WO2021227542A1 - 一种固定化酶电极、固定化酶传感器及其酶膜抗干扰的检测方法 - Google Patents

一种固定化酶电极、固定化酶传感器及其酶膜抗干扰的检测方法 Download PDF

Info

Publication number
WO2021227542A1
WO2021227542A1 PCT/CN2021/071159 CN2021071159W WO2021227542A1 WO 2021227542 A1 WO2021227542 A1 WO 2021227542A1 CN 2021071159 W CN2021071159 W CN 2021071159W WO 2021227542 A1 WO2021227542 A1 WO 2021227542A1
Authority
WO
WIPO (PCT)
Prior art keywords
electrode
enzyme
immobilized enzyme
immobilized
detection
Prior art date
Application number
PCT/CN2021/071159
Other languages
English (en)
French (fr)
Inventor
史建国
马耀宏
杨俊慧
王丙莲
高广恒
马润隆
郑岚
蔡雷
刘庆艾
Original Assignee
山东省科学院生物研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 山东省科学院生物研究所 filed Critical 山东省科学院生物研究所
Publication of WO2021227542A1 publication Critical patent/WO2021227542A1/zh

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • 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

Definitions

  • the invention belongs to the technical field of biosensors, and particularly relates to an immobilized enzyme electrode, an immobilized enzyme sensor and an anti-interference detection method of the enzyme membrane.
  • a biosensor is a detection instrument based on biomolecular recognition, using biologically active materials as sensitive elements, and converting the concentration of the measured substance into a digital signal through an appropriate physical and chemical transducer and signal amplification device.
  • the classic current type enzyme electrode using immobilized enzyme membrane is the earliest commercialized type of biosensor with the largest application market.
  • the enzyme membrane in the immobilized enzyme electrode adopts a three-layer structure ( Figure 1).
  • a three-layer structure Taking glucose oxidase (GOD) as an example, there are (1) immobilized GOD enzyme membrane including polycarbonate microporous membrane (carrier membrane), (2) immobilized enzyme layer and (3) cellulose acetate inner membrane three-layer membrane .
  • GOD is fixed on the carbonate microporous membrane.
  • Glucose and oxygen molecules in the sample can contact the GOD through the polycarbonate microporous membrane and undergo a catalytic reaction to generate hydrogen peroxide. Hydrogen peroxide diffuses through the cellulose acetate membrane to the surface of the electrode, generating an electric current.
  • the magnitude of the current is proportional to the concentration of glucose in the sample.
  • the enzyme membrane is adhered to a rubber ring to form an enzyme membrane device, which is convenient for storage and use, and also serves as a seal connected to the reaction cell. Maintaining the integrity of the enzyme membrane (inner membrane) is to prevent the interference of electroactive substances in the sample. key.
  • the inner membrane may be damaged or incomplete, resulting in a "leakage" of the inner membrane, which cannot prevent the diffusion of small molecules of electroactive interference to the electrode through the inner membrane. Surface, produce interference.
  • the residual H 2 O 2 in some food samples can pass through the inner membrane, which also causes deviations in the results.
  • the purpose of the present invention is to provide an immobilized enzyme electrode, an immobilized enzyme sensor and an anti-interference detection method of the enzyme membrane.
  • An immobilized enzyme electrode including a detection electrode and an auxiliary electrode; the auxiliary electrode includes a basic electrode, an immobilized inactivated enzyme layer and a carrier membrane that are sequentially connected, and the enzyme immobilized on the immobilized inactivated enzyme layer is inactivated Enzyme.
  • the detection electrode includes a basic electrode, a cellulose acetate membrane, an immobilized enzyme layer and a carrier membrane connected in sequence.
  • the carrier film is a polycarbonate microporous film.
  • the detection electrode includes a glucose oxidase electrode and/or a lactase oxidase electrode.
  • the basic electrode is a hydrogen peroxide electrode.
  • the present invention provides an immobilized enzyme sensor, including the immobilized enzyme electrode.
  • the detection electrode and the auxiliary electrode in the immobilized enzyme electrode are respectively installed on different sides of the reaction cell cavity of the immobilized enzyme reactor; the front ends of the detection electrode and the auxiliary electrode are respectively sealed with the reaction cell cavity by a rubber ring Connection; the back ends of the detection electrode and the auxiliary electrode are respectively connected to the host through electrode wires.
  • the detection electrode includes a glucose oxidase electrode and a lactase oxidase electrode.
  • the present invention provides a detection method for anti-interference of an immobilized enzyme electrode enzyme membrane, which comprises the following steps:
  • the standard solution is a solution that includes a target substance with a determined concentration
  • the electrical signal response value of the detection electrode to the sample is Xn
  • the electrical signal response value of the auxiliary electrode to the sample is X0
  • the standard electrode active material is potassium ferrocyanide solution
  • the target material includes glucose and lactic acid.
  • the present invention provides an immobilized enzyme electrode, including a detection electrode and an auxiliary electrode;
  • the auxiliary electrode includes a basic electrode, an immobilized inactivated enzyme layer, and a carrier film connected in sequence.
