Lactic acid sensor

Abstract

Provided is a lactic acid sensor exhibiting high sensitivity with a small amount of an enzyme, wherein deactivation of lactate oxidase on a support is prevented. The lactic acid sensor comprises an insulating substrate, an electrode formed on the insulating substrate, and a reagent layer formed on at least part of the electrode, wherein the reagent layer includes lactate oxidase, fibroin, and a support.

Description

Lactate Sensor

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

[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.

[0003] 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.

[0004] 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 effectively act as stabilizers for glucose oxidase and peroxidase.

[0005] Japanese Patent No. 3498105 Japanese Patent Application Laid-Open No. 2009-244013 Japanese Patent Application Laid-Open No. 2005-114368

[0006] 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, the stabilizers described in Patent Document 3 were insufficient to stabilize lactate oxidase, and a more effective stabilizer was desired.

[0007] In order to solve the above-mentioned problems, an object of the present disclosure is 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.

[0008] 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.

[0009] 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.

[0010] 1 is a schematic diagram showing an example of a lactate sensor according to a first embodiment of the present disclosure; FIG. 2 is a diagram showing steps of a method for manufacturing a lactate sensor according to a third embodiment of the present disclosure;

[0011] 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 3 and an electrode layer 2 thereon. The electrode layer is a layer including an electrode. The electrode does not need to be formed on the entire surface of the substrate. The lactate sensor further includes a reagent layer 1 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.

[0012] (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.

[0013] (Electrodes) 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.

[0014] The lactate sensor of the present disclosure has a measuring electrode and a counter electrode formed on the insulating substrate (two-electrode system), but 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.

[0015] 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 an oxidation current (response current) that flows when a potential is applied to a 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 the working electrode, relative to the reference electrode.

[0016] 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 to form 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 constituting each electrode (measurement electrode, counter electrode, 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.

[0017] 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.

[0018] 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 thereof is preferably 3 mm or less, and more preferably 2 mm or less.

[0019] 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, etc., 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.

[0020] (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 contains at least lactate oxidase, fibroin, and a carrier. In the reagent layer, lactate oxidase oxidizes lactate in the sample to pyruvic acid and reduces oxygen in the air to produce hydrogen peroxide. The generated hydrogen peroxide is instantly oxidized and electrolyzed by a voltage applied to the electrode. The concentration of lactate in the sample can be measured by measuring the oxidation current.

[0021] 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.

[0022] Known electron transfer substances can be used in the present disclosure, such as potassium ferricyanide, p-benzoquinone, phenazine methosulfate, methylene blue, and ferrocene derivatives.

[0023] 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 imposes 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.

[0024] 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 may contain a larger amount of lactate oxidase and fibroin, requiring a larger amount of sample, which may result in poor response to changes in sample concentration. When the reagent layer is primarily composed of a carrier supporting lactate oxidase and fibroin (a carrier supporting lactate oxidase and fibroin may sometimes be referred to as a "carrier-supported carrier" to distinguish it from a carrier not supporting lactate oxidase and fibroin), 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 supporting lactate oxidase and fibroin, various additives may be added to the reagent layer as needed.

[0025] (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.

[0026] In the present disclosure, the amount of lactate oxidase used is adjusted appropriately depending on the lactate concentration in the sample. ２ The 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.

[0027] 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 a carrier with the solution, and drying the impregnated carrier to form a carrier. Known methods can be used to dry the aqueous solution, but a low drying temperature is preferred from the perspective of preventing the inactivation of lactate oxidase. Drying temperatures generally used are from room temperature to about 60°C.

[0028] (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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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 (such as silk threads), and residual threads from processed cocoon thread products. In other words, in the present disclosure, fibroin derived from silk (silk) such as domesticated silkworm cocoons, cocoon threads, processed cocoon thread products (such as silk threads), and residual threads from processed cocoon thread products can be used. 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.

[0033] In the present disclosure, the molecular weight of the fibroin used is not particularly limited, 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).

[0034] 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.

[0035] 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, a sufficient stabilization effect of lactate oxidase cannot be obtained. On the other hand, if the amount of fibroin added is more than the above, although sufficient stabilization of lactate oxidase can be obtained, the concentration of lactate oxidase in the reagent layer will be relatively reduced, which may lead to a decrease in sensor sensitivity.

[0036] 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. Known methods can be used to dry the aqueous solution, but a low drying temperature is preferred from the perspective of preventing deactivation of lactate oxidase. Drying temperatures generally used are from room temperature to about 60°C. The above-mentioned addition method provides a sufficient stabilizing effect on lactate oxidase.

