WO2020019786A1 - 一种生物传感膜的制备方法、生物传感膜及监测装置 - Google Patents

一种生物传感膜的制备方法、生物传感膜及监测装置 Download PDF

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WO2020019786A1
WO2020019786A1 PCT/CN2019/084254 CN2019084254W WO2020019786A1 WO 2020019786 A1 WO2020019786 A1 WO 2020019786A1 CN 2019084254 W CN2019084254 W CN 2019084254W WO 2020019786 A1 WO2020019786 A1 WO 2020019786A1
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complex
preparation
carboxyl group
free amino
oxidoreductase
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PCT/CN2019/084254
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English (en)
French (fr)
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高志强
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三诺生物传感股份有限公司
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Priority to US17/044,016 priority Critical patent/US20210123882A1/en
Priority to KR1020207027653A priority patent/KR102541373B1/ko
Priority to JP2021500330A priority patent/JP7061224B2/ja
Priority to EP19840187.9A priority patent/EP3832296B1/en
Publication of WO2020019786A1 publication Critical patent/WO2020019786A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03004—Glucose oxidase (1.1.3.4)
    • 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
    • G01N27/31—Half-cells with permeable membranes, e.g. semi-porous or perm-selective membranes
    • 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
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • 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
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry

Definitions

  • the present application relates to the technical field of detection equipment, and in particular, to a method for preparing a biosensor film, a biosensor film, and a monitoring device.
  • An electrochemical biosensor is a device that is sensitive to biological substances and can convert its concentration into an electrical signal for detection.
  • Electrochemical biosensors include selective biological substances such as oxidoreductases and antibodies capable of identifying target substances, and electrodes and associated devices that convert the recognition signals into electrical signals.
  • oxidoreductase when used as a target recognition substance, the electronic exchange between it and an electrode is an important step in a biosensor device.
  • the redox active site of the redox enzyme does not exchange electrons with the electrode, and the electron transfer between the active site of the enzyme and the electrode is the limiting factor for most biosensors.
  • a redox small-molecule compound with excellent electrochemical performance can be introduced into the biosensor membrane, and an electronic channel can be established between the redox enzyme and the electrode, so that the electron exchange can be performed quickly.
  • a first object of the present invention is to provide a method for preparing a biosensor film; a second object of the present invention is to provide a biosensor film prepared by the above method; Three objectives are to provide a monitoring device prepared by the above-mentioned preparation method.
  • the preparation method provided by the present invention or the biosensor film and monitoring device prepared by the above preparation method are stable and durable, can be detected multiple times, and are particularly suitable for use as a biosensor film for a living body monitoring device.
  • a method for preparing a biosensor film After electrochemically modifying oxidoreductase, a chemical cross-linking agent is used for cross-linking treatment to coat the electrode surface to form a biosensor film.
  • the chemical cross-linking is formed.
  • the agent is glutaraldehyde or polyethylene glycol diglycidyl ether.
  • the oxidoreductase is any one or more of glucose oxidase, glucose dehydrogenase, lactate oxidase, lactate dehydrogenase, and catalase.
  • the oxidoreductase is electrochemically modified with a complex of ruthenium with a free amino or carboxyl group or a complex of osmium with a free amino or carboxyl group.
  • the specific step of electrochemically modifying the oxidoreductase is: combining a complex of ruthenium with a free amino group or a carboxyl group or a complex of osmium with a free amino group in a buffer solution with the oxidoreductase Mix well, add carbodiimide and N-hydroxysuccinimide in order, mix well, and react at 2-6 ° C for 12-48h, and then perform dialysis with ultrafiltration.
  • the oxidoreductase is modified twice with a complex of ruthenium with free amino or carboxyl groups or a complex with free amino sulfonium, the first modification uses ultrafiltration to cut the molecular weight of 5000-50000, and the second modification Ultrafiltration cuts molecular weights from 500 to 50,000.
  • the pyrene complex with a free amino or carboxyl group is specifically Os (bpy) 2 (3-aminopropylimidazole) Cl or Os (bpy) 2 (4-imidazolium butyric acid) Cl; the band The ruthenium complex of free amino or carboxyl group is specifically Ru (bpy) 2 (3-aminopropylimidazole) Cl or Os (bpy) 2 (4-imidazolium butyric acid) Cl.
  • the specific steps of the cross-linking treatment using the chemical cross-linking agent are: mixing the modified oxidoreductase with the chemical cross-linking agent in a buffer solution, and coating the electrode surface after 0.5-5 h to form a biological substance. Sensing film.
  • the surface is coated with a mixed solution of polyvinylpyridine and Nafion to form a biocompatible biosensor film.
  • biosensor film prepared by the preparation method according to any one of the above.
  • a monitoring device includes a sensor, and the sensor includes a biosensor film prepared by the preparation method according to any one of the foregoing.
