Method for developing electrochemical biosensor for real-time dynamic analysis of activity of intracellular choline oxidase

Abstract

The invention discloses a development method of an electrochemical biosensor for real-time dynamic analysis of intracellular choline oxidase activity, which comprises the following steps: firstly, aiming at the problems of low conversion rate of copper-based ferrite CuFe2O4 nano-enzyme and low catalytic property on H2O2, carrying out improvements in three aspects: introducing Cu0 to promote partial electron transfer in a material by virtue of the advantage of relatively high conductivity of Cu0; a surfactant sodium dodecylbenzene sulfonate (SDBS) is added to improve the dispersity; a two-dimensional material Bi2WO6 is introduced, so that the specific surface area is increased. The SDBSamp is obtained after improvement; and the Bi2WO6 coated Cu-CuFe2O4 has excellent catalytic activity on H2O2 (hydrogen peroxide). Besides, ChOx can oxidize Ch into betaine and H2O2, the electrochemical response current is continuously increased along with the increase of the concentration of Ch (ChOx), and the introduction of Pb < 2 + > can inhibit the activity of ChOx so as to cause the reduction of the electrochemical response current, so that the method can be applied to the analysis and detection of Ch (ChOx) in hepg2 cells and the screening of ChOx inhibitors, and a new idea is provided for choline-related clinical detection and drug development.

Description

Technical Field

[0001] The present invention relates to a method for developing an electrochemical biosensor and its application, in particular to a method for developing an electrochemical biosensor based on magnetic nanozyme for real-time dynamic analysis of intracellular choline oxidase activity, belonging to the fields of functional biomaterials and biosensing technology. Background Art

[0002] Choline (Ch) is a precursor of acetylcholine and has been identified as an essential nutrient for human health. It is necessary for the production of important membrane phospholipids, assisting transmembrane signal transduction, and providing cell membrane structure. Its main function is to transmit nerve impulses and form biological membranes. A lack of choline in humans can lead to various health problems, such as hemorrhagic renal necrosis, liver disease, fatty liver, and even neurological diseases. Therefore, the detection of choline and choline oxidase is very necessary for clinical analysis, biological science, feed additives, and the food industry. Conventional methods for choline analysis have been proposed, such as gas chromatography, amperometry, high-performance liquid chromatography (HPLC), etc. However, the above methods have many limitations, such as high cost, complex procedures, the need for experienced operators, and time-consuming. There is still a need to develop a simple, inexpensive, and easy-to-use choline detection sensor. The electrochemical method has the advantages of simple operation, rapid response, and high sensitivity, and stands out from many detection methods. Choline oxidase (ChOx) can catalyze choline to produce betaine and hydrogen peroxide (H2O2) under alkaline conditions with a pH of 8, which is closely related to the biochemical tests and drug treatments of choline diseases in clinical practice. Therefore, developing a new type of electrochemical dynamic analysis detection method to achieve the detection of choline and choline oxidase has very important scientific significance.

[0003] Compared with natural enzymes, artificial enzymes have the advantages of flexible design, low cost, good stability, etc. Ferrite can be easily extracted and separated from the solution by an external magnetic field and can be used as a nanozyme again. Due to its rich oxidation states and good conductivity, nano-ferrite has also been used as a heterogeneous catalyst in Fenton reaction, photocatalysis, gas sensors, treatment of heavy metal-containing waste and pigments, etc. In addition, the ability of nano-ferrite to generate reactive oxygen species can be used to simulate the catalytic activity of natural enzymes (catalase). Compared with other types of ferrites, CuFe2O4 has relatively high catalytic performance. Aiming at the problems of poor affinity of CuFe2O4 nanozyme for H2O2 and low catalytic activity, corresponding improvements have been made to CuFe2O4 nanozyme. So far, there have been a few reports on the improvement of CuFe2O4 nanozyme, but there is no case of simultaneously using the surfactant sodium dodecylbenzenesulfonate (SDBS) and two-dimensional thin-layer material Bi2WO6 (Bi2WO6) to improve the activity of CuFe2O4 nanozyme; in addition, there is no report on its use for the analysis and detection of H2O2, Ch, and ChOx and their inhibitors.

