An immobilized enzyme electrode, an immobilized enzyme sensor and a detection method for anti-interference of an enzyme membrane

Abstract

The present invention provides an immobilized enzyme electrode, an immobilized enzyme sensor and a detection method for anti-interference of an enzyme membrane, belonging to the technical field of biosensors. In the immobilized enzyme electrode of the present invention, it includes 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 enzyme immobilized in the immobilized inactivated enzyme layer is an inactivated enzyme. When measuring a sample in the present invention, the reference values of the detection electrode and the auxiliary electrode are tested, and the fluctuations and changes of the signals caused by interference are corrected simultaneously by the detection electrode and the auxiliary electrode, thereby avoiding the interference of the electrode signals caused by the "integrity" problem of the enzyme membrane and improving the measurement accuracy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and particularly relates to an immobilized enzyme electrode, an immobilized enzyme sensor, and a detection method for anti-interference of an enzyme membrane. Background Art

[0002] A biosensor is a detection instrument that is based on biomolecular recognition, uses bioactive materials as sensitive elements, and converts the concentration of a substance to be measured into a digital signal through an appropriate physicochemical transducer and signal amplification device. Among them, the classical amperometric enzyme electrode using an immobilized enzyme membrane is the type of biosensor with the earliest commercialization and the largest application market.

[0003] Amperometric enzyme electrodes mostly use immobilized oxidases (such as glucose oxidase membrane, lactate oxidase membrane, etc.) as recognition elements and a H2O2-based electrode as a transducer. Its significant advantage is high selectivity, and various components in the sample to be measured can be directly measured without pretreatment separation. However, due to different sample sources, the component types vary greatly. In particular, some electroactive components remaining in the sample will generate interference signals, affecting the accuracy of the measurement. For example, trace amounts of ascorbic acid in blood samples and residual H2O2 in food samples interfere with the detection results of glucose, lactate, etc.

[0004] To avoid interference from electroactive components remaining in the sample, the enzyme membrane in the immobilized enzyme electrode adopts a three-layer structure ( Figure 1 ). Taking glucose oxidase (GOD) as an example, (1) the immobilized GOD enzyme membrane includes a polycarbonate microporous membrane (carrier membrane), (2) an immobilized enzyme layer, and (3) a cellulose acetate inner membrane. GOD is fixed on the carbonate microporous membrane. Glucose and oxygen molecules in the sample can pass through the polycarbonate microporous membrane to contact GOD, and a catalytic reaction occurs to generate hydrogen peroxide. The hydrogen peroxide diffuses through the cellulose acetate membrane to the electrode surface to generate a current. The magnitude of the current has a proportional relationship with the glucose concentration in the sample. Usually, the enzyme membrane adheres to a rubber ring to form an enzyme membrane device, which is convenient for storage and use and also plays a sealing role in connecting with the reaction cell. Maintaining the integrity of the enzyme membrane (inner membrane) is the key to preventing interference from electroactive substances in the sample.

[0005] However, during the preparation, transportation, storage, installation, and use of the enzyme membrane, damage or incompleteness of the inner membrane may occur, forming an "inner membrane leakage", which cannot prevent small-molecule electroactive interferents from diffusing through the inner membrane to the electrode surface to generate interference. In addition, residual H2O2 in some food samples can pass through the inner membrane, also causing result deviation. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide an immobilized enzyme electrode, an immobilized enzyme sensor, and a detection method for anti-interference of an enzyme membrane.

[0007] To achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0008] An immobilized enzyme electrode includes a detection electrode and a counter electrode; the counter electrode includes a base electrode, an immobilized inactivated enzyme layer, and a carrier membrane connected in sequence, and the enzyme immobilized in the immobilized inactivated enzyme layer is an inactivated enzyme.

[0009] Preferably, the detection electrode includes a base electrode, a cellulose acetate membrane, an immobilized enzyme layer, and a carrier membrane connected in sequence.

