Biosensor

Abstract

To provide a biosensor capable of more accurately measuring the concentration of amino acids in a sample solution.SOLUTION: The present disclosure, in one aspect, relates to a biosensor including an enzyme electrode including a conductive layer containing a conductive material and a reagent layer adjacent to the conductive layer, the reagent layer including an amino acid oxidase and a polymer-type enzyme immobilizer. The polymer-type enzyme immobilizer is preferably a hydrogel.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a biosensor for electrochemically analyzing a specific organic substance contained in a solution to be measured. [Background technology]

[0002] In recent years, biosensors have been used to detect and measure the concentration of specific components in test fluids such as urine for the diagnosis and prevention of diseases. Urine, in particular, is a convenient test fluid for measurement because it can be collected without injuring the subject's body. However, test fluids that can be measured noninvasively tend to have lower concentrations of organic substances than blood, and therefore, sensors with higher sensitivity are required. Enzyme sensors, which utilize the high selectivity of enzymes and can selectively detect organic substances with high sensitivity, are considered to be promising candidates for this type of sensor. It is also known that free amino acids in urine may be a potential biomarker for chronic kidney disease, a leading cause of death in dogs and cats.

[0003] Patent Document 1 discloses an electrode having one-dimensional conductive properties and an enzyme on the working surface as an electrode to be used in a bioelectrochemical measurement system, and as an example, discloses an electrode in which D-amino acid oxidase is incorporated onto a depression electrode filled with tetrathiafulvalene TCNQ (mediator) using a dialysis membrane. Patent Document 2 discloses a pollen sensor comprising a first membrane formed by immobilizing amino acid oxidase on a porous polyacrylonitrile film, a second membrane formed by immobilizing a protease on a similar porous film laminated on the first membrane, an electrode for detecting hydrogen peroxide produced in the first membrane, and a drip device for supplying water or an aqueous solution to wet the second membrane. In the first membrane, for example, amino acid oxidase is immobilized on a porous polyacrylonitrile film with glutaraldehyde. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 61-269059 [Patent Document 2] Japanese Patent Application Publication No. 4-348270 Summary of the Invention [Problem to be solved by the invention]

[0005] There is a demand for more accurate measurement of amino acid concentrations.

[0006] In one aspect, the present disclosure provides a biosensor capable of measuring the concentration of an amino acid in a solution to be measured with high accuracy. [Means for solving the problem]

[0007] The present disclosure provides, in one aspect, The enzyme electrode includes a conductive layer containing a conductive material and a reagent layer adjacent to the conductive layer, the reagent layer contains an amino acid oxidase and a polymeric enzyme immobilizing agent, The polymeric enzyme immobilization agent is a hydrogel. Related to biosensors.

[0008] The present disclosure provides, in one aspect, a biosensor of the present disclosure; a control unit that controls application of a voltage to the biosensor; a detection unit that detects a current value obtained by applying a voltage to the biosensor; a calculation unit that calculates the amino acid concentration of the solution to be measured from the current value; an output unit that outputs the calculated amino concentration; The present invention relates to a measuring device, including:

[0009] In one aspect, the present disclosure provides a method for measuring an amino acid concentration using the measurement device of the present disclosure, supplying a test solution containing a test substance to the biosensor; The control unit applies a constant voltage to the biosensor, The detection unit detects a current value obtained from the biosensor, calculating the concentration of the substance to be measured from the detected current value by the calculation unit; The present invention relates to a method for measuring an amino acid concentration, comprising:

[0010] In one aspect, the present disclosure provides a method for testing a subject animal for renal dysfunction, comprising: The present invention relates to a method for testing for renal dysfunction, which comprises a step of measuring the urinary amino acid concentration of the subject animal by the method for measuring amino acid concentration of the present disclosure.

[0011] In one aspect, the present disclosure provides an amino acid-related information providing service system that provides amino acid-related information regarding a subject animal, the system comprising: A server; A user terminal; a measuring device comprising a biosensor; Equipped with The biosensor of the measurement device comprises: The enzyme electrode includes a conductive layer containing a conductive material and a reagent layer adjacent to the conductive layer, the reagent layer contains an amino acid oxidase and a polymeric enzyme immobilizing agent, the polymeric enzyme immobilization agent is a hydrogel, the user terminal and the measuring device are each identified; The user terminal is associated with a measuring device; the measuring device measures data on the amino acid concentration of a urine sample collected from the subject animal and transmits the measured data together with data on the timing of the measurement to the server; The server creates amino acid concentration-related information for the subject animal based on the data transmitted from the measuring device, and transmits the information to a user terminal associated with the measuring device; the user terminal outputs the amino acid concentration-related information for the subject animal transmitted from the server; The amino acid-related information about the subject animal includes information about the urinary amino acid concentration of the subject animal, or information about the renal function of the subject animal created based on the urinary amino acid concentration. System, related. [Effects of the Invention]

[0012] According to the present disclosure, a biosensor capable of measuring the concentration of amino acids in a solution to be measured with high accuracy can be provided. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a plan view of a biosensor according to one embodiment of the present disclosure. [Figure 2] FIG. 2 is a cross-sectional view of the biosensor shown in FIG. [Figure 3] FIG. 3 is a schematic diagram showing the state in which a potentiostat is connected to the biosensor shown in FIG. [Figure 4] FIG. 4 is a block diagram showing the configuration of a measurement device according to one embodiment of the present disclosure. [Figure 5] FIG. 5 shows an embodiment of an amino acid-related information providing service system. [Figure 6] FIG. 6 shows an example of the operation of the server 100, the user terminal 200, and the measurement system 300 in the amino acid-related information provision service system. [Figure 7] FIG. 7 is a graph showing the relationship between the current value measured using the biosensor of Example 1 and the D-amino acid concentration. [Figure 8] FIG. 8 is a graph showing the relationship between the current value measured using the biosensor of Comparative Example 1 and the D-amino acid concentration. [Figure 9] FIG. 9 is a graph showing the relationship between the current value measured using the biosensor of Comparative Example 2 and the D-amino acid concentration. DETAILED DESCRIPTION OF THE INVENTION

[0014] In one aspect, the biosensor of the present disclosure is an enzyme sensor for measuring the concentration of a substance to be measured (hereinafter also referred to as a "substrate") in a solution (hereinafter also referred to as a "solution to be measured") containing the substance to be measured. The inventors of the present invention have found that by configuring the reagent layer to contain not only amino acid oxidase (enzyme) but also a polymeric enzyme immobilization agent, the physical protection provided by the polymeric enzyme immobilization agent prevents the enzyme from eluting into water, thereby suppressing the decrease in reaction efficiency caused by the elution of the enzyme into water, and as a result, the substrate can be measured with high accuracy.

