Lactic acid sensor

Abstract

To provide a lactic acid sensor that prevents the deactivation of lactic acid oxidase on a carrier and has high sensitivity using a small amount of enzyme.SOLUTION: The lactic acid sensor includes an insulating substrate, an electrode formed on the insulating substrate, and a reagent layer formed on at least a part of the electrode, in which the reagent layer includes lactic acid oxidase, fibroin, and a carrier.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a lactate sensor that measures the concentration of lactate. [Background technology]

[0002] Methods for quantifying various biomarkers using enzyme electrodes are known. For example, Patent Document 1 proposes a disposable lactate sensor using lactate oxidase. Immobilizing an enzyme on an enzyme electrode enables continuous detection of changes in the concentration of a target biomarker. Various methods for immobilizing enzymes have been developed. Among them, methods using carriers are widely used from the viewpoint of ease of sensor manufacturing. Patent Document 2 discloses a sensor for measuring neutral fats, in which a neutral fat-degrading enzyme is supported on filter paper. In sensors such as those described in Patent Documents 1 and 2, an enzyme is immobilized on a carrier. A common method for immobilizing an enzyme on a carrier is to immerse the carrier in an aqueous enzyme solution and then dry it. However, there is a problem that some types of enzymes may be denatured and inactivated during this process. To address the above-mentioned issues, various stabilizers are used to maintain enzyme activity. Known stabilizers include proteins such as bovine serum albumin, amino acids such as sodium alginate, and polysaccharides such as trehalose. Patent Document 3 proposes that protein hydrolysates such as gelatin hydrolysates are effective stabilizers for glucose oxidase and peroxidase. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 3498105 [Patent Document 2] Japanese Patent Application Laid-Open No. 2009-244013 [Patent Document 3] Japanese Patent Application Laid-Open No. 2005-114368 Summary of the Invention [Problem to be solved by the invention]

[0004] Lactate oxidase used in lactic acid detection is easily inactivated, particularly during the process of impregnating a carrier and drying, and a large amount of enzyme was required to produce the lactate sensor disclosed in Patent Document 1. Furthermore, stabilizers such as those disclosed in Patent Document 3 were insufficient to stabilize lactate oxidase, and a more effective stabilizer was desired. [Means for solving the problem]

[0005] In order to solve the above-mentioned problems, the present disclosure aims to provide a lactate sensor that prevents the inactivation of lactate oxidase in the reagent layer and has high sensitivity with a small amount of enzyme. The lactate sensor of the present disclosure is a lactate sensor comprising an insulating substrate, an electrode formed on the insulating substrate, and a reagent layer formed on at least a portion of the electrode, wherein the reagent layer contains lactate oxidase, fibroin, and a carrier. [Effects of the Invention]

[0006] According to the present disclosure, it is possible to provide a lactate sensor that can suppress the deactivation of lactate oxidase in the reagent layer and achieve high sensitivity with a small amount of enzyme, and it is possible to measure the concentration of lactate simply, quickly, and accurately. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a schematic diagram illustrating an example of a lactate sensor according to a first embodiment of the present disclosure. [Figure 2] 10A to 10C are diagrams showing steps in a method for manufacturing a lactate sensor according to a third embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0008] As a first embodiment, the present disclosure provides a lactate sensor including an insulating substrate, an electrode formed on the insulating substrate, and a reagent layer formed on at least a portion of the electrode, the reagent layer including lactate oxidase, fibroin, and a carrier. As illustrated in FIG. 1 , the lactate sensor of this embodiment includes an insulating substrate 1 and an electrode layer 2 thereon. The electrode layer is a layer including an electrode. The electrode does not need to be formed over the entire surface of the substrate. The lactate sensor further includes a reagent layer 3 on at least a portion of the electrode. The reagent layer includes lactate oxidase, fibroin, and a carrier. This will be described in more detail below.

[0009] (insulating substrate) The lactate sensor of the present disclosure includes an insulating substrate as its base. The insulating substrate can be made of an insulating material such as plastic, paper, glass, or ceramic. Examples of the plastic include polyesters such as polyethylene terephthalate (PET), polystyrene, polypropylene, polycarbonate, polyimide, and acrylic resin. There are no particular limitations on the shape or size of the insulating substrate.

[0010] (electrode) The electrodes formed on the insulating substrate function as a potential application means for applying a potential to the sample solution in the reagent layer described below, and as a current detection means for detecting the current flowing in the sample solution, when the lactate sensor is in use.

