WO2024005192A1 - Electrode for glucose sensors - Google Patents
Electrode for glucose sensors Download PDFInfo
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- WO2024005192A1 WO2024005192A1 PCT/JP2023/024403 JP2023024403W WO2024005192A1 WO 2024005192 A1 WO2024005192 A1 WO 2024005192A1 JP 2023024403 W JP2023024403 W JP 2023024403W WO 2024005192 A1 WO2024005192 A1 WO 2024005192A1
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- Prior art keywords
- glucose
- electrode part
- electrode
- substrate
- glucose sensor
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 title claims abstract description 71
- 239000008103 glucose Substances 0.000 title claims abstract description 71
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
- A61B5/1468—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using chemical or electrochemical methods, e.g. by polarographic means
- A61B5/1473—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using chemical or electrochemical methods, e.g. by polarographic means invasive, e.g. introduced into the body by a catheter
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
- A61B5/1486—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using enzyme electrodes, e.g. with immobilised oxidase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
Definitions
- the present invention relates to an electrode for a glucose sensor. Specifically, the present invention relates to a glucose concentration sensing system using a microneedle array.
- Glucose levels in a patient's blood vary over time and typically depend on the individual's physical activity, food, drink, sugar intake, metabolic rate, etc.
- Glucose as a compound is difficult to directly measure electrochemically because it does not exhibit relatively significant changes in properties during oxidation and/or reduction processes.
- a preferred method is to use various enzymes and/or proteins to react specifically with glucose, and then quantitatively analyze the yield and/or byproducts to measure the glucose level. It's here. Therefore, there are many methods for measuring glucose, such as methods for quantitatively measuring glucose in blood using enzymes. However, these methods cannot be applied to in-vivo systems, and in most cases are difficult to apply even to simple in-vitro systems.
- Electrodes 1, 2, 3 Chemically improved electrodes have come to be used in electrochemical sensing mechanisms.
- a simple electrode is prepared by covalently bonding an enzyme or other protein quantification reagent to the electrode, and is used to contact the collected body fluid to measure the glucose concentration in the body fluid.
- Technology has further advanced in recent years.
- the current method is to insert an electrode part into the body percutaneously as a sensor, make it come into contact with blood or exudate in the body, and react with glucose to generate hydrogen peroxide, which is then electrochemically measured by measuring current or voltage. This is a mainstream device (Non-Patent Document 1).
- a first object of the invention is therefore to provide an improved electrode system capable of measuring glucose levels or concentrations in body fluids and which can be applied in-vitro or even in-vivo. be.
- a second object of the present invention is to provide an improved electrode means which is particularly suitable for measuring glucose concentrations in body fluids in-vitro or in-vivo and in which the sensing electrode is reliable, stable and strong. Our goal is to provide the following.
- Yet another object of the invention is to provide a method for preparing a glucose sensor capable of providing an electrical signal for glucose levels in body fluids and suitable for use with variable speed insulin pumps.
- an electrode for sensing glucose by means of an electrode that functions based on amperometric measurements.
- Such electrodes are formed by dividing the microneedle patch surface into two or three parts.
- the electrode consists of a working electrode part and a counter electrode part.
- the electrode consists of a working electrode part, a counter electrode part, and a reference electrode part. It is also possible to divide it into two parts to form a working electrode part and a reference electrode part.
- the working electrode part and the counter electrode part are formed by, for example, coating the microneedles and the substrate surface on which they stand with gold or platinum by electrolytic or electroless plating.
- Another coating method may be to cover the substrate surface with gold or platinum foil by pressure bonding.
- the platinum or gold that covers each divided electrode is coated so that they do not come into contact with each other.
- the reference electrode part in the three divisions is made into a silver/silver chloride electrode part by coating the microneedles and the substrate surface on which they stand with silver by electrolytic or electroless plating and chlorinating the surface.
- a calomel electrode may be used as the reference electrode section.
- the counter electrode part and the reference electrode part may be formed on the substrate part which does not have microneedles without dividing the patch surface on which the microneedles stand.
- Each electrode portion is coated with an immobilized enzyme that immobilizes glucose oxidase (GOD) and makes it insoluble in water.
- GOD glucose oxidase
- a covering layer such as a thin silicone rubber film, may be provided on the external surface of the GOD layer.
- GOD coating is essential for the working electrode portion, but is not essential for the counter electrode portion and the reference electrode portion.
- Lead wires are installed from the working electrode section, counter electrode section, and reference electrode section to extract electrical signals. Lead wire means an electrical signal extraction wire. The extracted electrical signals are processed in accordance with the methods used up to now. The present invention is as shown below. [1] A working electrode part, a counter electrode part, a reference electrode part, and an electric signal from each of these electrode parts on the surface of the substrate where the microneedles of the microneedle patch including microneedles made of a non-conductive material and a substrate are lined up.
- An immobilized enzyme sensor is provided with a lead-out line, and at least the working electrode part is coated with cross-linked glucose oxidase (even if the counter electrode part and the reference electrode part are also immobilized enzyme sensors coated with cross-linked glucose oxidase).
- Glucose sensors sense electrical signals generated in-vivo in response to the presence of glucose in body fluids, thereby measuring glucose concentration.
- a working electrode section, a reference electrode section, and electrical signal extraction lines from each electrode section are provided on the substrate surface where the microneedles of the microneedle patch including microneedles made of a non-conductive material and a substrate are lined up.
- Each electrode part is 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 glucose concentration.
- glucose sensor [3]
- the non-conductive material is from the group consisting of polyglycolic acid, poly(lactic acid-glycolic acid) copolymer, polycarbonate, polytetrafluoroethylene, polyoxymethylene, polyethylene terephthalate, and COP (cyclic olefin polymer).
- the glucose sensor according to [1] or [2], which is selected.
- the substrate of the microneedle patch has a circular shape with a diameter of 0.4 to 5 cm, and the microneedle has a needle length of 100 ⁇ m or more and 2,000 ⁇ m or less.
- the microneedle patch includes a base portion and a corridor portion.
- the working electrode portion and the counter electrode portion are covered with gold or platinum.
- the reference electrode portion is a silver/silver chloride electrode.
- the present invention is a sensor that detects glucose using a microneedle patch in which a large number of microneedles are lined up.
- the glucose sensor of the present invention can be easily introduced into the skin and can generate a large current value, so it is possible to provide a more stable system than conventional methods.
- the microneedle patch By applying the microneedle patch to the skin, the glucose concentration in the interstitial fluid can be expressed as a signal indication and is therefore useful as a blood glucose sensor.
- FIG. 1 is a plan view and a cross-sectional view of a microneedle patch for an immobilized enzyme electrode.
- FIG. 2 is a diagram of the completed immobilized enzyme electrode.
- FIG. 3 is a schematic diagram of an example of a microneedle patch used in the glucose sensor of the present invention.
- FIG. 4 is a plan view (B) and a cross-sectional view (A) of another example of a microneedle patch for an immobilized enzyme electrode. It has a working electrode part and a counter electrode part, and a silver/silver chloride electrode is used as a reference electrode part.
- the microneedle patch in the present invention has a structure including a substrate and a plurality of microneedles arranged on one side of the substrate, and the microneedle patch may be further lined with an adhesive sheet.
- Adhesive sheets typically use polyurethane, polyethylene, polyester, paper, etc. as a film base material, and coat the film with an acrylic or rubber adhesive in a thickness of 5 to 50 ⁇ m on a film formed to a thickness of about 5 to 50 ⁇ m. It has been applied to a certain extent.
- the shape of the pressure-sensitive adhesive sheet is not particularly limited, but it is preferably circular, oval, bead shape, etc. to resemble the shape of the microneedle array.
- the material, shape and size of the substrate of the microneedle patch are not particularly limited, and conventionally used ones can be used.
- the base material of the substrate and the base material of the microneedles are basically the same, but may be different.
- Examples of the base include non-conductive inorganic compounds such as silicone, silicon dioxide, ceramic, and glass, and non-conductive materials such as synthetic or natural resin materials that are water-insoluble.
- Synthetic or natural resin materials include biodegradable polymers such as polylactic acid, polyglycolic acid, poly(lactic acid-glycolic acid) copolymer, capronolactone, or nylon, polycarbonate, polytetrafluoroethylene, polyoxymethylene, Biodegradable polymers such as polyethylene terephthalate and COP (cyclic olefin polymer) can be mentioned.
- the substrate is made of the above-mentioned non-conductive material.
- 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). It is preferable.
- the shape of the substrate can be any shape.
- the shapes are basically circular, oval, triangular, quadrangular, polygonal, etc., and may be further modified according to the application site (skin).
- the size of the substrate is typically 0.2 to 5 cm, preferably 0.4 to 5 cm, and more preferably 0.5 to 3 cm, expressed in terms of diameter (major axis) or length of one side (long side). preferable.
- the shape of the substrate of the microneedle patch is preferably circular with a diameter of 0.4 to 5 cm, more preferably circular with a diameter of 0.5 to 3 cm.
- the area (usually a flat area) of the substrate is usually 0.05 to 100 cm 2 , preferably about 0.4 to 10 cm 2 , and more preferably about 0.6 to 5 cm 2 from the viewpoint of ease of handling.
- the thickness of the substrate is preferably 0.1 to 1.0 mm.
- Microneedles have a needle length of 100 ⁇ m or more and 2,000 ⁇ m or less, preferably 200 to 1,000 ⁇ m. When the size of the apex of the tip of the needle is expressed as a diameter, it is 80 ⁇ m or less, preferably 20 ⁇ m or more, considering ease and reliability of penetration into the skin.
- Individual microneedles can be cylindrical or conical with a circular base, elliptical cylinder or elliptical cone with an elliptical base, triangular prism or pyramid with a triangular base, or quadrangular prism or square with a square base.
- Examples include a quadrangular pyramid shape, a polygonal columnar shape or a polygonal pyramid shape whose bottom surface is a polygon.
- the size of the bottom surface is expressed by the major axis as the diameter, and the minor axis is shorter than the major axis as long as the ellipse can be formed.
- one side may be represented as a representative, or a diagonal may be represented as a representative.
- the diameter at the bottom is about 100 to 400 ⁇ m, preferably about 150 to 300 ⁇ m.
- the microneedle in the present invention may have a step.
- step refers to a step in which the cross-sectional area of the microneedle decreases discontinuously from a certain point toward the tip of the microneedle, creating a step-like shape.
- the basic form of a microneedle patch is that the microneedles stand directly on the substrate, but a base is provided on the substrate and the microneedles are placed on top of the base (called the patch plane). It may be something with a By providing the base portion, the skin puncturing ability of the microneedles becomes more reliable.
- the thickness of the base portion (height of the base surface) is preferably 0.2 mm to 4.0 mm above the substrate surface, more preferably 0.5 mm to 2.0 mm.
- a base part is provided for each electrode part, such as a working electrode part, a counter electrode part, and a reference electrode part.
- the three electrode sections should have approximately the same area, but there are no particular restrictions.
- the part of the substrate surrounded by the base parts of different electrode parts is called a corridor part. A schematic diagram of the above arrangement is shown in FIG.
- a molding method suitable for the material is used to mold the microneedle patch, but it is preferable for industrial production to use injection molding of biodegradable polymers and non-biodegradable polymers.
- a microneedle patch is coated with gold or platinum.
