Glucose measuring electrode and electrochemical sensor including the same

Abstract

To provide a glucose sensor which offers improved linearity between the glucose concentration and response current value and capability to stably make continuous measurement, and to provide an electrode that can be used for the same.SOLUTION: An electrode is provided, comprising an electrode layer containing an electrode material and a glucose redox enzyme and a permeation limiting layer covering the enzyme electrode layer, and also including a glucose diffusion layer, containing a hydrophilic substance, provided between the enzyme electrode layer and the permeation limiting layer.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an electrode for measuring glucose and an electrochemical sensor (glucose sensor) including the electrode. [Background technology]

[0002] Continuous Glucose Monitoring Using Conventional Electrochemical Sensors The system has at least four layers: a substrate, an electrode layer, an enzyme layer, and a permeation limiting layer. For example, Patent Document 1 discloses an electrochemical analyte sensor that includes an electrode and an analyte detection membrane, the analyte detection membrane including an obstacle layer and an enzyme layer, and a hydrophilic polymer layer disposed between the electrode and the obstacle layer or between the obstacle layer and the enzyme layer. Furthermore, Patent Document 2 discloses a biosensor characterized by forming a hydrophilic polymer membrane containing either an enzyme or an enzyme substrate on the surface of an electrode made of a hydrophobic material, and further discloses that the surface is covered with a permeation-limiting membrane. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO2010 / 107941 [Patent Document 2] Patent Publication No. 2002-221508 Summary of the Invention [Problem to be solved by the invention]

[0004] In blood glucose monitoring using a glucose sensor, it is important to monitor blood glucose levels accurately and continuously. Therefore, there has been a demand for glucose sensors that further improve the linearity of the detection current value relative to the glucose concentration and improve the stability of the electrodes so that the detection current value and linearity do not decrease even during long-term measurements. Therefore, the inventors evaluated a conventional glucose sensor equipped with an electrode having an enzyme electrode layer and a permeation-limiting layer covering it, and found that the linearity of the response current value decreased as the glucose concentration increased, and further that the response current value gradually decreased over long-term measurements. Therefore, the present invention aims to provide an enzyme electrode and a glucose sensor equipped with the same, which improves the linearity between the glucose concentration and the response current value and enables stable, continuous measurement by improving the conventional electrode having an enzyme electrode layer and a permeation-limiting layer covering it. [Means for solving the problem]

[0005] The present inventors have conducted research to solve the above problems. First, the inventors investigated the causes of the reduced linearity and stability of response current values ​​in conventional electrodes having an enzyme electrode layer and a permeation-limiting layer covering it, and hypothesized that the reduced linearity of response current values ​​was due to the non-uniformity of the polymer structure of the permeation-limiting layer, which resulted in only a portion of the enzyme molecules in the enzyme electrode layer being available for reaction. Based on this hypothesis, the inventors conducted trial and error to address the problems caused by the non-uniformity of the permeation-limiting layer, and found that by providing a glucose diffusion layer containing a hydrophilic substance between the enzyme electrode layer containing an electrode material and glucose oxidoreductase and the permeation-limiting layer covering the enzyme electrode layer, glucose is uniformly diffused within the glucose diffusion layer, allowing all of the enzyme molecules in the enzyme electrode layer to be used for reaction, thereby improving the linearity between glucose concentration and response current values. Furthermore, the inventors also found that providing a glucose diffusion layer between the enzyme electrode layer and the permeation-limiting layer resulted in a stable response current value over a long period of time. They also found that this electrode could be used in a glucose sensor capable of accurate and stable continuous measurement, leading to the completion of the present invention.

[0006] As mentioned above, Patent Document 1 discloses an electrochemical analyte sensor comprising an electrode and an analyte detection membrane, the analyte detection membrane including an obstacle layer and an enzyme layer, and a hydrophilic polymer layer disposed between the electrode and the obstacle layer or between the obstacle layer and the enzyme layer. However, it only discloses providing a hydrophilic polymer layer on the electrode side, and does not disclose providing a hydrophilic polymer layer on the permeation-limiting membrane side. There is no concept of diffusing glucose that has permeated the permeation-limiting membrane uniformly into the enzyme layer, and in fact, this sensor is based on the existence of an obstacle layer. Furthermore, as mentioned above, Patent Document 2 discloses a biosensor characterized by forming a hydrophilic polymer membrane containing either an enzyme or an enzyme substrate on the surface of an electrode made of a hydrophobic material, and further discloses that the surface is covered with a permeation-limiting membrane. However, there is a problem in that mixing an enzyme and a hydrophilic polymer in the same layer inhibits electron transfer, resulting in a decrease in the current value.

