WO2017057792A1 - Polyhydroxyethylmethacrylate-based hydrogel, method for producing same, and glucose sensor comprising same - Google Patents

Polyhydroxyethylmethacrylate-based hydrogel, method for producing same, and glucose sensor comprising same Download PDF

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WO2017057792A1
WO2017057792A1 PCT/KR2015/010935 KR2015010935W WO2017057792A1 WO 2017057792 A1 WO2017057792 A1 WO 2017057792A1 KR 2015010935 W KR2015010935 W KR 2015010935W WO 2017057792 A1 WO2017057792 A1 WO 2017057792A1
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glucose oxidase
hydrogel
region
polyhydroxyethyl methacrylate
concentration
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PCT/KR2015/010935
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French (fr)
Korean (ko)
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차준회
이지영
박하빈
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주식회사 엔게인
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L33/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Compositions of derivatives of such polymers
    • C08L33/04—Homopolymers or copolymers of esters
    • C08L33/06—Homopolymers or copolymers of esters of esters containing only carbon, hydrogen and oxygen, which oxygen atoms are present only as part of the carboxyl radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose

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  • the present invention relates to a polyhydroxyethyl methacrylate hydrogel, a preparation method thereof and a glucose sensor comprising the same.
  • the glucose sensor is a biosensor capable of measuring the amount of glucose in the blood.
  • the glucose sensor measures the amount of glucose in the blood using glucose oxidase.
  • Glucose oxidase oxidizes glucose in the blood, producing gluconic acid and reducing itself. Reduced glucose oxidase will produce hydrogen peroxide.
  • Korean Unexamined Patent Publication No. 10-2004-0079355 discloses a reverse ion osmosis type glucose sensor for glucose.
  • the present invention is to provide a polymer hydrogel containing a homogenous glucose oxidase, a preparation method thereof and a glucose sensor comprising the same.
  • the polyhydroxyethyl methacrylate hydrogel (hereinafter referred to as "pHEMA hydrogel") according to one embodiment comprises a glucose oxidase and satisfies the following formulas 1 and 2:
  • a is the concentration of glucose oxidase
  • W1 (a) is the weight of the first polyhydroxyethyl methacrylate hydrogel when the concentration of glucose oxidase is a
  • W2 (a ) Is the weight of the second polyhydroxyethyl methacrylate hydrogel when the concentration of glucose oxidase is a
  • CW1 (a) is the activity of the glucose oxidase in the first polyhydroxyethyl methacrylate hydrogel
  • CW2 (a) is the activity of glucose oxidase in the second polyhydroxyethyl methacrylate hydrogel.
  • the pHEMA hydrogel may include the glucose oxidase within a concentration range of 30 unit / mL to 667 unit / mL.
  • a method for preparing the pHEMA hydrogel is prepared by preparing a monomer composition solution by mixing the following components (A) to (F), by mixing glucose oxidase with the monomer composition solution and sonication. Preparing a homogeneous monomer composition solution and irradiating ultraviolet (UV) light to the homogeneous monomer composition solution to prepare a polymer hydrogel that satisfies Equation 1 and 2:
  • a glucose sensor includes a base film including a first region and a second region spatially separated from the first region, and a cathode disposed on the first region and on the second region. And an enzyme fixation membrane composed of an anode disposed on the electrode and the pHEMA hydrogel and disposed on the electrode.
  • the cathode may include a first-first region where a working electrode is disposed, a first-second region where a first extraction electrode is disposed and spatially separated from the first-first region to surround the first-first region, and a reference electrode. And a counter electrode disposed in and spaced apart from the first region and surrounding the first region, wherein the reference electrode and the counter electrode are within the region 1-3. Are spaced apart from each other.
  • the anode consists of a second extraction electrode.
  • 1 is a schematic plan view of a glucose sensor.
  • FIG. 2 is a schematic exploded view of a glucose sensor.
  • Figure 3 is a manufacturing process of the pHEMA hydrogel.
  • Figure 5 is a graph of the relationship between the weight and activity of the pHEMA hydrogel.
  • first, second, etc. are used to describe various components, these components are of course not limited by these terms. These terms are only used to distinguish one component from another. Therefore, of course, the first component mentioned below may be a second component within the technical spirit of the present invention.
  • a to B means A or more and B or less.
  • 1 is a schematic plan view of a glucose sensor.
  • 2 is a schematic exploded view of a glucose sensor.
  • the glucose sensor 100 includes a lower cover member 10, an upper cover member 20, a base film 30, a cathode C, an anode A, an insulating film 40, and Enzyme fixation membranes 50 are included.
  • the lower cover member 10 and the upper cover member 20 may be coupled to each other to form a housing of the glucose sensor 100.
  • the lower cover member 10 may include an accommodation space designed according to the shape of the base film 30, and the base film 30 may be disposed on the accommodation space.
  • the lower cover member 10 may be made of, for example, a synthetic resin having insulation.
  • the upper cover member 20 may include a through hole OP, and the cathode C and the anode A may be exposed to the outside through the through hole OP.
  • the upper cover member 10 may be composed of, for example, a clinical adhesive film.
  • the base film 30, the cathode C, the anode A, the insulating film 40, and the enzyme fixing films 50 may be mounted in a space between the lower cover member 10 and the upper cover member 20. have.
  • the base film 30 includes a first region and a second region spatially separated from the first region.
  • the first region is a region where the cathode C is disposed
  • the second region is a region where the anode A is disposed.
  • the base film 30 may be, for example, a polyethylene terephthalate (PET) film, but is not limited thereto.
  • the cathode C is composed of the working electrode WE, the first extraction electrode IE1, the reference electrode RE, and the counter electrode CE.
  • the first region may include a first-first region where the working electrode WE is disposed, a first-second region where the first extraction electrode IE1 is disposed, and a reference electrode RE and a counter electrode CE. Includes 1-3 areas.
  • the first-second area may be spatially separated from the first-first area, and may be configured to surround the outer periphery of the first-first area.
  • the 1-3 region may be spatially separated from the 1-2 region, and may be configured to surround the outside of the 1-2 region.
  • the reference electrode RE and the counter electrode CE may be spaced apart from each other, and the counter electrode CE may be the first extraction electrode IE1 not surrounded by the reference electrode RE.
  • the reference electrode RE may be configured to surround the outside of the first extraction electrode IE1 that is not surrounded by the counter electrode CE.
  • the anode A may be configured as the second extraction electrode IE2.
  • the working electrode CE, the first extraction electrode IE1, the second extraction electrode IE2, the counter electrode CE, and the reference electrode RE each have a circular enzyme fixation disposed in overlap with the enzyme fixation membranes 50. And a linear lead portion connected from the membrane placement portion and the circular enzyme fixation membrane placement portion and connected to an external electric terminal.
  • the shape of the enzyme fixation membrane arrangement portion and the lead portion is not limited to circular and linear, respectively, and may be variously modified.
  • the first extraction electrode IE1 and the second extraction electrode IE2 may extract glucose using reverse ion osmosis. Specifically, glucose may be promoted and diffused out of the cell membrane in accordance with the movement of sodium ions (Na + ).
  • the first extraction electrode IE1 and the second extraction electrode IE2 may be, for example, silver / silver chloride (Ag / AgCl) electrodes.
  • Glucose extracted using the reverse ion osmosis method accumulates in the enzyme fixation membranes 50 and generates hydrogen peroxide generated by glucose oxidase in the enzyme fixation membranes 50.
  • the working electrode WE, the counter electrode CE, and the reference electrode RE may serve as detection electrodes for detecting the electric coral generated by electrochemically oxidizing the generated hydrogen peroxide.
  • the working electrode WE may be, for example, a carbon electrode containing procian blue, which is a signal amplification medium
  • the counter electrode CE may be, for example, a silver / silver chloride (Ag / AgCl) electrode.
  • the reference electrode RE may be, for example, a silver / silver chloride (Ag / AgCl) electrode.
  • the insulating film 40 may be disposed on the remaining regions other than the first region and the second region of the base film 30, includes a through hole OP, and includes a cathode C and an anode A. May be exposed to the outside through the through hole OP.
  • the insulating film 40 may be, for example, an insulating paste coating film, but is not limited thereto.
  • the enzyme fixation membranes 50 may be disposed to overlap the enzyme fixation membrane arrangement of the cathode (C) and the anode (A).