[0004] The present invention designs a method for developing an electrochemical biosensor for real-time dynamic analysis of intracellular choline oxidase activity. First, aiming at the problems of low conversion rate of copper-based ferrite CuFe2O4 nanozyme and low catalysis for H2O2, there are three aspects of improvement: by virtue of the 0 advantage of higher electrical conductivity, introducing Cu 0 to promote the internal electron transfer of the material; adding the surfactant sodium dodecylbenzenesulfonate (SDBS) to improve its dispersibility; introducing the two-dimensional material Bi2WO6 to increase its specific surface area. The improved magnetic nanozyme (SDBS&Bi2WO6@Cu-CuFe2O4) has excellent catalytic activity for H2O2. As the concentration of H2O2 increases, the electrochemical response current continuously increases. Based on this, a real-time dynamic analysis and detection method is constructed. In addition, ChOx can oxidize Ch into betaine and H2O2. As the concentration of Ch(ChOx) increases, the electrochemical response current continuously increases, while the introduction of 2 + Pb will inhibit the activity of ChOx, resulting in a decrease in the electrochemical response current. Therefore, this method can be applied to the analysis and detection of Ch(ChOx) and the screening of ChOx inhibitors, providing a new idea for choline-related clinical detection and drug development. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for developing an electrochemical biosensor for real-time dynamic analysis of intracellular choline oxidase activity with high specificity, high sensitivity, accurate detection, stable signal, and low cost.

[0006] The technical solution adopted by the present invention to solve the above technical problems is as follows: A method for developing an electrochemical biosensor for real-time dynamic analysis of intracellular choline oxidase activity, and the specific steps are as follows:

[0007] (1) Preparation of magnetic nanozyme (SDBS&Bi2WO6@Cu-CuFe2O4)

[0008] 1.0 - 2.0 g of ferric nitrate nonahydrate (Fe(NO3)3·9H2O) and 0.1 - 0.5 g of copper nitrate trihydrate (Cu(NO3)2·3H2O) are successively added to 10 - 20 mL of ethylene glycol solution, and then 0.1 - 0.5 g of urea (CO(NH2)2) is added to the above solution. The above mixed solution is ultrasonically mixed evenly. Then, 0.1 - 0.3 g of Bi2WO6 two-dimensional material and 0.1 - 0.5 g of sodium dodecylbenzenesulfonate (SDBS) are successively added, and ultrasonically treated for 10 - 50 min until the solution is mixed evenly. Finally, the above solution is transferred to a 20 - 50 mL reaction kettle and reacted in an electrothermal constant temperature blast drying oven at 200 °C for 15 - 24 h. After the reaction is completed, the reaction kettle is taken out, and the above reaction mixture is transferred to a 10 mL centrifuge tube for centrifugation, and washed alternately with distilled water and ethanol 1 - 3 times. After centrifugation, it is dried in a vacuum drying oven at 40 - 80 °C for 5 - 10 h to obtain magnetic nanozyme (SDBS&Bi2WO6@Cu-CuFe2O4). The above dried material is added to 5 mL of distilled water and redispersed to prepare a 0.625 mg / mL solution for standby.

[0009] (2) Preparation of electrochemical biosensor

[0010] a. The magnetic glassy carbon electrode (MGCE, with a diameter of 3 mm) is polished on suede with alumina powder with particle sizes of 0.3 μm and 0.05 μm for 1 - 5 min. After polishing, the electrode is placed in an ultrasonic cleaner and ultrasonically cleaned with ultrapure water for 1 - 5 min, and then dried with N2, denoted as MGCE.

