Bis-enzyme active nanoscale enzyme, preparation method thereof and application thereof in profenofos bimodal detection

Abstract

The application discloses a kind of double-enzyme activity nano-enzyme and its preparation method and application in prothiofos bimodal detection, the double-enzyme activity nano-enzyme is UiO-66 (Ce) -Cu, with efficient and balanced phosphoric acid hydrolase and laccase activity, when self-cascading detection prothiofos, with high sensitivity and selectivity;The preparation method is to add copper modification on UiO-66 (Ce) using solvothermal method, the double-enzyme activity nano-enzyme can be effectively prepared;The application also constructs two kinds of methods for detecting prothiofos using the double-enzyme activity nano-enzyme by colorimetric method and fluorescence method, wherein, colorimetric detection method is simple in operation, process is efficient, and the detection limit is low, and the result is intuitive and clear;Fluorescence detection method is fast, time-consuming is short, and detection result can be obtained only in 4 minutes;Both of the two detection methods can sensitively, specifically and accurately detect prothiofos residue in vegetables.

Description

Technical Field

[0001] This invention relates to a nanozyme, its preparation method and application, and more particularly to a dual-enzyme active nanozyme, its preparation method and its application in the dual-modal detection of propargite. Background Technology

[0002] Profenofos (PFF) is a common organophosphorus pesticide widely used in agricultural production for pest control. However, PFF is toxic to humans and aquatic organisms. Long-term exposure to PFF-contaminated agricultural products can cause irreversible damage to the human central nervous system, seriously threatening human health. Furthermore, the high water solubility of organophosphorus pesticides makes PFF persistent in aquatic systems, difficult to eliminate through conventional processes. Therefore, its abuse and persistent residues also seriously endanger ecological and environmental safety.

[0003] Nanozymes are a class of biomimetic enzymes that possess the properties of nanomaterials and the catalytic function of enzymes. Due to their high catalytic activity and stability, they have become an ideal choice for biosensing applications. Thanks to their excellent characteristics such as high cost-effectiveness, tunable catalytic activity, and reusability, the colorimetric detection of organophosphorus compounds using nanozymes has been extensively studied and documented. However, colorimetric sensors based on single-activity nanozymes have limitations such as weak signal output and insufficient selectivity for target analytes.

[0004] To address this issue, nanozymes with dual-enzyme activity can be developed using the principle of multi-enzyme cascade catalysis. This transforms weak-signal substrates into strong-signal products, not only amplifying the signal and improving analytical sensitivity but also effectively increasing the utilization rate of intermediates by reducing diffusion between enzymes. Since phosphorylation-free phenol (PFF) can be hydrolyzed to produce the colorless bromide 4-bromo-2-chlorophenol, which can be further catalyzed by laccase to undergo an oxidative coupling reaction, nanozymes possessing both phosphorylation-hydrolase and laccase activities can overcome the limitations in sensitivity and selectivity of PFF detection through cascade catalysis. However, research on achieving effective substrate catalysis and signal amplification, and developing highly sensitive and selective PFF biosensors using nanozymes with cascaded phosphorylation-hydrolase and laccase activities, is currently scarce. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a dual-enzyme nanozyme that simultaneously possesses phosphorylase and laccase activities, its preparation method, and its application in the dual-modal detection of propargyl phosphide.

[0006] Technical solution: The dual-enzyme active nanozyme is copper-modified UiO-66(Ce), and the dual enzymes are phosphorylase and laccase.

[0007] There are two methods for detecting profenofos using the dual-enzyme active nanozyme (i.e., UiO-66(Ce)-Cu).

[0008] Approach 1: Colorimetric detection method based on the principle of dual-enzyme cascade. Detection principle: 4-bromo-2-chlorophenol produced by the hydrolysis of propargyl phosphatase (PFF) can be further catalyzed by laccase to undergo an oxidation reaction, thereby amplifying the signal and improving detection sensitivity.

[0009] Approach 2: Fluorescence detection method based on substrate inhibition principle. Detection principle: The non-fluorescent 4-MUP in the system can be converted into a fluorescent product. Profenofos in the system competes with 4-MUP for the activity of UiO-66(Ce)-Cu phosphatase, thereby reducing the fluorescent product of 4-MUP, thus establishing a quantitative relationship between profenofos concentration and fluorescence intensity.

[0010] The preparation method of the dual-enzyme active nanozyme includes the following steps:

[0011] Step 1: UiO-66(Ce) powder is prepared using terephthalic acid and cerium ammonium nitrate as precursors and N,N-diethylformamide as solvent.

[0012] Step 2: Disperse the UiO-66(Ce) obtained in Step 1 in N,N-diethylformamide, add copper acetate to the solution, and the ratio of UiO-66(Ce), N,N-diethylformamide to copper acetate is 8-11 mg:3 mL:8-11 mg; after ultrasonic treatment, heat the mixed solution; place the obtained solid in N,N-diethylformamide and acetone for immersion treatment in sequence; vacuum dry to obtain nanoenzyme UiO-66(Ce)-Cu powder, which is the dual-enzyme active nanoenzyme.

