Biosensor

Abstract

To provide a biosensor capable of measuring amino acid concentration in a solution with higher accuracy. [Solution] A biosensor comprising an enzyme electrode including a conductive layer and a reagent layer adjacent to the conductive layer, wherein the conductive layer contains a mediator, and the reagent layer contains an amino acid oxidase and a cured product of a photosensitive polymer.

Description

[Technical Field]

[0001] This invention relates to a biosensor for electrochemically analyzing amino acids contained in a solution. [Background technology]

[0002] In recent years, there has been a growing need to selectively detect organic substances in bodily fluids such as sweat, saliva, urine, tears, and blood for monitoring bodily conditions or for diagnosing and preventing diseases. Among these methods, non-invasive measurements that do not involve procedures that harm the subject's body, such as blood sampling, are attracting particular attention. Urine is useful as a bodily fluid to be measured because it can be easily collected without harming the subject's body. For example, free amino acids in urine are known to potentially serve as a biomarker for chronic kidney disease, a leading cause of death in dogs and cats. However, bodily fluids that can be measured non-invasively tend to have lower concentrations of organic substances than blood, thus requiring more sensitive sensors. Enzyme sensors, which utilize the high selectivity of enzymes to enable selective and highly sensitive detection of organic substances, are promising as such highly sensitive sensors.

[0003] Patent Document 1 discloses an electrode for use in a bioelectrochemical measurement system, which has one-dimensional conductive properties and an enzyme on its working surface. As an example, it discloses an electrode in which D-amino acid oxidase is incorporated into a tetrathiafulvalene TCNQ (mediator)-filled recess electrode using a dialysis membrane.

[0004] Patent Document 2 discloses a method for measuring the concentration of glycated proteins, which includes reacting a sample treated with protease with an enzyme immobilized on an electrode together with an artificial electron mediator using a water-soluble photocurable resin, and detecting the current value between the electrode on which the enzyme is immobilized and a predetermined detection electrode. An example of the water-soluble photocurable resin is a photocurable polyvinyl alcohol resin having an azide group as a photosensitive group. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Laid-Open No. 61-269059 [Patent Document 2] Japanese Patent Laid-Open No. 2009-171874 [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] In an enzyme sensor in which an enzyme is not immobilized on an electrode with an immobilizing agent as described in Patent Document 1, the enzyme on the electrode elutes into the measurement solution, and sufficient accuracy cannot be obtained for measuring the concentration of an organic substance. In Patent Document 2, although the reagent layer on the electrode contains an enzyme and a mediator together with a cured product of a photocurable resin, the mediator in the reagent layer permeates through the film of the cured photocurable resin and elutes and disperses into the measurement solution, resulting in a problem that the efficiency of the electron transfer reaction near the electrode decreases and sufficient accuracy cannot be obtained for measuring the concentration of an organic substance.

[0007] Improvement in the accuracy of measuring the concentration of an organic substance by an enzyme sensor is desired. The present invention provides a biosensor capable of measuring the amino acid concentration in a measurement solution with higher accuracy. [Means for Solving the Problems]

[0008] In one aspect, the present invention comprises an enzyme electrode including a conductive layer and a reagent layer adjacent to the conductive layer, the conductive layer contains a mediator, the reagent layer contains amino acid oxidase and a cured product of a photosensitive polymer, a biosensor, relates to.

[0009] In another aspect, the present invention the biosensor of the present invention and a control unit that controls voltage application to the biosensor. A detection unit that detects the current value obtained by applying voltage to the biosensor, A calculation unit that calculates the amino acid concentration of the solution being measured from the current value, An output unit that outputs the calculated amino acid concentration, A system for measuring the concentration of amino acids in a solution to be measured, Regarding.

[0010] In another embodiment, the present invention is described as follows: A method for measuring the amino acid concentration in a solution to be measured, To supply the solution to be measured to the biosensor of the measurement system of the present invention; The control unit of the measurement system applies a constant voltage to the biosensor; The detection unit of the measurement system detects the current value obtained from the biosensor; The calculation unit of the measurement system calculates the amino acid concentration in the solution being measured from the detected current value. Methods including Regarding.

[0011] In another embodiment, the present invention is described as follows: A method for testing renal dysfunction in non-human test animals, The present invention provides a method for measuring amino acid concentrations, which measures the amino acid concentration in a urine sample collected from a non-human animal subject for testing. Methods including Regarding.

[0012] In another embodiment, the present invention is described as follows: Use of the biosensor of the present invention in measuring the amino acid concentration in a solution to be measured. Regarding.

[0013] In another embodiment, the present invention is described as follows: An amino acid-related information provision service system that provides amino acid-related information on test animals, the system is Server and User terminal and Measuring device and Equipped with, The user terminal and the measuring device are each identified, The user terminal is associated with the measurement device. The measuring device is equipped with the biosensor of the present invention, measures data on the amino acid concentration of a urine sample collected from a test animal, and transmits the measured data, along with data on the timing of the measurement, to the server. The server creates amino acid-related information about the test animal based on the data transmitted from the measurement device, and transmits this information to the user terminal associated with the measurement device. The user terminal outputs amino acid-related information about the test animal transmitted from the server. The amino acid-related information for the test animal includes information on the urinary amino acid concentration of the test animal, or information on the renal function of the test animal prepared based on the urinary amino acid concentration. system, Regarding. [Effects of the Invention]

[0014] The biosensor of the present invention can measure the amino acid concentration in a solution with high accuracy. Furthermore, the biosensor of the present invention enables the quantitative determination of amino acids in a solution. The biosensor of the present invention can be used for diagnosing kidney disease by measuring amino acids in the urine of animals such as cats, diagnosing various diseases and evaluating physical condition by measuring amino acids in human bodily fluids, or measuring umami components and functional components by measuring amino acids in food and beverages. [Brief explanation of the drawing]

[0015] [Figure 1] Plan view of a biosensor relating to one embodiment of the present invention. [Figure 2] Figure 1 shows the II end view of the biosensor. [Figure 3] A schematic diagram showing a potentiostat connected to the biosensor shown in Figure 1. [Figure 4] A schematic diagram representing the measurement system including the biosensor shown in Figure 1. [Figure 5] One embodiment of an amino acid-related information provision service system. [Figure 6] An example of the operation of server 100, user terminal 200, and measurement system 300 in an amino acid-related information provision service system. [Figure 7] Relationship between current values ​​measured using the biosensor of Example 1 and D-amino acid concentration. [Figure 8] Relationship between current values ​​measured using the biosensor of Comparative Example 1 and D-amino acid concentration. [Figure 9] Relationship between current values ​​measured using the biosensor of Comparative Example 2 and D-amino acid concentration. [Modes for carrying out the invention]

[0016] (1. Biosensor) The present invention provides a biosensor for measuring the concentration of a substance to be measured in a solution that may contain the substance to be measured (hereinafter also referred to as the "solution to be measured"). The biosensor of the present invention is an enzyme sensor comprising an enzyme electrode including a conductive layer and a reagent layer. In the biosensor, the conductive layer and the reagent layer are adjacent to each other, the conductive layer includes a mediator, and the reagent layer includes an enzyme that reacts with the substance to be measured and a cured product of a photosensitive polymer (hereinafter also referred to as the "cured polymer" in this specification).

[0017] The substance to be measured by the biosensor of the present invention is an amino acid, preferably a D-amino acid. Examples of D-amino acids include D-alanine, D-serine, D-arginine, D-lysine, D-asparagine, D-histidine, D-ornithine, D-glutamine, D-threonine, D-tyrosine, D-valine, D-phenylalanine, D-methionine, D-glutamic acid, D-aspartic acid, D-leucine, D-tryptophan, D-cysteine, D-isoleucine, and D-proline, which are D-type free amino acids.