  • the auxiliary electrode does not include a cellulose acetate inner membrane, and the enzyme immobilized on the immobilized inactivated enzyme layer is an inactivated enzyme; when the sample is measured, the reference value of the detection electrode and the auxiliary electrode is tested, and the detection electrode and the auxiliary electrode The electrode simultaneously corrects the fluctuation and change of the signal caused by the interference, thus avoiding the interference of the electrode signal caused by the "integrity" problem of the enzyme membrane, and improving the measurement accuracy.
  • the invention uses the auxiliary electrode to carry out the anti-interference detection method has no obvious influence; it shows that the immobilized enzyme electrode and the anti-interference detection method provided by the invention have accurate detection results and are not affected by electrically active interference substances, which has significant advantages.
  • Figure 1 shows the structure of the immobilized enzyme membrane of the detection electrode and the basic electrode
  • Figure 2 shows the reaction cell and three-electrode structure of the immobilized enzyme sensor in the embodiment.
  • the upper figure is a front view and the lower figure is a cross-sectional view; among them, 1. reaction cell module; 2. reaction cell cavity; 3. 'Shaped ring; 4. Sample injection and waste outflow channel; 5. Reaction cell top cap; 6. Sample injection cap; 7. Sample injection light sensor; 8. Enzyme electrode; 9. Enzyme electrode knob; 10. Buffer inlet And waste liquid emptying tube; 11. Waste liquid suction tube; 12. Electromagnetic stirrer; 13. Auxiliary enzyme electrode; 14. Electrode lead.
  • the present invention provides an immobilized enzyme electrode, including a detection electrode and an auxiliary electrode; the auxiliary electrode includes a basic electrode, an immobilized inactivated enzyme layer, and a carrier membrane connected in sequence, and the immobilized inactivated enzyme layer has an immobilized enzyme It is an inactivated enzyme.
  • the auxiliary electrode includes a basic electrode, an immobilized inactivated enzyme layer and a carrier membrane connected in sequence; the carrier membrane is preferably a polycarbonate microporous membrane, and the immobilized inactivated enzyme layer has an immobilized enzyme To be an inactivated enzyme, the inactivated enzyme is preferably inactivated by heating at a high temperature.
  • the auxiliary electrode does not include a cellulose acetate membrane as an "inner membrane"; the function of the auxiliary electrode is to correct detection data, eliminate electrical active interferences, and improve the accuracy of detection results.
  • the detection electrode includes a basic electrode, a cellulose acetate membrane, an immobilized enzyme layer and a carrier membrane connected in sequence.
  • the carrier film is preferably a polycarbonate microporous film.
  • the structure of the detection electrode is preferably as shown in FIG. 1.
  • the detection electrode includes a glucose oxidase electrode and/or a lactase oxidase electrode, preferably a glucose oxidase electrode and a lactase oxidase electrode; when the detection electrode is a glucose oxidase electrode and a lactase oxidase electrode
  • the basic electrode is preferably a hydrogen peroxide electrode.
  • the immobilized enzyme layers of the glucose oxidase electrode and the lactase oxidase electrode in the detection electrode are respectively coated with active glucose oxidase and lactase oxidase.
  • the front end of the carrier film of the detection electrode further includes a rubber sealing ring, and the detection electrode is sealed to the reaction cell through the rubber sealing ring.
  • the present invention does not specifically limit the sources of the raw materials of the detection electrode and the auxiliary electrode, including the basic electrode, cellulose acetate membrane, immobilized enzyme layer and carrier membrane, and adopts conventional commercial products in the field or self-preparation.
  • the present invention provides an immobilized enzyme sensor, including the immobilized enzyme electrode.
  • the detection electrode and the auxiliary electrode in the immobilized enzyme electrode are respectively installed on different sides of the reaction cell cavity of the immobilized enzyme reactor; the front ends of the detection electrode and the auxiliary electrode are respectively connected to the reaction cell through a rubber ring The cavity is closed and connected; the rear ends of the detection electrode and the auxiliary electrode are respectively connected to the host through electrode wires.
  • the structure of the immobilized enzyme sensor is preferably as shown in Figure 2, where 1. Reaction cell module; 2. Reaction cell cavity; 3.'O' ring of enzyme membrane; 4. Sample injection and Waste liquid outflow channel; 5. Reaction cell top cap; 6. Sample injection cap; 7. Sample injection light sensor; 8. Enzyme electrode; 9. Enzyme electrode knob; 10. Buffer inlet and waste liquid emptying tube; 11. Waste liquid suction tube; 12. Electromagnetic stirrer; 13. Auxiliary enzyme electrode; 14. Electrode lead.
  • the reaction tank module is preferably made of a square plexiglass block; the reaction tank is at the center of the reaction tank module module, and the reaction tank is preferably a cylindrical cavity.
  • the cavity volume is preferably 300-500 ⁇ L, more preferably 400 ⁇ L.
  • a liquid inlet pipe is arranged at the bottom end of the reaction tank cavity, and the liquid inlet pipe doubles as a liquid discharge pipe; an overflow cavity and a waste liquid extraction pipe are arranged on the top end of the reaction tank.
  • a magnetic stirrer is provided at the bottom of the cavity of the reaction cell.