[0015] An example of a biosensor according to the present disclosure will be described below with reference to Figures 1 and 2. However, in the present disclosure, there are no particular limitations on the shape or relative positions of the three electrodes in the electrode system, which include an enzyme electrode 2, a counter electrode 3, and a reference electrode 4 (standard electrode), and these may be the same as those of a conventionally known electrochemical biosensor. The electrode system may also be a two-electrode system, which includes only the enzyme electrode 2 and the reference electrode 4, excluding the counter electrode 3. However, a three-electrode system is preferable from the viewpoint of high-precision measurement, because the reference electrode potential changes when a current flows through the reference electrode.

[0016] In one embodiment, the biosensor of the present disclosure includes an insulating substrate 5 and an electrode system disposed on the insulating substrate 5. The insulating substrate 5 supports the electrodes and wiring. The electrode system includes an enzyme electrode 2, a counter electrode 3, a reference electrode 4, and wiring 21, 31, and 41. As can be clearly seen in FIG. 1 , the enzyme electrode 2, the counter electrode 3, and the reference electrode 4 are electrically connected to the wiring 21, 31, and 41, respectively. In a plan view of the biosensor 1, 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 enzyme electrode 2 includes a conductive layer 2a, a portion of which is formed in contact with one end 21a of the wiring 21, and a reagent layer 2b covering the surface of the conductive layer 2a. In the present disclosure, the conductive layer 2a is also referred to as a working electrode 2a. As shown in FIG. 2 , the reagent layer 2b is layered on the working electrode 2a formed on the insulating substrate 5. A conductive layer is also disposed on one end 31a, 41a of each of the wirings 31, 41, with a portion of the wirings 31, 41 in contact with the end. The conductive layers are the counter electrode 3 and the reference electrode 4. The counter electrode 3 is arc-shaped, surrounds the enzyme electrode 2, and is formed so as to maintain a substantially equal distance from the enzyme electrode 2 along the longitudinal direction of the arc. The enzyme electrode 2 is an electrode that transfers electrons to and from the substance to be measured, the counter electrode 3 is an electrode that passes a current between the enzyme electrode 2 and the counter electrode 3, and the reference electrode 4 is an electrode that serves as a reference for the potential of the enzyme electrode 2.

[0017] The solution to be measured may contain impurities other than the amino acids to be measured. To eliminate the influence of impurities on the measurement, the reagent layer 2b may be covered with a protective layer. Examples of reagents used for the protective layer include carbon resins, sulfonic acid resins such as Nafion, polyester resins, acrylic resins, urethane resins, epoxy resins, and polyolefin resins, and one or more selected from these may be used.

[0018] There is no particular limitation on the area of ​​the surface of the working electrode 2a facing the reagent layer 2b, but from the viewpoint of forming a reagent layer 2b with a sufficient amount of enzyme, it is preferably 0.1 mm 2 From the viewpoint of miniaturization of the biosensor, it is preferable to use a 2The thickness of the reagent layer 2b is preferably 100 nm or more, more preferably 500 nm or more, and even more preferably 1000 nm or more, from the viewpoint of retaining a sufficient amount of reagent for the electrochemical reaction. The thickness is preferably 1000 μm or less, more preferably 750 μm or less, and even more preferably 500 μm or less, from the viewpoint of preventing the reagent layer from inhibiting efficient electrochemical reaction on the conductive layer surface. There are no particular restrictions on the widths of the wiring 21, 31, and 41, and the widths of the counter electrode 3 and the reference electrode (standard electrode) 4, but from the viewpoint of ensuring both output stability and miniaturization of the biosensor, they are preferably 0.1 to 100 mm. There are no particular restrictions on the distance between adjacent wirings, the distance between the enzyme electrode 2 and the counter electrode 3, and the distance between the enzyme electrode 2 and the reference electrode 3, but each is preferably 0.1 mm or more and 100 mm or less. The thickness of the wiring 21, 31, and 41 and each electrode is preferably 10 nm or more and 100 μm or less.

[0019] A portion of the electrode system is covered with an insulating layer 6. In a system using three electrodes, the insulating layer 6 has an opening 61, through which the entire enzyme electrode 2, the counter electrode 3, and a portion of the reference electrode 4 are exposed. The opening 61 forms a reaction chamber and also serves as an inlet for the test solution. The other end of each wire 21, 31, or 41 is a connection site between the main body of the measurement device, which can perform a predetermined voltage sweep operation, and the biosensor 1. The measurement device, which includes the main body of the measurement device and the biosensor 1, measures the current generated by the enzyme reaction via the wires 21, 31, or 41 and calculates the presence or absence of the test substance in the test solution, or its content. Similarly, in a two-electrode electrode system, the entire enzyme electrode 2 and a portion of the reference electrode 4 are exposed from the opening 61, and the other end of each wire 21 or 41 is a connection site between the main body of the measurement device, which can perform a predetermined voltage sweep operation, and the biosensor 1.

[0020] As shown in Figure 2, a reagent layer 2b containing amino acid oxidase is immobilized on the working electrode 2a. The amino acids in the sample solution react with the amino acid oxidase in the reagent layer 2b and are decomposed into hydrogen peroxide and keto acid ammonia. The hydrogen peroxide is converted to HO → 2H at the enzyme electrode 2.+ +O2+2e - is converted into a current as follows:

[0021] The analyte of the biosensor of the present disclosure is an amino acid, preferably a D-amino acid. More specifically, the D-amino acid is one or more D-type free amino acids selected from the group consisting of 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-glycine, D-isoleucine, and D-proline. Reagent layer 2b contains an amino acid oxidase and a polymeric enzyme immobilization agent. Reagent layer 2b may further contain a stabilizer, a polymer, a surfactant, or other reagents useful for measuring the amino acid concentration. It may also contain a mediator (electron transfer promoter) to promote electron transfer.

[0022] The amino acid oxidase varies depending on the type of analyte, 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. The amount of amino acid oxidase in the reagent layer 2b is preferably 0.2 μg / mm 2 More than 1mU / mm 2 More preferably, 0.7 μg / mm 2 ≥ 3.6 mU / mm 2 More preferably, 2 μg / mm 2 and 10mU / mm 2 From the viewpoint of suppressing inhibition of the enzyme reaction due to the enzyme itself becoming an electrical resistance, the concentration is preferably 30 μg / mm 2 Less than or equal to 150mU / mm 2 Less than 25 μg / mm 2 Less than or equal to 125mU / mm 2 or less, more preferably 15 μg / mm 2 Less than or equal to 75mU / mm2 In this specification, the amount of enzyme in the reagent layer 2b as described above is the amount of enzyme in the area of ​​the reagent layer 2b that can come into contact with the test solution per mm 2 This refers to the amount of enzyme per unit volume.