[0013] In step two, the ultrasound is preferably performed for 20-40 minutes; the mixed solution is preferably heated at a temperature of 90-120°C for 24 hours; the soaking treatment is preferably performed by changing the solution every 12 hours and repeating 2-3 times; and the vacuum drying temperature is preferably 60-80°C.

[0014] The dual-enzyme active nanozyme can be used for the detection of propargite. The concentration range for propargite detection is 0.05-50 μg / mL; the detection method for propargite is colorimetric detection or fluorescence detection.

[0015] The colorimetric detection method is as follows: Add a dual-enzyme active nanozyme (4-6 mg / mL) and the test sample to a Tris-HCl buffer solution with a concentration of 0.15-0.2 mol / L and a pH of 8.5-9.0. The dual-enzyme active nanozyme is dispersed in an aqueous solution, and the test sample is a solution obtained by extraction with an organic solvent. The volume ratio of Tris-HCl buffer, dual-enzyme active nanozyme, and test sample in the mixed solution is 35-40:2-3:2-3. After maintaining the mixed solution at 40-50℃ for 10-30 minutes, add a phosphate buffer solution with a concentration of 1.0-1.5 mol / L and a pH of 2.0-2.5, and a 4-AP solution with a concentration of 40-60 mmol / L, wherein the volume ratio of the mixed solution, phosphate buffer, and 4-AP solution is 39-46:4-5:2-3. After reacting for 15-25 minutes, filter the colorimetric product using a 0.22 μm filter membrane. The absorbance of the filtrate was measured at nm, and the content of propargite in the sample was calculated using a standard curve.

[0016] The colorimetric detection method corresponds to detection route one mentioned above. The 4-AP in this method refers to 4-aminoantipyrine.

[0017] The fluorescence detection method is as follows: The sample to be tested is added to a Tris-HCl buffer solution with a concentration of 0.15-0.2 mol / L and a pH of 8.5-9.0. The sample to be tested is a solution obtained by extraction with an organic solvent. The volume ratio of the sample to the Tris-HCl buffer solution is 2-3:40-45. Then, 4-MUP with a concentration of 0.8-1.2 mol / L and a dual-enzyme active nanozyme with a concentration of 4-6 mg / mL are added to the mixed solution. The dual-enzyme active nanozyme is dispersed in an aqueous solution. The volume ratio of the mixed solution, 4-MUP, and dual-enzyme active nanozyme is 84-96:2-5:1-3. After reacting for 4-5 min, the fluorescence emission intensity at 448 nm under a 360 nm excitation wavelength is recorded. The content of profenofos in the sample is calculated using a standard curve.

[0018] The fluorescence detection method corresponds to detection pathway two mentioned above. 4-MUP refers to 4-methylumbelliferyl ketone phosphate.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0020] 1. This invention utilizes an easily implemented preparation method and a copper modification strategy to modify the surface of the original MOF material UiO-66(Ce) with copper. The resulting UiO-66(Ce)-Cu exhibits both phosphorylase and laccase activities. The preparation method is simple and efficient. Compared to UiO-66(Ce) with only one activity, UiO-66(Ce)-Cu demonstrates highly efficient and balanced dual-enzyme activity. This superior characteristic can be used in the design of cascade detection systems for profenofos.

[0021] 2. The dual-enzyme active nanozyme UiO-66(Ce)-Cu described in this invention can sensitively, specifically, and accurately detect profenofos residues in vegetables using two methods: (1) a colorimetric detection method based on a dual-enzyme cascade: it has the advantages of simple operation, efficient process, and intuitive and clear results, and has a low detection limit of up to 44.8 μg / mL; (2) a fluorescence detection method based on substrate competition: it has the advantages of short time consumption, fast detection speed, and can meet the needs of rapid on-site screening. The recovery rate of profenofos by these two detection methods is between 85% and 126%, proving that the dual-enzyme active nanozyme UiO-66(Ce)-Cu has good practical applicability. This invention constructs a dual-modal profenofos detection method based on the dual-enzyme active nanozyme UiO-66(Ce)-Cu, which can effectively and sensitively detect profenofos in actual food, providing a good way for rapid screening and detection of profenofos in actual samples. Attached Figure Description

[0022] Figure 1 Scanning electron microscope images of UiO-66(Ce) and UiO-66(Ce)-Cu (A is UiO-66(Ce); B is UiO-66(Ce)-Cu).

[0023] Figure 2 The enzyme activities of UiO-66(Ce) and UiO-66(Ce)-Cu change over time (A is phosphorylase activity; B is laccase activity).