[0018] The solution to be measured to which the biosensor of the present invention is applied is not particularly limited as long as it is a solution that may contain the aforementioned amino acids. Examples of such solutions include biological fluids such as sweat, saliva, urine, tears, and blood, or sample solutions derived therefrom, beverages and foods, or their dilutions or suspensions.

[0019] In a preferred embodiment, the solution to be measured is a urine sample collected from a test animal. The urine sample can be one used in general urinalysis, such as urine collected directly from excreted urine, or urine collected from or held in a pet litter box or diaper. The urine sample may be fresh urine, stored urine, concentrated urine, or a diluted version thereof. The test animal may be a mammal, including humans and non-human animals, preferably a non-human animal, and more preferably a feline.

[0020] The enzyme contained in the reagent layer of the biosensor of the present invention is an amino acid oxidase. The amino acid oxidase used in the biosensor of the present invention may vary depending on the type of amino acid to be measured, but is preferably at least one of D-amino acid oxidase and L-amino acid oxidase, and more preferably D-amino acid oxidase. The amino acid in the solution to be measured reacts with the amino acid oxidase in the reagent layer to decompose into hydrogen peroxide, keto acid, and ammonia. Hydrogen peroxide is converted to H2O2 → 2H at the enzyme electrode. + +O2+2e - It is converted into electric current as shown.

[0021] The configuration of the biosensor of the present invention will be described below with reference to Figures 1 and 2. However, the biosensor shown in Figures 1 and 2 is an exemplary embodiment of the present invention and does not limit the scope of the present invention. For example, the shape and relative positions of the three electrodes, including the enzyme electrode 2, the counter electrode 3, and the reference electrode 4 (reference electrode), and other parts in the biosensor of the present invention are not limited to those shown in Figure 1 or 2, and may be the same as those of conventionally known electrochemical biosensors. Alternatively, the electrode system may be a three-electrode system including the enzyme electrode 2, the counter electrode 3, and the reference electrode 4, or a two-electrode system including only the enzyme electrode 2 and the reference electrode 4. Since the reference electrode potential changes when current flows through the reference electrode, it is preferable from the viewpoint of high-precision measurement that the electrode system be a three-electrode system.

[0022] Figure 1 is a plan view of a biosensor according to one embodiment of the present invention. Figure 2 is an end view II of the biosensor shown in Figure 1. The biosensor 1 includes an insulating substrate 5 and an electrode system disposed on the insulating substrate 5. The insulating substrate 5 supports each electrode and wiring. The electrode system includes an enzyme electrode 2, a counter electrode 3, a reference electrode 4, and wirings 21, 31, and 41. As shown in Figure 1, the enzyme electrode 2, the counter electrode 3, and the reference electrode 4 are electrically connected to the wirings 21, 31, and 41, respectively. The counter electrode 3 and the reference electrode 4 are formed on the insulating substrate 5 so as to sandwich the enzyme electrode 2 from both sides. The shape of the counter electrode 3 is arc-shaped, surrounding the enzyme electrode 2, and is formed to maintain approximately equidistant from the enzyme electrode 2 along the longitudinal direction of the arc. Enzyme electrode 2 is an electrode that exchanges electrons with the substance to be measured, counter electrode 3 is an electrode that conducts current between enzyme electrode 2 and counter electrode 3, and reference electrode 4 is an electrode that serves as a reference for the potential of enzyme electrode 2 (reference electrode).

[0023] A portion of the electrode system is covered by an insulating layer 6. The insulating layer 6 has an opening 61 through which the entire enzyme electrode 2, as well as portions of the counter electrode 3 and reference electrode 4, are exposed. The opening 61 forms a reaction chamber and also serves as an inlet for the solution to be measured. One end of the wirings 21, 31, and 41 is in contact with the enzyme electrode 2, the counter electrode 3, and the reference electrode 4, respectively. The other ends of the wirings 21, 31, and 41 are connection points for devices for voltage sweeping operations on the electrodes or for measuring the current generated in the electrodes. The signal of the current generated due to the reaction between the enzyme in the enzyme electrode 2 and the substance to be measured is measured by an external device via the wirings 21, 31, and 41. Based on the measured current, the presence or absence of the substance to be measured in the solution to be measured, or its content, can be calculated. Similarly, in the case of a two-electrode system, the entire enzyme electrode 2 and a portion of the reference electrode 4 are exposed through the opening 61, one end of the wiring 21 and 41 is in contact with the enzyme electrode 2 and the reference electrode 4, respectively, and the other end of the wiring 21 and 41 is a connection point to a device for voltage sweeping operations on the electrodes or a device for measuring the current generated in the electrodes.

[0024] The insulating substrate 5 is made of an insulating material so that the three electrodes (or two electrodes if there is no counter electrode) are not electrically connected. There are no particular restrictions on the insulating material, and it may be the same as that of an insulating substrate that constitutes a known electrochemical biosensor. Examples of the insulating material include films, paper, mica, ceramics, etc., made from materials such as polyimide, polystyrene, polycarbonate, polyvinyl chloride resin, or polyester such as polyethylene terephthalate (PET). There are no particular restrictions on the thickness of the insulating substrate 5, but it is preferably 25 to 1000 μm.

[0025] Examples of materials for forming the insulating layer 6 include silicon oxide, silicon nitride, aluminum oxide, and photoresist that is transparent to visible light. The insulating layer 6 can be formed by conventionally known methods such as screen printing, vacuum deposition, electron beam, sputtering, plating, CVD, ion plating coating, and inkjet. The thickness of the insulating layer 6 is preferably 10 nm to 100 μm.

[0026] As shown in Figure 2, the enzyme electrode 2 includes a conductive layer 2a and a reagent layer 2b that covers the surface of the conductive layer 2a. In this specification, the conductive layer 2a is also referred to as the working electrode 2a. The working electrode 2a is formed such that a portion of it is in contact with one end 21a of the wiring 21. Conductive layers are arranged on one end 31a and 41a of the wiring 31 and 41, respectively, with a portion of each in contact with them, and these conductive layers are the counter electrode 3 and the reference electrode 4.

[0027] The area of ​​the reagent layer 2b in contact with the working electrode 2a is not particularly limited, but from the viewpoint of forming a reagent layer 2b with a sufficient amount of enzyme, it is preferably 0.1 mm. 2 The above is true, and from the viewpoint of miniaturizing the biosensor, preferably 1000 mm 2 The following are the specifications: The widths of the wirings 21, 31, and 41, and the widths of the counter electrode 3 and the reference electrode 4 are not particularly limited, but from the viewpoint of ensuring the output stability of the biosensor and achieving miniaturization, they are preferably 0.1 to 100 mm. The spacing between adjacent wirings, the spacing between the enzyme electrode 2 and the counter electrode 3, and the spacing between the enzyme electrode 2 and the reference electrode 4 are not particularly limited, but each is preferably 0.1 mm or more and 100 mm or less. The thickness of the wirings 21, 31, and 41, and each electrode are not particularly limited, but each is preferably 10 nm or more and 100 μm or less.

[0028] The conductive layers of each electrode, i.e., the working electrode 2a, the counter electrode 3, and the reference electrode 4, as well as the wirings 21, 31, and 41, can be formed on the surface of the insulating substrate 5 by conventionally known methods such as screen printing, vacuum deposition, electron beam, sputtering, plating, CVD, ion plating coating, and inkjet, depending on the material. Typical materials for the wirings 21, 31, and 41 include conductive materials such as metals like gold, silver, palladium, platinum, rhodium, indium, or iridium, and conductive carbon materials, and preferably the wirings 21, 31, and 41 are made of silver. Examples of materials for the working electrode 2a and the counter electrode 3 include conductive carbon materials, platinum, gold, and other conductive materials, preferably conductive carbon materials. An example of the reference electrode 4 is a silver / silver chloride electrode.

