Preparation of DNA-driven nanozyme / bioenzyme composite materials and their application in the detection of methicillin
By immobilizing acetylcholinesterase on the surface of copper-based nanozymes and constructing a CuNFs-Apt-AChE cascade enzyme composite material, the stability and reaction condition incompatibility problems of the nanozyme-bioenzyme cascade system were solved, and efficient detection of cypermethrin was achieved.
Patent Information
- Application Number
- CN202411763784.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-03
- Publication Date
- 2025-09-16
- Estimated Expiration
- 2044-12-03
AI Technical Summary
The existing nanozyme-bioenzyme cascade system has problems in the sensor, such as insufficient enzyme stability, incompatible reaction conditions and low enzyme load, which affect the reaction rate and detection sensitivity of the sensor.
DNA tweezers were used to immobilize acetylcholinesterase on the surface of copper-based nanozymes to form a CuNFs-Apt-AChE cascade enzyme composite material. The base complementary pairing and high specific binding of DNA were combined with an alginate/calcium hydrogel system to construct a sensor for the detection of mefenamic acid.
The sensitivity and stability of the sensor are improved, and efficient and convenient detection of cypermethrin is achieved, with the characteristics of portability and visualization.
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Figure CN119500128B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biosensors, and particularly relates to a preparation method of a flower-like nanostructured cascade enzyme composite material (CuNFs-Apt-AChE) and its application in the rapid detection of metolacarb. Background Art
[0002] In the field of biosensor research, multi-enzyme cascade systems have attracted considerable attention due to their ability to mimic the multi-enzyme structure within biological cells. By integrating multiple catalytic reactions, these systems effectively reduce the number of substrate transport and intermediate diffusion steps, offering new solutions for fields such as biomedical diagnostics, environmental monitoring, and food safety. However, cascade catalytic systems composed of natural enzymes suffer from insufficient enzyme stability, limiting their practical applications. Compared to natural enzymes, nanozymes offer advantages such as simple preparation, excellent stability, and high cost-effectiveness. The design of nanozyme-bioenzyme cascade systems combines the stability of nanozymes with the specificity of bioenzymes, thereby improving the overall performance of biosensors. Typically, nanozyme cascade systems are simply mixed with natural enzymes and used independently, rather than forming a cascade system. This can lead to waste of intermediates, affecting the reaction rate and detection sensitivity of the sensor system. Furthermore, the reaction conditions of bioenzymes and nanozymes are often incompatible, preventing nanozyme-enzyme cascade hybrid systems from fully leveraging the advantages of both components and limiting their synergistic effects within the cascade system. To address these issues, the construction of immobilized nanozyme / bioenzyme cascade systems 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. DNA, due to its precisely defined sequence, programmable self-assembly and specific base pairing ability, can give nanomaterials the properties of adjustable activity and easy surface modification. DNA tweezers, due to their secondary and tertiary structures, have high specificity and affinity for molecular targets. 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] This study innovatively designed a copper-based nanozyme-acetylcholinesterase (CuNFs-Apt-AChE) composite to construct a visual sensing method for the detection of the pesticide methicillin. First, a nanoflower-like structured nanozyme (CuNFs) was synthesized using copper sulfate and DNA self-assembly at room temperature. The nanozyme exhibited excellent laccase-like activity and catalytic stability. Based on the principle of complementary base pairing and the specific recognition of aptamers for their molecular targets, acetylcholinesterase (AChE) was immobilized on the surface of the nanozyme using DNA tweezers, forming a CuNFs-Apt-AChE cascade enzyme composite. While the nanozyme amplifies the output signal, the synergistic effect of the enzyme cascade further enhances the sensor output, improving detection sensitivity and exhibiting excellent stability. Furthermore, the CuNFs-Apt-AChE cascade enzyme composite was immobilized using an alginate / calcium hydrogel system to fabricate a sensor for the instant detection of methicillin. The resulting sensor demonstrated excellent sensitivity and specificity in practical applications, while also being portable and visually accessible, providing a new platform for rapid on-site pesticide detection. 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 its application in the rapid detection of methicillin. Based on the complementary base pairing effect between 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 the carbamate pesticide methicillin on acetylcholinesterase activity, a hydrogel sensor based on the CuNFs-Apt-AChE cascade enzyme composite material was developed to achieve efficient and convenient detection of methicillin.
[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 methicillin are as follows:
[0006] Preparation method of flower-like 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 The mixture was then mixed with 150:1 (volume ratio) of 1% MgCl2 (pH = 7.4) 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.
[0007] Preparation method of CuNFs-Apt-AChE cascade enzyme composite material: DNA tweezers solution (0.01-5 μmol L -1 ) were 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 CuNFs-Apt mixture was washed three times by centrifugation and redissolved in ultrapure water. The CuNFs-Apt solution (0.01-1 mg mL -1 ) with 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. The obtained CuNFs-Apt-AChE precipitate was redispersed in ultrapure water to obtain a CuNFs-Apt-AChE solution.