[0007] According to one aspect of the present invention, there is provided an electrode comprising an enzyme electrode layer containing an electrode material and a glucose oxidoreductase, and a permeation-limiting layer covering the enzyme electrode layer, and a glucose diffusion layer containing a hydrophilic substance between the enzyme electrode layer and the permeation-limiting layer. Here, in one embodiment of the electrode, the enzyme electrode layer has a layer structure of a metal or carbon layer as the electrode material and the glucose oxidoreductase placed on the metal or carbon layer. In another embodiment of the electrode, the enzyme electrode layer is a layer containing a mixture of the conductive substance as the electrode material and the glucose oxidoreductase.

[0008] According to another aspect of the present invention, there is provided a glucose sensor including the electrode.

[0009] According to another aspect of the present invention, there is provided a method for manufacturing an electrode, comprising the steps of forming an enzyme electrode layer containing an electrode material and a glucose oxidoreductase, laminating a glucose diffusion layer on the enzyme electrode layer, and laminating a permeation-limiting layer on the glucose diffusion layer. [Effects of the Invention]

[0010] In the electrode of the present invention, by providing a glucose diffusion layer containing a hydrophilic substance between the permeation-limiting layer and the enzyme electrode layer, glucose that has passed through the permeation-limiting layer can be uniformly diffused, and glucose is uniformly supplied to all the enzyme molecules in the enzyme electrode layer, improving the linearity of the response current value relative to the glucose concentration. Furthermore, by providing a glucose diffusion layer containing a hydrophilic substance between the permeation-limiting layer and the enzyme electrode layer, the electrode of the present invention can maintain a high response current value for a long period of time. Therefore, the electrode of the present invention makes it possible to fabricate a glucose sensor that can perform accurate, long-term continuous measurement without requiring complex corrections. [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a schematic diagram showing one embodiment of an electrode of the present invention. [Figure 2] FIG. 1 is a diagram showing one embodiment of a glucose measurement system (continuous glucose measurement device A) including a glucose sensor of the present invention. [Figure 3] 1 is a graph showing the results of plotting glucose-responsive current values ​​using glucose sensors including electrodes of Example 1 and Comparative Example 1. [Figure 4] 1 is a graph showing the change over time in glucose response current values ​​when glucose sensors including the electrodes of Example 1 and Comparative Example 1 are used. [Figure 5] 1 is a graph showing the results of plotting glucose-responsive current values ​​using glucose sensors including the electrodes of Examples 1 to 4. [Figure 6] 1 is a graph showing the results of plotting glucose-responsive current values ​​using glucose sensors including electrodes of Example 5 and Comparative Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0012] <Electrode> The electrode of the present invention comprises: an enzyme electrode layer containing an electrode material and a glucose oxidoreductase; a permeation limiting layer covering the enzyme electrode layer, The device is characterized in that it has a glucose diffusion layer containing a hydrophilic substance between the enzyme electrode layer and the permeation limiting layer.

[0013] <Electrode material> Examples of electrode materials include metal materials and carbon materials. Examples of metal materials include gold (Au), platinum (Pt), silver (Ag), and palladium (Pd). Examples of carbon materials include carbon, such as graphite, carbon nanotubes, graphene, and mesoporous carbon.

[0014] <Glucose oxidoreductase> The glucose oxidoreductase may be any enzyme capable of oxidizing and reducing glucose, which is the substance to be measured, and examples thereof include glucose dehydrogenase (GDH) and glucose oxidase. The glucose oxidoreductase preferably contains at least one of pyrroloquinoline quinone (PQQ) and flavin adenine dinucleotide (FAD) as a catalytic subunit or catalytic domain. For example, a glucose oxidoreductase containing PQQ is PQQ glucose dehydrogenase (PQQGDH), and a glucose oxidase containing FAD is exemplified. (GOD) is one example.

[0015] Furthermore, the glucose oxidoreductase may contain an electron transfer subunit or an electron transfer domain. Examples of the electron transfer subunit include a subunit having a heme that has the function of donating and receiving electrons. Examples of oxidoreductases containing a heme subunit include glucose dehydrogenase containing a cytochrome and a fusion protein of PQQGDH and a cytochrome (International Publication No. WO2005 / 030807).