[0029] The conductive layer of the enzyme electrode 2, i.e., the working electrode 2a, contains a mediator (electron transfer promoter) from the viewpoint of promoting electron transfer. The mediator is not particularly limited as long as it is a redox substance that can transfer electrons to the electrode, and conventionally known reversibly redox compounds can be used. Preferably, the mediator is water-insoluble (i.e., its solubility in water at 25°C is 1 g / 100 mL or less). Examples of the mediator include metal complexes and their derivatives such as Prussian blue, ferrocene and its derivatives, osmium complexes and its derivatives (monomers, polymers), ruthenium complexes; quinones, dichloroindophenol, tetrathiafulvalene (TTF) and its derivatives; and tetracyanoquinodimethane. Among these, Prussian blue is preferred. The reduced form of Prussian blue reacts with hydrogen peroxide produced by the reaction between the enzyme and the substance to be measured to become the oxidized form of Prussian blue. This oxidized form accepts electrons from the conductive material of the conductive layer and returns to the reduced form, so that an electric current corresponding to the reaction between the enzyme and the substance to be measured is generated via the Prussian blue. The conductive layer containing this mediator can be obtained by forming a conductive layer on the surface of the insulating substrate 5 as described above using a conductive material containing the mediator (for example, Prussian blue-containing carbon paste).

[0030] In the biosensor of the present invention, the mediator is contained in a conductive layer (working electrode) 2a, and a reagent layer 2b containing a cured polymer holding an enzyme is formed on the conductive layer. With this configuration, the biosensor of the present invention prevents the mediator from eluting into the solution, and because the enzyme is in close proximity to the mediator in the conductive layer, an efficient electron transfer reaction is possible near the electrode, and therefore, even low-concentration amino acids can be measured with high accuracy. In contrast, if the mediator is contained in the reagent layer instead of the conductive layer, the mediator may pass through the cured polymer and elute into the solution, which can reduce the efficiency of the electron transfer reaction near the electrode and prevent sufficient measurement accuracy from being obtained. Also, if the mediator is contained in both the reagent layer and the conductive layer, electron transfer between the enzyme and the mediator in the conductive layer may be inhibited by the mediator in the reagent layer, making high-precision measurement impossible. Therefore, in the biosensor of the present invention, the mediator is not contained in the reagent layer 2b, but only in the conductive layer 2a.

[0031] The mediator content in the working electrode 2a is preferably 0.01% by mass or more, more preferably 0.05% by mass or more, and even more preferably 0.10% by mass or more, when the total amount of conductive material and mediator contained in the working electrode 2a is 100% by mass, from the viewpoint of promoting electron transfer, and preferably 10% by mass or less, more preferably 7.5% by mass or less, and even more preferably 5.0% by mass or less, from the viewpoint of suppressing the generation of noise during electrochemical measurements due to excessive electron transfer.

[0032] A reagent layer 2b is fixed on the working electrode 2a. The reagent layer 2b contains amino acid oxidase and a cured photosensitive polymer (cured polymer). The reagent layer 2b may further contain stabilizers, polymers, surfactants, and other reagents advantageous for measuring the concentration of amino acids.

[0033] The amino acid oxidase contained in the reagent layer 2b varies depending on the type of amino acid to be measured, but is preferably at least one enzyme selected from D-amino acid oxidase and L-amino acid oxidase, and more preferably D-amino acid oxidase. From the viewpoint of ensuring a sufficient redox reaction necessary for the quantification of amino acids, the amount of amino acid oxidase in the reagent layer 2b is preferably 0.2 μg / mm 2 or more, more preferably 0.7 μg / mm 2 or more, still more preferably 2 μg / mm 2 or more. On the other hand, from the viewpoint of suppressing the inhibition of the enzyme reaction due to the enzyme itself becoming an electric resistance, it is preferably 30 μg / mm 2 or less, more preferably 25 μg / mm 2 or less, still more preferably 15 μg / mm 2 or less. In the present specification, the amount of enzyme in the reagent layer 2b as described above refers to the amount of enzyme per 1 mm 2 of the region in the reagent layer 2b that can contact the measurement solution.

[0034] The cured polymer contained in the reagent layer 2b holds the enzyme contained in the reagent layer 2b, immobilizes the enzyme in the reagent layer 2b, inhibits the elution of the enzyme into the measurement solution, and suppresses the decrease in the reaction efficiency of the reagent layer 2b caused by the elution of the enzyme. With a reagent layer having such a configuration, the biosensor 1 can measure the amino acid concentration with high precision.

[0035] Since the photosensitive polymer used in the present invention has the property of being cured by light irradiation, a reagent layer 2b containing a cured polymer holding an enzyme adjacent to the conductive layer can be formed by applying a mixture of the photosensitive polymer and the enzyme onto the conductive layer and irradiating it with light. For example, the reagent layer 2b can be formed by dropping an aqueous solution containing amino acid oxidase and a photosensitive polymer onto the working electrode 2a formed on the insulating substrate 5 and curing it by UV irradiation.

[0036] The photosensitive polymer preferably has azide groups, from the viewpoint that it can form a cured polymer with sufficient strength to suppress enzyme elution upon light irradiation. Furthermore, since the photosensitive polymer needs to be stable and not decompose during the preparation of an aqueous solution containing the polymer and the enzyme, and during light irradiation, it is preferable that it has highly stable amide bonds.

[0037] Preferably, the photosensitive polymer includes repeating units I represented by the following formula (1). [ka]

[0038] In the above formula, n is an integer between 1 and 3; R 1 -NR 2 R 3 and; R 2 and R 3 Each of these is independently a linear or branched alkyl or alkenyl group having 1 to 6 carbon atoms, or R 2 and R 3 They combine to form a linear or branched alkylene group having 2 to 6 carbon atoms, or a linear or branched alkylene or alkenylene group having 3 to 8 carbon atoms, which may contain an oxygen atom or a nitrogen atom, or R 2 and R 3 Together with N, they form a heterocycle having 3 to 8 carbon atoms, which may be substituted with alkyl groups having 1 to 6 carbon atoms.

[0039] In one preferred embodiment, R 1 R that constitutes 2 and R 3 Each of these is independently a linear or branched alkyl or alkenyl group having 1 to 6 carbon atoms, preferably a linear or branched alkyl or alkenyl group having 1 to 4 carbon atoms. In another preferred embodiment, R 2 and R3 They combine to form a linear alkylene group having 2 to 6 carbon atoms. In another preferred embodiment, R 2 and R 3 Together with N, they form a heterocycle having 3 to 8 carbon atoms. In one embodiment, the heterocycle may contain further oxygen or nitrogen atoms. In another embodiment, the heterocycle may be substituted with an alkyl group. The alkyl group substituting the heterocycle is a linear or branched alkyl group having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, and more preferably a methyl or ethyl group.

[0040] In a more preferred embodiment, R 1 The group is selected from the following group: [ka]

[0041] More preferably, the repeating unit I is represented by the following formula (2). [ka]

[0042] Preferably, the photosensitive polymer is a copolymer polymer comprising at least a repeating unit I represented by formula (1) and a repeating unit II having water solubility. More specifically, the repeating unit II having water solubility in water at 25°C of 1 g / 100 mL or more when the degree of polymerization is 100. In a preferred embodiment, the repeating unit II is selected from the group consisting of the following: [ka]

[0043] In a more preferred embodiment, the repeating unit II is represented by the following formula (3). [ka]

[0044] The photosensitive polymer may be a copolymer comprising, in addition to the repeating unit I and repeating unit II represented by formula (1), another repeating unit. An example of the other repeating unit is a repeating unit derived from a vinyl compound. Examples of such vinyl compounds include vinyl acetate, diacetone acrylamide, acrylamide, and ethylene glycol. Another example of the other repeating unit is a repeating unit represented by the following formula (4). [ka]

[0045] Preferred examples of the photosensitive polymer include copolymer polymers having repeating units represented by formulas (2), (3), and (4). An example of such a photosensitive polymer is Biosurfine® AWP-MRH (manufactured by Toyo Gosei Kogyo Co., Ltd.).

