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WO2024250285A1 - Système d'électrode enzymatique et son utilisation - Google Patents

Système d'électrode enzymatique et son utilisation Download PDF

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Publication number
WO2024250285A1
WO2024250285A1 PCT/CN2023/099459 CN2023099459W WO2024250285A1 WO 2024250285 A1 WO2024250285 A1 WO 2024250285A1 CN 2023099459 W CN2023099459 W CN 2023099459W WO 2024250285 A1 WO2024250285 A1 WO 2024250285A1
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Prior art keywords
glucose
enzyme electrode
electrode system
enzyme
electrode
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Application number
PCT/CN2023/099459
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English (en)
Chinese (zh)
Inventor
范怡麟
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Oxsyns Technology Co Ltd
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Oxsyns Technology Co Ltd
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Priority to PCT/CN2023/099459 priority Critical patent/WO2024250285A1/fr
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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
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage

Definitions

  • FIG. 7 is a titration test of glucose concentration (0-15 mM) by the FNR-GDH enzyme electrochemical system of the present invention in a body fluid simulation environment, indicating the corresponding relationship between the glucose titration concentration and the reaction current.
  • 1 electrode supporting substrate
  • 2 conductive substrate
  • 3 coenzyme reductase
  • 4 glucose dehydrogenase
  • the present invention provides an enzyme electrode system, the enzyme electrode system comprising:
  • a working electrode a counter electrode, and optionally a reference electrode
  • the working electrode comprises an electrode supporting substrate and a conductive substrate located thereon, wherein The surface or interior of the conductive substrate contains glucose dehydrogenase, coenzyme reductase and optional cofactors.
  • glucose is first converted into gluconolactone under the catalysis of glucose dehydrogenase, and the oxidized NAD(P) + is reduced to NADPH
  • NAD(P)H is oxidized to oxidized NADP under the catalysis of coenzyme reductase.
  • the present invention shows that the glucose dehydrogenase NAD (P) type electron acceptor system can realize the detection function of glucose concentration.
  • P glucose dehydrogenase NAD
  • the unique direct electron transfer ability of the system eliminates the need for toxic artificial electron mediators, making this enzyme system an ideal choice for glucose detection methods. Reaction equation:
  • NAD(P) in the reaction formula refers to a reduced coenzyme that depends on NAD (coenzyme I) and NADP (coenzyme II).
  • the coenzyme reductase is an enzyme that can reduce the coenzyme, which is widely present in nature.
  • the present invention can directly use wild-type enzymes or genetically modified enzymes.
  • the coenzyme reductase is a diaphorase, that is, an enzyme that can catalyze reactions involving NADP (H) or NAD (H); preferably FNR.
  • FNR from various sources of Protein Data Bank have been tried, and all have response currents.
  • the role of diaphorase is to convert oxidized coenzyme I/II into reduced coenzyme I/II.
  • the glucose dehydrogenase and the coenzyme reductase can be placed on the surface and inside of the conductive substrate of the working electrode in any proportion.
  • the molar concentration ratio of the glucose dehydrogenase to the coenzyme reductase is 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1.
  • the surface or interior of the conductive substrate may also contain a cofactor.
  • the scientific name of the cofactor is nicotinamide adenine dinucleotide (NAD(P)), and its trade name is also coenzyme I/II.
  • the cofactor of the present invention is selected from one or more of NAD, NADP or FAD.
  • the added cofactor can be either oxidized or reduced, and both oxidized coenzyme I/II and reduced coenzyme I/II can generate effective current. Since the cofactor naturally exists in biological Chemical fluids include tissue fluid, cell fluid, plasma, platelets, blood, interstitial fluid, saliva, sweat, etc.
  • the coenzyme content used in the present invention is relatively small, the content of micromoles or even picomoles of the substance can react, so in actual detection, if the liquid already contains cofactors, the enzyme electrode does not need to add additional cofactors.
  • the amount of the cofactor added is 5 pM to 50 mM, preferably 10 ⁇ M-50 ⁇ M.
  • the surface microstructure of the working electrode is a smooth plane, a rough plane or a porous plane; in fact, any electrode that can fix and accommodate an enzyme can be a working electrode.
  • a working electrode with a porous plane is preferably used, so that the current density per unit area can be better improved under the same area.
  • (1) is an electrode support substrate, which provides support for the electrode system during the detection process.
  • the electrode support substrate can be conductive or insulating, including but not limited to metals (such as titanium and its alloys) and bioceramics (such as calcium phosphate (such as HAp and ⁇ , ⁇ -tricalcium phosphate [ ⁇ , ⁇ ])-TCP]), calcium carbonate, bioactive glass and glass ceramics, alumina and zirconium oxide) and natural (such as collagen, gelatin, silk protein [SF], chitosan, hyaluronic acid [HA], gellan gum [GG] and its derivatives, alginate and synthetic polymers (such as polyurethane [PU] and polycaprolactone [PCL]) and combinations thereof.
