Electrode for glucose sensors

Abstract

To provide an improved electrode system that can measure a glucose level or concentration in a body fluid and can be utilized in vitro or in vivo.SOLUTION: Provided is a glucose sensor having such a structure that a working electrode part, a counter electrode part and a reference electrode part and an electric signal lead-out wire from these electrode parts are provided on the surface of a substrate densely populated with microneedles of a microneedle patch, including the substrate and the microneedles made from a non-electroconductive material. Each of the electrode parts is an immobilized enzyme sensor coated by a crosslinked glucose oxidase and detects an electric signal generated in vivo in accordance with the presence of glucose in a body fluid, thereby measuring the concentration of glucose.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an electrode for a glucose sensor, and more particularly to a glucose concentration sensing system that utilizes a microneedle array. [Background technology]

[0002] The glucose level in a patient's blood varies over time and usually depends on the individual's physical activity, food, drink, sugar intake, metabolic rate, etc. Glucose as a compound is difficult to measure directly electrochemically because it does not show a significant change in properties during oxidation and / or reduction processes. For this reason, it has been preferred to measure glucose levels by using various enzymes and / or proteins to specifically react with glucose and quantitatively analyze the yield and / or by-products. Thus, there are many methods for measuring glucose, such as the method of quantitatively measuring glucose in blood using enzymes. However, these methods cannot be applied to in-vivo systems, and in most cases, they are difficult to apply even to simple in-vitro systems.

[0003] Chemically improved electrodes have come to be used in electrochemical sensing mechanisms (Patent Documents 1, 2, 3). In this case, an enzyme or other protein quantitative reagent is covalently bonded to the electrode to prepare a simple electrode, which is then used to measure the glucose concentration in a sampled body fluid by contacting it with the fluid. In recent years, further advances have been made, and the current mainstream device is one in which the electrode part is inserted percutaneously into the body as a sensor, and hydrogen peroxide is generated by contacting it with blood or exudate in the body and reacting with glucose, and the hydrogen peroxide is generated and electrochemically measured by measuring the current or voltage (Non-Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2-501679 [Patent Document 2] Japanese Patent Application Publication No. 9-80010 [Patent Document 3] JP 2013-53907 A [Non-patent literature]

[0005] [Non-Patent Document 1] Continuous glucose monitoring devices: A brief presentation EXPERIMENTAL AND THERAPEUTIC MEDICIE 23: 174,2022 Summary of the Invention [Problem to be solved by the invention]

[0006] The currently mainstream method of using devices requires a stainless steel needle to introduce the sensor probe for chemical detection into the skin, making the device as a whole very complex, the operation of attaching it to the patient is also complicated, and the system as a whole is expensive. It is therefore a first object of the present invention to provide an improved electrode system which is capable of measuring glucose levels or concentrations in body fluids and which can be applied in-vitro or in-vivo. A second object of the present invention is to provide an improved electrode means which is particularly suitable for measuring glucose concentration in body fluids in-vitro or in-vivo, and which sensing electrode is reliable, stable and robust. It is yet another object of the present invention to provide a method for preparing a glucose sensor capable of providing an electrical signal relative to the glucose level in a body fluid and suitable for use with a variable speed insulin pump. [Means for solving the problem]