[0046] The content of the repeating unit I in the photosensitive polymer is preferably 0.5% by mass or more, more preferably 1% by mass or more, and even more preferably 2% by mass or more, with the entire photosensitive polymer as 100% by mass, from the viewpoint of good retention of the enzyme in the reagent layer, while preferably 30% by mass or less, more preferably 25% by mass or less, and even more preferably 20% by mass or less, with the entire photosensitive polymer as 100% by mass. The content of the repeating unit II in the photosensitive polymer is preferably 40% by mass or more, more preferably 45% by mass or more, and even more preferably 50% by mass or more, with the entire photosensitive polymer as 100% by mass, from the viewpoint of good retention of the polymer aqueous solution in the reagent layer, while preferably 98% by mass or less, more preferably 95% by mass or less, and even more preferably 90% by mass or less, with the entire photosensitive polymer as 100% by mass.

[0047] The overall average degree of polymerization of the photosensitive polymer is preferably 100 or more, more preferably 200 or more, and even more preferably 300 or more, in order to obtain sufficient photosensitivity. On the other hand, from the viewpoint of suppressing deterioration of operability during the formation of the reagent layer due to increased viscosity of the polymer and inhibition of enzymatic reactions by the cured polymer, it is preferably 10,000 or less, more preferably 7,500 or less, and even more preferably 5,000 or less.

[0048] In the reagent layer 2b, the mass ratio of the enzyme to the cured polymer (enzyme / cured polymer) is preferably 0.05 or higher, more preferably 0.10 or higher, and even more preferably 0.20 or higher, from the viewpoint of suppressing inhibition of the enzyme reaction by the cured polymer, and preferably 10 or lower, more preferably 7.5 or lower, and even more preferably 5 or lower, from the viewpoint of good retention of the enzyme in the reagent layer.

[0049] From the viewpoint of effectively retaining the enzyme in the reagent layer 2b, the content of the cured polymer is preferably 0.2 μg / mm³. 2 More preferably 0.7 μg / mm³ 2 More preferably 2 μg / mm³ 2 Therefore, from the viewpoint of suppressing the inhibition of the enzymatic reaction by the cured polymer, 50 μg / mm³ is preferred. 2 More preferably 40 μg / mm³ 2 More preferably 30 μg / mm³ 2 The following applies: In this specification, the amount of cured polymer in the reagent layer 2b as described above refers to the 1 mm area of ​​the reagent layer 2b that is in contact with the solution being measured. 2 This refers to the amount of curing polymer per unit.

[0050] (2. Measurement System) The biosensor of the present invention is used to measure the amino acid concentration in a solution to be measured. The following describes a system for measuring the amino acid concentration in a solution to be measured using the biosensor of the present invention (hereinafter also referred to as "the measurement system of the present invention").

[0051] As an exemplary embodiment of the measurement system of the present invention, a measurement system for chronoamperometry (CA) measurement using a biosensor 1 will be described with reference to Figures 3 and 4. Figure 3 is a schematic diagram showing a potentiostat connected to the biosensor 1. In Figure 3, the wiring 21, 31, and 41 of the biosensor 1 are connected to the potentiostat 8, and the enzyme electrode 2, counter electrode 3, and reference electrode 4 are immersed in the solution to be measured. In chronoamperometry (CA) measurement, a constant potential is applied to the enzyme electrode 2 by the potentiostat 8, and the current value flowing through the enzyme electrode 2 is measured.

[0052] Figure 4 is a schematic diagram representing a measurement system including a biosensor 1. In the measurement system shown in Figure 4, a measuring device 10 is connected to the biosensor 1. The measuring device 10 includes a control unit 11 connected to the biosensor 1 and controlling the voltage applied to the biosensor 1, a detection unit 12 that detects the current obtained by the voltage applied to the biosensor 1, a calculation unit 13 that calculates the amino acid concentration of the solution to be measured from the current value, and an output unit 14 that outputs the calculated amino acid concentration.

[0053] The control unit 11 is electrically connected to the biosensor 1 and controls the applied voltage value and voltage application time. The detection unit 12 measures the current generated due to the enzymatic reaction of the enzyme electrode 2 and sends the measurement result to the calculation unit 13. The control unit 11 and the detection unit 12 may be potentiostats equipped with the above functions. The calculation unit 13 calculates and stores the concentration of the substance to be measured from the current value sent from the detection unit 12. For example, the calculation unit 13 may be an information processing terminal including a memory that stores a linear function between the concentration of the substance to be measured and the current value, and a central processing unit (CPU) that includes a calculation unit for calculating the absolute value of the concentration of the substance to be measured. The output unit 14 transmits the calculation result of the concentration of the substance to be measured by the calculation unit 13 to the display unit 15 through a communication interface. The communication interface may be wired or wireless, and examples of wireless communication interfaces include Wi-Fi and Bluetooth®. The display unit may be a digital display or an analog display.

[0054] (3. Amino Acid-Related Information Provision Service System) As an example of an application of the biosensor of the present invention, an amino acid-related information provision service system (hereinafter also referred to as "the service system of the present invention") will be described. The service system of the present invention measures the amino acid concentration in a urine sample (solution to be measured) collected from a test animal and provides amino acid-related information concerning the test animal to the user of the service system.

[0055] The amino acid-related information concerning test animals provided by the service system of the present invention includes the urinary amino acid concentration of the test animal and information on the renal function of the test animal created based on the urinary amino acid concentration. The information on renal function includes the degree of renal function in the test animal, information for preventing renal disease corresponding to the degree of renal function (including information on pet food and lifestyle habits that are effective for prevention), information on the risk and possibility of renal disease, as well as information on the presence or severity of renal disease and information on the treatment of renal disease (including information on treatment methods, drugs, veterinarians, hospital locations, etc.).

[0056] A user of the service system of the present invention is a person who wishes to receive amino acid-related information concerning the test animal. Examples of such users include, if the test animal is a non-human animal, a person who raises or manages the test animal, such as the owner of the test animal, a veterinarian, or an employee of an animal breeding facility such as a zoo (animal caretaker, veterinarian, facility manager). Alternatively, if the test animal is a human, the user may be the test subject themselves, or a family member or caregiver of the test subject may be the user.

[0057] In a typical embodiment, the service system of the present invention comprises a server, a user terminal, and a measuring device. Each user registered with the service system of the present invention uses at least one user terminal and at least one measuring device.

[0058] Each user terminal and each measuring device is identified. For example, each user terminal and each measuring device can be managed by assigning an ID number. Registered users can also be identified and managed by an ID number, etc. Each user terminal and each measuring device is associated with one of the registered users. In addition, each user terminal is associated with one or more of the measuring devices. The association between user terminals, measuring devices, and users can be performed and managed by their identification information (e.g., ID numbers).

[0059] A single user may use one measurement device and one user terminal. Alternatively, a single user may use multiple measurement devices, each associated with one or more user terminals. The number of users (or user terminals and measurement devices) that the service system may allow is not particularly limited and can be changed depending on, for example, the server's performance.

[0060] The aforementioned measuring device is equipped with the biosensor of the present invention and can measure data on the amino acid concentration of a urine sample collected from a test animal. The measuring device is also configured to transmit the measured data to the server. Therefore, the measuring device may include a communication unit for transmitting the data.

[0061] The server creates amino acid-related information about the test animal based on the data transmitted from the measurement device and transmits this information to the user terminal associated with the measurement device. The user terminal outputs the amino acid-related information about the test animal transmitted from the server.

[0062] Figure 5 shows one embodiment of the configuration of the service system of the present invention. The system shown in Figure 5 includes a server 100, user terminals 200, and a measuring device 300 including the biosensor of the present invention. The server 100 is connected to a plurality of user terminals 200 and a plurality of measuring devices 300, and can send and receive data in a timely manner. In Figure 5, this connection is via the Internet 50, but is not limited to this.

