Method for detecting enzyme activity of peroxidase in compost sample by using functional enzyme electrochemical sensor

Abstract

The invention discloses a method for detecting the enzyme activity of peroxidase in a compost sample by using a functional enzyme electrochemical sensor, which is used for detecting the enzyme activity of peroxidase in the compost sample by using the functional enzyme electrochemical sensor, the screen-printed electrode comprises a working electrode, and the surface of the reaction end of the working electrode is modified with UiO-66-NH2 through an adhesive. Compared with a conventional electrochemical sensor, the sensor disclosed by the invention has the following advantages of high detection speed, better adaptability, higher accuracy and simpler operation, so that when the sensor disclosed by the invention is used for detecting the enzyme activity of peroxidase in a compost sample, the sensitivity is high; the method has the advantages of low cost, simplicity in operation, low sample consumption, short detection time, wide detection range, high sensitivity, strong anti-interference capability and the like, and the enzyme activity of peroxidase in a compost sample can be quickly and accurately obtained, so that effective data can be timely provided for compost maturity analysis.

Description

Technical Field

[0001] This invention belongs to the field of enzyme activity concentration detection technology, and relates to a method for detecting peroxidase activity in compost samples, specifically a method for detecting peroxidase activity in compost samples using a functional enzyme electrochemical sensor. Background Technology

[0002] During composting, enzymes primarily originate from the metabolism of microorganisms. Since the numbers of bacteria, actinomycetes, and fungi vary at different times and under different conditions during composting, enzyme activity is closely related to the quantity and types of microorganisms in the compost pile, as well as their living environment. Changes in the microbial population and quantity directly affect the composting process and product quality; therefore, it is necessary to select appropriate enzymes as indicators of compost maturity. Peroxidase activity shows a certain correlation with the degradation of organic matter; its activity can be used to characterize the status of nutrients in the compost and also as an indicator of the degree of harmlessness in composting.

[0003] Currently, enzyme activity detection methods mainly include spectrophotometry and fluorescence detection. However, the accuracy of these two methods is greatly reduced when the sample liquid is colored or turbid, or when there are many absorbing and fluorescent compounds in the sample causing interference. Electrochemical methods, with their advantages of simple operation, short response time, and high sensitivity, have become one of the preferred methods for online rapid detection. Existing electrochemical methods for detecting peroxidase activity in compost use a glassy carbon electrode as the working electrode. They measure current changes under different enzyme activity conditions, establish a linear relationship between the current change slope and enzyme activity, and then calculate the peroxidase activity in the sample based on the current change data and the linear relationship between the current change slope and enzyme activity. However, this method has limitations, including a narrow detection range (2.27–29.79 U / L for lignin peroxidase and 0.085–1.37 U / L for manganese peroxidase), long detection time, and low correlation. In particular, existing electrochemical methods struggle to accurately determine peroxidase activity in low-concentration compost samples due to the low concentration of peroxidase. Therefore, developing a functional enzyme electrochemical sensor with fast reaction speed, good conductivity, and high current intensity is crucial for the rapid and accurate detection of peroxidase activity in compost samples, especially low-concentration compost samples. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method for detecting peroxidase activity in compost samples using a functional enzyme electrochemical sensor, which is low in cost, simple to operate, consumes less sample, has a short detection time, a wide detection range, high sensitivity, and strong anti-interference ability.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] A method for detecting peroxidase activity in compost samples using a functional enzyme electrochemical sensor, wherein the functional enzyme electrochemical sensor includes a screen-printed electrode, the screen-printed electrode includes a working electrode, and the reaction end surface of the working electrode is modified with a zirconium-based metal-organic framework material by an adhesive; the zirconium-based metal-organic framework material is UiO-66-NH2.

[0007] A further improvement to the above method, the preparation method of the functional enzyme electrochemical sensor includes the following steps:

[0008] S1. Disperse zirconium-based metal-organic framework materials in a mixed solution of ethanol and binder, and then ultrasonically disperse them to prepare a zirconium-based metal-organic framework material mixture.