- a coating method a vapor deposition method, an electrolytic plating method, an electroless plating method, etc. can be used.
- the reference electrode section is a silver/silver chloride electrode.
- the silver/silver chloride electrode is produced by electrolyzing silver or dipping it in an aqueous solution containing chloride ions to convert the silver surface into silver chloride.
- the working electrode section is coated with immobilized GOD.
- the counter electrode portion and the reference electrode portion may or may not be coated with GOD.
- glucose oxidase is insolubilized by reacting with a base polymer having an amino group using a polyfunctional aldehyde such as glutaraldehyde, and at the same time, the base is also insolubilized and fixed to the electrode portion.
- base polymers having amino groups include serum albumin, nucleic acids, chitosan, and the like.
- the platinum plating method using electroless plating will be described in detail below.
- the entire corridor and the reference electrode are covered with silicone resin, heated to form a film, and masked. Mask the sides and back in the same way.
- Electroless platinum plating is carried out using dinitrodiammine platinum, ammonia, and hydrazine at 60° C. for 1 hour.
- the obtained microneedle patch with a platinum film is washed with water and then dried. Thereafter, the working electrode part and the counter electrode part are masked with silicone, the masking of the reference electrode part is removed, and the reference electrode part is plated with electroless silver.
- the lead wire is installed on the microneedle side, but it may be installed on the back side instead of the microneedle side.
- a GOD film is formed on the working electrode portion and the other two electrodes, and further crosslinked with glutaraldehyde to obtain an immobilized enzyme electrode (glucose sensor).
- a known method is used to measure the glucose concentration in the interstitial fluid using the immobilized enzyme electrode produced in this way.
- An immobilized enzyme electrode senses an electrical signal generated in-vivo in response to the presence (abundance) of glucose in a body fluid, thereby making it possible to measure glucose concentration.
- the immobilized enzyme electrode microneedle patch is administered transdermally using an applicator.
- the method described in JP-A-2-501679 connects three or two lead wires to a voltage applying device, applies a voltage of 0.6 to 1.0 V, and measures the flowing current. All you have to do is follow.
- a transmitter is placed on the skin adjacent to the skin-administered microneedle, electrical operation is performed from there, and the obtained electrical signal is wirelessly transmitted to a monitor to transmit the electrical signal over time. It is also possible to monitor the glucose concentration in the fluid.
- Example 1 The mold was attached to an injection molding machine, polyglycolic acid was melted, and a microneedle patch as shown in FIG. 1 was formed by injection molding. Patch diameter: 12.3 mm. The details of this microneedle patch are as follows. Outer diameter of the base on which the needles stand on the patch board: 10.5 mm, width of the corridor: 1.2 mm. Height of the corridor part and peripheral part from the board surface: 0.5 mm. Base height: 1.3mm. Needle height: 0.9mm. Needle spacing: 0.6mm. A two-component curing type silicone resin (HYV-4000, manufactured by Engraving Japan Co., Ltd.) was used as the masking silicone resin.
- HYV-4000 manufactured by Engraving Japan Co., Ltd.
- the silicone resin was peeled off from the reference electrode part, the working electrode part and the counter electrode part were masked, and the reference electrode part was made into a silver/silver chloride electrode.
- the thickness of the platinum film and silver film was approximately 10 ⁇ m.
- Lead wires were attached to each of the three electrode parts.
- the patch was immersed in a 1% aqueous solution of equal amounts of GOD and human albumin, taken out, dried, and immersed in an aqueous glutaraldehyde solution to crosslink GOD and albumin, thereby forming an immobilized enzyme electrode for measuring glucose concentration.
- the thickness of the immobilized enzyme membrane was 30 ⁇ m.
- Example 2 Polyglycolic acid was melted and injection molded, and a milky white oval microneedle patch was taken out.
- the patch major axis was 12.3 mm, and the minor axis was 11.5 mm (FIG. 3).
- the details of this microneedle patch are as follows.
- the outer diameter of the base on which the needles stand on the patch board 10.5 mm, the inner diameter of the base: 6.0 mm.
- Corridor width 1.2mm. Height of the corridor section and center section from the board surface: 0.5 mm. Base height: 1.3mm. Needle height: 0.6mm. Total number of needles: 280.
- the base of this microneedle patch was divided into two parts, and the needle on one of the bases was covered with a platinum film without masking to serve as the working electrode.
- the thickness of the platinum film was 10 ⁇ m.
- the needle part on the other base part was similarly made into a silver/silver chloride electrode part.
- the thickness of the silver/silver chloride film was 10 ⁇ m.
- both electrode surfaces were covered with an immobilized enzyme membrane in the same manner as in Example 1 to obtain an immobilized enzyme electrode for measuring glucose concentration.
- the thickness of the immobilized enzyme membrane was 30 ⁇ m.
- a reference electrode is used as a counter electrode.
- Example 3 The mold was attached to an injection molding machine, polyglycolic acid was melted, and a microneedle patch as shown in FIG. 4 was formed by injection molding.
- the patch had an oval shape with a major axis of 16 mm and a minor axis of 12.3 mm.
- the details of this microneedle patch are as follows. Outer diameter of the base on which the needles stand on the patch board: 10.5 mm, width of the corridor: 1.2 mm. Height of the corridor part and peripheral part from the board surface: 0.5 mm. Base height: 1.3mm. Needle height: 0.9mm. Needle spacing: 0.6mm.
- the silicone resin for masking was a two-component curing type (HYV-4000, manufactured by Engraving Japan Co., Ltd.) to protect the substrate, the corridor, and the surrounding area, and after electroless platinum plating, the silicone resin was peeled off.
- a silver wire with a diameter of 0.3 mm was electrolytically oxidized in an iron chloride solution to obtain a silver/silver chloride electrode part.
- a platinum lead wire was attached to each of the three electrode parts.
- a conductive resin (Denacol EX-830, manufactured by Nagase ChemteX) was used for connection to the electrodes.
- the patch was immersed in an equal amount of 1% by mass aqueous solution of GOD and human albumin, taken out, dried, and immersed in an aqueous glutaraldehyde solution to crosslink GOD and albumin, thereby forming an immobilized enzyme electrode for measuring glucose concentration.
- the thickness of the immobilized enzyme film was 30 ⁇ m.
- the effect of the manufactured electrode was confirmed.
- the microneedle patch was left still facing upward, and a circular filter paper with a diameter of 1.5 cm was placed on the surface of the microneedle.
- a sample solution was prepared by adding glucose to a 1% by mass sodium chloride solution to a concentration of 200 mg/dl and 400 mg/dl, and 200 ⁇ l of the solution was dropped onto a filter paper to cover the three electrodes.
- a voltage of 0.7 V was applied to the working electrode section and the counter electrode section, and the value of the current flowing through both electrodes was measured. The results are shown in Table 1.
- Reference electrode part 2 Counter electrode part 3
- Working electrode part 4 Microneedle 5
- Corridor part 6 Base part 7
- Substrate part 8 Lead wire 9
- Immobilized enzyme film 10 Silver/silver chloride electrode part
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Abstract
The purpose of the present invention is 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. 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 on which microneedles of a microneedle patch including the substrate and microneedles made from a non-electroconductive material bristle, in which 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 to thereby measure the concentration of glucose.
Description
本発明は、グルコースセンサー用電極に関する。詳しくは、マイクロニードルアレイを利用したグルコース濃度の感知システムに関する。
The present invention relates to an electrode for a glucose sensor. Specifically, the present invention relates to a glucose concentration sensing system using a microneedle array.
患者の血液中のグルコースレベルは、時間に依って変化し、また通常、個人の肉体的活動、食べ物、飲物、糖の摂取、代謝速度などに依って変化する。化合物としてのグルコースは、酸化及び／又は還元工程において比較的顕著な特性変化を示さないために、電気化学的に直接測定するのが難しい。このため、各種の酵素及び／又は蛋白質を利用し、これらとグルコースとを特異的に反応せしめて収量及び／又は副生成物を量的に分析して、グルコースレベルを測定する方法が好ましく行なわれてきた。従って、酵素を用いて血液中のグルコースを量的に測定する方法などの、多くのグルコースの測定法がある。しかしながら、これらの方法は、ｉｎ－ｖｉｖｏの系に適用することができず、また、ほとんどの場合、簡単なｉｎ－ｖｉｔｒｏの系においても適用するのに困難を伴う。
Glucose levels in a patient's blood vary over time and typically depend on the individual's physical activity, food, drink, sugar intake, metabolic rate, etc. Glucose as a compound is difficult to directly measure electrochemically because it does not exhibit relatively significant changes in properties during oxidation and/or reduction processes. For this reason, a preferred method is to use various enzymes and/or proteins to react specifically with glucose, and then quantitatively analyze the yield and/or byproducts to measure the glucose level. It's here. Therefore, there are many methods for measuring glucose, such as methods for quantitatively measuring glucose in blood using enzymes. However, these methods cannot be applied to in-vivo systems, and in most cases are difficult to apply even to simple in-vitro systems.
化学的に改良された電極が電気化学的感知機構に使用されるようになった（特許文献１、２、３）。この場合、酵素あるいは他の蛋白定量試薬を電極に共有結合させて簡単な電極を調製し、これを用いて、採取した体液に接触させて体液中のグルコース濃度を測定する。近年さらに技術は進歩している。電極部をセンサーとして経皮的に体内に挿入し、体内の血液あるいは浸出液に接触させてグルコースと反応させて発生する過酸化水素を電流測定あるいは電圧測定により電気化学的測定を行なうのが現在の主流の装置である(非特許文献１)。
Chemically improved electrodes have come to be used in electrochemical sensing mechanisms ( Patent Documents 1, 2, 3). In this case, a simple electrode is prepared by covalently bonding an enzyme or other protein quantification reagent to the electrode, and is used to contact the collected body fluid to measure the glucose concentration in the body fluid. Technology has further advanced in recent years. The current method is to insert an electrode part into the body percutaneously as a sensor, make it come into contact with blood or exudate in the body, and react with glucose to generate hydrogen peroxide, which is then electrochemically measured by measuring current or voltage. This is a mainstream device (Non-Patent Document 1).
現在主流の装置を使用する方法においては、化学的検知のためのセンサープローブを皮内に導入するためにステンレスの導入用針が必要であり、装置全体として非常に複雑な物となり、患者への装着の操作もまた複雑であり、システムとしても高価となる。
しかして本発明の第１の目的は、体液中のグルコースレベル又は濃度を測定することができ、そしてｉｎ－ｖｉｔｒｏあるいはｉｎ－ｖｉｖｏにおいても適用することのできる改良された電極システムを提供することにある。
本発明の第２の目的は、ｉｎ－ｖｉｔｒｏあるいはｉｎ－ｖｉｖｏにおいて体液中のグルコース濃度を測定するのに特に好適であり、かつ、感知電極は信頼性があり安定で強い、改良された電極手段を提供することにある。
本発明の更に他の目的は、体液中のグルコースレベルに対する電気的信号を提示することができ、速度可変インシュリンポンプと共に使用するのに適した、グルコースセンサーを調製する方法を提供することにある。 The current method of using the mainstream device requires a stainless steel introduction needle to introduce the sensor probe for chemical detection into the skin, making the device as a whole very complicated and difficult for the patient. The mounting operation is also complicated and the system is expensive.