  • metals such as titanium and its alloys
  • bioceramics such as calcium phosphate (such as HAp and ⁇ , ⁇ -tricalcium phosphate [ ⁇ , ⁇ ])-TCP])
  • calcium carbonate such as HA
  • the hardness of the substrate is not limited.
  • the electrode support substrate is preferably made of a softer material to improve wearing comfort; when the enzyme electrode is applied to a test strip, the electrode support substrate is preferably made of a harder material.
  • (2) is a conductive substrate, and the conductive substrate is selected From precious metal nanoparticles, nanocarbon, conductive polymers and nanocomposites, such as ITO, graphene, carbon nanotubes, gold electrodes, silver electrodes, conductive hydrogels or conductors or semiconductors doped with nanoelectrodes.
  • the conductive substrate plays the role of fixing or accommodating the enzyme during the detection process and allowing electrons to flow through it and be conducted to the enzyme.
  • the material properties need to be conductive and have one or more forces of physical adsorption, chemical adsorption, and electrostatic adsorption on the enzyme, so that the enzyme is placed on the surface and inside of the conductive substrate without being lost.
  • (3) is coenzyme reductase.
  • (4) is glucose dehydrogenase.
  • the glucose dehydrogenase is used to undergo an oxidation-reduction reaction with glucose, and the coenzyme reductase is used to convert the reduced cofactor into an oxidized cofactor.
  • the cofactor plays an electron transfer role.
  • the starting end of the electron transfer chain of the induction process is the glucose in the sample, and the terminal is the electrode system.
  • the method of placing the glucose dehydrogenase, coenzyme reductase and optional coenzyme factors on the surface and inside of the conductive substrate to form a conductive layer includes but is not limited to physical adsorption, chemical crosslinking, embedding, electrostatic adsorption, bioengineering co-expression, etc.
  • the glucose dehydrogenase, coenzyme reductase and optional coenzyme factors are placed in the porous structure of the conductive substrate, such as on the conductive nanolayer or in the gap formed by the nanolayer.
  • Different combinations of supporting substrate materials and conductive substrate materials will have different current densities. According to different examples of the present invention, the current density is 5 picoamperes/square centimeter to 1 milliampere/square centimeter. Different materials will be selected according to different needs during commercialization.
  • the glucose dehydrogenase, coenzyme reductase and optional coenzyme factor are co-immobilized by various physical or chemical methods and then immobilized on the surface or inside of a conductive substrate.
  • the glucose dehydrogenase and the coenzyme reductase are placed in a conductive matrix to prevent the enzyme from falling off.
  • the conductive matrix has a porous structure; more preferably, the conductive matrix is a porous material with nanometer size.
  • the diameter of the pores is 20-100 nanometers, preferably 50 nanometers; the thickness of the conductive matrix is 100-5000 nanometers; preferably 1000 nanometers to 3000 nanometers, more preferably 2000 nanometers.
  • the present invention also provides a method for determining the glucose concentration in a sample, the method comprising:
  • the glucose concentration determination method can be used for but is not limited to continuous monitoring equipment (continues glucose monitoring, CGM), and can also be used on single detection devices of glucose test strips, such as made into test strips and used in conjunction with supporting detection instruments.
  • continuous monitoring equipment continuous glucose monitoring, CGM
  • single detection devices of glucose test strips such as made into test strips and used in conjunction with supporting detection instruments.
  • the present invention also provides a system for determining the glucose concentration in a sample, wherein the system determines the glucose concentration in the sample solution by contacting the sample solution.
  • the system comprises:
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • the three-electrode electrochemical reactor described in Example 1 was placed in an anaerobic glove box and in the air for reaction.
  • a constant voltage scan of -0.2V was performed under the control of the Ivium electrochemical workstation, and magnetic stirring was turned on at the same time (800 rpm).
  • the change in reaction rate is fed back by the reaction current, corresponding to the change in glucose concentration, and the reaction process is shown in Figure 4 below.
  • NADP-type FNR 1.5 ⁇ L of NADP-type FNR and 28.5 ⁇ L of NADP-type GDH were mixed to make the final concentration of FNR to be 0.04 mM and the final concentration of GDH to be 0.068 mM, and then evenly applied on the nanogold electrode (surface area 2.8 cm 2 ), allowed to stand in parallel and cooled for 25 min, and then the surface enzyme solution was rinsed off with pH 9 TAPS buffer to obtain the nanogold electrode loaded with FNR and GDH.
  • the above-mentioned nano-gold electrode sheet loaded with FNR and GDH was used as the working electrode, the platinum sheet was used as the counter electrode, and Ag/AgCl (3M KCl) was used as the reference electrode to assemble a three-electrode electrolytic cell.