[0009] S2. A mixture of zirconium-based metal-organic framework materials is dropped onto the working electrode surface of a screen-printed electrode and dried to obtain a functional enzyme electrochemical sensor.

[0010] In a further improvement to the above method, in step S1, the ratio of the zirconium-based metal-organic framework material dispersed in the mixed solution of ethanol and binder is 3 mg to 5 mg: 1000 μL; the volume ratio of anhydrous ethanol to binder in the mixed solution of ethanol and binder is 1:1; the binder is a Nafion solution; and the mass ratio of the Nafion solution is 0.5% to 2.0%.

[0011] In a further improvement to the above method, in step S1, the zirconium-based metal-organic framework material is prepared by the following method: zirconium chloride, 2-aminoterephthalic acid, N,N-dimethylformamide and deionized water are mixed, heated at 110℃~140℃ for 24h, centrifuged, washed, and dried to obtain the zirconium-based metal-organic framework material; the ratio of zirconium chloride, 2-aminoterephthalic acid and N,N-dimethylformamide is 0.1g~0.2g∶0.1g~0.2g∶25mL~75mL.

[0012] In a further improvement to the above method, in step S1, the ultrasonic dispersion time is 20 min to 30 min.

[0013] In a further improvement to the above method, in step S2, the drying time is 45 min to 60 min.

[0014] In a further improvement to the above method, the adhesive is a perfluorinated resin solution; the screen-printed electrode also includes a counter electrode and a reference electrode, and the working electrode, counter electrode, and reference electrode are respectively loaded on the substrate; the working electrode, counter electrode, and reference electrode are also respectively connected with lead wires.

[0015] A further improvement to the above method, which utilizes a functional enzyme electrochemical sensor to detect peroxidase activity in compost samples, includes the following steps:

[0016] (1) Prepare compost enzyme activation stock solution from compost samples;

[0017] (2) The compost enzyme activation stock solution was mixed with BR buffer containing hydroquinone and hydrogen peroxide to prepare the test solution;

[0018] (3) Place the screen-printed electrode in the solution to be tested, and use the differential pulse voltammetry method to scan the solution to be tested and collect the current value of the solution to be tested;

[0019] (4) Based on the current value of the solution to be tested, and combined with the linear regression equation of peroxidase activity and current value, calculate the peroxidase activity in the compost sample.

[0020] In a further improvement to the above method, in step (4), the linear regression equation between the peroxidase activity and the current value is as follows:

[0021] Y=1037.1607×X+0.1897 (1);

[0022] In equation (1), Y is the maximum current value obtained by differential pulse voltammetry minus the baseline current, and X is the total peroxidase activity in the compost sample, with a detection linear range of 0–2.5 × 10⁻⁶. -4 U / mL, detection limit is 2×10 -5 U / mL, linear correlation coefficient R 2 It is 0.998.

[0023] The above method is further improved in step (1), wherein the preparation method of the compost enzyme activation stock solution includes the following steps: mixing the compost sample with toluene, letting it stand for 15 min to 20 min, adding BR buffer to obtain the compost enzyme activation stock solution; the ratio of the compost sample to toluene is 0.1 g: 40 μL to 70 μL; the BR buffer is prepared by mixing acetic acid, phosphoric acid and boric acid; the molar ratio of acetic acid, phosphoric acid and boric acid is 1:1:1; the concentration of the BR buffer is 0.04 M; and the pH value of the BR buffer is 4 to 5.

[0024] The above method is further improved in step (2), wherein the preparation method of the test solution includes the following steps: mixing the compost enzyme activation stock solution with a BR buffer containing hydroquinone and hydrogen peroxide, reacting at 30°C for 2-3 hours, standing for 5 minutes under ice bath conditions, filtering, centrifuging at 10,000 rpm for 5 minutes, and taking the supernatant as the test solution; the BR buffer containing hydroquinone and hydrogen peroxide is prepared by mixing hydroquinone, hydrogen peroxide and BR buffer; the concentration of hydroquinone in the BR buffer containing hydroquinone and hydrogen peroxide is 0.5 mM-1 mM, and the concentration of hydrogen peroxide is 1 mM-2 mM; the BR buffer is prepared by mixing acetic acid, phosphoric acid and boric acid; the molar ratio of acetic acid, phosphoric acid and boric acid is 1:1:1; the pH value of the BR buffer is 4-5.