[0007] In accordance with the present invention, there is provided an electrode for sensing glucose with an electrode functioning on an amperometric basis. Such an electrode is formed by dividing the microneedle patch surface into two or three parts. In the case of dividing into two parts, the electrode consists of a working electrode part and a counter electrode part. In the case of dividing into three parts, the electrode consists of a working electrode part, a counter electrode part, and a reference electrode part. In the case of dividing into two parts, it is also possible to form a working electrode part and a reference electrode part. The working electrode part and the counter electrode part are formed by coating the microneedle and the substrate surface on which it stands with gold or platinum by electrolytic or electroless plating. As another coating method, the substrate surface may be coated with gold or platinum foil by pressure bonding. In the coating, the platinum or gold that covers each divided electrode is coated so as not to come into contact with each other. In the case of dividing into three parts, the reference electrode part is formed as a silver / silver chloride electrode part by coating the microneedle and the substrate surface on which it stands with silver by electrolytic or electroless plating and chlorinating the surface. A calomel electrode may be used as the reference electrode part. Instead of dividing the patch surface on which the microneedles stand, a counter electrode portion and a reference electrode portion may be formed on a substrate portion that does not have microneedles. Each electrode is further coated with an immobilized enzyme, glucose oxidase (GOD), which is immobilized and made water insoluble. A coating layer, such as a thin silicone rubber film, may be provided on the outer surface of the GOD layer. The GOD coating is essential for the working electrode, but not necessarily for the counter electrode and reference electrode. Lead wires are provided from the working electrode, counter electrode, and reference electrode to extract electrical signals. The lead wires refer to electrical signal extraction wires. The electrical signals extracted are processed in accordance with currently used methods. The present invention is as follows. [1] A glucose sensor comprising a microneedle patch including microneedles and a substrate made of a non-conductive material, a working electrode section, a counter electrode section, and a reference electrode section, as well as electrical signal output wires from each of these electrode sections, provided on the substrate surface on which the microneedles of the microneedle patch are forested, wherein at least the working electrode section is an immobilized enzyme sensor coated with cross-linked glucose oxidase (the counter electrode section and the reference electrode section may also be immobilized enzyme sensors coated with cross-linked glucose oxidase), and which senses an electrical signal generated in vivo in response to the presence of glucose in body fluids, thereby measuring the glucose concentration. [2] A glucose sensor comprising a microneedle patch including microneedles and a substrate made of a non-conductive material, the substrate surface on which the microneedles are forested being provided with a working electrode section, a reference electrode section, and wires for extracting electrical signals from each electrode section, each electrode section being an immobilized enzyme sensor coated with cross-linked glucose oxidase, which senses electrical signals generated in vivo in response to the presence of glucose in body fluids, thereby measuring the glucose concentration. [3] The glucose sensor according to [1] or [2], wherein the non-conductive material is selected from the group consisting of polyglycolic acid, poly(lactic acid-glycolic acid) copolymer, polycarbonate, polytetrafluoroethylene, polyoxymethylene, polyethylene terephthalate, and COP (cyclic olefin polymer). [4] The glucose sensor according to [1] or [2], wherein the shape of the substrate of the microneedle patch is a circle having a diameter of 0.4 to 5 cm, and the microneedle has a needle length of 100 μm or more and 2,000 μm or less. [5] The glucose sensor described in [1] or [2], wherein the microneedle patch further includes a base portion and a gallery portion. [6] The glucose sensor according to [1], wherein the working electrode and the counter electrode are covered with gold or platinum. [7] The glucose sensor according to [1] or [2], wherein the reference electrode portion is a silver / silver chloride electrode. [8] The glucose sensor according to [1] or [2], wherein the electrode portion is covered with a coating in which glucose oxidase is cross-linked. [9] The glucose sensor according to [1] or [2], wherein the electrode portion is covered with a coating formed by crosslinking a polymer having an amino group as a base with glucose oxidase.

[10] A monitoring system for glucose concentration in interstitial fluid, comprising the glucose sensor according to any one of [1] to [9], a transmitter, and a monitor, and transmitting an electrical signal obtained from the glucose sensor to the monitor via the transmitter. Effect of the Invention

[0008] The present invention is a sensor that detects glucose using a microneedle patch with many microneedles, which can be easily introduced into the skin and generates a large current value, making it possible to provide a more stable system than conventional methods. By applying the microneedle patch to the skin, the glucose concentration in the interstitial fluid can be displayed as a signal, and therefore the microneedle patch is useful as a blood glucose sensor. [Brief description of the drawings]