[0037] (Carrier) In the present disclosure, a carrier is used to immobilize lactate oxidase and fibroin on an electrode. The carrier preferably contains any material selected from the group consisting of glass, cellulose, and cellulose acetate. By using a carrier made of the above material, the stabilizing effect of fibroin on lactate oxidase is fully exerted.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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 an electrode material such as conductive ink can be formed 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.

[0047] 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.

[0048] 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.

[0049] 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 amount of lactate oxidase 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.

[0050] 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 and 60° C. or lower). Temperatures above 60° C. may result in the inactivation of lactate oxidase, so a temperature of 60° C. or lower is preferred.

[0051] 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.

[0052] 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.

[0053] <Preparation of fibroin aqueous solution A> (Scouring step) 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 domesticated silkworm cocoons (Tajima Shoji Co., Ltd.) chopped into approximately 1 cm cubes were added 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.

[0054] (Neutral salt dissolution step) 80.7 g of lithium bromide anhydrous (Kishida Chemical Co., Ltd.) was added to a measuring flask and the volume was increased to 100 mL to obtain a 9.3 mol / L lithium bromide aqueous 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 an oven at 60 ° C. for 2 hours to obtain a transparent neutral salt aqueous solution.

[0055] (Desalting step) 19 mL of the neutral salt aqueous solution prepared above was injected into a 30 mL dialysis cassette (manufactured by Thermo Scientific) with a molecular weight cutoff of 3,500 using a syringe, and the cassette was 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, for a total of 53 hours of dialysis and desalting. The resulting aqueous solution was centrifuged twice in a centrifuge CR7N (manufactured by Eppendorf Himac Technologies) at 11,000 rpm, 4 ° C, and 20 minutes to precipitate insoluble matter, and a fibroin aqueous solution A was obtained.

[0056] (Measurement of fibroin aqueous solution concentration) 0.5 mL of the above fibroin aqueous solution was placed in a tared glass container and dried in an oven adjusted to 60°C for more than 2 hours.The solids concentration was calculated from the change in weight before and after drying, and the solids concentration of the obtained fibroin aqueous solution A was 8%.

[0057] (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 (Agilent) under the following conditions. Microchip, separation matrix, fluorescent dye, electrophoresis buffer, molecular weight standard ladder: Agilent Protein 230 Kit. Control sample: Lyophilized bovine serum albumin powder, >96% (agarose gel electrophoresis) (Sigma-Aldrich, molecular weight 66.5 kDa). Dilutions and concentrations of silk fibroin aqueous solution and control sample: The silk fibroin aqueous solution was diluted to 1.0-1.5% by mass / volume, and the control sample was diluted to approximately 1.3% by mass / volume using an 8 M urea aqueous solution. Excitation wavelength: 630 nm. Detection wavelength: 680 nm. The molecular weight of silk fibroin was calculated using the dedicated 2100Expert 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 together with the sample. The band of 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.

[0058] <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 step of the fibroin aqueous solution A was changed from 30 minutes to 10 minutes. The molecular weight of fibroin aqueous solution B was 230 kDa.

[0059] <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 scouring step of the fibroin aqueous solution A was changed from 30 minutes to 150 minutes. The molecular weight of fibroin aqueous solution C was 90 kDa.

[0060] (Example 1) (Preparation of Reagent Layer) A lactate oxidase-fibroin aqueous solution containing lactate oxidase (Toyobo, LCO-301, activity value 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 (Whatman Grade 1, Cytiva, thickness 0.18 mm) and dried at room temperature (25°C) for 1 hour. The spot diameter at this time was 0.64 cm, and the lactate oxidase amount was 0.75 U / cm ２ is.

[0061] (Enzyme Activity Evaluation) The enzymatic 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: 0.1 M phosphate buffer (pH 7.4) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) 18.3 mL Reagent B: 8% aqueous solution of lithium DL-lactate (manufactured by Kishida Chemical Co., Ltd.) 3.7 mL Reagent C: 0.5% aqueous solution of 4-aminoantipyrine (manufactured by Kishida Chemical Co., Ltd.) 0.18 mL Reagent D: 40 mM aqueous solution of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.18 mL Reagent E: 500 U / mL aqueous solution of horseradish peroxidase (manufactured by Sigma-Aldrich) 0.18 mL Reagent F: 0.1% aqueous solution of sodium cholate (manufactured by Kishida Chemical Co., Ltd.) 0.75 mL A throw-in spectrophotometer (manufactured by JASCO Corporation, MV-3000, light 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 added to 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 enzymatic activity of the reagent layer was evaluated based on this measurement. The enzymatic activity was evaluated according to the following criteria, and an evaluation rank of A to B indicated that the enzymatic activity of the reagent layer was good. A: The enzymatic activity of the reagent layer was 75% or more of the standard. B: The enzymatic activity of the reagent layer was 50% or more but less than 75% of the standard. C: The enzymatic activity of the reagent layer was 25% or more but less than 50% of the standard. D: The enzymatic activity of the reagent layer was less than 25% of the standard.

