WO2023085617A1 - Structure comprising anthracenediboronic acid-based compound for detecting glucose, preparation method therefor, and use thereof - Google Patents

Structure comprising anthracenediboronic acid-based compound for detecting glucose, preparation method therefor, and use thereof Download PDF

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WO2023085617A1
WO2023085617A1 PCT/KR2022/015534 KR2022015534W WO2023085617A1 WO 2023085617 A1 WO2023085617 A1 WO 2023085617A1 KR 2022015534 W KR2022015534 W KR 2022015534W WO 2023085617 A1 WO2023085617 A1 WO 2023085617A1
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glucose
acid
electrode
group
based compound
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PCT/KR2022/015534
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French (fr)
Korean (ko)
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김중현
임흥섭
최홍식
박철순
송인혁
이찬희
김훈주
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재단법인 대구경북첨단의료산업진흥재단
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Priority claimed from KR1020220088272A external-priority patent/KR20230071048A/en
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Publication of WO2023085617A1 publication Critical patent/WO2023085617A1/en

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    • 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
    • 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 structure for detecting glucose by an electrochemical method, a method for preparing the same, and a use thereof.
  • Existing personal blood glucose measurement systems mainly use glucose oxidase to measure an increased current according to electrons generated when glucose is oxidized.
  • An enzyme-based electrochemical continuous blood glucose measurement system is used for more effective blood sugar management due to individual differences and fluctuations in blood glucose levels, but it is difficult to popularize it due to high cost and short period of use (less than 2 weeks).
  • the continuous blood glucose measurement system that optically measures blood glucose using diboronic acid can be used for up to 180 days.
  • the electrochemical measurement system is not only smaller and easier to manufacture than an optical system, but also has the advantage of being able to be used for a long time. As a result of studying a technology that can measure glucose by an electrochemical method using diboronic acid, completed the present invention.
  • the present inventors confirmed that the resistance of the electrode increases as the glucose concentration increases by immobilizing one boronic acid on the surface of the electrode, and as the diboronic acid binds to glucose, the three-dimensional electrical transmission material can be more easily transferred to the electrode.
  • a technology capable of detecting glucose was derived by using the fact that the resistance of the electrode surface decreases as the glucose concentration increases as the structure is modified.
  • an object of the present invention is an electrode; and a diboronic acid anthracene-based compound bonded to the surface of the electrode through a linker, to provide a structure for detecting glucose.
  • Another object of the present invention is to provide a method for preparing the structure for detecting glucose.
  • Another object of the present invention is to provide a use of the structure for detecting glucose.
  • the present invention is an electrode; and a diboronic acid anthracene-based compound bonded to the surface of the electrode through a linker.
  • the present invention (a) reacting by adding an acid to the alcohol solution; (b) washing the electrode with the reactant of (a); (c) reacting by adding a mixture of a diboronic acid anthracene-based compound and a coupling agent to the washed electrode; and (d) washing and drying the electrode after the reaction of (c).
  • the present invention provides a kit for detecting glucose comprising the structure for detecting glucose.
  • the present invention provides a glucose detection method comprising the step of reacting the glucose detection structure with a specimen.
  • the diboronic acid anthracene-based compound is bonded to the electrode surface as a linker, and when the diboronic acid anthracene-based compound reacts with glucose present in the specimen, the resistance of the electrode surface decreases depending on the glucose concentration, and the reaction time It is fast, can improve reversibility and repeatability, can be used for a long time compared to an enzyme-type blood glucose measurement sensor, and has excellent effects that are easier to manufacture than an optical blood glucose measurement sensor.
  • 1 is a view showing a synthetic route of diboronic acid anthracene-based compounds according to an embodiment of the present invention.
  • Figure 2 shows a process of immobilizing the diboronic acid anthracene-based compound on the electrode according to an embodiment of the present invention.
  • FIG 3 is a graph obtained through Cyclic Voltametry (CV) for each glucose concentration using a structure for detecting glucose according to an embodiment of the present invention.
  • FIG. 4 is a graph showing the maximum current value for each glucose concentration measured using the structure for detecting glucose according to an embodiment of the present invention.
  • NP Nyquist Plot
  • FIG. 6 is a graph showing a resistance change ratio for each glucose concentration measured using a structure for detecting glucose according to an embodiment of the present invention.
  • FIG. 7 is a diagram showing structural changes in diboronic acid anthracene-based compounds in which diboronic acid gathers towards anthracene by glucose binding according to an embodiment of the present invention.
  • the present invention provides an electrode; and a diboronic acid anthracene-based compound bonded to the surface of the electrode through a linker.
  • the electrode may be one in which a diboronic acid anthrancene-based compound is bonded to the surface of the electrode by a linker.
  • the diboronic acid anthracene-based compound may be a compound represented by Formula 1 below, but is not limited thereto:
  • the linker may be at least one selected from the group consisting of a hydroxyl group, an ester group, an ether group, a carbonyl group, an amine group, an amide group, a cyano group, a thiol group, a thioester group, and a thioether group. , but is not limited thereto.
  • the diboronic acid anthracene-based compound is the compound represented by Formula 1
  • the diboronic acid anthracene-based compound is formed in the electrode through an amide bond between an amino group of the compound and a carboxyl group present on the surface of the electrode. Can be bonded to and immobilized on a surface
  • the structure may further include a resistance measuring unit for measuring the resistance of the electrode surface.
  • the resistance measuring unit may measure the resistance of the electrode surface of the structure, and specifically, measure the change in resistance of the electrode surface when the diboronic acid anthracene-based compound of the structure reacts with glucose present in the specimen, More specifically, it may be to measure the degree of change in the reduced electrode surface resistance while the diboronic acid anthracene-based compound of the structure binds to the diol of glucose present in the sample.
  • the structure may further include an analysis unit for quantifying the content of glucose present in the specimen from the resistance measured by the resistance measurement unit.
  • a diboronic acid anthracene-based compound is bonded to the surface of an electrode by a linker, and when the diboronic acid anthracene-based compound reacts with glucose present in a specimen, an electron mediator ( electron mediator) is more easily accessible to the electrode surface, and the resistance of the electrode surface decreases in a glucose concentration-dependent manner, thereby confirming that glucose in the sample can be detected and specifically quantified.
  • the present invention comprises the steps of (a) immersing an electrode in an acid solution to immobilize an acid on the electrode; (b) reacting by adding a mixture of a diboronic acid anthracene-based compound and a coupling agent to the acid-immobilized electrode; and (c) washing and drying the electrode after the reaction of (b).
  • the electrode, the diboronic acid anthracene-based compound, and the structure for detecting glucose are as described above.
  • the acid may be at least one selected from the group consisting of mercaptobenzoic acid, mercaptohexanol, mercaptodecanoic acid, mercaptodecanol, thioglycolic acid, and mercaptopropionic acid, but is not limited thereto. .
  • the solvent in the acid solution may be an organic solvent such as alcohol, dimethyl sulfoxide (DMSO), or dimethyl formamide (DMF), and when the acid is water-soluble, water It can also be used as a solvent.
  • organic solvent such as alcohol, dimethyl sulfoxide (DMSO), or dimethyl formamide (DMF)
  • DMSO dimethyl sulfoxide
  • DMF dimethyl formamide
  • the reaction time in step (a) may be 10 minutes to 24 hours, specifically, 10 minutes or more, 20 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, 4 hours or more It can be more than 6 hours, more than 8 hours, more than 10 hours, more than 12 hours, more than 14 hours, more than 16 hours, more than 18 hours, more than 20 hours, or more than 22 hours, less than 24 hours, less than 22 hours, less than 20 hours.
  • the coupling agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) (1- (3-dimethylaminopropyl) -3-ethylcarbodimide, EDC) and N-hydroxysulfosuk Shinimide (N-hydroxysulfosuccinide, NHS) may be included.
