Biosensor

Abstract

A biosensor comprising an enzyme electrode that includes a conductive layer and a reagent layer located adjacent to the conductive layer, wherein the conducive layer contains a mediator, and the reagent layer contains an amino acid oxidase and an anionic compound.

Description

Biosensor

[0001] The present invention relates to a biosensor for electrochemically analyzing amino acids contained in a measurement target solution.

[0002] In recent years, there has been an increasing need to selectively detect organic substances in biological fluids such as sweat, saliva, urine, tears, and blood in order to monitor the body's condition or for the diagnosis and prevention of diseases. Among these, non-invasive measurements that do not involve procedures that damage the body, such as blood collection, have attracted attention. Urine is useful as a biological fluid for measurement because it can be easily collected without damaging the body. For example, it is known that free amino acids in urine may be biomarkers for chronic kidney disease, which is among the leading causes of death in dogs and cats. However, biological fluids that can be measured non-invasively tend to have lower organic substance concentrations than blood, and therefore, more sensitive sensors are required. As such a sensitive sensor, an enzyme sensor that utilizes the high selectivity of an enzyme and can selectively detect organic substances with high sensitivity is promising.

[0003] Patent Document 1 discloses a biosensor including a hydrogen peroxide electrode and a hydrogen peroxide selective permeation membrane and an enzyme membrane laminated thereon, wherein the hydrogen peroxide selective permeation membrane is a composite membrane composed of an electrolytic polymerization polymer and a non-electrolytic polymerization polymer. Patent Document 2 discloses a biosensor including a conductive support bonded to a hydrogel, wherein the hydrogel contains alginate and includes an electron mediator, a reducing agent, and a metabolic enzyme.

[0004] Patent Document 3 discloses a method for measuring glycated protein concentration, including 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 a current value between the electrode on which the enzyme is immobilized and a predetermined detection electrode. As the water-soluble photocurable resin, a photocurable polyvinyl alcohol resin having an azide group as a photosensitive group is exemplified.

[0005] (Patent Document 1) JP-A-2004-163386 (Patent Document 2) JP-T-2015-535077 (Patent Document 3) JP-A-2009-171874

[0006] In one embodiment, the present invention provides 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 an anionic compound.

[0007] In another embodiment, the present invention provides a system for measuring the amino acid concentration in a solution to be measured, comprising: a biosensor; a control unit for controlling the application of voltage to the biosensor; a detection unit for detecting a current value obtained by the application of voltage to the biosensor; a calculation unit for calculating the amino acid concentration of the solution to be measured from the current value; and an output unit for outputting the calculated amino acid concentration.

[0008] In another embodiment, the present invention provides a method for measuring the amino acid concentration in a solution to be measured, comprising: supplying the solution to be measured to a biosensor of a system for measuring the amino acid concentration in the solution to be measured; applying a constant voltage to the biosensor with a control unit of the measurement system; detecting a current value obtained from the biosensor with a detection unit of the measurement system; and calculating the amino acid concentration in the solution to be measured from the detected current value with a calculation unit of the measurement system.

[0009] In another embodiment, the present invention provides a method for testing renal dysfunction in a non-human test animal, comprising: measuring the amino acid concentration in a urine sample collected from the non-human test animal by a method for measuring the amino acid concentration in a solution to be measured.

[0010] In another embodiment, the present invention provides an amino acid-related information provision service system for providing amino acid-related information relating to a test animal, the system comprising: a server; a user terminal; and a measuring device equipped with the biosensor, wherein the user terminal and the measuring device are each identified, the user terminal is associated with the measuring device, the measuring device measures data on the amino acid concentration of a urine sample taken from the test animal and transmits the measured data along with the timing data of the measurement to the server, the server creates amino acid concentration-related information relating to the test animal based on the data transmitted from the measuring device and transmits the information to the user terminal associated with the measuring device, the user terminal outputs the amino acid concentration-related information relating to the test animal transmitted from the server, and the amino acid-related information relating to 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 created based on the urinary amino acid concentration.

[0011] A plan view of a biosensor according to one embodiment of the present invention. An I-I end view of the biosensor shown in Figure 1. A schematic diagram showing a potentiostat connected to the biosensor shown in Figure 1. A schematic diagram representing a measurement system including the biosensor shown in Figure 1. An embodiment of an amino acid-related information provision service system. An example of the operation of the server 100, user terminal 200, and measurement system 300 in the amino acid-related information provision service system. Relationship between the current value measured using the biosensor of Example 1 and the D-amino acid concentration. A: In the absence of creatinine, B: In the presence of creatinine. For the biosensors of Examples 1 and 2, and Comparative Examples 1 and 2, the slope of the regression line between the steady-state current value measured by the sensor and the D-alanine concentration in the measured solution, in the presence and absence of creatinine (current value per unit base mass). Detailed description of the invention

[0012] In the biosensor described in Patent Document 1, the enzyme layer is located on the electrode surface, making the enzyme susceptible to interference from impurities in the solution being measured, which can result in insufficient accuracy in measuring the concentration of organic substances. In the biosensors described in Patent Documents 2 and 3, the enzyme and mediator are contained in a polymer layer placed on the electrode. However, even with this configuration, the enzyme can still be affected by impurities in the solution being measured. Furthermore, the mediator permeates the polymer layer and dissolves and disperses into the measurement solution, reducing the efficiency of electron transfer reactions near the electrode, which can result in insufficient accuracy in measuring the concentration of the target organic substance.