[0025] In a further improvement to the above method, in step (3), the voltage is controlled to be -0.8V to 0V during the scanning process.

[0026] In this invention, the use of differential pulse voltammetry for detection can reduce errors caused by the presence of capacitance.

[0027] Compared with the prior art, the advantages of the present invention are as follows:

[0028] To address the shortcomings of existing electrochemical testing methods, such as long detection time, narrow detection range, and low sensitivity, which lead to difficulties in online detection and low detection accuracy, this invention creatively proposes a method for detecting peroxidase activity in compost samples using a functional enzyme electrochemical sensor. The method utilizes a functional enzyme electrochemical sensor to detect peroxidase activity in compost samples. The sensor includes a screen-printed electrode, which comprises a working electrode. The surface of the working electrode's reaction end is modified with a zirconium-based metal-organic framework (Zr-MOFs) using an adhesive (such as a perfluorinated resin solution, Nafion). The zirconium-based metal-organic framework is UiO-66-NH2. In this invention, by modifying the reaction end surface of the working electrode with UiO-66-NH2, on the one hand, the accumulated enzyme products can be pre-concentrated on the electrode surface by utilizing the π-π static interaction between the aromatic groups of the UiO-66-NH2 ligand and the aromatic groups of the enzyme products (such as p-benzoquinone). Furthermore, the large surface area and customizable highly ordered porosity of UiO-66-NH2 allow bioactive molecules (such as peroxidases) to diffuse into the porous structure, thereby increasing the reaction rate. On the other hand, the zirconium-based metal-organic framework material can be firmly fixed to the screen-printed electrode surface through the fixing effect of the adhesive, which improves both the stability and conductivity of the electrode. This results in a functional enzyme electrochemical sensor with fast reaction rate, good conductivity, and high current intensity, exhibiting stronger capabilities in detecting low concentrations of enzyme products. In addition, compared with conventional glassy carbon electrodes, the screen-printed electrode of this invention is disposable, inexpensive, easy to mass-produce, and highly reproducible. Using the screen-printed electrode as a substrate enhances the sensor's application prospects and expands its scope of use. Compared with conventional electrochemical sensors, the screen-printed electrode modified with UiO-66-NH2 used in this invention as a functional enzyme electrochemical sensor for detecting peroxidase activity in compost samples has the following advantages: (a) faster detection speed, allowing detection to be completed in a shorter time, with the detection time reduced to 90 seconds; (b) better adaptability, with a detection limit of 2 × 10⁻⁶. -5(c) It has higher accuracy, with a correlation performance of 0.998 within the linear range of actual compost sample detection, demonstrating excellent accuracy and better detection of peroxidase activity; (d) It is simpler to operate, with relatively simple pretreatment of actual samples and is not affected by light-absorbing substances or turbidity in the sample, making it suitable for online detection and showing better application prospects. Therefore, the method for detecting peroxidase activity in compost samples using a functional enzyme electrochemical sensor has the advantages of low cost, simple operation, low sample consumption, short detection time, wide detection range, high sensitivity, and strong anti-interference ability. It can quickly and accurately obtain the peroxidase activity in compost samples, thereby providing timely and effective data for compost maturity analysis. Attached Figure Description

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0030] Figure 1 This is a schematic diagram of the structure of the screen-printed electrode in Embodiment 1 of the present invention.

[0031] Figure 2 This is a scanning electron microscope (SEM) image of a screen-printed electrode with a zirconium-based metal-organic framework material on its surface, prepared in Example 1 of this invention.

[0032] Figure 3 This is a graph showing the changes in current values ​​of standard enzyme-catalyzed product solutions with different peroxidase activity concentrations in Example 1 of the present invention.