  • the reaction time in step (b) may be 1 to 24 hours, specifically 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 8 hours or more, 10 hours or more , 12 hours or more, 14 hours or more, 16 hours or more, 18 hours or more, 20 hours or more, or 22 hours or less, 24 hours or less, 22 hours or less, 20 hours or less, 18 hours or less, 16 hours or less, 14 hours or less , 12 hours or less, 10 hours or less, 8 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, or 2 hours or less, but is not limited thereto.
  • the drying step may be drying with gas, and the gas may be drying with one or more selected from the group consisting of an inert gas.
  • the present invention provides a kit for detecting glucose comprising the structure for detecting glucose.
  • the description of the structure for detecting glucose may also be applied to a kit for detecting glucose.
  • the present invention provides a method for detecting glucose comprising reacting the structure for detecting glucose with a specimen.
  • the description of the structure for detecting glucose can also be applied to a method for detecting glucose.
  • the detection method may be to detect glucose to a degree of change in electrode surface resistance reduced while the diboronic acid anthracene-based compound binds to a diol of glucose, but is not limited thereto, and the detection method and Resistance change measurement may use a method commonly used in the art.
  • Acetylated diboronic anthracene containing two primary amines was synthesized according to the synthetic route shown in FIG. 1 .
  • the detailed synthesis procedure is as follows.
  • reaction mixture was extracted with methylene chloride (600 mL). The organic layer of the mixture was separated and dried over anhydrous magnesium sulfate. The dried compound was stirred in 500 mL of hexane for 0.5 h until a precipitate formed. After filtering off the precipitate, an oily compound 1 (21.5 g, 89.9%) was obtained and purified by silica gel chromatography using dichloromethane/hexane (1:8, v/v) as an eluent.
  • 9,10-Dimethylanthracene (20.0 g, 0.1 mol) was dissolved in methylene chloride (600 mL). Acetyl chloride (0.4g, 0.12mol) and aluminum chloride (18.8g, 0.149mol) were added to the mixture and stirred at 0-5°C. After stirring for an additional 5 hours and refluxing for 1 hour, the reaction mixture was allowed to warm to room temperature. 1 kg of ice and 50 mL of hydrochloric acid were added to the reaction mixture. The mixture was extracted with methylene chloride and water.
  • the electrode After reacting with 1 mM mercaptobenzoic acid (MBA) in ethanol for 10 minutes, the electrode (220BT, Metrohm) was washed with ethanol. Then, in 0.1x PBS, 2 mM diboronic acid anthranthene compound (Compound 7 above), 100 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) (1-(3-dimethylaminopropyl)- 3-ethylcarbodimide, EDC) and 150 mM N-hydroxysulfosuccinimide (N-hydroxysulfosuccinide, NHS) were dispensed in 20 ul to the working electrode and reacted for 1 hour. Thereafter, the electrode was washed with distilled water and dried with nitrogen gas to prepare a structure in which the diboronic acid anthracene-based compound was fixed on the surface of the electrode.
  • MBA mercaptobenzoic acid
  • Electrochemical Impedence Spectroscopy was performed for each glucose concentration of 0, 40, 100, 200, 300, 400, 500, and 600 mg/dL using the glucose detection structure prepared in the above example, and the results are It is shown as a Nyquist Plot (NP) of the electrode.
  • the size of the semicircle decreased as the glucose concentration increased.
  • the size of the semicircle that is, the resistance, decreased as the glucose concentration increased.
  • the structure according to the present invention has a fast reaction time when reacting with glucose present in a specimen, can improve reversibility and repeatability, can be used for a long time compared to an enzyme-type blood glucose measurement sensor, and is easier to manufacture than an optical blood glucose measurement sensor. .

Abstract

The present invention relates to a structure for detecting glucose by an electrochemical method, a preparation method therefor, and use thereof. In the structure according to the present invention, an anthracenediboronic acid-based compound is bonded to an electrode surface via a linker, and thus when the anthracenediboronic acid-based compound reacts with glucose present in a specimen, resistance at the electrode surface is reduced in a glucose concentration-dependent manner, exhibiting excellent effects in that the reaction time is quick, reversibility and repeatability can be improved, it can be used for a long time as compared to an enzyme-based glucose measurement sensor, and manufacturing is easy compared to an optical-type glucose measurement sensor.

Description

디보론산 안트라센계 화합물을 포함하는 포도당 검출용 구조체, 이의 제조방법 및 용도Structure for detecting glucose containing diboronic acid anthracene-based compound, manufacturing method and use thereof
본 발명은 전기화학적인 방법으로 포도당을 검출하기 위한 구조체, 그 제조방법 및 용도에 관한 것이다.The present invention relates to a structure for detecting glucose by an electrochemical method, a method for preparing the same, and a use thereof.
기존의 개인 혈당 측정 시스템은 포도당 산화 효소를 이용하여 포도당이 산화되면서 발생되는 전자에 따라 증가된 전류를 측정하는 방식이 주를 이루고 있다. 개인별 차이 및 혈당 수치의 변동으로 더 효과적인 혈당관리를 위해 효소기반의 전기화학적 연속혈당측정시스템을 이용하고 있으나 높은 비용과 짧은 (2주미만) 사용기간으로 대중화가 어려운 실정이다. Existing personal blood glucose measurement systems mainly use glucose oxidase to measure an increased current according to electrons generated when glucose is oxidized. An enzyme-based electrochemical continuous blood glucose measurement system is used for more effective blood sugar management due to individual differences and fluctuations in blood glucose levels, but it is difficult to popularize it due to high cost and short period of use (less than 2 weeks).
이에 반하여 디보론산을 이용한 광학적으로 혈당을 측정하는 연속혈당 측정 시스템은 최대 180일 까지 이용이 가능하다. 전기화학적 측정 시스템은 광학적 시스템보다 소형화와 제작이 용이할 뿐만 아니라 장시간 사용이 가능한 장점을 가지고 있어, 본 발명자들은 디보론산을 이용하여 포도당을 전기화학적인 방법으로 측정할 수 있는 기술을 연구한 결과, 본 발명을 완성하였다.In contrast, the continuous blood glucose measurement system that optically measures blood glucose using diboronic acid can be used for up to 180 days. The electrochemical measurement system is not only smaller and easier to manufacture than an optical system, but also has the advantage of being able to be used for a long time. As a result of studying a technology that can measure glucose by an electrochemical method using diboronic acid, completed the present invention.
본 발명자들은 1개의 보론산을 전극표면에 고정화시켜 포도당 농도가 증가함에 따라 전극의 저항이 증가함을 확인하고, 디보로산이 포도당과 결합하면서 입체적으로 전기전달 물질이 전극으로 더 쉽게 전달될 수 있는 구조로 변형됨으로써 전극표면의 저항이 감소하여 포도당 농도 증가에 따라 전극표면 저항이 감소하는 것을 이용하여 포도당을 검출할 수 있는 기술을 도출하였다.The present inventors confirmed that the resistance of the electrode increases as the glucose concentration increases by immobilizing one boronic acid on the surface of the electrode, and as the diboronic acid binds to glucose, the three-dimensional electrical transmission material can be more easily transferred to the electrode. A technology capable of detecting glucose was derived by using the fact that the resistance of the electrode surface decreases as the glucose concentration increases as the structure is modified.
이에, 본 발명의 목적은, 전극; 및 상기 전극 표면에 링커를 통해 결합되어 있는 디보론산 안트라센계 화합물을 포함하는, 포도당 검출용 구조체를 제공하는 것이다.Accordingly, an object of the present invention is an electrode; and a diboronic acid anthracene-based compound bonded to the surface of the electrode through a linker, to provide a structure for detecting glucose.
본 발명의 다른 목적은, 상기 포도당 검출용 구조체를 제조하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for preparing the structure for detecting glucose.
본 발명의 또 다른 목적은, 상기 포도당 검출용 구조체의 용도를 제공하는 것이다.Another object of the present invention is to provide a use of the structure for detecting glucose.
상기의 목적을 달성하기 위하여, 본 발명은 전극; 및 상기 전극 표면에 링커를 통해 결합되어 있는 디보론산 안트라센계 화합물을 포함하는, 포도당 검출용 구조체를 제공한다.In order to achieve the above object, the present invention is an electrode; and a diboronic acid anthracene-based compound bonded to the surface of the electrode through a linker.