[0013] When measuring amino acid concentration in a solution using a biosensor, amine compounds present in the solution (e.g., urinary creatinine, urea, hexylamine, dimethylamine, trimethylamine, etc.) act as contaminants. Specifically, the reaction between the mediator in the electrode and the amine compound inhibits the redox reaction between the target amino acid and the mediator, leading to a decrease in the sensor's output.

[0014] There is a need to improve the accuracy of measuring the concentration of organic substances using enzyme sensors. This invention provides a biosensor that can measure the amino acid concentration in a solution with higher accuracy.

[0015] 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.

[0016] (1. Biosensor) The present invention provides a biosensor capable of measuring amino acid concentration with high accuracy. The biosensor of the present invention can measure the concentration of a substance to be measured in a solution that may contain the substance to be measured (amino acid) (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 an anionic compound.

[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, preferably a non-human animal, and more preferably a feline.

[0020] 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 that the electrode system be a three-electrode system from the viewpoint of high-precision measurement.

[0021] Figure 1 is a plan view of a biosensor according to one embodiment of the present invention. Figure 2 is an I-I end view 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. The enzyme electrode 2 is an electrode that exchanges electrons with the substance to be measured, the counter electrode 3 is an electrode that conducts current between it and the enzyme electrode 2, and the reference electrode 4 is an electrode that serves as a reference for the potential of the enzyme electrode 2 (reference electrode).

[0022] A portion of the electrode system is covered with 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 to 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.

[0023] 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.

[0024] 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 printing. The thickness of the insulating layer 6 is preferably 10 nm or more, more preferably 100 nm or more, even more preferably 1 μm or more, and 100 μm or less, more preferably 50 μm or less, and even more preferably 20 μm or less.

[0025] 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. The conductive layer 2a is formed such that a portion of it is in contact with one end 21a of the wiring 21. Conductive layers are also placed on one end 31a and 41a of the wiring 31 and 41, respectively, with a portion of each in contact with them. These conductive layers are the counter electrode 3 and the reference electrode 4.

[0026] The area of ​​the reagent layer 2b in contact with the conductive layer 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 applies: 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.

[0027] The conductive layer 2a of each electrode, the counter electrode 3 and the reference electrode 4, and 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 conductive layer 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.

[0028] The conductive layer 2a of the enzyme electrode 2 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 an 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 the 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, carbon mixed with the mediator, preferably Prussian blue-containing carbon paste).

[0029] In the biosensor of the present invention, the mediator is contained in a conductive layer 2a, and a reagent layer 2b containing an enzyme is formed on top of 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 can be made near the electrode, and thus accurate concentration measurement is possible even for amino acids in the low concentration range. In contrast, if the mediator is contained in the reagent layer instead of the conductive layer, the mediator may pass through the reagent layer 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 contained in the reagent layer, making high-precision measurement impossible. Therefore, in the biosensor of the present invention, the mediator may be contained in the reagent layer 2b, but preferably it is not contained in the reagent layer 2b and is contained only in the conductive layer 2a.

[0030] The mediator content in the conductive layer 2a 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 even more preferably 0.5% by mass or more, when the total amount of conductive material and mediator contained in the conductive layer 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.

[0031] A reagent layer 2b is fixed on the conductive layer 2a. The reagent layer 2b contains an enzyme and an anionic compound. The enzyme contained in the reagent layer 2b is 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 enzyme selected from 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 and is decomposed into hydrogen peroxide, keto acid, and ammonia. Hydrogen peroxide is converted into current as H2O2 → 2H ＋ + O2 + 2e － as shown.

[0032] From the viewpoint of ensuring a sufficient redox reaction necessary for the quantification of amino acids, the content 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 2.0 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 electrical 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, and even more preferably 10 μg / mm 2 or less. In this specification, the enzyme content in the reagent layer 2b as described above refers to the amount of enzyme per 1 mm 2 of the area in the reagent layer 2b that can come into contact with the solution to be measured.

[0033] The anionic compound contained in reagent layer 2b captures amine compounds (e.g., urinary creatinine, urea, hexylamine, dimethylamine, trimethylamine, etc.) contained in the solution being measured and inhibits their reaction with the mediator contained in conductive layer 2a. Amine compounds act as contaminants in amino acid concentration measurements. That is, amine compounds contained in the solution being measured can pass through the reagent layer and approach the conductive layer, where they may react with the mediator contained in the conductive layer. This inhibits the redox reaction between the target amino acid and the mediator, thereby reducing the output of the biosensor or decreasing the accuracy of amino acid concentration measurement by the biosensor. The anionic compound contained in reagent layer 2b can prevent the reduction in biosensor output or the decrease in the accuracy of amino acid concentration measurement caused by amine compounds.

[0034] The type of anionic compound contained in reagent layer 2b is not limited, as long as it has a charge capable of capturing amine compounds. Preferably, the pH (at 25°C) of a 1% by mass aqueous solution of the anionic compound (if not in salt form) is 7 or less. From the viewpoint of preventing elution into the solution to be measured and the capture efficiency of amine compounds, the anionic compound is preferably a polymer or copolymer having anionic repeating units. Examples of the anionic repeating units include repeating units having functional groups selected from the group consisting of carboxyl groups, sulfonic acid groups, and phosphate groups, such as acrylic acid and maleic acid. A polymer or copolymer consisting of one or more of the repeating units listed above can be used as the anionic compound. Preferred examples of the anionic compound include at least one selected from the group consisting of acrylic acid polymers or copolymers, maleic acid polymers or copolymers, and alginic acid polymers or copolymers, and more preferably polyacrylic acid and its salts, alginic acid and its salts.