[0011] b. Take 5 - 10 μL of the solution in (1) and drop it onto MGCE, and let it stand at room temperature for 10 - 50 min. Then, the electrode is slowly rinsed with ultrapure water, denoted as SDBS&Bi2WO6@Cu-CuFe2O4 / MGCE. Subsequently, it is tested by the current-time method, with the potential set at -0.8 V and the time at 400 s, and PBS (10 mM pH 7.0) containing a certain concentration of 10 μM 3 μL H2O2 (final concentration 0.3 μM) is used as the electrolyte solution to test the performance of the electrode.

[0012] c. ChOx Catalytic Reaction System: For a reaction solution with a total volume of 20 μL, 2 μL of 50000 nM Ch (final concentration: 5000 nM) and 2 μL of 1000 U / L ChOx (final concentration: 100 U / L) were successively added to a phosphate buffer (Na2HPO4 / NaH2PO4, 10 mM, pH 8.0). The above reaction solution was reacted in a 37 °C water bath for 10 - 50 min. Subsequently, following the steps in 2b above, the ChOx catalytic reaction solution was used instead of H2O2 to complete the experiment, and the current-time method was used for testing. The potential was set at -0.8 V and the time was 120 s to evaluate the possibility of this method for the analysis and testing of Ch and ChOx.

[0013] Detection and Analysis of Different Concentrations of H2O2: Based on the experimental steps in 2b, with the potential set at -0.8 V and the time at 120 s, the concentration of H2O2 was changed (final concentration: 0 - 8000 μM), and other steps were the same as above. Based on this, the analysis and detection of different concentrations of H2O2 can be achieved.

[0014] Detection and Analysis of Different Concentrations of Ch(ChOx): Based on the experimental steps in 2c, when the ChOx catalytic reaction was carried out, the concentration of Ch was changed (0 - 50000 nM), and other steps were the same as above. Based on this, the analysis and detection of Ch can be achieved; based on the experimental steps in 2c, when the ChOx catalytic reaction was carried out, the concentration of ChOx was changed (0 - 600 U / L), and other steps were the same as above. Based on this, the analysis and detection of ChOx can be achieved.

[0015] Analysis of Different Concentrations of Inhibitor Pb 2+ Analysis: Based on the experimental steps in 2c, different concentrations of Pb 2+ (final concentration: 0 - 800 nM) were introduced during the ChOx catalytic reaction, and after mixing, the reaction was carried out for 1 - 5 min. Other steps were the same as above. Based on this, the screening of ChOx inhibitors can be achieved.

[0016] This patent is a method for developing an electrochemical biosensor for real-time dynamic analysis of intracellular choline oxidase activity. Using the current-time method (Amperometric i-t Curve), with the potential set at -0.8 V and the time at 120 s, the electrochemical sensor prepared above was used to achieve the current response to H2O2; the corresponding current magnitude of H2O2 was measured in the electrolyte solution containing the ChOx reaction solution, and a quantitative relationship between Ch(ChOx) was established. According to the quantitative relationship between the two, the content of Ch(ChOx) in the sample to be tested can be detected and analyzed, and Pb 2+ can inhibit the reaction between Ch and ChOx. Based on this, the detection and analysis of Pb 2+ can be achieved.

[0017] Principle of the invention: The present invention is a method for developing an electrochemical biosensor for real-time dynamic analysis of intracellular choline oxidase activity. First, aiming at the problems of low conversion rate of magnetic nanozyme (CuFe2O4) and low affinity for H2O2, a highly efficient SDBS&Bi2WO6@Cu-CuFe2O4 nanozyme was synthesized. Secondly, the current-time method was used to detect the current responses of the sensor to different concentrations of Ch (ChOx) and its inhibitor Pb 2+ . Obviously, within a certain concentration range, when the concentration of ChOx is fixed, the greater the concentration of Ch, the more H2O2 is produced, and the more obvious the current response; similarly, when the concentration of Ch is fixed, the greater the concentration of ChOx, the more H2O2 is produced, and the more obvious the current response; when the concentrations of Ch and ChOx are fixed, within a certain concentration range, the greater the concentration of Pb 2+ added, the less H2O2 is produced, and the weaker the current response signal. And throughout the process, as the concentration of the target substance continuously increases, the current continuously increases, showing a dynamic change trend. The experimental results show that the magnitude of the final current and the concentration of Ch (ChOx) show a linear relationship within a certain range, and the dynamic analysis and detection of Ch (ChOx) can be realized. Its advantages are as follows:

[0018] (1) High catalytic activity. The modified magnetic nanozyme SDBS&Bi2WO6@Cu-CuFe2O4 has high catalytic activity for H2O2, and the generated electrochemical response signal is obvious. This nanozyme can be modified onto the electrode surface in one step, with a relatively fast speed (using a magnetic field), and generates a current signal by catalyzing H2O2, having good adhesion and catalytic performance.

[0019] (2) High sensitivity. Choline oxidase catalyzes the reaction of choline to produce H2O2. Based on this, a series of analytical sensing methods can be established. As the concentrations of H2O2, Ch, and ChOx continuously increase, the current continuously increases, showing a dynamic change trend, and three linear equations are obtained: The linear correlation equation between the current response and the H2O2 concentration is y = -6.60LogC H2O2 - 13.24, R 2 = 0.9969, and the detection limit is 5.8 nM; the linear correlation equation between the current response and the Ch concentration is: y = -38.59LogC Ch - 0.15, R 2 = 0.9938, and the detection limit is 0.49 nM; the linear correlation equation between the current response and the ChOx concentration is: y = -41.56LogC ChOx - 45.34, R 2 = 0.9956, and the detection limit is 0.065 U / L; for choline oxidase, Pb 2+ has a good inhibitory effect, IC 50is 2.71 nM, indicating that the sensor can achieve highly sensitive detection of H2O2, Ch, ChOx, and Pb. 2+ High sensitivity detection.

[0020] (3) High specificity. For the detection of H2O2, other control substances such as dopamine (DA), ascorbic acid (AA), glutathione (GSH), cysteine (Cys), citric acid (CA), and uric acid (UA) have no interference on the system; for the detection of Ch, other control substances such as cysteine (Cys), tyrosine (Tyr), arginine (Arg), glycine (Gly), uric acid (UA), and coenzyme A (CoA) have no interference on the system; for the detection of ChOx, other control substances such as acetylcholinesterase (AChE), alkaline phosphatase (ALP), inorganic pyrophosphatase (PPase), papain, lysozyme (LZM), and glucose oxidase (GOx) have no interference on the system.

[0021] (4) Simple equipment and low cost. The equipment required for this invention is only an electrochemical workstation. The steps for preparing the electrode are simple and fast, and the detection is very convenient. Only a small amount of reagents and materials are needed to achieve the analytical detection of H2O2, Ch, ChOx, and Pb. 2+ Analysis and detection.

[0022] In summary, this invention constructs a method for developing an electrochemical biosensor for real-time dynamic analysis of intracellular choline oxidase activity and applies it to the analysis and detection of H2O2, Ch, ChOx, and Pb. 2+ It has the advantages of high catalytic activity, sensitivity, specificity, and low cost, and has good application prospects. Brief description of the drawings

[0023] Figure 1 is the current response diagram of different types of magnetic nanozymes to H2O2 in this invention;

[0024] Figure 2 is the linear relationship diagram of the current response of the sensor of this invention to different concentrations of H2O2 versus concentration;

[0025] Figure 3 is the linear relationship diagram of the current response of the sensor of this invention to different concentrations of Ch versus concentration;

[0026] Figure 4 is the linear relationship diagram of the current response of the sensor of this invention to different concentrations of ChOx versus concentration;

[0027] Figure 5 is the curve relationship diagram of the current response of the sensor of this invention to different concentrations of Pb 2+ versus concentration;

[0028] Figure 6 Selectivity experiment diagrams of the sensor of the present invention for H2O2, Ch, and ChOx;

[0029] Figure 7 Monitoring and analysis experiment diagrams of the ChOx activity in liver cancer cells and normal cells by the sensor of the present invention. Specific implementation manners