[0011] The lactate sensor of the present disclosure has a measuring electrode and a counter electrode formed on the insulating substrate (two-electrode system), and may also have a reference electrode formed thereon if necessary (three-electrode system). From the viewpoint of more sensitive control of the electrode potential, the three-electrode system is preferable to the two-electrode system. In addition, the electrodes may include a sensing electrode for sensing the liquid volume.

[0012] The working electrode and counter electrode form a pair when the lactate sensor is in use, and function as a current measuring means for measuring the oxidation current (response current) that flows when a potential is applied to the sample solution in the reagent layer (described later). When the lactate sensor is in use, a predetermined potential is applied between the counter electrode and working electrode, with the reference electrode as the reference.

[0013] The electrode material that can be used to construct the lactate sensor of the present disclosure is not particularly limited as long as it can electrochemically detect the reaction between the analyte and the sample, and electrodes conventionally used in forming biosensor electrodes can be used as appropriate. However, from the viewpoint of further improving the response sensitivity of the lactate sensor, it is preferable that the electrodes be constructed from a material with a lower surface resistance value. Specific examples of electrodes include carbon electrodes, gold electrodes, silver electrodes, platinum electrodes, and palladium electrodes. The materials that constitute each electrode (measurement electrode, counter electrode, and reference electrode) may be the same or different. From the viewpoints of corrosion resistance and cost, it is preferable that the working electrode and counter electrode be constructed primarily from carbon. Furthermore, from the viewpoint of high stability of the applied potential, the reference electrode is preferably constructed from silver / silver chloride.

[0014] The method for forming the electrodes is not particularly limited, and they can be formed by conventionally known methods such as screen printing and sputtering. When forming electrodes by these methods, the material constituting the electrodes is provided in the form of a paste containing a resin binder such as polyester. For example, the paste is applied to an insulating substrate by screen printing or sputtering to form a coating film, and then a heat treatment is performed to harden the coating film, thereby forming the electrodes. The shape of the electrode is not particularly limited, and examples thereof include a circle, an ellipse, a rectangle, etc. For example, in the case of a circle, the radius is preferably 3 mm or less, and more preferably 2 mm or less.

[0015] In the present disclosure, the thickness of the insulating substrate and the electrodes is not particularly limited, but when the lactate sensor of the present disclosure is applied to a wearable sensor, the lactate sensor is preferably in sheet form, and the insulating substrate and the electrodes are preferably thin layers. The combined thickness of the insulating substrate and the electrodes is preferably 0.2 mm or less, more preferably 0.1 mm or less.

[0016] (reagent layer) The lactate sensor of the present disclosure has a reagent layer provided on at least a portion of the electrode, the reagent layer including at least lactate oxidase, fibroin, and a carrier. In the reagent layer, lactate oxidase oxidizes lactic acid in the sample to pyruvic acid and reduces oxygen in the air to produce hydrogen peroxide. The hydrogen peroxide produced is instantly oxidized and electrolyzed by the voltage applied to the electrodes. The concentration of lactate in the sample can be measured by measuring the oxidation current. Instead of oxygen, an electron carrier can be used as the target for reduction during the oxidation of lactic acid. The electron carrier is reduced from its oxidized form to its reduced form during the enzymatic reaction of lactic acid. The concentration of lactic acid can be measured by measuring the oxidation current generated when this reduced electron carrier is oxidized on an electrode. By using an electron carrier with a low oxidation potential, inhibition by easily oxidizable substances such as ascorbic acid, uric acid, and reduced glutathione can be reduced when they coexist in the sample.

[0017] Known electron transfer substances can be used in the present disclosure, such as potassium ferricyanide, p-benzoquinone, phenazine methosulfate, methylene blue, and ferrocene derivatives. Whether the target to be reduced is oxygen or an electron carrier is used, lactate oxidase itself is not consumed. Therefore, the lactate sensor of the present disclosure based on current measurement can be used repeatedly and can measure lactate in real time. Lactate is produced in conjunction with sugar metabolism and is said to be an indicator of the level of stress that exercise places on the body, for example. When measuring lactate while exercising, being able to measure it in real time is advantageous. Furthermore, if the sensor can be used repeatedly, it does not need to be disposable, which is preferable when used as a wearable sensor.