[0035] The weight-average molecular weight of the polymer or copolymer having the anionic repeating units is preferably 1000 or more, more preferably 2000 or more, and even more preferably 3000 or more, from the viewpoint of preventing elution into the solution to be measured, while from the viewpoint of suppressing deterioration of operability during the formation of the reagent layer due to increased viscosity of the polymer, it is preferably 1,000,000 or less, more preferably 750,000 or less, and even more preferably 500,000 or less.

[0036] From the viewpoint of the capture efficiency of amine compounds, the amount of anionic compound in the reagent layer 2b is preferably 1.0 μg / mm². 2 More preferably 2.0 μg / mm 2 More preferably, 3.0 μg / mm³ 2 The above is true, but from the viewpoint of suppressing the inhibition of enzymatic reactions by anionic compounds, 50 μg / mm³ is preferable. 2 More preferably, 40 μg / mm² 2 More preferably, 30 μg / mm² 2 The following applies: In this specification, the amount of anionic compound 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 to be measured. 2 This refers to the amount of anionic compound per unit area.

[0037] In the reagent layer 2b, the mass ratio of enzyme to anionic compound (enzyme / anionic compound) is preferably 0.01 or higher, more preferably 0.05 or higher, and even more preferably 0.10 or higher, from the viewpoint of suppressing inhibition of the enzymatic reaction by the anionic compound. On the other hand, from the viewpoint of the capture efficiency of amine compounds, it is preferably 30 or lower, more preferably 20 or lower, even more preferably 10 or lower, and even more preferably 5.0 or lower.

[0038] Preferably, the reagent layer 2b contains an enzyme immobilizer in addition to the enzyme and anionic compound. Preferably, the enzyme and anionic compound contained in the reagent layer 2b are contained in the enzyme immobilizer. The enzyme immobilizer plays a role in immobilizing the enzyme and anionic compound in the reagent layer 2b, thereby inhibiting their elution into the solution to be measured, and suppressing the decrease in the reaction efficiency of the reagent layer 2b caused by the elution of the enzyme. Examples of the enzyme immobilizer include polymeric enzyme immobilizers that can inhibit the elution of enzymes. Examples of polymeric enzyme immobilizers include hydrogels and cured polymers.

[0039] Examples of the hydrogels mentioned above include hydrogels of compounds such as chitosan, chitosan methacrylate, glycol chitosan, glycol chitosan methacrylate, alginic acid, alginic acid methacrylate, hyaluronic acid (HA), and HA methacrylate, as well as their salts. For example, a reagent layer 2b containing a hydrogel that holds an enzyme and an anionic compound can be formed by dropping an aqueous solution containing an amino acid oxidase, a hydrogel, and an anionic compound onto a conductive layer 2a formed on an insulating substrate 5 and drying it. Alternatively, a hydrogel of an anionic compound (e.g., alginic acid or its salt) can be used as a polymeric enzyme immobilizer.

[0040] Examples of the cured polymer include cured photosensitive polymers. Since photosensitive polymers have the property of curing upon light irradiation, a mixture of the photosensitive polymer, an enzyme, and an anionic compound can be applied to the conductive layer and irradiated with light to form a reagent layer 2b adjacent to the conductive layer, which contains a cured polymer holding the enzyme and anionic compound. For example, the reagent layer 2b can be formed by dropping an aqueous solution containing amino acid oxidase, an anionic compound, and a photosensitive polymer onto the conductive layer 2a formed on an insulating substrate 5, and curing it by UV irradiation.

[0041] The photosensitive polymer preferably has azide groups, from the viewpoint of being able to form a cured polymer with sufficient strength to suppress enzyme elution by light irradiation. Furthermore, since the photosensitive polymer needs to be stable and not decompose during the preparation of an aqueous solution containing the polymer, enzyme, and anionic compound, and during light irradiation, it is preferable that the photosensitive polymer has highly stable amide bonds.

[0042] Preferably, the photosensitive polymer includes repeating units I represented by the following formula (1).

[0043] In the above formula, n is an integer from 1 to 3; R 1 is, -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 an alkyl group having 1 to 6 carbon atoms.

[0044] In one preferred embodiment, R 1 R 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 R 3 Together they form a linear alkylene group having 2 to 6 carbon atoms. In another preferred embodiment, R 2 and R 3Together 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.

[0045] In a more preferred embodiment, R 1 The group is selected from the following group:

[0046] More preferably, the repeating unit I is represented by the following formula (2).

[0047] 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:

[0048] In a more preferred embodiment, the repeating unit II is represented by the following formula (3).

[0049] 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).

[0050] 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.).

[0051] 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 being 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 being 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 being 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 being 100% by mass.

[0052] The overall average degree of polymerization of the hydrogel or photosensitive polymer used as the enzyme immobilization agent is preferably 100 or more, more preferably 200 or more, and even more preferably 300 or more, in order to properly retain the enzyme in the reagent layer or for the photosensitive polymer to obtain sufficient photosensitivity. On the other hand, from the viewpoint of preventing deterioration of operability during the formation of the reagent layer due to increased viscosity of the polymer and inhibiting the enzyme reaction by the enzyme immobilization agent, it is preferably 10,000 or less, more preferably 7,500 or less, and even more preferably 5,000 or less.

[0053] From the viewpoint of effectively retaining the enzyme in the reagent layer 2b, the content of the enzyme immobilization agent is preferably 0.2 μg / mm³. 2 More preferably 0.7 μg / mm 2 More preferably 2.0 μg / mm 2Therefore, from the viewpoint of suppressing inhibition of the enzymatic reaction by the enzyme immobilizer, 50 μg / mm³ is preferred. 2 More preferably, 40 μg / mm² 2 More preferably, 30 μg / mm² 2 The following applies: In this specification, the amount of enzyme immobilizer 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 to be measured. 2 This refers to the amount of enzyme immobilizer used per unit.

