Preparation of DNA-driven nano-enzyme / bio-enzyme composite material and application of metolcarb detection

Abstract

The invention discloses preparation of a nano-enzyme / biological enzyme cascade composite material based on DNA tweezers driving and application of the nano-enzyme / biological enzyme cascade composite material in on-site rapid detection of metolcarb. The copper-based nano enzyme with the nanoflower structure, which is synthesized from copper sulfate and DNA (Deoxyribonucleic Acid) at room temperature, shows excellent laccase-imitating activity and stability; according to the base complementary pairing principle and the high affinity of an aptamer to a molecular target, DNA tweezers fix AChE on the surface of nano-enzyme to form a CuNFs-Apt-AChE cascade enzyme composite material. The output signal of the sensing system is further enhanced through the synergistic effect of the enzyme cascade system, so that the detection sensitivity is improved. Sodium alginate hydrogel is used for fixing a CuNFs-Apt-AChE cascade enzyme composite material, and a high-stability immediate response platform for detecting meta-tolcarb residues is prepared. The sensor shows good sensitivity, portability and stability in practical application, and an innovative platform is provided for on-site rapid detection.

Description

Technical Field

[0001] The invention belongs to the technical field of biosensors, and in particular relates to a preparation method of a flower-shaped nanostructured cascade enzyme composite material (CuNFs-Apt-AChE) and an application thereof in the rapid detection of medicarb. Background Art

[0002] In the field of biosensor research, multi-enzyme cascade systems have attracted much attention because of their ability to simulate the multi-enzyme structure in biological cells. This system effectively reduces the steps of substrate transport and intermediate diffusion by integrating multiple catalytic reactions, bringing new solutions to biomedical diagnosis, environmental monitoring, food safety and other fields. However, the cascade catalytic system composed of natural enzymes has the problem of insufficient enzyme stability, which is limited in practical applications. Compared with natural enzymes, nanozymes have the advantages of simple preparation, good stability and high cost-effectiveness. The design of the nanozyme-bioenzyme cascade system combines the stability of nanozymes and the specificity of bioenzymes, thereby improving the overall performance of biosensors. Usually, most nanozyme cascade systems are just mixed with natural enzymes and work independently, rather than building a cascade system, which may lead to the waste of intermediates and affect the reaction rate and detection sensitivity of the sensor system. In addition, the reaction conditions of bioenzymes and nanozymes are often incompatible, resulting in the inability of the nanozyme-enzyme cascade hybrid system to fully utilize the advantages of these two components, limiting their synergistic effect in the cascade system. In order to solve these problems, the construction of immobilized nanozyme / bioenzyme cascade system has become a research hotspot in the field of biosensors. Traditional enzyme immobilization methods, such as physical adsorption or covalent grafting, often result in low enzyme loading and enzyme inactivation, thereby reducing the activity of the cascade system. Therefore, it is necessary to develop a mild enzyme immobilization method to effectively connect nanozymes with natural enzymes in the cascade system. The DNA-based nanozyme synthesis method is simple and environmentally friendly, in which DNA can give nanomaterials the properties of adjustable activity and easy surface modification due to its precisely defined sequence, programmable self-assembly and specific base pairing ability. DNA tweezers have high specificity and affinity for molecular targets due to their secondary and tertiary structures. They can be used as identifiers for immobilized enzymes or as surface modification linkers to build a "bridge" between enzymes and nanozymes to form a hybrid cascade system.