  • the invention provides a method for preparing a biosensor film. After electrochemically activating and modifying an oxidoreductase, a chemical cross-linking agent is used for cross-linking and coating on the surface of an electrode to form a biosensor film.
  • the chemical crosslinking agent is glutaraldehyde or polyethylene glycol diglycidyl ether.
  • glutaraldehyde or polyethylene glycol diglycidyl ether as a chemical cross-linking agent
  • the modified redox enzyme is treated, and then coated on the electrode surface, and then the electrode surface can be formed.
  • Biosensor film The biosensor film formed by the cross-linking treatment of glutaraldehyde or polyethylene glycol diglycidyl ether is stable and durable and can be detected multiple times, and is especially suitable for use as a biosensor film for a living body monitoring device.
  • FIG. 1 is a cyclic voltammogram of glucose oxidase modified with Os (bpy) 2 (3-aminopropylimidazole) Cl and natural glucose oxidase in an embodiment of the present invention
  • (a) is a cyclic voltammogram of glucose oxidase modified with Os (bpy) 2 (3-aminopropylimidazole) Cl
  • (b) is a cyclic voltammogram of natural glucose oxidase.
  • glucose oxidase 2 is an ultraviolet-visible absorption spectrum of a glucose oxidase modified with Os (bpy) 2 (3-aminopropylimidazole) Cl and a natural glucose oxidase in an embodiment of the present invention
  • (a) is an ultraviolet-visible absorption spectrum of natural glucose oxidase
  • (b) is an ultraviolet-visible absorption spectrum of glucose oxidase modified with Os (bpy) 2 (3-aminopropylimidazole) Cl.
  • FIG. 3 is a cyclic voltammogram of a biosensor membrane containing modified glucose oxidase in a PBS (pH 7.4) buffer solution and a cyclic voltammogram after adding 5.0 mM glucose according to an embodiment of the present invention
  • (a) is a cyclic voltammogram of a biosensor membrane containing a modified glucose oxidase in a PBS (pH 7.4) buffer solution; (b) is a cyclic voltammogram after adding 5.0 mM glucose.
  • FIG. 4 is a schematic diagram showing the relationship between the electrochemical catalytic oxidation current of glucose and glucose concentration on a biosensor membrane in the embodiment of the present invention
  • FIG. 5 is a cyclic voltammogram of a biosensor membrane containing a modified lactate oxidase in a PBS (pH 7.4) buffer solution and a cyclic voltammogram after adding 5.0 mM lactose according to an embodiment of the present invention
  • (1) is the cyclic voltammogram of the modified lactate oxidase biosensor membrane in PBS (pH 7.4) buffer solution; (2) is the cyclic voltammogram after adding 5.0mM lactose.
  • FIG. 6 is a cyclic voltammogram of a biosensor membrane containing a modified glucose dehydrogenase in a PBS (pH 7.4) buffer solution and a cyclic voltammogram after adding 5.0 mM glucose;
  • (1) is the cyclic voltammogram of the modified glucose dehydrogenase biosensor membrane in PBS (pH 7.4) buffer solution; (2) is the cyclic voltammogram after adding 5.0mM glucose.
  • an embodiment of the present invention provides a method for preparing a biosensor film. After modifying the oxidoreductase, a chemical cross-linking agent is used for cross-linking treatment, and the electrode surface is coated to form The biosensor film, wherein the chemical cross-linking agent is glutaraldehyde or polyethylene glycol diglycidyl ether.
  • the invention provides a method for preparing a biosensor film. After electrochemically activating and modifying an oxidoreductase, a chemical cross-linking agent is used for cross-linking and coating on the surface of an electrode to form a biosensor film.
  • the chemical crosslinking agent is glutaraldehyde or polyethylene glycol diglycidyl ether.
  • glutaraldehyde or polyethylene glycol diglycidyl ether as a chemical cross-linking agent
  • the modified redox enzyme is treated, and then coated on the electrode surface, and then the electrode surface can be formed.
  • Biosensor film The biosensor film formed by the cross-linking treatment of glutaraldehyde or polyethylene glycol diglycidyl ether is stable and durable and can be detected multiple times, and is especially suitable for use as a biosensor film for a living body monitoring device.
  • the oxidoreductase is any one or more of glucose oxidase, glucose dehydrogenase, lactate oxidase, lactate dehydrogenase, and catalase.
  • the oxidoreductase is specifically any one or more of glucose oxidase, glucose dehydrogenase, lactate oxidase, lactate dehydrogenase, and catalase.
  • the oxidoreductase is electrochemically modified and then treated with a chemical cross-linking agent, a stable biosensor film can be formed on the electrode surface, and the target substance can be detected.
  • the oxidoreductase is electrochemically modified with a complex of ruthenium with a free amino or carboxyl group or a complex of osmium with a free amino or carboxyl group.
