Copper-based nano-enzyme array sensor based on cyano activation and application of copper-based nano-enzyme array sensor

Abstract

The invention is applicable to the technical field of pesticide detection, and provides a cyano activation-based copper-based nano enzyme array sensor and application thereof, the sensor comprises four independent sensing units, and each sensing unit is based on a copper-based nano enzyme, namely cytarabine-Cu, isophthalic acid-Cu, 5 '-guanylic acid-Cu and thiazole-5-formic acid-Cu. According to the method, four copper-based nano-enzymes are selected as sensing pathways to construct a colorimetric pathway, so that not only is extremely high selectivity achieved, rapid and convenient detection of the second-generation pyrethroid pesticide achieved, but also complex material design is not needed, and on-site convenient detection of the second-generation pyrethroid pesticide can be achieved.

Description

Technical Field

[0001] This invention belongs to the field of pesticide detection technology, and in particular relates to a copper-based nanozyme array sensor based on cyano activation and its application. Background Technology

[0002] Pyrethroid pesticides are a class of biomimetic insecticides synthesized by mimicking the components of natural pyrethroids. Due to their lipid solubility, they pose unknown hazards to the human body, causing certain damage to human hormone secretion and the nervous system. Long-term exposure to pyrethroid pesticides can cause adverse reactions in the male reproductive system, and children exposed to pyrethroid pesticides may have an increased risk of developing diseases such as tumors and leukemia.

[0003] Second-generation pyrethroid pesticides are synthesized by introducing α-cyanide ions into first-generation pyrethroid pesticides. Compared with first-generation pyrethroid pesticides, the introduction of cyanide ions enhances the photostability of pyrethroid pesticides, but also increases their toxicity to fish and other aquatic organisms, and exacerbates their potential environmental risks. Second-generation pyrethroid pesticides all exhibit high cytotoxicity; for example, deltamethrin has long-term toxicity in soil and can damage the central nervous system of fetuses, affecting children's intelligence; fenvalerate can cause metabolic disorders in lettuce, affecting lettuce yield. Therefore, developing rapid detection methods for second-generation pyrethroid pesticides is crucial.

[0004] Enzyme inhibition is a common rapid pesticide detection method. This method identifies pesticides by inhibiting the activity of acetylcholinesterase. However, this method can only detect organophosphates and carbamates, and has no response to pyrethroids. Currently, detection methods for second-generation pyrethroids include mass spectrometry, chromatography, and fluorescence methods. While mass spectrometry and chromatography can achieve accurate detection of second-generation pyrethroids, they require large instruments and skilled technicians, have certain technical requirements, and are complex to operate. The limitations of large instruments make it difficult to achieve convenient on-site detection of second-generation pyrethroids. Fluorescence methods can achieve rapid detection of second-generation pyrethroids, but current detection methods often rely on complex fluorescent material designs, resulting in high synthesis costs. The fluorescence signal is also affected and interfered with by ambient light, quenchers, or background fluorescence. Summary of the Invention

[0005] The purpose of this invention is to provide a copper-based nanozyme array sensor based on cyano activation, which aims to solve the problems mentioned in the background art.

[0006] The present invention is implemented as follows: a copper-based nanozyme array sensor based on cyano activation, the sensor comprising four independent sensing units, each sensing unit being based on a copper-based nanozyme, namely cytarabine-Cu, isophthalic acid-Cu, 5'-guanylic acid-Cu, and thiazolyl-5-carboxylic acid-Cu.