[0023] From the viewpoint of effectively suppressing the elution of the enzyme into water, the polymeric enzyme immobilizing agent is preferably a hydrogel, more preferably a hydrogel having a structure with repeating units, and even more preferably a cationic polymer having an average hydroxyl group content per unit of 1.5 or more. From the viewpoint of effectively suppressing the elution of the enzyme into water, the average hydroxyl group content per unit is preferably 1.7 or more, even more preferably 1.9 or more, and from the viewpoint of highly accurate measurement of amino acid concentration, it is preferably 5.0 or less, more preferably 4.0 or less.

[0024] Specific examples of polymeric enzyme immobilizing agents include chitosan, chitosan methacrylate, glycol chitosan, glycol chitosan methacrylate, alginate, alginate methacrylate, hyaluronic acid (HA), and HA methacrylate, and one or more selected from these may be used. Among these, chitosan is particularly preferred.

[0025] The mass ratio of the enzyme to the polymeric enzyme immobilization agent in the reagent layer 2b (enzyme / polymeric enzyme immobilization agent) is preferably 0.25 or more, more preferably 0.30 or more, and even more preferably 0.50 or more, from the viewpoint of preventing the polymeric enzyme immobilization agent from becoming an inhibitor of the enzymatic reaction, and is preferably 20 or less, more preferably 15 or less, and even more preferably 10 or less, from the viewpoint of good retention of the enzyme in the reagent layer 2b.

[0026] The content of the polymeric enzyme immobilizing agent in the reagent layer 2b is preferably 0.2 μg / mm 2 More preferably, 0.7 μg / mm 2 More preferably, 2 μg / mm 2From the viewpoint of preventing the polymeric enzyme immobilization agent from becoming an inhibitor of the enzyme reaction, the concentration is preferably 30 μg / mm 2 Less than 25 μg / mm 2 or less, more preferably 10 μg / mm 2 In this specification, the content of the polymeric enzyme immobilizing agent in the reagent layer 2b is the amount of the polymeric enzyme immobilizing agent in the area of ​​the reagent layer 2b that can come into contact with the solution to be measured. 2 The reagent layer 2b containing the polymeric enzyme immobilization agent can be formed, for example, by dropping an aqueous solution containing amino acid oxidase and a polymeric enzyme immobilization agent onto the working electrode 2a and drying it under an atmosphere at room temperature.

[0027] The insulating substrate 5 is made of an insulating material to prevent short-circuiting of the three electrodes (or the two electrodes if no counter electrode is provided). There are no particular limitations on the material, and it may be the same as that of insulating substrates constituting known electrochemical biosensors. Examples of materials for the insulating substrate 5 include films made of materials such as polyimide, polystyrene, polycarbonate, polyvinyl chloride resin, or polyesters such as polyethylene terephthalate (PET), as well as paper, mica, and ceramics, and one or more materials selected from these may be used. The thickness of the insulating substrate 5 is also not particularly limited, but is preferably 25 to 1000 μm.

[0028] The working electrode 2a, counter electrode 3, reference electrode 4, and wirings 21, 31, and 41 are formed on the surface of the insulating substrate 5 by conventional methods such as screen printing, vacuum deposition, electron beam deposition, sputtering, plating, CVD, ion plating coating, and inkjet printing, depending on the material. The wirings 21, 31, and 41 are typically made of one or more conductive materials selected from the group consisting of metals such as gold, silver, palladium, platinum, rhodium, indium, and iridium, and conductive carbon materials, preferably silver. The working electrode 2a and counter electrode 3 are made of, for example, one or more conductive materials selected from the group consisting of conductive carbon materials, platinum, and gold, preferably conductive carbon materials. The material forming the working electrode 2a preferably contains a mediator to promote electron transfer. The reference electrode 4 is made of, for example, silver / silver chloride.

[0029] The mediator is not particularly limited as long as it is a redox substance capable of transferring electrons to the electrode, and conventionally known compounds capable of reversible oxidation and reduction can be used. Examples of mediators include Prussian blue, potassium ferricyanide (potassium ferrocyanide), ferrocene and its derivatives, osmium complexes and their derivatives (monomers and polymers), metal complexes and their derivatives, such as ruthenium complexes, quinones, phenazine methosulfate and its derivatives, dichloroindophenol, redox dyes, such as methylene blue and methylene green, tetrathiafulvalene (TTF) and its derivatives, and tetracyanoquinodimethane. One or more mediators can be used. 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 analyte to become the oxidized form of Prussian blue. This oxidized form accepts electrons from the conductive material of the working electrode 2a and returns to the reduced form, generating a current corresponding to the reaction between the enzyme and the analyte.

[0030] When a mediator is contained in at least one of the conductive layer (working electrode 2a) and the reagent layer 2b constituting the enzyme electrode 2, the content of the mediator in the enzyme electrode 2 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, from the viewpoint of promoting electron transfer, where the sum of the content of the conductive material contained in the conductive layer and the content of the mediator contained in the enzyme electrode 2 is taken as 100% by mass, and is 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 measurement due to excessive electron transfer. Furthermore, from the viewpoint of improving measurement accuracy, it is more preferable that the mediator be contained only in the conductive layer.

[0031] The insulating layer 6 is preferably made of silicon oxide, silicon nitride, aluminum oxide, or a photoresist that is transparent to visible light, and at least one selected from these may be used. The insulating layer 6 is formed by a conventionally known method, such as screen printing, vacuum deposition, electron beam deposition, sputtering, plating, CVD, ion plating coating, or inkjet printing. The thickness of the insulating layer 6 is preferably 10 nm or more and 100 μm or less.

[0032] Next, one embodiment of a measuring device of the present disclosure including the biosensor 1 of the present disclosure will be described with reference to the drawings. 4 is a block diagram showing the configuration of a measurement device according to one embodiment of the present disclosure. The measurement device 10 includes a biosensor 1 according to the present disclosure, a control unit 11 that controls the application of a voltage to the biosensor 1, a detection unit 12 that detects a current value obtained by applying a voltage to the biosensor 1, a calculation unit 13 that calculates the amino acid concentration of the measurement solution from the current value, and an output unit 14 that outputs the calculated amino acid concentration.

[0033] The control unit 11 is electrically connected to the biosensor 1 and controls the applied voltage value, voltage application time, etc. The detection unit 12 measures the current generated by the enzyme reaction in the enzyme electrode 2 and sends the measurement results to the calculation unit 13. The control unit 11 and the detection unit 12 may be a potentiostat equipped with these functions. The calculation unit 13 calculates and stores the concentration of the analyte from the detected current value. The calculation unit 13 may be, for example, an information processing terminal including a memory storing a linear function of the concentration of the analyte and the current value, and a central processing unit (CPU) including a calculation unit for calculating the absolute value of the concentration of the analyte. The output unit 14 transmits the calculation result of the analyte concentration by the calculation unit 13 to the display unit 15 via a communication interface. The communication interface may be wired or wireless, and examples of wireless communication interfaces include technologies such as Wi-Fi and Bluetooth (registered trademark). The display may be digital or analog.