A first object of the invention is therefore to provide an improved electrode system capable of measuring glucose levels or concentrations in body fluids and which can be applied in-vitro or even in-vivo. be.
A second object of the present invention is to provide an improved electrode means which is particularly suitable for measuring glucose concentrations in body fluids in-vitro or in-vivo and in which the sensing electrode is reliable, stable and strong. Our goal is to provide the following.
Yet another object of the invention is to provide a method for preparing a glucose sensor capable of providing an electrical signal for glucose levels in body fluids and suitable for use with variable speed insulin pumps.
しかして本発明の第１の目的は、体液中のグルコースレベル又は濃度を測定することができ、そしてｉｎ－ｖｉｔｒｏあるいはｉｎ－ｖｉｖｏにおいても適用することのできる改良された電極システムを提供することにある。
本発明の第２の目的は、ｉｎ－ｖｉｔｒｏあるいはｉｎ－ｖｉｖｏにおいて体液中のグルコース濃度を測定するのに特に好適であり、かつ、感知電極は信頼性があり安定で強い、改良された電極手段を提供することにある。
本発明の更に他の目的は、体液中のグルコースレベルに対する電気的信号を提示することができ、速度可変インシュリンポンプと共に使用するのに適した、グルコースセンサーを調製する方法を提供することにある。 The current method of using the mainstream device requires a stainless steel introduction needle to introduce the sensor probe for chemical detection into the skin, making the device as a whole very complicated and difficult for the patient. The mounting operation is also complicated and the system is expensive.
A first object of the invention is therefore to provide an improved electrode system capable of measuring glucose levels or concentrations in body fluids and which can be applied in-vitro or even in-vivo. be.
A second object of the present invention is to provide an improved electrode means which is particularly suitable for measuring glucose concentrations in body fluids in-vitro or in-vivo and in which the sensing electrode is reliable, stable and strong. Our goal is to provide the following.
Yet another object of the invention is to provide a method for preparing a glucose sensor capable of providing an electrical signal for glucose levels in body fluids and suitable for use with variable speed insulin pumps.
本発明によれば、電流測定に基づいて機能する電極によりグルコースを感知する電極が提供される。
かかる電極は、マイクロニードルパッチ面を２分割あるいは３分割して形成する。２分割の場合は、電極は作用電極部と対極部とからなる。３分割の場合は、電極は作用電極部、対極部、及び参照電極部からなる。２分割において作用電極部及び参照電極部とすることも可能である。作用電極部及び対極部は、例えば、電解あるいは無電解めっきによってマイクロニードル及びそれが立つ基板面を金あるいは白金で被覆することで形成される。別の被覆法としては、基板面に金あるいは白金の箔を圧着により基板面被覆することもありえる。被覆にあたっては、分割された各電極を被覆する白金ないしは金は、互いに接触しないように被覆する。３分割における参照電極部は、電解あるいは無電解めっきによってマイクロニードル及びそれが立つ基板面を銀で被覆し表面を塩素化することで、銀／塩化銀電極部とする。参照電極部としては、カロメル電極を用いてもよい。
マイクロニードルが林立するパッチ面を分割しないで、マイクロニードルを有しない基板部に対極部や参照電極部を形成してもよい。
各電極部をグルコースオキシダーゼ（ＧＯＤ）を固定化して水不溶とした固定化酵素で被覆する。ＧＯＤ層の外部表面上には、薄いシリコーンラバーフィルムの如き被覆層を設けてもよい。ＧＯＤの被覆は作用電極部においては必須であるが、対極部及び参照電極部においては必須ではない。
作用電極部、対極部、参照電極部からはリード線を設置し電気信号の取り出しを設ける。リード線は、電気信号取り出し線を意味する。取り出した電気的信号の処理は、現在までに成されている方法に準じる。
本発明は、以下に示す通りである。
〔１〕 非導電性材料からなるマイクロニードル及び基板を含むマイクロニードルパッチのマイクロニードルが林立している基板面に作用電極部、対極部、及び参照電極部並びにこれらの各電極部からの電気信号取り出し線を設けてなり、少なくとも作用電極部は架橋されたグルコースオキシダーゼで被覆した固定化酵素センサーであり（対極部及び参照電極部も架橋されたグルコースオキシダーゼで被覆した固定化酵素センサーであってもよく）、体液中のグルコースの存在に応じてｉｎ－ｖｉｖｏで発生する電気的信号を感知し、それによりグルコース濃度を測定するグルコースセンサー。
〔２〕 非導電性材料からなるマイクロニードル及び基板を含むマイクロニードルパッチのマイクロニードルが林立している基板面に作用電極部、及び参照電極部並びに各電極部からの電気信号取り出し線を設けてなり、各電極部は架橋されたグルコースオキシダーゼで被覆した固定化酵素センサーであり、体液中のグルコースの存在に応じてｉｎ－ｖｉｖｏで発生する電気的信号を感知し、それによりグルコース濃度を測定するグルコースセンサー。
〔３〕 非導電性材料が、ポリグリコール酸、ポリ（乳酸－グリコール酸）共重合体、ポリカーボネート、ポリテトラフルオロエチレン、ポリオキシメチレン、ポリエチレンテレフタレート、及びＣＯＰ（サイクリックオレフィンポリマー）からなる群より選ばれる、〔１〕又は〔２〕に記載のグルコースセンサー。
〔４〕 前記マイクロニードルパッチの基板の形状が直径０．４～５ｃｍの円形であり、前記マイクロニードルが針長さ１００μｍ以上２，０００μｍ以下である、〔１〕～〔３〕のいずれかに記載のグルコースセンサー。
〔５〕 前記マイクロニードルパッチが土台部及び回廊部を含む、〔１〕～〔４〕のいずれかに記載のグルコースセンサー。
〔６〕 前記作用電極部及び対極部が金又は白金で覆われている、〔１〕に記載のグルコースセンサー。
〔７〕 前記参照電極部が銀／塩化銀電極である、〔１〕～〔５〕のいずれかに記載のグルコースセンサー。
〔８〕 前記作用電極部、対極部、及び参照電極部が、架橋されたグルコースオキシダーゼで被覆した固定化酵素センサーである、［１］に記載のグルコースセンサー。
［９］ 前記電極部がグルコースオキシダーゼを架橋させた皮膜で被覆されている、〔１〕～〔８〕のいずれかに記載のグルコースセンサー。
〔１０〕 前記電極部が基剤としてアミノ基を有する高分子とグルコースオキシダーゼとを架橋させた皮膜で被覆されている、〔１〕～〔９〕のいずれかに記載のグルコースセンサー。
〔１１〕 〔１〕～〔１０〕のいずれかに記載のグルコースセンサーと、トランスミッターと、モニターとを用いて、該グルコースセンサーから得られた電気信号を該トランスミッターにより該モニターにデータ送信する、間質液中のグルコース濃度のモニターシステム。 According to the present invention, an electrode is provided for sensing glucose by means of an electrode that functions based on amperometric measurements.
Such electrodes are formed by dividing the microneedle patch surface into two or three parts. In the case of two-part electrode, the electrode consists of a working electrode part and a counter electrode part. In the case of three parts, the electrode consists of a working electrode part, a counter electrode part, and a reference electrode part. It is also possible to divide it into two parts to form a working electrode part and a reference electrode part. The working electrode part and the counter electrode part are formed by, for example, coating the microneedles and the substrate surface on which they stand with gold or platinum by electrolytic or electroless plating. Another coating method may be to cover the substrate surface with gold or platinum foil by pressure bonding. In coating, the platinum or gold that covers each divided electrode is coated so that they do not come into contact with each other. The reference electrode part in the three divisions is made into a silver/silver chloride electrode part by coating the microneedles and the substrate surface on which they stand with silver by electrolytic or electroless plating and chlorinating the surface. A calomel electrode may be used as the reference electrode section.
The counter electrode part and the reference electrode part may be formed on the substrate part which does not have microneedles without dividing the patch surface on which the microneedles stand.
Each electrode portion is coated with an immobilized enzyme that immobilizes glucose oxidase (GOD) and makes it insoluble in water. A covering layer, such as a thin silicone rubber film, may be provided on the external surface of the GOD layer. GOD coating is essential for the working electrode portion, but is not essential for the counter electrode portion and the reference electrode portion.
Lead wires are installed from the working electrode section, counter electrode section, and reference electrode section to extract electrical signals. Lead wire means an electrical signal extraction wire. The extracted electrical signals are processed in accordance with the methods used up to now.
The present invention is as shown below.
[1] A working electrode part, a counter electrode part, a reference electrode part, and an electric signal from each of these electrode parts on the surface of the substrate where the microneedles of the microneedle patch including microneedles made of a non-conductive material and a substrate are lined up. An immobilized enzyme sensor is provided with a lead-out line, and at least the working electrode part is coated with cross-linked glucose oxidase (even if the counter electrode part and the reference electrode part are also immobilized enzyme sensors coated with cross-linked glucose oxidase). Glucose sensors sense electrical signals generated in-vivo in response to the presence of glucose in body fluids, thereby measuring glucose concentration.
[2] A working electrode section, a reference electrode section, and electrical signal extraction lines from each electrode section are provided on the substrate surface where the microneedles of the microneedle patch including microneedles made of a non-conductive material and a substrate are lined up. Each electrode part is 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 glucose concentration. glucose sensor.
[3] The non-conductive material is from the group consisting of polyglycolic acid, poly(lactic acid-glycolic acid) copolymer, polycarbonate, polytetrafluoroethylene, polyoxymethylene, polyethylene terephthalate, and COP (cyclic olefin polymer). The glucose sensor according to [1] or [2], which is selected.
[4] Any one of [1] to [3], wherein the substrate of the microneedle patch has a circular shape with 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. The glucose sensor described.
[5] The glucose sensor according to any one of [1] to [4], wherein the microneedle patch includes a base portion and a corridor portion.
[6] The glucose sensor according to [1], wherein the working electrode portion and the counter electrode portion are covered with gold or platinum.
[7] The glucose sensor according to any one of [1] to [5], wherein the reference electrode portion is a silver/silver chloride electrode.
[8] The glucose sensor according to [1], wherein the working electrode part, the counter electrode part, and the reference electrode part are immobilized enzyme sensors coated with crosslinked glucose oxidase.
[9] The glucose sensor according to any one of [1] to [8], wherein the electrode portion is coated with a film in which glucose oxidase is crosslinked.
[10] The glucose sensor according to any one of [1] to [9], wherein the electrode portion is coated with a film made by crosslinking glucose oxidase with a polymer having an amino group as a base.
[11] Using the glucose sensor according to any one of [1] to [10], a transmitter, and a monitor, and transmitting data of an electrical signal obtained from the glucose sensor to the monitor by the transmitter. Monitoring system for glucose concentration in quality fluid.