  • the specific step of modifying the oxidoreductase is: mixing a complex of ruthenium with a free amino group or a complex of osmium with a free amino group with the oxidoreductase in a buffer solution, and sequentially adding The carbodiimide and N-hydroxysuccinimide were mixed and reacted at 2-6 ° C for 12-48h, and the dialysis was performed by ultrafiltration.
  • a ruthenium complex with a free carboxyl group or a complex with a free carboxyl group is used to modify the oxidoreductase twice, the first modified ultrafiltration cuts the molecular weight 5000-50,000, and the second modified ultrafiltration The cutting molecular weight is 500-50000.
  • the complex of osmium with free amino or carboxyl group Os (bpy) 2 (3-aminopropylimidazole) Cl or Os (bpy) 2 (4-imidazolium butyric acid) Cl; Ru complexation
  • the substance is specifically Ru (bpy) 2 (3-aminopropylimidazole) Cl or Os (bpy) 2 (4-imidazole butyric acid) Cl. .
  • a complex of ruthenium with a free amino group or a complex of osmium with a free amino group is preferably used. More preferably, the modification is performed with Os (bpy) 2 (3-aminopropylimidazole) Cl or Ru (bpy) 2 (3-aminopropylimidazole) Cl. Where bpy refers to 2,2-bipyridine.
  • the specific steps for electrochemically modifying the oxidoreductase are: mixing the complex of ruthenium with free amino groups or the complex of osmium with free amino groups with the oxidoreductase in a buffer solution, and then adding carbonized Imine, N-hydroxysuccinimide, mix well, react at 2-6 ° C for 12-48h, and dialysis by ultrafiltration. More preferably, the oxidoreductase is modified twice with a complex of ruthenium with a free carboxyl group or a complex with a free carboxyl sulfonium, the first modification ultrafiltration cuts the molecular weight 5000-50,000, and the second modification Ultrafiltration cuts molecular weights from 500 to 50,000.
  • the electrochemical activation modification of the oxidoreductase twice can be performed by the following steps:
  • the concentration of the ruthenium complex with a free amino or carboxyl group or the osmium complex with a free amino or carboxyl group is preferably 0.1-50 mg / ml, more preferably 1-20 mg / ml.
  • the carbodiimide concentration used is preferably 0.1-50 mmol / L, and more preferably 0.1-25 mmol / L.
  • the concentration of the N-hydroxysuccinimide used is preferably 0.01 to 5 mmol / L. Spectrophotometric analysis can confirm that the oxidoreductase has been successfully modified.
  • the specific steps of the cross-linking treatment using the chemical cross-linking agent are: mixing the modified oxidoreductase with the chemical cross-linking agent in a buffer solution, and coating the electrode surface after 0.5-5 h to form a biological substance. Sensing film.
  • the surface is coated with a mixed solution of polyvinylpyridine and Nafion to form a biocompatible biosensor film.
  • the specific steps of cross-linking treatment using a chemical cross-linking agent are: mixing the modified oxidoreductase with the chemical cross-linking agent in a buffer solution, and then coating the electrode surface for 0.5-5 hours to form Biosensor film.
  • the surface is coated with a mixed solution of polyvinylpyridine and Nafion to form a biocompatible biosensor film.
  • the modified oxidoreductase is sufficiently mixed with a glutaraldehyde solution or a polyethylene glycol diglycidyl ether solution in a PBS buffer solution, and the reaction is performed for 0.5-5 hours, preferably 0.5-3 hours, and then a drop coating method is used. Or, the oxidoreductase after chemical cross-linking is coated on the electrode surface by a dipping and pulling method to make a biosensor film. After the biosensor film on the electrode is completely dried, the mixed solution of polyvinylpyridine and Nafion is coated on the surface of the biosensor film by the drop coating method or the dipping and pulling method to make a biocompatible film. Biosensor film.
  • the concentration of the chemical crosslinking agent glutaraldehyde solution or polyethylene glycol diglycidyl ether solution is 0.1-5%.
  • the concentration of the modified oxidoreductase used is preferably 5-150 mg / ml.
  • the concentration of the polyvinylpyridine solution used is preferably 20-300 mg / ml.
  • the concentration of the Nafion mixed alcohol solution used is preferably 0.1-5%, preferably a polyvinylpyridine and Nafion mixed ethanol solution, that is, a polyvinylpyridine and Nafion mixed ethanol solution obtained by dissolving and mixing polyvinylpyridine and Nafion in ethanol are mixed. .
  • oxidoreductases After chemical cross-linking, oxidoreductases still retain their direct electrochemical action.
  • glucose oxidase not only maintains its catalytic oxidation performance on glucose in biosensing membranes, but its catalytic oxidation efficiency of glucose through direct electrochemistry is 140 times higher than that of natural glucose oxidase through oxygen. .