[0007] Another objective of this invention is to provide a method for preparing a cyano-activated copper-based nanozyme array sensor, comprising the following steps: (1) Synthesis of copper-based nanozymes: Synthesis of cytarabine-Cu: An aqueous solution containing cytarabine and NaOH is mixed with an aqueous solution containing copper chloride and reacted at room temperature. After the reaction is completed, the mixture is centrifuged and washed several times with water. The resulting precipitate is then uniformly dispersed in ultrapure water to obtain a cytarabine-Cu nanozyme solution. Synthesis of isophthalic acid-Cu: Dissolve isophthalic acid in an aqueous solution containing NaOH, then mix it with an aqueous solution of copper nitrate trihydrate, react at room temperature, and after the reaction is complete, centrifuge and wash several times with water, and redisperse the resulting precipitate uniformly in ultrapure water to obtain isophthalic acid-Cu nanozyme solution. Synthesis of 5'-guanylic acid-Cu: Mix 5'-guanylic acid disodium aqueous solution, copper chloride solution, Tris-HCl buffer and ultrapure water, react at room temperature, after the reaction is completed, centrifuge and wash several times with water, and disperse the obtained precipitate in ultrapure water to obtain 5'-guanylic acid-Cu nanozyme solution. Synthesis of thiazol-5-carboxylic acid-Cu: Mix an aqueous solution of thiazol-5-carboxylic acid with an aqueous solution containing copper nitrate trihydrate, stir and react at 50°C. After the reaction is complete, centrifuge and wash several times with water, and disperse the resulting precipitate in ultrapure water to obtain a thiazol-5-carboxylic acid-Cu nanozyme solution. (2) Construction of detection system: Mix acetate-sodium acetate buffer, nanozyme solution, ultrapure water and TMB solution, add to the well of 96-well plate to complete the construction of one sensing unit. Create four sensing units according to the above method. The four sensing units are arranged in parallel to form a complete four-channel sensor array, thus obtaining the copper-based nanozyme array sensor based on cyano activation.

[0008] Another objective of this invention is to provide an application of a cyano-activated copper-based nanozyme array sensor in pesticide detection, wherein the pesticide is a second-generation pyrethroid pesticide.

[0009] The sensor proposed in this invention consists of four specific copper-based nanozyme sensing units: cytarabine-Cu (Arac-Cu), isophthalic acid-Cu (13-Cu), 5'-guanylic acid-Cu (GMP-Cu), and thiazolyl-5-carboxylic acid-Cu (T5CA-Cu). It utilizes the cyanide ions released by the hydrolysis of second-generation pyrethroid pesticides under alkaline conditions to specifically enhance the oxidase activity of copper-based nanozymes, thereby catalyzing the chromogenic substrate to generate a detectable optical signal, thus realizing the basis for the identification of this type of pesticide. Compared with existing second-generation pyrethroid pesticide detection technologies, the embodiments of the present invention have the following significant advantages and effects: High specificity and strong anti-interference ability: Utilizing the specific activation effect of cyanogen on copper-based nanozymes, it can accurately identify second-generation pyrethroid pesticides from complex systems after hydrolysis, effectively eliminating the influence of other types of pesticides, common metal ions, amino acids and sugars. Excellent classification and recognition performance: The sensor array, constructed based on four different copper-based nanozymes, can generate differentiated response signal patterns. Combined with chemometric analysis, it can accurately distinguish and identify five structurally similar second-generation pyrethroid pesticides (cypermethrin, fenvalerate, deltamethrin, deltamethrin, and cypermethrin). Rapid detection and simple operation: The entire detection process, including hydrolysis, centrifugation, array reaction and reading, can be completed in a short time without complicated pretreatment and expensive large instruments, making it suitable for rapid screening in the field or laboratory. The material is simple to synthesize and low in cost: the copper-based nanozyme synthesis method is simple, the conditions are mild, and the raw materials are readily available. The sensor array construction and detection system are low in cost, which facilitates large-scale preparation and application promotion. Highly adaptable to practical applications: By introducing steps such as centrifugation, it overcomes the turbidity interference caused by incomplete hydrolysis of pesticides in actual samples, and can still maintain reliable identification ability in complex matrices such as fruits and vegetables, showing good potential for practical application and scalability. Attached Figure Description

[0010] Figure 1 A comparison of the hydrolysis effects of NaOH solutions of different concentrations provided in the embodiments of the present invention; Figure 2 Differentiation charts of five second-generation pyrethroid pesticides provided in embodiments of the present invention; Figure 3 The fitting curve of linear discriminant analysis factor 1 as a function of cypermethrin residue concentration is provided in the embodiments of the present invention; Figure 4 The chromatograms show the differentiation of second-generation pyrethroid pesticides in actual samples provided in the embodiments of the present invention. Detailed Implementation