かかる電極は、マイクロニードルパッチ面を２分割あるいは３分割して形成する。２分割の場合は、電極は作用電極部と対極部とからなる。３分割の場合は、電極は作用電極部、対極部、及び参照電極部からなる。２分割において作用電極部及び参照電極部とすることも可能である。作用電極部及び対極部は、例えば、電解あるいは無電解めっきによってマイクロニードル及びそれが立つ基板面を金あるいは白金で被覆することで形成される。別の被覆法としては、基板面に金あるいは白金の箔を圧着により基板面被覆することもありえる。被覆にあたっては、分割された各電極を被覆する白金ないしは金は、互いに接触しないように被覆する。３分割における参照電極部は、電解あるいは無電解めっきによってマイクロニードル及びそれが立つ基板面を銀で被覆し表面を塩素化することで、銀／塩化銀電極部とする。参照電極部としては、カロメル電極を用いてもよい。
マイクロニードルが林立するパッチ面を分割しないで、マイクロニードルを有しない基板部に対極部や参照電極部を形成してもよい。
各電極部をグルコースオキシダーゼ（ＧＯＤ）を固定化して水不溶とした固定化酵素で被覆する。ＧＯＤ層の外部表面上には、薄いシリコーンラバーフィルムの如き被覆層を設けてもよい。ＧＯＤの被覆は作用電極部においては必須であるが、対極部及び参照電極部においては必須ではない。
作用電極部、対極部、参照電極部からはリード線を設置し電気信号の取り出しを設ける。リード線は、電気信号取り出し線を意味する。取り出した電気的信号の処理は、現在までに成されている方法に準じる。
本発明は、以下に示す通りである。
〔１〕 非導電性材料からなるマイクロニードル及び基板を含むマイクロニードルパッチのマイクロニードルが林立している基板面に作用電極部、対極部、及び参照電極部並びにこれらの各電極部からの電気信号取り出し線を設けてなり、少なくとも作用電極部は架橋されたグルコースオキシダーゼで被覆した固定化酵素センサーであり（対極部及び参照電極部も架橋されたグルコースオキシダーゼで被覆した固定化酵素センサーであってもよく）、体液中のグルコースの存在に応じてｉｎ－ｖｉｖｏで発生する電気的信号を感知し、それによりグルコース濃度を測定するグルコースセンサー。
〔２〕 非導電性材料からなるマイクロニードル及び基板を含むマイクロニードルパッチのマイクロニードルが林立している基板面に作用電極部、及び参照電極部並びに各電極部からの電気信号取り出し線を設けてなり、各電極部は架橋されたグルコースオキシダーゼで被覆した固定化酵素センサーであり、体液中のグルコースの存在に応じてｉｎ－ｖｉｖｏで発生する電気的信号を感知し、それによりグルコース濃度を測定するグルコースセンサー。
〔３〕 非導電性材料が、ポリグリコール酸、ポリ（乳酸－グリコール酸）共重合体、ポリカーボネート、ポリテトラフルオロエチレン、ポリオキシメチレン、ポリエチレンテレフタレート、及びＣＯＰ（サイクリックオレフィンポリマー）からなる群より選ばれる、〔１〕又は〔２〕に記載のグルコースセンサー。
〔４〕 前記マイクロニードルパッチの基板の形状が直径０．４～５ｃｍの円形であり、前記マイクロニードルが針長さ１００μｍ以上２，０００μｍ以下である、〔１〕～〔３〕のいずれかに記載のグルコースセンサー。
〔５〕 前記マイクロニードルパッチが土台部及び回廊部を含む、〔１〕～〔４〕のいずれかに記載のグルコースセンサー。
〔６〕 前記作用電極部及び対極部が金又は白金で覆われている、〔１〕に記載のグルコースセンサー。
〔７〕 前記参照電極部が銀／塩化銀電極である、〔１〕～〔５〕のいずれかに記載のグルコースセンサー。
〔８〕 前記作用電極部、対極部、及び参照電極部が、架橋されたグルコースオキシダーゼで被覆した固定化酵素センサーである、［１］に記載のグルコースセンサー。
［９］ 前記電極部がグルコースオキシダーゼを架橋させた皮膜で被覆されている、〔１〕～〔８〕のいずれかに記載のグルコースセンサー。
〔１０〕 前記電極部が基剤としてアミノ基を有する高分子とグルコースオキシダーゼとを架橋させた皮膜で被覆されている、〔１〕～〔９〕のいずれかに記載のグルコースセンサー。
〔１１〕 〔１〕～〔１０〕のいずれかに記載のグルコースセンサーと、トランスミッターと、モニターとを用いて、該グルコースセンサーから得られた電気信号を該トランスミッターにより該モニターにデータ送信する、間質液中のグルコース濃度のモニターシステム。 According to the present invention, an electrode is provided for sensing glucose by means of an electrode that functions based on amperometric measurements.
Such electrodes are formed by dividing the microneedle patch surface into two or three parts. In the case of two-part electrode, the electrode consists of a working electrode part and a counter electrode part. In the case of three parts, the electrode consists of a working electrode part, a counter electrode part, and a reference electrode part. It is also possible to divide it into two parts to form a working electrode part and a reference electrode part. The working electrode part and the counter electrode part are formed by, for example, coating the microneedles and the substrate surface on which they stand with gold or platinum by electrolytic or electroless plating. Another coating method may be to cover the substrate surface with gold or platinum foil by pressure bonding. In coating, the platinum or gold that covers each divided electrode is coated so that they do not come into contact with each other. The reference electrode part in the three divisions is made into a silver/silver chloride electrode part by coating the microneedles and the substrate surface on which they stand with silver by electrolytic or electroless plating and chlorinating the surface. A calomel electrode may be used as the reference electrode section.
The counter electrode part and the reference electrode part may be formed on the substrate part which does not have microneedles without dividing the patch surface on which the microneedles stand.
Each electrode portion is coated with an immobilized enzyme that immobilizes glucose oxidase (GOD) and makes it insoluble in water. A covering layer, such as a thin silicone rubber film, may be provided on the external surface of the GOD layer. GOD coating is essential for the working electrode portion, but is not essential for the counter electrode portion and the reference electrode portion.
Lead wires are installed from the working electrode section, counter electrode section, and reference electrode section to extract electrical signals. Lead wire means an electrical signal extraction wire. The extracted electrical signals are processed in accordance with the methods used up to now.
The present invention is as shown below.
[1] A working electrode part, a counter electrode part, a reference electrode part, and an electric signal from each of these electrode parts on the surface of the substrate where the microneedles of the microneedle patch including microneedles made of a non-conductive material and a substrate are lined up. An immobilized enzyme sensor is provided with a lead-out line, and at least the working electrode part is coated with cross-linked glucose oxidase (even if the counter electrode part and the reference electrode part are also immobilized enzyme sensors coated with cross-linked glucose oxidase). Glucose sensors sense electrical signals generated in-vivo in response to the presence of glucose in body fluids, thereby measuring glucose concentration.
[2] A working electrode section, a reference electrode section, and electrical signal extraction lines from each electrode section are provided on the substrate surface where the microneedles of the microneedle patch including microneedles made of a non-conductive material and a substrate are lined up. Each electrode part is 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 glucose concentration. glucose sensor.
[3] The non-conductive material is from the group consisting of polyglycolic acid, poly(lactic acid-glycolic acid) copolymer, polycarbonate, polytetrafluoroethylene, polyoxymethylene, polyethylene terephthalate, and COP (cyclic olefin polymer). The glucose sensor according to [1] or [2], which is selected.
[4] Any one of [1] to [3], wherein the substrate of the microneedle patch has a circular shape with 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. The glucose sensor described.
[5] The glucose sensor according to any one of [1] to [4], wherein the microneedle patch includes a base portion and a corridor portion.
[6] The glucose sensor according to [1], wherein the working electrode portion and the counter electrode portion are covered with gold or platinum.
[7] The glucose sensor according to any one of [1] to [5], wherein the reference electrode portion is a silver/silver chloride electrode.
[8] The glucose sensor according to [1], wherein the working electrode part, the counter electrode part, and the reference electrode part are immobilized enzyme sensors coated with crosslinked glucose oxidase.
[9] The glucose sensor according to any one of [1] to [8], wherein the electrode portion is coated with a film in which glucose oxidase is crosslinked.
[10] The glucose sensor according to any one of [1] to [9], wherein the electrode portion is coated with a film made by crosslinking glucose oxidase with a polymer having an amino group as a base.
[11] Using the glucose sensor according to any one of [1] to [10], a transmitter, and a monitor, and transmitting data of an electrical signal obtained from the glucose sensor to the monitor by the transmitter. Monitoring system for glucose concentration in quality fluid.
本発明は、多数のマイクロニードルが林立しているマイクロニードルパッチを用いてグルコースを検出するセンサーである。本発明のグルコースセンサーは、皮内導入が容易であり、また発生する電流値を大きくすることができるので、従来法に比べて安定したシステムを提供することが可能である。マイクロニードルパッチを皮膚に適用することにより、間質液中のグルコース濃度をシグナル表示として表わすことができ、それ故、血液グルコースセンサーとして有用である。
The present invention is a sensor that detects glucose using a microneedle patch in which a large number of microneedles are lined up. The glucose sensor of the present invention can be easily introduced into the skin and can generate a large current value, so it is 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 expressed as a signal indication and is therefore useful as a blood glucose sensor.
マイクロニードルパッチ
本発明におけるマイクロニードルパッチは、基板と、基板の片側に複数のマイクロニードルが林立している構成をいい、さらに粘着シートをもってマイクロニードルパッチを裏打ちしてもよい。粘着シートは、典型的には、フィルムの基材としてポリウレタン、ポリエチレン、ポリエステル、紙等を使用し、厚さ５～５０μｍ程度に成形したフィルム上に、アクリル系ないしはゴム系粘着剤を５～５０μｍ程度塗布したものである。粘着シートの形状は特に制限はないが、マイクロニードルアレイの形状に類似させて円形、楕円形、勾玉形等が好ましい。 Microneedle Patch The microneedle patch in the present invention has a structure including a substrate and a plurality of microneedles arranged on one side of the substrate, and the microneedle patch may be further lined with an adhesive sheet. Adhesive sheets typically use polyurethane, polyethylene, polyester, paper, etc. as a film base material, and coat the film with an acrylic or rubber adhesive in a thickness of 5 to 50 μm on a film formed to a thickness of about 5 to 50 μm. It has been applied to a certain extent. The shape of the pressure-sensitive adhesive sheet is not particularly limited, but it is preferably circular, oval, bead shape, etc. to resemble the shape of the microneedle array.
本発明におけるマイクロニードルパッチは、基板と、基板の片側に複数のマイクロニードルが林立している構成をいい、さらに粘着シートをもってマイクロニードルパッチを裏打ちしてもよい。粘着シートは、典型的には、フィルムの基材としてポリウレタン、ポリエチレン、ポリエステル、紙等を使用し、厚さ５～５０μｍ程度に成形したフィルム上に、アクリル系ないしはゴム系粘着剤を５～５０μｍ程度塗布したものである。粘着シートの形状は特に制限はないが、マイクロニードルアレイの形状に類似させて円形、楕円形、勾玉形等が好ましい。 Microneedle Patch The microneedle patch in the present invention has a structure including a substrate and a plurality of microneedles arranged on one side of the substrate, and the microneedle patch may be further lined with an adhesive sheet. Adhesive sheets typically use polyurethane, polyethylene, polyester, paper, etc. as a film base material, and coat the film with an acrylic or rubber adhesive in a thickness of 5 to 50 μm on a film formed to a thickness of about 5 to 50 μm. It has been applied to a certain extent. The shape of the pressure-sensitive adhesive sheet is not particularly limited, but it is preferably circular, oval, bead shape, etc. to resemble the shape of the microneedle array.