[0008] Preparation method of fluorescently labeled CuNFs-Apt-AChE cascade enzyme composite material: Carboxyfluorescein labeled DNA tweezers solution (0.01-5 μmol L -1 ) were 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 CuNFs-Apt mixture 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.
[0009] 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.
[0010] Study on the cascade activity of CuNFs-Apt-AChE cascade enzyme composite. DNA tweezers solution (0.01-1 μmolL -1 ) and AChE solution (0.01-10 mg mL -1 ) were mixed in a ratio of 1:1 and reacted 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 methicillin solution (0.001-100 μg mL -1 ), the ratio is 4:1:1, 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 (1-25mmol 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 ) were stirred evenly with a volume ratio of 2:7:1. The mixture was reacted at 20-80°C for 5-200 min, and then taken out. The absorbance at 470 nm was measured using a UV-visible spectrophotometer at room temperature, and the ratio of the absorbance to the absorbance of the blank group was calculated.
[0011] 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 thorough mixing, react at 35-37 ° C for 5-250 min, recorded as system A. 5,5'-dithiobis(2-nitrobenzoic acid) solution (0.1-10 mgmL) was added to system A. -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 that of the control group.
[0012] A hydrogel colorimetric method based on a CuNFs-Apt-AChE cascade enzyme composite was used for on-site detection of the pesticide methicillin. Weigh 10-200 mg of sodium alginate into a centrifuge tube and shake it appropriately to evenly distribute it on the tube wall. Add 1-30 mL of ultrapure water, shake it, and ultrasonicate it for 1-60 minutes before removing it from the tube and centrifuging it for later use. Add a CuNFs-Apt-AChE solution (1-10 μg mL) to the wells of the enzyme label strip. -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. MES buffer (30 mmol L -1 , pH = 6), epinephrine hydrochloride solution (0.1-10 mg mL -1) were stirred evenly in a volume ratio of 6:3:1. The mixture was allowed to react at 20-80°C for 5-200 minutes before removal. The absorbance at 470 nm generated by the colorimetric reaction between different concentrations of metolacarb solutions and the CuNFs-Apt-AChE enzyme cascade system in the sodium alginate gel was measured using a UV-Vis spectrophotometer at room temperature. Regression analysis was used to fit the absorbance ratios of several groups to the blank group and the logarithmic values of the corresponding pesticide concentrations, resulting in a linear relationship for the sensing system and enabling quantitative detection of metolacarb.
[0013] The mechanism of the present invention is as follows:
[0014] Using DNA and copper sulfate solution as raw materials, a nanozyme with laccase-mimicking activity was prepared by utilizing the abundant amide and amine groups in DNA to form complexes with copper ions. Based on the principle of complementary base pairing, the high affinity and specific binding ability of DNA tweezers for the target molecule was exploited to specifically capture acetylcholinesterase (AChE) and immobilize it on the nanozyme surface, resulting in a CuNFs-Apt-AChE cascade enzyme composite. In this enzyme cascade system, AChE catalyzes the conversion of thioacetylcholine to thiocholine, which competitively binds to the copper active site on the copper-based nanozyme, inhibiting the laccase-mimicking activity of the copper-based nanozyme and causing a change in the colorimetric signal. This enzyme cascade system can be used to construct a hydrogel reactor for rapid visualization and qualitative detection of metolacarb. Quantitative detection of metolacarb was achieved using a curve constructed by plotting the absorbance versus the logarithm of the standard pesticide concentration.
[0015] The present invention has the following characteristics:
[0016] Using DNA tweezers to gently immobilize acetylcholinesterase on nanozymes, a CuNFs-Apt-AChE cascade enzyme composite was designed and prepared for the first time;
[0017] 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.
[0018] The enzyme cascade hydrogel colorimetric sensor prepared by the present invention has good sensitivity, selectivity and stability, and the construction process is low-cost and simple to operate.
[0019] The present invention detects the content of methicillin 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 testing. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] 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.
[0021] Figure 2 This is the laccase-like activity of the CuNFs-Apt-AChE cascade enzyme composite material in Example 2.
[0022] Figure 3 This is the cascade activity of the CuNFs-Apt-AChE cascade enzyme composite material in Example 3.
[0023] Figure 4 This is the stability of the CuNFs-Apt-AChE cascade enzyme composite material in Example 4.
[0024] Figure 5 Schematic diagram of the method for detecting the sulfadiazol hydrogel in Example 5.
[0025] Figure 6 (A) is a photograph of the hydrogel colorimetric sensor described in Example 5 and the aqueous colorimetric sensor under natural light; (B) is the feasibility of the hydrogel colorimetric sensor described in Example 5 in detecting metolacarb; (C) is a linear relationship graph between the absorbance of the hydrogel colorimetric sensor described in Example 5 and the logarithm of the concentration of metolacarb. DETAILED DESCRIPTION
[0026] Example 1: Preparation of flower-like nanostructured CuNFs-Apt-AChE cascade enzyme composite material
[0027] Copper sulfate solution (120 mmol L -1 , 8 μL) was added with 0.5 μmol L -1 G-DNA PBS buffer solution (10 mmol L -1 , pH = 7.4, 1200 μL), then thoroughly mixed and incubated at 25°C for 3 days. The mixture was centrifuged (10,000 rpm, 5 min) and washed three times with ultrapure water, and the blue CuNFs precipitate was redispersed in 600 μL of ultrapure water.