[0018] In the present disclosure, the thickness of the reagent layer is not particularly limited, but is preferably 0.01 mm to 3.0 mm, and more preferably 0.1 mm to 1.0 mm. A thinner reagent layer than the above is preferable from the standpoint of miniaturizing, thinning, and flexibility of the sensor, but it may reduce mechanical strength and may not be able to immobilize a sufficient amount of lactate oxidase for lactate detection. On the other hand, a thicker reagent layer than the above will contain more lactate oxidase and fibroin, and a larger amount of sample will be required, potentially resulting in poor response to changes in sample concentration. When the reagent layer is primarily composed of a carrier carrying lactate oxidase and fibroin (carriers carrying lactate oxidase and fibroin are sometimes referred to as "carriers carrying lactate oxidase and fibroin" to distinguish them from carriers not carrying them), the thickness of the reagent layer depends on the thickness of the carrier and can be adjusted by the thickness of the carrier used. In addition to the carrier that supports lactate oxidase and fibroin, various additives may be added to the reagent layer as needed.

[0019] (lactate oxidase) The lactate oxidase used in the present disclosure is not particularly limited as long as it is a protein having lactate oxidase activity, but the lactate oxidase described in Japanese Patent No. 5593689 is preferred. In the present disclosure, the amount of lactate oxidase used is adjusted appropriately depending on the lactate concentration in the sample. 2The lactate oxidase content is preferably 0.1 U to 20 U, and more preferably 0.3 U to 5 U, per unit volume. If the amount of lactate oxidase is less than the above range, a sufficient response current cannot be obtained, resulting in a decrease in detection sensitivity. On the other hand, if the amount of lactate oxidase is large, the response current will be large, but the response current may saturate in high-concentration samples, resulting in a decrease in the linearity of the calibration curve. The method for applying lactate oxidase to the reagent layer is not particularly limited, but a preferred method involves preparing an aqueous solution containing lactate oxidase and preferably fibroin, impregnating the carrier with the solution, and drying the impregnated carrier to form a carrier. The aqueous solution can be dried using any known method, but a low drying temperature is preferred to prevent the inactivation of lactate oxidase. The drying temperature generally used is from room temperature to about 60°C.

[0020] (fibroin) The inventors of the present invention have found that when a carrier such as filter paper is impregnated with an aqueous solution of lactate oxidase and then dried, the enzymatic activity is significantly reduced, but that adding fibroin to the aqueous solution of lactate oxidase can prevent the inactivation of lactate oxidase in the above process. In the present disclosure, fibroin acts as a stabilizer for lactate oxidase and suppresses the inactivation of lactate oxidase when the carrier is impregnated with a lactate oxidase solution. Because fibroin can suppress the inactivation of lactate oxidase during impregnation, a highly sensitive lactate sensor can be obtained with a small amount of lactate oxidase.

[0021] Although the mechanism by which fibroin stabilizes lactate oxidase is unclear, it is thought that lactate oxidase is stabilized by the following mechanism. When a porous material such as filter paper is impregnated with an aqueous solution of lactate oxidase, the water is rapidly absorbed into the porous material, and the enzyme is thought to be unable to maintain its structure in the aqueous solution, denaturing and inactivating it. On the other hand, when fibroin is added to the enzyme aqueous solution, it is thought that the fibroin surrounds the enzyme in place of the water that is lost, thereby maintaining its structure in the aqueous solution and stabilizing the enzyme.

[0022] The fibroin used in the present disclosure is a fibrous protein derived from an organism classified in the order Lepidoptera, Hymenoptera, or Araneae. The fibroin is not limited to the organism from which it is derived, and may also be a fibroin whose sequence has been partially modified. Fibroin may also be purified from cocoons or the like, or may be produced by genetic engineering technology. Needless to say, commercially available products may also be used. From the viewpoint of easy availability of raw materials, fibroin derived from domesticated silkworm cocoons is preferred. The fibroin used in the present disclosure can be derived from domesticated silkworm cocoons, cocoon threads, processed cocoon thread products (e.g., silk threads), and residual threads from processed cocoon thread products. In other words, in the present disclosure, fibroin derived from silk (silk) can be used, such as domesticated silkworm cocoons, cocoon threads, processed cocoon thread products (e.g., silk threads), and residual threads from processed cocoon thread products. Fibroin can be obtained from these raw materials by removing sericin using a known refining method. The obtained fibroin can be dissolved in a highly concentrated aqueous solution of lithium bromide or calcium chloride, and then desalted by methods such as dialysis or ultrafiltration using a semipermeable membrane to obtain an aqueous solution. The obtained aqueous solution is unstable and forms a gel and solidifies when left at room temperature; therefore, it is preferable to store it refrigerated at around 4°C.