マイクロニードルパッチの基板
マイクロニードルパッチの基板の材料、形状及び大きさは、特に限定されず、従来用いられてきたものを使用することができる。基板の基剤とマイクロニードルの基剤とは同一であることを基本とするが、異なっていてもよい。
上記基剤としては、シリコーン、二酸化ケイ素、セラミック、ガラス、等の非導電性無機化合物、合成又は天然の樹脂素材で水不溶性材料等の非導電性材料が挙げられる。合成又は天然の樹脂素材としては、ポリ乳酸、ポリグリコール酸、ポリ（乳酸－グリコール酸）共重合体、カプロノラクトン、等の生体分解性ポリマー、又はナイロン、ポリカーボネート、ポリテトラフルオロエチレン、ポリオキシメチレン、ポリエチレンテレフタレート、ＣＯＰ（サイクリックオレフィンポリマー）等の生体非分解性ポリマーが挙げられる。基板は、上記非導電性材料からなることが好ましい。上記非導電性材料は、ポリグリコール酸、ポリ（乳酸－グリコール酸）共重合体、ポリカーボネート、ポリテトラフルオロエチレン、ポリオキシメチレン、ポリエチレンテレフタレート、及びＣＯＰ（サイクリックオレフィンポリマー）からなる群より選ばれることが好ましい。 Substrate of Microneedle Patch The material, shape and size of the substrate of the microneedle patch are not particularly limited, and conventionally used ones can be used. The base material of the substrate and the base material of the microneedles are basically the same, but may be different.
Examples of the base include non-conductive inorganic compounds such as silicone, silicon dioxide, ceramic, and glass, and non-conductive materials such as synthetic or natural resin materials that are water-insoluble. Synthetic or natural resin materials include biodegradable polymers such as polylactic acid, polyglycolic acid, poly(lactic acid-glycolic acid) copolymer, capronolactone, or nylon, polycarbonate, polytetrafluoroethylene, polyoxymethylene, Biodegradable polymers such as polyethylene terephthalate and COP (cyclic olefin polymer) can be mentioned. Preferably, the substrate is made of the above-mentioned non-conductive material. 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). It is preferable.
マイクロニードルパッチの基板の材料、形状及び大きさは、特に限定されず、従来用いられてきたものを使用することができる。基板の基剤とマイクロニードルの基剤とは同一であることを基本とするが、異なっていてもよい。
上記基剤としては、シリコーン、二酸化ケイ素、セラミック、ガラス、等の非導電性無機化合物、合成又は天然の樹脂素材で水不溶性材料等の非導電性材料が挙げられる。合成又は天然の樹脂素材としては、ポリ乳酸、ポリグリコール酸、ポリ（乳酸－グリコール酸）共重合体、カプロノラクトン、等の生体分解性ポリマー、又はナイロン、ポリカーボネート、ポリテトラフルオロエチレン、ポリオキシメチレン、ポリエチレンテレフタレート、ＣＯＰ（サイクリックオレフィンポリマー）等の生体非分解性ポリマーが挙げられる。基板は、上記非導電性材料からなることが好ましい。上記非導電性材料は、ポリグリコール酸、ポリ（乳酸－グリコール酸）共重合体、ポリカーボネート、ポリテトラフルオロエチレン、ポリオキシメチレン、ポリエチレンテレフタレート、及びＣＯＰ（サイクリックオレフィンポリマー）からなる群より選ばれることが好ましい。 Substrate of Microneedle Patch The material, shape and size of the substrate of the microneedle patch are not particularly limited, and conventionally used ones can be used. The base material of the substrate and the base material of the microneedles are basically the same, but may be different.
Examples of the base include non-conductive inorganic compounds such as silicone, silicon dioxide, ceramic, and glass, and non-conductive materials such as synthetic or natural resin materials that are water-insoluble. Synthetic or natural resin materials include biodegradable polymers such as polylactic acid, polyglycolic acid, poly(lactic acid-glycolic acid) copolymer, capronolactone, or nylon, polycarbonate, polytetrafluoroethylene, polyoxymethylene, Biodegradable polymers such as polyethylene terephthalate and COP (cyclic olefin polymer) can be mentioned. Preferably, the substrate is made of the above-mentioned non-conductive material. 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). It is preferable.
基板の形状は、任意の形状とすることができる。一例として、円形、楕円形、三角形、四角形、多角形等を基本とし、適用部位（皮膚）に合わせてさらに変形したものであってもよい。基板の大きさは、直径（長径）又は一辺（長辺）の長さで代表して表すと、通常０．２～５ｃｍであり、０．４～５ｃｍが好ましく、０．５～３ｃｍがより好ましい。上記マイクロニードルパッチの基板の形状は直径０．４～５ｃｍの円形であることが好ましく、直径０．５～３ｃｍの円形であることがより好ましい。
The shape of the substrate can be any shape. As an example, the shapes are basically circular, oval, triangular, quadrangular, polygonal, etc., and may be further modified according to the application site (skin). The size of the substrate is typically 0.2 to 5 cm, preferably 0.4 to 5 cm, and more preferably 0.5 to 3 cm, expressed in terms of diameter (major axis) or length of one side (long side). preferable. The shape of the substrate of the microneedle patch is preferably circular with a diameter of 0.4 to 5 cm, more preferably circular with a diameter of 0.5 to 3 cm.
基板の面積（通常、平面積）は、通常０．０５～１００ｃｍ２であり、取扱い易さの観点から、０．４～１０ｃｍ２程度が好ましく、０．６～５ｃｍ２程度がより好ましい。基板の厚さは０．１～１．０ｍｍが好ましい。
The area (usually a flat area) of the substrate is usually 0.05 to 100 cm 2 , preferably about 0.4 to 10 cm 2 , and more preferably about 0.6 to 5 cm 2 from the viewpoint of ease of handling. The thickness of the substrate is preferably 0.1 to 1.0 mm.
マイクロニードルの形状
マイクロニードルは、針長さが１００μｍ以上２，０００μｍ以下であり、好ましくは２００～１，０００μｍである。針の先端部頂点の大きさを直径として表すと、皮膚への刺し入れの容易性と確実性を考慮して、８０μｍ以下であり、２０μｍ以上が好ましい。
個々のマイクロニードルとしては、底面が円である円柱状もしくは円錐状、底面が楕円である楕円柱状もしくは楕円錐状、底面が三角形である三角柱状もしくは三角錐状、底面が四角形である四角柱状もしくは四角錐状、又は底面が多角形である多角柱状もしくは多角錐状が挙げられる。底面の大きさは、楕円の場合、長径を直径として表し、短径は楕円を形成できる限りにおいて長径より短い。三角形ないし多角形の場合、一辺を代表として表してもよく、対角線を代表として表してもよい。マイクロニードルが円錐状である場合には、その底面における直径は、１００～４００μｍ程度であり、１５０～３００μｍ程度が好ましい。 Shape of microneedles Microneedles have a needle length of 100 μm or more and 2,000 μm or less, preferably 200 to 1,000 μm. When the size of the apex of the tip of the needle is expressed as a diameter, it is 80 μm or less, preferably 20 μm or more, considering ease and reliability of penetration into the skin.
Individual microneedles can be cylindrical or conical with a circular base, elliptical cylinder or elliptical cone with an elliptical base, triangular prism or pyramid with a triangular base, or quadrangular prism or square with a square base. Examples include a quadrangular pyramid shape, a polygonal columnar shape or a polygonal pyramid shape whose bottom surface is a polygon. In the case of an ellipse, the size of the bottom surface is expressed by the major axis as the diameter, and the minor axis is shorter than the major axis as long as the ellipse can be formed. In the case of a triangle or polygon, one side may be represented as a representative, or a diagonal may be represented as a representative. When the microneedles are conical, the diameter at the bottom is about 100 to 400 μm, preferably about 150 to 300 μm.
マイクロニードルは、針長さが１００μｍ以上２，０００μｍ以下であり、好ましくは２００～１，０００μｍである。針の先端部頂点の大きさを直径として表すと、皮膚への刺し入れの容易性と確実性を考慮して、８０μｍ以下であり、２０μｍ以上が好ましい。
個々のマイクロニードルとしては、底面が円である円柱状もしくは円錐状、底面が楕円である楕円柱状もしくは楕円錐状、底面が三角形である三角柱状もしくは三角錐状、底面が四角形である四角柱状もしくは四角錐状、又は底面が多角形である多角柱状もしくは多角錐状が挙げられる。底面の大きさは、楕円の場合、長径を直径として表し、短径は楕円を形成できる限りにおいて長径より短い。三角形ないし多角形の場合、一辺を代表として表してもよく、対角線を代表として表してもよい。マイクロニードルが円錐状である場合には、その底面における直径は、１００～４００μｍ程度であり、１５０～３００μｍ程度が好ましい。 Shape of microneedles Microneedles have a needle length of 100 μm or more and 2,000 μm or less, preferably 200 to 1,000 μm. When the size of the apex of the tip of the needle is expressed as a diameter, it is 80 μm or less, preferably 20 μm or more, considering ease and reliability of penetration into the skin.
Individual microneedles can be cylindrical or conical with a circular base, elliptical cylinder or elliptical cone with an elliptical base, triangular prism or pyramid with a triangular base, or quadrangular prism or square with a square base. Examples include a quadrangular pyramid shape, a polygonal columnar shape or a polygonal pyramid shape whose bottom surface is a polygon. In the case of an ellipse, the size of the bottom surface is expressed by the major axis as the diameter, and the minor axis is shorter than the major axis as long as the ellipse can be formed. In the case of a triangle or polygon, one side may be represented as a representative, or a diagonal may be represented as a representative. When the microneedles are conical, the diameter at the bottom is about 100 to 400 μm, preferably about 150 to 300 μm.
本発明におけるマイクロニードルは、段差を有していてもよい。ここに段差とは、マイクロニードルのある点から先端方向に向かって、マイクロニードルの断面積が不連続的に縮小し、階段状を呈しているものをいう。
The microneedle in the present invention may have a step. Here, the term "step" refers to a step in which the cross-sectional area of the microneedle decreases discontinuously from a certain point toward the tip of the microneedle, creating a step-like shape.
基板上のマイクロニードルの配置
マイクロニードルパッチは、基板の上に直接マイクロニードルが立つのが基本形であるが、基板上に土台部を設けて、その土台部の上（パッチ平面という）にマイクロニードルが立ったものであってもよい。土台部を設けることにより、マイクロニードルの皮膚穿刺性がより確実になる。土台部の厚さ（土台面の高さ）は基板面上に０．２ｍｍ～４．０ｍｍであることが好ましく、より好ましくは０．５ｍｍ～２．０ｍｍである。
基板上に土台部を設ける場合、作用電極部、対極部、参照電極部の各電極部毎に土台部を設ける。３電極部の配置は、ほぼ同面積を原則とするが、特に制約はない。異なる電極部の土台部に囲まれた基板部分を回廊部とよぶ。上記の配置の模式図を図１に示す。 Placement of microneedles on a substrate The basic form of a microneedle patch is that the microneedles stand directly on the substrate, but a base is provided on the substrate and the microneedles are placed on top of the base (called the patch plane). It may be something with a By providing the base portion, the skin puncturing ability of the microneedles becomes more reliable. The thickness of the base portion (height of the base surface) is preferably 0.2 mm to 4.0 mm above the substrate surface, more preferably 0.5 mm to 2.0 mm.