[0016] <Enzyme electrode layer> The enzyme electrode layer contains the above-mentioned electrode material and glucose oxidoreductase.

[0017] The enzyme electrode layer may contain other optional components to efficiently promote the glucose oxidation-reduction reaction. The other optional components may be one or more selected from the group consisting of conductive materials, crosslinking agents, sugars, etc. The above are some examples.

[0018] Examples of the conductive substance include conductive polymers and conductive particles. The conductive polymer is preferably water-soluble, and examples thereof include polyacetylene, polyparaphenylene, polyaniline, polythiophene, polyparaphenylene vinylene, polypyrrole, polyacene, graphene, and polyethylenedioxythiophene.

[0019] Examples of conductive particles include metal particles and high-order structures made of carbon. Examples of metal particles include gold, silver, copper, nickel, vanadium, platinum, palladium, iron, aluminum, titanium, zinc, tin, tungsten, and chromium. Higher-order structures made of carbon may include carbon particles or fine carbon particles, such as conductive carbon black, carbon nanotubes (CNTs), and fullerenes. Examples of conductive carbon black include oil furnace black, ketjen black, black pearl, and acetylene black.

[0020] The enzyme electrode layer may also contain a cross-linking agent. The type of cross-linking agent may be selected based on the enzyme and the conductive material. The crosslinking agent is not particularly limited as long as it can be crosslinked, and may be, for example, a compound containing at least one functional group selected from the group consisting of an aldehyde group, a maleimide group, a carbodiimide group, an oxazoline group, and an epoxy group. The form of the crosslinking agent is also not limited, and may be either a monomer or a polymer.

[0021] The enzyme electrode layer may also contain a sugar. The type of sugar is not particularly limited as long as it can stabilize glucose oxidoreductase, and examples thereof include trehalose, sucrose, maltose, mannose, fructose, lactose, maltotriose, and raffinose.

[0022] According to one embodiment of the electrode of the present invention, the enzyme electrode layer has a layer structure including a layer of metal or carbon as the electrode material and a layer containing the glucose oxidoreductase placed on the metal or carbon layer. For example, an enzyme electrode layer can be formed by forming a layer of metal or carbon (electrode material) on a substrate, and then forming a layer containing glucose oxidoreductase (also called an enzyme layer or reagent layer) on the metal or carbon layer.

[0023] According to another embodiment of the electrode of the present invention, the enzyme electrode layer is a layer containing a mixture of the conductive substance as the electrode material and the glucose oxidoreductase. For example, an enzyme electrode layer can be formed by mixing a reagent containing glucose oxidoreductase, a conductive substance, and carbon (electrode material) and applying the mixture onto a substrate.

[0024] <Substrate> The electrodes are preferably provided on a substrate. Substrates commonly used in electrochemical sensors can be used as the substrate, including substrates made of thermoplastic resins, various resins (plastics), and insulating materials. Examples of thermoplastic resins include polyetherimide (PEI), polyetheretherketone (PEEK), polyethylene terephthalate (PET), and polyethylene (PE). Examples of various resins (plastics) include polyimide resins and epoxy resins. Examples of insulating materials include glass, ceramic, and paper. Among these, substrates made of insulating materials are preferred.

[0025] <Permeation limiting layer> The permeation limiting layer covers the enzyme electrode layer and is provided to limit the permeation of glucose into the glucose diffusion layer and the enzyme electrode layer and to regulate the amount of glucose that reaches the glucose diffusion layer and the enzyme electrode layer in order to prevent the sensor sensitivity from quickly saturating, i.e., to prevent the sensitivity from saturating before the upper limit of the glucose concentration that the sensor can detect is reached.

[0026] The permeation-limiting layer is a material that allows glucose to permeate while restricting its permeation, and is preferably formed of an insoluble material, for example, a polymer containing hydrophilic groups or a porous material. Specific examples include polyethylene co-tetrafluoroethylene, polyolefin, polyester, polycarbonate, polytetrafluoroethylene, polytetrafluoroethylene homopolymer, copolymer, and terpolymer, polypropylene, polyvinyl chloride, polyvinylidene fluoride, polybutylene terephthalate, polymethyl methacrylate, polyether ether ketone, polyurethane, cellulose polymer, polysulfone, silicone polymer (polysiloxane), cellulose acetate, HEMA (hydroxyethyl methacrylate), copolymers containing these, and polyvinylpyridine. do. Among these, one or more selected from cellulose polymers, polyurethanes, and polyvinylpyridines are preferred. The thickness of the permeation limiting layer may be any thickness that allows glucose to pass through while restricting it, and is preferably 1 μm or more and 100 μm or less, more preferably 5 μm or more and 50 μm or less. do.