[0010] Preferably, the carrier membrane is a polycarbonate microporous membrane.

[0011] Preferably, the detection electrode includes a glucose oxidase electrode and / or a lactate oxidase electrode.

[0012] Preferably, the base electrode is a hydrogen peroxide electrode.

[0013] The present invention provides an immobilized enzyme sensor including the above-mentioned immobilized enzyme electrode.

[0014] Preferably, the detection electrode and the counter 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 counter electrode are respectively hermetically connected to the reaction cell cavity through rubber gaskets; the rear ends of the detection electrode and the counter electrode are respectively connected to the main unit through electrode wires.

[0015] Preferably, the detection electrode includes a glucose oxidase electrode and a lactate oxidase electrode.

[0016] The present invention provides a method for detecting anti-interference of an immobilized enzyme electrode enzyme membrane, including the following steps:

[0017] 1) Use a solution of a standard electrode active substance to perform an integrity test on the inner membrane of the electrode. The electro-signal response value of the detection electrode to the standard electrode active substance is An, and the electro-signal response value of the counter electrode to the standard electrode active substance is A0;

[0018] 2) Calibrate with a standard solution; the standard solution is a solution including a target substance with a determined concentration;

[0019] 3) Detect the sample. The electro-signal response value of the detection electrode to the sample is Xn, and the electro-signal response value of the counter electrode to the sample is X0. Calculate the content of the target substance in the sample according to the electro-signal response value; the content of the target substance in the sample = Xn×(100 / Sn) - (A0 / An)×(100 / Sn); where n is a natural number greater than or equal to 1.

[0020] Preferably, the standard electrode active substance is a potassium ferrocyanide solution, and the target substances include glucose and lactic acid.

[0021] Advantages of the present invention: 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. In the present invention, the auxiliary electrode does not include an inner cellulose acetate membrane, and the enzyme immobilized in the immobilized inactivated enzyme layer is an inactivated enzyme; during sample determination, the reference values of the detection electrode and the auxiliary electrode are measured, and the fluctuations and changes of the signals caused by interference are corrected simultaneously by the detection electrode and the auxiliary electrode, thereby avoiding the interference of the electrode signal caused by the "integrity" problem of the enzyme membrane and improving the measurement accuracy. According to the records of the embodiments, there is no obvious difference in the measurement results of the glucose and lactic acid contents in the experimental samples with electroactive interferents and the two indicators of the control samples without interferents, that is, the presence of electroactive interferents has no obvious influence on the anti-interference detection method using the auxiliary electrode in the present invention; it shows that the immobilized enzyme electrode and the anti-interference detection method provided by the present invention have accurate detection results, are not affected by electroactive interfering substances, and have significant advantages. Description of the Drawings

[0022] Figure 1 For the structure of the immobilized enzyme membrane of the detection electrode and the basic electrode;

[0023] Figure 2 For the reaction cell and the three-electrode structure of the immobilized enzyme sensor in the embodiment, where the upper figure is the front view and the lower figure is the cross-sectional view; where 1. reaction cell module; 2. reaction cell cavity; 3. 'O'-ring of the enzyme membrane; 4. sample injection and waste liquid outflow channel; 5. reaction cell top cap; 6. sample injection cap; 7. sample injection optical sensor; 8. enzyme electrode; 9. enzyme electrode knob; 10. buffer inlet and waste liquid drain pipe; 11. waste liquid suction pipe; 12. electromagnetic stirrer; 13. auxiliary enzyme electrode; 14. electrode wire. Detailed Embodiments

[0024] 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 enzyme immobilized in the immobilized inactivated enzyme layer is an inactivated enzyme.

[0025] In the present invention, 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, the enzyme immobilized in the immobilized inactivated enzyme layer is an inactivated enzyme, and the inactivated enzyme is preferably inactivated by high-temperature heating. In the present invention, the auxiliary electrode does not include a cellulose acetate membrane as the "inner membrane"; the function of the auxiliary electrode is to correct the detection data, eliminate electroactive interferents, and improve the accuracy of the detection results.