[0009] [Figure 1] 1A and 1B are plan and cross-sectional views of a microneedle patch for an immobilized enzyme electrode. [Diagram 2] A completed diagram of the immobilized enzyme electrode. [Diagram 3] 1 is a schematic diagram of an example of a microneedle patch used in the glucose sensor of the present invention. [Figure 4] A plan view (B) and a cross-sectional view (A) of another example of a microneedle patch for an immobilized enzyme electrode. The patch has a working electrode portion and a counter electrode portion, and a silver / silver chloride electrode is used as a reference electrode portion. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Microneedle patch The microneedle patch in the present invention refers to a structure in which a substrate and a plurality of microneedles stand on one side of the substrate, and the microneedle patch may be backed with an adhesive sheet. The adhesive sheet is typically a film made of polyurethane, polyethylene, polyester, paper, etc. as a film base material, and is formed to a thickness of about 5 to 50 μm, on which an acrylic or rubber-based adhesive is applied to a thickness of about 5 to 50 μm. The shape of the adhesive sheet is not particularly limited, but is preferably a circular, elliptical, or bead-shaped shape similar to the shape of the microneedle array.

[0011] Microneedle patch substrate The material, shape and size of the substrate of the microneedle patch are not particularly limited, and any conventionally used materials can be used. Basically, the substrate and the base material of the microneedle are the same, but they may be different. The base material may be a non-conductive inorganic compound such as silicone, silicon dioxide, ceramic, glass, etc., or a water-insoluble synthetic or natural resin material, etc. The synthetic or natural resin material may be a biodegradable polymer such as polylactic acid, polyglycolic acid, poly(lactic acid-glycolic acid) copolymer, capronolactone, etc., or a non-biodegradable polymer such as nylon, polycarbonate, polytetrafluoroethylene, polyoxymethylene, polyethylene terephthalate, COP (cyclic olefin polymer), etc.

[0012] The shape of the substrate can be any shape. For example, it may be a circle, an ellipse, a triangle, a rectangle, a polygon, etc., which is further modified according to the application site (skin). The size of the substrate, expressed typically by the diameter (major axis) or the length of one side (long side), is usually 0.2 to 5 cm, or 0.4 to 5 cm, and preferably 0.5 to 3 cm.

[0013] The area of ​​the substrate is usually 0.05 to 100 cm 2 From the viewpoint of ease of handling, the thickness should be 0.4 to 10 cm. 2 Approximately 0.6 to 5 cm is preferable. 2The thickness of the substrate is preferably 0.1 to 1.0 mm.

[0014] Microneedle shape The microneedle has a needle length of 100 μm to 2,000 μm, preferably 200 to 1,000 μm. The size of the tip of the needle, expressed as a diameter, is 80 μm or less, preferably 20 μm or more, taking into consideration ease and certainty of piercing the skin. Examples of individual microneedles include a cylindrical or conical shape with a circular base, an elliptical cylindrical or elliptical cone shape with an elliptical base, a triangular prism or triangular pyramid shape with a triangular base, a quadrangular prism or quadrangular pyramid shape with a square base, or a polygonal prism or polygonal pyramid shape with a polygonal base. In the case of an ellipse, the size of the base is expressed as the major axis as the diameter, and the minor axis is shorter than the major axis as long as an ellipse can be formed. In the case of a triangle or polygon, one side may be expressed as a representative, or the diagonal may be expressed as a representative. When the microneedle is conical, the diameter of the base is about 100 to 400 μm, preferably about 150 to 300 μm.

[0015] The microneedle of the present invention may have a step. The step here refers to a step-like shape in which the cross-sectional area of ​​the microneedle discontinuously decreases from a certain point of the microneedle toward the tip.