[0027] <Glucose diffusion layer> The glucose diffusion layer is present between the permeation-limiting layer and the enzyme electrode layer to diffuse glucose that has passed through the permeation-limiting layer and ensure that it is uniformly supplied to the enzyme electrode layer. The glucose diffusion layer contains a hydrophilic substance. The glucose diffusion layer has a superior glucose diffusion ability compared to the permeation-limiting layer due to the hydrophilic substance. Since glucose does not pass through the permeation-limiting layer uniformly, in the case of a conventional electrode that uses only a permeation-limiting layer, the area of ​​the electrode surface that glucose reaches is limited. This causes only a portion of the enzyme in the enzyme electrode to be used for the reaction, which is thought to lead to a decrease in the linearity of the response current value relative to the glucose concentration. For example, when a polymer such as polyurethane is used as the permeation-limiting layer, it is thought that the polymer contains irregular hydrophilic and hydrophobic portions, and glucose passes through the more hydrophilic portions of the polymer. If a permeation-limiting layer using the polymer is formed directly on the enzyme layer, glucose passes only through the more hydrophilic portions of the polymer, and does not spread throughout the entire enzyme electrode layer, so it can only react with some of the enzymes in the enzyme electrode layer. In contrast, the electrode of the present invention includes a glucose diffusion layer containing a hydrophilic substance between the permeation-limiting layer and the enzyme electrode layer, which homogenizes the glucose that has passed through the permeation-limiting layer within the glucose diffusion layer. As a result, glucose can reach the entire enzyme electrode layer evenly, improving the linearity of the response current value relative to the glucose concentration. In order to achieve this effect, the glucose diffusion layer is preferably provided so as to cover the entire surface of the enzyme electrode layer.

[0028] The hydrophilic substance is not particularly limited as long as it can diffuse glucose, and examples thereof include hydrophilic polymers and other substances. Examples of hydrophilic polymers include polyvinylpyrrolidone (PVP), poly-N-vinyl-3-ethyl-2-pyrrolidone, poly- N-vinyl-4,5-dimethyl-2-pyrrolidone, polyacrylamide, poly-N,N-dimethyl Polyacrylamide, Polyvinyl alcohol, Polyethylene glycol, pendent Examples of other substances include polymers having ionic groups, and copolymers or blends thereof. Examples of other substances include hydrophilic substances other than hydrophilic polymers such as sodium alginate, methyl acrylate, chitosan, carboxymethyl cellulose, carboxyethyl cellulose, and sodium acrylate. Among these, one or more selected from polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol and sodium alginate are preferred.

[0029] The hydrophilic substance in the glucose diffusion layer is preferably cross-linked so that it is not dissolved by the specimen (sample) liquid. The crosslinking treatment can be carried out, for example, by using a crosslinking agent. The type of crosslinking agent is not particularly limited as long as it is a substance that can crosslink a hydrophilic substance to make it insoluble in the sample liquid, and is appropriately selected depending on the type of hydrophilic substance. Examples of the crosslinking agent include crosslinking agents that can target carboxyl groups for crosslinking, such as crosslinking agents having an oxazoline group, crosslinking agents having an epoxy group, and crosslinking agents having a carbodiimide group. For example, an aqueous solution containing a crosslinking agent and a hydrophilic substance can be applied to the enzyme electrode layer, and then the crosslinking reaction can be promoted by heating or UV irradiation, thereby forming a glucose diffusion layer containing a crosslinked hydrophilic substance on the enzyme electrode layer.

[0030] The amount of the hydrophilic substance in the glucose diffusion layer may be any amount sufficient to sufficiently diffuse glucose, but is preferably 1.19 μg / cm 2 More than 1.19 g / cm 2 Less than 6 μg / cm 2 More than 119 mg / cm 2 or less, more preferably 11.9 μg / cm 2 More than 11.9 mg / cm 2 The following is the result.