  • the enzyme fixation membranes 50 are composed of a pHEMA hydrogel, and the pHEMA hydrogel contains glucose oxidase.
  • the pHEMA hydrogel is a transparent polymer hydrogel, in which the glucose oxidase is homogeneously distributed.
  • the pHEMA hydrogel may contain a high concentration of glucose oxidase (for example, a concentration that can be readily oxidized when glucose enters the hydrogel: 667 units / mL), and a high concentration of glucose oxidase is homogeneously distributed in the hydrogel. It may have a high elasticity and swelling compared to polyhydroxypropyl methacrylate hydrogel (hereinafter, referred to as "pHPMA hydrogel").
  • the concentration of the glucose oxidase contained in the pHEMA hydrogel may be, for example, 30 unit / mL to 667 unit / mL, and when the concentration of the glucose oxidase is 667 unit / mL, into the pHEMA hydrogel Incoming glucose can be oxidized immediately.
  • the pHPMA-based hydrogel is a polymer hydrogel having an opaque property, and hydroxypropyl methacrylate (HPMA), dimethylamine (DMA), and ethylene glycol (EG) are first mixed for 5 minutes, and triethylene is mixed into the primary mixture. Glycol dimethacrylate (TEGDMA) and tetramethylethylenediamine (TEMED) are added to the mixture for 2 minutes for 5 minutes, glucose oxidase is added to the mixture for 3 minutes for 5 minutes and ammonium is added to the mixture.
  • Persulfate (APS) may be prepared by adding a fourth mixture for 1 minute and then reacting the fourth mixture in a gelatin chamber at 25 ° C. for 3 hours. However, in the method for producing the pHPMA hydrogel, the total production time was over 3 hours, and the yield was about 10%.
  • Figure 3 shows a manufacturing process of the pHEMA hydrogel, the manufacturing method of the pHEMA hydrogel, it is possible to shorten the manufacturing time compared to the manufacturing method of the pHPMA hydrogel, and to produce a pHEMA hydrogel with a high yield Can be.
  • the experimental groups prepared in the following experimental examples took only 21 minutes to produce the pHEMA-based hydrogel, and the yield was 97%.
  • step (S) to prepare a monomer composition solution by mixing the following components (A) (S1), the glucose oxidase is mixed with the monomer composition solution And performing a sonication (S2) to prepare a homogeneous monomer composition solution (S2) and irradiating ultraviolet (UV) to the homogeneous monomer composition solution to prepare the pHEMA-based hydrogel (S3).
  • the method for preparing the pHEMA hydrogel may further include the step (S4) of aging and washing the pHEMA hydrogel.
  • Experiment Sonication S1 Mixing time: 10 minutes (A) component 4.8 mL 4.8 mL (B) component 2.4 mL 2.4 mL (C) component 0.3 mL 0.3 mL (D) component 0.1g 0.1g (E) component 3.25 mL 3.25 mL (F) component 4.25 mL 4.25 mL
  • S2 Mixing time: 5 minutes Glucose oxidase 667 unit / mL 667 unit / mL Sound Processing: 3 minutes practice Not carried S3
  • UV irradiation time 3 minutes
  • control group not treated with sonication is judged to have a lower homogeneity of the monomer composition solution than the sonicated experimental group.
  • Figure 4 shows that the homogeneity difference between the experimental group and the control group affects the activity of glucose oxidase contained in the pHEMA hydrogel.
  • the experimental group was evenly measured without significant deviation in the range of activity (OD) from about 0.5 (au) to about 1.0 (au), while the control group had an activity (OD) of about 0.5 (au). ), With a large deviation in the range between about 2.0 (au).
  • the activity of the experimental group and the control group was measured by the absorbance of the pHEMA hydrogels reacted with glucose oxidase.
  • Control Group 1 Control Group 2 (Condition 2) Control Group 3 (Condition 3)
  • S1 Mixing time: 10 minutes (A) component 4.8 mL 4.8 mL 4.8 mL 4.8 mL 4.8 mL (B) component 2.4 mL 1.5 mL 0.75 mL 0 mL (C) component 0.3 mL 0.3 mL 0.3 mL 0.3 mL (D) component 0.1g 0.1g 0.1g 0.1g 0.1g (E) component 3.25 mL 3.25 mL 3.25 mL 3.25 mL 3.25 mL (F) component 4.25 mL 4.25 mL 4.25 mL 4.25 mL S2 Mixing time: 5 minutes Glucose oxidase 667 unit / mL 667 unit / mL 667 unit / mL 667 unit / mL 667 unit / mL Sound Processing: 3 minutes practice S3 UV irradiation time: 3 minutes S4
  • the inventors of the present application visually observed the experimental group and the control group, the experimental group with a dimethylamine content of more than 0 mL, the control group 1, the control group 2 was transparent, while the control group 3 with a dimethylamine content of 0 mL was opaque.
  • Table 3 summarizes the results of measuring the content of glucose oxidase in each of the experimental group, control group 1, control group 2 and control group 3.
  • the content of glucose oxidase was measured using the megazyme method.
  • Control (Unit / gram) Control Group 1 (Condition 1) (Unit / gram) Control 2 (condition 2) (Unit / gram) Control Group 3 (Condition 3) (Unit / gram) 0.97 0.77 0.43 0.65 0.84 0.75 0.50 0.73 0.82 0.93 0.43 0.65 0.87 0.92 0.48 0.71 0.98 0.75 0.49 0.71 Average: 0.90 Average: 0.82 Average: 0.47 Average: 0.69
  • control group 3 In the case of the control group 3 containing no dimethylamine, the content of glucose oxidase was higher than that of the control group 3, but as shown in the visual observation, the control group 3 was mixed in an opaque manner, and the conditions were not suitable for the hydrogel, and the glucose To calculate the oxidase content activity, absorbance, weight, and extracted volume are required.
  • Control 3 had less swelling compared to experimental group, control group 1, and control group 2. It was 1/3 or more smaller than this experimental group, the control group 1, and the control group 2. Taking these points together, control 3 is expected to have a heterogeneous distribution of glucose oxidases.
  • Figure 5 is a graph of the relationship between the weight and activity of the pHEMA hydrogel.
  • the control is a blank (blank) solution used to measure the change in absorbance
  • Experimental Group 1 Experimental Group 2
  • Experimental Group 3 is 0.05g, 0.1g, 0.15g pHEMA-based hydrogel prepared under the conditions of Table 4 Samples taken at the weight of
  • a is the concentration of glucose oxidase
  • W1 (a) is the weight of the first polyhydroxyethyl methacrylate hydrogel when the concentration of glucose oxidase is a
  • W2 (a ) Is the weight of the second polyhydroxyethyl methacrylate hydrogel when the concentration of glucose oxidase is a
  • CW1 (a) is the activity of the glucose oxidase in the first polyhydroxyethyl methacrylate hydrogel
  • CW2 (a) is the activity of glucose oxidase in the second polyhydroxyethyl methacrylate hydrogel.
  • the present invention can provide a polymer hydrogel containing glucose oxidase homogeneously, a method for preparing the same, and a glucose sensor including the same.

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Abstract

A polyhydroxyethylmethacrylate-based hydrogel contains glucose oxidase and satisfies both formulas 1 and 2 below: (formula 1) W1(a)〈 W2(a), (formula 2) CW1(a)〈 CW2(a) (a, W1(a), W2(a), CW1(a), and CW2(a) are as defined in the specification.)

Description

폴리히드록시에틸메타크릴레이트계 수화겔, 이의 제조방법 및 이를 포함하는 글루코스 센서Polyhydroxyethyl methacrylate hydrogel, preparation method thereof and glucose sensor comprising the same
발명은 폴리히드록시에틸메타크릴레이트계 수화겔, 이의 제조방법 및 이를 포함하는 글루코스 센서에 대한 것이다.The present invention relates to a polyhydroxyethyl methacrylate hydrogel, a preparation method thereof and a glucose sensor comprising the same.