  • the solution in the electrolytic cell was 6 mL of human body fluid simulation fluid SBF with a pH of 7.4 (ingredients were NaCl 135 mM, KCl 5 mM, MgCl 2 1.5 mM, CaCl 2 2.5 mM, Na 2 HPO 4 1 mM, Na 2 SO 4 0.5 mM, NaHCO 3 4.2 mM, Tris 5 mM), and 5 ⁇ M of coenzyme NADP + was added.
  • the three-electrode electrolytic cell described in Example 2 was placed in an anaerobic glove box and subjected to a constant voltage scan of -0.1 V under the control of an Ivium electrochemical workstation. (800 rpm). After the reaction starts, wait until the current is stable, and use a needle to add a concentrated glucose solution for titration, so that the glucose concentration gradient in the reaction solution increases, ranging from 0-15mM. The change in reaction rate is fed back by the reaction current, corresponding to the change in glucose concentration.
  • the reaction process is shown in Figure 5, where Figure 5a shows the curve of current change over time, and the magnitude of the current reflects the rate of the reaction. This reaction is a glucose oxidation reaction.
  • Figure 5b plots the correspondence between the glucose titration concentration and the reaction current; the figure reflects that within the concentration range of glucose in a simulated body fluid environment, there is a good correspondence between the current and the concentration.
  • Embodiment 3 is a diagrammatic representation of Embodiment 3
  • NADP-type FNR 1.8 ⁇ L of NADP-type FNR and 10.2 ⁇ L of NADP-type glucose dehydrogenase GDH solution were mixed to make the final concentration of FNR to 0.048 mM and the final concentration of GDH to 0.024 mM, and then drop-coated on the nano-gold-coated C electrode (surface area 0.2 cm2), left to stand and cool for 5 min in parallel, and then rinsed off the surface enzyme solution with pH 9 TAPS buffer to obtain a nano-gold-coated electrode loaded with FNR and GDH.
  • solution 1 contains human body fluid simulation liquid SBF with a pH of 7.4 (ingredients are NaCl 135mM, KCl 5mM, MgCl 2 1.5mM, CaCl 2 2.5mM, Na 2 HPO 4 1mM, Na 2 SO 4 0.5mM, NaHCO 3 4.2mM, Tris 5mM) and 5 ⁇ M coenzyme NADP + ; solution 2 is 15mM glucose, which is dissolved in solution 1.
  • the enzyme electrode system described in Example 2 was reacted in air.
  • a constant voltage scan of -0.2V was performed under the control of an Ivium electrochemical workstation. After the reaction started, the current was stabilized, and the operation of adding solution 1 and solution 2 was performed multiple times. The change in reaction rate was fed back by the reaction current, corresponding to the change in glucose concentration.
  • the specific operation was as follows:
  • Solution 2 is continuously added to simulate the flow of body fluids to continuously supply a stable concentration level of glucose
  • Solution 2 was added once to the stand-alone system to simulate the situation where the glucose concentration increased again;
  • Solution 1 was added for rinsing to simulate the situation where the glucose concentration decreased again.
  • Figure 6 shows the test process of the flow system under the condition of real-time dynamic changes in glucose, proving the feasibility of the system for glucose monitoring.
  • Embodiment 4 is a diagrammatic representation of Embodiment 4:
  • 2.2 ⁇ L of NAD-type FNR, 2.2 ⁇ L of NADP-type FNR, 7.5 ⁇ L of NAD-type GDH, 7.5 ⁇ L of NADP-type GDH and 8.6 ⁇ L of TAPS buffer at pH 9 were mixed to make the final concentration of FNR to be 0.12 mM and the final concentration of GDH to be 0.036 mM.
  • the mixture was evenly applied on a nanogold electrode (surface area 2.8 cm 2 ), allowed to stand and cooled for 25 min in parallel, and then the surface enzyme solution was rinsed off with TAPS buffer at pH 9 to obtain a nanogold electrode loaded with NAD/NADP mixed-dependent FNR and NAD/NADP mixed-dependent GDH.
  • nano-gold electrode sheet loaded with FNR and GDH was used as the working electrode
  • platinum sheet was used as the counter electrode
  • Ag/AgCl (3M KCl) was used as the reference electrode to assemble a three-electrode electrolytic cell.
  • the solution in the electrolytic cell was 6 mL of human body fluid simulation liquid SBF with a pH of 7.4 (ingredients were NaCl 135mM, KCl 5mM, MgCl 2 1.5mM, CaCl 2 2.5mM, Na 2 HPO 4 1mM, Na 2 SO 4 0.5mM, NaHCO 3 4.2mM, Tris 5mM), and 2.5 ⁇ M of coenzyme NADP + and 2.5 ⁇ M of coenzyme NAD + were additionally added.
  • Example 1 The three-electrode electrolytic cell described in Example 1 was placed in an anaerobic glove box, and a constant voltage scan of -0.2V was performed under the control of the Ivium electrochemical workstation, and magnetic stirring (800 rpm) was turned on at the same time. After the reaction started, when the current was stable, a concentrated glucose solution was added with a needle for titration to increase the glucose concentration gradient in the reaction solution to a range of 0-15mM.
  • the change in the reaction rate was fed back by the reaction current, corresponding to the change in glucose concentration, and the corresponding relationship between the glucose titration concentration and the reaction current was plotted (as shown in Figure 7); the figure reflects that within the concentration range of glucose in the simulated body fluid environment, the current and concentration generated by the mixed cofactor of NAD/NADP have a good correspondence.