[0031] The thickness of the glucose diffusion layer may be any thickness sufficient to allow glucose to diffuse sufficiently within the layer, and is, for example, preferably 10 nm to 10 mm, more preferably 50 nm to 1 mm, and even more preferably 100 nm to 100 μm.

[0032] <Example of electrode> An electrode according to one embodiment of the present invention will be described with reference to FIG. An electrode layer (2) made of an electrode material such as metal or carbon is provided on a substrate (1), and an enzyme layer (3) containing glucose oxidoreductase is provided on the electrode layer (2). A glucose diffusion layer (4) is provided to cover the enzyme layer (3), and a permeation limiting layer (5) is provided to cover the glucose diffusion layer (4). Here, the enzyme layer (3) may contain, in addition to glucose oxidoreductase, the above-mentioned conductive substance, crosslinking agent, sugar, etc. The glucose diffusion layer (4) is preferably provided so as to cover the entire enzyme layer (3), but may be provided only on the enzyme layer (3).

[0033] In addition, when an enzyme and an electrode material are mixed to form an enzyme electrode layer on a substrate, a single enzyme electrode layer containing the enzyme and the electrode material is formed instead of the electrode layer (2) and the enzyme layer (3), and a glucose diffusion layer is formed on top of that, and a permeation-limiting layer is further formed. In this case, the enzyme electrode layer may contain, in addition to the electrode material and glucose oxidoreductase, the conductive substance, crosslinker, sugar, etc., as described above. Furthermore, the glucose diffusion layer (4) is preferably provided so as to cover the entire enzyme electrode layer, but may also be provided only on the enzyme electrode layer.

[0034] When a liquid sample containing glucose is added onto the permeation limiting layer (5), the glucose permeates the permeation limiting layer (5) and reaches the glucose diffusion layer (4). Due to the presence of hydrophilic substances, the glucose is uniformly diffused within the glucose diffusion layer (4), reaching all of the glucose oxidoreductase molecules in the enzyme layer (3) evenly, causing an oxidation (reduction) reaction. The electrons generated by this reaction move to the electrode layer (2), and by applying a voltage to the electrode, a current that accurately reflects the glucose concentration in the liquid sample flows. The glucose oxidoreductase and the electrode material are mixed together to form a single When the enzyme electrode layer (1) is formed, the glucose diffused uniformly in the glucose diffusion layer (4) due to the presence of the hydrophilic substance reaches the glucose oxidoreductase molecules in the enzyme electrode layer evenly, causing an oxidation (reduction) reaction. The electrons generated by this reaction then move to the electrode material in the enzyme electrode layer, and by applying a voltage to the electrode, a current that accurately reflects the glucose concentration in the liquid sample flows.

[0035] <Example of electrode fabrication method> The above-described electrodes are fabricated, for example, as follows. That is, a layer of electrode material such as metal or carbon is formed on one side of an insulating substrate. For example, carbon ink can be screen-printed on one side of a film-like insulating substrate with a predetermined thickness (e.g., about 100 μm) to form a carbon film with a desired thickness (e.g., about 10 μm). Instead of a carbon layer, a metal layer with a desired thickness (e.g., about 30 nm) can also be formed by depositing a metal material by physical vapor deposition (PVD, e.g., sputtering) or chemical vapor deposition (CVD). Next, an enzyme layer is formed on the electrode. For example, a solution containing an oxidoreductase and a conductive substance is prepared, and the solution is dropped onto the surface of the electrode. The solution is dried and solidified on the electrode, forming an enzyme layer on the electrode. Next, a glucose diffusion layer containing a hydrophilic substance is formed on the enzyme layer. A glucose diffusion layer containing a hydrophilic substance can be formed on the enzyme layer by applying a solution of the hydrophilic substance onto the layer and drying it. Alternatively, a glucose diffusion layer containing a hydrophilic substance can be formed by spin coating, dip coating, drop coating, or the like using a solution of the hydrophilic substance. In the glucose diffusion layer, it is preferable to crosslink the hydrophilic substance. Then, a permeation-limiting layer is formed on the glucose diffusion layer. For example, a solution of cellulose polymer, polyurethane, polyvinylpyridine, or the like is applied to the glucose diffusion layer and then dried to form the permeation-limiting layer on the glucose diffusion layer. Alternatively, the permeation-limiting layer can be formed by spin coating, dip coating, drop coating, or the like using a solution of cellulose polymer, polyurethane, polyvinylpyridine, or the like.