[0003] The present invention innovatively designs a copper-based nanozyme-acetylcholinesterase composite material (CuNFs-Apt-AChE) to construct a visual sensing method for the detection of cypermethrin pesticide. First, nano-flower-like nanozyme materials (CuNFs) are synthesized by self-assembly of copper sulfate and DNA at room temperature, which exhibit excellent laccase-like activity and catalytic stability; based on the principle of base complementary pairing and the recognition specificity of aptamers to molecular targets, acetylcholinesterase (AChE) is fixed on the surface of nanozymes with DNA tweezers to form a CuNFs-Apt-AChE cascade enzyme composite material. On the basis of nanozyme amplification of the output signal, the synergistic effect of the enzyme cascade system further enhances the output signal of the sensing system, improves the sensitivity of detection, and exhibits excellent stability; at the same time, the CuNFs-Apt-AChE cascade enzyme composite material is fixed with an alginate / calcium hydrogel system to prepare a sensor for instant detection of cypermethrin. The constructed sensor exhibits excellent sensitivity and specificity in practical applications, and is also portable and visual, providing a new platform for rapid on-site detection of pesticides. Summary of the invention

[0004] The present invention is dedicated to providing a method for preparing a CuNFs-Apt-AChE cascade enzyme composite material and a rapid detection application of cypermethrin. Based on the complementary base pairing effect of DNA tweezers and DNA sequences and the highly specific binding to the target molecule enzyme, a nanozyme-bioenzyme cascade nanocomposite material with high specificity and excellent stability is prepared in a gentle manner. The enzyme cascade system shortens the distance between AChE and CuNFs nanozymes, promotes the transfer of intermediates, and thus enhances the catalytic effect of the cascade enzyme. Utilizing the inhibitory effect of carbamate pesticide cypermethrin on the activity of acetylcholinesterase, a hydrogel sensor based on the CuNFs-Apt-AChE cascade enzyme composite material is developed to achieve efficient and convenient detection of cypermethrin.

[0005] The preparation method of the CuNFs-Apt-AChE cascade enzyme composite material with flower-like nanostructures and the establishment of a hydrogel colorimetric method for on-site detection of cypermethrin are as follows: Preparation method of flower-shaped CuNFs nanozymes. Copper sulfate solution (50-500 mmol L -1 ) and G-DNA (0.05-5 μmol L -1 ) PBS buffer solution (10 mmol L -1 , pH = 7.4) in a volume ratio of 150:1 and incubated at 20-25°C for 1 hour to 5 days. The CuNFs mixture was washed three times by centrifugation and then redispersed in ultrapure water to obtain a CuNFs nanoflower solution.

[0006] Preparation method of CuNFs-Apt-AChE cascade enzyme composite material: DNA tweezers solution (0.01-5 μmol L -1 ) was denatured at 65-95°C for 2-20 min, cooled to room temperature, and allowed to stand at 4-35°C for 1-50 min to stabilize its spatial conformation. CuNFs solution (0.01-1 mg mL -1 ) were mixed thoroughly and reacted at 20-25°C for 5-100 min. After the reaction was completed, the mixture CuNFs-Apt was washed three times by centrifugation and redissolved in ultrapure water. The CuNFs-Apt solution (0.01-1 mg mL -1 ) and AChE solution (0.1-5 mg mL -1 ) were mixed in a ratio of 1:1, reacted at 30-40°C for 10-250 min, and after incubation, the mixture was centrifuged and washed three times, and the obtained CuNFs-Apt-AChE precipitate was redispersed in ultrapure water to obtain a CuNFs-Apt-AChE solution.

[0007] Preparation method of fluorescently labeled CuNFs-Apt-AChE cascade enzyme composite material: Carboxyfluorescein-labeled DNA tweezers solution (0.01-5 μmol L -1 ) was denatured at 65-95°C for 2-20 min, cooled to room temperature, and allowed to stand at 4-35°C for 1-50 min to stabilize its spatial conformation. CuNFs solution (0.01-1 mg mL -1 ) were mixed thoroughly and reacted at 20-25°C for 5-100 min. After the reaction was completed, the mixture CuNFs-Apt was washed three times by centrifugation and redissolved in ultrapure water. The CuNFs-Apt solution (0.01-1 mg mL -1 ) and Rhodamine B-stained AChE solution (0.1-5 mg mL -1 ) were mixed in a ratio of 1:1 and reacted at 25-37°C for 10-250 min. After incubation, the mixture was centrifuged and washed three times. The obtained CuNFs-Apt-AChE precipitate was redispersed in ultrapure water to obtain a fluorescently labeled CuNFs-Apt-AChE solution, which was characterized by laser scanning confocal microscopy.