[0023] In the present disclosure, the fibroin used is not particularly limited in terms of molecular weight, but the higher the molecular weight of the fibroin, the greater the stabilizing effect on lactate oxidase, and fibroin with a molecular weight of 100,000 or more is preferred. In the present disclosure, the fibroin preferably has a molecular weight of 100,000 or more, more preferably 150,000 or more. Furthermore, since the molecular weight of fibroin derived from domesticated silkworms is 350,000, the upper limit of the molecular weight of the fibroin used in this embodiment is 350,000. In other words, the preferred molecular weight of fibroin is 100,000 or more and 350,000 or less (100 kDa or more and 350 kDa or less). The molecular weight of fibroin can be controlled by the temperature and time during degumming. Fibroin derived from domesticated silkworms has a molecular weight of around 350,000, but fibroin of the desired molecular weight can be obtained by changing the degumming temperature and time. Generally, the higher the degumming temperature, the lower the molecular weight of fibroin obtained, and the longer the degumming time, the lower the molecular weight of fibroin obtained.

[0024] In the present disclosure, the amount of fibroin added is preferably 5 μg to 1000 μg, more preferably 40 μg to 400 μg, per 1 U of lactate oxidase. If the amount of fibroin added is less than the above, the lactate oxidase cannot be sufficiently stabilized. On the other hand, if the amount of fibroin added is more than the above, although sufficient stabilization of lactate oxidase is achieved, the lactate oxidase concentration in the reagent layer will be relatively reduced, which may lead to a decrease in sensor sensitivity. The method for applying fibroin to the reagent layer is not particularly limited, but a preferred method involves mixing an aqueous fibroin solution with the aqueous lactate oxidase solution used when applying lactate oxidase to the reagent layer, impregnating a carrier with this, and drying the impregnated carrier to form a carrier. The aqueous solution can be dried using known methods, but a low drying temperature is preferred to prevent deactivation of lactate oxidase. Drying temperatures generally range from room temperature to approximately 60°C. The above-mentioned addition method provides a sufficient stabilizing effect on lactate oxidase.

[0025] (Carrier) In the present disclosure, the carrier is used to immobilize lactate oxidase and fibroin on the electrode. The carrier preferably contains any material selected from the group consisting of glass, cellulose, and cellulose acetate. By using a carrier made of such a material, the stabilization effect of fibroin against lactate oxidase can be fully exerted. When the carrier contains glass, the glass is preferably fibrous, and glass fiber filter paper, etc., can be used as the carrier. Examples of glass fibers that can be used include borosilicate glass fibers and silica glass (quartz glass) fibers, and the glass fibers may contain a binder as needed. When the carrier contains cellulose, the cellulose is preferably fibrous or porous, and cellulose filter paper, regenerated cellulose membrane, etc. can be used as the carrier. When the carrier contains cellulose acetate, the cellulose acetate is preferably fibrous or porous, and a cellulose acetate membrane or the like can be used as the carrier. In the present disclosure, the carrier may be composed of any of the above materials alone or may be a composite containing a plurality of materials. Furthermore, the carrier may contain materials other than the above materials, such as various additives, as necessary. In the present disclosure, the thickness of the carrier is not particularly limited, but is preferably adjusted according to the design of the layer thickness of the reagent layer. The thickness of the carrier is usually preferably 0.01 mm or more and 3.0 mm or less, and more preferably 0.1 mm or more and 1.0 mm or less.

[0026] As a second embodiment, the present invention provides an article including the lactate sensor according to the first embodiment. Examples of the article include clothing such as T-shirts, hats, pants, and underwear, armbands, wristwatches, smartwatches, smartphones, storage cases for smartphones and the like, bags, medical measuring devices, and simple measuring devices, all of which include a lactate sensor.

[0027] An article including a lactate sensor can include an electrode analyzer, an ammeter, an electrometer, etc. in addition to the lactate sensor according to the first embodiment.

[0028] In a third embodiment, the present invention provides a method for manufacturing a lactate sensor, comprising the steps of obtaining an electrode substrate having an electrode formed on an insulating substrate, preparing an aqueous solution containing lactate oxidase and fibroin, impregnating a carrier with the aqueous solution to obtain an impregnated carrier, drying the impregnated carrier impregnated with the aqueous solution to obtain a carrier carrying the lactate oxidase and the fibroin, and placing the carrier on the electrode.

[0029] The manufacturing method according to this embodiment is shown in Figure 2. Step S1 for obtaining an electrode substrate is as described in the section on electrodes, and can be performed by forming an electrode material such as conductive ink on an insulating material such as plastic, paper, glass, or ceramic by a conventionally known method such as screen printing or sputtering. It is also possible to purchase a commercially available electrode substrate such as a printed electrode.

[0030] Step S2 of preparing an aqueous solution containing lactate oxidase and fibroin, step S3 of impregnating a carrier with the aqueous solution to obtain an impregnated carrier, and step S4 of drying the impregnated carrier to obtain a loaded carrier are as described in the sections on lactate oxidase and fibroin.