[0062] (Lactate sensor evaluation) The above-mentioned reagent layer was printed on a printed electrode (Biodevice Technology, round carbon electrode EP, electrode area 2.64 mm ２) to fabricate 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 +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 value stabilized. A PBS(-) solution of 350 mM DL-lithium lactate was added dropwise to the solution so that the lithium lactate concentration ranged from 0.5 to 50 mM. Measurements were continued until the change in response current after the addition of the lithium lactate solution reached equilibrium, and the integrated current value for 10 seconds after equilibrium was recorded as the response current at each lactate concentration. Lactate sensors were evaluated according to the following criteria. A rating of A to B indicated that the lactate sensor had good sensitivity. A: The response current value at a lactate concentration of 10 mM is 2 μA or more. B: The response current value at a lactate concentration of 10 mM is 1 μA or more but less than 2 μA. C: The response current value at a lactate concentration of 10 mM is 0.5 μA or more but less than 1 μA. D: The response current value at a lactate concentration of 10 mM is less than 0.5 μA.

[0063] Example 2 A reagent layer and a lactate sensor of Example 2 were fabricated by using fibroin aqueous solution B instead of fibroin aqueous solution A of Example 1.

[0064] Example 3 A reagent layer and a lactate sensor of Example 3 were fabricated by using fibroin aqueous solution C instead of fibroin aqueous solution A of Example 1.

[0065] Example 4 A reagent layer and a 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.

[0066] Example 5 A reagent layer and a 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.

[0067] Example 6 A reagent layer and a 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.

[0068] Example 7 A reagent layer and a lactate sensor of Example 7 were fabricated by changing the concentration of the aqueous lactate oxidase solution of Example 1 to 24 U / mL.

[0069] Example 8 A reagent layer and a lactate sensor of Example 8 were fabricated by changing the concentration of the aqueous lactate oxidase solution of Example 1 to 2400 U / mL.

[0070] Example 9 A reagent layer and a lactate sensor of Example 9 were fabricated by adjusting the concentration of the fibroin aqueous solution A of Example 1 to 1 mg / mL.

[0071] Example 10 A reagent layer and a lactate sensor of Example 10 were fabricated by changing the concentration of the fibroin aqueous solution A of Example 1 to 200 mg / mL.

[0072] Example 11 A 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).

[0073] 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.

[0074] 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.

[0075] 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.

[0076] Comparative Example 4 A reagent layer and a lactate sensor of Comparative Example 4 were fabricated by using gelatin hydrolysate (manufactured by Nitta Gelatin Co., Ltd.) instead of the fibroin aqueous solution A in Example 1.

[0077] The evaluation results of the reagent layer and lactate sensor fabricated as described above are summarized in Table 1.

[0078]

[0079] As shown in Table 1, the use of a carrier made of fibroin, cellulose, glass, and cellulose acetate, which is a constituent element of the present disclosure, suppresses the deactivation of lactate oxidase. Furthermore, it is clear that a lactate sensor having a reagent layer constructed as described above becomes a highly sensitive lactate sensor with a small amount of enzyme.

[0080] The present invention is not limited to the above-described embodiments, and various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the following claims are appended to apprise the public of the scope of the present invention.

[0081] This application claims priority based on Japanese Patent Application No. 2024-011098, filed January 29, 2024, the entire contents of which are incorporated herein by reference.

[0082] 1 Reagent layer 2 Electrode layer 3 Insulating substrate

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

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

4. A lactate sensor according to any one of claims 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.

5. The reagent layer is formed by dissolving the lactate oxidase in 1 cm of the carrier. ２ The lactate sensor according to claim 1 , wherein the lactate sensor contains 0.1 U or more and 20 U or less per unit area.

6. A lactate sensor according to any one of claims 1 to 5, wherein the reagent layer contains 5 μg to 1000 μg of fibroin per 1 U of the lactate oxidase.

7. The lactate sensor according to any one of claims 1 to 6, wherein the reagent layer further contains an electron transfer substance.

8. The lactate sensor according to any one of claims 1 to 7, wherein the electrodes include a measuring electrode and a counter electrode.

9. The lactate sensor according to any one of claims 1 to 8, which is in the form of a sheet.

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

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

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

13. A method for manufacturing a lactate sensor, comprising: a step of obtaining an electrode substrate having an electrode formed on an insulating substrate; a step of preparing an aqueous solution containing lactate oxidase and fibroin; a step of 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 a step of placing the carrier on the electrode.

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