A whole-cell based urea electrochemical sensor and a method for preparing the same

Abstract

This invention provides a whole-cell-based urea electrochemical sensor and its preparation method, belonging to the field of electrochemical sensor technology. The whole-cell-based urea electrochemical sensor includes: an ITO conductive glass substrate, a working electrode, a counter electrode, and a reference electrode; the working electrode, the counter electrode, and the reference electrode are formed on the ITO conductive glass substrate; the working electrode, the counter electrode, and the reference electrode are used to detect urea; the surface of the working electrode is modified with three-dimensional graphene aerogel and immobilized Bacillus subtilis; the reference electrode is an Ag / AgCl electrode; and the counter electrode is a carbon electrode. This invention uses whole-cell Bacillus subtilis as a biorecognition element, utilizing its self-renewal and strong adaptability to overcome the defects of easy enzyme inactivation and short lifespan in traditional biosensors, significantly improving the stability and environmental adaptability of the system.

Description

Technical Field

[0001] This invention relates to the field of electrochemical sensor technology, and in particular to a whole-cell-based urea electrochemical sensor and its preparation method. Background Technology

[0002] In recent years, the rapid development of biosensor technology has promoted the research process of new electrochemical detection platforms, providing important technical support for real-time monitoring of biomolecules. As a common nitrogen-containing organic compound, urea has important detection value in agriculture, environment and medicine. Specifically: (1) In agricultural production, urea is the most widely used nitrogen fertilizer, and its content directly affects crop growth efficiency and soil nitrogen cycle; (2) In medical testing, urea concentration is an important indicator reflecting human metabolic function and kidney excretion function. Abnormal blood urea levels may be closely related to diseases such as kidney damage and metabolic disorders; (3) In the environmental field, excessive urea content can cause eutrophication and ecological pollution of water bodies. Therefore, the development of sensitive, rapid and portable urea detection methods is of great significance for agricultural production management, environmental monitoring and clinical diagnosis.

[0003] Currently, conventional methods for urea detection mainly include colorimetry, spectrophotometry, ion-selective electrode methods, and enzyme-based sensor methods. However, these traditional detection methods generally suffer from a series of problems such as complex operation procedures, slow response speed, poor long-term stability, and high cost. In particular, urease-based electrochemical biosensors, which are a research hotspot, show good performance in terms of selectivity and response sensitivity, but the activity of their core biological component—the enzyme—is easily affected by changes in external environment such as temperature and pH, leading to inactivation. In addition, the enzyme immobilization process on the electrode surface is cumbersome. These factors together limit the large-scale production and practical application of such sensors.

[0004] In recent years, whole-cell biosensors have gradually attracted attention as an emerging detection technology. Unlike traditional enzyme sensors, whole-cell sensors use living cells as biorecognition elements, relying on their metabolic reactions to directly convert target substances into detectable signals. These sensors have advantages such as self-renewal, strong environmental adaptability, and long lifespan, making them particularly suitable for real-time monitoring in complex environments. Existing research has shown that both engineered strains and natural bacteria can be used for the detection of nitrogen compounds, but most methods rely on expensive microfluidic platforms or complex immobilization materials, making it difficult to balance cost and stability.

[0005] In addition, existing electrochemical detection technologies for urea detection still face the following technical bottlenecks: (1) the stability of urease-based detection methods is insufficient, limiting their long-term application; (2) some material-modified electrodes suffer from poor conductivity and limited specific surface area during detection, resulting in low signal intensity; (3) traditional immobilization methods are insufficient in maintaining bacterial activity, easily causing signal attenuation. Therefore, it is urgent to develop a novel electrochemical sensing platform with simple structure, high sensitivity, excellent stability, and low cost to achieve rapid and reliable urea detection, providing a more efficient detection method for agricultural production, environmental protection, and medical diagnosis. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a whole-cell-based urea electrochemical sensor and its preparation method, which can solve the technical problems of insufficient stability, poor conductivity, limited specific surface area, insufficient preservation of bacterial activity, and easy attenuation of detection signal in the prior art.