[0026] In the present invention, the detection electrode comprises a base electrode, a cellulose acetate membrane, an immobilized enzyme layer and a carrier membrane which are connected in sequence. In the present invention, the carrier membrane is preferably a polycarbonate microporous membrane. In the present invention, the structure of the detection electrode is preferably as Figure 1 shown.

[0027] In the present invention, the detection electrode comprises a glucose oxidase electrode and / or a lactate oxidase electrode, preferably comprising a glucose oxidase electrode and a lactate oxidase electrode; when the detection electrode is a glucose oxidase electrode and a lactate oxidase electrode, the base electrode is preferably a hydrogen peroxide electrode. In the present invention, the immobilized enzyme layers of the glucose oxidase electrode and the lactate oxidase electrode in the detection electrode are respectively coated with active glucose oxidase and lactate oxidase.

[0028] In the present invention, the front end of the carrier membrane of the detection electrode further comprises a rubber sealing ring, and the detection electrode is hermetically connected to the reaction cell through the rubber sealing ring.

[0029] The present invention has no special limitation on the sources of the raw materials of the detection electrode and the auxiliary electrode, including the base electrode, the cellulose acetate membrane, the immobilized enzyme layer and the carrier membrane, and they can be obtained by using conventional commercially available products in the art or by self-preparation.

[0030] The present invention provides an immobilized enzyme sensor comprising the immobilized enzyme electrode as described above. In the present invention, 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 hermetically connected to the reaction cell cavity through rubber rings; the rear ends of the detection electrode and the auxiliary electrode are respectively connected to the main unit through electrode wires.

[0031] In the present invention, the structure of the immobilized enzyme sensor is preferably as Figure 2 shown, where 1. reaction cell module; 2. reaction cell cavity; 3. 'O'-ring of the enzyme membrane; 4. sampling and waste liquid outflow channel; 5. reaction cell top cap; 6. sampling cap; 7. sampling optical sensor; 8. enzyme electrode; 9. enzyme electrode knob; 10. buffer inlet and waste liquid drain pipe; 11. waste liquid suction pipe; 12. electromagnetic stirrer; 13. auxiliary enzyme electrode; 14. electrode wire.

[0032] In the present invention, the reaction cell module is preferably made of a square plexiglass block; the reaction cell is located at the center of the reaction cell module, and the reaction cell is preferably a cylindrical cavity, and the cavity volume of the cavity is preferably 300-500 μL, more preferably 400 μL. In the present invention, a liquid inlet pipe is provided at the bottom end of the reaction cell cavity, and the liquid inlet pipe also serves as a liquid discharge pipe; an overflow cavity and a waste liquid extraction pipe are provided at the top end of the reaction cell. In the present invention, a magnetic stirrer is provided at the bottom of the cavity of the reaction cell. In the present invention, when the detection electrode includes a glucose oxidase electrode and a lactate oxidase electrode, the glucose oxidase electrode, the lactate oxidase electrode and the auxiliary electrode are respectively installed on different sides of the reaction cell cavity of the immobilized enzyme reactor; the front ends of the glucose oxidase electrode, the lactate oxidase electrode and the auxiliary electrode are respectively sealed and connected to the reaction cell cavity through a sealing rubber ring.

[0033] The present invention also provides a method for detecting anti-interference of the enzyme membrane of the immobilized enzyme electrode, including the following steps: 1) performing an integrity test on the inner membrane of the electrode using a solution of a standard electrode active substance, and the electro-signal response value of the detection electrode to the standard electrode active substance is An, and the electro-signal response value of the auxiliary electrode to the standard electrode active substance is A0; 2) performing calibration using a standard product solution; the standard product solution is a solution including a target substance with a determined concentration; 3) detecting the sample, the electro-signal response value of the detection electrode to the sample is Xn, and the electro-signal response value of the auxiliary electrode to the sample is X0, and calculating the content of the target substance in the sample according to the electro-signal response value, the content of the target substance in the sample = Xn×(100 / Sn)-(A0 / An)×(100 / Sn); where n is a natural number greater than or equal to 1.