[0036] <Glucose sensor> The electrode of the present invention can be used as a working electrode of a glucose sensor. That is, the glucose sensor of the present invention includes the electrode of the present invention, and preferably further includes a counter electrode. The counter electrode may be any electrode that is generally usable as a counter electrode for a glucose sensor, for example, a silver / silver chloride electrode or a carbon electrode. The counter electrode can also be formed on a substrate by physical vapor deposition (PVD, e.g., sputtering), chemical vapor deposition (CVD), or screen printing. The glucose sensor may also be a three-electrode system that further includes a reference electrode. A glucose sensor can be produced by forming, on a substrate, the electrode of the present invention as a working electrode and a silver / silver chloride electrode or a carbon electrode as a counter electrode. The glucose sensor including the electrode of the present invention may be an implantable sensor, a batch type sensor, or a sensor for measurement at the time of use. Preferably, the sensor is for continuous glucose measurement and is an implantable sensor that is placed subcutaneously or the like.

[0037] The glucose sensor of the present invention can be used to measure the glucose concentration in a sample. The sample is not particularly limited as long as it contains glucose, and examples thereof include biological samples. Examples of biological samples include blood, interstitial fluid, plasma, lymph, cerebrospinal fluid, urine, sweat, and saliva, with blood, interstitial fluid, and plasma being preferred.

[0038] <How to measure glucose> The glucose measurement method of the present invention comprises: contacting a glucose sensor with a sample containing glucose; applying a voltage to the electrodes of the glucose sensor and measuring the value of the current that flows due to the oxidation-reduction reaction of glucose by the glucose oxidoreductase; The method includes a step of calculating the glucose concentration based on the current value.

[0039] The step of contacting the glucose sensor with a sample containing glucose can be carried out by contacting the sample with the permeation limiting layer of the electrode of the glucose sensor. In the case of an implantable sensor, this also includes a mode in which the sensor is implanted in the body and placed in a state in which a sample such as blood or interstitial fluid comes into contact with the permeation limiting layer of the electrode of the glucose sensor.

[0040] When a sample containing glucose is brought into contact with the permeation limiting layer of the glucose sensor, the glucose in the sample passes through the permeation limiting layer, is diffused in the diffusion layer, and uniformly reaches the enzyme electrode layer. The glucose oxidizing reaction occurs due to the glucose oxidizing enzyme, and electrons are transferred to the electrodes. When a potential is applied to the electrodes, a current corresponding to the amount of glucose flows between the electrodes. Since the current value depends on the concentration of glucose in the sample, the amount of glucose in the sample can be calculated based on the measured current value. The glucose concentration can be calculated. The voltage applied to the electrode is preferably 0 mV to 800 mV, and more preferably 40 mV to 200 mV, relative to a silver / silver chloride electrode serving as a reference electrode.

[0041] <Glucose measurement system> A glucose measurement system according to one embodiment of the present invention includes at least a glucose sensor including an electrode of the present invention, a power source that applies a voltage to the electrode of the glucose sensor, a detection unit that detects a response current, and a calculation unit that calculates a glucose concentration from the response current value.

[0042] One embodiment of a glucose measurement system (continuous glucose measurement device A) including the glucose sensor of the present invention will be described with reference to FIG. As shown in FIG. 2, the circuit board 10 includes a communication unit 11, a power supply 12, a control unit 13, a calculation unit 14, a storage unit 15, and a connection unit 16. The connection part 16 is electrically connected to the electrode of the present invention of the sensor part 17, i.e., the enzyme electrode layer, and is used to apply a voltage to the sensor part 17 to obtain a response current. The sensor part 17 corresponds to the glucose sensor of the present invention.

[0043] The communication unit 11 performs data communication between the continuous glucose monitoring device A and an external information processing device. The communication unit 11 includes at least a transmitting unit, and may also include a receiving unit as necessary. The data communication means may be, for example, a wireless communication means such as infrared communication or Bluetooth (registered trademark). Alternatively, the continuous glucose monitoring device A may be configured to perform data communication via a cable. In this case, a cable connection unit is provided in the communication unit 11 of the continuous glucose monitoring device A, and data communication is performed by connecting to an external information processing device or the like via a cable.

[0044] Examples of external information processing devices include an insulin supply device that administers insulin to the human body, a simple blood glucose level measuring device, a wristwatch-type display device, and a personal computer.

[0045] The power supply 12 is a DC power supply for supplying power to the circuit board 10 and the sensor unit 17, and may be, for example, a battery with a power supply voltage of 1 to 3V.

