to repeatedly reduce pressure in the four-neck flask and supply Ar gas 10 times to degas the sample in the flask. After the degassing process, the four-neck flask was sealed and the polymerization reaction shown in Figure 10 was carried out at a reaction temperature of 60°C for 24 hours. The progress and completion of the polymerization were monitored by 1 The disappearance of the monomer was confirmed by measuring it with H-NMR. After the reaction was completed, the reaction solution was placed in a dialysis membrane (MWCO: 3500) and dialyzed for 3 days. The solution was replaced twice a day. After the dialysis was completed, the reaction product was freeze-dried to obtain polymer A. Polymer A corresponds to the polymer in the above formula (7) where n:m:l=21.2:8.1:70.7. The molar ratio of each monomer was calculated from the integral ratio of the signals in the NMR spectrum. The same applies to the following polymers B, C, E, and F. In addition, as a result of measurement by gel permeation chromatography (GPC), polymer A had a number average molecular weight Mn of 89642 and a weight average molecular weight Mw of 502171.

[0079] (Synthesis of Polymer B) FIG. 11 is a diagram showing the polymerization reaction of polymer B. First, monomer a and (vinylbenzyl)trimethylammonium chloride (monomer d) were prepared. Then, 7.36 mmol of monomer a, 7.36 mmol of monomer d, 0.07 mmol of V-50, and 616 mmol of ion-exchanged water were charged into a four-neck flask. Thereafter, the same treatment as in the case of polymer A was carried out, and polymer B was obtained by carrying out the polymerization reaction shown in FIG. 11. Polymer B corresponds to the polymer in the above formula (4) where n:m=48.5:51.5.

[0080] (Synthesis of Polymer C) FIG. 12 is a diagram showing the polymerization reaction of polymer C. First, monomer a and monomer c were prepared. Then, 1.14 mmol of monomer a, 10.22 mmol of monomer c, 0.06 mmol of V-50, and ion-exchanged water were charged into a four-neck flask. Thereafter, the same treatment as in the case of polymer A was carried out, and the polymerization reaction shown in FIG. 12 was carried out to obtain polymer C (the reaction time was appropriately adjusted as necessary). Polymer C corresponds to the polymer in the above formula (6) where n:m=13.3:86.7.

[0081] (Preparation of Mediator-bound Polymer D as a Comparative Example) Phenazine was used as a mediator, specifically, 5-{[(2,5-dioxopyridin-1-yl)oxy]-5-oxopentyl}-1-methoxyphenazinium nitrate (Tokyo Chemical Industry Co., Ltd.). 0.47 mg of this mediator was weighed out and dissolved in 47 μL of MiliQ water. Poly(L-lysine) hydrochloride (PLKC800: Allamanda Polymers) was also prepared as polymer D of the comparative example. This polymer D corresponds to the polymer not having the second binding site 48 for glucose dehydrogenase used in the preparation of the reagent described below. 0.513 mg of polymer D was weighed out and dissolved in 51.3 μL of MiliQ water. 9.59 mg of a condensation agent (WSC W001: Dojindo Chemical Co., Ltd.) was also weighed out and dissolved in 479.3 μL of MiliQ.

[0082] These three solutions were mixed with 96 μL of 250 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.0), and the total volume was adjusted to 1200 μL with MiliQ water. The reaction was then allowed to proceed for about 20 hours at room temperature while stirring the mixture. After the reaction was completed, the reaction solution was ultrafiltered several times using a centrifugal ultrafiltration filter (Amicon Ultra-4 50k: Merck Millipore), and the solution from which low molecular weight compounds had been removed was collected as a solution of mediator-bound polymer D (poly(L-lysine)-bound phenazine).

[0083] (Preparation of mediator-bound polymer A) A solution of mediator-bound polymer A was prepared in the same manner as for the preparation of mediator-bound polymer D, except that 2.63 mg of polymer A was weighed out instead of polymer D and dissolved in 175.4 μL of MiliQ water.

[0084] (Preparation of mediator-bound polymer B) A solution of mediator-bound polymer B was prepared in the same manner as for the preparation of mediator-bound polymer D, except that 1.56 mg of polymer B was weighed out instead of polymer D and dissolved in 104.1 μL of MiliQ water.

[0085] (Preparation of mediator-bound polymer C) A solution of mediator-bound polymer C was prepared in the same manner as for the preparation of mediator-bound polymer D, except that 6.08 mg of polymer C was weighed out instead of polymer D and dissolved in 405.2 μL of MiliQ water.

[0086] Each mediator-bound polymer solution was diluted 25-fold, and 100 μL of each was added to a microplate. Then, the absorption spectrum of each solution was measured using a plate reader. The results are shown in FIG. 13. The microplate used was a UV-Star (registered trademark) 96 Well F-Boden (manufactured by Greiner Bio-One). The plate reader used was an infinite (registered trademark) M200 Pro (manufactured by Tecan).