[0054] In the reagent layer 2b, the mass ratio of the enzyme to the enzyme immobilizer (enzyme / enzyme immobilizer) 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 enzymatic reaction by the enzyme immobilizer, and preferably 10 or lower, more preferably 7.5 or lower, even more preferably 5 or lower, and even more preferably 2 or lower, from the viewpoint of good retention of the enzyme in the reagent layer.

[0055] The mediator content in the reagent layer 2b is preferably 0.1 μg / mm³. ２ More preferably, 0.01 μg / mm ２ The following, and more preferably, the reagent layer 2b does not contain the mediator.

[0056] The reagent layer 2b may further contain stabilizers, other polymers, surfactants, and other reagents advantageous for measuring amino acid concentrations.

[0057] (2. Amino Acid Concentration Measurement System) The biosensor of the present invention is used to measure the amino acid concentration in a solution to be measured. The 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") will be described below.

[0058] As an exemplary embodiment of the measurement system of the present invention, a measurement system for chronoamperometry (CA) measurement using the biosensor 1 shown in Figure 1 or Figure 2 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 16. 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.

[0059] 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 comprises a control unit 11 connected to the biosensor 1 and controlling the voltage application to the biosensor 1; a detection unit 12 that detects the current obtained by the voltage application to the biosensor 1; a calculation unit 13 that calculates the amino acid concentration of the solution to be measured 16 from the current value; and an output unit 14 that outputs the calculated amino acid concentration.

[0060] 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 the amino acid concentration in the solution to be measured 16 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 amino acid concentration and current value, and a central processing unit (CPU) that includes a calculation unit for calculating the absolute value of amino acid concentration. The output unit 14 may be included in the same information processing terminal as the calculation unit 13.

[0061] In one embodiment, the measurement system of the present invention further includes a display unit 15 that displays the amino acid concentration output from the output unit 14. The output unit 14 transmits the calculation result of the amino acid concentration by the calculation unit 13 to the display unit 15. The measurement system shown in Figure 4 includes the display unit 15. The display by the display unit 15 may be a digital display or an analog display. In another embodiment, the measurement system of the present invention includes a storage unit 17, and the calculation result of the amino acid concentration output from the output unit 14 is stored in the storage unit 17.

[0062] The connections between the detection unit 12 and the calculation unit 13, the calculation unit 13 and the output unit 14, and the output unit 14 and the display unit 15 or storage unit 17 may be wired or wireless. Examples of wireless connection interfaces include Wi-Fi and Bluetooth®.

[0063] (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.

[0064] 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.).

[0065] Users of the service system of the present invention are those who wish to receive amino acid-related information concerning test animals. Examples of such users include those who raise or manage test animals, such as the owners of test animals, veterinarians, and employees of animal breeding facilities such as zoos (animal keepers, veterinarians, facility managers, etc.).

[0066] 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.

[0067] 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).

[0068] A user may use one measurement device and one user terminal. Alternatively, a 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.

[0069] 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.

[0070] 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 concentration-related information about the test animal transmitted from the server.

[0071] 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.

[0072] Figure 5 shows that there are multiple users A, B, and C who utilize the service system of the present invention, each of whom keeps a test animal (e.g., a cat). 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] The user terminals 200 (200A, 200B, 200C, etc.) receive and output amino acid-related information concerning the test animals from the server 100. The format of the output is not particularly limited and may include screen display, printing, or recording in a database. In a preferred embodiment, the user terminal 200 has an application program (hereinafter also referred to as "app") corresponding to the amino acid-related information provision service system of the present invention installed, and the user terminal 200 may output the amino acid-related information provided from the server 100 via the app. As the user terminal 200, for example, an information processing terminal such as a smartphone, mobile phone, tablet PC (Personal Computer), notebook PC, or desktop PC with the necessary app installed can be used.

[0078] The measuring device 300 (300A, 300B, 300C, etc.) 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.

[0079] 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.

[0080] 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.

[0081] [Example of operation of the amino acid-related information provision service system]

[0082] 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 hereinafter referred to as the "sensor ID").

[0083] 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, 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).

[0084] Next, the server 100 determines whether multiple amino acid concentration data sets associated with the same sensor ID and different measurement timings are stored (S13). If multiple amino acid concentration data sets 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).

[0085] 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).

[0086] 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).

[0087] 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.

[0088] (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.

[0089] The measurement method of the present invention includes: supplying the solution to be measured to a biosensor of the measurement system of the present invention; applying a constant voltage to the biosensor with the control unit of the measurement system; detecting the current value obtained from the biosensor with the detection unit of the measurement system; and calculating the amino acid concentration in the solution to be measured from the detected current value with the calculation unit of the measurement system.

[0090] 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: Supplying the solution to be measured to the reagent layer 2b, counter electrode 3, and reference electrode 4 of the biosensor 1; Step B: Applying a constant voltage to the biosensor 1 with the control unit 11; Step C: Detecting the current value obtained from the biosensor 1 with the detection unit 12; Step D: Calculating the concentration of the substance to be measured with the calculation unit 13 from the detected current value.

[0091] In step A described above, the solution to be measured is supplied to the enzyme electrode 2 (reagent layer 2b), the counter electrode 3, and the reference electrode 4 through the opening 61 in the insulating layer 6.

[0092] 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.

[0093] 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.

[0094] The measurement method of the present invention allows for the detection of kidney disease in a test animal by measuring the amino acid concentration in a urine sample collected from 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.

[0095] 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.