[0046] The control unit 13 controls the entire continuous glucose monitoring device A. For example, it controls the timing of voltage application, sampling of response current, calculation processing of glucose concentration, communication with an external information processing device, and the like.

[0047] The calculation unit 14 executes various calculations required for processing in the continuous glucose monitoring device A, including, for example, calculation of the glucose concentration.

[0048] The memory unit 15 stores various programs executed by the control unit 13, such as a program for executing a test mode processing routine described below, as well as data such as a calibration curve, correction data, and applied voltage pattern used in the calculations of the calculation unit 14. The memory unit 15 also stores data on the response current detected by the sensor unit 17 and the glucose concentration calculated by the calculation unit 14.

[0049] The control unit 13, the calculation unit 14, and the storage unit 15 are configured by electronic components such as a CPU or MPU, a ROM, and a RAM mounted on the circuit board 10. [Example]

[0050] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the embodiments of the following examples.

[0051] <Electrode Manufacturing Example: Example 1> A metal (gold) layer was formed on a substrate made of polyetheretherketone by sputtering, and a portion of the metal was removed by etching to form an electrode pattern.

[0052] 12 nL of the following reagents (dissolved in water to achieve the respective final concentrations) was applied onto the electrode (metal layer), and the mixture was dried and heated to form an enzyme layer (reagent layer). Reagent Final Concentration Phosphate buffer 10mM Trehalose 0.43wt% GDH 8.5 mg / mL Crosslinker 5% Carbon black dispersion 0.4wt%

[0053] Next, the part of the electrode containing the enzyme layer was soaked in a 6 w / w% aqueous solution of polyvinyl alcohol containing a crosslinking agent. The enzyme layer was coated with polyvinyl alcohol by immersing the enzyme layer in water, forming a glucose diffusion layer on the enzyme layer. The glucose diffusion layer was then crosslinked by drying and heating.

[0054] Next, the portion of the electrode containing the enzyme layer coated with the glucose diffusion layer was immersed in a dimethylformamide solution of polyurethane to coat the glucose diffusion layer with polyurethane and form a permeation-limiting layer on the glucose diffusion layer, thereby producing an electrode.

[0055] <Electrode Manufacturing Example: Comparative Example 1> The same procedure as in Example 1 was carried out except that the glucose diffusion layer containing polyvinyl alcohol was not provided. , electrodes were fabricated.

[0056] <Electrode Evaluation: Example 1, Comparative Example 1> Using the electrodes of Example 1 and Comparative Example 1 obtained as described above, a phosphate buffered saline solution containing glucose was added as a sample onto the permeation limiting layer, and then the sample was applied to an Ag / AgCl reference electrode. A voltage of 150 mV was applied to the electrode, and the current flowing through the electrode was measured using an electrochemical measurement device. Measurements were started at a glucose concentration of 70 mg / dL, increased to 180 mg / dL after about 20 hours, and increased to 500 mg / dL after another hour. Measurements were then continued for 14 days. The response current plots for glucose concentrations of 70, 180, and 500 mg / dl in this evaluation are shown in Figure 3. The time course of the response values ​​measured over 14 days is shown in FIG.

[0057] As can be seen from FIG. 3, in Example 1, the response current value was linear up to the high glucose concentration side. On the other hand, in Comparative Example 1, the response value was 0.01 at the glucose concentration of 180 mg / dL. It was confirmed that the increase was not linear. Furthermore, as can be seen from FIG. 4, in Example 1, the response current value during the 14-day measurement was reduced by about 10% compared to the response value one day after the start of measurement, while in Comparative Example 1, the reduction was about 90%.

[0058] <Investigation of polyvinyl alcohol concentration in the glucose diffusion layer> The concentration of the polyvinyl alcohol solution was set to about 4 w / w %, and the glucose diffusion layer was formed. Other than that, an electrode was produced in the same manner as in Example 1 (Example 2).

[0059] The concentration of the polyvinyl alcohol solution was set to about 8 w / w %, and the glucose diffusion layer was formed. Other than that, an electrode was produced in the same manner as in Example 1 (Example 3).

[0060] An electrode was prepared in the same manner as in Example 1, except that the glucose diffusion layer was formed using a polyvinyl alcohol solution with a concentration of about 10 w / w % (Example 4).