또한, 본 발명은 (a) 알코올 용액에 산을 첨가하는 반응시키는 단계; (b) 상기 (a)의 반응물로 전극을 세척하는 단계; (c) 상기 세척된 전극에 디보론산 안트라센계 화합물 및 커플링제의 혼합물을 첨가하여 반응하는 단계; 및 (d) 상기 (c)의 반응 후 전극을 세척하고 건조하는 단계;를 포함하는 포도당 검출용 구조체의 제조방법을 제공한다.In addition, the present invention (a) reacting by adding an acid to the alcohol solution; (b) washing the electrode with the reactant of (a); (c) reacting by adding a mixture of a diboronic acid anthracene-based compound and a coupling agent to the washed electrode; and (d) washing and drying the electrode after the reaction of (c).
또한, 본 발명은 상기 포도당 검출용 구조체를 포함하는 포도당 검출용 키트를 제공한다.In addition, the present invention provides a kit for detecting glucose comprising the structure for detecting glucose.
또한, 본 발명은 상기 포도당 검출용 구조체를 검체와 반응시키는 단계를 포함하는 포도당의 검출방법을 제공한다.In addition, the present invention provides a glucose detection method comprising the step of reacting the glucose detection structure with a specimen.
본 발명에 따른 구조체는 전극 표면에 디보론산 안트라센계 화합물이 링커로 결합되어, 상기 디보론산 안트라센계 화합물이 검체에 존재하는 포도당과 반응 시 포도당 농도 의존적으로 전극 표면의 저항이 감소하는바, 반응시간이 빠르고 가역성과 반복성을 향상시킬 수 있고, 효소식 혈당 측정 센서에 비하여 장시간 사용이 가능하고, 광학식 혈당 측정 센서에 비해 제작이 용이한 우수한 효과가 있다.In the structure according to the present invention, the diboronic acid anthracene-based compound is bonded to the electrode surface as a linker, and when the diboronic acid anthracene-based compound reacts with glucose present in the specimen, the resistance of the electrode surface decreases depending on the glucose concentration, and the reaction time It is fast, can improve reversibility and repeatability, can be used for a long time compared to an enzyme-type blood glucose measurement sensor, and has excellent effects that are easier to manufacture than an optical blood glucose measurement sensor.
도 1은 본 발명의 일 실시예에 따른 디보론산 안트라센계 화합물의 합성 경로를 나타낸 도면이다. 1 is a view showing a synthetic route of diboronic acid anthracene-based compounds according to an embodiment of the present invention.
도 2는 본 발명의 일 실시예에 따른 전극에 디보론산 안트라센계 화합물을 고정화하는 과정을 나타낸 것이다.Figure 2 shows a process of immobilizing the diboronic acid anthracene-based compound on the electrode according to an embodiment of the present invention.
도 3은 본 발명의 일 실시예에 따른 포도당 검출용 구조체를 이용하여 포도당 농도별 순환 전압전류법(Cyclic Voltametry, CV)을 통해 얻은 그래프이다.3 is a graph obtained through Cyclic Voltametry (CV) for each glucose concentration using a structure for detecting glucose according to an embodiment of the present invention.
도 4는 본 발명의 일 실시예에 따른 포도당 검출용 구조체를 이용하여 측정된 포도당 농도별 최대 전류값을 나타낸 그래프이다.4 is a graph showing the maximum current value for each glucose concentration measured using the structure for detecting glucose according to an embodiment of the present invention.
도 5는 본 발명의 일 실시예에 따른 포도당 검출용 구조체를 이용하여 측정된 포도당 농도별 전극의 나이퀴스트 플롯(Nyquist Plot, NP)을 나타낸 것이다.5 shows a Nyquist Plot (NP) of an electrode for each glucose concentration measured using a structure for detecting glucose according to an embodiment of the present invention.
도 6은 본 발명의 일 실시예에 따른 포도당 검출용 구조체를 이용하여 측정된 포도당 농도별 저항 변화 비율을 나타낸 그래프이다.6 is a graph showing a resistance change ratio for each glucose concentration measured using a structure for detecting glucose according to an embodiment of the present invention.
도 7은 본 발명의 일 실시예에 따른 디보론산 안트라센계 화합물이 포도당 결합에 의해 디보론산이 안트라센 쪽으로 모이는 구조의 변화를 나타낸 도이다.7 is a diagram showing structural changes in diboronic acid anthracene-based compounds in which diboronic acid gathers towards anthracene by glucose binding according to an embodiment of the present invention.
이하, 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
일 측면에서, 본 발명은 전극; 및 상기 전극 표면에 링커를 통해 결합되어 있는 디보론산 안트라센계 화합물을 포함하는, 포도당 검출용 구조체를 제공한다.In one aspect, the present invention provides an electrode; and a diboronic acid anthracene-based compound bonded to the surface of the electrode through a linker.
본 발명의 일 측면에서, 상기 전극은 표면에 디보론산 안트란센계 화합물이 링커로 결합되어 있는 것일 수 있다.In one aspect of the present invention, the electrode may be one in which a diboronic acid anthrancene-based compound is bonded to the surface of the electrode by a linker.
본 발명의 일 측면에서, 상기 디보론산 안트라센계 화합물은 하기 화학식 1로 표시되는 화합물일 수 있으나, 이에 한정되는 것은 아니다:In one aspect of the present invention, the diboronic acid anthracene-based compound may be a compound represented by Formula 1 below, but is not limited thereto:
[화학식 1][Formula 1]
Figure PCTKR2022015534-appb-img-000001
Figure PCTKR2022015534-appb-img-000001
본 발명의 일 측면에서, 상기 링커는 히드록실기, 에스테르기, 에테르기, 카르보닐기, 아민기, 아미드기, 시아노기, 티올기, 티오에스테르기 및 티오에테르기로 이루어진 군에서 선택되는 하나 이상일 수 있으나, 이에 한정되는 것은 아니다. 본 발명의 일 실시예에 따르면, 상기 디보론산 안트라센계 화합물이 상기 화학식 1로 표시되는 화합물인 경우, 상기 화합물의 아미노기와 전극 표면에 존재하는 카르복실기의 아마이드 결합을 통해 상기 디보론산 안트라센계 화합물이 전극 표면에 결합되어 고정화될 수 있다In one aspect of the present invention, the linker may be at least one selected from the group consisting of a hydroxyl group, an ester group, an ether group, a carbonyl group, an amine group, an amide group, a cyano group, a thiol group, a thioester group, and a thioether group. , but is not limited thereto. According to one embodiment of the present invention, when the diboronic acid anthracene-based compound is the compound represented by Formula 1, the diboronic acid anthracene-based compound is formed in the electrode through an amide bond between an amino group of the compound and a carboxyl group present on the surface of the electrode. Can be bonded to and immobilized on a surface
본 발명의 일 측면에서, 상기 구조체는 상기 전극 표면의 저항을 측정하는 저항 측정부를 추가로 포함할 수 있다. 상기 저항 측정부는 상기 구조체의 전극 표면의 저항을 측정하는 것일 수 있고, 구체적으로, 상기 구조체의 디보론산 안트라센계 화합물이 검체 내 존재하는 포도당과 반응 시 전극 표면의 저항 변화를 측정하는 것일 수 있으며, 보다 구체적으로 상기 구조체의 디보론산 안트라센계 화합물이 검체 내 존재하는 포도당의 디올과 결합하면서 감소된 전극 표면 저항의 변화 정도를 측정하는 것일 수 있다.In one aspect of the present invention, the structure may further include a resistance measuring unit for measuring the resistance of the electrode surface. The resistance measuring unit may measure the resistance of the electrode surface of the structure, and specifically, measure the change in resistance of the electrode surface when the diboronic acid anthracene-based compound of the structure reacts with glucose present in the specimen, More specifically, it may be to measure the degree of change in the reduced electrode surface resistance while the diboronic acid anthracene-based compound of the structure binds to the diol of glucose present in the sample.