[0007] In a first aspect, the present invention provides a whole-cell-based urea electrochemical sensor, comprising: an ITO conductive glass substrate, a working electrode, a counter electrode, and a reference electrode; The working electrode, the counter electrode, and the reference electrode are formed on the ITO conductive glass substrate; The working electrode, the counter electrode, and the reference electrode are used to detect urea; The surface of the working electrode is modified with three-dimensional graphene aerogel and immobilized Bacillus subtilis; The reference electrode is an Ag / AgCl electrode; The counter electrode is a carbon electrode.

[0008] A second aspect of this invention provides a method for preparing a whole-cell-based urea electrochemical sensor, comprising: Step 1: Form the working electrode, counter electrode, and reference electrode on the ITO conductive glass substrate; Step 2: Modify the working electrode with three-dimensional graphene aerogel to form a three-dimensional graphene aerogel working electrode; Step 3: Immobilize Bacillus subtilis on the three-dimensional graphene aerogel working electrode and assemble it with the reaction chamber to complete the fabrication of the urea electrochemical sensor.

[0009] The beneficial effects of the technical solutions provided by the embodiments of the present invention include at least the following: In this embodiment of the invention, whole cells of Bacillus subtilis are used as biorecognition elements. Leveraging their self-renewal and adaptability, the invention overcomes the shortcomings of traditional biosensors, such as easy enzyme inactivation and short lifespan, significantly improving the system's stability and environmental adaptability. The sensor's working electrode is modified with three-dimensional graphene aerogel. Its large specific surface area and excellent electronic conductivity effectively enhance the transmission efficiency of electrochemical signals, providing an ideal immobilization carrier for cells, thereby improving overall detection sensitivity and response speed, and enabling rapid detection of urea concentration. Attached Figure Description

[0010] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0011] Figure 1 This is a schematic diagram of the structure of a whole-cell-based urea electrochemical sensor provided in an embodiment of the present invention.

[0012] Figure 2 This is a schematic diagram of an electrochemical sensor electrode for detecting urea concentration provided in an embodiment of the present invention.

[0013] Figure 3 This is a SEM image of a three-dimensional graphene aerogel provided in an embodiment of the present invention.

[0014] Figure 4 This is a cyclic voltammetry curve of urea at different concentrations provided in an embodiment of the present invention.

[0015] Figure 5 This is a linear fitting graph of urea concentration and current provided in an embodiment of the present invention.

[0016] Explanation of reference numerals in the attached figures: 1-ITO conductive glass substrate; 2-Working electrode; 3-Counter electrode; 4-Reference electrode; 5-Three-dimensional graphene aerogel; 6-Reaction chamber. Detailed Implementation

[0017] To enable those skilled in the art to better understand the technical solutions in the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. It should be understood that these descriptions are merely exemplary and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0018] Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts disclosed in this invention.

[0019] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention.

[0020] like Figure 1 and Figure 2 As shown in the figure, the structure of a whole-cell-based urea electrochemical sensor provided in this embodiment of the invention includes: an ITO conductive glass substrate 1, a working electrode 2, a counter electrode 3, and a reference electrode 4.

[0021] A working electrode 2, a counter electrode 3, and a reference electrode 4 are formed on an ITO conductive glass substrate 1.

[0022] Working electrode 2, counter electrode 3, and reference electrode 4 are used to detect urea.

[0023] The working electrode 2 is patterned on an ITO glass surface using conductive silver paste, exhibiting excellent electrical conductivity stability. The surface of the working electrode 2 is modified with three-dimensional graphene aerogel 5 and immobilized Bacillus subtilis. The three-dimensional graphene aerogel material possesses a large specific surface area, high conductivity, and a three-dimensional porous structure, which not only significantly enhances the electrode's electron transport capability and electrochemical activity but also provides a stable and permeable support platform for cell immobilization, effectively achieving signal amplification and efficient integration with the biological interface.

[0024] Reference electrode 4 is an Ag / AgCl electrode, which uses Ag / AgCl electrode paste and is formed on the substrate by screen printing technology. It has good repeatability and batch processing. The reference electrode provides a stable potential reference point in the electrochemical reaction, ensuring the accuracy and reproducibility of the signal response during the measurement process. It is a key link in the potential control of the entire system.