글루코스 센서는 혈중 글루코스의 양을 측정할 수 있는 바이오 센서로서, 일반적으로 글루코스 산화효소를 이용하여 혈중 글루코스의 양을 측정한다. 글루코스 산화효소는 혈액 내의 글루코스를 산화시켜 글루콘산을 생성시키고 자신은 환원된다. 환원된 글루코스 산화효소는 과산화수소를 생성시키게 된다. 대한민국 공개특허공보 제 10-2004-0079355호(2004.09.14) 에는 역이온삼투 방식의 글루코스용 바이온 센서가 개시되어 있다.The glucose sensor is a biosensor capable of measuring the amount of glucose in the blood. In general, the glucose sensor measures the amount of glucose in the blood using glucose oxidase. Glucose oxidase oxidizes glucose in the blood, producing gluconic acid and reducing itself. Reduced glucose oxidase will produce hydrogen peroxide. Korean Unexamined Patent Publication No. 10-2004-0079355 (September 14, 2004) discloses a reverse ion osmosis type glucose sensor for glucose.
발명은 글루코스 산화효소가 균질하게 함유된 고분자 수화겔, 이의 제조방법 및 이를 포함하는 글루코스 센서를 제공하고자 한다.The present invention is to provide a polymer hydrogel containing a homogenous glucose oxidase, a preparation method thereof and a glucose sensor comprising the same.
발명이 해결하고자 하는 과제는 이상에서 언급한 기술적 과제로 제한되지 않으며, 언급되지 않은 또 다른 기술적 과제들은 아래의 기재로부터 본 기술분야에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.The problem to be solved by the invention is not limited to the above-mentioned technical problem, and other technical problems not mentioned will be clearly understood by those skilled in the art from the following description.
일 실시예에 따른 폴리히드록시에틸메타크릴레이트계 수화겔(이하, "pHEMA계 수화겔"이라 한다)은 글루코스 산화효소를 포함하고 하기 식 1 및 2 를 만족한다:The polyhydroxyethyl methacrylate hydrogel (hereinafter referred to as "pHEMA hydrogel") according to one embodiment comprises a glucose oxidase and satisfies the following formulas 1 and 2:
(식 1) W1(a)〈 W2(a) (1) W1 ( a ) 〈 W2 ( a )
(식 2) CW1(a)〈 CW2(a) (Equation 2) CW1 ( a ) 〈 CW2 ( a )
상기 식 1 내지 식 2에서, a 는 글루코스 산화효소의 농도이고, W1(a) 는 글루코스 산화효소의 농도가 a 인 때, 제1 폴리히드록시에틸메타크릴레이트계 수화겔의 중량이며, W2(a) 는 글루코스 산화효소의 농도가 a 인 때, 제2 폴리히드록시에틸메타크릴레이트계 수화겔의 중량이고, CW1(a)는 제1 폴리히드록시에틸메타크릴레이트계 수화겔 내의 글루코스 산화효소의 활성도이며, CW2(a)는 제2 폴리히드록시에틸메타크릴레이트계 수화겔 내의 글루코스 산화효소의 활성도이다. In Formula 1 to Formula 2, a is the concentration of glucose oxidase, W1 (a) is the weight of the first polyhydroxyethyl methacrylate hydrogel when the concentration of glucose oxidase is a, W2 (a ) Is the weight of the second polyhydroxyethyl methacrylate hydrogel when the concentration of glucose oxidase is a, and CW1 (a) is the activity of the glucose oxidase in the first polyhydroxyethyl methacrylate hydrogel , CW2 (a) is the activity of glucose oxidase in the second polyhydroxyethyl methacrylate hydrogel.
상기 pHEMA계 수화겔은 상기 글루코스 산화효소를 30 unit/mL 내지 667 unit/mL 의 농도범위 내에서 포함할 수 있다.The pHEMA hydrogel may include the glucose oxidase within a concentration range of 30 unit / mL to 667 unit / mL.
다른 실시예에 따른 상기 pHEMA계 수화겔의 제조방법은 하기의 (A) 성분 내지 (F) 성분을 혼합하여 단량체 조성물 용액을 제조하는 단계, 상기 단량체 조성물 용액에 글루코스 산화효소를 혼합하고 음파처리(sonication)하여 균질 단량체 조성물 용액을 제조하는 단계 및 상기 균질 단량체 조성물 용액에 자외선(UV)을 조사하여 상기 식 1 및 2를 만족하는 고분자 수화겔을 제조하는 단계를 포함한다:According to another embodiment of the present invention, a method for preparing the pHEMA hydrogel is prepared by preparing a monomer composition solution by mixing the following components (A) to (F), by mixing glucose oxidase with the monomer composition solution and sonication. Preparing a homogeneous monomer composition solution and irradiating ultraviolet (UV) light to the homogeneous monomer composition solution to prepare a polymer hydrogel that satisfies Equation 1 and 2:
(A) 성분: 히드록시에틸메타크릴레이트(A) component: hydroxyethyl methacrylate
(B) 성분: 디메틸아민(B) Component: Dimethylamine
(C) 성분: 트리에틸렌글리콜디메타크릴레이트(C) component: triethylene glycol dimethacrylate
(D) 성분: 광중합 개시제(D) component: photoinitiator
(E) 성분: 에틸렌 글리콜(E) Component: Ethylene Glycol
(F) 성분: 물(F) Ingredient: Water
또 다른 실시예에 따른 글루코스 센서는, 제1 영역과 상기 제1 영역과 공간적으로 분리된 제2 영역을 포함하는 베이스 필름, 상기 제1 영역 상에 배치된 캐소드(cathode)와 상기 제2 영역 상에 배치된 애노드(anode)로 구성된 전극부 및 상기 pHEMA계 수화겔로 구성되고 상기 전극부 상에 배치된 효소 고정막을 포함한다.According to yet another embodiment, a glucose sensor includes a base film including a first region and a second region spatially separated from the first region, and a cathode disposed on the first region and on the second region. And an enzyme fixation membrane composed of an anode disposed on the electrode and the pHEMA hydrogel and disposed on the electrode.
상기 캐소드는, 작업전극이 배치되는 제1-1 영역, 제1 추출전극이 배치되고 상기 제1-1 영역과 공간적으로 분리되어 상기 제1-1 영역을 둘러싸고 있는 제1-2 영역, 기준전극과 상대전극이 배치되고 상기 제1-2 영역과 공간적으로 분리되어 상기 제1-2 영역을 둘러싸고 있는 제1-3 영역을 포함하고, 상기 기준전극과 상기 상대전극은 상기 제1-3 영역 내에서 상호 이격 배치된다. 상기 애노드는 제2 추출 전극으로 구성된다. The cathode may include a first-first region where a working electrode is disposed, a first-second region where a first extraction electrode is disposed and spatially separated from the first-first region to surround the first-first region, and a reference electrode. And a counter electrode disposed in and spaced apart from the first region and surrounding the first region, wherein the reference electrode and the counter electrode are within the region 1-3. Are spaced apart from each other. The anode consists of a second extraction electrode.
기타 실시예들의 구체적인 사항은 상세한 설명 및 도면들에 포함되어 있다.Specific details of other embodiments are included in the detailed description and the drawings.
도 1은 글루코스 센서의 개략적인 평면도이다.1 is a schematic plan view of a glucose sensor.
도 2는 글루코스 센서의 개략적인 분해도이다.2 is a schematic exploded view of a glucose sensor.
도 3은 pHEMA계 수화겔의 제조 공정도이다.Figure 3 is a manufacturing process of the pHEMA hydrogel.
도 4는 음파처리 유무에 따른 글루코스 산화효소의 활성도 비교 그래프이다.4 is a graph comparing the activity of glucose oxidase according to the presence or absence of sonication.
도 5는 pHEMA계 수화겔의 무게와 활성도의 관계 그래프이다.Figure 5 is a graph of the relationship between the weight and activity of the pHEMA hydrogel.
발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 첨부되는 도면과 함께 상세하게 후술되어 있는 실험예들을 참조하면 명확해질 것이다. Advantages and features of the invention, and methods of achieving them will be apparent with reference to the experimental examples described below in detail in conjunction with the accompanying drawings.
그러나 발명은 이하에서 개시되는 실시예들에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 것이며, 단지 실시예들은 발명의 개시가 완전하도록 하며, 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이며, 발명은 청구항의 범주에 의해 정의될 뿐이다. However, the invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, only the embodiments to complete the disclosure of the invention, to those skilled in the art to which the invention pertains It is provided to fully inform the scope of the invention, and the invention is defined only by the scope of the claims.
도면에서 층 및 영역들의 크기 및 상대적인 크기는 설명의 명료성을 위해 과장된 것일 수 있다.In the drawings, the sizes and relative sizes of layers and regions may be exaggerated for clarity.