  • biosensor film prepared by the preparation method according to any one of the above.
  • a monitoring device includes a sensor, and the sensor includes a biosensor film prepared by the preparation method according to any one of the foregoing.
  • the present invention provides a biosensor film prepared by the preparation method according to any one of the above.
  • a monitoring device is also provided, including a sensor, the sensor including a biosensor film prepared by the preparation method according to any one of the above.
  • the monitoring device is preferably an implantable continuous monitoring device, which can stably operate in a living body (such as a human body) and can detect a target substance multiple times for a long time.
  • An implantable continuous monitoring device is a method in which a sensor with a fixed biosensor film is implanted under the skin and the data returned by the sensor is received in real time by a receiver or a mobile device to achieve long-term continuous monitoring of the concentration of the target substance.
  • an implantable monitoring device for blood glucose can be developed. While monitoring blood glucose, it can also be used with insulin pump products to supplement insulin in time to regulate blood glucose levels in the body.
  • the 5-150 mg / ml modified oxidoreductase is thoroughly mixed with a 0.1-5% glutaraldehyde solution in a PBS buffer solution, and after reacting for 0.5-3 hours, the solution is applied by a drop coating method or a dipping and pulling method.
  • the chemically crosslinked oxidoreductase is coated on the electrode surface to make a biosensor film. After the biosensor film on the electrode is completely dried, the solution containing 20-300mg / ml polyvinylpyridine and 0.1-5% Nafion mixed alcohol solution is coated on the biosensor film by the drop coating method or the dipping and pulling method. Surface, made of biocompatible biosensor film.
  • glucose oxidase but also other oxidoreductases such as lactate oxidase and glucose dehydrogenase can be successfully modified and chemically cross-linked with glutaraldehyde.