  • the detection electrode when the detection electrode includes a glucose oxidase electrode and a lactase oxidase electrode, the glucose oxidase electrode, the lactase oxidase electrode and the auxiliary electrode are respectively installed in the reaction cell cavity of the immobilized enzyme reactor
  • the front ends of the glucose oxidase electrode, lactase oxidase electrode and auxiliary electrode are respectively connected to the reaction cell cavity in a sealed manner through a sealing rubber ring.
  • the immobilized enzyme sensor before the immobilized enzyme sensor is used, it is preferable to install the immobilized enzyme sensor in an SBA biosensing analyzer for pretreatment; the pretreatment includes turning on the power, turning on, and Turn on the cleaning pump and empty the pump to ensure that the buffer solution with a pH of 7.0 required by the sensing system fills the reaction tank to ensure the stability of the biosensing system.
  • a standard electrode active material solution is used to test the integrity of the electrode inner membrane, the electrical signal response value of the detection electrode to the standard electrode active material is An, and the electrical signal response value of the auxiliary electrode to the standard electrode active material is A0.
  • the electrical signal response value of the glucose oxidase electrode to the sample is A1
  • the electrical signal response value of the lactate oxidase electrode to the sample is A2.
  • the standard electrode active material is preferably a potassium ferrocyanide solution
  • the concentration of the potassium ferrocyanide solution is preferably 0.04 to 0.06 mol/L, more preferably 0.05 mol/L
  • the volume of the potassium ferricyanide solution is preferably 20-30 ⁇ L, more preferably 25 ⁇ L.
  • it is preferable that the potassium ferrocyanide is placed in the reaction cell cavity for 20-30 seconds, and then the electrical signal response values of the detection electrode and the auxiliary electrode are measured.
  • the detection electrode when the detection electrode generates an electrical signal response value, it indicates that the inner membrane of the detection electrode is incomplete; the correction is performed based on the ratio of the signal generated by the auxiliary electrode and the detection electrode to the active material of the electrode.
  • the standard product is calibrated after the completion of the test of the inner membrane of the electrode; when the target substance includes glucose and lactic acid, the standard product is a mixed solution of glucose and lactic acid; in the present invention, The concentration of glucose in the standard product is preferably 0.8-1.2 mg/mL, more preferably 1.0 mg/mL; the concentration of lactic acid in the standard product is preferably 0.8-1.2 mg/mL, more preferably 1.0 mg/mL.
  • the standard product is placed in the reaction cell, and the electrical signal response value is detected 20 seconds later. In the present invention, it is preferable to continuously measure 3 to 5 times. When the results of two consecutive measurements, the error of the response values of the glucose oxidase detection electrode and the lactate oxidase detection electrode are both ⁇ 1%, and the calibration is passed.
  • the sample is detected.
  • the sample is placed in the reaction cell, and the electrical signal response value is detected 20 seconds later.
  • the content of the target substance in the sample Xn ⁇ (100/Sn)-(A0/An) ⁇ (100/Sn); where n is a natural number ⁇ 1.
  • the electrical signal response value of the glucose oxidase electrode to the sample is X1
  • the electrical signal response value of the lactate oxidase electrode to the sample is X2
  • the electrical signal response value of the auxiliary electrode to the sample is X0.
  • the number of detections is preferably 1 to 5, and more preferably 3; the electrical signal response value of the detection is preferably an average value of 1 to 5 detections.
  • the program uses the last electrical signal response value to test the standard value.
  • the glucose electrode S1 and the lactic acid electrode S2 are recorded as 1214 and 2099, respectively.
  • Example 1 (1) According to the steps (1), (2), and (3) in Example 1, the glucose electrode, the lactate electrode and the auxiliary enzyme electrode were prepared, and the anti-interference performance of the inner membrane of the two electrodes was tested. target.
  • Example 2 Take the fermentation broth described in Example 1 and divide it into two equal volumes, one of which is added with an equal volume of a mixture of glucose and lactic acid with a concentration of 2g/L to make a spiked sample, and the other fermentation broth As a control sample.
  • Example 2 Take the fermentation broth described in Example 1 and divide it into three parts, and one part is used as a control sample to directly determine the content of glucose and lactic acid. The other two were added with ascorbic acid, H 2 O 2 and other easily residual electrical activity interference substances to prepare experimental samples with final concentrations of ascorbic acid and H 2 O 2 of 50 mg/100 ml and 20 mg/100 ml, respectively, for standby testing.
  • the present invention adds an auxiliary enzyme electrode to test the reference value of the detection electrode and the auxiliary electrode, and simultaneously corrects the fluctuation and change of the signal caused by the interference by the two electrodes, which can avoid the interference of the active interfering substances and improve the detection efficiency. Accuracy.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Electrochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