[0063] Figure 5 shows multiple users A, B, and C who utilize the service system of the present invention. In the embodiment shown in Figure 5, users A, B, and C each own a test animal (e.g., a cat). In another embodiment, if the test animal is a human, users A, B, and C may be the test subject, or family members or caregivers of the test subject. Users A, B, and C each possess a user terminal (200A, 200B, and 200C in the figure) and a measurement device (300A, 300B, and 300C in the figure), which are each connected to the server 100.

[0064] Server 100 is an information processing device managed by the service operator. Server 100 receives data measured by the measurement device 300, creates amino acid-related information about the test animal, such as urinary amino acid concentration and information on renal function, from the received data, and transmits it to the user terminal 200.

[0065] The server 100 may include a communication interface for sending and receiving data with a user terminal 200 or a measuring device 300, a calculation unit for creating amino acid-related information about the test animal, such as urinary amino acid concentration and information on renal function, from data received from the measuring device 300, and a control unit for controlling the operation of the communication interface and the calculation unit.

[0066] Furthermore, the server 100 may include a storage unit for storing information necessary for calculations performed by the calculation unit, or for storing the calculation results. Alternatively, the storage unit may be installed outside the server 100. The storage unit may store various applications for executing the amino acid-related information provision service system according to the present invention, amino acid-related information about test animals created by the server 100, and various databases used to create the amino acid-related information. Examples of such databases, though not limited to them, include a user information database that stores attribute information about users, test animals, and measurement devices owned by each user; a renal function information database that stores information on the association between amino acid concentration and renal function, and information on the preservation of renal function or the prevention or treatment of renal diseases; and an amino acid-related information database that stores amino acid-related information about test animals for each user. These databases can be referenced from each other as needed when the server 100 creates amino acid-related information.

[0067] In the embodiment shown in Figure 5, only one server 100 is shown, but the server 100 may be composed of multiple information processing devices, and the processing performed by the server 100 may be distributed and executed by the multiple information processing devices.

[0068] User terminals 200 (200A, 200B, 200C, etc.) receive and output amino acid-related information concerning test animals from server 100. The format of the output is not particularly limited and may include screen display, printing, or recording to a database. In a preferred embodiment, an application program (hereinafter also referred to as "app") corresponding to the amino acid-related information provision service system of the present invention is installed on the user terminal 200, and the user terminal 200 may output the amino acid-related information provided from server 100 via the app. As user terminals 200, for example, information processing terminals such as smartphones, mobile phones, tablet PCs (personal computers), notebook PCs, and desktop PCs can be used, with the necessary app installed.

[0069] The measuring device 300 (300A, 300B, 300C...) is equipped with the biosensor of the present invention described above and measures data on the amino acid concentration in a urine sample from a test animal. In one embodiment, the measuring device 300 may include a biosensor 1 as shown in Figures 3 and 4, a control unit 11, a detection unit 12, and a calculation unit 13 as needed. In one embodiment, data of a current value reflecting the amino acid concentration in the urine sample detected by the detection unit 12 of the measuring device 300 is sent to the server 100 via the Internet 50, and the server 100 calculates the amino acid concentration in the urine sample from the test animal from the current value. In another embodiment, the amino acid concentration in the urine sample is calculated by the calculation unit 13 of the measuring device 300, and the calculated amino acid concentration data is sent to the server 100 via the Internet 50.

[0070] In a preferred embodiment, the measuring device 300 acquires data on the timing (e.g., measurement date and time) of the measurement of data on the amino acid concentration in the urine sample, and sends the measurement timing data along with the data on the amino acid concentration to the server 100.

[0071] The user terminal 200 and the measuring device 300 are equipped with a communication unit for wired or wireless communication, and data is transmitted and received between the user terminal 200 or the measuring device 300 and the server 100 via this communication unit. The measuring device 300 may be directly connected to the server 100 via the internet 50, as shown in Figure 5, or the measuring device 300 may be connected to the user terminal 200 and connected to the internet 50 via the user terminal 200.

[0072] [Example of operation of the amino acid-related information provision service system]

[0073] Figure 6 illustrates an example of the operation of the server 100, user terminal 200, and measurement device 300 in the service system of the present invention. As a prerequisite for the processing in this example, each user is assumed to possess a user terminal 200 and a measurement device 300 for the test animal. The measurement device 300 is, for example, loaned or transferred by the operator of an amino acid-related information provision service. Each user terminal and measurement device, or the user and test animal using them, are registered and managed on the server 100 (in this example, the identification information of the measurement device is referred to as the "sensor ID" below).

[0074] The user collects urine from the test animal, measures the amino acid concentration in the urine sample using the measuring device 300 (S31), and transmits the measured amino acid concentration data, along with the measurement timing data, to the server 100 (S32). The server 100 receives the amino acid concentration data associated with the measurement timing and sensor ID (S11) and stores this information (S12).

[0075] Next, the server 100 determines whether multiple amino acid concentration data associated with the same sensor ID but different measurement timings are stored (S13). If multiple amino acid concentration data associated with different measurement timings are stored (Yes in S13), the server 100 generates amino acid-related information, including renal function level information that shows the degree of renal function of the test animal over time (S14).

[0076] On the other hand, if multiple amino acid concentration data associated with different measurement timings are not stored (No in S13), the server 100 generates amino acid-related information, including information on the degree of renal function, which shows the correspondence between the degree of renal function of the test animal and a single measurement timing, similar to S14 (S15).

[0077] Next, the server 100 sends the created amino acid-related information to the user terminal 200 (S16). The user terminal 200 receives the amino acid-related information (S21) and displays the amino acid-related information (S22).

[0078] As described above, in the amino acid-related information provision service system of the present invention, in addition to being able to accurately measure urinary amino acid concentration using the measuring device 300, the server 100 can create and provide the user with the aforementioned information on the degree of renal function based on the amino acid concentration information accumulated in the server. This makes it possible for users without experimental equipment or testing technology to easily obtain information on urinary amino acid concentration and renal function of test animals.

[0079] (4. Method for measuring amino acid concentration) Next, a method for measuring the amino acid concentration in a solution to be measured using the biosensor of the present invention (hereinafter also referred to as "the measurement method of the present invention") will be described.

[0080] The measurement method of the present invention includes the following: To supply the solution to be measured to the biosensor of the measurement system of the present invention; The control unit of the measurement system applies a constant voltage to the biosensor; The detection unit of the measurement system detects the current value obtained from the biosensor; The calculation unit of the measurement system calculates the amino acid concentration in the solution being measured from the detected current value.

[0081] As an exemplary embodiment of the measurement method of the present invention, a method for measuring the amino acid concentration in a solution to be measured using the measurement system shown in Figure 4 will be described. This method includes the following steps A to D. Step A: Supply the solution to be measured to the reagent layer 2b, counter electrode 3, and reference electrode 4 of the biosensor 1; Step B: The control unit 11 applies a constant voltage to the biosensor 1; Step C: The detection unit 12 detects the current value obtained from the biosensor 1; Step D: The calculation unit 13 calculates the concentration of the substance to be measured from the detected current value.

[0082] In step A described above, the solution to be measured is supplied to the reagent layer 2b, the counter electrode 3, and the reference electrode 4 through the opening 61 in the insulating layer 6.

[0083] Steps B to C described above are steps for detecting electrical signals using chronoamperometry (CA) measurement. When the enzyme electrode 2 and the counter electrode 3 are electrically connected and a predetermined voltage is applied between the enzyme electrode 2 and the counter electrode 3, the current value flowing between the enzyme electrode 2 and the counter electrode 3 is detected.

[0084] In step D, the absolute value of the concentration of the substance to be measured corresponding to the current value is calculated from the current value detected in step C, for example, based on a linear function of the concentration of the substance to be measured and the current value that is pre-stored in the measuring device.