  • Biosensor membranes containing modified lactate oxidase and modified glucose dehydrogenase showed good electrochemical performance on the electrodes (as shown in Figure 5).
  • the cyclic voltammogram of the biosensor membrane clearly shows the typical electrochemical catalytic process (as shown in Figure 6). This result indicates that they retain their catalytic oxidation performance through direct electrochemistry in biosensor membranes.

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Abstract

一种生物传感膜的制备方法，对氧化还原酶进行电化学活化修饰后，使用化学交联剂交联处理，涂覆在电极表面，即形成生物传感膜，其中，化学交联剂为戊二醛或聚乙二醇二缩水甘油醚。还公开了一种制备得到的生物传感膜及监测装置。提供的制备方法或制备方法制得的生物传感膜及监测装置，稳定耐用，可以多次检测，尤其适用于作为活体监测装置的生物传感膜。

Description

一种生物传感膜的制备方法、生物传感膜及监测装置 技术领域
本申请涉及检测设备技术领域，特别是涉及一种生物传感膜的制备方法、生物传感膜及监测装置。
背景技术
电化学生物传感器是一种对生物物质敏感并能将其浓度转化为电信号进行检测的装置。电化学生物传感器包含能够识别目标物质的具有选择性的生物物质如氧化还原酶和抗体，以及将该识别信号转换为电信号的电极及其附属装置。例如，当氧化还原酶被用作目标识别物质时，其和电极之间的电子交换是生物传感装置的重要步骤。然而，通常情况下，氧化还原酶的氧化还原活性位点不与电极交换电子，酶的活性位点与电极之间的电子转移是大多数生物传感器的限制因素。为克服电子转移效率低的问题，可以在生物传感膜引入电化学性能优秀的氧化还原小分子化合物，在氧化还原酶与电极之间建立电子通道，使电子交换快速进行。
但引入氧化还原小分子化合物后，如何与氧化还原酶一起制成稳定的生物传感膜，并固定在电极上，能够稳定进行多次检测，特别是植入活体后还能够稳定进行多次检测，并保持检测结果的稳定，是本领域技术人员亟待解决的技术问题。
发明概述
技术问题
问题的解决方案
技术解决方案
为解决上述技术问题，本发明的第一个目的为提供一种生物传感膜的制备方法；本发明的第二个目的为提供一种上述制备方法制备的生物传感膜；本发明的第三个目的为提供上述制备方法制备的监测装置。本发明提供的制备方法或上述制备方法制得的生物传感膜及监测装置，稳定耐用，可以多次检测，尤其适 用于作为活体监测装置的生物传感膜。
本发明提供的技术方案如下：
一种生物传感膜的制备方法，对氧化还原酶进行电化学活化修饰后，使用化学交联剂交联处理，涂覆在电极表面，即形成生物传感膜，其中，所述化学交联剂为戊二醛或聚乙二醇二缩水甘油醚。
优选地，所述氧化还原酶为葡萄糖氧化酶、葡萄糖脱氢酶、乳酸氧化酶、乳酸脱氢酶、过氧化氢酶中的任意一种或多种。
优选地，采用带游离氨基或羧基的钌的络合物或带游离氨基或羧基的锇的络合物，对氧化还原酶进行电化学活化修饰。
优选地，所述对氧化还原酶进行电化学活化修饰的具体步骤为：将带游离氨基或羧基的钌的络合物或带游离氨基的锇的络合物，与氧化还原酶在缓冲溶液中混匀，依次加入碳化二亚胺、N-羟基琥珀酰亚胺，混匀，2-6℃反应12-48h，超滤透析。
优选地，采用带游离氨基或羧基的钌的络合物或带游离氨基锇的络合物，对氧化还原酶进行两次修饰，第一次修饰超滤切割分子量5000-50000，第二次修饰超滤切割分子量500-50000。
优选地，所述带游离氨基或羧基的锇的络合物具体为Os(bpy) 2(3-氨丙基咪唑)Cl或Os(bpy) 2(4-咪唑丁酸)Cl；所述带游离氨基或羧基的钌的络合物具体为Ru(bpy) 2(3-氨丙基咪唑)Cl或Os(bpy) 2(4-咪唑丁酸)Cl。
优选地，使用化学交联剂交联处理的具体步骤为：将修饰后的氧化还原酶，与化学交联剂在缓冲溶液中混匀，反应0.5-5h后，涂覆在电极表面，形成生物传感膜。
优选地，待交联处理后形成的生物传感膜干燥后，在其表面涂覆聚乙烯吡啶和Nafion混合醇溶液，形成具有生物相容性的生物传感膜。
上述任意一项所述的制备方法制得的生物传感膜。
一种监测装置，包括传感器，所述传感器包括上述任意一项所述的制备方法制得的生物传感膜。