[0034] In the present invention, before the immobilized enzyme sensor is used, it is preferably placed in an SBA biosensing analyzer for pretreatment; the pretreatment includes turning on the power supply, starting up, turning on the cleaning pump and the evacuation pump to ensure that the reaction cell is filled with a buffer solution with a pH value of 7.0 required by the sensing system and ensuring the stability of the biosensing system. In the present invention, a solution of a standard electrode active substance is used to test the integrity of the inner membrane of the electrode, and the electro-signal response value of the detection electrode to the standard electrode active substance is An, and the electro-signal response value of the auxiliary electrode to the standard electrode active substance is A0. In the present invention, the electro-signal response value of the glucose oxidase electrode to the sample is A1, and the electro-signal response value of the lactate oxidase electrode to the sample is A2. In the present invention, the standard electrode active substance is preferably a potassium ferrocyanide solution, and the concentration of the potassium ferrocyanide solution is preferably 0.04 - 0.06 mol / L, more preferably 0.05 mol / L; the volume of the potassium ferrocyanide solution is preferably 20 - 30 μL, more preferably 25 μL. In the specific implementation process of the present invention, preferably, after the potassium ferrocyanide is placed in the reaction cell cavity for 20 - 30 s, the electro-signal response values of the detection electrode and the auxiliary electrode are measured. In the present invention, when the detection electrode generates an electro-signal response value, it indicates that the inner membrane of the detection electrode is incomplete; calibration is performed according to the signal ratio relationship between the auxiliary electrode and the detection electrode to the electrode active substance.

[0035] After the completion of the test on the integrity of the inner membrane of the electrode in the present invention, calibration of the standard product is carried out; when the target substances include glucose and lactate, the standard product is a mixed solution of glucose and lactate; 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 lactate in the standard product is preferably 0.8 - 1.2 mg / mL, more preferably 1.0 mg / mL. In the present invention, the standard product is placed in the reaction cell, and the electro-signal response value is detected after 20 s. In the present invention, it is preferably measured continuously 3 - 5 times, and when the response value errors of the glucose oxidase detection electrode and the lactate oxidase detection electrode in the results of two consecutive measurements are both ≤ 1%, the calibration passes.

[0036] After the calibration passes in the present invention, the sample is detected. In the present invention, the sample is placed in the reaction cell, and the electro-signal response value is detected after 20 s. The content of the target substance in the sample is calculated according to the electro-signal response value, and the content of the target substance in the sample = Xn × (100 / Sn) - (A0 / An) × (100 / Sn); where n is a natural number ≥ 1. In the present invention, the electro-signal response value of the glucose oxidase electrode to the sample is X1, the electro-signal response value of the lactate oxidase electrode to the sample is X2; the electro-signal response value of the auxiliary electrode to the sample is X0.

[0037] In the present invention, the number of detections is preferably 1 to 5 times, more preferably 3 times; the detected electrical signal response value is preferably the average value of 1 to 5 detections.

[0038] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] Example 1

[0040] (1) Preparation of enzyme electrodes: Prepare glucose oxidase electrodes and lactate oxidase electrodes according to the Figure 1 structure. The auxiliary enzyme electrode is not coated with the cellulose acetate membrane as the "inner membrane", and the used oxidase is inactivated by heating. After installing the three electrodes according to the Figure 2 reaction cell structure, install them in the SBA biosensor analyzer, turn on the power, start the machine, turn on the cleaning pump and the evacuation pump, and fill the reaction cell with the pH 7.0 buffer solution required by the sensing system to stabilize the biosensing system. The pH 7.0 buffer solution is 0.1 mmol phosphate buffer solution; stored at room temperature, with a validity period of 24 months.