본 발명의 일 측면에서, 상기 구조체는 상기 저항 측정부에서 측정된 저항으로부터 상기 검체 내 존재하는 포도당의 함량을 정량화하는 분석부를 추가로 포함할 수 있다.In one aspect of the present invention, the structure may further include an analysis unit for quantifying the content of glucose present in the specimen from the resistance measured by the resistance measurement unit.
본 발명의 일 실시예에 따르면, 본 발명에 따른 포도당 검출용 구조체는 전극 표면에 디보론산 안트라센계 화합물이 링커로 결합되어, 상기 디보론산 안트라센계 화합물이 검체에 존재하는 포도당과 반응 시 전자 매개체(electron mediator)가 전극 표면으로 더 쉽게 접근이 가능하여 포도당 농도 의존적으로 전극 표면의 저항이 감소하여, 이로부터 검체 내 포도당을 검출, 구체적으로 포도당 함량을 정량화할 수 있음을 확인하였다.According to one embodiment of the present invention, in the structure for detecting glucose according to the present invention, a diboronic acid anthracene-based compound is bonded to the surface of an electrode by a linker, and when the diboronic acid anthracene-based compound reacts with glucose present in a specimen, an electron mediator ( electron mediator) is more easily accessible to the electrode surface, and the resistance of the electrode surface decreases in a glucose concentration-dependent manner, thereby confirming that glucose in the sample can be detected and specifically quantified.
다른 측면에서, 본 발명은 (a) 전극을 산 용액에 침지하여 상기 전극에 산을 고정화하는 단계; (b) 상기 산이 고정화된 전극에 디보론산 안트라센계 화합물 및 커플링제의 혼합물을 첨가하여 반응하는 단계; 및 (c) 상기 (b)의 반응 후 전극을 세척하고 건조하는 단계;를 포함하는 포도당 검출용 구조체의 제조방법을 제공한다. 상기 전극, 디보론산 안트라센계 화합물, 포도당 검출용 구조체에 대한 설명은 상술한 바와 같다.In another aspect, the present invention comprises the steps of (a) immersing an electrode in an acid solution to immobilize an acid on the electrode; (b) reacting by adding a mixture of a diboronic acid anthracene-based compound and a coupling agent to the acid-immobilized electrode; and (c) washing and drying the electrode after the reaction of (b). Descriptions of the electrode, the diboronic acid anthracene-based compound, and the structure for detecting glucose are as described above.
본 발명의 일 측면에서, 상기 산은 머캅토벤조산, 머캅토헥산올, 머캅토운데칸산, 머캅토운데칸올, 티오글리콜산, 및 머캅토프로피온산으로 이루어진 군으로부터 선택된 하나 이상일 수 있으나, 이에 제한되지 않는다.In one aspect of the present invention, the acid may be at least one selected from the group consisting of mercaptobenzoic acid, mercaptohexanol, mercaptodecanoic acid, mercaptodecanol, thioglycolic acid, and mercaptopropionic acid, but is not limited thereto. .
본 발명의 일 측면에서, 상기 산 용액 내 용매는 알코올, 디메틸설폭사이드(dimethyl sulfoxide, DMSO), 디메틸포름아미드(dimethyl formamide, DMF) 등의 유기 용매일 수 있으며, 상기 산이 수용성인 경우에는 물을 용매로도 사용할 수 있다. In one aspect of the present invention, the solvent in the acid solution may be an organic solvent such as alcohol, dimethyl sulfoxide (DMSO), or dimethyl formamide (DMF), and when the acid is water-soluble, water It can also be used as a solvent.
본 발명의 일 측면에서, 상기 (a) 단계에서의 반응 시간은 10분 내지 24시간일 수 있고, 구체적으로, 10 분 이상, 20 분 이상, 30 분 이상, 1 시간 이상, 2 시간 이상, 4 시간 이상, 6 시간 이상, 8 시간 이상, 10 시간 이상, 12 시간 이상, 14 시간 이상, 16 시간 이상, 18 시간 이상, 20 시간 이상 또는 22 시간 이상일 수 있고, 24 시간 이하, 22 시간 이하, 20 시간 이하, 18 시간 이하, 16 시간 이하, 14 시간 이하, 12 시간 이하, 10 시간 이하, 8 시간 이하, 6 시간 이하, 4 시간 이하, 2 시간 이하, 1 시간 이하, 50 분 이하, 40 분 이하, 30 분 이하, 20 분 이하 또는 10 분 이하일 수 있으나, 이에 제한되지 않는다.In one aspect of the present invention, the reaction time in step (a) may be 10 minutes to 24 hours, specifically, 10 minutes or more, 20 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, 4 hours or more It can be more than 6 hours, more than 8 hours, more than 10 hours, more than 12 hours, more than 14 hours, more than 16 hours, more than 18 hours, more than 20 hours, or more than 22 hours, less than 24 hours, less than 22 hours, less than 20 hours. 18 hours or less, 16 hours or less, 14 hours or less, 12 hours or less, 10 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, 1 hour or less, 50 minutes or less, 40 minutes or less , 30 minutes or less, 20 minutes or less, or 10 minutes or less, but is not limited thereto.
본 발명의 일 측면에서, 상기 커플링제는 1-(3-디메틸아미노프로필)-3-에틸카르보디미드)(1-(3-dimethylaminopropyl)-3-ethylcarbodimide, EDC) 및 N-히드록시설포숙신이미드(N-hydroxysulfosuccinide, NHS)를 포함할 수 있다.In one aspect of the present invention, the coupling agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) (1- (3-dimethylaminopropyl) -3-ethylcarbodimide, EDC) and N-hydroxysulfosuk Shinimide (N-hydroxysulfosuccinide, NHS) may be included.
본 발명의 일 측면에서, 상기 (b) 단계에서의 반응 시간은 1 내지 24 시간일 수 있고, 구체적으로 1 시간 이상, 2 시간 이상, 4 시간 이상, 6 시간 이상, 8 시간 이상, 10 시간 이상, 12 시간 이상, 14 시간 이상, 16 시간 이상, 18 시간 이상, 20 시간 이상 또는 22 시간 이상일 수 있고, 24 시간 이하, 22 시간 이하, 20 시간 이하, 18 시간 이하, 16 시간 이하, 14 시간 이하, 12 시간 이하, 10 시간 이하, 8 시간 이하, 6 시간 이하, 5 시간 이하, 4 시간 이하, 3 시간 이하 또는 2 시간 이하일 수 있으나, 이에 제한되지 않는다. In one aspect of the present invention, the reaction time in step (b) may be 1 to 24 hours, specifically 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 8 hours or more, 10 hours or more , 12 hours or more, 14 hours or more, 16 hours or more, 18 hours or more, 20 hours or more, or 22 hours or less, 24 hours or less, 22 hours or less, 20 hours or less, 18 hours or less, 16 hours or less, 14 hours or less , 12 hours or less, 10 hours or less, 8 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, or 2 hours or less, but is not limited thereto.
본 발명의 일 측면에서, 상기 건조 단계는 가스로 건조하는 것일 수 있고, 상기 가스는 비활성 기체로 이루어진 군으로부터 선택된 하나 이상으로 건조하는 것일 수 있다.In one aspect of the present invention, the drying step may be drying with gas, and the gas may be drying with one or more selected from the group consisting of an inert gas.
또 다른 측면에서, 본 발명은 상기 포도당 검출용 구조체를 포함하는 포도당 검출용 키트를 제공한다. 상기 포도당 검출용 구조체에 대한 설명은 포도당 검출용 키트에도 적용될 수 있다.In another aspect, the present invention provides a kit for detecting glucose comprising the structure for detecting glucose. The description of the structure for detecting glucose may also be applied to a kit for detecting glucose.
또 다른 측면에서, 본 발명은 상기 포도당 검출용 구조체를 검체와 반응시키는 단계를 포함하는 포도당의 검출방법을 제공한다. 상기 포도당 검출용 구조체에 대한 설명은 포도당 검출방법에도 적용될 수 있다.In another aspect, the present invention provides a method for detecting glucose comprising reacting the structure for detecting glucose with a specimen. The description of the structure for detecting glucose can also be applied to a method for detecting glucose.