[0008] Study on the enzyme activity of CuNFs-Apt-AChE. -1 ), CuNFs-Apt solution (1-20 μg mL -1 ) and CuNFs-Apt-AChE solution (1-20 μg mL -1) were mixed with epinephrine hydrochloride solution (0.1-5 mg mL -1 ) and MES buffer (30 mmol L -1 , pH = 6) in a ratio of 1:1:8, react at 20-80°C for 5-200 min, and measure the visible absorption spectra of the substrates using a UV-visible spectrophotometer.

[0009] Study on the cascade activity of CuNFs-Apt-AChE cascade enzyme composites. DNA tweezers solution (0.01-1 μmolL -1 ) and AChE solution (0.01-10 mg mL -1 ) in a ratio of 1:1 and react at 35-37°C for 10-250 min to obtain Apt-AChE. -1 ) was recorded as solution a, and CuNFs (1-50 μg mL -1 ) and Apt-AChE were mixed in equal volumes and recorded as solution b. CuNFs (1-50 μg mL -1 ) and AChE (0.01-10 mg mL -1 ) were mixed in equal volumes and recorded as solution c. Add solutions a, b, and c to three centrifuge tubes respectively, and then add Tris-HCl buffer (10 mmol L -1 , pH = 7.5) and mefenamic acid solution (0.001-100 μg mL -1 ), the ratio is 4:1:1, stirred evenly, reacted at 35-37℃ for 5-400 min, and obtained system A. Tris-HCl buffer (10 mmol L -1 , pH = 7.5) and thioacetylcholine solution (1-25mmol L -1 ) were stirred evenly with a volume ratio of 6:1:1, and reacted at 35-37°C for 5-250 min to obtain system B. MES buffer (30 mmol L -1 , pH = 6), epinephrine hydrochloride solution (0.1-10 mg mL -1 ) and stir evenly with a volume ratio of 2:7:1. After reacting at 20-80°C for 5-200 min, take out and measure the absorbance at 470 nm using a UV-visible spectrophotometer at room temperature, and calculate the ratio of the absorbance to the absorbance of the blank group.

[0010] The stability of CuNFs-Apt-AChE cascade enzyme composites was studied. -1) and CuNFs-Apt-AChE solution (0.5-10 mg mL -1 ) were added with trypsin solution (0.1-1 mg mL -1 ), mix thoroughly, react at 35-37℃ for 5-400min, and record it as solution a. Take solution a, add water and PBS buffer solution (10 mmol L -1 , pH = 7.4), thioacetylcholine solution (1-10 mmol L -1 ), the ratio is 5:9:5:5, after fully mixing, react at 35-37℃ for 5-250 min, recorded as system A. Add 5,5'-dithiobis(2-nitrobenzoic acid) solution (0.1-10 mgmL -1 ), the ratio was 1:1, the reaction was carried out at 10-40℃ for 1-100 min, and the absorbance at 405nm was measured using a UV-visible spectrophotometer at room temperature and compared with the control group.