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Abstract

L'invention concerne un système d'électrode enzymatique et une utilisation de celui-ci. Le système d'électrode enzymatique comprend une électrode de travail, une contre-électrode et une électrode de référence optionnelle. L'électrode de travail comprend une base de support d'électrode (1) et une base conductrice (2) située sur celle-ci, la surface ou l'intérieur de la base conductrice (2) contenant de la glucose déshydrogénase (4), une coenzyme réductase (3) et un cofacteur facultatif. La glucose déshydrogénase (4), la coenzyme réductase (3) et le cofacteur forment conjointement un système complet d'électrode enzymatique pour tester le glucose dans un échantillon.
PCT/CN2023/099459 2023-06-09 2023-06-09 Système d'électrode enzymatique et son utilisation Pending WO2024250285A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1452717A (zh) * 2000-09-25 2003-10-29 旭化成株式会社 酶电极
JP2011050300A (ja) * 2009-09-01 2011-03-17 Toyobo Co Ltd グルコースセンサ
WO2019224628A1 (fr) * 2018-05-21 2019-11-28 King Abdullah University Of Science And Technology Capteurs de métabolites électrochimiques imprimés par jet d'encre
CN111896600A (zh) * 2020-02-07 2020-11-06 山东省科学院生物研究所 一种葡萄糖脱氢酶电极及其制备方法和应用
CN112986354A (zh) * 2019-12-02 2021-06-18 中国科学院天津工业生物技术研究所 木糖检测用酶电极，电化学生物传感器及其制备方法和应用
CN115768349A (zh) * 2020-05-29 2023-03-07 美国雅培糖尿病护理公司 用于检测黄递酶抑制剂的分析物传感器和传感方法
CN116698945A (zh) * 2023-06-09 2023-09-05 深圳津合生物有限公司 一种酶电极系统及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1452717A (zh) * 2000-09-25 2003-10-29 旭化成株式会社 酶电极
JP2011050300A (ja) * 2009-09-01 2011-03-17 Toyobo Co Ltd グルコースセンサ
WO2019224628A1 (fr) * 2018-05-21 2019-11-28 King Abdullah University Of Science And Technology Capteurs de métabolites électrochimiques imprimés par jet d'encre
CN112986354A (zh) * 2019-12-02 2021-06-18 中国科学院天津工业生物技术研究所 木糖检测用酶电极，电化学生物传感器及其制备方法和应用
CN111896600A (zh) * 2020-02-07 2020-11-06 山东省科学院生物研究所 一种葡萄糖脱氢酶电极及其制备方法和应用
CN115768349A (zh) * 2020-05-29 2023-03-07 美国雅培糖尿病护理公司 用于检测黄递酶抑制剂的分析物传感器和传感方法
CN116698945A (zh) * 2023-06-09 2023-09-05 深圳津合生物有限公司 一种酶电极系统及其应用

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