[0025] The counter electrode 3 is a carbon electrode, which is formed by printing conductive carbon paste on the surface of ITO. The carbon material itself has good chemical inertness, structural stability and electrical conductivity. In the system, it mainly plays the role of closing the circuit and conducting current, ensuring the current balance and system stability during the electrochemical measurement process.

[0026] Optionally, the ITO conductive glass substrate 1 has an overall size of 46mm×42mm and a thickness of 1.1mm. ITO has excellent electrical conductivity, chemical stability and optical transparency. A three-dimensional graphene aerogel structure is modified on the surface of the ITO. Bacillus subtilis is fixed inside the three-dimensional graphene aerogel, which can produce ammonia and carbon dioxide by metabolizing urea, thereby triggering local electrochemical signal changes.

[0027] Furthermore, the urea electrochemical sensor is encapsulated in a reaction chamber 6 prepared by photopolymerization 3D printing to form a standardized detection space, ensuring signal consistency and reproducibility.

[0028] This invention provides a method for preparing a whole-cell-based urea electrochemical sensor, comprising: Step 1: Form the working electrode, counter electrode, and reference electrode on the ITO conductive glass substrate.

[0029] Step 2: Modify the working electrode with three-dimensional graphene aerogel to form a three-dimensional graphene aerogel working electrode.

[0030] In one possible implementation, step 2 specifically includes sub-steps 201 to 205: Sub-step 201: Add pyrrole to the graphene oxide dispersion and stir the mixture with a magnetic stirrer to form a homogeneous reduced graphene oxide solution. The concentration of the graphene oxide dispersion is 1.5 mg / mL, and the purity of the pyrrole reagent is not less than 99.7%. The mass ratio of the graphene oxide dispersion to pyrrole is 1:0.5.

[0031] Sub-step 202: The reduced graphene oxide solution is added dropwise to the acrylic mold using a pipette. The mold is then sealed and placed in a constant temperature water bath. The mixture is heated at 80°C for 24 hours to allow the system to undergo a self-assembly reaction and form a three-dimensional porous grid structure of graphene hydrogel.

[0032] Sub-step 203: Place the graphene hydrogel in a frost-free refrigerator and freeze it overnight to allow the water molecules inside the hydrogel to form solid ice crystals.

[0033] Sub-step 204: The three-dimensional hydrogel, after being frozen overnight, is placed in a vacuum freeze dryer for low-temperature drying to remove internal ice crystals. The resulting dried product is a three-dimensional graphene aerogel with a porous structure.

[0034] Sub-step 205: Apply conductive silver paste to the working electrode area of ​​the ITO conductive glass substrate to enhance the bonding force between the graphene aerogel and the substrate. Then, modify the surface of the working electrode with the three-dimensional graphene aerogel to obtain the three-dimensional graphene aerogel working electrode.

[0035] like Figure 3 The image shown is a SEM image of a three-dimensional graphene aerogel. Figure 3 (a) is a scale of 1:200 μm. Figure 3 (b) is a scale bar of 1:100 μm. The figure clearly shows that the prepared graphene aerogel material exhibits a typical multilayer sheet structure, with graphene sheets stacked and interleaved at the micro-nano scale to construct a stable three-dimensional porous network framework. This three-dimensional structure not only effectively avoids excessive aggregation between graphene sheets but also enhances the material's mechanical strength and spatial support, laying the foundation for constructing high-performance electrode materials. These unique three-dimensional surface structures are due, on the one hand, to the self-assembly behavior during GO reduction, which causes single-layer graphene molecules to stack and form a three-dimensional porous structure. On the other hand, the sublimation of water during freeze-drying perfectly preserves the three-dimensional porous structure, thus forming an aerogel. These unique surface morphologies and three-dimensional features make the prepared three-dimensional graphene aerogel an excellent electrochemical biodetection electrode material. It not only retains the good conductivity of graphene but also provides greater convenience for charge transport at the electrode-electrolyte interface, fixation of Bacillus subtilis, and the introduction of composite materials.

[0036] The three-dimensional graphene aerogel electrode prepared through the above steps has good porosity and conductivity, which can provide a stable and effective material basis for subsequent biological immobilization and electrochemical detection.