[0016] Placement of microneedles on the substrate The basic form of the microneedle patch is that the microneedles stand directly on the substrate, but a base part may be provided on the substrate and the microneedles stand on the base part (called the patch plane). By providing the base part, the skin puncture of the microneedles becomes more reliable. The thickness of the base part (height of the base surface) is preferably 0.2 mm to 4.0 mm above the substrate surface, and more preferably 0.5 mm to 2.0 mm. When a base is provided on the substrate, it is provided for each electrode part: the working electrode part, the counter electrode part, and the reference electrode part. The layout of the three electrodes should be approximately the same area as a rule, but there are no particular restrictions. The substrate part surrounded by the bases of the different electrodes is called a corridor part. A schematic diagram of the above layout is shown in Figure 1.

[0017] Microneedle patches are formed using a molding method suited to the material, but for industrial production, injection molding of biodegradable and non-biodegradable polymers is preferred.

[0018] Glucose sensor fabrication The working electrode and counter electrode are microneedle patches coated with gold or platinum. Coating methods that can be used include vapor deposition, electrolytic plating, and electroless plating. Furthermore, gold or platinum foil can be pierced with a needle and then adhesively applied to the substrate surface. The reference electrode is a silver / silver chloride electrode. A silver / silver chloride electrode is made by electrolyzing silver or immersing it in an aqueous solution containing chloride ions to convert the silver surface into silver chloride. The working electrode is coated with immobilized GOD. The counter electrode and reference electrode may or may not be coated with GOD. GOD coating is performed by reacting glucose oxidase with a base polymer having an amino group using a multifunctional aldehyde such as glutaraldehyde to insolubilize it, and at the same time insolubilizing the base, thereby immobilizing it on the electrode. Examples of base polymers having amino groups include serum albumin, nucleic acid, and chitosan.

[0019] The method of platinum plating by electroless plating is described in detail below. In order to platinum plate the working electrode and the counter electrode, the entire corridor and the reference electrode are covered with silicone resin, heated to form a film, and masked. The sides and back are also masked in the same way. Platinum electroless plating is performed with dinitrodiamine platinum, ammonia, and hydrazine at 60°C for 1 hour. The obtained microneedle patch with platinum film is washed with water and then dried. Then, the working electrode and the counter electrode are masked with silicone, the masking of the reference electrode is removed, and the reference electrode is electrolessly silver plated. Then, it is immersed in a saturated iron (III) chloride solution to form an Ag / AgCl electrode. The masking of the working electrode, the counter electrode, and the corridor is removed, and the three electrodes are each connected to a lead wire, and the lead wire is led from the corridor to the outside. The potential and current value applied between the electrodes can be measured externally. In FIG. 2, the lead wire is installed on the microneedle side, but it may be installed on the back side instead of the microneedle side. After that, a GOD film is formed on the working electrode and the other two electrodes, and then cross-linked with glutaraldehyde to form an immobilized enzyme electrode (glucose sensor). When preparing the GOD film, it is preferable to have a hydrophilic polymer with an amino group other than GOD, such as albumin, present in order to prevent glucose from penetrating from the interstitial fluid and to improve the stability of GOD. An example of an immobilized enzyme electrode prepared by the above process is shown in Figure 2.

[0020] The above is a basic immobilized enzyme electrode with three electrodes, but it is also possible to use the reference electrode instead of the counter electrode. In that case, the electrode unit will be two.

[0021] The glucose concentration in the interstitial fluid can be measured using the immobilized enzyme electrode thus prepared by a known method. The immobilized enzyme electrode microneedle patch is administered transdermally by an applicator. In order to stably hold the microneedle on the skin, the back of the substrate is covered with adhesive tape and adhered to the skin. Three or two lead wires are connected to a voltage application device, a voltage of 0.6 to 1.0 V is applied, and the flowing current is measured, for example, according to the method described in JP-A-2-501679. It is also possible to install a transmitter on the skin adjacent to the microneedle administered to the skin without connecting it to an external device, perform electrical operation from there, and further wirelessly transmit the obtained electrical signal to a monitor to monitor the glucose concentration in the interstitial fluid over time. EXAMPLES

[0022] The present invention will now be described in more detail with reference to the following examples. These examples are merely illustrative of the present invention, and the scope of the present invention is not limited to these examples.