[0034] Next, an amino acid concentration measurement method (hereinafter sometimes abbreviated as "the measurement method of the present disclosure") using the measurement device of the present disclosure will be described. In one embodiment, the measurement method of the present disclosure comprises: supplying a test solution containing a test substance to a biosensor of the present disclosure; The control unit applies a constant voltage to the biosensor, The detection unit detects a current value obtained from the biosensor, The calculation unit calculates the concentration of the substance to be measured from the detected current value.

[0035] In the following, a case where three electrodes are used will be described. In one embodiment, the measurement method of the present disclosure includes the following steps A to D. Step A: supplying a test solution containing a test substance to the reagent layer 2b, the counter electrode 3, and the reference electrode 4; Step B: applying a constant voltage to the biosensor 1 by the control unit 11; Step C: detecting a current obtained from the biosensor 1 in the detection unit 12; Step D: Calculating the concentration of the substance to be measured from the detected current value in the calculation unit 13.

[0036] In the above step A, the solution to be measured is supplied to the reagent layer 2 b, the counter electrode 3 and the reference electrode 4 through the opening 61 of the insulating layer 6 .

[0037] The above steps B to C are a method for measuring an electrical signal using an electrochemical technique called chronoamperometry (CA measurement). The enzyme electrode 2 and the counter electrode 3 are electrically connected, and when a predetermined voltage is applied between the enzyme electrode 2 and the counter electrode 3, the value of the current flowing between the enzyme electrode 2 and the counter electrode 3 is measured.

[0038] In step D, the current value is used to calculate the absolute value of the concentration of the substance to be measured corresponding to the current value, for example, from a linear function of the concentration of the substance to be measured and the current value, which is stored in advance in the measurement device.

[0039] In some embodiments of the present disclosure, the test solution is preferably mammalian urine, more preferably feline urine, and even more preferably cat urine. In some embodiments of the present disclosure, the test animal is preferably a mammal, more preferably a feline, and even more preferably a cat.

[0040] The present disclosure further relates to a method for testing a subject animal for renal dysfunction, comprising measuring the amino acid concentration in the subject animal's urine, which is a test solution, by the amino acid concentration measurement method of the present disclosure. A cat urine with a total D-amino acid concentration of approximately 400 μM or less is considered to be suspected of having a renal disease. According to the present disclosure, the amino acid concentration in the test solution can be measured with high accuracy even in the low concentration range (1000 μM or less), and therefore the present disclosure can be used to easily determine renal disease in cats.

[0041] The present application further discloses a method for testing the presence or absence of renal disease in a subject animal by measuring the urinary amino acid concentration of the subject animal using the above-mentioned measuring device. Here, the present application will be described by taking the case where cat urine is used as the subject animal urine and a three-electrode biosensor is used.

[0042] In one embodiment, the above-described inspection method of the present disclosure includes the following steps A to E. Step A: Supplying cat urine to the reagent layer 2b, the counter electrode 3, and the reference electrode 4; Step B: applying a constant voltage to the biosensor 1 by the control unit 11; Step C: detecting a current value obtained from the biosensor 1 by the detection unit 12; Step D: calculating the concentration of urinary amino acids from the detected current value in the calculation unit 13; Step E: Examining the presence or absence of renal dysfunction in the cat based on the calculated concentration of the urinary amino acids.

[0043] As an application example of the biosensor of the present disclosure, an amino acid-related information providing service system (hereinafter also referred to as the "service system of the present disclosure") will be described. The service system of the present disclosure measures the amino acid concentration in a urine sample (measured solution) collected from a subject animal, and provides amino acid-related information about the subject animal to a user of the service system.

[0044] The amino acid-related information on the subject animal provided by the service system of the present disclosure includes the urinary amino acid concentration of the subject animal, information on the renal function of the subject animal created based on the urinary amino acid concentration, etc. The information on renal function includes the degree of renal function of the subject animal, information for preventing renal disease corresponding to the degree of renal function (including information on pet foods and lifestyle habits effective for prevention), information on the risk and possibility of renal disease, information on the presence or severity of renal disease, information on the treatment of renal disease (including information on treatment methods, therapeutic drugs, veterinarians, hospital locations, etc.), etc.

[0045] A user of the service system of the present disclosure is a person who wishes to receive amino acid-related information about a subject animal. Examples of such users include those who raise or manage the subject animals, such as the subject animal's owner, a veterinarian, or an employee of an animal care facility such as a zoo (keeper, veterinarian, or facility manager).

[0046] In an exemplary embodiment, the service system of the present disclosure includes a server, a user terminal, and a measurement device. Each user registered in the service system of the present disclosure uses at least one user terminal and at least one measurement device.

[0047] Each user terminal and each measuring device is identified. For example, each user terminal and each measuring device can be assigned an ID number and managed. Registered users can also be identified and managed by ID numbers, etc. Each user terminal and each measuring device is associated with one of the registered users. Furthermore, 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).

[0048] One user may use one measurement device and one user terminal, or one 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 can accept is not particularly limited and can be changed depending on, for example, the performance of the server.

[0049] The measuring device includes the biosensor of the present disclosure and is capable of measuring 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.

[0050] The server creates amino acid-related information for the subject animal based on the data transmitted from the measuring device and transmits the information to a user terminal associated with the measuring device, and the user terminal outputs the amino acid concentration-related information for the subject animal transmitted from the server.

[0051] Figure 5 shows one embodiment of the configuration of the service system of the present disclosure. The system shown in Figure 5 includes a server 100, a user terminal 200, and a measuring device 300 including a biosensor of the present disclosure. The server 100 is connected to a plurality of user terminals 200 and a plurality of measuring devices 300, and is capable of transmitting and receiving data as needed. In Figure 5, this connection is via the Internet 50, but is not limited to this.

[0052] 5 shows a plurality of users A, B, and C who use the service system of the present disclosure, and each of them keeps a test animal (e.g., a cat). Users A, B, and C each have 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 server 100.

[0053] The server 100 is an information processing device managed by the service operator. The server 100 receives data measured by the measuring device 300, and creates amino acid-related information about the subject animal, such as urinary amino acid concentrations and information about renal function, from the received data, and transmits the information to the user terminal 200.

[0054] The server 100 may include a communication interface for sending and receiving data to and from the user terminal 200 or the measuring device 300, a calculation unit for creating amino acid-related information about the subject animal, such as information about urinary amino acid concentrations and renal function, from data received from the measuring device 300, and a control unit for controlling the operation of the communication interface and calculation unit.