[0030] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

[0031] Example 1 Preparation of magnetic nanozyme (SDBS&Bi2WO6@Cu-CuFe2O4)

[0032] 1.3 g of ferric nitrate nonahydrate (Fe(NO3)3·9H2O) and 0.4 g of copper nitrate trihydrate (Cu(NO3)2·3H2O) were successively added to 15 mL of ethylene glycol solution, and then 0.3 g of urea (CO(NH2)2) was added to the above solution. The above mixed solution was ultrasonically mixed evenly, and then 0.2 g of two-dimensional Bi2WO6 material and 0.25 g of sodium dodecylbenzenesulfonate (SDBS) were successively added, and ultrasonically treated for 30 min until the solution was mixed evenly. Then the above solution was transferred to a 20 mL reaction kettle and reacted in an electrothermal constant temperature blast drying oven at 200 °C for 22 h. After the reaction was completed, the reaction kettle was taken out, and the above reaction mixture was transferred to a 10 mL centrifuge tube for centrifugation, and washed alternately with distilled water and ethanol 3 times. After centrifugation, it was dried in a vacuum drying oven at 60 °C for 6 h to obtain magnetic nanozyme (SDBS&Bi2WO6@Cu-CuFe2O4). The above dried material was added to 5 mL of water and redispersed to prepare a 0.625 mg / mL solution for standby.

[0033] Example 2 Preparation of electrochemical biosensor

[0034] a. The magnetic glassy carbon electrode (MGCE, with a diameter of 3 mm) was polished on suede with alumina powder with particle sizes of 0.3 μm and 0.05 μm for 2 min. After polishing, the electrode was placed in an ultrasonic cleaner and ultrasonically cleaned with ultrapure water for 3 min, and then dried with N2, denoted as MGCE.

[0035] b. 6 μL of the solution in Example 1 was dropped onto MGCE and left to stand at room temperature for 20 min, and then the electrode was slowly rinsed with ultrapure water, denoted as SDBS&Bi2WO6@Cu-CuFe2O4 / MGCE. Subsequently, the current-time method was used for testing, the potential was set to -0.8 V and the time was 400 s, and PBS (10 mM pH 7.0) containing a certain concentration of 10 μM 3 μL H2O2 (final concentration 0.3 μM) was used as the electrolyte solution to test the performance of the electrode.

[0036] c. ChOx catalytic reaction system: For a reaction solution with a total volume of 20 μL, 2 μL of Ch (final concentration 5000 nM) at 50000 nM and 2 μL of ChOx (final concentration 100 U / L) at 1000 U / L were successively added to a phosphate buffer (Na2HPO4 / NaH2PO4, 10 mM, pH 8.0). The above reaction solution was reacted in a 37 °C water bath for 40 min. Subsequently, following step 2b above, the ChOx catalytic reaction solution was used instead of H2O2 to complete the experiment, and the current-time method was used for testing. The potential was set at -0.8 V and the time was 120 s to evaluate the possibility of this method for the analysis and testing of Ch and ChOx.

[0037] According to the above Example 2b, different types of electrodes were prepared respectively, namely MGCE, CuFe2O4 / MGCE, Cu-CuFe2O4 / MGCE, SDBS&Cu-CuFe2O4 / MGCE, SDBS&Bi2WO6@Cu-CuFe2O4 / MGCE. The other experimental steps in the above Examples 2a and 2b remained unchanged.

[0038] The results are as Figure 1 shown. The experimental phenomena show that the electrode modified with SDBS&Bi2WO6@Cu-CuFe2O4 has the largest current response to H2O2, while the bare magnetic glassy carbon electrode has almost no response to H2O2. The electrodes modified with Cu-CuFe2O4 and SDBS&Cu-CuFe2O4 have smaller current responses to H2O2 than the electrode modified with SDBS&Bi2WO6@Cu-CuFe2O4. This proves that SDBS&Bi2WO6@Cu-CuFe2O4 has a stronger catalytic ability for H2O2, thus proving that it is feasible to catalytically generate a current signal by H2O2 in this experiment theoretically.