[0011] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0012] Nanozymes are a class of nanomaterials with enzyme-like activity. Compared with natural enzymes, nanozymes are simpler to synthesize, cheaper, more stable, and more adaptable to the environment, maintaining activity under a wider range of pH and temperature conditions. In recent years, nanozymes have found extensive applications in the detection of environmental pollutants, including pesticides, antibiotics, and phenolic pollutants. An array sensor is an intelligent detection system based on multi-channel signal acquisition and pattern recognition analysis. Inspired by the olfactory or gustatory systems of mammals, it is also defined as an "electronic nose" or "electronic tongue." Because the different pathways in the array respond differently to the detected objects, the cross-response of multiple non-specific sensing units, combined with various data processing methods such as linear discriminant analysis (LDA) and hierarchical clustering analysis (PCA), generates feature point clusters (fingerprints) corresponding to different detected objects. This enables the simultaneous detection of multiple detected objects. Traditional one-to-one detection cannot be used to distinguish and identify multiple detected objects, affecting its detection efficiency and sensitivity. However, one-to-many detection can identify multiple detected objects and generate specificity by using fewer sensor pathways to identify multiple analytes. Constructing colorimetric arrays using nanozymes enables simultaneous detection of multiple substances while exhibiting high anti-interference performance. This invention presents a sensing array based on copper-based nanozymes and oxidase activity, which not only achieves highly sensitive and specific detection of pyrethroid pesticides but also enables precise identification of second-generation pyrethroid pesticide molecules. Specifically: The release of cyanide ions (CN) through hydrolysis of second-generation pyrethroid pesticides under alkaline conditions. - ), CN -Strong coordination with copper ions in specific copper-based nanozymes significantly enhances the oxidase activity of the nanozymes, thereby catalyzing the oxidation of the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB), resulting in a color change (the solution turns blue with a characteristic absorption peak at 652 nm). By constructing a sensor array containing four different copper-based nanozymes (Arac-Cu, 13-Cu, GMP-Cu, T5CA-Cu), the differential absorbance signals after their reaction with different target substances are collected. Combined with pattern recognition algorithms (such as linear discriminant analysis and hierarchical clustering analysis) to process multidimensional data, highly selective identification, accurate classification, and quantitative analysis of various second-generation pyrethroid pesticides (cypermethrin, deltamethrin, fenvalerate, deltamethrin, and lambda-cyhalothrin) can be achieved. It can effectively eliminate the influence of other types of pesticides, metal ions, amino acids, sugars, and other common interfering substances, and has good detection specificity.

[0013] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0014] Example 1: A copper-based nanozyme array sensor based on cyano activation, consisting of four independent sensing units, each based on a specific copper-based nanozyme. The sensing carrier is typically a 96-well plate (fully transparent microplate). Its preparation method includes the following steps: (1) Synthesis of copper-based nanozymes: Cytarabine-Cu (Arac-Cu): 5.2 mL of aqueous solution containing 0.126 g cytarabine and 0.02 g NaOH was mixed with 5.2 mL of aqueous solution containing 0.08 g copper chloride (CuCl2). The mixture was reacted at room temperature for 30 minutes. After the reaction was completed, the mixture was centrifuged and washed three times with water to remove unreacted ligands and metals. Finally, the precipitate was redispersed uniformly in ultrapure water to obtain the Arac-Cu nanozyme stock solution. Isophthalic acid-Cu (13-Cu): 1 mmol of isophthalic acid was dissolved in an aqueous solution containing 0.08 g of NaOH, and then mixed with an aqueous solution of 1 mmol of copper nitrate trihydrate [Cu(NO3)2·3H2O]. The mixture was reacted at room temperature for 2 hours. After the reaction was completed, the mixture was centrifuged and washed with water 3 times to remove unreacted ligands and metals. Finally, the precipitate was redispersed uniformly in ultrapure water to obtain 13-Cu nanozyme stock solution. 5'-Guanylic Acid-Cu (GMP-Cu): 5'-Guanylic Acid Disodium Aqueous Solution (10 mM), Copper Chloride Solution (50 mM), Tris-HCl Buffer (100 mM, pH 8.5) and Ultrapure Water were mixed at a volume ratio of 2:1:1:6 and reacted at room temperature. After the reaction was completed, the mixture was centrifuged and washed three times with water to remove unreacted ligands and metals. The precipitate was then dispersed in ultrapure water to obtain GMP-Cu nanozyme stock solution. Thiazole-5-carboxylic acid-Cu (T5CA-Cu): 10 mL of aqueous solution containing 0.4 mmol thiazol-5-carboxylic acid was mixed with 10 mL of aqueous solution containing 0.2 mmol copper nitrate trihydrate [Cu(NO3)2·3H2O]. The mixture was stirred at 50 °C for 1 hour. After the reaction was completed, the mixture was centrifuged and washed with water 3 times to disperse the precipitate and obtain T5CA-Cu nanozyme stock solution. The concentration of all synthesized nanozyme stock solutions was adjusted to 1 mg / mL; (2) Construction of the detection system: The reaction system of each sensing unit is constructed in the same way: take acetate-sodium acetate buffer (60 mM, pH 5.0), nanozyme solution (1 mg / mL), ultrapure water and TMB solution (20 mM, dissolved in acetone), mix them in a volume ratio of 5:2:8:1, and add them to the corresponding wells of the 96-well plate in sequence to complete the construction of one sensing unit (one detection channel). Four sensing units are arranged in parallel to form a complete four-channel sensor array.