[0031] In step S2 of preparing an aqueous solution containing lactate oxidase and fibroin, an aqueous solution containing lactate oxidase and fibroin is prepared. The lactate oxidase-fibroin aqueous solution preferably contains 50 U / mL to 500 U / mL of lactate oxidase and 4 mg / mL to 40 mg / mL of fibroin. The aqueous solution may also contain various additives as needed. Preferably, the fibroin is derived from any material selected from the group consisting of domesticated silkworm cocoons, cocoon filaments, and processed cocoon filaments, and has a molecular weight of 100,000 to 350,000. The lactate oxidase is not particularly limited as long as it is a protein having lactate oxidase activity, and commercially available products may be used.

[0032] In step S3 of impregnating a carrier with an aqueous solution to obtain an impregnated carrier, the carrier is impregnated with an aqueous solution containing lactate oxidase and fibroin. The carrier is preferably made of a material such as glass, cellulose, or cellulose acetate, and is preferably fibrous or porous, such as filter paper or glass fiber filter paper. The thickness of the carrier is preferably 0.01 mm or more and 3.0 mm or less, more preferably 0.1 mm or more and 1.0 mm or less. In addition, the thickness of the carrier is preferably 0.01 mm or more and 3.0 mm or less, more preferably 0.1 mm or more and 1.0 mm or less. 2 It is preferable to adjust the amount of the carrier so that the lactate oxidase concentration is preferably 0.1 U or more and 20 U or less, more preferably 0.3 U or more and 5 U or less, per 100 ml of the solution.

[0033] In step S4, the impregnated carrier is dried to obtain a carrier loaded with lactate oxidase, and the impregnated carrier is dried by a known method. The drying temperature is not particularly limited, but can be, for example, room temperature (20°C or higher) to 60°C or lower. Temperatures above 60°C may cause the activity of lactate oxidase to be inactivated, so a temperature of 60°C or lower is preferred.

[0034] In step S5 of placing the carrier on the electrode, the carrier is placed so as to cover at least the counter electrode and the measurement electrode of the electrode substrate. If a reference electrode is present, the carrier does not need to cover the reference electrode. The carrier may be used as a reagent layer, or, if necessary, an additive or the like may be added to form the reagent layer. [Example]

[0035] The present disclosure will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples as long as it does not depart from the gist of the present invention. Note that, with regard to the amount of components, "parts" and "%" are by mass unless otherwise specified.

[0036] <Preparation of fibroin aqueous solution A> (scouring process) 4.5 L of ultrapure water was heated to a boil in a 5 L glass beaker, and then 8.48 g of sodium carbonate (Kishida Chemical Co., Ltd.) was added to prepare a 0.02 mol / L sodium carbonate solution. 10 g of cut cocoons (Tajima Shoji Co., Ltd.) cut into approximately 1 cm cubes were added to the mixture and heated for 30 minutes to obtain fibroin from which sericin had been removed. The fibroin was washed with cold ultrapure water, drained, and dried overnight in a fume hood to obtain refined fibroin.

[0037] (Neutral salt dissolution process) 80.7 g of lithium bromide anhydrous (Kishida Chemical Co., Ltd.) was added to a measuring flask and the volume was adjusted to 100 mL to obtain a 9.3 mol / L lithium bromide solution. 3.0 g of refined fibroin was placed in a 100 mL glass beaker, and 14.8 mL of 9.3 mol / L LiBr solution was added so that the refined fibroin was completely immersed. Dissolution was carried out in a 60°C oven for 2 hours, yielding a clear neutral salt solution.

[0038] (Desalination process) A syringe was used to inject 19 mL of the neutral salt solution prepared above into a 30 mL dialysis cassette (Thermo Scientific) with a molecular weight cutoff of 3500. The cassette was then immersed in 2 L of ultrapure water for dialysis. The water was replaced 1 hour and 4 hours after the start of dialysis, and then every 8 hours thereafter. Dialysis was continued for a total of 53 hours, resulting in desalting. The resulting solution was centrifuged twice in a CR7N centrifuge (Eppendorf-Himac Technologies) at 11,000 rpm at 4°C for 20 minutes to precipitate the insoluble matter, yielding fibroin aqueous solution A.

[0039] (Measurement of fibroin aqueous solution concentration) 0.5 mL of the above fibroin aqueous solution was placed in a tared glass container and dried for more than 2 hours in an oven adjusted to 60°C.The solid concentration was calculated from the change in weight before and after drying, and the solid concentration of the resulting fibroin aqueous solution A was found to be 8%.