[0085] The measurement method of the present invention can be used to measure the amino acid concentration in a urine sample collected from a test animal, thereby testing for the presence or absence of kidney disease in the test animal. For example, in cats, it is known that kidney disease is suspected when the total concentration of D-amino acids in the urine is approximately 400 μM or less. The measurement method of the present invention enables highly accurate concentration measurement of amino acids in the solution being measured, even in the low concentration range (1000 μM or less), and can therefore be used for a simple diagnosis of kidney disease in cats.

[0086] Therefore, an example of the application of the measurement method of the present invention is a method for examining renal dysfunction in test animals. In this examination method, a urine sample collected from the test animal is used as the solution to be measured. This examination method includes measuring the amino acid concentration in the urine sample using the measurement method of the present invention.

[0087] As one embodiment, a method for testing for renal dysfunction in cats using the measurement system shown in Figure 4 will be described. In this method, cat urine is used as the urine sample. The method includes the following steps A to E. Step A: Supply cat urine to the reagent layer 2b, counter electrode 3, and reference electrode 4 of the biosensor 1. Step B: The control unit 11 applies a constant voltage to the biosensor 1; Step C: The detection unit 12 detects the current value obtained from the biosensor 1; Step D: The calculation unit 13 calculates the amino acid concentration in the urine based on the detected current value; Step E: Test the cat for renal dysfunction based on the calculated amino acid concentration in the urine.

[0088] As exemplary embodiments of the present invention, the following substances, manufacturing methods, uses, or methods are further disclosed herein. However, the present invention is not limited to these embodiments.

[0089] [1] An enzyme electrode comprising a conductive layer and a reagent layer adjacent to the conductive layer, The conductive layer contains a mediator, The reagent layer comprises an amino acid oxidase and a cured photosensitive polymer. Biosensor. [2] Preferably, the biosensor according to [1], wherein the photosensitive polymer has an azide group. [3] Preferably, the biosensor according to [1] or [2], wherein the photosensitive polymer has an amide bond. [4] Preferably, the photosensitive polymer comprises a repeating unit I represented by formula (1), according to any one of [1] to [3], the biosensor. [5] Preferably, in formula (1), R 2 and R 3 However, each is independently a linear or branched alkyl or alkenyl group having 1 to 4 carbon atoms; R 2 and R 3 They combine to form a linear alkylene group having 2 to 6 carbon atoms; or R 2 and R 3 Together with N, they form a heterocycle having 3 to 8 carbon atoms, which may be substituted with alkyl groups. Here, The heterocycle may contain additional oxygen or nitrogen atoms. The alkyl group substituting the heterocycle is preferably a linear or branched alkyl group having 1 to 4 carbon atoms, and more preferably a methyl group or an ethyl group. [4] The biosensor described below. [6] Preferably, in formula (1), R 1 is a group selected from the following group: [ka] [4] The biosensor described below. [7] Preferably, the biosensor according to any one of [4] to [6], wherein the repeating unit I is represented by formula (2). [8] Preferably, the photosensitive polymer is a copolymer polymer comprising at least the repeating unit I and the repeating unit II having water solubility, according to any one of [4] to [7]. [9] The repeating unit II is Preferably, selected from the group consisting of the following: [ka] More preferably represented by formula (3), [8] The biosensor described.

[10] Preferably, the photosensitive polymer is a copolymer polymer further comprising repeating units represented by formula (4) according to [8] or [9].

[11] The content of the repeating unit I in the photosensitive polymer is preferably 0.5% by mass or more, more preferably 1% by mass or more, even more preferably 2% by mass or more, and preferably 30% by mass or less, more preferably 25% by mass or less, even more preferably 20% by mass or less, based on 100% by mass of the entire photosensitive polymer. A biosensor as described in any one of items [4] to

[10] .

[12] The content of the repeating unit II in the photosensitive polymer is preferably 40% by mass or more, more preferably 45% by mass or more, even more preferably 50% by mass or more, and preferably 98% by mass or less, more preferably 95% by mass or less, and even more preferably 90% by mass or less, based on 100% by mass of the entire photosensitive polymer.

[11] The biosensor described below.

[13] The total average degree of polymerization of the photosensitive polymer is preferably 100 or more, more preferably 200 or more, even more preferably 300 or more, and preferably 10000 or less, more preferably 7500 or less, even more preferably 5000 or less. A biosensor as described in any one of items [4] to

[12] .

[14] The amount of amino acid oxidase in the reagent layer is preferably 0.2 μg / mm³ 2 More preferably 0.7 μg / mm³ 2 More preferably 2 μg / mm³ 2 The above is required, and preferably 30 μg / mm³ 2 More preferably 25 μg / mm³ 2 More preferably, 15 μg / mm³ 2 The following is: A biosensor as described in any one of items [1] to

[13] .

[15] The content of the cured photosensitive polymer in the reagent layer is preferably 0.2 μg / mm 2 More preferably 0.7 μg / mm³ 2 More preferably 2 μg / mm³ 2 The above is required, and preferably 50 μg / mm³ 2 More preferably 40 μg / mm³ 2 More preferably 30 μg / mm³ 2 The following is: A biosensor as described in any one of items [1] to

[14] .

[16] The mass ratio of the amino acid oxidase to the cured photosensitive polymer in the reagent layer (amino acid oxidase / cured product) is preferably 0.05 or more, more preferably 0.10 or more, even more preferably 0.20 or more, and preferably 10 or less, more preferably 7.5 or less, and even more preferably 5 or less. A biosensor as described in any one of items [1] to

[15] .

[17] Preferably, the mediator is insoluble in water, the biosensor according to any one of [1] to

[16] .

[18] Preferably, the mediator is Prussian blue, the biosensor according to

[17] .

[19] The mediator content in the conductive layer is preferably 0.01% by mass or more, more preferably 0.05% by mass or more, even more preferably 0.10% by mass or more, and preferably 10% by mass or less, more preferably 7.5% by mass or less, and even more preferably 5.0% by mass or less, when the total amount of conductive material and mediator contained in the conductive layer is 100% by mass. A biosensor as described in any one of items [1] to

[18] .

[20] The area of ​​the reagent layer in contact with the conductive layer is preferably 0.1 to 1000 mm². 2 A biosensor as described in any one of items [1] to

[19] . A biosensor as described in any one of items

[21] , [1] to

[20] , A control unit that controls the voltage applied to the biosensor, A detection unit that detects the current value obtained by applying voltage to the biosensor, A calculation unit that calculates the amino acid concentration of the solution being measured from the current value, An output unit that outputs the calculated amino acid concentration, A system for measuring the concentration of amino acids in a solution to be measured, comprising the following components.

[22] Preferably, the measurement system according to

[21] , wherein the solution to be measured is a urine sample collected from a test animal.

[23] Preferably, the measurement system according to

[22] , wherein the test animal is a feline.

[24] A method for measuring the amino acid concentration in a solution to be measured,

[21] Supply the solution to be measured to the biosensor of the measurement system described; The control unit of the measurement system applies a constant voltage to the biosensor; The detection unit of the measurement system detects the current value obtained from the biosensor; The calculation unit of the measurement system calculates the amino acid concentration in the solution being measured from the detected current value. Methods that include...

[25] Preferably, the method according to

[24] , wherein the solution to be measured is a urine sample collected from the test animal.

[26] Preferably, the method according to

[25] , wherein the test animal is a feline.

[27] A method for testing renal dysfunction in non-human test animals,

[24] The amino acid concentration in a urine sample collected from a non-human test animal is measured using the measurement method described above. Methods that include...

[28] Preferably, the method according to

[27] , wherein the non-human animal being tested is a feline.

[29] Use of a biosensor described in any one of items [1] to

[20] for measuring the amino acid concentration in the solution to be measured.