When a base part is provided on the substrate, a base part is provided for each electrode part, such as a working electrode part, a counter electrode part, and a reference electrode part. As a general rule, the three electrode sections should have approximately the same area, but there are no particular restrictions. The part of the substrate surrounded by the base parts of different electrode parts is called a corridor part. A schematic diagram of the above arrangement is shown in FIG.
マイクロニードルパッチは、基板の上に直接マイクロニードルが立つのが基本形であるが、基板上に土台部を設けて、その土台部の上（パッチ平面という）にマイクロニードルが立ったものであってもよい。土台部を設けることにより、マイクロニードルの皮膚穿刺性がより確実になる。土台部の厚さ（土台面の高さ）は基板面上に０．２ｍｍ～４．０ｍｍであることが好ましく、より好ましくは０．５ｍｍ～２．０ｍｍである。
基板上に土台部を設ける場合、作用電極部、対極部、参照電極部の各電極部毎に土台部を設ける。３電極部の配置は、ほぼ同面積を原則とするが、特に制約はない。異なる電極部の土台部に囲まれた基板部分を回廊部とよぶ。上記の配置の模式図を図１に示す。 Placement of microneedles on a substrate The basic form of a microneedle patch is that the microneedles stand directly on the substrate, but a base is provided on the substrate and the microneedles are placed on top of the base (called the patch plane). It may be something with a By providing the base portion, the skin puncturing ability of the microneedles becomes more reliable. The thickness of the base portion (height of the base surface) is preferably 0.2 mm to 4.0 mm above the substrate surface, more preferably 0.5 mm to 2.0 mm.
When a base part is provided on the substrate, a base part is provided for each electrode part, such as a working electrode part, a counter electrode part, and a reference electrode part. As a general rule, the three electrode sections should have approximately the same area, but there are no particular restrictions. The part of the substrate surrounded by the base parts of different electrode parts is called a corridor part. A schematic diagram of the above arrangement is shown in FIG.
マイクロニードルパッチの成形は、材料に適した成形法を採用するが、生体分解性ポリマー、生体非分解性ポリマーを射出成形法により行うことが工業生産上好ましい。
A molding method suitable for the material is used to mold the microneedle patch, but it is preferable for industrial production to use injection molding of biodegradable polymers and non-biodegradable polymers.
グルコースセンサー作製
作用電極部及び対極部は、マイクロニードルパッチを金あるいは白金で被覆する。被覆の方法は、蒸着法、電解メッキ法、無電解メッキ法などを用いることができる。さらには、金箔あるいは白金箔を針で突き刺して、基板面に接着塗布することでも可能である。参照電極部は、銀／塩化銀電極とする。銀／塩化銀電極は、銀を電解あるいは塩素イオン含有水溶液浸漬により銀表面を塩化銀とすることにより、作製する。
作用電極部を固定化ＧＯＤで被覆する。対極部及び参照電極部は、ＧＯＤ被覆してもしなくてもよい。ＧＯＤ被覆は、グルコースオキシダーゼをグルタルアルデヒドなどの多官能性アルデヒドでアミノ基を有する基剤高分子と反応させて不溶化し、同時に基剤をも不溶化して電極部に固定する。アミノ基を有する基剤高分子として、血清アルブミン、核酸、キトサン、等が例示される。 For the electrode part and counter electrode part for producing a glucose sensor , a microneedle patch is coated with gold or platinum. As a coating method, a vapor deposition method, an electrolytic plating method, an electroless plating method, etc. can be used. Furthermore, it is also possible to pierce gold or platinum foil with a needle and apply the adhesive onto the substrate surface. The reference electrode section is a silver/silver chloride electrode. The silver/silver chloride electrode is produced by electrolyzing silver or dipping it in an aqueous solution containing chloride ions to convert the silver surface into silver chloride.
The working electrode section is coated with immobilized GOD. The counter electrode portion and the reference electrode portion may or may not be coated with GOD. In the GOD coating, glucose oxidase is insolubilized by reacting with a base polymer having an amino group using a polyfunctional aldehyde such as glutaraldehyde, and at the same time, the base is also insolubilized and fixed to the electrode portion. Examples of base polymers having amino groups include serum albumin, nucleic acids, chitosan, and the like.
作用電極部及び対極部は、マイクロニードルパッチを金あるいは白金で被覆する。被覆の方法は、蒸着法、電解メッキ法、無電解メッキ法などを用いることができる。さらには、金箔あるいは白金箔を針で突き刺して、基板面に接着塗布することでも可能である。参照電極部は、銀／塩化銀電極とする。銀／塩化銀電極は、銀を電解あるいは塩素イオン含有水溶液浸漬により銀表面を塩化銀とすることにより、作製する。
作用電極部を固定化ＧＯＤで被覆する。対極部及び参照電極部は、ＧＯＤ被覆してもしなくてもよい。ＧＯＤ被覆は、グルコースオキシダーゼをグルタルアルデヒドなどの多官能性アルデヒドでアミノ基を有する基剤高分子と反応させて不溶化し、同時に基剤をも不溶化して電極部に固定する。アミノ基を有する基剤高分子として、血清アルブミン、核酸、キトサン、等が例示される。 For the electrode part and counter electrode part for producing a glucose sensor , a microneedle patch is coated with gold or platinum. As a coating method, a vapor deposition method, an electrolytic plating method, an electroless plating method, etc. can be used. Furthermore, it is also possible to pierce gold or platinum foil with a needle and apply the adhesive onto the substrate surface. The reference electrode section is a silver/silver chloride electrode. The silver/silver chloride electrode is produced by electrolyzing silver or dipping it in an aqueous solution containing chloride ions to convert the silver surface into silver chloride.
The working electrode section is coated with immobilized GOD. The counter electrode portion and the reference electrode portion may or may not be coated with GOD. In the GOD coating, glucose oxidase is insolubilized by reacting with a base polymer having an amino group using a polyfunctional aldehyde such as glutaraldehyde, and at the same time, the base is also insolubilized and fixed to the electrode portion. Examples of base polymers having amino groups include serum albumin, nucleic acids, chitosan, and the like.
以下に、無電解メッキ法による白金メッキの方法に関して詳述する。作用電極部及び対極部を白金メッキするために回廊部の全て及び参照電極部をシリコーン樹脂で覆い、加熱して皮膜とし、マスキングする。側面及び裏面も同様にマスキングする。白金無電解めっきは、ジニトロジアンミン白金、アンモニア、ヒドラジン、により６０℃において１時間実施する。得られた白金膜付きマイクロニードルパッチを水洗いした後、乾燥させる。その後、作用電極部、対極部をシリコーンマスキングし、参照電極部のマスキングを剥がし参照電極部を無電解銀メッキする。その後、飽和塩化鉄（III）溶液に浸すことにより、Ag/AgCl電極とする。作用電極、対極及び回廊部のマスキングを取り去って、３電極部をそれぞれリード線に接続し、リード線を回廊部から外部に導く。電極間に与える電位、電流値を外部計測できるようにする。図２においては、リード線をマイクロニードル側に設置したが、マイクロニードル側でなく裏側に設置してもよい。その後、作用電極部及びその他の２電極上にＧＯＤ皮膜を作り、さらにグルタルアルデヒドにより架橋させ、固定化酵素電極（グルコースセンサー）とする。ＧＯＤ皮膜を作製するに当たっては、例えばアルブミンのようなＧＯＤ以外のアミノ基を有する親水性高分子を共存させることがグルコースの間質液からの浸透、及びＧＯＤの安定性の面から望ましい。以上のプロセスにより作製する固定化酵素電極の例を図２に示す。
The platinum plating method using electroless plating will be described in detail below. In order to plate the working electrode and counter electrode with platinum, the entire corridor and the reference electrode are covered with silicone resin, heated to form a film, and masked. Mask the sides and back in the same way. Electroless platinum plating is carried out using dinitrodiammine platinum, ammonia, and hydrazine at 60° C. for 1 hour. The obtained microneedle patch with a platinum film is washed with water and then dried. Thereafter, the working electrode part and the counter electrode part are masked with silicone, the masking of the reference electrode part is removed, and the reference electrode part is plated with electroless silver. Thereafter, it is immersed in a saturated iron(III) chloride solution to form an Ag/AgCl electrode. The masking of the working electrode, counter electrode, and corridor section is removed, and the three electrode sections are connected to respective lead wires, and the lead wires are guided to the outside from the corridor section. Make it possible to externally measure the potential and current value applied between the electrodes. 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. Thereafter, a GOD film is formed on the working electrode portion and the other two electrodes, and further crosslinked with glutaraldehyde to obtain an immobilized enzyme electrode (glucose sensor). When preparing a GOD film, it is desirable to coexist a hydrophilic polymer having an amino group other than GOD, such as albumin, from the viewpoints of penetration of glucose from the interstitial fluid and stability of GOD. An example of an immobilized enzyme electrode produced by the above process is shown in FIG.
以上は３電極を有する基本的な固定化酵素電極であるが、対極を設けず参照電極に対極の役割をさせることも可能である。その場合、電極部は２つとなる。
Although the above is a basic immobilized enzyme electrode having three electrodes, it is also possible to have a reference electrode serve as a counter electrode without providing a counter electrode. In that case, there will be two electrode parts.
このようにして作製した固定化酵素電極を用いて間質液中のグルコース濃度を測定するには、既に公知の方法による。固定化酵素電極が体液中のグルコースの存在（存在量）に応じてｉｎ－ｖｉｖｏで発生する電気的信号を感知し、それによりグルコース濃度を測定することができる。固定化酵素電極マイクロニードルパッチをアプリケーターにより経皮投与する。安定的に皮膚に保持するためには、マイクロニードルを基板の背面から粘着テープで覆い、皮膚に密着させる。３本あるいは２本のリード線を電圧付与装置に接続し、０．６～１．０Ｖの電圧を与え、流れる電流を計測するのは、例えば、特開平２－５０１６７９号公報に記載の方法によればよい。また、外部装置に接続することなく、皮膚投与マイクロニードルに隣接した皮膚上にトランスミッターを設置し、そこから電気的操作を行い、さらに得られた電気信号をモニターに無線送信して経時的に間質液中のグルコース濃度をモニターすることも可能である。
A known method is used to measure the glucose concentration in the interstitial fluid using the immobilized enzyme electrode produced in this way. An immobilized enzyme electrode senses an electrical signal generated in-vivo in response to the presence (abundance) of glucose in a body fluid, thereby making it possible to measure glucose concentration. The immobilized enzyme electrode microneedle patch is administered transdermally using an applicator. In order to stably hold the microneedles on the skin, cover the back of the substrate with adhesive tape so that the microneedles stick to the skin. For example, the method described in JP-A-2-501679 connects three or two lead wires to a voltage applying device, applies a voltage of 0.6 to 1.0 V, and measures the flowing current. All you have to do is follow. In addition, without connecting to an external device, a transmitter is placed on the skin adjacent to the skin-administered microneedle, electrical operation is performed from there, and the obtained electrical signal is wirelessly transmitted to a monitor to transmit the electrical signal over time. It is also possible to monitor the glucose concentration in the fluid.