소자(elements) 또는 층이 다른 소자 또는 층의 "위(on)" 또는 "상(on)"으로 지칭되는 것은 다른 소자 중간에 다른 층 또는 다른 소자를 개재한 경우를 모두 포함한다. When an element or layer is referred to as "on" or "on" of another element or layer, it includes all cases where another element or layer is interposed between other elements or layers.
비록 제1, 제2 등이 다양한 구성요소들을 서술하기 위해서 사용되나, 이들 구성요소들은 이들 용어에 의해 제한되지 않음은 물론이다. 이들 용어들은 단지 하나의 구성요소를 다른 구성요소와 구별하기 위하여 사용하는 것이다. 따라서, 이하에서 언급되는 제1 구성요소는 본 발명의 기술적 사상 내에서 제2 구성요소일 수도 있음은 물론이다.Although the first, second, etc. are used to describe various components, these components are of course not limited by these terms. These terms are only used to distinguish one component from another. Therefore, of course, the first component mentioned below may be a second component within the technical spirit of the present invention.
본 명세서에서, "A 내지 B" 는 A 이상 내지 B 이하를 의미한다. In the present specification, "A to B" means A or more and B or less.
도 1은 글루코스 센서의 개략적인 평면도이다. 도 2는 글루코스 센서의 개략적인 분해도이다. 1 is a schematic plan view of a glucose sensor. 2 is a schematic exploded view of a glucose sensor.
도 1 내지 도 2를 참고하면, 글루코스 센서(100)는 하부 커버부재(10), 상부 커버부재(20), 베이스 필름(30), 캐소드(C), 애노드(A), 절연막(40) 및 효소 고정막들(50)을 포함하여 구성된다. 1 to 2, the glucose sensor 100 includes a lower cover member 10, an upper cover member 20, a base film 30, a cathode C, an anode A, an insulating film 40, and Enzyme fixation membranes 50 are included.
하부 커버부재(10)와 상부 커버부재(20)는 상호 결합되어 글루코스 센서(100)의 하우징을 형성할 수 있다. The lower cover member 10 and the upper cover member 20 may be coupled to each other to form a housing of the glucose sensor 100.
하부 커버부재(10)는 베이스 필름(30)의 형상에 따라 설계된 수납 공간을 포함할 수 있고, 베이스 필름(30)은 상기 수납 공간 상에 배치될 수 있다. 하부 커버부재(10)는, 예를 들어, 절연성을 갖는 합성 수지로 구성될 수 있다. The lower cover member 10 may include an accommodation space designed according to the shape of the base film 30, and the base film 30 may be disposed on the accommodation space. The lower cover member 10 may be made of, for example, a synthetic resin having insulation.
상부 커버부재(20)는, 관통홀(OP)을 포함할 수 있고, 캐소드(C)와 애노드(A)는 관통홀(OP)을 통해 외부로 노출될 수 있다. 상부 커버부재(10)는, 예를 들어, 임상용 점착필름으로 구성될 수 있다.The upper cover member 20 may include a through hole OP, and the cathode C and the anode A may be exposed to the outside through the through hole OP. The upper cover member 10 may be composed of, for example, a clinical adhesive film.
베이스 필름(30), 캐소드(C), 애노드(A), 절연막(40) 및 효소 고정막들(50)은 하부 커버부재(10)와 상부 커버부재(20)의 사이의 공간에 장착될 수 있다. The base film 30, the cathode C, the anode A, the insulating film 40, and the enzyme fixing films 50 may be mounted in a space between the lower cover member 10 and the upper cover member 20. have.
베이스 필름(30)은 제1 영역과 상기 제1 영역과 공간적으로 분리된 제2 영역을 포함한다. 제1 영역은 캐소드(C)가 배치되는 영역이고, 제2 영역은 애노드(A)가 배치되는 영역이다. 베이스 필름(30)은, 예를 들어, 폴리에틸렌 테레프탈레이트(PET) 필름일 수 있으나 이것만으로 한정되는 것은 아니다.The base film 30 includes a first region and a second region spatially separated from the first region. The first region is a region where the cathode C is disposed, and the second region is a region where the anode A is disposed. The base film 30 may be, for example, a polyethylene terephthalate (PET) film, but is not limited thereto.
캐소드(C)는 작업전극(WE), 제1 추출전극(IE1), 기준전극(RE) 및 상대전극(CE)로 구성된다. 상기 제1 영역은 작업전극(WE)이 배치되는 제1-1 영역, 제1 추출전극(IE1)이 배치되는 제1-2 영역, 기준전극(RE)과 상대전극(CE)가 배치되는 제1-3 영역을 포함한다. 상기 제1-2 영역은 상기 제1-1 영역과 공간적으로 분리되어 있고, 상기 제1-1 영역의 외곽을 둘러싸도록 구성될 수 있다. 상기 제1-3 영역은 상기 제1-2 영역과 공간적으로 분리되어 있고, 상기 제1-2 영역의 외곽을 둘러싸도록 구성될 수 있다. 상기 제1-3 영역 내에서, 기준전극(RE)과 상대전극(CE)은 서로 이격되어 배치될 수 있고, 상대전극(CE)은 기준전극(RE)으로 둘러싸이지 않은 제1 추출전극(IE1)의 외곽을 둘러싸도록 구성될 수 있고, 기준전극(RE)은 상대전극(CE)으로 둘러싸이지 않은 제1 추출전극(IE1)의 외곽을 둘러싸도록 구성될 수 있다.The cathode C is composed of the working electrode WE, the first extraction electrode IE1, the reference electrode RE, and the counter electrode CE. The first region may include a first-first region where the working electrode WE is disposed, a first-second region where the first extraction electrode IE1 is disposed, and a reference electrode RE and a counter electrode CE. Includes 1-3 areas. The first-second area may be spatially separated from the first-first area, and may be configured to surround the outer periphery of the first-first area. The 1-3 region may be spatially separated from the 1-2 region, and may be configured to surround the outside of the 1-2 region. In the region 1-3, the reference electrode RE and the counter electrode CE may be spaced apart from each other, and the counter electrode CE may be the first extraction electrode IE1 not surrounded by the reference electrode RE. The reference electrode RE may be configured to surround the outside of the first extraction electrode IE1 that is not surrounded by the counter electrode CE.
애노드(A)는 제2 추출전극(IE2)으로 구성될 수 있다. The anode A may be configured as the second extraction electrode IE2.
작업전극(CE), 제1 추출전극(IE1), 제2 추출전극(IE2), 상대전극(CE)과 기준전극(RE)은 각각 효소 고정막들(50)과 중첩 배치되는 원형의 효소 고정막 배치부와 상기 원형의 효소 고정막 배치부로부터 연결되고 외부 전기단자와 연결되는 선상의 리드부를 포함하여 구성될 수 있다. 다만, 상기 효소 고정막 배치부와 상기 리드부의 형상은 각각 원형과 선상으로 한정되는 것은 아니고, 다양하게 변형될 수 있다. The working electrode CE, the first extraction electrode IE1, the second extraction electrode IE2, the counter electrode CE, and the reference electrode RE each have a circular enzyme fixation disposed in overlap with the enzyme fixation membranes 50. And a linear lead portion connected from the membrane placement portion and the circular enzyme fixation membrane placement portion and connected to an external electric terminal. However, the shape of the enzyme fixation membrane arrangement portion and the lead portion is not limited to circular and linear, respectively, and may be variously modified.
제1 추출전극(IE1)과 제2 추출전극(IE2)은 역이온삼투압법을 이용하여 글루코스를 추출할 수 있다. 구체적으로, 글루코스는 나트륨 이온(Na+)의 이동에 따라 촉진확산되어 세포막 밖으로 배출될 수 있다. 제1 추출전극(IE1)과 제2 추출전극(IE2)은, 예를 들어, 은/염화은(Ag/AgCl) 전극일 수 있다. The first extraction electrode IE1 and the second extraction electrode IE2 may extract glucose using reverse ion osmosis. Specifically, glucose may be promoted and diffused out of the cell membrane in accordance with the movement of sodium ions (Na + ). The first extraction electrode IE1 and the second extraction electrode IE2 may be, for example, silver / silver chloride (Ag / AgCl) electrodes.
역이온삼투압법을 이용하여 추출한 글루코스는 효소 고정막들(50) 내에 축적되고, 효소 고정막들(50)내의 글루코스 산화효소에 의해 발생한 과산화수소를 생성시킨다. Glucose extracted using the reverse ion osmosis method accumulates in the enzyme fixation membranes 50 and generates hydrogen peroxide generated by glucose oxidase in the enzyme fixation membranes 50.