[0033] Figure 4 This is a graph showing the linear relationship between peroxidase activity and current value in Example 1 of the present invention.

[0034] Figure 5 This is a comparison chart of the current values ​​of the unmodified screen-printed electrode (Bare) and the screen-printed electrode modified with zirconium-based metal-organic framework material (UiO-66-NH2) in Example 1 of the present invention for HRP enzyme activation stock solution.

[0035] Legend:

[0036] 1. Counter electrode; 2. Working electrode; 3. Reference electrode; 4. Substrate; 5. Lead wire. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0038] Example 1

[0039] A method for detecting peroxidase activity in compost samples using a functional enzyme electrochemical sensor, specifically involving the detection of peroxidase activity in compost samples using a functional enzyme electrochemical sensor, includes the following steps:

[0040] (1) Mix 0.1g of sieved compost sample with 60μL of toluene. After mixing thoroughly, let stand at room temperature for 15min. Then add BR buffer solution with a concentration of 0.04M and a pH of 4-5 to obtain compost enzyme activation stock solution.

[0041] In step (1), the compost sample is an aerobic compost sample. The BR buffer used is prepared by mixing acetic acid, phosphoric acid and boric acid and adjusting the pH value to 4-5 with sodium hydroxide, wherein the molar ratio of acetic acid, phosphoric acid and boric acid is 1:1:1.

[0042] (2) Mix the compost enzyme activation stock solution with BR buffer containing hydroquinone and hydrogen peroxide, shake and react at 30°C for 3 hours, let stand in an ice bath for 5 minutes, filter, centrifuge at 10000 rpm for 5 minutes, take the supernatant to obtain the test solution.

[0043] In step (2), the BR buffer containing hydroquinone and hydrogen peroxide is prepared by mixing hydroquinone, hydrogen peroxide, and BR buffer. The concentration of hydroquinone in this BR buffer is 1 mM, and the concentration of hydrogen peroxide is 2 mM. The BR buffer used is prepared by mixing acetic acid, phosphoric acid, and boric acid and adjusting the pH to 4-5 with sodium hydroxide, wherein the molar ratio of acetic acid, phosphoric acid, and boric acid is 1:1:1.

[0044] (3) Place the screen-printed electrode in the solution to be tested, and use differential pulse voltammetry (DPV) to scan the solution to be tested in the range of -0.8V to 0V for 90s, and collect the current value of the solution to be tested.

[0045] In step (3), such as Figure 1 As shown, the screen-printed electrode includes a substrate 4, leads 5, and a three-electrode system consisting of a counter electrode 1, a working electrode 2, and a reference electrode 3. The counter electrode 1, working electrode 2, and reference electrode 3 are each placed on the substrate. The working electrode surface is modified with UiO-66-NH2. Carbon is used as the leads 5 for the counter electrode 1, working electrode 2, and reference electrode 3. The screen-printed electrode is connected to an electrochemical workstation to construct a functional enzyme electrochemical sensor.

[0046] (4) Based on the current value of the solution to be tested, and combined with the linear regression equation of peroxidase activity and current value, the peroxidase activity in the compost sample was calculated, and the results are shown in Table 1.

[0047] In this embodiment, the preparation method of the functional enzyme electrochemical sensor includes the following steps:

[0048] S1. Disperse 4 mg of zirconium-based metal-organic framework material in 1000 μL of a mixed solution of ethanol and Nafion solution, and sonicate for 30 min to prepare a zirconium-based metal-organic framework material mixture. In this step, the volume ratio of anhydrous ethanol to Nafion solution in the mixed solution is 1:1, and the mass ratio of Nafion solution is 1.0%.

[0049] S2. Use a pipette to take 6 μL of zirconium-based metal-organic framework material mixture and drop it onto the working electrode surface of the screen-printed electrode. Dry it at room temperature for 60 min to modify the working electrode surface of the screen-printed electrode with zirconium-based metal-organic framework material, and obtain a functional enzyme electrochemical sensor.