[0055] Furthermore, the server 100 may include a memory unit for storing information necessary for the calculations performed by the calculation unit or the calculation results. Alternatively, the memory unit may be installed outside the server 100. The memory unit may store various applications for executing the amino acid-related information provision service system according to the present disclosure, amino acid-related information about test subjects created by the server 100, and various databases used to create the amino acid-related information. Examples of such databases include, but are not limited to, a user information database that stores attribute information about users, test subjects, and measurement devices owned by each user; a renal function information database that stores information relating to the association between amino acid concentrations and renal function, and information on maintaining renal function and preventing or treating renal disease; and an amino acid-related information database that stores amino acid-related information about test subjects for each user. These databases may be mutually referenced as necessary when the server 100 creates the amino acid-related information.

[0056] In the embodiment shown in FIG. 5, only one server 100 is shown, but the server 100 may be configured from a plurality of information processing devices, and the processing executed by the server 100 may be distributed and executed by the plurality of information processing devices.

[0057] The user terminals 200 (200A, 200B, 200C, etc.) receive and output amino acid-related information related to the subject animal from the server 100. The output format is not particularly limited, and may include screen display, printing, recording in a database, etc. In a preferred embodiment, an application program (hereinafter also referred to as an app) compatible with the amino acid-related information provision service system of the present disclosure is installed in the user terminal 200, and the user terminal 200 may output the amino acid-related information provided from the server 100 via the app. The user terminal 200 may be, for example, an information processing terminal such as a smartphone, mobile phone, tablet PC (Personal Computer), notebook PC, or desktop PC, on which the necessary app is installed.

[0058] The measuring device 300 (300A, 300B, 300C, etc.) is equipped with the biosensor of the present disclosure 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 be equipped with a biosensor 1 as shown in Figures 3 and 4, a control unit 11, a detection unit 12, and, if necessary, a calculation unit 13. In one embodiment, data on the current value that reflects 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 data on the calculated amino acid concentration is sent to the server 100 via the Internet 50.

[0059] In a preferred embodiment, the measuring device 300 acquires data on the timing (e.g., the date and time of measurement) at which data on the amino acid concentration in the urine sample was measured, and sends the data on the timing of measurement to the server 100 together with the data on the amino acid concentration.

[0060] 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 the communication unit. The measuring device 300 may be directly connected to the server 100 via the Internet 50, as shown in Fig. 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.

[0061] An example of the operation of the server 100, user terminal 200, and measuring device 300 in the service system of the present disclosure will be described with reference to Figure 6. As a premise for the processing of this operation example, it is assumed that each user possesses a user terminal 200 and a measuring device 300 for a test animal. The measuring device 300 is, for example, loaned or transferred from the operator of the amino acid-related information providing service. Each user terminal and measuring device, or the user and test animal using them, are ID-registered and managed on the server 100 (in this operation example, the identification information of the measuring device is hereinafter referred to as "sensor ID").

[0062] The user collects urine from a 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 together with data on the measurement timing to the server 100 (S32). The server 100 receives the amino acid concentration data associated with the measurement timing and the sensor ID (S11) and stores this information (S12).

[0063] Next, the server 100 determines whether or not a plurality of amino acid concentration data items associated with the same sensor ID and different measurement times are stored (S13). If a plurality of amino acid concentration data items associated with different measurement times are stored (Yes in S13), the server 100 generates amino acid-related information including renal function level information indicating the level of renal function of the subject animal over time (S14).

[0064] On the other hand, if multiple amino acid concentration data corresponding to 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 that indicates the correspondence between the degree of renal function of the subject animal and a single measurement timing, as in S14 (S15).

[0065] Next, the server 100 transmits 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).

[0066] As described above, in the amino acid-related information providing service system of the present disclosure, in addition to being able to accurately measure urinary amino acid concentrations using the measuring device 300, the server 100 can also generate and provide to the user the above-mentioned information on the degree of renal function based on the amino acid concentration information accumulated therein. This allows even users without laboratory equipment or testing skills to easily obtain information on the urinary amino acid concentrations and renal function of test animals.

[0067] Exemplary, non-limiting aspects of the present disclosure are disclosed below.