[0028] 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. 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).
[0029] 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 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).
[0030] Example 2: Study on laccase-like activity of CuNFs-Apt-AChE cascade enzyme composite material
[0031] 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 epinephrine 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 ).
[0032] Example 3: Study on the cascade activity of CuNFs-Apt-AChE cascade enzyme composite material
[0033] DNA tweezers solution (0.06 μmol L -1 , 200 μL) and AChE solution (0.016 mg mL -1 , 200 μL) were mixed 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 μg mL -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 methicillin 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 After the mixture was fully mixed and reacted at 75°C for 60 min, the sample was taken out and 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 ).
[0034] Example 4: Study on the stability of CuNFs-Apt-AChE cascade enzyme composite material.
[0035] 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, 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 added 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 ).
[0036] Example 5: CuNFs-Apt-AChE-based hydrogel colorimetric method for on-site detection of the pesticide methicillin
[0037] Weigh sodium alginate (60 mg) and pour it into a 15 mL centrifuge tube. Shake it properly 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. Remove it 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 After stirring evenly, the sample was reacted at 75°C for 60 minutes. The absorbance at 470 nm generated by the colorimetric reaction of different concentrations of metolacarb solutions with the CuNFs-Apt-AChE enzyme cascade system in the sodium alginate gel was measured using a UV-visible spectrophotometer at room temperature. Regression analysis was used to fit the relationship curve based on the ratio of the absorbance of several groups to that of the blank group and the logarithmic value of the corresponding pesticide concentration, thereby obtaining the linear relationship of the sensing system and achieving quantitative detection of metolacarb ( Figure 5 、 Figure 6 ).
[0038] Example 6: Detection of Metformin in Actual Samples
[0039] To evaluate the practicality of the metolacarb detection strategy, experiments were conducted on environmental samples (tap water, paddy water) and biological samples (apple juice). Different concentrations of metolacarb standard solution (50, 100, 200, and 500 ng mL -1 ) to simulate the actual 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 factors were 1, 1, and 20, respectively. As shown in Table 1, the recovery rate of sulfamethoxazole in actual samples ranged from 87.63% to 109.56%, and the relative standard deviation was less than 1.67%. This result shows the applicability and accuracy of the sulfamethoxazole detection method in the detection of actual samples, providing reliable technical support for the environmental monitoring of sulfamethoxazole. 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).
[0040] Table 1: Detection of metolacarb in actual samples based on UV-visible method
[0041] .
Claims
1. A method for preparing a DNA tweezers-driven nanozyme / biological enzyme cascade composite material, characterized in that: The copper sulfate solution and the PBS buffer solution containing G-DNA were mixed in a volume ratio of 150:1 and incubated at 20-25°C for 1 hour to 5 days. The CuNFs mixture was centrifuged and washed three times and then redispersed in ultrapure water to obtain a CuNFs nanoflower solution. The DNA tweezers solution was denatured at 65-95°C for 2-20 min and allowed to stand at 4-35°C for 1-50 min to stabilize its spatial conformation. The CuNFs solution was added in a volume ratio of 1:1 and mixed thoroughly and reacted at 20-25°C for 5-100 min. The DNA tweezers and the DNA in the CuNFs nanoflower solution were hydrogen bonded by the principle of base complementary pairing to obtain a mixture CuNFs-Apt. The CuNFs-Apt mixture was centrifuged and washed three times and redissolved in ultrapure water. The CuNFs-Apt solution was mixed with the acetylcholinesterase AChE solution in a volume ratio of 1:1 and reacted at 30-40°C for 10-250 min. min, using the high affinity and highly specific binding ability of DNA tweezers to target molecules, natural enzymes were captured to construct an enzyme cascade system, resulting in a mixture of CuNFs-Apt-AChE. The mixture of CuNFs-Apt-AChE was centrifuged and washed three times and then redispersed in ultrapure water to obtain a CuNFs-Apt-AChE cascade enzyme composite material.
2. The preparation method according to claim 1, characterized in that The concentration of the copper sulfate solution is 50-500 mmol / L -1 The G-DNA concentration was 0.05-5 μmol L -1 The PBS buffer solution had a pH of 7.4 and a concentration of 10 mmol L - 1 The concentration of DNA tweezers solution is 0.01-5 μmol L -1 , the concentration of CuNFs solution was 0.01-1 mg mL -1 , the concentration of CuNFs-Apt solution was 0.01-1 mg mL -1 , AChE solution concentration is 0.1-5 mg mL -1 .
3. Use of a composite material obtained by the preparation method according to claim 1 or 2, characterized in that: The colorimetric sensor made of the composite material was used to quickly detect metolacarb.