一种固定化酶电极、固定化酶传感器及其酶膜抗干扰的检测方法，属于生物传感器技术领域。固定化酶电极包括检测电极和辅助电极；辅助电极包括依次连接的基础电极、固定化失活酶层和载体膜，固定化失活酶层固定的酶为失活的酶。在样品测定时，测试检测电极和辅助电极基准值，由检测电极和辅助电极同时修正干扰所引起的信号的波动和变化，避免了由于酶膜"完整性"问题所造成的电极信号的干扰，提高测定准确性。

Description

一种固定化酶电极、固定化酶传感器及其酶膜抗干扰的检测方法 技术领域
本发明属于生物传感器技术领域，尤其涉及一种固定化酶电极、固定化酶传感器及其酶膜抗干扰的检测方法。
背景技术
生物传感器是以生物分子识别为基础、以生物活性材料为敏感元件，通过适当的理化换能器及信号放大装置将被测物质的浓度转换为数字信号的检测仪器。其中，采用固定化酶膜的经典电流型酶电极是商品化最早、应用市场最大的生物传感器类型。
电流型酶电极多采用固定化氧化酶(葡萄糖氧化酶膜、乳酸氧化酶膜等)为识别元件、以H 2O 2基础电极为换能器。其显著优势是具有很高的选择性，被测样品中多种组分可以不经前处理分离就可直接测定。但由于样品来源不同，组分种类差别很大，特别是一些样品中残存的电活性组分会产生干扰信号，影响测定的准确性，如血液样品中微量的抗坏血酸、食品样品中残留的H 2O 2等对葡萄糖、乳酸等检测结果有干扰。
为避免样品中残存电活性组分的干扰，固定化酶电极中的酶膜采用了三层结构(图1)。以葡萄糖氧化酶(GOD)为例，有(1)固定化GOD酶膜包括聚碳酸酯微孔膜(载体膜)、(2)固定化酶层和(3)醋酸纤维素内膜三层膜。GOD固定在碳酸酯微孔膜上，样品中的葡萄糖和氧分子可透过聚碳酸酯微孔膜与GOD接触，发生催化反应，产生过氧化氢。过氧化氢透过醋酸纤维膜扩散到电极表面，产生电流。电流大小与样品中的葡萄糖浓度有比例关系。通常酶膜粘附在一个橡胶圈上，形成酶膜器件，便于储存和使用，也起到与反应池连接的密封作用，保持酶膜(内膜)的完整性是防止样品中电活性物质干扰的关键。
但在酶膜制备或运输、储藏、安装、使用过程中，可能会造成内膜的损伤、或不完 整，形成内膜“泄露”，不能阻止小分子的电活性干扰物通过内膜扩散到电极表面，产生干扰。另外，有些食品样品中残存H 2O 2能穿过内膜，也造成结果偏差。
发明内容
有鉴于此，本发明的目的在于提供一种固定化酶电极、固定化酶传感器及其酶膜抗干扰的检测方法。
为了实现上述发明目的，本发明提供了以下技术方案：
一种固定化酶电极，包括检测电极和辅助电极；所述辅助电极包括依次连接的基础电极、固定化失活酶层和载体膜，所述固定化失活酶层固定的酶为失活的酶。
优选的，所述检测电极包括依次连接的基础电极、醋酸纤维素膜、固定化酶层和载体膜。
优选的，所述载体膜为聚碳酸酯微孔膜。
优选的，所述检测电极包括葡萄糖氧化酶电极和/或乳酸酶氧化酶电极。
优选的，所述基础电极为过氧化氢电极。
本发明提供了一种固定化酶传感器，包括所述的固定化酶电极。
优选的，所述固定化酶电极中的检测电极和辅助电极分别安装在固定化酶反应器的反应池腔的不同侧面；所述检测电极、辅助电极的前端分别通过橡胶圈与反应池腔封闭连接；所述检测电极、辅助电极的后端分别通过电极导线与主机连接。
优选的，所述检测电极包括葡萄糖氧化酶电极和乳酸酶氧化酶电极。
本发明提供了一种固定化酶电极酶膜抗干扰的检测方法，包括以下步骤：
1)采用标准电极活性物的溶液进行电极内膜完整性测试，检测电极对标准电极活性物的电信号响应值为An，辅助电极对标准电极活性物的电信号响应值为A0；
2)采用标准品溶液进行定标；所述标准品溶液为包括确定浓度目标物质的溶液；
3)对样品进行检测，检测电极对样品的电信号响应值为Xn，辅助电极对样品的电信 号响应值为X0，根据电信号响应值计算样品中目标物质的含量；样品中目标物质的含量＝Xn×(100/Sn)-(A0/An)×(100/Sn)；其中n为≥1的自然数。
优选的，所述标准电极活性物为亚铁氰化钾溶液，所述目标物质包括葡萄糖和乳酸。
本发明的有益效果：本发明提供一种固定化酶电极，包括检测电极和辅助电极；所述辅助电极包括依次连接的基础电极、固定化失活酶层和载体膜。本发明中辅助电极不包括醋酸纤维素内膜，并且所述固定化失活酶层固定的酶为失活的酶；在样品测定时，测试检测电极和辅助电极基准值，由检测电极和辅助电极同时修正干扰所引起的信号的波动和变化，从而避免了由于酶膜“完整性”问题所造成的电极信号的干扰，提高测定准确性。根据实施例记载，在加入电活性干扰物的实验样品中葡萄糖、乳酸含量的测定结果与未加干扰物的对照样品的两指标的测定结果无明显差异，即电活性干扰物的存在，对于本发明利用辅助电极的进行的抗干扰检测方法没有明显影响；说明本发明提供的固定化酶电极和抗干扰检测方法，检测结果准确，不受电活性干扰物质的影响，具有显著优势。