[0040] (Measurement of fibroin molecular weight) The molecular weight of the obtained fibroin aqueous solution was measured using a microchip electrophoresis device, Agilent 2100 Bioanalyzer Electrophoresis System (manufactured by Agilent) under the following conditions. Microchip, separation matrix, fluorescent dye, electrophoresis buffer, molecular weight standard ladder: Agilent Protein 230 Kit Control sample: Bovine serum albumin lyophilized powder, >96% (agarose gel electrophoresis) (Sigma-Aldrich, molecular weight 66.5 kDa) Dilution and concentration of silk fibroin aqueous solution and control sample: The silk fibroin aqueous solution was diluted to 1.0-1.5% by mass / volume using an 8M urea aqueous solution, and the control sample was diluted to approximately 1.3% by mass / volume. Excitation wavelength: 630nm Detection wavelength: 680nm The molecular weight of silk fibroin was calculated using the dedicated 2100 Expert software. The molecular weight of silk fibroin was calculated using a molecular weight calibration curve obtained from the data of the molecular weight standard ladder measured along with the sample. The band in the electrophoresis used for molecular weight calculation was the band with the darkest color. The molecular weight of fibroin aqueous solution A determined by the above method was 150 kDa.

[0041] <Preparation of fibroin aqueous solution B> Fibroin aqueous solution B was prepared in the same manner as fibroin aqueous solution A, except that the heating time in the scouring process of fibroin aqueous solution A was changed from 30 minutes to 10 minutes. The molecular weight of fibroin aqueous solution B was 230 kDa.

[0042] <Preparation of fibroin aqueous solution C> Fibroin aqueous solution C was prepared in the same manner as fibroin aqueous solution A, except that the heating time in the refining process of fibroin aqueous solution A was changed from 30 minutes to 150 minutes. The molecular weight of fibroin aqueous solution C was 90 kDa.

[0043] Example 1 (Preparation of reagent layer) A lactate oxidase-fibroin aqueous solution containing lactate oxidase (Toyobo, LCO-301, activity 108 U / mg) and the above fibroin aqueous solution A was prepared using ultrapure water. The lactate oxidase-fibroin aqueous solution contained 240 U / mL of lactate oxidase and 20 mg / mL of fibroin. 1 μL of the prepared aqueous solution was spotted on cellulose filter paper (Cytiva, Whatman Grade 1, thickness 0.18 mm) and dried at room temperature (25°C) for 1 hour. The spot diameter was 0.64 cm, and the lactate oxidase concentration was 0.75 U / cm. 2 is.

[0044] (Enzyme activity evaluation) The enzyme activity of lactate oxidase in the reagent layer prepared as above was evaluated by the following method. The following reagents A to F were mixed to prepare a reaction solution. Reagent A: 0.1M phosphate buffer (pH 7.4) (Fujifilm Wako Pure Chemical Industries, Ltd.) 18.3mL Reagent B: 3.7 mL of 8% aqueous solution of DL-lithium lactate (Kishida Chemical Co., Ltd.) Reagent C: 0.5% 4-aminoantipyrine (Kishida Chemical) aqueous solution 0.18 mL Reagent D: 0.18 mL of 40 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (Tokyo Chemical Industry Co., Ltd.) aqueous solution Reagent E: 0.18 mL of 500 U / mL horseradish peroxidase (Sigma-Aldrich) aqueous solution Reagent F: 0.1% sodium cholate (Kishida Chemical) aqueous solution 0.75 mL A throw-in spectrophotometer (JASCO Corporation, MV-3000, optical path length 1 cm) was placed in the prepared reaction solution, and the temperature was adjusted to 37°C while stirring. The reagent layer prepared above was placed in the reaction solution, and the change in absorbance at a measurement wavelength of 555 nm was measured for 5 minutes. The lactate oxidase activity of the reagent layer was calculated from the slope of the change in absorbance over time. Separately, the change in absorbance when 1 μL of a lactate oxidase-fibroin aqueous solution was added was measured, and the enzyme activity of the reagent layer was evaluated based on this measurement. The enzyme activity was evaluated according to the following criteria, and if the evaluation rank was A to B, the enzyme activity of the reagent layer was judged to be good. A: The enzyme activity in the reagent layer is 75% or more of the standard. B: Enzyme activity in the reagent layer is 50% or more but less than 75% of the standard. C: Enzyme activity in the reagent layer is 25% or more but less than 50% of the standard. D: Enzyme activity in the reagent layer is less than 25% of the standard.