[0023] Example 1 The mold was attached to an injection molding machine, and polyglycolic acid was melted and injection molded to form a microneedle patch as shown in Figure 1. Patch diameter: 12.3 mm. The details of this microneedle patch are as follows. The outer diameter of the base on which the needle stands on the patch board is 10.5 mm, the width of the corridor is 1.2 mm. The height of the corridor and the surrounding area from the board surface is 0.5 mm. The height of the base surface is 1.3 mm. The needle height is 0.9 mm. The needle spacing is 0.6 mm. The silicone resin used for masking was a two-liquid curing type (HYV-4000, manufactured by Engraving Japan). After platinum electroless plating, the silicone resin was peeled off from the reference electrode, and the working electrode and counter electrode were masked, with the reference electrode being a silver / silver chloride electrode. The thickness of the platinum and silver films was approximately 10 μm. Lead wires were attached to each of the three electrodes. The patch was then immersed in an aqueous solution of 1% GOD and human albumin, taken out, dried, and then immersed in an aqueous solution of glutaraldehyde to crosslink GOD and albumin to prepare an immobilized enzyme electrode for measuring glucose concentration. The thickness of the immobilized enzyme membrane was 30 μm.

[0024] Example 2 Polyglycolic acid was melted and injection molded to produce a milky white oval microneedle patch. The patch had a major axis of 12.3 mm and a minor axis of 11.5 mm (Figure 3). The details of this microneedle patch are as follows: The outer diameter of the base on which the needles stand on the patch substrate was 10.5 mm, and the inner diameter of the base was 6.0 mm. The width of the corridor was 1.2 mm. The height of the corridor and the center from the substrate surface was 0.5 mm. The height of the base surface was 1.3 mm. The needle height was 0.6 mm. The total number of needles was 280.

[0025] This microneedle patch has a base divided into two, and the needle part on one of the base parts is covered with a platinum film without masking to form a working electrode part. The platinum film has a thickness of 10 μm. The needle part on the other base part is similarly formed as a silver / silver chloride electrode part. The silver / silver chloride film has a thickness of 10 μm. After the lead wire is installed, both electrode surfaces are covered with an immobilized enzyme film as in Example 1 to form an immobilized enzyme electrode for measuring glucose concentration. The immobilized enzyme film has a thickness of 30 μm. In this electrode, a reference electrode is used as a counter electrode.

[0026] Example 3 The mold was attached to an injection molding machine, and polyglycolic acid was melted and injection molded to form a microneedle patch as shown in Figure 4. The patch was elliptical with a long diameter of 16 mm and a short diameter of 12.3 mm. The details of this microneedle patch are as follows: Outer diameter of base part on which needles stand on patch substrate: 10.5 mm, width of corridor part: 1.2 mm. Height of corridor part and surrounding part from substrate surface: 0.5 mm. Height of base surface: 1.3 mm. Needle height: 0.9 mm. Needle spacing: 0.6 mm. The masking silicone resin was a two-liquid curing type (HYV-4000, Engraving Japan) that was used to protect the substrate, corridor, and periphery, and the silicone resin was peeled off after platinum electroless plating. A silver wire with a diameter of 0.3 mm was electrolytically oxidized in a ferric chloride solution to form a silver / silver chloride electrode as a reference electrode. Platinum lead wires were attached to each of the three electrodes. A conductive resin (Denacol EX-830, Nagase Chemtex) was used to connect to the electrodes. The patch was then immersed in an aqueous solution of equal amounts of GOD and human albumin (1% by weight), taken out, dried, and then immersed in an aqueous solution of glutaraldehyde to crosslink GOD and albumin to prepare an immobilized enzyme electrode for measuring glucose concentration. The thickness of the immobilized enzyme film was 30 μm.