[0015] Example 2: Optimization analysis of alkaline hydrolysis conditions for second-generation pyrethroid pesticides (aiming to determine the optimal conditions for the decomposition of cyanide ions (CN)). - The optimal alkaline hydrolysis conditions for the complete release of pesticide molecules include the following steps: (1) Sample hydrolysis and treatment: Prepare a standard solution of a certain second-generation pyrethroid pesticide (taking high-efficiency cypermethrin as an example) with a concentration of 100 μg / mL. Mix it with a series of sodium hydroxide (NaOH) solutions of different concentrations (e.g., 0.01 M, 0.05 M, 0.1 M, 0.2 M, 0.5 M) in equal volumes and react at room temperature for 10 minutes. After the reaction is completed, immediately neutralize each reaction system to neutral with hydrochloric acid solution of equal concentration. Since the pesticide that is not completely hydrolyzed may cause the solution to be turbid, centrifuge all the neutralized reaction solutions at 10000 rpm for 1 minute and take the clear supernatant as the test solution. (2) Detection and signal acquisition: Take the test solution corresponding to each concentration of NaOH hydrolysis and add it to the corresponding well of the four-channel sensor array constructed in Example 1. React at room temperature for 5 minutes and use an enzyme-linked immunosorbent assay reader to read the absorbance value of each well at a wavelength of 652 nm. The experiment was performed in parallel 5 times under each NaOH concentration condition. (3) Data Analysis and Results: The absorbance data from the five parallel experiments were averaged and imported into Origin software. A line graph was plotted with sodium hydroxide concentration on the x-axis and the average or sum of the absorbance of the four sensing units on the y-axis. The results are shown below. Figure 1 As shown, by analyzing the plateau interval of the curve, the NaOH concentration corresponding to the maximum and stable signal response was determined, i.e., the optimal hydrolysis concentration. The results show that 0.1 M NaOH can effectively hydrolyze pesticides and release CN in this system. - It produces a stable colorimetric signal, so it was selected as the actual hydrolysis concentration.

[0016] Example 3: Classification and identification of five second-generation pyrethroid pesticides (used to verify the ability of the array sensor prepared in Example 1 to distinguish between different target pesticides with similar structures), including the following steps: (1) Standard preparation and detection: Five second-generation pyrethroid pesticide standard solutions with a concentration of 100 μg / mL were prepared: high-efficiency cypermethrin, fenvalerate, deltamethrin, fenvalerate, and high-efficiency flucypermethrin. Each standard solution was mixed with 0.1 M NaOH solution at room temperature and reacted for 10 minutes (to hydrolyze and break the cyano group in the pesticide molecule and release cyanide ions). After the reaction, the solution was neutralized with HCl solution of equal concentration. In order to eliminate the interference of turbidity on subsequent optical detection, the reaction solution was centrifuged (centrifuged at 10000 rpm for 1 minute). The clear supernatant was used as the pretreatment solution to be tested. (2) Array reaction and data acquisition: The pretreatment solutions of the five pesticides were added to independent sensor arrays. After reacting at room temperature for 5 minutes, the absorbance value of each well at 652 nm was read using an enzyme-linked immunosorbent assay (ELISA) reader. Each pesticide was tested 5 times (n=5). (3) Pattern recognition and classification: The obtained raw data (the absorbance values ​​of 4 channels for each sample constitute a 4-dimensional response vector) were imported into SPSS statistical software. Linear discriminant analysis (LDA) was performed on all data (5 pesticides × 5 replicates = 25 samples) to extract the scores of the first two linear discriminant functions (factor 1 and factor 2). Results Display: A two-dimensional discriminant score graph was plotted with factor 1 score as the x-axis and factor 2 score as the y-axis, as shown below. Figure 2 As shown, the data points of the five pesticides each form independent and tightly clustered clusters, and the clusters of different pesticides are clearly separated. This indicates that the array sensor and detection application prepared based on the embodiments of the present invention can clearly and accurately classify and identify five structurally similar second-generation pyrethroid pesticides, and has extremely high distinguishing ability.