[30] Preferably, the use according to

[29] , wherein the solution to be measured is a urine sample collected from the non-human animal subject to the test.

[31] Preferably, the use described in

[30] , wherein the non-human animal being tested is a feline.

[32] An amino acid-related information provision service system that provides amino acid-related information concerning test animals, the system is Server and User terminal and Measuring device and Equipped with, The user terminal and the measuring device are each identified, The user terminal is associated with the measurement device. The measuring device comprises a biosensor as described in any one of items [1] to

[20] , measures data on the amino acid concentration of a urine sample collected from a test animal, and transmits the measured data, along with data on the timing of the measurement, to the server. The server creates amino acid-related information about the test animal based on the data transmitted from the measurement device, and transmits this information to the user terminal associated with the measurement device. The user terminal outputs amino acid-related information about the test animal transmitted from the server. The amino acid-related information for the test animal includes information on the urinary amino acid concentration of the test animal, or information on the renal function of the test animal prepared based on the urinary amino acid concentration. system. [Examples]

[0090] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0091] (Example 1) Biosensor 1, shown in Figures 1 and 2, was fabricated.

[0092] [Fabrication of electrode systems] On an insulating substrate 5 (AS ONE Corporation, polyimide film, model number: HJA-A4-225μm, thickness 225μm), silver paste (SUN CHEMICAL Corporation, product number C2080415P2) was screen printed through a screen mask, and sintered at 130°C for 10 minutes to form wiring 21 for the enzyme electrode, wiring 31 for the counter electrode, and wiring 41 for the reference electrode. Next, on one end of the wiring 21, a portion of which overlaps, is placed, with a diameter of 3.5 mm (area of ​​9.6 mm). 2A circular conductive layer was formed. Specifically, a Prussian blue-containing carbon paste (manufactured by SUN CHEMICAL, product number C2070424D2) was screen printed through a screen mask and dried at 80°C for 10 minutes to form a working electrode 2a containing 1% by mass of Prussian blue in the conductive layer. Next, a counter electrode 3 was formed by screen printing carbon paste (SUN CHEMICAL, product number C2030519P4) through a screen mask onto one end of wiring 31, overlapping a portion of it, and drying it at 80°C for 10 minutes. Then, a reference electrode 4 was formed by screen printing silver / silver chloride paste (SUN CHEMICAL, product number C2130809D5) through a screen mask onto one end of wiring 41, overlapping a portion of it, and sintering it at 80°C for 10 minutes. Next, an insulating layer 6 was formed by screen printing an insulating paste (manufactured by SUN CHEMICAL, product number D2070423D5) through a screen mask so that it covered parts of wiring 21, wiring 31, and wiring 41, and then drying it at 130°C for 10 minutes. In the procedure described above, the silver paste, Prussian blue-containing carbon paste, carbon paste, silver / silver chloride paste, and insulating paste were each applied by screen printing to a thickness of 10 μm after firing or drying.

[0093] [Enzyme modification] Next, a mixed solution was prepared containing 50 U / mL (10 mg / mL) of D-amino acid oxidase (enzyme, manufactured by SIGMA-Aldrich, D-Amino Acid Oxidase from porcine kidney) and 3% by mass of Biosurfine-AWP-MRH (manufactured by Toyo Gosei Co., Ltd.) in PBS solution (0.1 mol / L phosphate-buffered saline, pH=8.0, 25°C; manufactured by Fujifilm Wako Pure Chemical Industries). Then, 7 μL of the obtained mixed solution was dropped onto the working electrode 2a, dried at 25°C for 24 hours, and further exposed to UV light (100 mJ / cm²). 2By doing so, a reagent layer 2b containing an enzyme and a cured photosensitive polymer was formed on the working electrode 2a. Following the above procedure, a biosensor 1 was obtained comprising an enzyme electrode 2 containing a working electrode 2a with a mediator and an adjacent reagent layer 2b. The D-amino acid oxidase content in the reagent layer 2b was 7.3 μg / mm³. 2 The cured product content of Biosurfine-AWP-MRH is 21.9 μg / mm³. 2 Furthermore, the mass ratio of these (D-amino acid oxidase / Biosurfine-AWP-MRH cured product) is 0.33.

[0094] When the prepared enzyme electrode 2 is immersed in any D-amino acid solution, the redox substance (Prussian blue) is oxidized by the hydrogen peroxide produced by the enzymatic reaction.

[0095] [Measurement of amino acid concentration] The following experiment was conducted to obtain a correlation equation between the D-amino acid concentration and the steady-state current value of enzyme electrode 2. D-alanine was used as the D-amino acid. As shown in Figure 3, the wiring 21 for the enzyme electrode, the wiring 41 for the reference electrode, and the wiring 31 for the counter electrode of the biosensor 1 were connected to the potentiostat 8, and each electrode was immersed in 10 mL of PBS solution containing D-alanine (manufactured by Peptide Laboratories, 0.1 mol / L phosphate-buffered saline, pH=8.0, 25°C; manufactured by Fujifilm Wako Pure Chemical Industries). Next, chronoamperometry (CA) was performed by applying a constant potential of -0.20 V to the enzyme electrode 2 using the potentiostat 8, and the steady-state current value of the enzyme electrode 2 (150 seconds after the start of measurement) was measured. The concentration of D-alanine in the solution was 100 μM, 200 μM, 400 μM, or 800 μM.

[0096] As shown in Figure 7, the steady-state current value decreased as the concentration of D-alanine in the solution under test increased, confirming a negative linear relationship between the D-alanine concentration and the steady-state current value. This indicates that the D-alanine concentration of the solution under test can be quantified from the current value obtained by CA measurement. The linear correlation equation between the D-alanine concentration and the steady-state current value is expressed by the following equation (5). In the following equation (5), y is the quantitative current value (A) and x is the concentration of D-alanine (μM). y = -1.71 × 10 -09 x + 6.08 × 10 -08 (5) In the correlation equation, the correlation coefficient R in the low concentration region where the concentration of D-alanine is 1000 μM or less. 2 The value was 0.988, which was very close to 1. When the concentration of D-alanine in the solution being measured was in the range of 0 to 800 μM, the current value per unit concentration of D-alanine was -1.71 nA / μM. These results confirm that by using the biosensor of Example 1, the concentration of amino acids in the solution being measured can be measured with sufficient accuracy even in the low concentration range, and a large output can be obtained relative to the amino acid concentration.

[0097] (Comparative Example 1) [Fabrication of electrode systems] The procedure was carried out in the same manner as in Example 1 [Fabrication of the electrode system]. [Enzyme modification] A biosensor equipped with an enzyme electrode was obtained in the same manner as in Example 1's [Enzyme Modification], except that 7 μL of the PBS solution (pH=8.0) containing 50 U / mL (10 mg / mL) of D-amino acid oxidase was dropped onto the working electrode 2a instead of the mixed solution used in Example 1's [Enzyme Modification].

[0098] [Measurement of amino acid concentration] When the [amino acid concentration measurement] was performed in the same manner as in Example 1, the relationship between D-alanine concentration and steady-state current value as shown in Figure 8 was obtained. As can be seen from the comparison between Figure 7 and Figure 8, the output with respect to D-alanine concentration was smaller in Comparative Example 1 compared to Example 1. In Comparative Example 1, when the D-alanine concentration in the solution being measured was in the range of 0 to 800 μM, the current value per unit concentration of D-alanine was -0.0548 nA / μM. Furthermore, the correlation coefficient R in the low concentration region of D-alanine below 1000 μM was also obtained. 2 The value was 0.909, which was lower than that of Example 1.

[0099] (Comparative Example 2) [Fabrication of electrode systems] The procedure was carried out in the same manner as in Example 1 [Fabrication of the electrode system]. [Enzyme modification] A biosensor equipped with an enzyme electrode was obtained in the same manner as in the [enzyme modification] of Example 1, except that 7 μL of the PBS solution (pH=8.0) containing 50 U / mL (10 mg / mL) of D-amino acid oxidase and 1% by mass of glutaraldehyde (manufactured by Fujifilm Wako Pure Chemical Industries) was dropped onto the working electrode 2a, instead of the mixed solution used in the [enzyme modification] of Example 1.