[0050] In this embodiment, the zirconium-based metal-organic framework material used is UiO-66-NH2, which is prepared by the following method: 0.2g zirconium chloride, 0.155g 2-aminoterephthalic acid, 50mL N,N-dimethylformamide and 140μL deionized water are mixed, heated at 120℃ for 24h, centrifuged, washed and dried to obtain the zirconium-based metal-organic framework material.

[0051] Figure 2 This is a scanning electron microscope (SEM) image of a screen-printed electrode with a surface modified with zirconium-based metal-organic framework material, prepared in Example 1 of this invention. Figure 2 As shown, the material is octahedral in shape with an average diameter of about 100 nm, indicating that UiO-66-NH2 was successfully prepared and fixed on the electrode surface by Nafion.

[0052] In this embodiment, the linear regression equation between peroxidase activity and current value is as follows:

[0053] Y=1037.1607×X+0.1897 (1);

[0054] In equation (1), Y is the maximum current value obtained by differential pulse voltammetry minus the baseline current, and X is the total peroxidase activity in the compost sample, with a detection linear range of 0–2.5 × 10⁻⁶. -4 U / mL, detection limit is 2×10 -5 U / mL, linear correlation coefficient R 2 It is 0.998.

[0055] In this embodiment, the method for constructing the linear regression equation between peroxidase activity and current value is as follows:

[0056] (a) Standard enzyme-catalyzed product solutions with different peroxidase activity concentrations were prepared. The total peroxidase activity concentration was 2.29 × 10⁻⁶. -4 U / mL, 2.03×10 -4 U / mL, 1.78×10 -4 U / mL, 1.27×10 -4 U / mL, 7.62×10 -5 U / mL, 5.08×10 -5 U / mL.

[0057] (b) The standard enzyme products with different peroxidase activity concentrations were mixed with BR buffer containing hydroquinone and hydrogen peroxide (the same as in step (1)). The mixture was shaken at 30°C for 3 h, allowed to stand in an ice bath for 5 min, filtered, centrifuged at 10000 rpm for 5 min, and the supernatant was collected to obtain the standard solutions to be tested with different peroxidase activity concentrations.

[0058] (c) Place the screen-printed electrode from step (2) into the test standard solutions with different peroxidase activity concentrations, and use differential pulse voltammetry (DPV) to scan the test standard solutions with different peroxidase activity concentrations in the range of -0.8V to 0V for 90s. Collect the current values ​​of the test standard solutions with different peroxidase activity concentrations, establish the linear relationship between peroxidase activity and current value, and obtain the linear regression equation between peroxidase activity and current value.

[0059] Figure 3 This is a graph showing the changes in current values ​​of standard enzymatically synthesized product solutions with different peroxidase activity concentrations in Example 1 of the present invention. Figure 3 It can be seen that the measured reduction current peak is positively correlated with the concentration of the enzyme-catalyzed product p-benzoquinone in the solution within a certain range. Therefore, the enzyme activity can be indirectly reflected by the p-benzoquinone reduction current peak.

[0060] Figure 4 This is a graph showing the linear relationship between peroxidase activity and current value in Example 1 of the present invention.

[0061] In this embodiment, the detection results of peroxidase activity in compost samples using commercial kits were also examined, as shown in Table 1.

[0062] Table 1. Detection and verification results of peroxidase in compost samples.

[0063]

[0064] As shown in Table 1, the total peroxidase activity detected by the screen-printed electrode modified with UiO-66-NH2 in this invention is basically consistent with the enzyme activity detected by commercial kits, indicating that the electrochemical method of this invention has high reliability. Furthermore, Table 1 shows that the relative standard deviation is 8.11%, which is within the acceptable range of 10%. Considering the presence of many interfering substances in compost samples, this relative standard deviation basically meets the requirements for enzyme activity detection in compost samples. In addition, compared with commercial kits, the electrochemical method of this invention is simpler and faster to operate, and can eliminate some of the errors caused by colorimetric interference in commercial kits, resulting in higher accuracy. Therefore, the method of detecting peroxidase activity in compost samples using a functional enzyme electrochemical sensor of this invention can be used for the detection of peroxidase in compost samples.