[0068] <1> The enzyme electrode includes a conductive layer containing a conductive material and a reagent layer adjacent to the conductive layer, the reagent layer contains an amino acid oxidase and a polymeric enzyme immobilizing agent, The polymeric enzyme immobilization agent is a hydrogel. Biosensor. <2> Preferably, the hydrogel is a cationic polymer comprising the repeating unit <1> The biosensor described. <3> The average hydroxy group content per unit of the cationic polymer is preferably 1.5 or more, more preferably 1.7 or more, even more preferably 1.9 or more, and is preferably 5.0 or less, more preferably 4.0 or less. <2> The biosensor described. <4> The polymeric enzyme immobilizing agent is preferably one or more selected from the group consisting of chitosan, chitosan methacrylate, glycol chitosan, glycol chitosan methacrylate, alginate, alginate methacrylate, hyaluronic acid (HA), and HA methacrylate. <1> ~ <3> The biosensor according to any one of the preceding claims. <5> the mass ratio of the amino acid oxidase to the polymeric enzyme immobilization agent in the reagent layer (amino acid oxidase / polymeric enzyme immobilization agent) is preferably 0.25 or more, more preferably 0.30 or more, even more preferably 0.50 or more, and is preferably 20 or less, more preferably 15 or less, even more preferably 10 or less; <1> ~ <4> The biosensor according to any one of the preceding claims. <6> The content of the polymeric enzyme immobilizing agent in the reagent layer is preferably 0.2 μg / mm 2 More preferably, 0.7 μg / mm 2 More preferably, 2 μg / mm 2 or more, and preferably 30 μg / mm 2 Less than 25 μg / mm 2 or less, more preferably 10 μg / mm 2 Below is the <1> ~ <5> The biosensor according to any one of the preceding claims. <7> The substance to be measured by the biosensor is Preferably, it is an amino acid, More preferably, the amino acid oxidase is one or more selected from the group consisting of 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-glycine, D-isoleucine, and D-proline, and the amino acid oxidase is a D-amino acid oxidase. <1> ~ <6> The biosensor according to any one of the preceding claims. <8> The amount of the amino acid oxidase in the reagent layer is preferably 0.2 μg / mm 2 More than 1mU / mm 2 More preferably, 0.7 μg / mm 2 ≥ 3.6 mU / mm 2 More preferably, 2 μg / mm 2 and 10mU / mm 2 or more, and preferably 30 μg / mm 2Less than or equal to 150mU / mm 2 Less than 25 μg / mm 2 Less than or equal to 125mU / mm 2 or less, more preferably 15 μg / mm 2 Less than or equal to 75mU / mm 2 Below is the <1> ~ <7> The biosensor according to any one of the preceding claims. <9> Preferably, the device comprises an insulating substrate and an electrode system disposed on the insulating substrate; the electrode system includes an enzyme electrode; The enzyme electrode includes the conductive layer and the reagent layer, and Preferably, the reagent layer is laminated on the conductive layer. <1> ~ <8> The biosensor according to any one of the preceding claims. <10> Preferably, the material of the insulating substrate is a film made of one or more materials selected from the group consisting of polyimide, polystyrene, polycarbonate, polyvinyl chloride resin, and polyester, paper, mica, or ceramics. <9> The biosensor described. <11> Preferably, the electrode system includes the enzyme electrode, a counter electrode, a reference electrode, and wiring, and Preferably, the working electrode, the counter electrode, the reference electrode, and the wiring of the enzyme electrode are formed on the surface of the insulating substrate by screen printing, vacuum deposition, electron beam deposition, sputtering, plating, CVD, ion plating coating, or inkjet printing. <9> or <10> The biosensor described. <12> Preferably, the working electrode, the counter electrode, and the wiring are made of one or more conductive materials selected from the group consisting of metals and conductive carbon materials, and The metal is preferably one or more selected from the group consisting of gold, silver, palladium, platinum, rhodium, indium, and iridium. <9> ~ <11> The biosensor according to any one of the preceding claims. <13> Preferably, the reference electrode is made of silver / silver chloride. <11> or <12> The biosensor described. <14> Preferably, the enzyme electrode further contains a mediator. <1> ~ <13> The biosensor according to any one of the preceding claims. <15> Preferably, the conductive layer further contains a mediator. <14> The biosensor described. <16> Preferably, the mediator is at least one selected from the group consisting of Prussian blue, potassium ferricyanide (potassium ferrocyanide), ferrocene and its derivatives, osmium complexes and their derivatives (monomers and polymers), ruthenium complexes, quinones, phenazine methosulfate and its derivatives, dichloroindophenol, methylene blue, methylene green, tetrathiafulvalene (TTF) and its derivatives, and tetracyanoquinodimethane. <14> or <15> The biosensor described. <17> The content of the mediator in the enzyme electrode 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 is preferably 10% by mass or less, more preferably 7.5% by mass or less, even more preferably 5.0% by mass or less, when the total of the content of the conductive material contained in the working electrode and the content of the mediator contained in the enzyme electrode is 100% by mass. <14> ~ <16> The biosensor according to any one of the preceding claims. <18> A portion of the electrode system is covered with an insulating layer. <9> ~ <17> The biosensor according to any one of the preceding claims. <19> Preferably, the material forming the insulating layer is one or more selected from the group consisting of silicon oxide, silicon nitride, aluminum oxide, and photoresist that is transparent to visible light. <18> The biosensor described. <20> Preferably, the thickness of the insulating layer is 10 nm or more and 100 μm or less. <18> or <19> The biosensor described.

[0069] <21> <1> ~ <20> The biosensor according to any one of the above items, a control unit that controls application of a voltage to the biosensor; a detection unit that detects a current value obtained by applying a voltage to the biosensor; a calculation unit that calculates the amino acid concentration of the solution to be measured from the current value; an output unit that outputs the calculated amino concentration; a measuring device comprising: <22> Preferably, the control unit and the detection unit are potentiostats. <21> The measuring device described. <23> Preferably, the calculation unit is an information processing terminal including a memory in which a linear function of the amino acid concentration and the current value is stored, and a central processing unit (CPU) including a calculation unit for calculating the absolute value of the amino acid concentration. <21> or <22> The measuring device described. <24> Preferably, the output unit transmits the calculation result of the amino acid concentration by the calculation unit 13 to a display unit through a communication interface, and Preferably, the communication interface is wired or wireless. <21> ~ <23> 10. The measuring device according to claim 9, <25> Preferably, the display is a digital display or an analog display. <24> The measuring device described.

[0070] <26> <21> ~ <25> A method for measuring an amino acid concentration using the measurement device according to any one of the above, comprising: <1> ~ <20> supplying a test solution containing an amino acid to the biosensor according to any one of the preceding claims; applying a constant voltage to the biosensor by the control unit; detecting a current value obtained from the biosensor by the detection unit; calculating the amino acid concentration from the detected current value by the calculation unit; A method comprising: <27> Preferably, the method includes the following steps A to D: <26> How to write: Step A: supplying a test solution containing an amino acid to the reagent layer, the counter electrode, and the reference electrode; Step B: applying a constant voltage to the biosensor using the control unit; Step C: detecting a current obtained from the biosensor in the detection unit; Step D: Calculating the amino acid concentration from the detected current value in the calculation unit. <28> Preferably, steps B to C are measurements of electrical signals by chronoamperometry (CA measurement). <27> The method described. <29> The test solution is preferably mammalian urine, more preferably feline urine. <26> ~ <28> 10. The measuring device according to claim 9,

[0071] <30> A method for testing renal dysfunction in a subject animal, comprising: <26> ~ <28> measuring the urinary amino acid concentration of the subject animal by the method for measuring an amino acid concentration according to any one of the above. method. <31> Preferably, the subject animal urine is cat urine. <30> The method described. <32> Preferably, the method includes the following steps A to E: <31> How to write: Step A: Supplying cat urine to the reagent layer, the counter electrode, and the reference electrode; Step B: applying a constant voltage to the biosensor using the control unit; Step C: detecting a current value obtained from the biosensor with the detection unit; Step D: calculating the concentration of urinary amino acids from the detected current value by the calculation unit; Step E: Examining the presence or absence of renal dysfunction in the cat based on the calculated concentration of the urinary amino acids.