[0037] Step 3: Immobilize Bacillus subtilis on the three-dimensional graphene aerogel working electrode and assemble it with the reaction chamber to complete the fabrication of the urea electrochemical sensor.

[0038] Optionally, the reaction chamber is prepared by photopolymerization 3D printing technology. The preparation steps of the reaction chamber include: modeling, slicing, printing, and ultrasonic cleaning with anhydrous ethanol.

[0039] Specifically, the steps for creating the reaction chamber include: building a 3D model of the reaction chamber using SolidWorks software and saving it as an STL file; importing the model file into BMF slicing software for slicing; printing using a photopolymer 3D printer; and removing the model after printing. Finally, placing the printed mold into a beaker containing anhydrous ethanol and ultrasonically cleaning it for 20 minutes to remove residual resin.

[0040] In one possible implementation, step 3 specifically includes sub-steps 301 to 304: Sub-step 301: Take a concentration of 10 8 100 μL of Bacillus subtilis suspension (CFU / mL) was uniformly dropped onto the surface of the three-dimensional graphene aerogel electrode, allowing the bacterial solution to fully penetrate the porous structure of the aerogel.

[0041] Sub-step 302: After adding the bacterial solution, the electrode is placed in a refrigerated environment of 0~6℃ and incubated for 4 hours. Under low temperature conditions, the bacteria are gradually adsorbed and embedded in the aerogel pore structure to achieve physical adsorption and pore embedding and fixation of the bacteria.

[0042] Sub-step 303: After incubation, slowly rinse the electrode surface three times with PBS buffer to remove any loosely bound bacteria and ensure the uniformity and stability of the immobilized layer.

[0043] Sub-step 304: The reaction chamber is bonded to the ITO conductive glass with double-sided adhesive, and then sealed and reinforced with silicone sealant after assembly to complete the fabrication of the urea electrochemical sensor. This achieves the effect of forming a stable microcavity electrode platform, which is beneficial for subsequent liquid sample injection and reaction control.

[0044] Through the above immobilization steps, Bacillus subtilis can form a stable bond with three-dimensional graphene aerogel while maintaining its metabolic activity, providing a reliable biorecognition interface for the subsequent electrochemical detection of urea.

[0045] In one possible implementation, the ITO conductive glass substrate is further cleaned and hydrophilically treated before step 1: The ITO glass was ultrasonically cleaned in sequence with acetone, isopropanol, and deionized water.

[0046] Hydrophilization treatment was carried out by incubating the solution of hydrogen peroxide, ammonia and deionized water in a volume ratio of 1:1:5 at 90°C for 60 minutes.

[0047] Wash with deionized water and dry with nitrogen, then dry in an oven at 60°C for later use.

[0048] In one possible implementation, the preparation method further includes: Step 4: Perform electrochemical tests on the electrochemical sensor using cyclic voltammetry to obtain the relationship curve between urea concentration and current response.

[0049] Further, step 4 specifically includes: adding 3 mL of the urea solution to be tested into the reaction chamber, with a concentration range of 2-6 mM, and performing cyclic voltammetry scanning using an electrochemical workstation under constant temperature conditions of 25°C.

[0050] Optionally, the potential scan range of the cyclic voltammetry test is set to -0.2V to +0.4V, the potential sampling interval is 1mV, the scan rate is 0.01V / s, and the number of cycles is 10; based on the oxidation peak current values ​​measured under different concentrations of urea solution, the peak current-urea concentration relationship curve is plotted.