[0096] As one embodiment, a method for testing for renal dysfunction in cats using the measurement system shown in Figure 4 will be described. Here, cat urine is used as the urine sample. The method includes the following steps A to D. Step A: Supplying cat urine to the reagent layer 2b, counter electrode 3, and reference electrode 4 of the biosensor 1; Step B: Applying a constant voltage to the biosensor 1 with the control unit 11; Step C: Detecting the current value obtained from the biosensor 1 with the detection unit 12; Step D: Calculating the amino acid concentration in the urine with the calculation unit 13 from the detected current value; Step E: Testing for the presence or absence of renal dysfunction in the cat based on the calculated amino acid concentration in the urine. The specific processes of steps A to D are the same as the method for measuring the amino acid concentration in the solution to be measured described above.

[0097] 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.

[0098] [1] 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 an anionic compound. [2] The biosensor according to [1], preferably wherein the anionic compound is a polymer or copolymer having anionic repeating units. [3] The biosensor according to [2], preferably wherein the anionic repeating units are repeating units having a functional group selected from the group consisting of a carboxyl group, a sulfonic acid group and a phosphate group, and more preferably selected from the group consisting of acrylic acid, maleic acid and maleic acid esters. [4] The biosensor according to any one of [1] to [3], wherein the anionic compound is preferably a polymer or copolymer consisting of one or more repeating units having a carboxyl group, repeating units having a sulfonic acid group, and repeating units having a phosphate group, more preferably at least one selected from the group consisting of acrylic acid polymers or copolymers, maleic acid polymers or copolymers, and alginic acid polymers or copolymers, and even more preferably polyacrylic acid, alginic acid, or a salt thereof. [5] The biosensor according to any one of [2] to [4], wherein the weight-average molecular weight of the polymer or copolymer having the anionic repeating unit is preferably 1000 or more, more preferably 2000 or more, even more preferably 3000 or more, and preferably 1,000,000 or less, more preferably 750,000 or less, and even more preferably 500,000 or less. [6] Preferably, the mediator has a solubility in water of 1 g / 100 mL or less at 25°C, and more preferably, the mediator is Prussian blue, the biosensor according to any one of [1] to [5]. [7] Preferably, the content of the mediator in the reagent layer is 0.1 μg / mm³.２ More preferably, 0.01 μg / mm ２ The biosensor according to any one of [1] to [6], wherein, more preferably, the reagent layer 2b does not contain the mediator. [8] Preferably, the biosensor according to any one of [1] to [7], wherein the reagent layer further contains an enzyme immobilizer. [9] Preferably, the biosensor according to [8], wherein the enzyme immobilizer contains the amino acid oxidase and the anionic compound.

[10] Preferably, the biosensor according to [8] or [9], wherein the enzyme immobilizer is a polymeric enzyme immobilizer, more preferably a hydrogel or a cured photosensitive polymer.

[11] Preferably, the biosensor according to

[10] , wherein the photosensitive polymer has an azide group and / or the photosensitive polymer has an amide bond.

[12] Preferably, the biosensor according to

[10] , wherein the hydrogel is a hydrogel of alginic acid or a salt thereof.

[13] Preferably, the photosensitive polymer comprises a repeating unit I represented by formula (1) according to any one of

[10] to

[12] .

[14] 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 The carbon atoms together with N form a heterocycle having 3 to 8 carbon atoms, which may be substituted with an alkyl group, where the heterocycle may contain an additional oxygen or nitrogen atom, and the alkyl group substituting the heterocycle is preferably a linear or branched alkyl group having 1 to 4 carbon atoms, more preferably a methyl group or an ethyl group, as described in

[13] .

[15] Preferably, in formula (1), R 1 is a group selected from the following group:

[13] The biosensor according to

[16] Preferably, the biosensor according to any one of

[13] to

[15] , wherein the repeating unit I is represented by formula (2).

[17] Preferably, the biosensor according to any one of

[13] to

[16] , wherein the photosensitive polymer is a copolymer polymer comprising at least the repeating unit I and a repeating unit II having water solubility.

[18] Preferably, the repeating unit II is selected from the group consisting of the following: A biosensor according to

[17] , more preferably represented by formula (3).

[19] A biosensor according to

[17] or

[18] , preferably the photosensitive polymer is a copolymer polymer further comprising a repeating unit represented by formula (4).

[20] A biosensor according to any one of

[13] to

[19] , wherein 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, and even more preferably 20% by mass or less, based on 100% by mass of the entire photosensitive polymer.

[21] A biosensor according to

[20] , wherein 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.

[22] The biosensor according to any one of

[10] to

[21] , wherein 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, and even more preferably 5000 or less.

[23] The content of the amino acid oxidase in the reagent layer is preferably 0.2 μg / mm 2 More preferably 0.7 μg / mm 2 More preferably 2.0 μg / mm 2 The above is required, and preferably 30 μg / mm². 2More preferably, 25 μg / mm² 2 More preferably, 15 μg / mm² 2 More preferably, 10 μg / mm² 2 The biosensor according to any one of the following items [1] to

[22] :

[24] The content of the anionic compound in the reagent layer is preferably 1.0 μg / mm 2 More preferably 2.0 μg / mm 2 More preferably, 3.0 μg / mm³ 2 The above is required, and preferably 50 μg / mm². 2 More preferably, 40 μg / mm² 2 More preferably, 30 μg / mm² 2 The biosensor according to any one of the following items [1] to

[23] :

[25] The biosensor according to any one of the following items [1] to

[24] , wherein the mass ratio of the amino acid oxidase to the anionic compound in the reagent layer (amino acid oxidase / anionic compound) is preferably 0.01 or more, more preferably 0.05 or more, even more preferably 0.10 or more, and preferably 30 or less, more preferably 20 or less, even more preferably 10 or less, and even more preferably 5.0 or less.