[0045] (Lactic acid sensor evaluation) The above reagent layer was printed on a printed electrode (Biodevice Technology, round carbon electrode EP, electrode area 2.64 mm 2 ) to create a prototype lactate sensor. The prototype lactate sensor was evaluated according to the following method. The prototype electrode was connected to an electrochemical analyzer (BAS, ALS660D), and a voltage of +0.8 V was applied relative to the Ag / AgCl reference electrode on the electrode. The response current was measured by chronoamperometry. The electrode was immersed in 0.7 mL of PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.) and left to stand with stirring until the current stabilized. A 350 mM DL-lithium lactate solution in PBS(-) was added dropwise to the electrode so that the lithium lactate concentration ranged from 0.5 to 50 mM. The response current was measured until equilibrium was reached after the addition of the lithium lactate solution. The integrated current value for 10 seconds after equilibrium was recorded as the response current at each lactate concentration. The lactate sensors were evaluated according to the following criteria, and if the evaluation rank was A to B, the sensitivity of the lactate sensor was judged to be good. A: Response current value of 2 μA or more at a lactate concentration of 10 mM B: Response current value of 1 μA or more and less than 2 μA at lactate concentration of 10 mM C: Response current value at lactate concentration of 10 mM is 0.5 μA or more and less than 1 μA D: Response current value of 10 mM lactate is less than 0.5 μA

[0046] Example 2 The reagent layer and lactate sensor of Example 2 were fabricated by using fibroin aqueous solution B instead of fibroin aqueous solution A of Example 1.

[0047] Example 3 The reagent layer and lactate sensor of Example 3 were fabricated by using fibroin aqueous solution C instead of fibroin aqueous solution A of Example 1.

[0048] Example 4 The reagent layer and lactate sensor of Example 4 were fabricated by using glass fiber filter paper (Whatman GF / A, manufactured by Cytiva, thickness 0.26 mm) instead of the cellulose filter paper of Example 1.

[0049] Example 5 The reagent layer and lactate sensor of Example 5 were fabricated by using a cellulose acetate membrane (ST69 manufactured by Cytiva, thickness 0.14 mm) instead of the cellulose filter paper of Example 1.

[0050] Example 6 The reagent layer and lactate sensor of Example 6 were fabricated by using a cellulose mixed ester membrane (ME29 manufactured by Cytiva, thickness 0.15 mm) instead of the cellulose filter paper of Example 1.

[0051] Example 7 The reagent layer and lactate sensor of Example 7 were fabricated by changing the concentration of the aqueous lactate oxidase solution of Example 1 to 24 U / mL.

[0052] Example 8 The concentration of the aqueous lactate oxidase solution of Example 1 was adjusted to 2400 U / mL to prepare the reagent layer and lactate sensor of Example 8.

[0053] Example 9 The reagent layer and lactate sensor of Example 9 were fabricated by adjusting the concentration of the fibroin aqueous solution A of Example 1 to 1 mg / mL.

[0054] Example 10 The reagent layer and lactate sensor of Example 10 were fabricated by adjusting the concentration of the fibroin aqueous solution A of Example 1 to 200 mg / mL.

[0055] Example 11 The lactate sensor of Example 11 was fabricated by changing the substrate of the printed electrode of Example 1 to a PET film (thickness: 0.075 mm).

[0056] (Comparative Example 1) A reagent layer and a lactate sensor of Comparative Example 1 were prepared by omitting the fibroin aqueous solution A in Example 1.

[0057] (Comparative Example 2) A reagent layer and a lactate sensor of Comparative Example 2 were fabricated by using trehalose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) instead of the fibroin aqueous solution A in Example 1.

[0058] (Comparative Example 3) A reagent layer and a lactate sensor of Comparative Example 3 were fabricated by using bovine serum albumin (BSA, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) instead of the fibroin aqueous solution A in Example 1.

[0059] Comparative Example 4 A reagent layer and a lactate sensor of Comparative Example 4 were prepared by using gelatin hydrolysate (manufactured by Nitta Gelatin Co., Ltd.) instead of the fibroin aqueous solution A in Example 1.

[0060] The evaluation results of the reagent layer and lactate sensor fabricated as described above are summarized in Table 1. [Table 1]

[0061] As shown in Table 1, the use of a carrier consisting of fibroin, cellulose, glass, and cellulose acetate, which is a constituent element of the present disclosure, prevents the inactivation of lactate oxidase. Furthermore, it is clear that a lactate sensor having a reagent layer configured as described above can be a highly sensitive lactate sensor with a small amount of enzyme.