작업전극(WE), 상대전극(CE)과 기준전극(RE)은, 생성된 과산화수소를 전기화학적으로 산화시켜 발생된 전기적 산호를 검출하는 검출 전극으로서 역할을 할 수 있다. 작업전극(WE)은, 예를 들어, 신호증폭 매개체인 프로시안 블루를 함유하고 있는 탄소 전극일 수 있고, 상대전극(CE)은, 예를 들어, 은/염화은(Ag/AgCl) 전극일 수 있으며, 기준전극(RE)은, 예를 들어, 은/염화은(Ag/AgCl) 전극일 수 있다. The working electrode WE, the counter electrode CE, and the reference electrode RE may serve as detection electrodes for detecting the electric coral generated by electrochemically oxidizing the generated hydrogen peroxide. The working electrode WE may be, for example, a carbon electrode containing procian blue, which is a signal amplification medium, and the counter electrode CE may be, for example, a silver / silver chloride (Ag / AgCl) electrode. The reference electrode RE may be, for example, a silver / silver chloride (Ag / AgCl) electrode.
절연막(40)은, 베이스 필름(30)의 상기 제1 영역과 상기 제2 영역 이외의 나머지 영역 상에 배치될 수 있고, 관통홀(OP)을 포함하며, 캐소드(C)와 애노드(A)는 관통홀(OP)을 통해 외부로 노출될 수 있다. 절연막(40)는, 예를 들어, 절연 페이스트 코팅막일 수 있으나, 이것만으로 한정되는 것은 아니다.The insulating film 40 may be disposed on the remaining regions other than the first region and the second region of the base film 30, includes a through hole OP, and includes a cathode C and an anode A. May be exposed to the outside through the through hole OP. The insulating film 40 may be, for example, an insulating paste coating film, but is not limited thereto.
효소 고정막들(50)은 캐소드(C)와 애노드(A)의 상기 효소 고정막 배치부와 중첩되게 배치될 수 있다. 효소 고정막들(50)은 pHEMA계 수화겔로 구성되고, 상기 pHEMA계 수화겔은 글루코스 산화효소를 포함한다. 상기 pHEMA계 수화겔은 투명한 고분자 수화겔로서, 상기 글루코스 산화효소가 균질하게 분포되어 있다. The enzyme fixation membranes 50 may be disposed to overlap the enzyme fixation membrane arrangement of the cathode (C) and the anode (A). The enzyme fixation membranes 50 are composed of a pHEMA hydrogel, and the pHEMA hydrogel contains glucose oxidase. The pHEMA hydrogel is a transparent polymer hydrogel, in which the glucose oxidase is homogeneously distributed.
상기 pHEMA계 수화겔은 고농도의 글루코스 산화효소(예를 들어, 글루코스가 수화겔 내로 들어오면 즉시 산화될 수 있는 농도: 667 unit/mL)를 함유할 수 있고, 고농도의 글루코스 산화효소가 수화겔 내에 균질하게 분포되어 있을 수 있으며, 폴리히드록시프로필메타크릴레이트계 수화겔(이하, "pHPMA계 수화겔"이라 한다)에 비해 탄성과 팽윤(swelling)이 우수하다. 상기 pHEMA계 수화겔 내에 함유된 상기 글루코스 산화효소의 농도는 예를 들어, 30 unit/mL 내지 667 unit/mL 일 수 있고, 상기 글루코스 산화효소의 농도가 667 unit/mL 인 때, 상기 pHEMA계 수화겔 내로 들어온 글루코스는 즉시 산화될 수 있다. The pHEMA hydrogel may contain a high concentration of glucose oxidase (for example, a concentration that can be readily oxidized when glucose enters the hydrogel: 667 units / mL), and a high concentration of glucose oxidase is homogeneously distributed in the hydrogel. It may have a high elasticity and swelling compared to polyhydroxypropyl methacrylate hydrogel (hereinafter, referred to as "pHPMA hydrogel"). The concentration of the glucose oxidase contained in the pHEMA hydrogel may be, for example, 30 unit / mL to 667 unit / mL, and when the concentration of the glucose oxidase is 667 unit / mL, into the pHEMA hydrogel Incoming glucose can be oxidized immediately.
상기 pHPMA계 수화겔은 불투명한 성상을 갖는 고분자 수화겔로서, 히드록시프로필메타크릴레이트(HPMA), 디메틸아민(DMA), 에틸렌글리콜(EG)을 5분 동안 1차 혼합하고, 1차 혼합물에 트리에틸렌글리콜디메타크릴레이트(TEGDMA)와 테트라메틸에틸렌디아민 (TEMED)을 첨가하여 5 분 동안 2차 혼합하며, 2차 혼합물에 글루코스 산화효소를 첨가하여 5 분 동안 3차 혼합하고, 3차 혼합물에 암모늄퍼설페이트(APS)를 첨가하여 1분 동안 4 차 혼합한 뒤, 4차 혼합물을 3 시간 동안 25 ℃의 젤라틴 챔버 내에서 반응시키는 과정을 통해 제조될 수 있다. 그러나, 상기 pHPMA계 수화겔의 제조방법은, 총 제조 시간이 3 시간이 넘고, 수율 또한 10% 정도였다. The pHPMA-based hydrogel is a polymer hydrogel having an opaque property, and hydroxypropyl methacrylate (HPMA), dimethylamine (DMA), and ethylene glycol (EG) are first mixed for 5 minutes, and triethylene is mixed into the primary mixture. Glycol dimethacrylate (TEGDMA) and tetramethylethylenediamine (TEMED) are added to the mixture for 2 minutes for 5 minutes, glucose oxidase is added to the mixture for 3 minutes for 5 minutes and ammonium is added to the mixture. Persulfate (APS) may be prepared by adding a fourth mixture for 1 minute and then reacting the fourth mixture in a gelatin chamber at 25 ° C. for 3 hours. However, in the method for producing the pHPMA hydrogel, the total production time was over 3 hours, and the yield was about 10%.
도 3에는 상기 pHEMA계 수화겔의 제조 공정도가 도시되어 있는데, 상기 pHEMA계 수화겔의 제조방법은, 상기 pHPMA계 수화겔의 제조방법에 비해 제조시간을 단축할 수 있고, 높은 수율로 pHEMA계 수화겔을 제조할 수 있다. 예를 들어, 하기 실험예들에서 제조된 실험군들은 상기 pHEMA계 수화겔을 제조하는데 있어서 단 21 분 밖에 소요되지 않았고, 수율이 97% 였다. Figure 3 shows a manufacturing process of the pHEMA hydrogel, the manufacturing method of the pHEMA hydrogel, it is possible to shorten the manufacturing time compared to the manufacturing method of the pHPMA hydrogel, and to produce a pHEMA hydrogel with a high yield Can be. For example, the experimental groups prepared in the following experimental examples took only 21 minutes to produce the pHEMA-based hydrogel, and the yield was 97%.
도 3을 참고하면, 상기 pHEMA계 수화겔의 제조방법은, 하기의 (A) 성분 내지 (F) 성분을 혼합하여 단량체 조성물 용액을 제조하는 단계(S1), 상기 단량체 조성물 용액에 글루코스 산화효소를 혼합하고 음파처리(sonication)하여 균질 단량체 조성물 용액을 제조하는 단계(S2) 및 상기 균질 단량체 조성물 용액에 자외선(UV)을 조사하여 상기 pHEMA계 수화겔을 제조하는 단계(S3)를 포함한다. Referring to Figure 3, the method for producing a pHEMA hydrogel, step (S) to prepare a monomer composition solution by mixing the following components (A) (S1), the glucose oxidase is mixed with the monomer composition solution And performing a sonication (S2) to prepare a homogeneous monomer composition solution (S2) and irradiating ultraviolet (UV) to the homogeneous monomer composition solution to prepare the pHEMA-based hydrogel (S3).