以下、本発明を下記実施例によりさらに詳しく説明する。これら実施例は、単に本発明を具体的に説明するための例であり、本発明の範囲がこれら実施例に限定されるものではない。
Hereinafter, the present invention will be explained in more detail with reference to the following examples. These Examples are merely examples for specifically explaining the present invention, and the scope of the present invention is not limited to these Examples.
実施例１
金型を射出成形機に取付け、ポリグリコール酸を溶融して射出成形により図１のようなマイクロニードルパッチを成形した。パッチ直径：１２．３ｍｍであった。本マイクロニードルパッチの詳細は、以下のようである。
パッチ基板上針が立つ土台部外径：１０．５ｍｍ、回廊部幅：１．２ｍｍ。回廊部及び周辺部の基板面からの高さ：０．５ｍｍ。土台面の高さ：１．３ｍｍ。針高さ：０．９ｍｍ。針間隔：０．６ｍｍ。
マスキングするシリコーン樹脂は、２液性硬化型（ＨＹＶ－４０００，エングラービングジャパン社製）を用いた。白金無電解めっき後、シリコーン樹脂を参照電極部から剥がし、作用電極部、対極部をマスキングし、参照電極部を銀／塩化銀電極とした。白金膜、銀膜の厚みは約１０μｍであった。３電極部にそれぞれリード線を取り付けた。
次いで、ＧＯＤ及びヒトアルブミンの等量１％水溶液にパッチを浸漬し、取り出し、乾燥後、グルタルアルデヒド水溶液に浸漬して、ＧＯＤ及びアルブミンを架橋し、グルコース濃度測定用固定化酵素電極とした。固定化酵素膜の厚みは３０μｍであった。 Example 1
The mold was attached to an injection molding machine, polyglycolic acid was melted, and a microneedle patch as shown in FIG. 1 was formed by injection molding. Patch diameter: 12.3 mm. The details of this microneedle patch are as follows.
Outer diameter of the base on which the needles stand on the patch board: 10.5 mm, width of the corridor: 1.2 mm. Height of the corridor part and peripheral part from the board surface: 0.5 mm. Base height: 1.3mm. Needle height: 0.9mm. Needle spacing: 0.6mm.
A two-component curing type silicone resin (HYV-4000, manufactured by Engraving Japan Co., Ltd.) was used as the masking silicone resin. After electroless platinum plating, the silicone resin was peeled off from the reference electrode part, the working electrode part and the counter electrode part were masked, and the reference electrode part was made into a silver/silver chloride electrode. The thickness of the platinum film and silver film was approximately 10 μm. Lead wires were attached to each of the three electrode parts.
Next, the patch was immersed in a 1% aqueous solution of equal amounts of GOD and human albumin, taken out, dried, and immersed in an aqueous glutaraldehyde solution to crosslink GOD and albumin, thereby forming an immobilized enzyme electrode for measuring glucose concentration. The thickness of the immobilized enzyme membrane was 30 μm.
金型を射出成形機に取付け、ポリグリコール酸を溶融して射出成形により図１のようなマイクロニードルパッチを成形した。パッチ直径：１２．３ｍｍであった。本マイクロニードルパッチの詳細は、以下のようである。
パッチ基板上針が立つ土台部外径：１０．５ｍｍ、回廊部幅：１．２ｍｍ。回廊部及び周辺部の基板面からの高さ：０．５ｍｍ。土台面の高さ：１．３ｍｍ。針高さ：０．９ｍｍ。針間隔：０．６ｍｍ。
マスキングするシリコーン樹脂は、２液性硬化型（ＨＹＶ－４０００，エングラービングジャパン社製）を用いた。白金無電解めっき後、シリコーン樹脂を参照電極部から剥がし、作用電極部、対極部をマスキングし、参照電極部を銀／塩化銀電極とした。白金膜、銀膜の厚みは約１０μｍであった。３電極部にそれぞれリード線を取り付けた。
次いで、ＧＯＤ及びヒトアルブミンの等量１％水溶液にパッチを浸漬し、取り出し、乾燥後、グルタルアルデヒド水溶液に浸漬して、ＧＯＤ及びアルブミンを架橋し、グルコース濃度測定用固定化酵素電極とした。固定化酵素膜の厚みは３０μｍであった。 Example 1
The mold was attached to an injection molding machine, polyglycolic acid was melted, and a microneedle patch as shown in FIG. 1 was formed by injection molding. Patch diameter: 12.3 mm. The details of this microneedle patch are as follows.
Outer diameter of the base on which the needles stand on the patch board: 10.5 mm, width of the corridor: 1.2 mm. Height of the corridor part and peripheral part from the board surface: 0.5 mm. Base height: 1.3mm. Needle height: 0.9mm. Needle spacing: 0.6mm.
A two-component curing type silicone resin (HYV-4000, manufactured by Engraving Japan Co., Ltd.) was used as the masking silicone resin. After electroless platinum plating, the silicone resin was peeled off from the reference electrode part, the working electrode part and the counter electrode part were masked, and the reference electrode part was made into a silver/silver chloride electrode. The thickness of the platinum film and silver film was approximately 10 μm. Lead wires were attached to each of the three electrode parts.
Next, the patch was immersed in a 1% aqueous solution of equal amounts of GOD and human albumin, taken out, dried, and immersed in an aqueous glutaraldehyde solution to crosslink GOD and albumin, thereby forming an immobilized enzyme electrode for measuring glucose concentration. The thickness of the immobilized enzyme membrane was 30 μm.
実施例２
ポリグリコール酸を溶融して射出成形を行ない、乳白色の楕円形のマイクロニードルパッチを取り出した。パッチ長径：１２．３ｍｍ、短径：１１．５ｍｍであった（図３）。本マイクロニードルパッチの詳細は、以下のようである。
パッチ基板上針が立つ土台部外径：１０．５ｍｍ、土台部内径：６．０ｍｍ。回廊部幅：１．２ｍｍ。回廊部及び中央部の基板面からの高さ：０．５ｍｍ。土台面の高さ：１．３ｍｍ。針高さ：０．６ｍｍ。針総本数：２８０本であった。 Example 2
Polyglycolic acid was melted and injection molded, and a milky white oval microneedle patch was taken out. The patch major axis was 12.3 mm, and the minor axis was 11.5 mm (FIG. 3). The details of this microneedle patch are as follows.
The outer diameter of the base on which the needles stand on the patch board: 10.5 mm, the inner diameter of the base: 6.0 mm. Corridor width: 1.2mm. Height of the corridor section and center section from the board surface: 0.5 mm. Base height: 1.3mm. Needle height: 0.6mm. Total number of needles: 280.
ポリグリコール酸を溶融して射出成形を行ない、乳白色の楕円形のマイクロニードルパッチを取り出した。パッチ長径：１２．３ｍｍ、短径：１１．５ｍｍであった（図３）。本マイクロニードルパッチの詳細は、以下のようである。
パッチ基板上針が立つ土台部外径：１０．５ｍｍ、土台部内径：６．０ｍｍ。回廊部幅：１．２ｍｍ。回廊部及び中央部の基板面からの高さ：０．５ｍｍ。土台面の高さ：１．３ｍｍ。針高さ：０．６ｍｍ。針総本数：２８０本であった。 Example 2
Polyglycolic acid was melted and injection molded, and a milky white oval microneedle patch was taken out. The patch major axis was 12.3 mm, and the minor axis was 11.5 mm (FIG. 3). The details of this microneedle patch are as follows.
The outer diameter of the base on which the needles stand on the patch board: 10.5 mm, the inner diameter of the base: 6.0 mm. Corridor width: 1.2mm. Height of the corridor section and center section from the board surface: 0.5 mm. Base height: 1.3mm. Needle height: 0.6mm. Total number of needles: 280.
本マイクロニードルパッチは、土台部が２つに分かれ、その片方の土台部上の針部をマスキングすることなく白金膜で覆って、作用電極部とした。白金膜の厚さは１０μｍであった。もう片方の土台部上の針部を同様に銀／塩化銀電極部とした。銀／塩化銀膜の厚さは１０μｍであった。リード線を設置後、両電極面を実施例１と同様に固定化酵素膜で覆って、グルコース濃度測定用固定化酵素電極とした。固定化酵素膜の厚みは３０μｍであった。本電極では参照電極を対極として用いる。
The base of this microneedle patch was divided into two parts, and the needle on one of the bases was covered with a platinum film without masking to serve as the working electrode. The thickness of the platinum film was 10 μm. The needle part on the other base part was similarly made into a silver/silver chloride electrode part. The thickness of the silver/silver chloride film was 10 μm. After the lead wires were installed, both electrode surfaces were covered with an immobilized enzyme membrane in the same manner as in Example 1 to obtain an immobilized enzyme electrode for measuring glucose concentration. The thickness of the immobilized enzyme membrane was 30 μm. In this electrode, a reference electrode is used as a counter electrode.
実施例３
金型を射出成形機に取付け、ポリグリコール酸を溶融して射出成形により図４のようなマイクロニードルパッチを成形した。パッチは長径：１６ｍｍ、短径：１２．３ｍｍの楕円形であった。本マイクロニードルパッチの詳細は、以下のようである。パッチ基板上針が立つ土台部外径：１０．５ｍｍ、回廊部幅：１．２ｍｍ。回廊部及び周辺部の基板面からの高さ：０．５ｍｍ。土台面の高さ：１．３ｍｍ。針高さ：０．９ｍｍ。針間隔：０．６ｍｍ。
マスキングするシリコーン樹脂は、２液性硬化型（ＨＹＶ－４０００，エングラービングジャパン社製）を用いて基板部、回廊部、周辺部を保護して白金無電解めっき後、シリコーン樹脂を剥がした。参照電極部として、直径０．３ｍｍの銀線を塩化鉄溶液中で電解酸化して銀／塩化銀電極部とした。３電極部にそれぞれ白金リード線を取り付けた。電極との接続は導電性樹脂（デナコールEX-830，ナガセケムテックス社製）を用いた。
次いで、ＧＯＤ及びヒトアルブミンの等量１質量％水溶液にパッチを浸漬し、取り出し、乾燥後、グルタルアルデヒド水溶液に浸漬して、ＧＯＤ及びアルブミンを架橋し、グルコース濃度測定用固定化酵素電極とした。固定化酵素皮膜の厚みは３０μｍであった。 Example 3
The mold was attached to an injection molding machine, polyglycolic acid was melted, and a microneedle patch as shown in FIG. 4 was formed by injection molding. The patch had an oval shape with a major axis of 16 mm and a minor axis of 12.3 mm. The details of this microneedle patch are as follows. Outer diameter of the base on which the needles stand on the patch board: 10.5 mm, width of the corridor: 1.2 mm. Height of the corridor part and peripheral part from the board surface: 0.5 mm. Base height: 1.3mm. Needle height: 0.9mm. Needle spacing: 0.6mm.
The silicone resin for masking was a two-component curing type (HYV-4000, manufactured by Engraving Japan Co., Ltd.) to protect the substrate, the corridor, and the surrounding area, and after electroless platinum plating, the silicone resin was peeled off. As a reference electrode part, a silver wire with a diameter of 0.3 mm was electrolytically oxidized in an iron chloride solution to obtain a silver/silver chloride electrode part. A platinum lead wire was attached to each of the three electrode parts. A conductive resin (Denacol EX-830, manufactured by Nagase ChemteX) was used for connection to the electrodes.