[0065] This embodiment also investigated the current values ​​of unmodified screen-printed electrodes and metal-organic framework-modified screen-printed electrodes for HRP enzyme activation stock solution, specifically:

[0066] (1) Mix the horseradish peroxidase (HRP) standard enzyme activity stock solution with BR buffer containing hydroquinone and hydrogen peroxide, and react at 20°C for 2 min to obtain the enzyme product solution, which is used as the test solution.

[0067] (2) The unmodified screen-printed electrode (Bare) and the metal-organic framework-modified screen-printed electrode (UiO-66-NH2) were placed in the enzyme product solution, respectively. Differential pulse voltammetry (DPV) was used to scan the test solution in the range of -0.8V to 0V for 90s. The current values ​​of the test solution under different functional enzyme electrochemical sensors were collected, and the results are as follows: Figure 5 As shown;

[0068] Figure 5 This is a comparison graph showing the current values ​​of the unmodified screen-printed electrode (Bare) and the screen-printed electrode modified with zirconium-based metal-organic framework material (UiO-66-NH2) in Example 1 of this invention against the HRP enzyme activity stock solution. Figure 5 As can be seen, under constant time, constant substrate concentration, and constant HRP concentration, the current of the screen-printed electrode modified with UiO-66-NH2 is significantly higher than that of the blank screen-printed electrode, with an improvement rate as high as 33%. Therefore, compared to the blank screen-printed electrode, this invention, by modifying the working electrode surface with UiO-66-NH2, can significantly increase the concentration of the enzymatic reactant p-benzoquinone near the electrode, thereby generating a higher reduction current during the electrochemical reduction process, ultimately improving the detection linear range and sensitivity.

[0069] The results above show that, compared with conventional electrochemical sensors, the screen-printed electrode modified with UiO-66-NH2 used in this invention as a functional enzyme electrochemical sensor for detecting peroxidase activity in compost samples has the following advantages: (a) faster detection speed, allowing detection to be completed in a shorter time, with the detection time reduced to 90 seconds; (b) better adaptability, with a detection limit of 2 × 10⁻⁶. -5 (c) It has higher accuracy, with a correlation performance of 0.998 within the linear range of actual compost sample detection, demonstrating excellent accuracy and better detection of peroxidase activity; (d) It is simpler to operate, with relatively simple pretreatment of actual samples and is not affected by light-absorbing substances or turbidity in the sample, making it suitable for online detection and showing better application prospects. Therefore, the method for detecting peroxidase activity in compost samples using a functional enzyme electrochemical sensor has the advantages of low cost, simple operation, low sample consumption, short detection time, wide detection range, high sensitivity, and strong anti-interference ability. It can quickly and accurately obtain the peroxidase activity in compost samples, thereby providing timely and effective data for compost maturity analysis.

[0070] The above embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting peroxidase enzyme activity in a compost sample using a functional enzyme electrochemical sensor, characterized in that, The method is for detecting peroxidase enzyme activity in a compost sample by using a functional enzyme electrochemical sensor; the functional enzyme electrochemical sensor comprises a screen-printed electrode, and the screen-printed electrode comprises a working electrode, and a reaction end surface of the working electrode is modified with a zirconium-based metal organic framework material by an adhesive; the zirconium-based metal organic framework material is UiO-66-NH2.

2. The method of claim 1, wherein, The preparation method of the functional enzyme electrochemical sensor comprises the following steps: S1, dispersing the zirconium-based metal organic framework material in a mixed solution of ethanol and adhesive, and ultrasonic dispersion to prepare a zirconium-based metal organic framework material mixture; S2, dropping the zirconium-based metal organic framework material mixture onto a surface of a working electrode of a screen-printed electrode, and drying to obtain a functional enzyme electrochemical sensor.