[0039] Example 3 Analysis and Detection of H2O2, Ch and ChOx

[0040] Based on the steps in Example 2b, the concentration of H2O2 in Example 2b was changed (final concentrations: 0, 0.005, 0.01, 0.03, 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000, 3000, 5000, 8000 μM), the potential was set at -0.8 V, the time was 120 s, and the other steps remained unchanged, enabling the detection of H2O2. The results are as Figure 2 shown. The sensor prepared according to the above steps shows a good linear relationship between the current response to H2O2 and the concentration. The linear equation between the current response and H2O2 is y = -6.60LogC H2O2 - 13.24, R 2= 0.9969, the detection limit was 5.8 nM. It can be seen from the figure that within the linear range of 0.01 - 3000 μM, as the concentration of H2O2 increased, its current signal gradually enhanced. Based on the above changes in the current signal, the detection and analysis of H2O2 can be achieved.

[0041] Based on the experimental steps in step 2c, when the ChOx catalytic reaction was carried out, the concentration of Ch was changed (0, 0.5, 1, 2, 3, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10000, 20000, 50000 nM), and other steps were the same as above. Based on this, the analysis and detection of Ch can be achieved. The results are as Figure 3 shown. The current response of the sensor prepared according to the above steps and the concentration of Ch showed a good linear relationship. The linear correlation equation between the current response and the concentration of Ch was: y = -38.59LogC Ch - 0.51, R 2 = 0.9938, the detection limit was 0.49 nM. It can be seen from the figure that with the concentration of ChOx fixed, within the concentration range of 1 - 10000 nM, as the concentration of added Ch increased, the intensity of the generated current signal increased, indicating that the amount of H2O2 produced by Ch and ChOx increased. Based on the above changes in the current signal, the detection and analysis of Ch can be achieved; based on the experimental steps in step 2c, when the ChOx catalytic reaction was carried out, the concentration of ChOx was changed (0, 0.05, 0.1, 0.2, 0.4, 0.6, 1, 2, 4, 6, 10, 20, 40, 60, 100, 200, 400, 600 U / L), and other steps were the same as above. Based on this, the analysis and detection of ChOx can be achieved. The results are as Figure 4 shown. The current response of the sensor prepared according to the above steps and the concentration of ChOx showed a good linear relationship. The linear correlation equation between the current response and the concentration of ChOx was: y = -41.56LogC ChOx - 45.34, R 2 = 0.9956, the detection limit was 0.065 U / L. It can be seen from the figure that with the concentration of Ch fixed, within the concentration range of 0.1 - 200 U / L, as the concentration of added ChOx increased, the intensity of the generated current signal increased, indicating that the amount of H2O2 produced by Ch and ChOx increased. Based on the above changes in the current signal, the detection and analysis of ChOx can be achieved.

[0042] Example 4 Analysis and detection of inhibitor Pb 2+ of

[0043] Based on the experimental steps in step 2c, different concentrations of Pb were introduced during the ChOx catalytic reaction 2+(Final concentrations: 0, 0.1, 0.2, 0.3, 0.5, 0.8, 1, 2, 5, 8, 10, 20, 50, 100, 200, 500, 800 nM). After mixing the above reaction solutions, react for 1 min. Other steps are the same as above. Based on this, the screening of ChOx inhibitors can be achieved. The results are as Figure 5 shown. In the concentration range of 0.1 - 800 nM, it can be seen from the figure that as the concentration of added Pb 2+ increases, the corresponding generated current signal decreases. Because Pb 2+ inhibits the enzymatic activity of ChOx, resulting in a decrease in the amount of H2O2 generated, and further leading to a weakening of the current signal. The above phenomenon indicates that Pb 2+ has a good inhibitory effect on the activity of ChOx, and the IC 50 is 2.71 nM.