Next, the patch was immersed in an equal amount of 1% by mass aqueous solution of GOD and human albumin, taken out, dried, and immersed in an aqueous glutaraldehyde solution to crosslink GOD and albumin, thereby forming an immobilized enzyme electrode for measuring glucose concentration. The thickness of the immobilized enzyme film was 30 μm.
金型を射出成形機に取付け、ポリグリコール酸を溶融して射出成形により図４のようなマイクロニードルパッチを成形した。パッチは長径：１６ｍｍ、短径：１２．３ｍｍの楕円形であった。本マイクロニードルパッチの詳細は、以下のようである。パッチ基板上針が立つ土台部外径：１０．５ｍｍ、回廊部幅：１．２ｍｍ。回廊部及び周辺部の基板面からの高さ：０．５ｍｍ。土台面の高さ：１．３ｍｍ。針高さ：０．９ｍｍ。針間隔：０．６ｍｍ。
マスキングするシリコーン樹脂は、２液性硬化型（ＨＹＶ－４０００，エングラービングジャパン社製）を用いて基板部、回廊部、周辺部を保護して白金無電解めっき後、シリコーン樹脂を剥がした。参照電極部として、直径０．３ｍｍの銀線を塩化鉄溶液中で電解酸化して銀／塩化銀電極部とした。３電極部にそれぞれ白金リード線を取り付けた。電極との接続は導電性樹脂（デナコールEX-830，ナガセケムテックス社製）を用いた。
次いで、ＧＯＤ及びヒトアルブミンの等量１質量％水溶液にパッチを浸漬し、取り出し、乾燥後、グルタルアルデヒド水溶液に浸漬して、ＧＯＤ及びアルブミンを架橋し、グルコース濃度測定用固定化酵素電極とした。固定化酵素皮膜の厚みは３０μｍであった。 Example 3
The mold was attached to an injection molding machine, polyglycolic acid was melted, and a microneedle patch as shown in FIG. 4 was formed by injection molding. The patch had an oval shape with a major axis of 16 mm and a minor axis of 12.3 mm. The details of this microneedle patch are as follows. Outer diameter of the base on which the needles stand on the patch board: 10.5 mm, width of the corridor: 1.2 mm. Height of the corridor part and peripheral part from the board surface: 0.5 mm. Base height: 1.3mm. Needle height: 0.9mm. Needle spacing: 0.6mm.
The silicone resin for masking was a two-component curing type (HYV-4000, manufactured by Engraving Japan Co., Ltd.) to protect the substrate, the corridor, and the surrounding area, and after electroless platinum plating, the silicone resin was peeled off. As a reference electrode part, a silver wire with a diameter of 0.3 mm was electrolytically oxidized in an iron chloride solution to obtain a silver/silver chloride electrode part. A platinum lead wire was attached to each of the three electrode parts. A conductive resin (Denacol EX-830, manufactured by Nagase ChemteX) was used for connection to the electrodes.
Next, the patch was immersed in an equal amount of 1% by mass aqueous solution of GOD and human albumin, taken out, dried, and immersed in an aqueous glutaraldehyde solution to crosslink GOD and albumin, thereby forming an immobilized enzyme electrode for measuring glucose concentration. The thickness of the immobilized enzyme film was 30 μm.
製造した電極の作用を確認した。上記マイクロニードルパッチを上向きに静置しマイクロニードル面に直径１．５ｃｍの円形濾紙を置いた。試料溶液として１質量％の塩化ナトリウム溶液に２００ｍｇ／ｄｌ、及び４００ｍｇ／ｄｌになるようグルコースを添加した溶液を作製し、その２００μｌを濾紙上に滴下し３電極を覆った。作用電極部及び対極部に０．７Ｖを付加し、両電極を流れる電流値を測定した。結果を表１に示す。
The effect of the manufactured electrode was confirmed. The microneedle patch was left still facing upward, and a circular filter paper with a diameter of 1.5 cm was placed on the surface of the microneedle. A sample solution was prepared by adding glucose to a 1% by mass sodium chloride solution to a concentration of 200 mg/dl and 400 mg/dl, and 200 μl of the solution was dropped onto a filter paper to cover the three electrodes. A voltage of 0.7 V was applied to the working electrode section and the counter electrode section, and the value of the current flowing through both electrodes was measured. The results are shown in Table 1.
１ 参照電極部
２ 対極部
３ 作用電極部
４ マイクロニードル
５ 回廊部
６ 土台部
７ 基板部
８ リード線
９ 固定化酵素皮膜
１０ 銀／塩化銀電極部 1Reference electrode part 2 Counter electrode part 3 Working electrode part 4 Microneedle 5 Corridor part 6 Base part 7 Substrate part 8 Lead wire 9 Immobilized enzyme film 10 Silver/silver chloride electrode part
２ 対極部
３ 作用電極部
４ マイクロニードル
５ 回廊部
６ 土台部
７ 基板部
８ リード線
９ 固定化酵素皮膜
１０ 銀／塩化銀電極部 1
Claims (11)
- 非導電性材料からなるマイクロニードル及び基板を含むマイクロニードルパッチのマイクロニードルが林立している基板面に作用電極部、対極部、及び参照電極部並びにこれらの各電極部からの電気信号取り出し線を設けてなり、少なくとも作用電極部は架橋されたグルコースオキシダーゼで被覆した固定化酵素センサーであり、体液中のグルコースの存在に応じてｉｎ－ｖｉｖｏで発生する電気的信号を感知し、それによりグルコース濃度を測定するグルコースセンサー。 A working electrode part, a counter electrode part, a reference electrode part, and electrical signal extraction lines from each of these electrode parts are connected to the surface of the substrate where the microneedles of the microneedle patch containing microneedles and a substrate made of a non-conductive material are lined up. wherein at least the working electrode portion is an immobilized enzyme sensor coated with cross-linked glucose oxidase to sense electrical signals generated in-vivo in response to the presence of glucose in body fluids, thereby determining glucose concentration. A glucose sensor that measures
- 非導電性材料からなるマイクロニードル及び基板を含むマイクロニードルパッチのマイクロニードルが林立している基板面に作用電極部、及び参照電極部並びに各電極部からの電気信号取り出し線を設けてなり、各電極部は架橋されたグルコースオキシダーゼで被覆した固定化酵素センサーであり、体液中のグルコースの存在に応じてｉｎ－ｖｉｖｏで発生する電気的信号を感知し、それによりグルコース濃度を測定するグルコースセンサー。 A working electrode section, a reference electrode section, and electrical signal extraction lines from each electrode section are provided on the surface of the substrate on which the microneedles of the microneedle patch containing microneedles made of a non-conductive material and a substrate are arranged. The electrode part is an immobilized enzyme sensor coated with cross-linked glucose oxidase, and the glucose sensor senses an electrical signal generated in-vivo in response to the presence of glucose in a body fluid, thereby measuring glucose concentration.
- 非導電性材料が、ポリグリコール酸、ポリ（乳酸－グリコール酸）共重合体、ポリカーボネート、ポリテトラフルオロエチレン、ポリオキシメチレン、ポリエチレンテレフタレート、及びＣＯＰ（サイクリックオレフィンポリマー）からなる群より選ばれる、請求項１又は２に記載のグルコースセンサー。 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); The glucose sensor according to claim 1 or 2.
- 前記マイクロニードルパッチの基板の形状が直径０．４～５ｃｍの円形であり、前記マイクロニードルが針長さ１００μｍ以上２，０００μｍ以下である、請求項１～３のいずれか１項に記載のグルコースセンサー。 The glucose according to any one of claims 1 to 3, wherein the substrate of the microneedle patch has a circular shape with 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. sensor.
- 前記マイクロニードルパッチが土台部及び回廊部を含む、請求項１～４のいずれか１項に記載のグルコースセンサー。 The glucose sensor according to any one of claims 1 to 4, wherein the microneedle patch includes a base portion and a corridor portion.
- 前記作用電極部及び対極部が金又は白金で覆われている、請求項１に記載のグルコースセンサー。 The glucose sensor according to claim 1, wherein the working electrode part and the counter electrode part are covered with gold or platinum.
- 前記参照電極部が銀／塩化銀電極である、請求項１～５のいずれか１項に記載のグルコースセンサー。 The glucose sensor according to any one of claims 1 to 5, wherein the reference electrode part is a silver/silver chloride electrode.
- 前記作用電極部、対極部、及び参照電極部が、架橋されたグルコースオキシダーゼで被覆した固定化酵素センサーである、請求項１に記載のグルコースセンサー。 The glucose sensor according to claim 1, wherein the working electrode part, the counter electrode part, and the reference electrode part are immobilized enzyme sensors coated with crosslinked glucose oxidase.
- 前記電極部がグルコースオキシダーゼを架橋させた皮膜で被覆されている、請求項１～８のいずれか１項に記載のグルコースセンサー。 The glucose sensor according to any one of claims 1 to 8, wherein the electrode portion is coated with a film in which glucose oxidase is crosslinked.
- 前記電極部が基剤としてアミノ基を有する高分子とグルコースオキシダーゼとを架橋させた皮膜で被覆されている、請求項１～９のいずれか１項に記載のグルコースセンサー。 The glucose sensor according to any one of claims 1 to 9, wherein the electrode portion is coated with a film made by crosslinking glucose oxidase with a polymer having an amino group as a base.
- 請求項１～１０のいずれか１項に記載のグルコースセンサーと、トランスミッターと、モニターとを用いて、該グルコースセンサーから得られた電気信号を該トランスミッターにより該モニターにデータ送信する、間質液中のグルコース濃度のモニターシステム。 The glucose sensor according to any one of claims 1 to 10, a transmitter, and a monitor are used to transmit an electrical signal obtained from the glucose sensor to the monitor by the transmitter, in an interstitial fluid. glucose concentration monitoring system.
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| JP2012509138A (en) * | 2008-11-20 | 2012-04-19 | アーカル・メディカル，インコーポレーテッド | Devices, systems, methods, and tools for continuous analyte monitoring |
| WO2020100854A1 (en) * | 2018-11-13 | 2020-05-22 | コスメディ製薬株式会社 | Water-repellent-coated micro needle array |
| WO2021232109A1 (en) * | 2020-05-20 | 2021-11-25 | Commonwealth Scientific And Industrial Research Organisation | Bioanalyte detection and monitoring |
| JP2022508576A (en) * | 2018-10-02 | 2022-01-19 | ウェアオプティモ ピーティーワイ リミテッド | Electrode equipment |
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| JP2012509138A (en) * | 2008-11-20 | 2012-04-19 | アーカル・メディカル，インコーポレーテッド | Devices, systems, methods, and tools for continuous analyte monitoring |
| JP2022508576A (en) * | 2018-10-02 | 2022-01-19 | ウェアオプティモ ピーティーワイ リミテッド | Electrode equipment |
| WO2020100854A1 (en) * | 2018-11-13 | 2020-05-22 | コスメディ製薬株式会社 | Water-repellent-coated micro needle array |
| WO2021232109A1 (en) * | 2020-05-20 | 2021-11-25 | Commonwealth Scientific And Industrial Research Organisation | Bioanalyte detection and monitoring |
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