3. The method of claim 2, wherein, In step S1, the ratio of the zirconium-based metal organic framework material to the mixed solution of ethanol and adhesive is 3mg-5mg:1000μL; the volume ratio of anhydrous ethanol to adhesive in the mixed solution of ethanol and adhesive is 1:1; the adhesive is a Nafion solution; and the mass ratio of the Nafion solution is 0.5%-2.0%.

4. The method of claim 3, wherein, In step S1, the zirconium-based metal organic framework material is prepared by the following method: mixing zirconium chloride, 2-amino terephthalic acid, N,N-dimethylformamide and deionized water, heating at 110-140℃ for 24h, centrifugation, washing, and drying to obtain the zirconium-based metal organic framework material; and the ratio of the zirconium chloride, 2-amino terephthalic acid and N,N-dimethylformamide is 0.1g-0.2g:0.1g-0.2g:25mL-75mL.

5. The method of claim 4, wherein, In step S1, the ultrasonic dispersion time is 20-30min. In step S2, the drying time is 45-60min.

6. The method of claim 1, wherein, The adhesive is a perfluorinated resin solution; the screen-printed electrode further comprises a counter electrode and a reference electrode, and the working electrode, the counter electrode and the reference electrode are respectively loaded on a substrate; and the working electrode, the counter electrode and the reference electrode are respectively connected with lead wires.

7. The method according to any one of claims 1 to 6, characterized in that, When the functional enzyme electrochemical sensor is used to detect peroxidase enzyme activity in a compost sample, the following steps are included: (1) preparing a compost enzyme activity stock solution from the compost sample; (2) mixing the compost enzyme activity stock solution with a BR buffer containing hydroquinone and hydrogen peroxide to prepare a test solution; (3) placing the screen-printed electrode in the test solution, scanning the test solution by using a differential pulse voltammetry method, and collecting current values of the test solution; (4) calculating the enzyme activity of peroxidase in the compost sample according to the current values of the test solution and a linear regression equation of peroxidase enzyme activity and current values.

8. The method of claim 7, wherein, In step (4), the linear regression equation of peroxidase enzyme activity and current values is: Y=1037.1607×X+0.1897 (1). In formula (1), Y is the maximum current value of the current minus the baseline current measured by differential pulse voltammetry, X is the total peroxidase enzyme activity in the compost sample, the linear range of detection is 0-2.5×10 -4 U / mL, the detection limit is 2×10 -5 U / mL, and the linear correlation coefficient R 2 is 0.

998.

9. The method of claim 8, wherein, In step (1), the preparation method of the compost enzyme activation stock solution includes the following steps: mixing the compost sample with toluene, letting it stand for 15 min to 20 min, adding BR buffer to obtain the compost enzyme activation stock solution; the ratio of the compost sample to toluene is 0.1 g: 40 to 70 μL; the BR buffer is prepared by mixing acetic acid, phosphoric acid and boric acid; the molar ratio of acetic acid, phosphoric acid and boric acid is 1:1:1; the concentration of the BR buffer is 0.04 M; the pH value of the BR buffer is 4 to 5; In step (2), the preparation method of the test solution includes the following steps: mixing the compost enzyme activation stock solution with a BR buffer containing hydroquinone and hydrogen peroxide, reacting at 30°C for 2-3 hours, standing for 5 minutes under ice bath conditions, filtering, centrifuging at 10,000 rpm for 5 minutes, and taking the supernatant as the test solution; the BR buffer containing hydroquinone and hydrogen peroxide is prepared by mixing hydroquinone, hydrogen peroxide and BR buffer; the concentration of hydroquinone in the BR buffer containing hydroquinone and hydrogen peroxide is 0.5 mM-1 mM, and the concentration of hydrogen peroxide is 1 mM-2 mM; the BR buffer is prepared by mixing acetic acid, phosphoric acid and boric acid; the molar ratio of acetic acid, phosphoric acid and boric acid is 1:1:1; the pH value of the BR buffer is 4-5.

10. The method of claim 9, wherein, In step (3), the control potential during the scanning process is -0.8V to 0V.

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