[0024] Figure 3 The absorbance of the gradient concentration PFF solution at different corresponding wavelengths in performance test 2 of Example 2;

[0025] Figure 4 This is the standard curve of PFF concentration-absorbance change at 510 nm wavelength in performance test 2 of Example 2;

[0026] Figure 5 The colorimetric changes of the reaction system with different concentrations of PFF in Example 2 were measured to determine the performance of the system.

[0027] Figure 6The absorbance test results are from the anti-interference test in performance test 3 of Example 2.

[0028] Figure 7 This is the standard curve of PFF concentration-fluorescence intensity change at 448 nm wavelength in performance test 5 of Example 3;

[0029] Figure 8 Two pathways and schematic diagrams for the detection of profenofos by the dual-enzyme nanozyme UiO-66(Ce)-Cu. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0031] Example 1

[0032] This embodiment provides a dual-enzyme active nanoenzyme UiO-66(Ce)-Cu, such as Figure 1 As shown in (B), the enzyme is copper-modified UiO-66(Ce), and the dual enzymes are phosphatase and laccase. UiO-66(Ce) is as follows... Figure 1 As shown in (A).

[0033] Example 2

[0034] This embodiment provides a method for preparing the dual-enzyme active nanozyme UiO-66(Ce)-Cu described in Example 1. The specific steps are as follows:

[0035] Step 1: Preparation of nanozyme UiO-66(Ce): 350 mg of terephthalic acid (H2BDC) powder was added to 10 mL of N,N-diethylformamide (DMF) and dissolved by sonication. Then, 5 mL of 0.533 mol / L cerium ammonium nitrate ((NH4)2Ce(NO3)6) aqueous solution was added dropwise to the H2BDC solution. The mixed solution was maintained at 100℃ in a water bath for 15 min. The synthesized solid was obtained by centrifugation and washed three times with DMF and acetone (Ace), respectively. Then, the crystals were vacuum dried overnight at 60℃ to obtain UiO-66(Ce) powder.

[0036] Step 2, Preparation of the nanozyme UiO-66(Ce)-Cu: 100 mg of UiO-66(Ce) obtained in Step 1 was dispersed in a 30 mL container of DMF. Then, 100 mg of copper acetate was added to the solution. After sonication for 30 min, the mixture was heated at 100 °C for 24 h. The solid was then immersed in DMF and Ace sequentially, with the solution changed every 12 h, and the above operation was repeated 3 times. Finally, the nanozyme UiO-66(Ce)-Cu powder was obtained by vacuum drying at 60 °C overnight.

[0037] Performance Test 1: Dual Enzyme Activity Assay.

[0038] The phosphorylase activity and laccase activity of the nanozyme UiO-66(Ce)-Cu were determined by colorimetric detection.

[0039] Phosphohydrolase activity assay: p-nitrophenyl phosphate (p-NPP) was used as a model substrate to study phosphohydrolase-like activity. The yellow product of p-NPP hydrolysis showed significant absorbance at 400 nm. Therefore, 50 μL of 10 mmol / L p-NPP and 20 μL of 5 mg / mL nanozyme UiO-66(Ce)-Cu (control group with added nanozyme UiO-66(Ce)) were added to 930 μL of 0.2 mol / L Tris buffer at pH 9.0, and the absorbance at 400 nm was measured over time.

[0040] The results are as follows Figure 2 As shown in (A), the absorbance at 400 nm gradually increases over time, indicating that the phosphorylase activity of nanozyme UiO-66(Ce)-Cu is comparable to that of nanozyme UiO-66(Ce).

[0041] Laccase activity assay: 2,4-Dichlorophenol (2,4-DP) was used as a model substrate to verify laccase activity. Specifically, 50 μL of 10 mmol / L 2,4-DP and 50 μL of 40 mmol / L 4-AP nanozyme UiO-66(Ce)-Cu (UiO-66(Ce) nanozyme was added to 850 μL Tris buffer, and the absorbance was measured at 510 nm over time.

[0042] The results are as follows Figure 2 As shown in (B), the laccase activity of the nanozyme UiO-66(Ce)-Cu is higher than that of the nanozyme UiO-66(Ce).

[0043] Performance Test 2: Construction of PFF colorimetric detection system, namely a colorimetric detection method for profenofos based on the dual-enzyme active nanozyme UiO-66(Ce)-Cu described in Example 1.

[0044] 50 μL of the nanozyme UiO-66(Ce)-Cu and 50 μL of PFF at different concentrations (0.05-50 μg / mL) were added to 790 μL of 0.2 mol / L Tris-HCl buffer at pH 9, and the mixture was then incubated at 45 °C for 20 min. Afterward, 100 μL of 1 mol / L phosphate buffer at pH 2 and 10 μL of 40 mmol / L 4-AP solution were added to the mixture to adjust the pH and generate a signal. After 10 min of reaction, the colorimetric product was filtered through a 0.22 μm filter membrane, and the absorbance of the filtrate was measured at 510 nm using a UV-Vis spectrophotometer. All samples were repeated three times.