[26] The content of the enzyme immobilizer in the reagent layer is preferably 0.2 μg / mm 2 More preferably 0.7 μg / mm 2 More preferably 2.0 μg / mm 2 The above is required, and preferably 50 μg / mm². 2 More preferably, 40 μg / mm² 2 More preferably, 30 μg / mm² 2The biosensor according to any one of the following items [8] to

[25] :

[27] The biosensor according to any one of the following items [8] to

[26] , wherein the mass ratio of the amino acid oxidase to the enzyme immobilizer in the reagent layer (amino acid oxidase / enzyme immobilizer) 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, even more preferably 5 or less, and even more preferably 2 or less.

[28] The biosensor according to any one of the following items [1] to

[27] , wherein the content of the mediator 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, even more preferably 0.5% 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 the mediator contained in the conductive layer is 100% by mass.

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

[28] .

[0099] A system for measuring the amino acid concentration in a solution to be measured, comprising: a biosensor according to any one of items [1] to

[29] ; a control unit for controlling the application of voltage to the biosensor; a detection unit for detecting a current value obtained by applying voltage to the biosensor; a calculation unit for calculating the amino acid concentration of the solution to be measured from the current value; and an output unit for outputting the calculated amino acid concentration.

[31] Preferably, the measurement system according to

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

[32] Preferably, the measurement system according to

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

[0100]

[33] A method for measuring the amino acid concentration in a solution to be measured, comprising: supplying the solution to be measured to a biosensor of the measurement system described in

[30] ; applying a constant voltage to the biosensor with the control unit of the measurement system; detecting a current value obtained from the biosensor with the detection unit of the measurement system; and calculating the amino acid concentration in the solution to be measured from the detected current value with the calculation unit of the measurement system.

[34] The method according to

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

[35] The method according to

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

[0101]

[36] A method for testing renal dysfunction in a non-human test animal, comprising measuring the amino acid concentration in a urine sample taken from the non-human test animal by the measurement method described in

[33] .

[37] The method according to

[36] , wherein the non-human test animal is a feline.

[0102]

[38] An amino acid-related information provision service system that provides amino acid-related information relating to a test animal, the system comprising: a server; a user terminal; and a measuring device equipped with a biosensor as described in any one of [1] to

[29] , wherein the user terminal and the measuring device are each identified, the user terminal is associated with the measuring device, the measuring device measures data on the amino acid concentration of a urine sample taken from a test animal and transmits the measured data along with the timing data of the measurement to the server, the server creates amino acid concentration-related information relating to the test animal based on the data transmitted from the measuring device and transmits the information to the user terminal associated with the measuring device, the user terminal outputs the amino acid concentration-related information relating to the test animal transmitted from the server, and the amino acid-related information relating to 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 created based on the urinary amino acid concentration.

[0103] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0104] 1. Fabrication of Biosensors (Example 1) The biosensors shown in Figures 1 and 2 were fabricated.

[0105] [Fabrication of the electrode system] Silver paste (manufactured by SUN CHEMICAL, product number C2080415P2) was screen printed onto an insulating substrate 5 (AS ONE Corporation, polyimide film, model number: HJA-A4-225μm, thickness 225μm) using 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 overlapped, was attached, with a diameter of 3.5 mm (area 9.6 mm). 2A circular conductive layer was formed. Specifically, a conductive layer 2a containing 1% by mass of Prussian blue was formed by screen printing a Prussian blue-containing carbon paste (manufactured by SUN CHEMICAL, product number C2070424D2) through a screen mask and drying at 80°C for 10 minutes. Next, a counter electrode 3 was formed by screen printing a carbon paste (manufactured by SUN CHEMICAL, product number C2030519P4) through a screen mask onto one end of the wiring 31, with a portion overlapping, and drying at 80°C for 10 minutes. Next, a reference electrode 4 was formed by screen printing a silver / silver chloride paste (manufactured by SUN CHEMICAL, product number C2130809D5) through a screen mask onto one end of the wiring 41, with a portion overlapping, and sintering at 80°C for 10 minutes. Next, insulating paste (manufactured by SUN CHEMICAL, product number D2070423D5) was screen printed through a screen mask so that it covered parts of wiring 21, wiring 31, and wiring 41, and an insulating layer 6 was formed by drying at 130°C for 10 minutes. In the above procedure, 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 their thickness after firing or drying was 10 μm.

[0106] [Enzyme Modification] Next, an enzyme solution was prepared by mixing 50 U / mL (10 mg / mL) of D-amino acid oxidase (enzyme, manufactured by SIGMA-Aldrich, D-amino Acid Oxidase from porcine kidney), 3% by mass of anionic polymer (polyacrylic acid (PAA); manufactured by Fujifilm Wako Pure Chemical Industries, weight-average molecular weight 5000), and 3% by mass of photosensitive polymer (Biosurfine-AWP-MRH; manufactured by Toyo Gosei Co., Ltd.) in a PBS solution (0.1 mol / L phosphate-buffered saline, pH = 8.0, 25°C). Next, 7 μL of the obtained enzyme solution was dropped onto the conductive layer 2a, dried at 25°C for 24 hours, and then subjected to UV exposure (100 mJ / cm²). 2By heating for 1 minute, a reagent layer 2b containing an enzyme, an anionic polymer, and a cured photosensitive polymer was formed on the conductive layer 2a. Following this procedure, a biosensor was obtained comprising an enzyme electrode 2 containing a conductive layer 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 PAA content is 21.9 μg / mm³. 2 The content of the cured product of Biosurfine-AWP-MRH is 21.9 μg / mm³. 2 Furthermore, the mass ratio of the enzyme to PAA (D-amino acid oxidase / PAA) was 0.33, and the mass ratio of the enzyme to the cured photosensitive polymer (D-amino acid oxidase / Cured Biosurfine-AWP-MRH) was 0.33.