[0051] Specifically, the steps for generating the electrochemical sensor operating curve include: the three-dimensional graphene aerogel material not only has high electrical conductivity but also a high specific surface area, which can significantly enhance the electrochemical response of urea. The modified three-dimensional graphene aerogel working electrode is used as the detection electrode, a carbon electrode is selected as the counter electrode, and an Ag / AgCl electrode is used as the reference electrode to ensure the stability and accuracy of the entire electrochemical system. Electrochemical testing of the working electrode is performed at room temperature using cyclic voltammetry (CV), with the urea solution to be tested added to the reaction chamber at a concentration range of 2–6 mM. CV (Chemical Vibration) is a real-time method for monitoring redox behavior on the electrode surface, particularly suitable for studying charge changes induced by whole-cell interactions with urea, providing intuitive data for sensing mechanisms. In this project, Bacillus subtilis metabolizes urea to produce NH3 and CO2, altering local pH and charge environment, thereby changing the redox reaction behavior on the electrode surface. CV can sensitively capture these subtle changes. As a label-free detection method, CV eliminates the need for additional indicators or enzyme-labeled substrates, reducing detection complexity and cost, making it suitable for whole-cell activity studies and principle validation. CV can be used to determine the electrochemical activity, conductivity, and stability of graphene aerogel electrodes; for example, peak shape symmetry and peak current magnitude can be used to assess their electron transfer capabilities. The test parameters were set as follows: potential scan range of –0.2 V to +0.4 V, potential sampling interval of 1 mV, scan rate of 0.01 V / s, and 10 cycles. Electrochemical working curves were plotted based on the relationship between the obtained current response and the concentration of the urea solution.

[0052] In one possible implementation, the preparation method further includes: Step 5: Change the concentration of the urea solution to be tested, and fit the peak current-urea concentration relationship curve to establish a quantitative detection model of urea concentration by the sensor.

[0053] The results are as follows Figure 4 and 5 As shown; by Figure 4 It can be seen that when the urea concentration is detected, the peak current position appears in 0.1-0.12V, and when the concentration of the urea solution is changed (2-6mM), the current signal of urea shows a positive correlation trend, and the magnitude of the peak current increases with the increase of urea concentration.

[0054] Figure 5The graph shows a linear fit between urea concentration and current. As can be seen from the graph, there is a good linear relationship between the concentration of the urea to be measured and the peak current. The linear equation is y = 0.418x - 0.3917, with an R² of 0.972. This indicates that the electrochemical sensor described in this invention has good linear range and sensitivity, as well as good reliability. The detection range is 2–6 mM; the detection sensitivity is 0.418 mA / mM, and the calculated detection limit (LOD) is 0.368 mM.

[0055] In this embodiment of the invention, whole cells of Bacillus subtilis are used as biorecognition elements. Leveraging their self-renewal and adaptability, the invention overcomes the shortcomings of traditional biosensors, such as easy enzyme inactivation and short lifespan, significantly improving the system's stability and environmental adaptability. The sensor's working electrode is modified with three-dimensional graphene aerogel. Its large specific surface area and excellent electronic conductivity effectively enhance the transmission efficiency of electrochemical signals, providing an ideal immobilization carrier for cells, thereby improving overall detection sensitivity and response speed, and enabling rapid detection of urea concentration.

[0056] Unit explanation: M (mol per liter): represents the solution concentration, 1 M = 1 mol / L, that is, 1 mole of solute is contained in each liter of solution; mM (millimoles per liter): 1 mM = 10⁻ 3 mol / L means 1 millimole of solute per liter of solution; mL: milliliter; mg / mL: milligrams per milliliter, indicating solution concentration; mmol: millimole; µL: microliter; wt%: weight percent; PBS: phosphate-buffered saline solution.

[0057] This invention encompasses any substitutions, modifications, equivalent methods, and solutions made within the spirit and scope of this invention. To provide the public with a thorough understanding of this invention, specific details are described in detail in the preferred embodiments, while those skilled in the art will fully understand the invention even without these details. Furthermore, to avoid unnecessary misunderstanding of the essence of this invention, well-known methods, processes, procedures, components, and circuits are not described in detail.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the embodiments of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the protection scope of the present invention.

Claims

1. A whole-cell-based urea electrochemical sensor, characterized in that, include: ITO conductive glass substrate, working electrode, counter electrode and reference electrode; The working electrode, the counter electrode, and the reference electrode are formed on the ITO conductive glass substrate; The working electrode, the counter electrode, and the reference electrode are used to detect urea; The surface of the working electrode is modified with three-dimensional graphene aerogel and immobilized Bacillus subtilis; The reference electrode is an Ag / AgCl electrode; The counter electrode is a carbon electrode.