附图说明
图1为检测电极固定化酶膜与基础电极结构；
图2为实施例中固定化酶传感器的反应池及三电极结构，其中上图为主视图，下图为剖视图；其中1.反应池模块；2.反应池腔；3.酶膜的‘o’型圈；4.进样和废液流出通道；5.反应池顶帽；6.进样帽；7.进样光传感器；8.酶电极；9.酶电极旋钮；10.缓冲液进口和废液排空管；11.废液吸出管；12.电磁搅拌子；13.辅助酶电极；14.电极导线。
具体实施方式
本发明提供了一种固定化酶电极，包括检测电极和辅助电极；所述辅助电极包括依次连接的基础电极、固定化失活酶层和载体膜，所述固定化失活酶层固定的酶为失活的酶。
在本发明中，所述辅助电极包括依次连接的基础电极、固定化失活酶层和载体膜； 所述载体膜优选为聚碳酸酯微孔膜，所述固定化失活酶层固定的酶为失活的酶，所述失活的酶优选的通过高温加热灭活。在本发明中，所述辅助电极不包括作为“内膜”的醋酸纤维素膜；所述辅助电极的作用为校正检测数据，排除电活性干扰物，提高检测结果的准确度。
在本发明中，所述检测电极包括依次连接的基础电极、醋酸纤维素膜、固定化酶层和载体膜。在本发明中，所述载体膜优选为聚碳酸酯微孔膜。在本发明中，所述检测电极的结构优选的如图1所示。
在本发明中，所述检测电极包括葡萄糖氧化酶电极和/或乳酸酶氧化酶电极，优选的包括葡萄糖氧化酶电极和乳酸酶氧化酶电极；当所述检测电极为葡萄糖氧化酶电极和乳酸酶氧化酶电极时，所述基础电极优选为过氧化氢电极。在本发明中，所述检测电极中的葡萄糖氧化酶电极和乳酸酶氧化酶电极的固定化酶层分别涂布有活性的葡萄糖氧化酶和乳酸酶氧化酶。
在本发明中，所述检测电极的载体膜前端还包括橡胶密封圈，所述检测电极通过所述橡胶密封圈与反应池密封连接。
本发明对所述检测电极和辅助电极的原料包括基础电极、醋酸纤维素膜、固定化酶层和载体膜的来源没有特殊限定，采用本领域常规的市售产品或自行制备获得。
本发明提供了一种固定化酶传感器，包括所述的固定化酶电极。在本发明中，所述固定化酶电极中的检测电极和辅助电极分别安装在固定化酶反应器的反应池腔的不同侧面；所述检测电极、辅助电极的前端分别通过橡胶圈与反应池腔封闭连接；所述检测电极、辅助电极的后端分别通过电极导线与主机连接。
在本发明中，所述固定化酶传感器的结构优选的如图2所示，其中1.反应池模块；2.反应池腔；3.酶膜的‘o’型圈；4.进样和废液流出通道；5.反应池顶帽；6.进样帽； 7.进样光传感器；8.酶电极；9.酶电极旋钮；10.缓冲液进口和废液排空管；11.废液吸出管；12.电磁搅拌子；13.辅助酶电极；14.电极导线。
在本发明中，所述反应池模块优选的采用方形有机玻璃块制成；所述反应池在反应池模块模块的中心位置，所述反应池优选为一个圆柱形空腔，所述空腔的腔体积优选为300～500μL，更优选为400μL。在本发明中，所述反应池腔的底端设置有进液管，所述进液管兼作排液管；所述反应池的顶端设置有溢流腔和废液抽出管。在本发明中，所述反应池的腔底部设置有磁性搅拌子。在本发明中，当所述检测电极包括葡萄糖氧化酶电极和乳酸酶氧化酶电极时，所述葡萄糖氧化酶电极、乳酸酶氧化酶电极和辅助电极分别安装在固定化酶反应器的反应池腔的不同侧面；所述葡萄糖氧化酶电极、乳酸酶氧化酶电极和辅助电极的前端分别通过密封橡胶圈与反应池腔密封连接。
本发明还提供了一种固定化酶电极酶膜抗干扰的检测方法，包括以下步骤：1)采用标准电极活性物的溶液进行电极内膜完整性测试，检测电极对标准电极活性物的电信号响应值为An，辅助电极对标准电极活性物的电信号响应值为A0；2)采用标准品溶液进行定标；所述标准品溶液为包括确定浓度目标物质的溶液；3)对样品进行检测，检测电极对样品的电信号响应值为Xn，辅助电极对样品的电信号响应值为X0，根据电信号响应值计算样品中目标物质的含量，样品中目标物质的含量＝Xn×(100/Sn)-(A0/An)×(100/Sn)；其中n为≥1的自然数。
在本发明中，在所述固定化酶传感器使用之前，优选的将所述所述固定化酶传感器装于SBA生物传感分析仪中进行预处理；所述预处理包括接通电源、开机、开启清洗泵、排空泵，确保传感系统所需的pH值为7.0的缓冲液充满反应池，确保生物传感系统稳定。在本发明中，采用标准电极活性物的溶液进行电极内膜完整性测试，检测电极对标准电极活性物的电信号响应值为An，辅助电极对标准电极活性物的电信号响应值为A0。在本发明中，所述葡萄糖氧化酶电极对样品的电信号响应值为A1，所述乳酸氧化酶电极对样品的电信号响应值为A2。在本发明中，所述标准电极活性物优选为亚铁氰化钾溶液，所 述亚铁氰化钾溶液的浓度优选为0.04～0.06mol/L，更优选为0.05mol/L；所述亚铁氰化钾溶液的体积优选为20～30μL，更优选为25μL。在本发明具体实施过程中，优选的将所述亚铁氰化钾置于反应池腔体内20～30s后，测定所述检测电极和辅助电极的电信号响应值。在本发明中，当所述检测电极产生电信号响应值时，说明检测电极的内膜不完整；根据辅助电极与检测电极对电极活性物质产生的信号比例关系，进行校正。