[0062] Embodiments of the present disclosure include the following configurations and methods. (Configuration 1) A lactate sensor comprising an insulating substrate, an electrode formed on the insulating substrate, and a reagent layer formed on at least a portion of the electrode, the reagent layer comprising lactate oxidase, fibroin, and a carrier. (Configuration 2) 2. The lactate sensor according to claim 1, wherein the support includes at least one selected from the group consisting of glass, cellulose, and cellulose acetate. (Configuration 3) 3. The lactate sensor according to claim 1, wherein the molecular weight of the fibroin is 100,000 or more and 350,000 or less. (Configuration 4) 4. The lactate sensor according to any one of configurations 1 to 3, wherein the fibroin is derived from any one selected from the group consisting of domesticated silkworm cocoons, cocoon threads, and processed cocoon threads. (Configuration 5) The reagent layer is formed by dissolving the lactate oxidase in a solution of 1 cm 2 5. The lactate sensor according to any one of configurations 1 to 4, containing 0.1 U or more and 20 U or less per unit area. (Configuration 6) 6. The lactate sensor according to any one of configurations 1 to 5, wherein the reagent layer contains 5 μg to 1000 μg of the fibroin per 1 U of the lactate oxidase. (Configuration 7) 7. The lactate sensor according to any one of configurations 1 to 6, wherein the reagent layer further contains an electron transfer substance. (Configuration 8) 8. The lactate sensor according to any one of configurations 1 to 7, wherein the electrodes include a measurement electrode and a counter electrode. (Configuration 9) 9. The lactate sensor according to any one of configurations 1 to 8, which is in the form of a sheet. (Configuration 10) 10. The lactate sensor according to any one of configurations 1 to 9, wherein the insulating substrate and the electrodes have a combined thickness of 0.1 mm or less. (Configuration 11) 11. The lactate sensor according to any one of configurations 1 to 10, wherein the reagent layer has a thickness of 0.01 mm or more and 3.0 mm or less. (Configuration 12) 12. An article comprising the lactate sensor according to any one of claims 1 to 11. (Method 1) A process for obtaining an electrode substrate by forming electrodes on an insulating substrate. providing an aqueous solution containing lactate oxidase and fibroin; impregnating a carrier with the aqueous solution to obtain an impregnated carrier; a step of drying the impregnated carrier impregnated with the aqueous solution to obtain a carrier carrying the lactate oxidase and the fibroin; and placing the support carrier on the electrode; A method for manufacturing a lactate sensor comprising the steps of: [Explanation of symbols]

[0063] 1. Insulating substrate 2 electrode layer 3 Reagent layer

Claims

1. A lactate sensor comprising an insulating substrate, an electrode formed on the insulating substrate, and a reagent layer formed on at least a portion of the electrode, the reagent layer comprising lactate oxidase, fibroin, and a carrier.

2. 2. The lactate sensor according to claim 1, wherein the carrier includes at least one selected from the group consisting of glass, cellulose, and cellulose acetate.

3. 2. The lactate sensor according to claim 1, wherein the molecular weight of the fibroin is 100,000 or more and 350,000 or less.

4. 2. The lactate sensor according to claim 1, wherein the fibroin is derived from any one selected from the group consisting of domesticated silkworm cocoons, cocoon filaments, and processed cocoon filaments.

5. The reagent layer is a layer of the carrier containing the lactate oxidase. ２ The lactate sensor according to claim 4 , wherein the lactate sensor contains 0.1 U or more and 20 U or less per unit area.

6. 2. The lactate sensor according to claim 1, wherein the reagent layer contains 5 μg to 1000 μg of fibroin per 1 U of the lactate oxidase.

7. The lactate sensor of claim 1 , wherein the reagent layer further comprises an electron mediator.

8. The lactate sensor according to claim 1 , wherein the electrodes include a measurement electrode and a counter electrode.

9. 2. The lactate sensor according to claim 1, which is in the form of a sheet.

10. 10. The lactate sensor according to claim 9, wherein the insulating substrate and the electrodes have a combined thickness of 0.1 mm or less.

11. 10. The lactate sensor according to claim 9, wherein the reagent layer has a thickness of 0.01 mm or more and 3.0 mm or less.

12. An article comprising the lactate sensor of any one of claims 1 to 11.

13. A process for obtaining an electrode substrate by forming electrodes on an insulating substrate. providing an aqueous solution containing lactate oxidase and fibroin; impregnating a carrier with the aqueous solution to obtain an impregnated carrier; a step of drying the impregnated carrier impregnated with the aqueous solution to obtain a carrier carrying the lactate oxidase and the fibroin; and placing the support carrier on the electrode; A method for manufacturing a lactate sensor comprising the steps of:

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