[0061] For the electrodes of Examples 1, 2, 3, and 4, a phosphate buffered saline solution containing glucose was added as a sample onto the permeation limiting layer of each electrode, and then a voltage of 150 mV was applied to the Ag / AgCl reference electrode, and the current flowing through the electrode was measured using an electrochemical measurement device. The glucose concentration started at 70 mg / dl, and after about 20 hours, it increased to 180 mg / dl. The glucose concentration was increased to 500 mg / dL one hour later, and measurements were continued for 14 days. The response plots for glucose concentrations of 70, 180, and 500 mg / dl in this evaluation are shown in Figure 5. vinegar.

[0062] As can be seen from Figure 5, it was confirmed that the response current to glucose improved as the concentration of polyvinyl alcohol used increased. This is presumably because the diffusion of glucose increased as the thickness of the glucose diffusion layer increased.

[0063] <Example using sodium alginate as glucose diffusion layer> 6 w / w % sodium alginate instead of polyvinyl alcohol as the sensing material diffusion layer An electrode was prepared in the same manner as in Example 1 except that the electrode was cross-linked with calcium chloride. 5).

[0064] For the electrodes of Example 5 and Comparative Example 1, phosphate buffered saline containing glucose was used as a sample. After adding it to each permeation limiting layer, a voltage of 150 mV was applied to the Ag / AgCl reference electrode. A voltage was applied to the electrode, and the current value of the electrode was measured using an electrochemical measurement device. The measurement was started at a glucose concentration of 70 mg / dL, then increased to 180 mg / dL, and further increased to 500 mg / dL. The current plots for glucose concentrations of 70, 180, and 500 mg / dl in this evaluation are shown in Figure 6. vinegar.

[0065] As can be seen from FIG. 6, the use of sodium alginate resulted in a higher glucose tolerance than Comparative Example 1. It was confirmed that the response current value to the source was improved, and it was found that sodium alginate can also be used as a glucose diffusion layer. [Explanation of symbols]

[0066] 1 Substrate, 2 Electrode layer, 3 Enzyme layer, 4 Glucose diffusion layer, 5 Permeation limiting layer A continuous glucose monitoring device, 10 circuit board, 11 communication unit, 12 power supply, 13 control unit, 14 calculation unit, 15 memory unit, 16 connection unit, 17 sensor unit

Claims

1. an enzyme electrode layer containing an electrode material and a glucose oxidoreductase; a permeation limiting layer covering the enzyme electrode layer; and a glucose diffusion layer containing a hydrophilic polymer between the enzyme electrode layer and the permeation limiting layer; electrode.

2. 2. The electrode according to claim 1, wherein the enzyme electrode layer has a layer structure consisting of a layer of metal or carbon as the electrode material and the glucose oxidoreductase placed on the layer of metal or carbon.

3. 2. The electrode according to claim 1, wherein the enzyme electrode layer is a layer containing a mixture of a conductive substance as the electrode material and the glucose oxidoreductase.

4. 4. The method according to claim 1, wherein the glucose oxidoreductase is a glucose dehydrogenase. Item 1. The electrode according to item 1.

5. The hydrophilic polymer is at least one selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, and sodium alginate.

5. An electrode according to any one of claims 4 to 4.

6. The electrode according to any one of claims 1 to 5, wherein the hydrophilic polymer is crosslinked.

7. The amount of the hydrophilic polymer is 1.19 μg / cm ２ 1.19g / cm or more ２ 7. The electrode according to claim 1, wherein:

8. 8. Any one of claims 1 to 7, wherein the thickness of the glucose diffusion layer is 10 nm or more and 10 mm or less.

3. The electrode according to claim 1.

9. 9. The method according to claim 1, wherein the glucose diffusion layer is provided only on the enzyme electrode layer.

3. The electrode according to claim 1.

10. 10. The electrode according to claim 1, wherein the permeation limiting layer comprises one or more selected from the group consisting of cellulose polymers, polyurethanes, and polyvinylpyridines.

11. A glucose sensor comprising the electrode according to any one of claims 1 to 10.

12. The glucose sensor according to claim 11, which is for continuous glucose measurement.

13. forming an enzyme electrode layer containing an electrode material and a glucose oxidoreductase; A step of laminating a glucose diffusion layer containing a hydrophilic polymer on the enzyme electrode layer; and laminating a permeation-limiting layer on the glucose diffusion layer; A method for manufacturing an electrode, comprising:

Images