(A) 성분: 히드록시에틸메타크릴레이트(A) component: hydroxyethyl methacrylate
(B) 성분: 디메틸아민(B) Component: Dimethylamine
(C) 성분: 트리에틸렌글리콜디메타크릴레이트(C) component: triethylene glycol dimethacrylate
(D) 성분: 광중합 개시제(D) component: photoinitiator
(E) 성분: 에틸렌 글리콜(E) Component: Ethylene Glycol
(F) 성분: 물(F) Ingredient: Water
상기 pHEMA계 수화겔의 제조방법은, 상기 pHEMA계 수화겔을 에이징 및 세척하는 단계(S4)를 더 포함할 수 있다.The method for preparing the pHEMA hydrogel may further include the step (S4) of aging and washing the pHEMA hydrogel.
실험예 1_음파처리 유무에 따른 균질도 및 글루코스 산화효소의 활성도 비 교 Experimental Example 1 Comparison of homogeneity and glucose oxidase activity with or without sonication
도 4는 음파처리 유무에 따른 글루코스 산화효소의 활성도 비교 그래프이다. 4 is a graph comparing the activity of glucose oxidase according to the presence or absence of sonication.
실험예 1에 사용된 실험군과 대조군은 하기 표 1의 조건에 따라 제조되었다.Experimental groups and controls used in Experimental Example 1 were prepared according to the conditions of Table 1 below.
공정fair 공정조건Process conditions 성분ingredient 실험군(Control)Experiment 대조군(Sonication)Sonication
S1S1 혼합시간: 10 분Mixing time: 10 minutes (A) 성분(A) component 4.8 mL4.8 mL 4.8 mL 4.8 mL
(B) 성분(B) component 2.4 mL 2.4 mL 2.4 mL 2.4 mL
(C) 성분(C) component 0.3 mL 0.3 mL 0.3 mL 0.3 mL
(D) 성분(D) component 0.1g0.1g 0.1g0.1g
(E) 성분(E) component 3.25 mL 3.25 mL 3.25 mL3.25 mL
(F) 성분(F) component 4.25 mL 4.25 mL 4.25 mL4.25 mL
S2S2 혼합시간: 5분 Mixing time: 5 minutes 글루코스 산화효소Glucose oxidase 667 unit/ mL667 unit / mL 667 unit/ mL 667 unit / mL
음파처리: 3분Sound Processing: 3 minutes 실시practice 미실시Not carried
S3S3 UV 조사시간: 3분UV irradiation time: 3 minutes
S4S4 에이징(aging) 조건: 3 시간 동안 4 ℃ 냉장고에서 숙성세척 조건: 4 ℃의 증류수로 6 번 세척한 후, 인산완충식염수로 12 시간 동안 세척Aging condition: Aged in a refrigerator at 4 ° C. for 3 hours Washing condition: 6 times with distilled water at 4 ° C., followed by 12 hours with phosphate buffered saline
본 출원의 발명자들이 음파처리를 실시한 실험군과 음파처이를 실시하지 않은 대조군을 육안으로 관찰한 결과, 음파처리를 실시한 실험군과 달리 음파처리를 실시하지 않은 대조군은 버블(bubble)이 관찰되었다. 이를 기반으로, 음파처리를 하지 않은 대조군은 음파처리를 한 실험군에 비해 단량체 조성물 용액의 균질도가 낮은 것으로 판단된다. When the inventors of the present application visually observed the experimental group subjected to sonication and the control group not subjected to sonication, bubbles were observed in the control group not subjected to sonication, unlike the experimental group subjected to sonication. Based on this, the control group not treated with sonication is judged to have a lower homogeneity of the monomer composition solution than the sonicated experimental group.
도 4는 실험군과 대조군의 균질도 차이가 pHEMA계 수화겔 내에 함유된 글루코스 산화효소의 활성도에 영향을 미친다는 것을 보여준다. 도 4를 참고하면, 실험군은 활성도(OD)가 약 0.5(au) 내지 약 1.0(au)의 사이의 범위에서 큰 편차가 없이 고르게 측정된 반면에, 대조군은 활성도(OD)가 약 0.5(au) 내지 약 2.0(au)의 사이의 범위에서 큰 편차를 보이면서 측정되었다. 실험군과 대조군의 활성도는 글루코스 산화효소와 반응시킨 pHEMA계 수화겔의 흡광도를 측정한 것이다.Figure 4 shows that the homogeneity difference between the experimental group and the control group affects the activity of glucose oxidase contained in the pHEMA hydrogel. Referring to FIG. 4, the experimental group was evenly measured without significant deviation in the range of activity (OD) from about 0.5 (au) to about 1.0 (au), while the control group had an activity (OD) of about 0.5 (au). ), With a large deviation in the range between about 2.0 (au). The activity of the experimental group and the control group was measured by the absorbance of the pHEMA hydrogels reacted with glucose oxidase.
상기 육안관찰결과 및 도 4를 참고하면, 음파처리 유무에 따라 상기 단량체 조성물 용액의 균질도 및 pHEMA계 수화겔 내에 함유된 글루코스 산화효소의 활성도가 현저한 차이를 보임을 알 수 있다. Referring to the visual observation results and FIG. 4, it can be seen that the homogeneity of the monomer composition solution and the activity of glucose oxidase contained in the pHEMA hydrogel are significantly different depending on the presence or absence of sonication.
실험예 2_디메틸아민의 함량 차이에 따른 균질도 및 글루코스 산화효소의 활성도 비교 Experimental Example 2 Comparison of Homogeneity and Glucose Oxidase Activity According to Contents of Dimethylamine
실험예 2에 사용된 실험군과 대조군들은 하기 표 2의 조건에 따라 제조되었다.Experimental groups and controls used in Experimental Example 2 were prepared according to the conditions of Table 2 below.
공정fair 공정조건Process conditions 성분ingredient 실험군(Control)Control group 대조군 1(조건 1)Control Group 1 (Condition 1) 대조군 2(조건 2)Control Group 2 (Condition 2) 대조군 3(조건 3)Control Group 3 (Condition 3)
S1S1 혼합시간: 10 분Mixing time: 10 minutes (A) 성분(A) component 4.8 mL4.8 mL 4.8 mL4.8 mL 4.8 mL4.8 mL 4.8 mL4.8 mL
(B) 성분(B) component 2.4 mL2.4 mL 1.5 mL1.5 mL 0.75 mL0.75 mL 0 mL0 mL
(C) 성분(C) component 0.3 mL0.3 mL 0.3 mL0.3 mL 0.3 mL0.3 mL 0.3 mL0.3 mL
(D) 성분(D) component 0.1g0.1g 0.1g0.1g 0.1g0.1g 0.1g0.1g
(E) 성분(E) component 3.25 mL3.25 mL 3.25 mL3.25 mL 3.25 mL3.25 mL 3.25 mL3.25 mL
(F) 성분(F) component 4.25 mL4.25 mL 4.25 mL4.25 mL 4.25 mL4.25 mL 4.25 mL4.25 mL
S2S2 혼합시간: 5분 Mixing time: 5 minutes 글루코스 산화효소Glucose oxidase 667 unit/mL667 unit / mL 667 unit/mL667 unit / mL 667 unit/mL667 unit / mL 667 unit/mL667 unit / mL
음파처리: 3분Sound Processing: 3 minutes 실시practice
S3S3 UV 조사시간: 3분UV irradiation time: 3 minutes
S4S4 에이징(aging) 조건: 3 시간 동안 4 ℃ 냉장고에서 숙성세척 조건: 4 ℃의 증류수로 6 번 세척한 후, 인산완충식염수로 12 시간 동안 세척Aging condition: Aged in a refrigerator at 4 ° C. for 3 hours Washing condition: 6 times with distilled water at 4 ° C., followed by 12 hours with phosphate buffered saline
본 출원의 발명자들이 실험군과 대조군들을 육안으로 관찰한 결과, 디메틸아민의 함량이 0 mL 초과인 실험군, 대조군 1, 대조군 2 은 투명한 반면에, 디메틸아민의 함량이 0 mL 인 대조군 3은 불투명하였다. The inventors of the present application visually observed the experimental group and the control group, the experimental group with a dimethylamine content of more than 0 mL, the control group 1, the control group 2 was transparent, while the control group 3 with a dimethylamine content of 0 mL was opaque.
하기 표 3에는 실험군, 대조군 1, 대조군 2, 대조군 3 각각의 글루코스 산화효소의 함량 측정 결과가 정리되어 있다. 글루코스 산화효소의 함량은 메가자임법(megazyme method)를 이용하여 측정되었다. Table 3 below summarizes the results of measuring the content of glucose oxidase in each of the experimental group, control group 1, control group 2 and control group 3. The content of glucose oxidase was measured using the megazyme method.