本发明在所述电极内膜完整性测试完成后，进行标准品的定标；当所述目标物质包括葡萄糖和乳酸时，所述标准品为葡萄糖和乳酸的混合溶液；在本发明中，所述标准品中葡萄糖的浓度优选为0.8～1.2mg/mL，更优选为1.0mg/mL；所述标准品中乳酸的浓度优选为0.8～1.2mg/mL，更优选为1.0mg/mL。在本发明中，将标准品置于反应池中，20s后检测电信号响应值。在本发明中，优选的连续测定3～5次，当连续两次测定的结果，葡萄糖氧化酶检测电极和乳酸氧化酶检测电极的响应值误差均≤1％，定标通过。
本发明在所述定标通过后，进行样品的检测。在本发明中，将所述样品置于反应池中，20s后检测电信号响应值。根据电信号响应值计算样品中目标物质的含量，样品中目标物质的含量＝Xn×(100/Sn)-(A0/An)×(100/Sn)；其中n为≥1的自然数。在本发明中，所述葡萄糖氧化酶电极对样品的电信号响应值为X1，所述乳酸氧化酶电极对样品的电信号响应值为X2；辅助电极对样品的电信号响应值为X0。
在本发明中，所述检测的次数优选为1～5次，更优选为3次；所述检测的电信号响应值优选为1～5次检测的平均值。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
(1)酶电极制备：按图1结构制备葡萄糖氧化酶电极、乳酸氧化酶电极。辅助酶电极不涂布作为“内膜”的醋酸纤维素膜，且所用氧化酶经加热灭活。将三个电极按图2反应池结构安装后，装于SBA生物传感分析仪，接通电源，开机，开启清洗泵、排空泵， 传感系统所需pH值为7.0缓冲液充满反应池，生物传感系统稳定。所述pH值为7.0缓冲液为0.1mmol磷酸缓冲液；常温保存，有效期为24个月。
(2)酶电极内膜完整测试：吸取25μL、0.05mol/L亚铁氰化钾注入反应池，20s反应结束，分别记录葡萄糖氧化酶电极、乳酸氧化酶电极、辅助酶电极所对应电信号，葡萄糖氧化酶电极电信号响应值为A1，乳酸氧化酶电极电信号响应值为A2，辅助电极电信号响应值为A0，重复三次，取平均值，
Figure PCTCN2021071159-appb-000001
分别为15、18、138，以
Figure PCTCN2021071159-appb-000002
Figure PCTCN2021071159-appb-000003
分别作为葡萄糖电极内膜、乳酸电极内膜完整性及抗干扰性能指标。
(3)定标：准确吸取25μL葡萄糖(100mg/100ml)+乳酸(100mg/100ml)混合标准液注入反应池。20s反应结束后，仪器分别自动记录葡萄糖电极电信号值S1(1238)、乳酸电极电信号值S2(2146)、辅助电极电信号值A0(程序后台记录值并比对)。连续测定3～5次，连续两次进样葡萄糖电极响应值分别为S1(1220、1214)乳酸电极响应值分别为S2(2120、2099)，两电极响应值误差≤1％，定标通过。
表1电极定标检测结果
Figure PCTCN2021071159-appb-000004
程序以最后一次电信号响应值测试标准值，本发明中葡萄糖电极S1、乳酸电极S2分别记为1214、2099。
(4)葡萄糖、乳酸样品测定：运行检测样品程序，定标通过后，向反应池中注入25μL含葡萄糖、乳酸底物的发酵液。20s反应结束，葡萄糖氧化酶电极对样品的响应值为X1，乳酸氧化酶电极对样品的响应值为X2，重复测定3次，通过如下公式：
Figure PCTCN2021071159-appb-000005
Figure PCTCN2021071159-appb-000006
计算并显示测定结果，结果如表2。
表2样品测定结果
Figure PCTCN2021071159-appb-000007
实施例2
(1)按实施例1中(1)、(2)、(3)步骤所述，制备葡萄糖电极、乳酸电极以及辅助酶电极，并测试了两电极内膜抗干扰性能，后对仪器进行了定标。
(2)取实施例1中所述发酵液，分成等体积的两份，其中一份加入等体积，浓度为2g/L的葡萄糖、乳酸混合液，制成加标样品，另一份发酵液作为对照样品。
(3)运行检测样品程序，定标通过后，向反应池中注入25μL对照样品。经20s反应结束，仪器记录并显示电信号响应值，重复测定3次，得平均值
Figure PCTCN2021071159-appb-000008
再次启动样品测定程序，注入25μL加标样品，由仪器记录并显示信号响应值，重复测定3次，得平均值