[0041] (2) Integrity test of the inner membrane of the enzyme electrode: Pipette 25 μL of 0.05 mol / L potassium ferrocyanide into the reaction cell. After the reaction ends in 20 s, record the electrical signals corresponding to the glucose oxidase electrode, lactate oxidase electrode, and auxiliary enzyme electrode respectively. The electrical signal response value of the glucose oxidase electrode is A1, the electrical signal response value of the lactate oxidase electrode is A2, and the electrical signal response value of the auxiliary electrode is A0. Repeat three times and take the average value. They are 15, 18, and 138 respectively. Take them as the integrity and anti-interference performance indicators of the inner membrane of the glucose electrode and the inner membrane of the lactate electrode respectively.

[0042] (3) Calibration: Accurately pipette 25 μL of the glucose (100 mg / 100 ml) + lactate (100 mg / 100 ml) mixed standard solution into the reaction cell. After the reaction ends in 20 s, the instrument automatically records the electrical signal value S1 (1238) of the glucose electrode, the electrical signal value S2 (2146) of the lactate electrode, and the electrical signal value A0 of the auxiliary electrode (recorded in the program background and compared). Continuously measure 3 to 5 times. The response values of the glucose electrode for two consecutive injections are S1 (1220, 1214) and the response values of the lactate electrode are S2 (2120, 2099) respectively. The error of the response values of the two electrodes is ≤1%, and the calibration is passed.

[0043] Table 1 Detection results of electrode calibration

[0044]

[0045] The program tests the standard value with the last electrical signal response value. In the present invention, the glucose electrode S1 and the lactate electrode S2 are denoted as 1214 and 2099 respectively.

[0046] (4) Determination of glucose and lactate samples: Run the test sample program. After calibration is passed, inject 25 μL of the fermentation broth containing glucose and lactate substrates into the reaction cell. After 20 s, the reaction ends. The response value of the glucose oxidase electrode to the sample is X1, and the response value of the lactate oxidase electrode to the sample is X2. Repeat the determination 3 times. Through the following formula:

[0047]

[0048]

[0049] Calculate and display the determination results, as shown in Table 2.

[0050] Table 2 Sample determination results

[0051]

[0052] Example 2

[0053] (1) According to the steps (1), (2), and (3) in Example 1, prepare the glucose electrode, lactate electrode, and auxiliary enzyme electrode, and test the anti-interference performance of the inner membrane of the two electrodes. Then, calibrate the instrument.

[0054] (2) Take the fermentation broth described in Example 1 and divide it into two equal volumes. Add an equal volume of a glucose and lactate mixture with a concentration of 2 g / L to one portion to make a spiked sample, and use the other portion of the fermentation broth as a control sample.

[0055] (3) Run the test sample program. After calibration is passed, inject 25 μL of the control sample into the reaction cell. After 20 s, the reaction ends. The instrument records and displays the electrical signal response value. Repeat the determination 3 times to obtain the average value Start the sample determination program again, inject 25 μL of the spiked sample, and the instrument records and displays the signal response value. Repeat the determination 3 times to obtain the average value Through the formula:

[0056]

[0057]

[0058] Calculate the glucose and lactate contents in the control sample and the spiked sample respectively, and calculate the spike recovery rate. The results are shown in Table 3.

[0059] Table 3 Spiked sample determination results

[0060]

[0061] Example 3

[0062] (1) Prepare a glucose electrode, a lactic acid electrode, and an auxiliary enzyme electrode according to the steps (1), (2), and (3) in Example 1. Test the anti-interference performance of the inner membrane of the two electrodes. After calibrating the instrument, it is ready for use.

[0063] (2) Take the fermentation broth described in Example 1 and divide it into three parts. One part is used as a control sample for direct determination of the glucose and lactic acid contents. The other two parts are respectively added with electroactive interfering substances such as ascorbic acid and H2O2 to prepare experimental samples with final concentrations of ascorbic acid and H2O2 of 50 mg / 100 ml and 20 mg / 100 ml respectively, and are reserved for future measurement.