실험군 (Control)(Unit/gram)Experiment Group (Control) (Unit / gram) 대조군 1 (조건 1)(Unit/gram)Control Group 1 (Condition 1) (Unit / gram) 대조군 2 (조건 2)(Unit/gram)Control 2 (condition 2) (Unit / gram) 대조군 3 (조건 3)(Unit/gram)Control Group 3 (Condition 3) (Unit / gram)
0.970.97 0.770.77 0.430.43 0.650.65
0.840.84 0.750.75 0.500.50 0.730.73
0.820.82 0.930.93 0.430.43 0.650.65
0.870.87 0.920.92 0.480.48 0.710.71
0.980.98 0.750.75 0.490.49 0.710.71
평균: 0.90Average: 0.90 평균: 0.82Average: 0.82 평균: 0.47Average: 0.47 평균: 0.69Average: 0.69
상기 표 3을 참고하면, 글루코스 산화효소의 함량이 동일한 경우에도, 디메틸아민의 함량의 증가에 비례하여 글루코스 산화효소의 함량이 증가하였다. 즉, pHEMA계 수화겔의 중량의 증가에 비례하여 글루코스 산화효소의 함량이 증가하였다. 이로부터, pHEMA계 수화겔이 디메틸아민을 함유하는 경우, 글루코스 산화효소가 pHEMA계 수화겔 내에서 균질하게 분포함을 알 수 있었다. 디메틸아민을 함유하지 않는 대조군 3의 경우, 글루코스 산화효소의 함량이 대조군 3에 비해 높은 것으로 나타났으나, 상기 육안관찰결과에서와 같이, 대조군 3은 불투명하게 혼합되어 수화겔로서는 조건이 부적합하였고, 글루코스 산화효소의 함량 활성도 계산을 위해서는, 흡광도, 무게, 추출 부피(Extracted volume)이 필요한데, 대조군 3은 실험군, 대조군 1, 대조군 2에 비해 팽윤이 덜 되었고, 흡광도, 무게, 추출 부피(Extracted volume) 값이 실험군, 대조군 1, 대조군 2에 비해 1/3 이상 작았다. 이러한 점들을 종합적으로 고려해보면, 대조군 3은 글루코스 산화효소들이 불균일하게 분포되어 있는 것으로 예상된다. Referring to Table 3, even when the content of glucose oxidase is the same, the content of glucose oxidase increased in proportion to the increase in the content of dimethylamine. That is, the content of glucose oxidase increased in proportion to the increase in the weight of the pHEMA hydrogel. From this, it was found that when the pHEMA hydrogel contains dimethylamine, the glucose oxidase was distributed homogeneously in the pHEMA hydrogel. In the case of the control group 3 containing no dimethylamine, the content of glucose oxidase was higher than that of the control group 3, but as shown in the visual observation, the control group 3 was mixed in an opaque manner, and the conditions were not suitable for the hydrogel, and the glucose To calculate the oxidase content activity, absorbance, weight, and extracted volume are required.Control 3 had less swelling compared to experimental group, control group 1, and control group 2. It was 1/3 or more smaller than this experimental group, the control group 1, and the control group 2. Taking these points together, control 3 is expected to have a heterogeneous distribution of glucose oxidases.
도 5는 pHEMA계 수화겔의 무게와 활성도의 관계 그래프이다. 도 5에서, 대조군은 흡광도 변화를 측정하기 위해 사용된 블랭크(blank) 용액이고, 실험군 1, 실험군 2, 실험군 3은 하기 표 4의 조건으로 제조된 pHEMA계 수화겔을 0.05g, 0.1g, 0.15g 의 무게로 채취한 시료들이다.Figure 5 is a graph of the relationship between the weight and activity of the pHEMA hydrogel. In Figure 5, the control is a blank (blank) solution used to measure the change in absorbance, Experimental Group 1, Experimental Group 2, Experimental Group 3 is 0.05g, 0.1g, 0.15g pHEMA-based hydrogel prepared under the conditions of Table 4 Samples taken at the weight of
공정fair 공정조건Process conditions 성분ingredient 함량content
S1S1 혼합시간: 10 분Mixing time: 10 minutes (A) 성분(A) component 4.8 mL4.8 mL
(B) 성분(B) component 2.4 mL2.4 mL
(C) 성분(C) component 0.3 mL0.3 mL
(D) 성분(D) component 0.1g0.1g
(E) 성분(E) component 3.25 mL3.25 mL
(F) 성분(F) component 4.25 mL4.25 mL
S2S2 혼합시간: 5분 Mixing time: 5 minutes 글루코스 산화효소Glucose oxidase 667 unit/mL667 unit / mL
음파처리: 3분Sound Processing: 3 minutes 실시practice
S3S3 UV 조사시간: 3분UV irradiation time: 3 minutes
S4S4 에이징(aging) 조건: 3 시간 동안 4 ℃ 냉장고에서 숙성세척 조건: 4 ℃의 증류수로 6 번 세척한 후, 인산완충식염수로 12 시간 동안 세척Aging condition: Aged in a refrigerator at 4 ° C. for 3 hours Washing condition: 6 times with distilled water at 4 ° C., followed by 12 hours with phosphate buffered saline
하기 표 5에는 실험결과가 정리되어 있다. Table 5 summarizes the experimental results.
대조군 흡광도(AU)Control Absorbance (AU) 실험군 1흡광도(AU) Experimental group 1 absorbance (AU) 실험군 2흡광도(AU) Experimental group 2 absorbance (AU) 실험군 3흡광도(AU)Experimental group 3 absorbance (AU)
글루코스 용액과 반응 전Before reaction with glucose solution 0.1060.106 0.2420.242 0.2220.222 0.2160.216
글루코스 용액과 20분 동안 반응 후 After reaction for 20 minutes with glucose solution 0.2480.248 0.5610.561 1.0951.095 1.5451.545
△O.D.ΔO.D. 0.1420.142 0.3190.319 0.8730.873 1.3291.329
도 5 및 표 5를 참고하면, pHEMA계 수화겔의 무게의 증가에 비례하여 글루코스 산화효소의 활성도가 증가하는 것을 확인할 수 있다. 이로부터 글루코스 산화효소가 pHEMA계 수화겔 내에 균질하게 분포되어 있음을 알 수 있었다.5 and Table 5, it can be seen that the activity of glucose oxidase increases in proportion to the increase in the weight of the pHEMA hydrogel. From this, it was found that glucose oxidase was homogeneously distributed in the pHEMA hydrogel.
이를 식으로 표현하면, 상기 pHEMA계 수화겔은 하기 식 1 및 2 를 만족한다:Expressed in this formula, the pHEMA-based hydrogel satisfies Equations 1 and 2:
(식 1) W1(a)〈 W2(a) (1) W1 ( a ) 〈 W2 ( a )
(식 2) CW1(a)〈 CW2(a) (Equation 2) CW1 ( a ) 〈 CW2 ( a )
상기 식 1 내지 식 2에서, a 는 글루코스 산화효소의 농도이고, W1(a) 는 글루코스 산화효소의 농도가 a 인 때, 제1 폴리히드록시에틸메타크릴레이트계 수화겔의 중량이며, W2(a) 는 글루코스 산화효소의 농도가 a 인 때, 제2 폴리히드록시에틸메타크릴레이트계 수화겔의 중량이고, CW1(a)는 제1 폴리히드록시에틸메타크릴레이트계 수화겔 내의 글루코스 산화효소의 활성도이며, CW2(a)는 제2 폴리히드록시에틸메타크릴레이트계 수화겔 내의 글루코스 산화효소의 활성도이다. In Formula 1 to Formula 2, a is the concentration of glucose oxidase, W1 (a) is the weight of the first polyhydroxyethyl methacrylate hydrogel when the concentration of glucose oxidase is a, W2 (a ) Is the weight of the second polyhydroxyethyl methacrylate hydrogel when the concentration of glucose oxidase is a, and CW1 (a) is the activity of the glucose oxidase in the first polyhydroxyethyl methacrylate hydrogel , CW2 (a) is the activity of glucose oxidase in the second polyhydroxyethyl methacrylate hydrogel.