[0017] Example 4: Concentration response and quantitative analysis of second-generation pyrethroid pesticides (used to verify the concentration response characteristics of the method to the target pesticide and to establish a standard curve for semi-quantitative analysis), including the following steps: (1) Preparation and treatment of concentration gradient samples: Taking high-efficiency cypermethrin as an example, a series of standard solutions with concentrations of 1, 2.5, 5, 10, 25, 50, 100, 125, 150 and 200 μg / mL were accurately prepared. Each standard solution was mixed with 0.1 M NaOH solution, hydrolyzed at room temperature for 10 minutes, neutralized with hydrochloric acid of equal concentration, and centrifuged to obtain the pretreatment solution to be tested at that concentration point; (2) Detection and data acquisition: The clarified pretreatment solution corresponding to each concentration gradient was added to the sensor array prepared in Example 1. After reacting at room temperature for 5 minutes, the absorbance value of each well at 652 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. Five parallel experiments were performed for each concentration point. (3) Data analysis and standard curve establishment: The absorbance values ​​obtained by each sample (each concentration point) on the four sensing units are combined into a 4-dimensional response vector. All concentration gradient data (10 concentrations × 5 repetitions = 50 samples) are imported into SPSS software for linear discriminant analysis (LDA). After analysis, the score of the first linear discriminant function (LD1) is extracted. The standard concentration of high efficiency cypermethrin is used as the x-axis and the average score of the corresponding factor 1 is used as the y-axis for linear fitting. The results are as follows Figure 3 As shown, within the concentration range of 1 to 200 μg / mL, there is a good linear relationship between the concentration of high-efficiency cypermethrin and the factor 1 score obtained from LDA analysis. The establishment of this linear standard curve indicates that the method of the present invention can not only be used for qualitative identification, but also for semi-quantitative detection of target pesticides, thus expanding its application scope.

[0018] Example 5: Spiked recovery and identification verification of second-generation pyrethroid pesticides in actual environmental water samples (used to verify the ability of the method to directly distinguish and identify target pesticides in actual complex environmental matrices), including the following steps: (1) Preparation of actual water sample spiked samples: The pretreated lake water was used as the actual sample matrix. Five different second-generation pyrethroid pesticide standards were added to multiple equal-volume lake water samples to prepare a series of spiked simulated positive samples. The concentration of the added pesticides (high-efficiency cypermethrin, fenvalerate, deltamethrin, fenvalerate, and high-efficiency flucypermethrin) was 100 μg / mL. (2) Sample preparation and detection: The spiked lake water samples prepared above were subjected to alkaline hydrolysis (0.1 M NaOH, room temperature, 10 min), neutralization and centrifugation according to the process determined in Example 2 to obtain the test solution. Each test solution was added to an independent sensor array. After reacting at room temperature for 5 minutes, the absorbance value of each well at 652 nm was read using an ELISA reader. Each spiked sample was tested in 5 parallel experiments. (3) Data processing and analysis: The absorbance values ​​of each spiked sample obtained on the four sensing units are combined into a 4-dimensional response vector. The response data of all spiked samples (5 pesticides × 5 replicates = 25 samples) are imported into SPSS software for linear discriminant analysis (LDA) and the scores of the first two linear discriminant functions (factor 1 and factor 2) are extracted. A two-dimensional discriminant score plot was created with factor 1 score as the x-axis and factor 2 score as the y-axis. The result is shown below. Figure 4 As shown, the results indicate that the response data points of the five different pesticide-spikened lake water samples each form independent and well-separated clusters. This directly proves that the array sensor and detection application constructed in this embodiment of the invention can effectively overcome the interference of the actual water sample matrix and achieve accurate differentiation and identification of different types of second-generation pyrethroid pesticide residues.

[0019] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A copper-based nanozyme array sensor based on cyano activation, characterized in that, The sensor comprises four independent sensing units, each based on a copper-based nanozyme, namely cytarabine-Cu, isophthalic acid-Cu, 5'-guanylic acid-Cu, and thiazolyl-5-carboxylic acid-Cu.