본 발명의 일 측면에서, 상기 검출방법은 상기 디보론산 안트라센계 화합물이 포도당의 디올과 결합하면서 감소된 전극 표면 저항의 변화 정도로 포도당을 검출하는 것일 수 있으나, 이에 제한되는 것은 아니며, 상기 검출방법 및 저항 변화 측정은 당해 분야에서 통상적으로 사용하는 방법을 이용할 수 있다.In one aspect of the present invention, the detection method may be to detect glucose to a degree of change in electrode surface resistance reduced while the diboronic acid anthracene-based compound binds to a diol of glucose, but is not limited thereto, and the detection method and Resistance change measurement may use a method commonly used in the art.
이하, 본 발명을 실시예 및 실험예를 통하여 더욱 상세히 설명한다. 그러나, 하기 실시예 및 실험예는 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이에 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, the following examples and experimental examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.
<실시예> <Example>
1. 디보론산 안트라센계 화합물의 합성1. Synthesis of diboronic acid anthracene-based compounds
도 1에 도시한 합성 경로에 따라 2개의 1차 아민을 포함하는 아세틸화 디보론산 안트라센(acetylated diboronic anthracene)을 합성하였다. 상세한 합성 절차는 다음과 같다. Acetylated diboronic anthracene containing two primary amines was synthesized according to the synthetic route shown in FIG. 1 . The detailed synthesis procedure is as follows.
(1)4,4,5,5-tetramethyl-2-(2-methyl-4-(trifluoromethyl) phenyl)-1,3,2-dioxobor- olane (화합물 1)의 합성(1) Synthesis of 4,4,5,5-tetramethyl-2-(2-methyl-4-(trifluoromethyl) phenyl)-1,3,2-dioxobor- olane (Compound 1)
600mL의 디옥산 중에서 1-브로모-2-메틸-4-(트리플루오로메틸)벤젠 (20.00g, 0.084mol) 및 4,4,4',4',5,5,5',5'-옥타메틸-2,2'의 화합물 -비스(1,3,2-디옥소보롤란) (42.5g, 0.167mol), 아세트산칼륨 (32.85g, 0.335mol) 및 [1,1'비스(디페닐포스피노)페로센]디클로로팔라듐(II) (6.12g, 0.0084mol)을 100℃에서 1시간 동안 교반하였다. 용액을 실온으로 냉각시킨 후, DI water 600mL를 혼합물에 첨가하였다. 반응 혼합물을 메틸렌 클로라이드(600mL)를 사용하여 추출하였다. 혼합물의 유기층을 분리하고 무수 황산마그네슘으로 건조시켰다. 건조된 화합물을 침전이 형성될 때까지 500mL의 헥산에서 0.5시간 동안 교반하였다. 침전물을 여과 제거한 후 오일상 화합물 1 (21.5g, 89.9%)을 얻었고 디클로로메탄/헥산(1:8, v/v)을 용리액으로 하여 실리카겔 크로마토그래피로 정제하였다. 1-bromo-2-methyl-4-(trifluoromethyl)benzene (20.00 g, 0.084 mol) and 4,4,4',4',5,5,5',5' in 600 mL of dioxane -Octamethyl-2,2' compound -bis(1,3,2-dioxoborolane) (42.5g, 0.167mol), potassium acetate (32.85g, 0.335mol) and [1,1'bis(dioxoborolane) Phenylphosphino)ferrocene]dichloropalladium(II) (6.12 g, 0.0084 mol) was stirred at 100° C. for 1 hour. After cooling the solution to room temperature, 600 mL of DI water was added to the mixture. The reaction mixture was extracted with methylene chloride (600 mL). The organic layer of the mixture was separated and dried over anhydrous magnesium sulfate. The dried compound was stirred in 500 mL of hexane for 0.5 h until a precipitate formed. After filtering off the precipitate, an oily compound 1 (21.5 g, 89.9%) was obtained and purified by silica gel chromatography using dichloromethane/hexane (1:8, v/v) as an eluent.
[¹H NMR (400 MHz, Chloroform-d): δ 7.88 (d, 1H), 7.43-7.41 (m,2H), 2.61 (s, 3H), 1.38 (s. 12H).][¹H NMR (400 MHz, Chloroform-d): δ 7.88 (d, 1H), 7.43-7.41 (m, 2H), 2.61 (s, 3H), 1.38 (s. 12H).]
(2)2-(2-(bromomethyl)-4-(trifluoromethyl) phenyl)-4,4,5,5-tetramethyl-1,3,2- dioxoborolane (화합물 2)의 합성Synthesis of (2)2-(2-(bromomethyl)-4-(trifluoromethyl) phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxoborolane (Compound 2)
4,4,5,5-테트라메틸-2-(2-메틸-4-(트리플루오로메틸)페닐)-1,3,2-디옥소보롤란 (21.5g, 0.075mol) 및 N-브로모숙신이미드(NBS) (14.7g, 0.083mol)을 1,2-디클로로에탄 390mL에 용해시켰다. 2,2-아조비스이소부티로니트릴(AIBN) (1.23g, 0.075mol)을 혼합물에 첨가하고 80℃에서 3시간 동안 교반하였다. 반응 혼합물을 실온으로 냉각시킨 후 여과하였다. 감압하에 용매를 제거한 후, 혼합물을 헥산 200mL에서 0.5시간 동안 교반하였다. 여과된 침전물의 용매를 진공하에서 증발시켜 황색 오일상 화합물 2를 106%의 수율(29.00g)로 얻었다. 4,4,5,5-tetramethyl-2-(2-methyl-4-(trifluoromethyl)phenyl)-1,3,2-dioxoborolane (21.5 g, 0.075 mol) and N-bromide Mosuccinimide (NBS) (14.7 g, 0.083 mol) was dissolved in 390 mL of 1,2-dichloroethane. 2,2-Azobisisobutyronitrile (AIBN) (1.23 g, 0.075 mol) was added to the mixture and stirred at 80° C. for 3 hours. The reaction mixture was cooled to room temperature and then filtered. After removing the solvent under reduced pressure, the mixture was stirred in 200 mL of hexane for 0.5 hour. The solvent of the filtered precipitate was evaporated under vacuum to obtain a yellow oily compound 2 in 106% yield (29.00 g).
[¹H NMR (400 MHz, Chloroform-d): δ 7.93 (d, 1H), 7.63 (s, 1H), 7.52(d, 1H), 4.92 (s, 2H), 1.39(s, 12H).][¹H NMR (400 MHz, Chloroform-d): δ 7.93 (d, 1H), 7.63 (s, 1H), 7.52 (d, 1H), 4.92 (s, 2H), 1.39 (s, 12H).]
(3) 2-acetyl-9,10-dimethylanthracene (화합물 3)의 합성(3) Synthesis of 2-acetyl-9,10-dimethylanthracene (Compound 3)
9,10-디메틸안트라센 (20.0g, 0.1mol)을 메틸렌 클로라이드 (600mL)에 용해시켰다. 아세틸 클로라이드 (0.4g, 0.12mol) 및 알루미늄 클로라이드 (18.8g, 0.149mol)를 혼합물에 첨가하고 0~5℃에서 교반하였다. 추가로 5시간 동안 교반하고 1시간 동안 환류시킨 후, 반응 혼합물을 실온으로 가온시켰다. 반응 혼합물에 얼음 1kg과 염산 50mL를 첨가하였다. 혼합물을 메틸렌 클로라이드 및 물로 추출하였다. 감압하에 용매를 제거한 후 에틸아세테이트/헥산(1:10, v/v)을 용리액으로 하여 실리카겔 컬럼 크로마토그래피를 이용하여 생성물을 정제하여 황색 분말의 화합물 3을 56.5% 수율(14.0g)로 얻었다. 9,10-Dimethylanthracene (20.0 g, 0.1 mol) was dissolved in methylene chloride (600 mL). Acetyl chloride (0.4g, 0.12mol) and aluminum chloride (18.8g, 0.149mol) were added to the mixture and stirred at 0-5°C. After stirring for an additional 5 hours and refluxing for 1 hour, the reaction mixture was allowed to warm to room temperature. 1 kg of ice and 50 mL of hydrochloric acid were added to the reaction mixture. The mixture was extracted with methylene chloride and water. After removing the solvent under reduced pressure, the product was purified using silica gel column chromatography using ethyl acetate/hexane (1:10, v/v) as an eluent to obtain compound 3 as a yellow powder in a yield of 56.5% (14.0g).