[0027] The function of the manufactured electrode was confirmed. The above microneedle patch was placed facing upwards, and a circular filter paper with a diameter of 1.5 cm was placed on the microneedle surface. A sample solution was prepared by adding glucose to a 1% by mass sodium chloride solution to make it 200 mg / dl and 400 mg / dl, and 200 μl of the solution was dropped onto the filter paper to cover the three electrodes. 0.7 V was applied to the working electrode and the counter electrode, and the current flowing through both electrodes was measured. The results are shown in Table 1.

[0028] [Table 1] [Explanation of symbols]

[0029] 1 Reference electrode part 2 Counterelectrode 3 Working electrode part 4. Microneedle 5 Corridor 6 Base 7. Circuit Board 8 Lead Wire 9 Immobilized enzyme membrane 10 Silver / silver chloride electrode

Claims

1. A glucose sensor comprising a microneedle patch including a substrate and microneedles made of a non-conductive material, wherein the substrate surface on which the microneedles are arranged is provided with a working electrode portion, a counter electrode portion, a reference electrode portion, and electrical signal extraction lines from each of these electrode portions, the microneedle patch further comprising a base portion and a corridor portion, wherein the base portion is provided on the substrate surface and is provided for each electrode portion, the microneedles are arranged on the base portion, the height of the base portion is 0.2 mm to 4.0 mm, the corridor portion is a substrate portion surrounded by the base portion of different electrode portions, at least the working electrode portion is an immobilized enzyme sensor coated with cross-linked glucose oxidase, and the glucose sensor detects an electrical signal generated in vivo in response to the presence of glucose in body fluids and thereby measures the glucose concentration.

2. A glucose sensor comprising a microneedle patch including a substrate and microneedles made of a non-conductive material, wherein the substrate surface on which the microneedles are arranged is provided with an active electrode section, a reference electrode section, and electrical signal extraction lines from each electrode section, the microneedle patch further comprising a base section and a corridor section, wherein the base section is provided on the substrate surface and is provided for each electrode section, the microneedles are arranged on the base section, the height of the base section is 0.2 mm to 4.0 mm, the corridor section is a substrate portion surrounded by the base section of different electrode sections, each electrode section is an immobilized enzyme sensor coated with cross-linked glucose oxidase, and the glucose sensor detects an electrical signal generated in vivo in response to the presence of glucose in body fluids and thereby measures the glucose concentration.

3. The glucose sensor according to claim 1 or 2, wherein the nonconductive material is selected from the group consisting of polyglycolic acid, poly(lactic acid-glycolic acid) copolymer, polycarbonate, polytetrafluoroethylene, polyoxymethylene, polyethylene terephthalate, and COP (cyclic olefin polymer).

4. The glucose sensor according to claim 1 or 2, wherein the substrate shape of the microneedle patch is circular with a diameter of 0.4 to 5 cm, and the microneedles have a needle length of 100 μm or more and 2,000 μm or less.

5. The glucose sensor according to claim 1 or 2, further comprising a silicone rubber film on the outer surface of a coating layer made of crosslinked glucose oxidase.

6. The glucose sensor according to claim 1, wherein the working electrode and the counter electrode are covered with gold or platinum.

7. The glucose sensor according to claim 1 or 2, wherein the reference electrode portion is a silver / silver chloride electrode.

8. The glucose sensor according to claim 1, wherein the working electrode portion, counter electrode portion, and reference electrode portion are coated with cross-linked glucose oxidase, and the glucose sensor is an immobilized enzyme sensor.

9. The glucose sensor according to claim 1 or 2, wherein the electrode portion is covered with a film crosslinked with glucose oxidase.

10. The glucose sensor according to claim 1 or 2, wherein the electrode portion is coated with a film formed by crosslinking a polymer having an amino group as a base with glucose oxidase.

11. A monitoring system for glucose concentration in interstitial fluid, comprising a glucose sensor according to claim 1 or 2, a transmitter, and a monitor, wherein an electrical signal obtained from the glucose sensor is transmitted as data to the monitor via the transmitter.

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