[0011] The hydrogel colorimetric method based on the CuNFs-Apt-AChE cascade enzyme composite material was used for on-site detection of the pesticide cypermethrin. Weigh 10-200 mg of sodium alginate in a centrifuge tube, shake it appropriately to evenly distribute it on the wall of the centrifuge tube, add 1-30 mL of ultrapure water, shake it and ultrasonicate it for 1-60 min, remove it and centrifuge it for later use. Add CuNFs-Apt-AChE solution (1-10 μg mL -1 ) and sodium alginate to make the composite material evenly distributed, and then add calcium chloride solution (0.1-10mg mL -1 ) to form a gel, and then Tris-HCl buffer (10 mmol L -1 , pH = 7.5) and different concentrations (0 μg mL -1 -100 μg mL -1 ) and cypermethrin in a volume ratio of 2:1:1, stirred evenly and reacted at 35-37°C for 5-400 min to obtain system A. Tris-HCl buffer (10 mmol L -1 , pH = 7.5) and thioacetylcholine solution (1-20mmol L -1 ) were stirred evenly with a volume ratio of 4:1:1, and reacted at 35-37°C for 5-250 min to obtain system B. Then, MES buffer (30 mmol L -1 , pH = 6), epinephrine hydrochloride solution (0.1-10 mg mL -1) were stirred evenly with a volume ratio of 6:3:1, and reacted at 20-80°C for 5-200 min before being taken out. The absorbance at 470 nm generated by the color reaction of different concentrations of cypermethrin solutions and the CuNFs-Apt-AChE enzyme cascade system in the sodium alginate gel was measured using a UV-visible spectrophotometer at room temperature. According to the ratio of the absorbance of several groups to the absorbance of the blank group and the corresponding logarithmic value of the pesticide concentration, the relationship curve was fitted using regression analysis, thereby obtaining the linear relationship of the sensing system and realizing the quantitative detection of cypermethrin.

[0012] The mechanism of the present invention is as follows: Using DNA and copper sulfate solution as raw materials, the abundant amide and amine groups in DNA form complexes with copper ions to prepare nanozymes with laccase-like activity. Based on the principle of complementary base pairing, the high affinity and high specific binding ability of DNA tweezers to target molecules are used to specifically capture acetylcholinesterase (AChE) and fix it on the surface of nanozymes to prepare CuNFs-Apt-AChE cascade enzyme composite materials. In the enzyme cascade system, acetylcholinesterase catalyzes thioacetylcholine to generate thiocholine, which competitively binds to the copper active sites on the copper-based nanozymes, thereby inhibiting the laccase-like activity of the copper-based nanozymes and causing changes in the colorimetric signal. The enzyme cascade system can be used to construct a hydrogel reactor to achieve rapid visualization and qualitative detection of cypermethrin. The quantitative detection of cypermethrin was achieved through the relationship curve constructed by the logarithm of absorbance and standard pesticide concentration.

[0013] The present invention has the following characteristics: Using DNA tweezers to immobilize acetylcholinesterase on nanozymes in a gentle manner, the CuNFs-Apt-AChE cascade enzyme composite was designed and prepared for the first time; The enzyme cascade system shortens the distance between enzymes and effectively promotes the transfer of intermediates, thereby improving the enzyme catalytic effect. At the same time, nanozymes enhance the stability of the sensing system. The enzyme cascade hydrogel colorimetric sensor prepared by the invention has good sensitivity, selectivity and stability, and the construction process is low in cost and simple in operation.

[0014] The present invention detects the content of cypermethrin based on an enzyme cascade hydrogel colorimetric sensor, and shows significant application prospects in improving the sensitivity of pesticide detection and developing stable portable detection equipment to achieve rapid on-site inspection. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1(A) is a scanning electron microscope characterization image of the flower-like nanostructured CuNFs-Apt-AChE cascade enzyme composite material in Example 1; (B) is a laser scanning confocal microscope image of the fluorescently labeled CuNFs-Apt-AChE cascade enzyme composite material in Example 1.

[0016] Figure 2 This is the laccase-like activity of the CuNFs-Apt-AChE cascade enzyme composite material in Example 2.

[0017] Figure 3 This is the cascade activity of the CuNFs-Apt-AChE cascade enzyme composite material in Example 3.

[0018] Figure 4 This is the stability of the CuNFs-Apt-AChE cascade enzyme composite material in Example 4.

[0019] Figure 5 This is a schematic diagram of the detection method of the sulfadiazine hydrogel in Example 2.