发明的有益效果
有益效果
本发明提供一种生物传感膜的制备方法，对氧化还原酶进行电化学活化修饰后，使用化学交联剂交联处理，涂覆在电极表面，即形成生物传感膜，其中，所述化学交联剂为戊二醛或聚乙二醇二缩水甘油醚。本发明提供的制备方法，通过使用戊二醛或聚乙二醇二缩水甘油醚作为化学交联剂，对修饰后的氧化还原酶进行处理，然后涂覆在电极表面，即可在电极表面形成生物传感膜。经戊二醛或聚乙二醇二缩水甘油醚交联处理形成的生物传感膜，稳定耐用，可以多次检测，尤其适用于作为活体监测装置的生物传感膜。
对附图的简要说明
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请中记载的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例中经Os(bpy) 2(3-氨丙基咪唑)Cl修饰的葡萄糖氧化酶和天然葡萄糖氧化酶的循环伏安图；
其中，(a)为经Os(bpy) 2(3-氨丙基咪唑)Cl修饰的葡萄糖氧化酶的循环伏安图，(b)为天然葡萄糖氧化酶的循环伏安图。
图2为本发明实施例中经Os(bpy) 2(3-氨丙基咪唑)Cl修饰的葡萄糖氧化酶和天然葡萄糖氧化酶的紫外-可见吸收光谱；
其中，(a)为天然葡萄糖氧化酶的紫外-可见吸收光谱；(b)为经Os(bpy) 2(3-氨丙基咪唑)Cl修饰的葡萄糖氧化酶的紫外-可见吸收光谱。
图3为本发明实施例中含有已修饰的葡萄糖氧化酶的生物传感膜在PBS(pH 7.4)缓冲溶液中的循环伏安图和加入5.0mM葡萄糖后的循环伏安图；
其中，(a)为含有已修饰的葡萄糖氧化酶的生物传感膜在PBS(pH 7.4)缓冲溶液中的循环伏安图；(b)为加入5.0mM葡萄糖后的循环伏安图。
图4为本发明实施例中葡萄糖在生物传感膜上的电化学催化氧化电流与葡萄糖浓度的关系示意图；
图5为本发明实施例中含有经过修饰的乳酸氧化酶的生物传感膜在PBS(pH 7.4)缓冲溶液中的循环伏安图和加入5.0mM乳糖后的循环伏安图；
其中，(1)为经过修饰的乳酸氧化酶的生物传感膜在PBS(pH 7.4)缓冲溶液中的循环伏安图；(2)为加入5.0mM乳糖后的循环伏安图。
图6为含有经过修饰的葡萄糖脱氢酶的生物传感膜在PBS(pH 7.4)缓冲溶液中的循环伏安图和加入5.0mM葡萄糖后的循环伏安图；
其中，(1)为经过修饰的葡萄糖脱氢酶的生物传感膜在PBS(pH 7.4)缓冲溶液中的循环伏安图；(2)为加入5.0mM葡萄糖后的循环伏安图。
实施该发明的最佳实施例
本发明的最佳实施方式
为了使本技术领域的人员更好地理解本申请中的技术方案，下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本申请保护的范围。
请如图1至图6所示，本发明实施例提供一种生物传感膜的制备方法，对氧化还原酶进行修饰后，使用化学交联剂交联处理，涂覆在电极表面，即形成生物传感膜，其中，所述化学交联剂为戊二醛或聚乙二醇二缩水甘油醚。
本发明提供一种生物传感膜的制备方法，对氧化还原酶进行电化学活化修饰后，使用化学交联剂交联处理，涂覆在电极表面，即形成生物传感膜，其中，所述化学交联剂为戊二醛或聚乙二醇二缩水甘油醚。本发明提供的制备方法，通过使用戊二醛或聚乙二醇二缩水甘油醚作为化学交联剂，对修饰后的氧化还原酶进行处理，然后涂覆在电极表面，即可在电极表面形成生物传感膜。经戊二醛或聚乙二醇二缩水甘油醚交联处理形成的生物传感膜，稳定耐用，可以多次检测，尤其适用于作为活体监测装置的生物传感膜。
优选地，所述氧化还原酶为葡萄糖氧化酶、葡萄糖脱氢酶、乳酸氧化酶、乳酸脱氢酶、过氧化氢酶中的任意一种或多种。
本发明中，氧化还原酶具体为葡萄糖氧化酶、葡萄糖脱氢酶、乳酸氧化酶、乳 酸脱氢酶、过氧化氢酶中的任意一种或多种。上述氧化还原酶经电化学活化修饰后，再经过化学交联剂处理，都能够在电极表面形成稳定的生物传感膜，斌对目标物质进行检测。
优选地，采用带游离氨基或羧基的钌的络合物或带游离氨基或羧基的锇的络合物，对氧化还原酶进行电化学活化修饰。
优选地，所述对氧化还原酶进行修饰的具体步骤为：将带游离氨基的钌的络合物或带游离氨基的锇的络合物，与氧化还原酶在缓冲溶液中混匀，依次加入碳化二亚胺、N-羟基琥珀酰亚胺，混匀，2-6℃反应12-48h，超滤透析。
优选地，采用带游离羧基的钌的络合物或带游离羧基锇的络合物，对氧化还原酶进行两次修饰，第一次修饰超滤切割分子量5000-50000，第二次修饰超滤切割分子量500-50000。
优选地，所述带游离氨基或羧基的锇的络合物Os(bpy) 2(3-氨丙基咪唑)Cl或Os(bpy) 2(4-咪唑丁酸)Cl；的钌的络合物具体为Ru(bpy) 2(3-氨丙基咪唑)Cl或Os(bpy) 2(4-咪唑丁酸)Cl。。