2. A method for preparing a whole-cell-based urea electrochemical sensor, characterized in that, include: Step 1: Form the working electrode, counter electrode, and reference electrode on the ITO conductive glass substrate; Step 2: Modify the working electrode with three-dimensional graphene aerogel to form a three-dimensional graphene aerogel working electrode; Step 3: Immobilize Bacillus subtilis on the three-dimensional graphene aerogel working electrode and assemble it with the reaction chamber to complete the fabrication of the urea electrochemical sensor.

3. The preparation method according to claim 2, characterized in that, Step 2 specifically includes: Sub-step 201: Pyrrole is added to the graphene oxide dispersion, and the reaction is stirred using a magnetic stirrer to form a uniform reduced graphene oxide solution; the concentration of the graphene oxide dispersion is 1.5 mg / mL, and the purity of the pyrrole reagent is not less than 99.7%; the mass ratio of the graphene oxide dispersion to the pyrrole is 1:0.

5. Sub-step 202: The reduced graphene oxide solution is added dropwise to an acrylic mold using a pipette. After sealing the mold, it is placed in a constant temperature water bath and heated at 80°C for 24 hours to form a three-dimensional porous grid structure graphene hydrogel. Sub-step 203: Place the graphene hydrogel in a frost-free refrigerator and freeze it overnight to allow the water molecules inside the hydrogel to form solid ice crystals; Sub-step 204: The three-dimensional hydrogel after being frozen overnight is placed in a vacuum freeze dryer and dried at low temperature to obtain a three-dimensional graphene aerogel with a porous structure. Sub-step 205: Apply conductive silver paste to the working electrode area of ​​the ITO conductive glass substrate, and then modify the surface of the working electrode with the three-dimensional graphene aerogel to obtain the three-dimensional graphene aerogel working electrode.

4. The preparation method according to claim 2, characterized in that, Step 3 specifically includes: Sub-step 301 : Take 100 μL of Bacillus subtilis suspension with a concentration of 10 8 CFU / mL, and drop it evenly on the surface of the three-dimensional graphene aerogel electrode; Sub-step 302: After adding the bacterial solution, the electrode is placed in a refrigerated environment at 0~6℃ for 4 hours to achieve physical adsorption and pore embedding and fixation of the bacteria. Sub-step 303: After incubation, slowly rinse the electrode surface three times with PBS buffer to remove any loosely bound bacteria. Sub-step 304: The reaction chamber is bonded to the ITO conductive glass with double-sided adhesive, and then sealed and reinforced with silicone sealant after assembly to complete the fabrication of the urea electrochemical sensor.

5. The preparation method according to claim 2, characterized in that, Prior to step 1, the ITO conductive glass substrate is also cleaned and hydrophilicated. The ITO glass was ultrasonically cleaned sequentially using acetone, isopropanol, and deionized water. Hydrophilization treatment was carried out by incubating the solution of hydrogen peroxide, ammonia and deionized water in a volume ratio of 1:1:5 at 90°C for 60 minutes. Wash with deionized water and dry with nitrogen, then dry in an oven at 60°C for later use.

6. The preparation method according to claim 2, characterized in that, The reaction chamber is prepared by photopolymerization 3D printing technology. The preparation steps of the reaction chamber include: modeling, slicing, printing, and ultrasonic cleaning with anhydrous ethanol.

7. The preparation method according to claim 2, characterized in that, Also includes: Step 4: Perform electrochemical tests on the electrochemical sensor using cyclic voltammetry to obtain the relationship curve between urea concentration and current response.

8. The preparation method according to claim 7, characterized in that, Step 4 specifically includes: Add 3 mL of the urea solution to be tested, with a concentration range of 2-6 mM, into the reaction chamber, and perform cyclic voltammetry scanning using an electrochemical workstation under constant temperature of 25 °C.

9. The preparation method according to claim 8, characterized in that, The potential scan range of the cyclic voltammetry test was set to -0.2V to +0.4V, the potential sampling interval was 1mV, the scan rate was 0.01V / s, and the number of cycles was 10. Based on the oxidation peak current values ​​measured under different concentrations of urea solution, the peak current-urea concentration relationship curve was plotted.

10. The preparation method according to claim 9, characterized in that, Also includes: Step 5: Change the concentration of the urea solution to be tested, and fit the peak current-urea concentration relationship curve to establish a quantitative detection model of urea concentration by the sensor.

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