[0020] Figure 6 (A) is a photograph of the hydrogel colorimetric sensor described in Example 2 and the water colorimetric sensor under natural light; (B) is the feasibility of the hydrogel colorimetric sensor described in Example 2 in detecting mefenamic acid; (C) is a linear relationship diagram between the absorbance of the hydrogel colorimetric sensor described in Example 3 and the logarithm of the concentration of mefenamic acid. DETAILED DESCRIPTION

[0022] Example 1: Preparation method of CuNFs-Apt-AChE cascade enzyme composite material with flower-like nanostructure Copper sulfate solution (120 mmol L -1 , 8 μL) was added with 0.5 μmol L -1 G-DNA in PBS buffer (10 mmol L -1 , pH = 7.4, 1200 μL), then mixed thoroughly and incubated at 25 ° C for 3 days. The mixture was centrifuged (10000 rpm, 5 min) and washed three times with ultrapure water, and the blue CuNFs precipitate was redispersed in 600 μL ultrapure water.

[0023] DNA tweezers solution (0.5 μmol L -1, 240 mL) was denatured at 95 °C for 5 min, cooled to room temperature, and allowed to stand at 25 °C for 30 min to stabilize its spatial conformation. Subsequently, CuNFs solution (240 mL) was added, mixed thoroughly, and reacted at 25 °C for 60 min. After the reaction was completed, the mixture was centrifuged (10000 rpm, 5 min) and washed three times with ultrapure water to obtain CuNFs-Apt and redissolved in 800 μL ultrapure water. The CuNFs-Apt solution (200 μL) was mixed with AChE solution (0.8 mgmL -1 , 200 μL) and reacted at 37°C for 120 min. After incubation, the mixture was centrifuged (10000 rpm, 5 min) and washed three times with ultrapure water to obtain CuNFs-Apt-AChE. The precipitate was then redispersed in 800 μL ultrapure water to obtain a CuNFs-Apt-AChE solution (24 μg mL -1 ), characterized by scanning electron microscopy ( Figure 1 A).

[0024] Carboxycellulose-labeled DNA tweezers solution (0.5 μmol L -1 , 240 mL) was denatured at 95 °C for 5 min, cooled to room temperature, and allowed to stand at 25 °C for 30 min to stabilize its spatial conformation. Subsequently, CuNFs solution (240 mL) was added, mixed thoroughly, and reacted at 25 °C for 60 min. After the reaction was completed, the mixture was centrifuged (10000 rpm, 5 min) and washed three times with ultrapure water to obtain CuNFs-Apt and redissolved in 800 μL ultrapure water. The CuNFs-Apt solution (200 μL) was mixed with a rhodamine B-stained AChE solution (0.8 mg mL -1 , 200 μL) and reacted at 37°C for 120 min. After incubation, the mixture was centrifuged (10000 rpm, 5 min) and washed three times with ultrapure water to obtain CuNFs-Apt-AChE. The precipitate was then redispersed in 800 μL ultrapure water to obtain a fluorescently labeled CuNFs-Apt-AChE solution (24 μg mL -1 ) and characterized using laser scanning confocal microscopy ( Figure 1 B).

[0025] Example 2: Study on the laccase activity of CuNFs-Apt-AChE cascade enzyme composite material The CuNFs solution (8 μg mL -1 , 100 μL), CuNFs-Apt solution (8 μg mL -1, 100 μL) and CuNFs-Apt-AChE solution (8 μg mL -1 , 100 μL) were mixed with adrenaline hydrochloride solution (1 mg mL -1 , 100 μL) and MES buffer (30 mmol L -1 , pH=6, 800 μL) were mixed and reacted at 75°C for 60 min, and the visible absorption spectra of the substrates were measured by UV-visible spectrophotometer ( Figure 2 ).