[0087] Fig. 13 shows the absorption spectrum of each mediator-bound polymer. As shown in Fig. 13, in each mediator-bound polymer, peaks were observed in the wavelength range of 230 nm to 330 nm and in the wavelength range of 330 nm to 430 nm. These two peaks were derived from phenazine. This confirmed that phenazine was bound to each polymer.

[0088] (Preparation of reagent solutions) The following reagents were mixed to the following final concentrations and reacted for about 1 hour to prepare a reagent solution. The carbon dispersion was treated in an ultrasonic bath for about 10 minutes before use. Sodium phosphate buffer (pH 6.5) final concentration 8 mM FAD-dependent glucose dehydrogenase (GDH GLD1: BBI International) final concentration 2000U / mL Glutaraldehyde 25% solution (Wako Pure Chemical Industries, Ltd.) final concentration 0.005% (wt / v) Each mediator-bound polymer final concentration: 1 (absorbance at 386 nm is equivalent to 1) Carbon dispersion final concentration 4mg / mL

[0089] The sodium phosphate buffer was prepared with disodium hydrogen phosphate (Wako Pure Chemical Industries, Ltd.) and sodium dihydrogen phosphate (Wako Pure Chemical Industries, Ltd.). The carbon dispersion was prepared by mixing Ketjen Black (EC300J: Lion Specialty Chemicals Co., Ltd.) with 5 mg / mL hydroxypropyl cellulose (NISSO HPCL, Nippon Soda Co., Ltd.) solution to a carbon concentration of 16 mg / mL, and treating with an ultrasonic homogenizer for 3 minutes or more. The concentration of each mediator-bound polymer was adjusted based on the concentration of each polymer in the solution, which was confirmed by diluting the solution by 25 times and adding 100 μL to a microplate and measuring the absorption spectrum with a plate reader.

[0090] (Preparation of polymer solution for protective film) The following reagents were mixed to the following final concentrations to prepare a polymer solution for the protective film. Poly(tert-butyl methacrylate-b-4-vinylpyridine) (Polymer Source, hereafter referred to as tBuMA4VP) final concentration 7.11% Random copolymer of polypropylene glycol methyl ether-styrene-4-vinylpyridine (Polymer Source, hereafter referred to as PGMAS4VP) final concentration 0.89% Poly(ethylene glycol) dimethyl ether (Sigma-Aldrich, hereafter referred to as PEGDGE) final concentration 0.98% · HEPES buffer (pH8.0) final concentration 5mM

[0091] tBuMA4VP, PGMAS4VP and PEGDGE were dissolved in ethanol before use. The number average molecular weight Mn of tBuMA4VP is 80,000 for poly(tert-butyl methacrylate) and 77,000 for poly(4-vinylpyridine). The Mw / Mn of tBuMA4VP is 1.15. PGMAS4VP has a polypropylene glycol methyl ether:styrene:4-vinylpyridine=24:16:60 ratio, a number average molecular weight Mn of 52,000, a weight average molecular weight Mw of 94,000 and a Mw / Mn of 1.8. The number average molecular weight Mn of PEGDGE is 1,000. The HEPES buffer solution was prepared using 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (manufactured by Dojindo Chemical Industries).

[0092] (Preparation of electrodes for sensors) Gold electrodes were formed on an insulating substrate by sputtering. 0.4 μL of the reagent solution containing each mediator-bound polymer was applied to separate gold electrodes and dried for about 15 minutes. 0.4 μL of the reagent solution was again applied to each gold electrode, and then dried overnight. This formed a reagent layer on each gold electrode. Each gold electrode with the reagent layer formed was immersed in a protective film polymer solution, pulled out, and dried multiple times. After this operation was completed, the electrodes were dried for one day to form a protective film on each gold electrode. Through the above steps, a sensor electrode was obtained that had a reagent layer containing each mediator-bound polymer and a protective film covering the reagent layer.

[0093] (Sensor durability evaluation) The sensor electrode 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 BAS) was used as the reference electrode to fabricate a three-electrode electrode.Then, a potentiostat (manufactured by BAS) was used to measure the time change of the current in phosphate buffered saline by the amperometric method.

[0094] Specifically, glucose was added every 500 seconds from 1000 seconds after the start of measurement to reach theoretical values ​​of 50 mg / dL, 150 mg / dL, 300 mg / dL, and 500 mg / dL, and the current response value was continuously measured. After the measurement, the electrode was stored in phosphate buffered saline at 37°C. After storing for one day, the same measurement was performed. The current values ​​at glucose concentrations of 50 mg / dL, 150 mg / dL, and 300 mg / dL were calculated as the average value calculated from the five measured values ​​5 seconds, 10 seconds, 15 seconds, 20 seconds, and 25 seconds before the next glucose addition after the glucose to be measured was added.

[0095] The current value at the final concentration was the average value calculated from the measurements taken at five points 480 seconds, 485 seconds, 490 seconds, 495 seconds, and 500 seconds after adding a glucose solution so that the glucose concentration was 500 mg / dL. The current value at each glucose concentration was the current value after background correction processing was performed to subtract the current value at a glucose concentration of 0 mg / dL. The results are shown in Figure 14.