[0107] 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.

[0108] (Example 2) [Preparation of Electrode System] The procedure was carried out in the same manner as in Example 1 [Preparation of Electrode System]. [Enzyme Modification] Instead of the mixed solution used in Example 1 [Enzyme Modification], an enzyme solution was prepared by mixing 50 U / mL of D-amino acid oxidase and 0.67% by mass of anionic polymer (80-120% sodium alginate; manufactured by Fujifilm Wako Pure Chemical Industries) in PBS solution (0.1 mol / L phosphate-buffered saline, pH = 8.0, 25°C). 7 μL of the obtained enzyme solution was dropped onto the conductive layer 2a and dried at 25°C for 24 hours to form a reagent layer 2b containing the enzyme and anionic polymer on the conductive layer 2a. The D-amino acid oxidase content in the reagent layer 2b was 7.3 μg / mm³. ２ The sodium alginate content is 4.9 μg / mm³. ２ Furthermore, the mass ratio of the enzyme to sodium alginate (D-amino acid oxidase / sodium alginate) was 1.49.

[0109] (Comparative Example 1) [Electrode System Preparation] The same procedure as in Example 1 was followed for the preparation of the electrode system. [Enzyme Modification] In Comparative Example 1, a biosensor equipped with an enzyme electrode was obtained in the same manner as in Example 1's [Enzyme Modification], except that an anionic polymer was not added to the enzyme solution dropped onto the conductive layer 2a.

[0110] (Comparative Example 2) [Fabrication of Electrode System] The procedure was the same as in Example 1, except that the Prussian blue-containing carbon paste (manufactured by SUN CHEMICAL, product number C2070424D2) used to form the conductive layer 2a was replaced with a Prussian blue-free carbon paste (manufactured by SUN CHEMICAL, product number C2030519P4). [Enzyme Modification] A biosensor equipped with an enzyme electrode was obtained in the same manner as in Example 1, except that 1 mM Prussian blue (manufactured by Merck) was added to the enzyme solution dropped onto the conductive layer 2a.

[0111] 2. Measurement of amino acid concentration in the solution to be measured using a biosensor (Test 1) The correlation between the measured value from the biosensor in Example 1 and the amino acid concentration in the solution to be measured was investigated.

[0112] [Measurement of Amino Acid Concentration] D-alanine was used as the amino acid. A PBS solution (0.1 mol / L phosphate-buffered saline, pH = 8.0, 25°C; Fujifilm Wako Pure Chemical Industries) containing D-alanine (manufactured by Peptide Laboratories) and creatinine (manufactured by Fujifilm Wako Pure Chemical Industries) as an impurity was prepared as the solution to be measured. The concentration of D-alanine in the solution to be measured was 0 μM, 200 μM, 500 μM, or 1000 μM, and the concentration of creatinine was 0 or 500 mg / dL.

[0113] As shown in Figure 3, the wiring 21 for the enzyme electrode of the biosensor, the wiring 41 for the reference electrode, and the wiring 31 for the counter electrode were connected to the potentiostat 8, and each electrode was immersed in 10 mL of the solution to be measured. 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.

[0114] Figure 7A shows the steady-state current value measured from a creatinine-free solution using the biosensor of Example 1. The regression line between the D-alanine concentration and the steady-state current value in Figure 7A is y = -8.35 × 10 -10 x - 1.03 × 10 -8 (y: quantitative current value (A), x: D-alanine concentration (μM)), and the correlation coefficient R of this correlation equation 2 The value was 0.988. It was confirmed that the steady-state current value measured with the biosensor of Example 1 had a high correlation with the concentration of D-alanine in the solution being measured.

[0115] Figure 7B shows the steady-state current value measured from the creatinine-containing solution using the biosensor of Example 1. The regression line between the D-alanine concentration and the steady-state current value in Figure 7B is y = -8.23 × 10 -10 x + 1.99 × 10 -8 (y: quantitative current value (A), x: D-alanine concentration (μM)), and the correlation coefficient R of this correlation equation 2 The value was 0.989. Even in the presence of creatinine, an impurity, the steady-state current value measured by the biosensor in Example 1 and the concentration of D-alanine in the measured solution showed a high correlation similar to that in the absence of creatinine.

[0116] The results above demonstrate that by using the biosensor of Example 1, it is possible to quantify the amino acid concentration in the solution being measured even in the presence of creatinine, an impurity, and that low amino acid concentrations of 1000 μM or less can be measured with high accuracy.

[0117] (Test 2) The measurement accuracy of the biosensors of Examples 1 and 2, and Comparative Examples 1 and 2, was compared to that of the biosensors used in the test solution to measure amino acid concentration. Using the same procedure as in Test 1 [Measurement of Amino Acid Concentration], the amino acid concentration in the test solution was measured in the presence and absence of creatinine using the biosensors of Example 2 and Comparative Examples 1 and 2, and a regression line was obtained between the steady-state current value measured by the biosensor and the D-alanine concentration in the test solution.