[0044] Example 5 Specific Detection

[0045] As Figure 6 shown, compared with other small molecule substances and enzyme substances, this sensor has good electrochemical responses to the three target substances (H2O2, Ch, ChOx) in the present invention respectively. The electrochemical responses generated by other comparison substances are relatively small, showing good selectivity.

[0046] Example 6 Analysis of Actual Samples

[0047] Currently, choline oxidase analysis sensors are extremely rare, especially for intracellular analysis and detection. To test the practicality of the method involved in the patent, intracellular choline oxidase analysis was completed using laboratory liver cancer hepg2 cells. First, make hepg2 cell suspensions of 1000 cells / mL, 5000 cells / mL, and 10000 cells / mL, and then extract the nuclear proteins in hepg2 cells using a kit. As Figure 7 shown, as the number of cells increases, the electrochemical signal shows an increasing trend, indicating that there is a certain amount of ChOx in the cells. As the inhibitor Pb 2+ is added, the signal decreases again, indicating a good inhibitory effect of the inhibitor on ChOx, and proving the activity of ChOx from the side. This is beneficial for the diagnosis and treatment of liver cancer and drug development in clinical practice, and has certain guiding significance.

[0048] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples either. Changes, modifications, additions, or substitutions made by those of ordinary skill in the art within the essence of the present invention should also fall within the protection scope of the present invention.

Claims

1. A method for developing an electrochemical biosensor for real-time dynamic analysis of intracellular choline oxidase activity, the mechanism of which is as follows: The present invention is a method for developing an electrochemical biosensor for real-time dynamic analysis of intracellular choline oxidase activity. First, aiming at the problems of low conversion rate of magnetic nanozyme (CuFe2O4) and low affinity for H2O2, a highly efficient SDBS&Bi2WO6@Cu-CuFe2O4 nanozyme was synthesized. Secondly, the amperometric-time method was used to detect the electrochemical responses of the sensor to H2O2, Ch (ChOx) and its inhibitor Pb with different concentrations 2+ in the whole process, as the concentration of the target substance increases continuously, the current increases continuously, showing a dynamic change trend. The experimental results show that the magnitude of the final current and the concentration of Ch (ChOx) show a linear relationship within a certain range, and finally the intracellular analysis and detection of ChOx are realized.

2. The method for developing an electrochemical biosensor for real-time dynamic analysis of intracellular choline oxidase activity according to claim 1, wherein: First, an electrochemical analysis sensing method is constructed by combining the current-time method, SDBS&Bi2WO6@Cu-CuFe2O4, H2O2, Ch, ChOx and Pb, and finally the sensitive analysis and detection of H2O2, Ch, ChOx and Pb are realized. 2+ This method presents a dynamic analysis mode, in which the current changes with time, and is suitable for real-time dynamic analysis in biological organisms, overcoming the weaknesses of static analysis or transient analysis. 2+ The current changes with time, which is suitable for real-time dynamic analysis in biological organisms and overcomes the weaknesses of static analysis or transient analysis.

3. The method for developing an electrochemical biosensor for real-time dynamic analysis of intracellular choline oxidase activity according to claims 1-2, characterized in that: Set the potential at -0.8 V and the time at 120 s, and use the current-time method to achieve the analysis and detection of H2O2, Ch, ChOx, and Pb at different concentrations 2+ The electrochemical method has good sensitivity and specificity. The detection limit of H2O2 is 5.8 nM, the detection limit of Ch is 0.49 nM, the detection limit of ChOx is 0.065 U / L, and the IC 2+ of Pb 50 is 2.71 nM.

4. A method for developing an electrochemical biosensor for real-time dynamic analysis of intracellular choline oxidase activity according to claims 1-3, characterized in that: Currently, there are very few ChOx analysis sensors, especially for intracellular analysis and detection. This method effectively realizes the monitoring of intracellular ChOx and the screening of its inhibitors, which is beneficial to the diagnosis and treatment of liver cancer and drug development in clinical practice and has certain guiding significance.

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