[¹H NMR (400 MHz, Chloroform-d): δ 9.00 (m, 7H), 3.15(d, 6H), 2.80 (s, 3H)][¹H NMR (400 MHz, Chloroform-d): δ 9.00 (m, 7H), 3.15 (d, 6H), 2.80 (s, 3H)]
(4) 2-acetyl-9,10-bis(bromomethyl)anthracene (화합물 4)의 합성(4) Synthesis of 2-acetyl-9,10-bis(bromomethyl)anthracene (Compound 4)
2-아세틸-9,10-디메틸안트라센 (14.0g, 0.056mol)을 1,2-디클로로에탄 (300mL)에 용해시켰다. N-브로모숙신이미드 (22.1g, 0.124mol)를 첨가한 후, 반응액을 85℃에서 1시간 동안 교반하였다. 반응 혼합물을 실온으로 냉각시키고 용매를 감압 하에 증발시킨 다음 침전이 형성될 때까지 0.5시간 동안 150mL의 메탄올 중에서 교반하였다. 침전물을 여과하고, 메탄올로 세척하고, 40℃에서 12시간 동안 진공 건조시켜 밝은 노란색 분말의 화합물 4를 33.6%의 수율(7.7g)로 얻었다. 2-Acetyl-9,10-dimethylanthracene (14.0 g, 0.056 mol) was dissolved in 1,2-dichloroethane (300 mL). After adding N-bromosuccinimide (22.1 g, 0.124 mol), the reaction solution was stirred at 85° C. for 1 hour. The reaction mixture was cooled to room temperature, the solvent was evaporated under reduced pressure and stirred in 150 mL of methanol for 0.5 h until a precipitate formed. The precipitate was filtered, washed with methanol, and vacuum dried at 40° C. for 12 hours to obtain Compound 4 as a bright yellow powder in a yield of 33.6% (7.7g).
[¹H NMR (400 MHz, Chloroform-d): δ 9.00-7.50 (m, 7H), 5.50 (d, 4H), 2.80 (s, 3H).][¹H NMR (400 MHz, Chloroform-d): δ 9.00-7.50 (m, 7H), 5.50 (d, 4H), 2.80 (s, 3H).]
(5)di-tert-butyl ((((2-acetylanthracene-9,10-diyl) bis(methylene))bis(azanediyl)) bis(hexane-6,1-diyl))dicarbamate (화합물 5)의 합성(5) Synthesis of di-tert-butyl ((((2-acetylanthracene-9,10-diyl) bis(methylene))bis(azanediyl)) bis(hexane-6,1-diyl))dicarbamate (Compound 5)
화합물 4 (5.0g, 0.012mol), 터트-부틸(6-아미노헥실)카바메이트 (13.32g, 0.062mol), 부틸화 하이드록시-톨루엔(BHT) (0.25g, 0.001mol) 및 NN-디이소프로필에틸아민(DIPEA) (21.4 mL, 0.123mol)을 150mL의 클로로포름에 30°C에서 18시간 동안 용해시켰다. 용매를 감압 증발시킨 후 증류수(3회 x 200mL)로 세척하고 MgSO4로 건조시켰다. 얻어진 조 생성물을 디클로로메탄/메탄올(9:1, v/v)을 용리액으로 실리카겔 컬럼 크로마토그래피를 사용하여 정제해 63.6%의 수율(5.3g)로 황색 분말을 얻었다. Compound 4 (5.0 g, 0.012 mol), tert-butyl (6-aminohexyl) carbamate (13.32 g, 0.062 mol), butylated hydroxy-toluene (BHT) (0.25 g, 0.001 mol) and NN-diiso Propylethylamine (DIPEA) (21.4 mL, 0.123 mol) was dissolved in 150 mL of chloroform at 30 °C for 18 h. After evaporating the solvent under reduced pressure, the mixture was washed with distilled water (3 times x 200mL) and dried over MgSO 4 . The obtained crude product was purified using silica gel column chromatography with dichloromethane/methanol (9:1, v/v) as an eluent to obtain a yellow powder in a yield of 63.6% (5.3g).
[¹H NMR (400 MHz, Chloroform-d): δ 9.10-7.56(m, 7H), 4.77(s, 2H), 4.69(s, 2H), 4.64-4.52(m, 2H), 3.44-2.53(m, 11H), 1.61-1.35(s, 36H). LC MS: Calcd. for C40H60N4O5 m/z: 676.94; found m/z: 677 [M+H]+.][¹H NMR (400 MHz, Chloroform-d): δ 9.10-7.56 (m, 7H), 4.77 (s, 2H), 4.69 (s, 2H), 4.64-4.52 (m, 2H), 3.44-2.53 (m , 11H), 1.61-1.35 (s, 36H). LC MS: Calcd. for C40H60N4O5 m/z: 676.94; found m/z: 677 [M+H] + .]
(6)(((((2-acetylanthracene-9,10-diyl) bis(methylene))bis((6-((tert-butoxycarb- onyl) amino)hexyl)azanediyl)) bis(methylene))bis(4-(trifluoromethyl)-2,1-phenylene)) diboronic acid (화합물 6)의 합성(6)((((2-acetylanthracene-9,10-diyl) bis(methylene))bis((6-((tert-butoxycarb-onyl) amino)hexyl)azanediyl)) bis(methylene))bis( Synthesis of 4-(trifluoromethyl)-2,1-phenylene)) diboronic acid (Compound 6)
20mL의 클로로포름 중에서 화합물 5 (5.3g, 0.0078mol) 및 NN-디이소프로필에틸아민(DIPEA) (27.3mL, 0.156mol)을 실온에서 교반하였다. 부틸화 히드록시톨루엔(BHT) (0.3g, 0.001mol), 2-(2-(브로모메틸)-4-(트리플루오로메틸)페닐)-4,4,5,5-테트라메틸-1 및 3,2-디옥소보롤란 (14.29g, 0.039mol)을 반응 혼합물에 첨가한 다음, 실온에서 24시간 이상 동안 교반하였다. 감압하에 용매를 제거한 후 이소프로필에테르 20mL에 녹이고 인산 완충액(0.2M, pH 7.0) 20mL로 3회 세척하였다. 용매를 제거한 후, 디클로로메탄/메탄올(98:2, v/v)를 용리액으로 사용하여 실리카겔 컬럼 크로마토그래피를 사용하여 생성물을 정제하여 노란색 분말을 141.8%의 수율(12.0g)로 얻었다.Compound 5 (5.3 g, 0.0078 mol) and NN-diisopropylethylamine (DIPEA) (27.3 mL, 0.156 mol) were stirred at room temperature in 20 mL of chloroform. Butylated hydroxytoluene (BHT) (0.3 g, 0.001 mol), 2-(2-(bromomethyl)-4-(trifluoromethyl)phenyl)-4,4,5,5-tetramethyl-1 and 3,2-dioxoborolane (14.29 g, 0.039 mol) were added to the reaction mixture, which was then stirred at room temperature for at least 24 hours. After removing the solvent under reduced pressure, it was dissolved in 20 mL of isopropyl ether and washed three times with 20 mL of phosphate buffer (0.2M, pH 7.0). After removing the solvent, the product was purified using silica gel column chromatography using dichloromethane/methanol (98:2, v/v) as an eluent to obtain a yellow powder in a yield of 141.8% (12.0 g).