[0100] [Measurement of amino acid concentration] When the [amino acid concentration measurement] was performed in the same manner as in Example 1, the relationship between D-alanine concentration and steady-state current value as shown in Figure 9 was obtained. As can be seen from the comparison between Figure 7 and Figure 9, the output with respect to D-alanine concentration was smaller in Comparative Example 2 compared to Example 1. In Comparative Example 2, when the D-alanine concentration in the solution being measured was in the range of 0 to 800 μM, the current value per unit concentration of D-alanine was -0.128 nA / μM. Furthermore, the correlation coefficient R in the low concentration region of D-alanine below 1000 μM was obtained. 2 The value was 0.840, which was lower than that of Example 1.

[0101] (Example 2) [Fabrication of biosensors] The electrode system was fabricated and enzyme modified in the same manner as in Example 1.

[0102] [Measurement of amino acid concentration] As shown in Figure 3, the potentiostat 8 was connected to the enzyme electrode wiring 21, the reference electrode wiring 41, and the counter electrode wiring 31, respectively, and each electrode was immersed in the solution to be measured. Next, chronoamperometry (CA) was performed by applying a constant potential of -0.20V to the enzyme electrode 2 using the potentiostat 8, and the steady-state current value of the enzyme electrode 2 (150 seconds after the start of measurement) was measured. For the measurement solution, buffered cat urine was prepared by adding 93.6 μL of 1M sodium hydroxide (SIGMA-Aldrich) and 13.6 mg of potassium dihydrogen phosphate (Fujifilm Wako Pure Chemical Industries) to 10 mL of actual cat urine to adjust the pH to 8.0 (25°C). Three samples of actual cat urine were prepared: two from healthy cats and one from a cat with renal disease.

[0103] [Measurement results] The steady-state current values ​​obtained in this embodiment were substituted into the linear correlation equation (5) above to calculate the concentration of D-alanine in cat urine, which is shown in Table 1 below as a sensor-converted value. Table 1 also shows the D-amino acid concentration measured in the same solution using LC-MS (liquid chromatography).

[0104] [Table 1]

[0105] The D-alanine concentrations obtained by substituting the steady-state current values ​​obtained in Example 2 into the linear correlation equation (5) were 722 μM and 648 μM, respectively, for healthy cat urine, and 262 μM for cat urine with renal disease. In both cases, the D-alanine concentration (sensor-converted value) calculated from the linear correlation equation (5) was within 10% of the D-alanine concentration (LC-MS measurement value) obtained by LC-MS measurement. From the above, it was shown that the biosensor of Example 1 can differentiate between healthy cat urine and cat urine with renal disease. [Explanation of Symbols]

[0106] 1. Biosensor 2. Enzyme electrodes 2a Conductive layer (working electrode) 2b Reagent layer 3. Opposite 4 Reference pole Wiring 21, 31, 41 5. Insulating substrate 6. Insulating layer 61 Aperture 8. Potentiostat 10 Measuring device 11 Control Unit 12 Detection unit 13 Arithmetic section 14 Output section 15 Display

Claims

1. The enzyme electrode comprises a conductive layer and a reagent layer adjacent to the conductive layer, The conductive layer contains a mediator, The reagent layer comprises an amino acid oxidase and a cured photosensitive polymer. Biosensor.

2. The biosensor according to claim 1, wherein the photosensitive polymer has an azide group.

3. The biosensor according to claim 1, wherein the photosensitive polymer has an amide bond.

4. The biosensor according to claim 1, wherein the photosensitive polymer includes a repeating unit I represented by the following formula (1): 【Chemistry 1】 (In the formula, n is an integer between 1 and 3; R 1 -NR 2 R 3 And; R 2 and R 3 Each of these is independently a linear or branched alkyl or alkenyl group having 1 to 6 carbon atoms, or R 2 and R 3 They combine to form a linear or branched alkylene group having 2 to 6 carbon atoms, or a linear or branched alkylene or alkenylene group having 3 to 8 carbon atoms, which may contain an oxygen atom or a nitrogen atom, or R 2 and R 3 together with N form a heterocyclic ring having 3 to 8 carbon atoms which may be substituted with an alkyl group having 1 to 6 carbon atoms).

5. R 1 The biosensor according to claim 4, wherein the group is selected from the group consisting of the following. 【Chemistry 2】

6. The biosensor according to claim 4, wherein the repeating unit I is represented by the following formula (2). 【Transformation 3】

7. The biosensor according to claim 4, wherein the photosensitive polymer is a copolymer polymer comprising at least the repeating unit I and the water-soluble repeating unit II.

8. The biosensor according to claim 7, wherein the repeating unit II is selected from the group consisting of the following. 【Chemistry 4】

9. The biosensor according to claim 7, wherein the repeating unit II is represented by the following formula (3). 【Transformation 5】

10. The biosensor according to claim 1, wherein the mediator is insoluble in water.

11. The biosensor according to claim 10, wherein the mediator is Prussian blue.

12. The biosensor according to claim 1, wherein the mediator content in the conductive layer is 0.01% by mass or more and 10% by mass or less, when the total amount of conductive material and mediator contained in the conductive layer is 100% by mass.

13. The biosensor according to claim 1, wherein the mass ratio of the amino acid oxidase to the cured product of the photosensitive polymer in the reagent layer is 0.05 or more and 10 or less.

14. The content of the cured photosensitive polymer in the reagent layer is 0.2 μg / mm³. 2 50μg / mm or more 2 The biosensor according to claim 1, which is as follows:

15. A biosensor according to any one of claims 1 to 14, A control unit that controls the voltage applied to the biosensor, A detection unit that detects the current value obtained by applying voltage to the biosensor, A calculation unit that calculates the amino acid concentration of the solution being measured from the current value, An output unit that outputs the calculated amino acid concentration, A system for measuring the concentration of amino acids in a solution to be measured, comprising the following components.

16. The measurement system according to claim 15, wherein the solution to be measured is a urine sample collected from a test animal.

17. The measurement system according to claim 16, wherein the animal being tested is a feline.

18. A method for measuring the amino acid concentration in a solution to be measured, Supplying the solution to be measured to the biosensor of the measurement system according to claim 15; The control unit of the measurement system applies a constant voltage to the biosensor; The detection unit of the measurement system detects the current value obtained from the biosensor; The calculation unit of the measurement system calculates the amino acid concentration in the solution being measured from the detected current value. Methods that include...

19. The method according to claim 18, wherein the solution to be measured is a urine sample collected from a test animal.

20. The method according to claim 19, wherein the test subject animal is a feline.

21. A method for testing renal dysfunction in non-human test animals, The measurement method described in claim 18 is used to measure the amino acid concentration in a urine sample collected from a non-human animal subject for testing. Methods that include...

22. The method according to claim 21, wherein the non-human animal subject to the test is a feline.

23. An amino acid-related information provision service system that provides amino acid-related information on test animals, the system is Server and User terminal and Measuring device and Equipped with, The user terminal and the measuring device are each identified, The user terminal is associated with the measurement device. The measuring device comprises a biosensor according to any one of claims 1 to 14, measures data on the amino acid concentration of a urine sample collected from a test animal, and transmits the measured data, along with data on the timing of the measurement, to the server. The server creates amino acid-related information about the test animal based on the data transmitted from the measurement device, and transmits this information to the user terminal associated with the measurement device. The user terminal outputs amino acid-related information about the test animal transmitted from the server. The amino acid-related information for the test animal includes information on the urinary amino acid concentration of the test animal, or information on the renal function of the test animal prepared based on the urinary amino acid concentration. system.

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