[0026] Example 3: Study on cascade activity of CuNFs-Apt-AChE cascade enzyme composite material The DNA tweezers solution (0.06 μmol L -1 , 200 μL) and AChE solution (0.016 mg mL -1 , 200 μL) and reacted at 37°C for 120 min to obtain Apt-AChE. -1 , 100 μL) was recorded as solution A, and CuNFs (16 μg mL -1 , 50 μL) and Apt-AChE (50 μL) were mixed in equal volumes as solution B, and CuNFs (16 μgmL -1 , 50 μL) and AChE (0.008 mg mL -1 , 50 μL) and mixed in equal volumes as solution C. Add solutions A, B, and C to three centrifuge tubes, respectively, and then add Tris-HCl buffer (10 mmol L -1 , pH = 7.5, 25 μL) and mefenamic acid solution (0.2 μg mL -1 , 25 μL), mixed thoroughly and reacted at 37°C for 35 min. Then Tris-HCl buffer (10 mmol L -1 , pH = 7.5, 25 μL) and thioacetylcholine solution (8 mmol L -1 , 25 μL) were mixed thoroughly and reacted at 37°C for 25 min. Then, MES buffer (30 mmol L -1 , pH = 6, 700 μL), epinephrine hydrochloride solution (1 mg mL -1 , 100 μL) was fully mixed and reacted at 75°C for 60 min. The absorbance at 470 nm was measured using a UV-visible spectrophotometer at room temperature. The ratio of the absorbance to the blank group was calculated ( Figure 3 ).

[0027] Example 4: Study on the stability of CuNFs-Apt-AChE cascade enzyme composite material.

[0028] The CuNFs-Apt solution (0.1 mg mL -1 , 800 μL) and AChE solution (0.8 mg mL -1 , 800 μL) and reacted at 37°C for 120 min. After incubation, the mixture was centrifuged (10000 rpm, 5 min) and washed three times with ultrapure water to obtain CuNFs-Apt-AChE. The precipitate was then redispersed in 100 μL ultrapure water to obtain a CuNFs-Apt-AChE solution (1.3 mg mL -1 ). In AChE solution (1 mg mL -1 , 50 μL) and CuNFs-Apt-AChE solution (1.3 mg mL -1 , 50 μL) were added with trypsin solution (0.1 mg mL -1 , 50 μL), mix thoroughly, react at 37°C for 30 min, and then take 25 μL and add water (45 μL), PBS buffer solution (10 mmol L -1 , pH = 7.4, 25 μL), thioacetylcholine solution (4 mmol L -1 , 25 μL), mixed thoroughly, reacted at 37°C for 30 min, and then 5,5'-dithiobis(2-nitrobenzoic acid) solution (0.4 mg mL -1 , 120 μL), reacted at 25°C for 10 min, and the absorbance at 405 nm was measured using a UV-visible spectrophotometer at room temperature and compared with the control group ( Figure 4 ).

[0029] Example 5: CuNFs-Apt-AChE-based hydrogel colorimetric method for on-site detection of the pesticide cypermethrin Weigh sodium alginate (60 mg) and pour it into a 15 mL centrifuge tube. Shake it appropriately to make it evenly distributed on the wall of the centrifuge tube. Add ultrapure water (3 mL). Shake it and ultrasonicate it for 10 min. Take it out and centrifuge it for later use. Add CuNFs-Apt-AChE solution (10 μg mL -1 , 10 μL) and sodium alginate (32 μL) were stirred thoroughly to make the composite material evenly distributed, and then calcium chloride solution (5 mg mL -1 , 8 μL) to form a gel, and then Tris-HCl buffer (10 mmol L -1, pH = 7.5, 25 μL) and different concentrations of mefenamic acid (0 μg mL -1 -10 μg mL -1 , 25 μL), stirred evenly and reacted at 37°C for 35 min. Then Tris-HCl buffer (10 mmol L -1 , pH = 7.5, 25 μL) and thioacetylcholine solution (2 mmolL -1 , 25 μL) and stirred evenly, and then reacted at 37°C for 25 min. Then, MES buffer (30 mmol L -1 , pH = 6, 75 μL), epinephrine hydrochloride solution (1 mg mL -1 , 25 μL) was stirred evenly and reacted at 75℃ for 60 min before being taken out. The absorbance generated by the color reaction of different concentrations of cypermethrin solutions and the CuNFs-Apt-AChE enzyme cascade system in the sodium alginate gel at 470 nm was measured by UV-visible spectrophotometer at room temperature. According to the ratio of the absorbance of several groups to the absorbance of the blank group and the logarithmic value of the corresponding pesticide concentration, the relationship curve was fitted by regression analysis, thereby obtaining the linear relationship of the sensing system, and realizing the quantitative detection of cypermethrin ( Figure 5 , Figure 6 ).