[0072] <33> An amino acid-related information providing service system that provides amino acid-related information related to a subject animal, the system comprising: A server; A user terminal; a measuring device comprising a biosensor; Equipped with The biosensor of the measurement device comprises: The enzyme electrode includes a conductive layer containing a conductive material and a reagent layer adjacent to the conductive layer, the reagent layer contains an amino acid oxidase and a polymeric enzyme immobilizing agent, the polymeric enzyme immobilization agent is a hydrogel, the user terminal and the measuring device are each identified; The user terminal is associated with a measuring device; the measuring device measures data on the amino acid concentration of a urine sample collected from the subject animal and transmits the measured data together with data on the timing of the measurement to the server; The server creates amino acid concentration-related information for the subject animal based on the data transmitted from the measuring device, and transmits the information to a user terminal associated with the measuring device; the user terminal outputs the amino acid concentration-related information for the subject animal transmitted from the server; The amino acid-related information about the subject animal includes information about the urinary amino acid concentration of the subject animal, or information about the renal function of the subject animal created based on the urinary amino acid concentration. system. <34> An amino acid-related information providing service system that provides amino acid-related information related to a subject animal, the system comprising: A server; A user terminal; a measuring device; Equipped with the user terminal and the measuring device are each identified; The user terminal is associated with a measuring device; The measuring device comprises: <1> ~ <20> a method for measuring amino acid concentrations in a urine sample collected from a subject animal, and transmitting the measured data together with data on the timing of the measurement to the server; The server creates amino acid concentration-related information for the subject animal based on the data transmitted from the measuring device, and transmits the information to a user terminal associated with the measuring device; the user terminal outputs the amino acid concentration-related information for the subject animal transmitted from the server; The amino acid-related information about the subject animal includes information about the urinary amino acid concentration of the subject animal, or information about the renal function of the subject animal created based on the urinary amino acid concentration. system. [Example]

[0073] The present disclosure will be described in more detail below with reference to examples, but these are merely illustrative examples and the present disclosure is not limited to these examples.

[0074] Example 1 [Fabrication of the electrode system] The electrode system shown in Figures 1 and 2 was fabricated on an insulating substrate (As One Corporation, polyimide film, model number: HJA-A4-225 μm, thickness: 225 μm). Specifically, silver paste (Sun Chemical Corporation, product number C2080415P2) was screen-printed on the insulating substrate 5 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, a wire with a diameter of 3.5 mm (area of ​​9.6 mm) was placed on one end of the wiring 21 so that it partially overlapped the wire. 2 Specifically, a Prussian blue-containing carbon paste (manufactured by SUN CHEMICAL Corporation, 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 wt% of Prussian blue in the conductive layer. Next, a carbon paste (manufactured by SUN CHEMICAL Corporation, product number C2030519P4) was screen-printed through a screen mask onto one end of the wiring 31 so that it partially overlapped, and then dried at 80°C for 10 minutes to form a counter electrode 3. Next, a silver / silver chloride paste (manufactured by SUN CHEMICAL Corporation, product number C2130809D5) was screen-printed through a screen mask onto one end of the reference electrode wiring 41 so that it partially overlapped, and then sintered at 80°C for 10 minutes to form the reference electrode 4. Finally, insulating paste (manufactured by SUN CHEMICAL, product number D2070423D5) was screen-printed through a screen mask so as to cover parts of wiring 21, wiring 31, and wiring 41, and then dried at 130°C for 10 minutes to form insulating layer 6. The silver paste, Prussian blue-containing carbon paste, carbon paste, silver / silver chloride paste, and insulating paste were each applied by screen printing so that the thickness after firing or drying would be 10 μm.

[0075] [Enzyme modification] Next, a PBS solution (0.1 mol / L phosphate-buffered saline, pH = 8.0, Fujifilm Wako Pure Chemical Industries, Ltd.) containing 100 U / mL (20 mg / mL) of D-amino acid oxidase (enzyme, SIGMA-Aldrich, D-Amino Acid Oxidase from porcine kidney) was mixed with a chitosan-containing acetic acid solution (prepared by dissolving acetic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) in ion-exchanged water at a concentration of 2% by weight and adding chitosan (SIGMA-Aldrich, chitosan low molecular weight) to the acetic acid solution at a concentration of 2% by weight) at a volume ratio of 1:1. Next, 7 μL of the resulting mixed solution was dropped onto a working electrode 2a made of Prussian blue-containing carbon paste and dried at 25 °C for 24 hours to obtain an enzyme electrode 2 including a reagent layer 2b and a working electrode 2a. The D-amino acid oxidase and chitosan contents in the reagent layer 2b were both 7.3 μg / mm 2 and the mass ratio (D-amino acid oxidase / chitosan) is 1.

[0076] When the prepared enzyme electrode 2 is immersed in a solution of any D-amino acid, the redox substance (Prussian blue) is oxidized by hydrogen peroxide produced by the enzyme reaction.

[0077] [Method for measuring substrate concentration] The following experiment was carried out to obtain a correlation equation between the D-amino acid concentration and the steady-state current value of the enzyme electrode 2. D-alanine was used as the D-amino acid. As shown in Figure 3, the enzyme electrode wire 21, reference electrode wire 41, and counter electrode wire 31 were connected to potentiostat 8, and each electrode was immersed in 10 mL of the PBS solution (measurement solution, pH = 8.0, 25°C) containing D-alanine (manufactured by Peptide Institute). Next, chronoamperometry (CA) measurement was performed by applying a constant potential of -0.20 V to enzyme electrode 2 using potentiostat 8, and the steady-state current value of enzyme electrode 2 (150 seconds after the start of measurement) was measured. The concentrations of D-alanine in the measurement solution were 100 µM, 200 µM, 400 µM, or 800 µM.

[0078] As shown in Figure 7, the steady-state current value increased as the D-alanine concentration in the test solution increased, confirming a positive linear relationship between the D-alanine concentration and the steady-state current value. This indicates that the D-alanine acid concentration in the test solution 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 given by the following equation (1): In equation (1), y is the quantitative current value (A) and x is the D-alanine concentration (μM). y=-1.79E-09x+2.45E-08 (1) In addition, the correlation coefficient R in the low concentration range of D-alanine below 1000 μM 2 was 0.997, a value very close to 1. When the concentration of D-alanine in the measurement solution was 0 to 800 μM, the current value per unit mass of D-alanine was −1.79 nA / μM. These results confirmed that the use of the biosensor of Example 1 makes it possible to measure the concentration of amino acids such as D-alanine with sufficient accuracy even in the low concentration range, and that the output relative to the concentration of the substance to be measured is also large.

[0079] (Comparative Example 1) [Fabrication of the electrode system] [Fabrication of electrode system] was carried out in the same manner as in Example 1. [Enzyme modification] An enzyme electrode was obtained in the same manner as in [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 was dropped onto the working electrode 2a instead of the mixed solution used in [Enzyme Modification] of Example 1.

[0080] The method for measuring the substrate concentration was carried out in the same manner as in Example 1. As can be seen from a comparison between Figures 7 and 8, the output relative to the concentration of D-alanine was smaller in Comparative Example 1 than in Example 1. In Comparative Example 1, the current value per unit mass of D-alanine when the concentration of D-alanine in the measurement solution was 0 to 800 µM was -0.0548 nA / µM. In addition, the correlation coefficient R in the low concentration range of D-alanine below 1000 μM 2 was 0.909, which was a lower value than that of Example 1.