[0064] (3) Run the detection sample program. After the calibration passes, inject 25 μL of the control sample into the reaction pool. After 20 s, the reaction ends, and the instrument records and displays the electro-signal response value, and repeat the measurement 3 times. Start the sample measurement program again, inject 25 μL of the experimental sample, and the instrument records and displays the signal response value, and repeat the measurement 3 times. Through the formula:

[0065]

[0066]

[0067] Calculate the glucose and lactic acid contents in the control sample and the experimental sample respectively, calculate the standard deviation, and compare the differences between the control sample and the experimental sample through the t-test. The results are shown in Table 4.

[0068] Table 4 Influence of electroactive interfering substances on the detection results

[0069]

[0070] In this example, there is no obvious difference between the measurement results of the glucose and lactic acid contents in the experimental sample added with electroactive interfering substances and the two indicators of the control sample without interfering substances. That is, the presence of electroactive interfering substances has no obvious influence on the anti-interference detection method of the present invention by adding an auxiliary enzyme electrode.

[0071] As can be seen from the above examples, by adding an auxiliary enzyme electrode in the present invention, testing the reference values of the detection electrode and the auxiliary electrode, and simultaneously correcting the fluctuations and changes of the signals caused by interference by the two electrodes, it is possible to avoid the interference of electroactive interfering substances and improve the accuracy of detection.

[0072] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An immobilized enzyme sensor, characterized in that, It includes a detection electrode and an auxiliary electrode; The detection electrode includes a base electrode, a cellulose acetate membrane, an immobilized enzyme layer, and a carrier membrane connected in sequence; the auxiliary electrode includes a base electrode, an immobilized inactivated enzyme layer, and a carrier membrane connected in sequence, and the enzyme immobilized in the immobilized inactivated enzyme layer is an inactivated enzyme; The base electrode is a hydrogen peroxide electrode; The carrier membrane is a polycarbonate microporous membrane; The detection electrode includes a glucose oxidase electrode and a lactate oxidase electrode; The immobilized enzyme layers of the glucose oxidase electrode and the lactate oxidase electrode in the detection electrode are respectively coated with active glucose oxidase and lactate oxidase; The front end of the carrier membrane of the detection electrode further includes a rubber sealing ring, and the detection electrode is hermetically connected to the reaction cell through the rubber sealing ring; The auxiliary electrode does not include a cellulose acetate membrane as the inner membrane; the function of the auxiliary electrode is to correct the detection data, exclude electroactive interfering substances, and improve the accuracy of the detection result; The detection electrode and the auxiliary 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 hermetically connected to the reaction cell cavity through rubber rings; the rear ends of the detection electrode and the auxiliary electrode are respectively connected to the main machine through electrode wires.

2. A detection method for anti-interference of an immobilized enzyme electrode enzyme membrane, using the immobilized enzyme sensor as described in claim 1, including the following steps: 1) Use a solution of a standard electrode active substance to test the integrity of the inner membrane of the electrode. The electro-signal response value of the detection electrode to the standard electrode active substance is An, and the electro-signal response value of the auxiliary electrode to the standard electrode active substance is A0; 2) Calibrate with a standard solution; the standard solution is a solution including a target substance with a determined concentration, and the electro-signal response value of the detection electrode to the standard solution is Sn; 3) Detect the sample. The electro-signal response value of the detection electrode to the sample is Xn, and the electro-signal response value of the auxiliary electrode to the sample is X0. Calculate the content of the target substance in the sample according to the electro-signal response value. The content of the target substance in the sample = Xn × (100 / Sn) - (A0 / An) × (100 / Sn); where n is a natural number greater than or equal to 1.

3. The detection method according to claim 2, wherein, The standard electrode active substance is a potassium ferrocyanide solution, and the target substances include glucose and lactate.

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