상기한 제조예, 실험예 등은 발명의 이해를 돕기 위한 것으로서 발명의 기술적 사상이 상기한 실험시료의 제조예, 실험예 등으로만 제한되는 것은 아니다. 발명의 기술적 사상을 해치지 않는 범위 내에서의 균등물의 치환, 다른 구성요소의 부가, 삭제 등은 여전히 본 명세서 내에 합체되어 발명의 내용을 구성한다 할 것이다. The preparation examples, experimental examples and the like are intended to help the understanding of the invention, the technical spirit of the invention is not limited only to the preparation examples, experimental examples and the like of the above-described experimental sample. Substitution of equivalents, addition of other components, deletion, and the like within the scope of not impairing the technical idea of the present invention will still be incorporated in the present specification to constitute the present invention.
발명은 글루코스 산화효소가 균질하게 함유된 고분자 수화겔, 이의 제조방법 및 이를 포함하는 글루코스 센서를 제공할 수 있다.The present invention can provide a polymer hydrogel containing glucose oxidase homogeneously, a method for preparing the same, and a glucose sensor including the same.
본 발명에 따른 효과는 이상에서 예시된 내용에 의해 제한되지 않으며, 더욱 다양한 효과들이 본 명세서 내에 포함되어 있다.The effects according to the present invention are not limited by the contents exemplified above, and more various effects are included in the present specification.

Claims (6)

  1. 글루코스 산화효소를 포함하고 하기 식 1 및 2 를 만족하는 폴리히드록시에틸메타크릴레이트계 수화겔:Polyhydroxyethyl methacrylate hydrogel containing glucose oxidase and satisfying the following formulas 1 and 2:
    (식 1) W1(a)〈 W2(a) (1) W1 ( a ) 〈 W2 ( a )
    (식 2) CW1(a)〈 CW2(a) (Equation 2) CW1 ( a ) 〈 CW2 ( a )
    상기 식 1 내지 식 2에서, In Formula 1 to Formula 2,
    a 는 글루코스 산화효소의 농도이고, a is the concentration of glucose oxidase,
    W1(a) 는 글루코스 산화효소의 농도가 a 인 때, 제1 폴리히드록시에틸메타크릴레이트계 수화겔의 중량이며, W1 (a) is the weight of the first polyhydroxyethyl methacrylate hydrogel when the concentration of glucose oxidase is a,
    W2(a) 는 글루코스 산화효소의 농도가 a 인 때, 제2 폴리히드록시에틸메타크릴레이트계 수화겔의 중량이고, W2 (a) is the weight of the second polyhydroxyethyl methacrylate hydrogel when the concentration of glucose oxidase is a,
    CW1(a)는 제1 폴리히드록시에틸메타크릴레이트계 수화겔 내의 글루코스 산화효소의 활성도이며, CW1 (a) is the activity of glucose oxidase in the first polyhydroxyethyl methacrylate hydrogel,
    CW2(a)는 제2 폴리히드록시에틸메타크릴레이트계 수화겔 내의 글루코스 산화효소의 활성도이다. CW2 (a) is the activity of glucose oxidase in the second polyhydroxyethyl methacrylate hydrogel.
  2. 제1 항에 있어서, The method of claim 1,
    상기 글루코스 산화효소의 농도는 30 unit/mL 내지 667 unit/mL 인 폴리히드록시에틸메타크릴레이트계 수화겔.The concentration of glucose oxidase is 30 unit / mL to 667 unit / mL polyhydroxyethyl methacrylate hydrogel.
  3. 하기의 (A) 성분 내지 (F) 성분을 혼합하여 단량체 조성물 용액을 제조하는 단계;Preparing a monomer composition solution by mixing the following (A) to (F) components;
    상기 단량체 조성물 용액에 글루코스 산화효소를 혼합하고 음파처리(sonication)하여 균질 단량체 조성물 용액을 제조하는 단계; 및 Preparing a homogeneous monomer composition solution by mixing and sonicating glucose oxidase to the monomer composition solution; And
    상기 균질 단량체 조성물 용액에 자외선(UV)을 조사하여 하기 식 1 및 2를 만족하는 고분자 수화겔을 제조하는 단계;Irradiating ultraviolet (UV) to the homogeneous monomer composition solution to prepare a polymer hydrogel that satisfies Equations 1 and 2 below;
    를 포함하는 폴리히드록시에틸메타크릴레이트계 고분자 수화겔의 제조방법:Method for producing a polyhydroxyethyl methacrylate-based polymer hydrogel containing:
    (식 1) W1(a)〈 W2(a) (Equation 1) W1(a)〈 W2(a) 
    (식 2) CW1(a)〈 CW2(a) (Equation 2) CW1 ( a ) 〈 CW2 ( a )
    상기 식 1 내지 식 2에서, In Formula 1 to Formula 2,
    a 는 글루코스 산화효소의 농도이고, a is the concentration of glucose oxidase,
    W1(a) 는 글루코스 산화효소의 농도가 a 인 때, 제1 폴리히드록시에틸메타크릴레이트계 수화겔의 중량이며, W1 (a) is the weight of the first polyhydroxyethyl methacrylate hydrogel when the concentration of glucose oxidase is a,
    W2(a) 는 글루코스 산화효소의 농도가 a 인 때, 제2 폴리히드록시에틸메타크릴레이트계 수화겔의 중량이고, W2 (a) is the weight of the second polyhydroxyethyl methacrylate hydrogel when the concentration of glucose oxidase is a,
    CW1(a)는 제1 폴리히드록시에틸메타크릴레이트계 수화겔 내의 글루코스 산화효소의 활성도이며, CW1 (a) is the activity of glucose oxidase in the first polyhydroxyethyl methacrylate hydrogel,
    CW2(a)는 제2 폴리히드록시에틸메타크릴레이트계 수화겔 내의 글루코스 산화효소의 활성도이다. CW2 (a) is the activity of glucose oxidase in the second polyhydroxyethyl methacrylate hydrogel.
    (A) 성분: 히드록시에틸메타크릴레이트(A) component: hydroxyethyl methacrylate
    (B) 성분: 디메틸아민(B) Component: Dimethylamine
    (C) 성분: 트리에틸렌글리콜디메타크릴레이트(C) component: triethylene glycol dimethacrylate
    (D) 성분: 광중합 개시제(D) component: photoinitiator
    (E) 성분: 에틸렌 글리콜(E) Component: Ethylene Glycol
    (F) 성분: 물(F) Ingredient: Water
  4. 제3 항에 있어서, The method of claim 3, wherein
    상기 글루코스 산화효소의 농도는 30 unit/mL 내지 667 unit/mL 인 폴리히드록시에틸메타크릴레이트계 수화겔의 제조방법.The concentration of the glucose oxidase is 30 unit / mL to 667 unit / mL polyhydroxyethyl methacrylate hydrogel manufacturing method.
  5. 베이스 필름;Base film;
    공간적으로 분리된 상기 베이스 필름 상의 제1 영역과 제2 영역에 각각 배치된 캐소드(cathode)와 애노드(anode)로 구성된 전극부; 및An electrode portion including a cathode and an anode disposed in a first region and a second region on the spatially separated base film; And
    제1 항 또는 제2 항에 따른 폴리히드록시에틸메타크릴레이트계 수화물로 구성되고 상기 전극부 상에 배치된 효소 고정막;An enzyme fixation membrane composed of the polyhydroxyethyl methacrylate hydrate according to claim 1 and disposed on the electrode portion;
    을 포함하는 글루코스 센서.Glucose sensor comprising a.
  6. 제5 항에 있어서, The method of claim 5,
    상기 캐소드는, 작업전극이 배치되는 제1-1 영역, 제1 추출전극이 배치되고 상기 제1-1 영역과 공간적으로 분리되어 상기 제1-1 영역을 둘러싸고 있는 제1-2 영역, 기준전극과 상대전극이 배치되고 상기 제1-2 영역과 공간적으로 분리되어 상기 제1-2 영역을 둘러싸고 있는 제1-3 영역을 포함하고, 상기 기준전극과 상기 상대전극은 상기 제1-3 영역 내에서 상호 이격 배치되며, 상기 애노드는 제2 추출 전극으로 구성된 글루코스 센서.The cathode may include a first-first region where a working electrode is disposed, a first-second region where a first extraction electrode is disposed and spatially separated from the first-first region to surround the first-first region, and a reference electrode. And a counter electrode disposed in and spaced apart from the first region and surrounding the first region, wherein the reference electrode and the counter electrode are within the region 1-3. Are spaced apart from each other, and the anode comprises a second extraction electrode.
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