Figure PCTCN2021071159-appb-000009
通过公式：
Figure PCTCN2021071159-appb-000010
Figure PCTCN2021071159-appb-000011
分别计算对照样品、加标样品中葡萄糖、乳酸含量，并计算加标回收率。结果如表3所示。
表3加标样品测定结果
Figure PCTCN2021071159-appb-000012
实施例3
(1)按实施例1中(1)、(2)、(3)步骤所述，制备葡萄糖电极、乳酸电极以及辅助酶电极，测试两电极内膜抗干扰性能，标定仪器后待用。
(2)取实施例1中所述发酵液，分为三份，一份用作对照样品直接进行葡萄糖、乳酸含量测定。另两份分别加入抗坏血酸、H 2O 2等易残留的电活性干扰物，制备抗坏血酸、H 2O 2终浓度分别为50mg/100ml、20mg/100ml的实验样品，备用待测。
(3)运行检测样品程序，定标通过后，向反应池中注入25μL对照样品。经20s反应结束，仪器记录并显示电信号响应值，重复测定3次。再次启动样品测定程序，注入25μL实验样品，由仪器记录并显示信号响应值，重复测定3次。通过公式：
Figure PCTCN2021071159-appb-000013
Figure PCTCN2021071159-appb-000014
分别计算对照样品、实验样品中葡萄糖、乳酸含量，计算标准差，并通过t检验比较对照样品和实验样品的差异性，结果如表4所示。
表4电活性干扰物质对检测结果的影响
Figure PCTCN2021071159-appb-000015
本实施例中，加入电活性干扰物的实验样品中葡萄糖、乳酸含量的测定结果与未加干扰物的对照样品的两指标的测定结果无明显差异，即电活性干扰物的存在，对于本发 明通过添加辅助酶电极的抗干扰检测方法没有明显影响。
由上述实施例可知，本发明通过加入辅助酶电极，测试检测电极、辅助电极基准值，由两电极同时修正干扰所引起的信号的波动和变化，能够避免点活性干扰物质的干扰，提高检测的准确度。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种固定化酶电极，其特征在于，包括检测电极和辅助电极；所述辅助电极包括依次连接的基础电极、固定化失活酶层和载体膜，所述固定化失活酶层固定的酶为失活的酶。
  2. 根据权利要求1所述的固定化酶电极，其特征在于，所述检测电极包括依次连接的基础电极、醋酸纤维素膜、固定化酶层和载体膜。
  3. 根据权利要求1或2所述的固定化酶电极，其特征在于，所述载体膜为聚碳酸酯微孔膜。
  4. 根据权利要求1所述的固定化酶电极，其特征在于，所述检测电极包括葡萄糖氧化酶电极和/或乳酸酶氧化酶电极。
  5. 根据权利要求1或4所述的固定化酶电极，其特征在于，所述基础电极为过氧化氢电极。
  6. 一种固定化酶传感器，其特征在于，包括权利要求1～5任意一项所述的固定化酶电极。
  7. 根据权利要求6所述的固定化酶传感器，其特征在于，所述固定化酶电极中的检测电极和辅助电极分别安装在固定化酶反应器的反应池腔的不同侧面；所述检测电极、辅助电极的前端分别通过橡胶圈与反应池腔封闭连接；所述检测电极、辅助电极的后端分别通过电极导线与主机连接。
  8. 根据权利要求7所述的固定化酶传感器，其特征在于，所述检测电极包括葡萄糖氧化酶电极和乳酸酶氧化酶电极。
  9. 一种固定化酶电极酶膜抗干扰的检测方法，包括以下步骤：
    1)采用标准电极活性物的溶液进行电极内膜完整性测试，检测电极对标准电极活性物的电信号响应值为An，辅助电极对标准电极活性物的电信号响应值为A0；
    2)采用标准品溶液进行定标；所述标准品溶液为包括确定浓度目标物质的溶液；
    3)对样品进行检测，检测电极对样品的电信号响应值为Xn，辅助电极对样品的电信号响应值为X0，根据电信号响应值计算样品中目标物质的含量，样品中目标物质的含量＝Xn×(100/Sn)-(A0/An)×(100/Sn)；其中n为≥1的自然数。
  10. 根据权利要求9所述的检测方法，其特征在于，所述标准电极活性物为亚铁氰化钾溶液，所述目标物质包括葡萄糖和乳酸。
PCT/CN2021/071159 2020-05-12 2021-01-12 一种固定化酶电极、固定化酶传感器及其酶膜抗干扰的检测方法 WO2021227542A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010396177.3A CN111398386A (zh) 2020-05-12 2020-05-12 一种固定化酶电极、固定化酶传感器及其酶膜抗干扰的检测方法
CN202010396177.3 2020-05-12