[0096] Fig. 14 shows the results of durability evaluation of the sensor. As shown in Fig. 14, compared with mediator-bound polymer D (comparative example), mediator-bound polymers A to C showed a smaller change in current value between the first day and after one day of storage. In particular, in the high concentration range of 300 to 500 mg / dL, the decrease in current value was significantly suppressed. From this, it was confirmed that mediator-bound polymers A to C can suppress the outflow of oxidoreductase (protein) and redox mediator, thereby improving the durability of the sensor.

[0097] The inventors also actually synthesized polymer E and polymer F. The synthesis procedures for each polymer are described below.

[0098] (Synthesis of Polymer E) FIG. 15 is a diagram showing the polymerization reaction of polymer E. First, monomer b and monomer c were prepared. Then, 0.85 mmol of monomer b, 7.61 mmol of monomer c, and 0.25 mmol of 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (hereinafter referred to as V-70) were added to a four-neck flask. Thereafter, the same treatment as in the case of polymer A was carried out, and the polymerization reaction shown in FIG. 15 was carried out to obtain polymer E (the reaction temperature and reaction time were appropriately adjusted as necessary). Polymer E corresponds to the polymer in the above formula (5) where n:m=15.0:85.0.

[0099] (Synthesis of Polymer F) FIG. 16 is a diagram showing the polymerization reaction of polymer F. First, allylamine hydrochloride (monomer e) and monomer c were prepared. Then, 92.22 mmol of monomer e, 9.22 mmol of monomer c, 66.1 mmol of V-50, and 590 mmol of ion-exchanged water were charged into a four-neck flask. Thereafter, the same treatment as in the case of polymer A was carried out, and the polymerization reaction shown in FIG. 16 was carried out to obtain polymer F. Polymer F corresponds to the polymer in the above formula (3) where n:m=5.8:94.2. [Industrial Applicability]

[0100] The present disclosure may be utilized in polymers, reagent layers and sensors. [Explanation of symbols]

[0101] 1 sensor, 6 protective film, 26 working electrode, 30 counter electrode, 32 reagent layer, 34 electrode portion, 36 polymer, 38 protein, 40 redox mediator, 42 first crosslinker, 44 second crosslinker, 46 first binding site, 48 second binding site, 50 third binding site, 52 fourth binding site, 54 third crosslinker.

Claims

1. A reagent layer of a sensor for measuring an analyte in a sample, comprising: A polymer; a protein that binds to the polymer and acts on the analyte; The polymer is a first binding site that is covalently attached to the protein; a second binding site that binds to the protein through electrostatic interaction; Formula (1): 【Chemistry 1】 A first monomer represented by the formula (1): [in which A 1 is styryl, (meth)acryloyl or a group derived from an olefin, R 1 is an alkylene group, X is a group selected from primary amines and salts thereof, and when there are a plurality of first monomers, A 1 , R 1 and X may be the same or different]; and Formula (2): 【Chemistry 2】 [in formula (2), A 2 is styryl, (meth)acryloyl or a group derived from an olefin, R 2 is an alkylene group, Y is a quaternary ammonium group, and when there are a plurality of second monomers, A 2 , R 2 and Y may be the same or different], The protein is an oxidoreductase. Reagent layer.

2. a first cross-linker linking the oxidoreductase and the first binding site; The reagent layer of claim 1 .

3. The polymer has a third binding site that is covalently bonded to a redox mediator capable of transferring electrons; the reagent layer comprises the redox mediator bound to the polymer; The reagent layer of claim 1 .

4. a second cross-linker that links the redox mediator and the third binding site; The reagent layer of claim 3 .

5. a first cross-linking agent that links the oxidoreductase and the first binding site; The first crosslinker and the second crosslinker are the same crosslinker. The reagent layer of claim 4.

6. the reagent layer comprises a plurality of the polymers; each polymer has a fourth binding site covalently bonded to another said polymer; the reagent layer includes a third cross-linking agent that bonds the fourth binding sites together; The reagent layer of claim 1 .

7. The polymer has a third binding site that is covalently bonded to a redox mediator capable of transferring electrons; At least two of the first binding site, the third binding site and the fourth binding site have the same functional group; The reagent layer of claim 6.

8. The polymer has a third binding site that is covalently bonded to a redox mediator capable of transferring electrons; the reagent layer comprises a first crosslinker that bonds the oxidoreductase and the first binding site, and a second crosslinker that bonds the redox mediator and the third binding site; At least two of the first crosslinking agent, the second crosslinking agent, and the third crosslinking agent are the same crosslinking agent. The reagent layer of claim 6.

9. An electrode portion including a working electrode and a counter electrode; The reagent layer according to any one of claims 1 to 8, which is disposed so as to be in contact with the working electrode. Sensor.

10. A protective film is provided to cover the reagent layer. The sensor of claim 9.

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