[0118] Table 1 shows the slope (current value per unit mass) and coefficient of determination of the regression lines for Examples 1 and 2, and Comparative Examples 1 and 2. In Examples 1 and 2, the current values ​​output from the sensor were almost the same in the presence and absence of creatinine, and the coefficient of determination of the regression line was close to 1 in both cases. In contrast, in Comparative Examples 1 and 2, the output from the sensor decreased significantly in the presence of creatinine compared to the absence of creatinine, and the coefficient of determination of the regression line also decreased significantly. Figure 8 shows the slope (current value per unit mass) of the regression line between the steady-state current value measured by the sensor and the D-alanine concentration in the measured solution for the biosensors of Examples 1 and 2, and Comparative Examples 1 and 2, in the presence and absence of creatinine. It was shown that the accuracy of amino acid concentration measurement was greatly reduced in the biosensors of Comparative Examples 1 and 2 due to the influence of creatinine, an impurity.

[0119]

[0120] 1. Biosensor 2. Enzyme electrode 2a. Conductive layer 2b. Reagent layer 3. Counter electrode 4. Reference electrode 21, 31, 41. Wiring 5. Insulating substrate 6. Insulating layer 61. Aperture 8. Potentiostat 10. Measuring device 11. Control unit 12. Detection unit 13. Calculation unit 14. Output unit 15. Display unit 16. Solution to be measured

Claims

1. 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 an anionic compound.

2. The biosensor according to claim 1, wherein the anionic compound is a polymer or copolymer having anionic repeating units.

3. The biosensor according to claim 2, wherein the anionic repeating unit is a repeating unit having a functional group selected from the group consisting of a carboxyl group, a sulfonic acid group, and a phosphate group.

4. The biosensor according to claim 2, wherein the anionic compound is at least one selected from the group consisting of acrylic acid polymers or copolymers, maleic acid polymers or copolymers, and alginic acid polymers or copolymers.

5. The biosensor according to claim 4, wherein the anionic compound is polyacrylic acid, alginic acid, or a salt thereof.

6. The biosensor according to claim 1, wherein the mediator has a solubility in water of 1 g / 100 mL or less at 25°C.

7. The biosensor according to claim 6, wherein the mediator is Prussian blue.

8. The biosensor according to claim 1, wherein the reagent layer further contains an enzyme immobilizer.

9. The biosensor according to claim 8, wherein the enzyme immobilization agent contains the amino acid oxidase and the anionic compound.

10. The biosensor according to claim 8, wherein the enzyme immobilizing agent is a hydrogel or a cured photosensitive polymer.

11. The biosensor according to claim 10, wherein the photosensitive polymer contains a repeating unit I represented by the following formula (1): (In the formula, n is an integer of 1 to 3; R 1 is -NR 2 R 3 ; R 2 and R 3 are each independently a linear or branched alkyl group or alkenyl group having 1 to 6 carbon atoms, or R 2 and R 3 together form a linear or branched alkylene group having 2 to 6 carbon atoms, or a linear or branched alkylene group 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).

12. The biosensor according to claim 11, wherein the repeating unit I is represented by the following formula (2).

13. The biosensor according to claim 11, wherein the photosensitive polymer is a copolymer polymer comprising at least the repeating unit I and the water-soluble repeating unit II.

14. The biosensor according to claim 13, wherein the repeating unit II is as follows.

15. The content of the amino acid oxidase in the reagent layer is 0.2 μg / mm³. 2 30 μg / mm or more 2 The biosensor according to claim 1, which is as follows:

16. The content of the anionic compound in the reagent layer is 1.0 μg / mm³. 2 50 μg / mm or more 2 The biosensor according to claim 1, which is as follows:

17. The content of the enzyme immobilizer in the reagent layer is 0.2 μg / mm³. 2 50 μg / mm or more 2 The biosensor according to claim 8, which is as follows:

18. 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.

19. A system for measuring the amino acid concentration in a solution to be measured, comprising: a biosensor according to any one of claims 1 to 18; a control unit for controlling the application of voltage to the biosensor; a detection unit for detecting a current value obtained by applying voltage to the biosensor; a calculation unit for calculating the amino acid concentration of the solution to be measured from the current value; and an output unit for outputting the calculated amino acid concentration.

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

21. The measurement system according to claim 20, wherein the test subject animal is a feline.

22. A method for measuring the amino acid concentration in a solution to be measured, comprising: supplying the solution to be measured to a biosensor of a measurement system according to claim 19; applying a constant voltage to the biosensor with a control unit of the measurement system; detecting a current value obtained from the biosensor with a detection unit of the measurement system; and calculating the amino acid concentration in the solution to be measured from the detected current value with a calculation unit of the measurement system.

23. The method according to claim 22, wherein the solution to be measured is a urine sample collected from a test animal.

24. The method according to claim 23, wherein the test subject is a feline.

25. A method for testing renal dysfunction in a non-human test animal, comprising measuring the amino acid concentration in a urine sample collected from the non-human test animal by the measurement method described in claim 22.

26. The method according to claim 25, wherein the non-human animal subject to the test is a feline.

27. An amino acid-related information provision service system that provides amino acid-related information concerning a test animal, the system comprising: a server; a user terminal; and a measuring device equipped with a biosensor according to any one of claims 1 to 18, wherein the user terminal and the measuring device are each identified, the user terminal is associated with the measuring device, the measuring device measures data on the amino acid concentration of a urine sample taken from a test animal, and transmits the measured data along with the timing data of the measurement to the server, the server creates amino acid concentration-related information for the test animal based on the data transmitted from the measuring device, and transmits the information to the user terminal associated with the measuring device, the user terminal outputs the amino acid concentration-related information for the test animal transmitted from the server, and 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 created based on the urinary amino acid concentration.

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