[¹H NMR (400 MHz, Chloroform-d): δ 9.12-7.58(m, 13H), 4.80(s, 2H), 4.72(m, 3H), 4.66(s, 2H), 4.58(m, 3H), 3.44-2.53(m, 15H), 1.61-1.35(m, 34H). LC-MS: Calcd. for C56H72B2F6N4O9 m/z: 1080.82; found m/z: 1081 [M+H]+; 1063 [M-H2O+H]+, 1045 [M-2H2O+H]+.][¹H NMR (400 MHz, Chloroform-d): δ 9.12-7.58 (m, 13H), 4.80 (s, 2H), 4.72 (m, 3H), 4.66 (s, 2H), 4.58 (m, 3H), 3.44-2.53 (m, 15H), 1.61-1.35 (m, 34H). LC-MS: Calcd. for C 56 H 72 B 2 F 6 N 4 O 9 m/z: 1080.82; found m/z: 1081 [M+H] + ; 1063 [MH 2 O+H] + , 1045 [M-2H 2 O+H] + .]
(7)(((((2-acetylanthracene-9,10-diyl) bis(methylene))bis((6-aminohexyl)azaned- iyl))bis(methylene))bis(4-(trifluoromethyl)-2,1-phenylene))diboronic acid (화합물 7)의 합성(7)((((2-acetylanthracene-9,10-diyl) bis(methylene))bis((6-aminohexyl)azaned-iyl))bis(methylene))bis(4-(trifluoromethyl)-2, Synthesis of 1-phenylene))diboronic acid (Compound 7)
20중량% 트리플루오로아세트산을 함유하는 메틸렌 클로라이드 5mL 중에서 화합물 6(3.0g, 0.005mol)을 실온에서 20시간 동안 교반하였다. 진공 하에 용매를 증발시킨 후, 생성물을 클로로포름/메탄올(95:5, v/v)을 용리액으로 실리카겔 컬럼 크로마토그래피를 사용하여 정제해 노란색 분말을 43.5%의 수율(0.074g)로 얻었다.Compound 6 (3.0 g, 0.005 mol) was stirred at room temperature for 20 hours in 5 mL of methylene chloride containing 20% trifluoroacetic acid by weight. After evaporating the solvent in vacuo, the product was purified using silica gel column chromatography with chloroform/methanol (95:5, v/v) as the eluent to obtain a yellow powder in a yield of 43.5% (0.074 g).
[¹H NMR (400 MHz, Chloroform-d): δ 9.21-7.51(m, 13H), 4.82(s, 2H), 4.74(s, 2H), 4.68(s, 2H), 4.60(s, 2H), 3.51-2.49(m, 15H), 1.55-1.30(m, 20H). LC-MS: Calcd. for C46H56B2F6N4O5 m/z: 880.59; found m/z: 881 [M+H]+; 863 [M-H2O+H]+, 845[M-2H2O+H]+.][¹H NMR (400 MHz, Chloroform-d): δ 9.21-7.51 (m, 13H), 4.82 (s, 2H), 4.74 (s, 2H), 4.68 (s, 2H), 4.60 (s, 2H), 3.51-2.49 (m, 15H), 1.55-1.30 (m, 20H). LC-MS: Calcd. for C 46 H 56 B 2 F 6 N 4 O 5 m/z: 880.59; found m/z: 881 [M+H] + ; 863 [MH 2 O+H] + , 845 [M-2H 2 O+H] + .]
2. 전극에 디보론산 안트라센계 화합물이 고정된 포도당 검출용 구조체 제조2. Preparation of structure for glucose detection with diboronic acid anthracene-based compound immobilized on electrode
에탄올에서 1mM의 머캅토벤조산(Mercaptobenzoic Acid, MBA)을 10 분동안 반응한 후, 에탄올로 전극(220BT, Metrohm)을 세척하였다. 그런 다음, 0.1x PBS에서 2mM의 디보론산 안트란센계 화합물(상기 화합물 7), 100 mM의 1-(3-디메틸아미노프로필)-3-에틸카르보디미드)(1-(3-dimethylaminopropyl)-3-ethylcarbodimide, EDC) 및 150 mM의 N-히드록시설포숙신이미드(N-hydroxysulfosuccinide, NHS)의 혼합물을 상기 전극의 작업 전극에 20 ul 분주한 후 1시간 동안 반응시켰다. 이후, 상기 전극을 증류수로 세척하고 질소 가스로 건조시켜, 전극 표면에 디보론산 안트라센계 화합물이 고정된 구조체를 제조하였다.After reacting with 1 mM mercaptobenzoic acid (MBA) in ethanol for 10 minutes, the electrode (220BT, Metrohm) was washed with ethanol. Then, in 0.1x PBS, 2 mM diboronic acid anthranthene compound (Compound 7 above), 100 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) (1-(3-dimethylaminopropyl)- 3-ethylcarbodimide, EDC) and 150 mM N-hydroxysulfosuccinimide (N-hydroxysulfosuccinide, NHS) were dispensed in 20 ul to the working electrode and reacted for 1 hour. Thereafter, the electrode was washed with distilled water and dried with nitrogen gas to prepare a structure in which the diboronic acid anthracene-based compound was fixed on the surface of the electrode.
<실험예 1> 포도당 검출용 구조체를 이용한 포도당 농도별 전류 전위곡선 도출<Experimental Example 1> Derivation of current potential curve for each glucose concentration using a structure for detecting glucose
상기 실시예에서 제조한 포도당 검출용 구조체를 이용하여 포도당 농도 0, 40, 100, 200, 300, 400, 500, 600 mg/dL 별로 순환 전압전류법(Cyclic Voltametry, CV)을 통해 전류 전위곡선을 얻었다.Using the glucose detection structure prepared in the above example, a current potential curve was obtained by cyclic voltammetry (CV) for each glucose concentration of 0, 40, 100, 200, 300, 400, 500, and 600 mg/dL. got it
구체적으로, 상기 구조체의 전극, 즉 DA가 고정화된 전극에 5mM [Fe(CN)6]4-/[Fe(CN)6]3- 0.1 M KCl이 포함된 0.1x PBS (pH 7.4)에 포도당 농도를 0에서부터 600 mg/dL까지 각 농도별로 1시간 동안 인큐베이션한 후 -0.3V~0.6V 범위에서 스캔 속도 50 mV/s로 순환 전압전류법(CV)을 수행하여, 전류 전위곡선을 얻었으며, 그 결과는 도 3에 나타내었다. 또한, 상기 전류 전위곡선(CV 그래프)의 포도당 농도별 최대 포텐셜(potential) 값을 계산하였으며, 그 결과는 도 4와 같다.Specifically, 5mM [Fe(CN) 6 ] 4- /[Fe(CN) 6 ] 3- Glucose in 0.1x PBS (pH 7.4) containing 0.1 M KCl was added to the electrode of the structure, that is, the electrode on which DA was immobilized. After incubation for 1 hour at each concentration from 0 to 600 mg/dL, cyclic voltammetry (CV) was performed at a scan rate of 50 mV/s in the range of -0.3V to 0.6V to obtain a current potential curve. , and the results are shown in FIG. 3 . In addition, the maximum potential value for each glucose concentration of the current potential curve (CV graph) was calculated, and the result is shown in FIG. 4 .
도 3 및 도 4에 나타난 바와 같이, 최대 Potential 값이 포도당 농도가 증가함에 따라 증가함을 확인하였다.As shown in Figures 3 and 4, it was confirmed that the maximum potential value increased as the glucose concentration increased.
<실험예 2> 포도당 검출용 구조체를 이용한 포도당 농도별 전기화학적 임피던스 분광법(Electrochemical Impedence Spectroscopy) 수행<Experimental Example 2> Conducting electrochemical impedance spectroscopy by glucose concentration using a structure for detecting glucose
상기 실시예에서 제조한 포도당 검출용 구조체를 이용하여 포도당 농도 0, 40, 100, 200, 300, 400, 500, 600 mg/dL 별로 전기화학적 임피던스 분광법(Electrochemical Impedence Spectroscopy)을 수행하였으며, 그 결과를 전극의 나이퀴스트 플롯(Nyquist Plot, NP)으로 나타내었다.Electrochemical Impedence Spectroscopy was performed for each glucose concentration of 0, 40, 100, 200, 300, 400, 500, and 600 mg/dL using the glucose detection structure prepared in the above example, and the results are It is shown as a Nyquist Plot (NP) of the electrode.