Publications (1)

Publication Number Publication Date
WO2021227542A1 true WO2021227542A1 (zh) 2021-11-18

Family

ID=71435741

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/071159 WO2021227542A1 (zh) 2020-05-12 2021-01-12 一种固定化酶电极、固定化酶传感器及其酶膜抗干扰的检测方法

Country Status (2)

Country Link
CN (1) CN111398386A (zh)
WO (1) WO2021227542A1 (zh)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111398386A (zh) * 2020-05-12 2020-07-10 山东省科学院生物研究所 一种固定化酶电极、固定化酶传感器及其酶膜抗干扰的检测方法
CN112415062B (zh) * 2020-11-27 2022-09-16 山东省科学院生物研究所 一种快速检测乙醇的电极系统及使用电极系统对乙醇检测的方法
CN112505122B (zh) * 2020-12-17 2024-03-15 山东省科学院生物研究所 一种双指标酶电极检测装置对底物和产物的测试方法
CN113758982B (zh) * 2021-05-24 2023-06-02 中国科学院天津工业生物技术研究所 一种基于消除小分子电极的电化学装置

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6135786A (ja) * 1984-07-26 1986-02-20 Nec Corp 固定化酵素膜の部分的失活方法
JPS6275346A (ja) * 1985-09-30 1987-04-07 Nok Corp 酵素センサ−
JPS6350748A (ja) * 1986-08-20 1988-03-03 Sanyo Electric Co Ltd 酵素電極システム
JPS63186139A (ja) * 1987-01-29 1988-08-01 Nec Corp 酵素固定化膜の選択的失活方法
JPH08327587A (ja) * 1995-05-31 1996-12-13 Nec Corp バイオセンサ素子の製造方法
CN110514704A (zh) * 2018-05-22 2019-11-29 爱科来株式会社 新型生物传感方法
CN111398386A (zh) * 2020-05-12 2020-07-10 山东省科学院生物研究所 一种固定化酶电极、固定化酶传感器及其酶膜抗干扰的检测方法
CN211955306U (zh) * 2020-05-12 2020-11-17 山东省科学院生物研究所 一种固定化酶电极和固定化酶传感器

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6135786A (ja) * 1984-07-26 1986-02-20 Nec Corp 固定化酵素膜の部分的失活方法
JPS6275346A (ja) * 1985-09-30 1987-04-07 Nok Corp 酵素センサ−
JPS6350748A (ja) * 1986-08-20 1988-03-03 Sanyo Electric Co Ltd 酵素電極システム
JPS63186139A (ja) * 1987-01-29 1988-08-01 Nec Corp 酵素固定化膜の選択的失活方法
JPH08327587A (ja) * 1995-05-31 1996-12-13 Nec Corp バイオセンサ素子の製造方法
CN110514704A (zh) * 2018-05-22 2019-11-29 爱科来株式会社 新型生物传感方法
CN111398386A (zh) * 2020-05-12 2020-07-10 山东省科学院生物研究所 一种固定化酶电极、固定化酶传感器及其酶膜抗干扰的检测方法
CN211955306U (zh) * 2020-05-12 2020-11-17 山东省科学院生物研究所 一种固定化酶电极和固定化酶传感器

Also Published As

Publication number Publication date
CN111398386A (zh) 2020-07-10

Similar Documents

Publication Publication Date Title
WO2021227542A1 (zh) 一种固定化酶电极、固定化酶传感器及其酶膜抗干扰的检测方法
US4467811A (en) Method of polarographic analysis of lactic acid and lactate
WO2021223316A1 (zh) 基于锌配位黑磷纳米片复合物与生物酶催化的比率电化学尿酸传感器的制备方法
Zhang et al. Amperometric biosensor for uric acid based on uricase-immobilized silk fibroin membrane
JPH05164724A (ja) バイオセンサーおよびそれを用いた分離定量方法
RU2602170C2 (ru) Способ измерения аналита и система с компенсацией гематокрита
TW201350840A (zh) 血液樣本之血糖值的校正方法及其檢測試片與校正系統
US20240085362A1 (en) Method for improving stability of electrochemical sensor
Wu et al. Chemiluminescence biosensor system for lactic acid using natural animal tissue as recognition element
CN108627559B (zh) 基于铝-普鲁士蓝自供能电化学传感器构建的免疫分析方法
Kihara et al. Determination of creatinine with a sensor based on immobilized glutamate dehydrogenase and creatinine deiminase
CN211955306U (zh) 一种固定化酶电极和固定化酶传感器
Petersson Amperometric assay of glucose and lactic acid by flow injection analysis
WO2023246298A1 (zh) 电化学生物传感器及其制备方法
TW201719161A (zh) 用於補償基於測試條的極化效應的樣本相關測量之系統及方法
CN214310287U (zh) 一种双指标酶电极检测装置
CN112505122A (zh) 一种双指标酶电极检测装置和对生物反应器内底物和产物的在线测试方法
JPH029302B2 (zh)
US7794778B2 (en) Amperometric sensor for uric acid and method for the same
CN103163190A (zh) 用于单用途生物反应器/混合器的可拆装式溶解氧传感器
US20090120808A1 (en) Device for Detecting Methanol Concentration and the Method Thereof
Vokhmyanina et al. Prussian Blue-Based Thin-Layer Flow-Injection Multibiosensor for Simultaneous Determination of Glucose and Lactate
CN110988079A (zh) 体外诊断测试条制备方法以及诊断测试方法
US5320939A (en) Measuring apparatus of two components using enzyme electrodes and the measuring method thereof
CN215218663U (zh) 带有红细胞压积补偿的电化学试纸及其检测系统

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21803541

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 16/03/2023)

122 Ep: pct application non-entry in european phase

Ref document number: 21803541

Country of ref document: EP

Kind code of ref document: A1