[0081] (Comparative Example 2) [Fabrication of the electrode system] [Fabrication of electrode system] was carried out in the same manner as in Example 1. [Enzyme modification] An enzyme electrode 2 was obtained in the same manner as in [Enzyme modification] of Example 1, except that instead of the mixed solution used in [Enzyme modification] of Example 1, 7 μL of a PBS solution (pH = 8.0) containing D-amino acid oxidase and glutaraldehyde (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), with a D-amino acid oxidase content of 50 U / mL (10 mg / mL) and a glutaraldehyde content of 1 mass %, was dropped onto the working electrode 2a.

[0082] The method for measuring the substrate concentration was carried out in the same manner as in Example 1. As can be seen from a comparison between Figures 7 and 9, the output relative to the concentration of D-alanine was smaller in Comparative Example 2 than in Example 1. In Comparative Example 2, the current value per unit mass of D-alanine when the concentration of D-alanine in the measurement solution was 0 to 800 µM was -0.128 nA / µM. In addition, the correlation coefficient R in the low concentration range of D-alanine below 1000 μM 2 was 0.840, which was a lower value than that of Example 1.

[0083] Example 2 [Electrode preparation] [Enzyme modification] [Preparation of electrode system] and [enzyme modification] were carried out in the same manner as in Example 1. [Method for measuring substrate concentration] After connecting the enzyme electrode wire 21, the reference electrode wire 41, and the counter electrode wire 31 to the potentiostat 8, each electrode was immersed in the solution to be measured as shown in Fig. 3. Next, a chronoamperometry (CA) measurement was performed in which a constant potential of -0.20 V was applied to the enzyme electrode 2 by the potentiostat 8, and the steady-state current value of the enzyme electrode 2 (150 seconds after the start of the measurement) was measured. The test solution was buffered to pH 8.0 (25°C) by adding 93.6 μL of 1 M sodium hydroxide (Sigma-Aldrich) and 13.6 mg of potassium dihydrogen phosphate (Fujifilm Wako Pure Chemical Industries, Ltd.) to 10 mL of actual cat urine. Three samples of actual cat urine were prepared: two samples from healthy cats and one sample from a cat with renal disease.

[0084] [Measurement results] The steady-state current values ​​obtained in this example were substituted into the linear correlation equation (1) to calculate the D-alanine concentration in cat urine, which is shown as a sensor-equivalent value in Table 1. Table 1 also shows the D-amino acid concentration measured in the same test solution using LC-MS (liquid chromatography).

[0085] [Table 1]

[0086] The steady-state current values ​​obtained in Example 2 were substituted into the linear correlation equation (1) to obtain the D-alanine concentrations of 608 μM and 600 μM for the urine of healthy cats, and 308 μM for the urine of cats with renal disease. Furthermore, in both the urine of healthy cats and the urine of cats with renal disease, the D-alanine concentration (sensor-equivalent value) calculated from the linear correlation function (1) had an error of within 10% from the D-alanine concentration (LC-MS measurement value) obtained by LC-MS measurement, demonstrating that the biosensor of this example can distinguish between urine of healthy cats and urine of cats with renal disease. [Industrial Applicability]

[0087] The biosensor disclosed herein can quantify D-amino acids in a test solution, and can therefore be used to diagnose kidney disease by measuring amino acids in cat urine, as well as to diagnose dementia by measuring amino acids in human blood, and to measure umami components and functional components by measuring amino acids in food and beverages. [Explanation of symbols]

[0088] 1. Biosensor 2. Enzyme electrode 2a conductive layer 2b Reagent layer 3. Opposite 4 Reference pole 21, 31, 41 Wiring 5. Insulating substrate 6 insulating layer 61 Aperture 8. Potentiostat 10. Measuring equipment 11 Control section 12 Detector 13 Arithmetic section 14 Output section 15 Display

Claims

1. The enzyme electrode includes a conductive layer containing a conductive material and a reagent layer adjacent to the conductive layer, the reagent layer contains an amino acid oxidase and a polymeric enzyme immobilizing agent, The polymeric enzyme immobilization agent is a hydrogel. Biosensor.

2. The biosensor of claim 1 , wherein the hydrogel is a cationic polymer containing repeating units, and the average hydroxyl group content per repeating unit is 1.5 or more.

3. The biosensor of claim 2 , wherein the cationic polymer is chitosan.

4. 2. The biosensor according to claim 1, wherein the enzyme electrode further contains a mediator.

5. The biosensor of claim 4 , wherein the mediator is Prussian blue.

6. The biosensor of claim 1 , wherein the reagent layer is laminated on the conductive layer.

7. The biosensor according to claim 1, wherein the amino acid oxidase is a D-amino acid oxidase.

8. 2. The biosensor according to claim 1, wherein the mass ratio of the amino acid oxidase to the polymeric enzyme immobilization agent is 0.25 or more and 20 or less.

9. The content of the polymeric enzyme immobilization agent in the reagent layer is 0.2 μg / mm ２ 30μg / mm or more ２ 2. The biosensor of claim 1, wherein:

10. The biosensor according to any one of claims 1 to 9, a control unit that controls application of a voltage to the biosensor; a detection unit that detects a current value obtained by applying a voltage to the biosensor; a calculation unit that calculates the amino acid concentration of the solution to be measured from the current value; an output unit that outputs the calculated amino concentration; a measuring device comprising:

11. 11. The measuring device according to claim 10, wherein the solution to be measured is mammalian urine.

12. A method for measuring an amino acid concentration using the measurement device according to claim 10, supplying a test solution containing a test substance to the biosensor; The control unit applies a constant voltage to the biosensor, The detection unit detects a current value obtained from the biosensor, calculating the concentration of the substance to be measured from the detected current value in the calculation unit.

13. A method for testing a subject animal for renal dysfunction, comprising a step of measuring a urinary amino acid concentration in the subject animal by the method for measuring an amino acid concentration according to claim 12.

14. An amino acid-related information providing service system that provides amino acid-related information related to a subject animal, the system comprising: A server; A user terminal; a measuring device comprising a biosensor; Equipped with The biosensor of the measurement device comprises: The enzyme electrode includes a conductive layer containing a conductive material and a reagent layer adjacent to the conductive layer, the reagent layer contains an amino acid oxidase and a polymeric enzyme immobilizing agent, the polymeric enzyme immobilization agent is a hydrogel, the user terminal and the measuring device are each identified; The user terminal is associated with a measuring device; the measuring device measures data on the amino acid concentration of a urine sample collected from the subject animal and transmits the measured data together with data on the timing of the measurement to the server; The server creates amino acid concentration-related information for the subject animal based on the data transmitted from the measuring device, and transmits the information to a user terminal associated with the measuring device; the user terminal outputs the amino acid concentration-related information for the subject animal transmitted from the server; The amino acid-related information about the subject animal includes information about the urinary amino acid concentration of the subject animal, or information about the renal function of the subject animal created based on the urinary amino acid concentration. system.

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