本发明中，对氧化还原酶进行修饰，优选采用带游离氨基的钌的络合物或带游离氨基的锇的络合物。更优选采用Os(bpy) 2(3-氨丙基咪唑)Cl或Ru(bpy) 2(3-氨丙基咪唑)Cl进行修饰。其中bpy是指2，2-联吡啶。
对氧化还原酶进行电化学活化修饰的具体步骤为：将带游离氨基的钌的络合物或带游离氨基的锇的络合物，与氧化还原酶在缓冲溶液中混匀，依次加入碳化二亚胺、N-羟基琥珀酰亚胺，混匀，2-6℃反应12-48h，超滤透析。更优选的是，采用带游离羧基的钌的络合物或带游离羧基锇的络合物，对氧化还原酶进行两次修饰，第一次修饰超滤切割分子量5000-50000，第二次修饰超滤切割分子量500-50000。
具体而言，采用带游离氨基或羧基的钌的络合物或带游离氨基或羧基锇的络合物，对氧化还原酶进行两次电化学活化修饰的可采用以下步骤：
将带有游离氨基的钌的络合物，或带有游离氨基的锇的络合物，与氧化还原酶(如葡萄糖氧化酶)在PBS缓冲溶液进行充分混合，然后依次加入碳化二亚胺和N-羟基琥珀酰亚胺，充分混合后，2-6℃反应12-48h。然后利用超滤袋透析，切 割分子量5000-50000，对第一次修饰后的氧化还原酶进行分离和提纯。然后在提纯后的氧化还原酶溶液中加入游离羧基的钌的络合物，或带有游离羧基的锇的络合物，然后依次加入碳化二亚胺和N-羟基琥珀酰亚胺，充分混合后2-6℃反应12-48h，反应结束后，再次利用超滤袋透析，切割分子量500-50000，对第二次修饰后的氧化还原酶进行分离和提纯。其中，使用的带有游离氨基或羧基的钌的络合物，或带有游离氨基或羧基的锇的络合物，其浓度优选为0.1-50mg/ml，更优选为1-20mg/ml。使用的碳化二亚胺浓度优选为0.1-50mmol/L，更优选0.1-25mmol/L。使用的N-羟基琥珀酰亚胺的浓度优选为0.01-5mmol/L。经分光光度法分析，可以证实氧化还原酶已被成功地修饰。
优选地，使用化学交联剂交联处理的具体步骤为：将修饰后的氧化还原酶，与化学交联剂在缓冲溶液中混匀，反应0.5-5h后，涂覆在电极表面，形成生物传感膜。
优选地，待交联处理后形成的生物传感膜干燥后，在其表面涂覆聚乙烯吡啶和Nafion混合醇溶液，形成具有生物相容性的生物传感膜。
本发明中，使用化学交联剂交联处理的具体步骤为：将修饰后的氧化还原酶，与化学交联剂在缓冲溶液中混匀，反应0.5-5h后，涂覆在电极表面，形成生物传感膜。优选待交联处理后形成的生物传感膜干燥后，在其表面涂覆聚乙烯吡啶和Nafion混合醇溶液，形成具有生物相容性的生物传感膜。
具体而言，将修饰后的氧化还原酶，与化学交联剂反应可采用以下步骤：
将修饰后的氧化还原酶，在PBS缓冲溶液中，与戊二醛溶液或聚乙二醇二缩水甘油醚溶液进行充分混合，反应0.5-5h、优选0.5-3h后，用滴落涂布法或浸渍提拉法将化学交联后的氧化还原酶涂覆在电极表面，制成生物传感膜。待电极上的生物传感膜完全干燥后，再用滴落涂布法或浸渍提拉法将聚乙烯吡啶和Nafion混合醇溶液涂覆在生物传感膜表面，制成具有生物相容性的生物传感膜。优选地，使用的化学交联剂戊二醛溶液或聚乙二醇二缩水甘油醚溶液的浓度为0.1-5％。使用的修饰后的氧化还原酶的浓度优选为5-150mg/ml。使用的聚乙烯吡啶溶液的浓度优选为20-300mg/ml。使用的Nafion混合醇溶液的浓度优选为0.1-5％，优选使用聚乙烯吡啶和Nafion混合乙醇溶液，即，将聚乙烯吡啶和Nafion在乙醇中 溶解并混合得到的聚乙烯吡啶和Nafion混合乙醇溶液。
经过化学交联后，氧化还原酶仍然保持着它们的直接电化学作用。实验表明葡萄糖氧化酶的在生物传感膜中不仅保持了其对葡萄糖的催化氧化性能，其通过直接电化学对葡萄糖的催化氧化效率比天然葡萄糖氧化酶通过氧气对葡萄糖的催化氧化效率提高140倍。
上述任意一项所述的制备方法制得的生物传感膜。
一种监测装置，包括传感器，所述传感器包括上述任意一项所述的制备方法制得的生物传感膜。
本发明提供上述任意一项所述的制备方法制得的生物传感膜。还提供一种监测装置，包括传感器，所述传感器包括上述任意一项所述的制备方法制得的生物传感膜。
所述监测装置，优选为植入式持续监测装置，能够在植入生命体内(如人体内)稳定运行，并能长时间多次检测目标物质。植入式持续监测装置，是将固定了生物传感膜的传感器植入皮下，并通过接收器或移动设备实时接收传感器传回的数据，以实现对目标物质浓度的长期持续监测。例如，可以开发血糖的植入式监测装置，在对血糖进行监测的同时，还能配合胰岛素泵的产品，及时补充胰岛素，以调节体内血糖含量。
实施例1