[0030] Example 6: Detection of Metformin in Actual Samples To evaluate the practicality of the methicillin detection strategy, environmental samples (tap water, paddy field water) and biological samples (apple juice) were tested. Different concentrations of methicillin standard solution (50, 100, 200 and 500 ng mL -1 ), to simulate the real detection environment. In the experimental analysis, in order to eliminate the background interference of the samples, tap water, paddy water and apple juice samples were diluted with deionized water according to the characteristics of the samples, and the dilution multiples were 1, 1, and 20, respectively. As shown in Table 1, the recovery rate of sulphuric acid in actual samples ranged from 87.63 to 109.56%, and the relative standard deviation was less than 1.67%. This result shows that the applicability and accuracy of the sulphuric acid detection method in the detection of actual samples provide reliable technical support for the environmental monitoring of sulphuric acid. In addition, the sensing method shows good selectivity and anti-interference ability for interfering substances (different types of pesticides, ions, proteins, amino acids, vitamins, sugars) (Figure 7).

[0031] Table 1: Detection of mefenamic acid in actual samples based on UV-visible method .

Claims

1. A method for preparing a DNA tweezers-driven nanozyme / biological enzyme cascade composite material, characterized in that: DNA tweezers use the principle of base complementary pairing to bind to the DNA in the nanozyme through hydrogen bonds, and use the high affinity and high specificity of DNA tweezers to target molecules to capture natural enzymes to construct an enzyme cascade system.

2. The DNA tweezers driven nanozyme / biological enzyme cascade composite material according to claim 1, characterized in that: The captured native enzyme is acetylcholinesterase.

3. The DNA tweezers driven nanozyme / biological enzyme cascade composite material according to claim 1, characterized in that: The expression of the cascade enzyme composite material is CuNFs-Apt-AChE cascade enzyme composite material.

4. The CuNFs-Apt-AChE cascade enzyme composite material according to claim 1, characterized in that: The composite material has a nano-flower-like structure.

5. The CuNFs-Apt-AChE cascade enzyme composite material according to claim 1, characterized in that: The prepared composite material has laccase-like activity.

6. The CuNFs-Apt-AChE cascade enzyme composite material according to claim 1, characterized in that: The prepared composite material improved the enzyme cascade activity and the stability of AChE.

7. The CuNFs-Apt-AChE cascade enzyme composite material according to claim 1, characterized in that: The specific preparation method is as follows.

8. Add copper sulfate solution (50-500 mmol L -1 ) and G-DNA (0.05-5 μmol L -1 ) PBS buffer solution (10 mmol L -1 , pH = 7.4) in a volume ratio of 150:1 and incubated at 20-25°C for 1 hour to 5 days. The CuNFs mixture was washed three times by centrifugation and then redispersed in ultrapure water to obtain a CuNFs nanoflower solution.

9. Add DNA tweezers solution (0.01-5 μmol L -1 ) was denatured at 65-95°C for 2-20 min, cooled to room temperature, and allowed to stand at 4-35°C for 1-50 min to stabilize its spatial conformation. CuNFs solution (0.01-1 mg mL -1 ) were mixed thoroughly and reacted at 20-25°C for 5-100 min. After the reaction was completed, the mixture CuNFs-Apt was washed three times by centrifugation and redissolved in ultrapure water. The CuNFs-Apt solution (0.01 -1 mg mL -1 ) and AChE solution (0.1-5 mg mL -1 ) were mixed in a ratio of 1:1, reacted at 30-40°C for 10-250 min, and after incubation, the mixture was centrifuged and washed three times, and the obtained CuNFs-Apt-AChE precipitate was redispersed in ultrapure water to obtain a CuNFs-Apt-AChE solution.

10. A hydrogel colorimetric sensor based on CuNFs-Apt-AChE cascade enzyme composite material for pesticide detection, characterized in that: Application of enzyme cascade composite material to rapid detection of mefenamic acid.

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