2. A method for preparing a copper-based nanozyme array sensor based on cyano activation as described in claim 1, characterized in that, Includes the following steps: (1) Synthesis of copper-based nanozymes: Synthesis of cytarabine-Cu: An aqueous solution containing cytarabine and NaOH is mixed with an aqueous solution containing copper chloride and reacted at room temperature. After the reaction is completed, the mixture is centrifuged and washed several times with water. The resulting precipitate is then uniformly dispersed in ultrapure water to obtain a cytarabine-Cu nanozyme solution. Synthesis of isophthalic acid-Cu: Dissolve isophthalic acid in an aqueous solution containing NaOH, then mix it with an aqueous solution of copper nitrate trihydrate, react at room temperature, and after the reaction is complete, centrifuge and wash several times with water, and redisperse the resulting precipitate uniformly in ultrapure water to obtain isophthalic acid-Cu nanozyme solution. Synthesis of 5'-guanylic acid-Cu: Mix 5'-guanylic acid disodium aqueous solution, copper chloride solution, Tris-HCl buffer and ultrapure water, react at room temperature, after the reaction is completed, centrifuge and wash several times with water, and disperse the obtained precipitate in ultrapure water to obtain 5'-guanylic acid-Cu nanozyme solution. Synthesis of thiazol-5-carboxylic acid-Cu: Mix an aqueous solution of thiazol-5-carboxylic acid with an aqueous solution containing copper nitrate trihydrate, stir and react at 50°C. After the reaction is complete, centrifuge and wash several times with water, and disperse the resulting precipitate in ultrapure water to obtain a thiazol-5-carboxylic acid-Cu nanozyme solution. (2) Construction of detection system: Mix acetate-sodium acetate buffer, nanozyme solution, ultrapure water and TMB solution, add to the well of 96-well plate to complete the construction of one sensing unit. Create four sensing units according to the above method. The four sensing units are arranged in parallel to form a complete four-channel sensor array, thus obtaining the copper-based nanozyme array sensor based on cyano activation.

3. The method for preparing a cyano-activated copper-based nanozyme array sensor according to claim 2, characterized in that, In the step of synthesizing cytarabine-Cu, the mass ratio of cytarabine, NaOH and copper chloride is 6.3:1:

4.

4. The method for preparing a cyano-activated copper-based nanozyme array sensor according to claim 2, characterized in that, In the step of synthesizing isophthalic acid-Cu, the molar ratio of isophthalic acid to copper nitrate trihydrate is 1:

1.

5. The method for preparing a cyano-activated copper-based nanozyme array sensor according to claim 2, characterized in that, In the step of synthesizing 5'-guanylic acid-Cu, the concentration of the 5'-guanylic acid disodium aqueous solution is 10 mM, the concentration of the copper chloride solution is 50 mM, the concentration of the Tris-HCl buffer is 100 mM, the pH is 8, and the volume ratio of the 5'-guanylic acid disodium aqueous solution, copper chloride solution, Tris-HCl buffer and ultrapure water is 2:1:1:

6.

6. The method for preparing a cyano-activated copper-based nanozyme array sensor according to claim 2, characterized in that, In the step of synthesizing thiazole-5-carboxylic acid-Cu, the molar ratio of thiazole-5-carboxylic acid to copper nitrate trihydrate is 2:

1.

7. The method for preparing a cyano-activated copper-based nanozyme array sensor according to claim 2, characterized in that, In the steps of constructing the detection system, the concentration of the acetate-sodium acetate buffer is 60 mM, the pH is 5.0, the concentration of the nanozyme solution is 1 mg / mL, the concentration of the TMB solution is 20 mM, and the volume ratio of the acetate-sodium acetate buffer, the nanozyme solution, the ultrapure water, and the TMB solution is 5:2:8:

1.

8. The application of a cyano-activated copper-based nanozyme array sensor as described in claim 1 in pesticide detection, characterized in that, The pesticide in question is a second-generation pyrethroid pesticide.

9. The application according to claim 8, characterized in that, Includes the following steps: (1) Alkaline hydrolysis and pretreatment of pesticide samples: The second-generation pyrethroid pesticide sample to be tested was mixed with NaOH solution at room temperature and reacted. After the reaction was completed, HCl solution of equal concentration was added to neutralize to neutrality, and then centrifuged. The supernatant was taken as the clear pretreatment solution to be tested. (2) Array reaction and signal generation: The pretreatment solution to be tested was added to each reaction well of the copper nanozyme array sensor based on cyano activation and reacted at room temperature. The absorbance value of each well at a wavelength of 652 nm was read using an enzyme-linked immunosorbent assay (ELISA) reader. The absorbance value of each pesticide sample was obtained on four channels and imported into statistical analysis software for linear discriminant analysis.

10. The application according to claim 9, characterized in that, In step (1), the concentration of the NaOH solution is 0.1M.

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