구체적으로, 상기 구조체의 전극, 즉 DA가 고정화된 전극에 5mM [Fe(CN)6]4-/[Fe(CN)6]3- 0.1 M KCl이 포함된 0.1x PBS (pH 7.4)에 포도당 농도를 0에서부터 600 mg/dL까지 각 농도별로 1시간 동안 인큐베이션한 후 10,000 Hz에서 0.1 Hz까지 frequency Number 50으로 측정하여, 나이퀴스트 플롯을 얻었으며, 그 결과는 도 5에 나타내었다. 또한, 상기 나이퀴스트 플롯의 반원의 반지름을 포도당 농도별로 나타내어 포도당 농도별 저항 변화 비율을 계산하였으며, 그 결과는 도 6과 같다.Specifically, 5mM [Fe(CN) 6 ] 4- /[Fe(CN) 6 ] 3- Glucose in 0.1x PBS (pH 7.4) containing 0.1 M KCl was added to the electrode of the structure, that is, the electrode on which DA was immobilized. The concentration was incubated for 1 hour for each concentration from 0 to 600 mg/dL, and then measured by frequency number 50 from 10,000 Hz to 0.1 Hz to obtain a Nyquist plot, and the results are shown in FIG. 5 . In addition, the radius of the semicircle of the Nyquist plot was shown for each glucose concentration to calculate the resistance change ratio for each glucose concentration, and the results are shown in FIG. 6 .
도 5에 나타난 바와 같이, 포도당 농도가 증가함에 따라 반원의 크기가 줄어들었다. 또한, 도 6에 나타난 바와 같이, 포도당 농도가 증가함에 따라 반원의 크기, 즉 저항이 감소함을 확인하였다.As shown in Figure 5, the size of the semicircle decreased as the glucose concentration increased. In addition, as shown in FIG. 6, it was confirmed that the size of the semicircle, that is, the resistance, decreased as the glucose concentration increased.
이상으로 본 발명의 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시예일뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다.As above, specific parts of the content of the present invention have been described in detail, and for those skilled in the art, these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. It will be clear.
따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다. 본 발명의 단순한 변형 내지 변경은 이 분야의 통상의 지식을 가진 자에 의하여 용이하게 이용될 수 있으며, 이러한 변형이나 변경은 모두 본 발명의 영역에 포함되는 것으로 볼 수 있다.Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents. Simple modifications or changes of the present invention can be easily used by those skilled in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.
본 발명에 따른 구조체는 검체에 존재하는 포도당과 반응 시 반응시간이 빠르고 가역성과 반복성을 향상시킬 수 있고, 효소식 혈당 측정 센서에 비하여 장시간 사용이 가능하고, 광학식 혈당 측정 센서에 비해 제작이 용이하다.The structure according to the present invention has a fast reaction time when reacting with glucose present in a specimen, can improve reversibility and repeatability, can be used for a long time compared to an enzyme-type blood glucose measurement sensor, and is easier to manufacture than an optical blood glucose measurement sensor. .

Claims (11)

  1. 전극; 및 상기 전극 표면에 링커를 통해 결합되어 있는 디보론산 안트라센계 화합물을 포함하는, 포도당 검출용 구조체.electrode; and a diboronic acid anthracene-based compound bonded to the surface of the electrode through a linker.
  2. 제1항에 있어서, 상기 디보론산 안트라센계 화합물은 하기 화학식 1로 표시되는 화합물인, 포도당 검출용 구조체:The structure for glucose detection according to claim 1, wherein the diboronic acid anthracene-based compound is a compound represented by Formula 1 below:
    [화학식 1][Formula 1]
    Figure PCTKR2022015534-appb-img-000002
    .
    Figure PCTKR2022015534-appb-img-000002
    .
  3. 제1항에 있어서, 상기 링커는 히드록실기, 에스테르기, 에테르기, 카르보닐기, 아민기, 아미드기, 시아노기, 티올기, 티오에스테르기 및 티오에테르기로 이루어진 군에서 선택되는 하나 이상인, 포도당 검출용 구조체.The method of claim 1, wherein the linker is at least one selected from the group consisting of a hydroxyl group, an ester group, an ether group, a carbonyl group, an amine group, an amide group, a cyano group, a thiol group, a thioester group, and a thioether group, glucose detection dragon structure.
  4. 제1항에 있어서, 상기 구조체는 상기 전극 표면의 저항을 측정하는 저항 측정부를 추가로 포함하는, 포도당 검출용 구조체.According to claim 1, wherein the structure further comprises a resistance measuring unit for measuring the resistance of the surface of the electrode, the structure for detecting glucose.
  5. (a) 전극을 산 용액에 침지하여 상기 전극에 산을 고정화하는 단계;(a) immersing the electrode in an acid solution to immobilize the acid on the electrode;
    (b) 상기 산이 고정화된 전극에 디보론산 안트라센계 화합물 및 커플링제의 혼합물을 첨가하여 반응하는 단계; 및(b) reacting by adding a mixture of a diboronic acid anthracene-based compound and a coupling agent to the acid-immobilized electrode; and
    (c) 상기 (b)의 반응 후 전극을 세척하고 건조하는 단계; (c) washing and drying the electrode after the reaction of (b);
    를 포함하는 제1항 내지 제4항 중 어느 한 항에 따른 포도당 검출용 구조체의 제조방법.A method of manufacturing a structure for detecting glucose according to any one of claims 1 to 4 comprising a.
  6. 제5항에 있어서, 상기 산은 머캅토벤조산, 머캅토헥산올, 머캅토운데칸산, 머캅토운데칸올, 티오글리콜산, 및 머캅토프로피온산으로 이루어진 군으로부터 선택된 하나 이상인, 제조방법.The method of claim 5, wherein the acid is at least one selected from the group consisting of mercaptobenzoic acid, mercaptohexanol, mercaptodecanoic acid, mercaptodecanol, thioglycolic acid, and mercaptopropionic acid.
  7. 제5항에 있어서, 상기 알코올은 메탄올, 에탄올, 프로판올 및 부탄올로 이루어진 군에서 선택되는 하나 이상인, 제조방법.The method of claim 5, wherein the alcohol is at least one selected from the group consisting of methanol, ethanol, propanol and butanol.
  8. 제5항에 있어서, 상기 커플링제는 1-(3-디메틸아미노프로필)-3-에틸카르보디미드)(1-(3-dimethylaminopropyl)-3-ethylcarbodimide, EDC) 및 N-히드록시설포숙신이미드(N-hydroxysulfosuccinide, NHS)를 포함하는, 제조방법.The method of claim 5, wherein the coupling agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) (1- (3-dimethylaminopropyl) -3-ethylcarbodimide, EDC) and N-hydroxysulfosuccine A manufacturing method comprising mid (N-hydroxysulfosuccinide, NHS).
  9. 제1항 내지 제4항 중 어느 한 항의 포도당 검출용 구조체를 포함하는 포도당 검출용 키트.A kit for detecting glucose comprising the structure for detecting glucose according to any one of claims 1 to 4.
  10. 제1항 내지 제4항 중 어느 한 항의 포도당 검출용 구조체를 검체와 반응시키는 단계를 포함하는 포도당의 검출방법.A method for detecting glucose comprising reacting the structure for detecting glucose according to any one of claims 1 to 4 with a specimen.
  11. 제10항에 있어서,According to claim 10,
    상기 검출방법은 상기 디보론산 안트라센계 화합물이 포도당의 디올과 결합하면서 감소된 전극 표면 저항의 변화 정도로 포도당을 검출하는 것인, 검출방법.Wherein the detection method detects glucose to a degree of change in electrode surface resistance reduced while the diboronic acid anthracene-based compound binds to a diol of glucose.
PCT/KR2022/015534 2021-11-15 2022-10-13 Structure comprising anthracenediboronic acid-based compound for detecting glucose, preparation method therefor, and use thereof WO2023085617A1 (en)

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