将1-20mg/ml的Os(bpy) 2(3-氨丙基咪唑)Cl，与葡萄糖氧化酶在PBS缓冲溶液进行充分混合，然后依次加入0.1-25mmol/L碳化二亚胺和0.01-5mmol/L的N-羟基琥珀酰亚胺，充分混合后，4℃反应24h。然后利用超滤袋透析，切割分子量5000-50000，对第一次修饰后的氧化还原酶进行分离和提纯。然后在提纯后的氧化还原酶溶液中加入1-20mg/ml的Os(bpy) 2(4-咪唑丁酸)Cl，然后依次加入0.1-25mmol/L碳化二亚胺和0.01-5mmol/L N-羟基琥珀酰亚胺，充分混合后4℃反应24h，反应结束后，再次利用超滤袋透析，切割分子量500-50000，对第二次修饰后的氧化还原酶进行分离和提纯。葡萄糖氧化酶在经过修饰后，其催化活性中心就可以直接与电极进行非常快速电子交换(如附图1所示)。利用分光光度法进行分析，证实葡萄糖氧化酶已被成功地修饰 (如附图2所示)。
将5-150mg/ml修饰后的氧化还原酶，在PBS缓冲溶液中，与0.1-5％戊二醛溶液进行充分混合，反应0.5-3h后，用滴落涂布法或浸渍提拉法将化学交联后的氧化还原酶涂覆在电极表面，制成生物传感膜。待电极上的生物传感膜完全干燥后，再用滴落涂布法或浸渍提拉法将含有20-300mg/ml聚乙烯吡啶和0.1-5％Nafion混合醇溶液涂覆在生物传感膜表面，制成具有生物相容性的生物传感膜。经过戊二醛化学交联处理后，上述修饰后的葡萄糖氧化酶仍然保持着直接电化学作用，制成的生物传感膜在电极上呈现出良好的电化学性能(附图3中(a))。当在PBS缓冲溶液中加入5.0mM的葡萄糖后，生物传感膜的循环伏安图清晰地展示了一个典型的电化学催化过程(附图3中(b)).进一步的实验表明葡萄糖氧化酶的在生物传感膜中不仅保持了其对葡萄糖的催化氧化性能，其通过直接电化学对葡萄糖的催化氧化效率比天然葡萄糖氧化酶通过氧气对葡萄糖的催化氧化效率提高140倍。
在此基础上，我们上述生物传感膜应用于植入式持续葡萄糖监测系统。初步试验结果表明其工作曲线在2.0到32mM之间呈良好的线性(如附图4所示)，是目前线性范围最宽的持续葡萄糖监测系统。其稳定性也得到了显著的改善，在连续两个星期的试验中，灵敏度没有明显的变化。
不仅是葡萄糖氧化酶，其它氧化还原酶如乳酸氧化酶，葡萄糖脱氢酶等均可被成功地修饰，并和戊二醛化学交联。
含有经过修饰的乳酸氧化酶，和含有修饰后的葡萄糖脱氢酶的生物传感膜在电极上都呈现出良好的电化学性能(如附图5所示)。当在PBS缓冲溶液中加入5.0mM的乳糖或葡萄糖后，生物传感膜的循环伏安图清晰地展示了典型的电化学催化过程(如附图6所示)。这一结果表明它们在生物传感膜中保持了其通过直接电化学的催化氧化性能。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符 合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (10)

  1. 一种生物传感膜的制备方法，其特征在于，对氧化还原酶进行电化学活化修饰后，使用化学交联剂交联处理，涂覆在电极表面，即形成生物传感膜，其中，所述化学交联剂为戊二醛或聚乙二醇二缩水甘油醚。
  2. 根据权利要求1所述的制备方法，其特征在于，所述氧化还原酶为葡萄糖氧化酶、葡萄糖脱氢酶、乳酸氧化酶、乳酸脱氢酶、过氧化氢酶中的任意一种或多种。
  3. 根据权利要求2所述的制备方法，其特征在于，采用带游离氨基或羧基的钌的络合物或带游离氨基或羧基的锇的络合物，对氧化还原酶进行电化学活化修饰。
  4. 根据权利要求3所述的制备方法，其特征在于，所述对氧化还原酶进行电化学活化修饰的具体步骤为：将带游离氨基或羧基的钌的络合物或带游离氨基或羧基的锇的络合物，与氧化还原酶在缓冲溶液中混匀，依次加入碳化二亚胺、N-羟基琥珀酰亚胺，混匀，2-6℃反应12-48h，超滤透析。
  5. 根据权利要求4所述的制备方法，其特征在于，采用带游离氨基或羧基的钌的络合物或带游离氨基或羧基的锇的络合物，对氧化还原酶进行两次电化学活化修饰，第一次修饰超滤切割分子量500-50000，第二次修饰超滤切割分子量5000-50000。
  6. 根据权利要求5所述的制备方法，其特征在于，所述带游离氨基或羧基的锇的络合物具体为Os(bpy) 2(3-氨丙基咪唑)Cl，带游离羧基的络合物具体为Os(bpy) 2(4-咪唑丁酸)Cl；所述带游离氨基的钌的络合物具体为Ru(bpy) 2(3-氨丙基咪唑)Cl，带游离羧基的络合物具体为如Ru(bpy) 2(4-咪唑丁酸)Cl。
  7. 根据权利要求1-6中任一项所述的制备方法，其特征在于，使用化学交联剂交联处理的具体步骤为：将修饰后的氧化还原酶，与化学交联剂在缓冲溶液中混匀，反应0.5-5h后，涂覆在电极表面，形 成生物传感膜。
  8. 根据权利要求7所述的制备方法，其特征在于，待交联处理后形成的生物传感膜干燥后，在其表面涂覆聚乙烯吡啶和Nafion混合醇溶液，形成具有生物相容性的生物传感膜。
  9. 根据权利要求1-8中任意一项所述的制备方法制得的生物传感膜。
  10. 一种监测装置，包括传感器，其特征在于，所述传感器包括根据权利要求1-8中任意一项所述的制备方法制得的生物传感膜。
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