[0045] See results Figure 3-5 The established colorimetric detection system can detect profenofos at concentrations of 0.05-50 μg / mL.

[0046] Performance Test 3: Anti-interference test.

[0047] The anti-interference ability of the colorimetric detection method was tested using different interfering substances. Different types of pesticides were selected, including methyl parathion, fenitrothion, methyl thiophanate, triazophos, dimethoate, chlorpyrifos, methamidophos, bromoxynil, and promethazine. Their colorimetric signals were detected using the performance test method 2. The above pesticides were added to the reaction system to replace PFF, and the absorbance of the filtrate in each system was measured at 510 nm.

[0048] See results Figure 6 The "PFF present" group indicates that PFF and the aforementioned interfering substances coexist; it can be seen that the colorimetric detection method has good anti-interference ability and can specifically detect PFF in the sample.

[0049] Performance Test 4: Colorimetric detection method for testing actual samples.

[0050] To evaluate the application of a dual-enzyme nanozyme cascade catalytic strategy in the detection of pesticide residues (PFF) in real samples, a spiked experiment was conducted. Cabbage, bell peppers, and tomatoes were used as real samples to detect PFF pesticide residues in vegetables.

[0051] Test method: Crush cabbage, bell pepper and tomato, weigh 5.0 g of crushed sample, add 20 ml of methanol / water (v:v=2:1), extract by sonication for 20 min, centrifuge at 8000 rpm for 5 min, transfer the supernatant, filter the supernatant through a 0.22 μm filter membrane, add a known concentration of profenofos to the filtered solution for spiked test detection.

[0052] The average recovery rate was obtained through three measurements, as shown in Table 1.

[0053] Table 1. Application of the dual-enzyme activity PFF sensor in colorimetric detection of actual samples.

[0054] Chinese cabbage 0.09-0.11 90%-110% 0.47-0.52 94%-104% 0.95-1.02 95%-101% Bell peppers 0.11-0.12 110%-120% 0.52-0.55 104%-110% 0.91-1.23 91%-123% tomato 0.09-0.12 90%-120% 0.49-0.51 98%-102% 1.02-1.16 102%-116%

[0055] Example 3

[0056] This embodiment provides a method for preparing a dual-enzyme active nanozyme UiO-66(Ce)-Cu, which differs from that in Example 2, and provides a fluorescence detection method for detecting propargite using the nanozyme.

[0057] The preparation method is as follows.

[0058] Step 1: Same as Step 1 in Example 2.

[0059] Step 2, Preparation of the nanozyme UiO-66(Ce)-Cu: 80 mg of UiO-66(Ce) obtained in Step 1 was dispersed in a 30 mL container of DMF. Then, 110 mg of copper acetate was added to the solution. After sonication for 30 min, the mixture was heated at 100 °C for 24 h. The solid was then immersed in DMF and Ace sequentially, with the solution changed every 12 h, and the above operation was repeated 3 times. Finally, the nanozyme UiO-66(Ce)-Cu powder was obtained by vacuum drying at 60 °C overnight.

[0060] Performance Test 5: A fluorescence detection method for the detection of profenofos by the dual-enzyme active nanozyme UiO-66(Ce)-Cu.

[0061] 50 μL of PFF at different concentrations was added to 890 μL of 0.15 mol / L Tris-HCl buffer (pH 9.0) (final PFF concentration: 1 μg / mL-20 μg / mL). Then, 40 μL of 1 mol / L 4-MUP and 20 μL of 5 mg / mL UiO-66(Ce)-Cu were added to the above solution. After reacting for 4 min, the fluorescence emission intensity at 448 nm under a 360 nm excitation wavelength was recorded.

[0062] See results Figure 7 The above-mentioned fluorescence detection method can detect profenofos at concentrations of 1-20 μg / mL.

[0063] Performance Test 6: Fluorescence detection method for testing actual samples.

[0064] The actual sample preparation followed the colorimetric detection method described in Performance Test 4 of Example 2. The test results are shown in Table 2.

[0065] Table 2. Application of the dual-enzyme activity PFF sensor in fluorescence detection of actual samples.

[0066] Bell peppers 2.28-3.01 91.7%-120% 4.25-4.92 85.0%-98.4% Chinese cabbage 2.85-3.14 114%-126% 4.63-5.26 92.6%-105% tomato 2.14-3.10 85.6%-124% 4.25-5.10 85.0%-102%

[0067] In summary, the preparation method provided by this invention is easy to implement, and the obtained dual-enzyme active nanozyme UiO-66(Ce)-Cu exhibits excellent performance in detecting profenofos with high sensitivity and good selectivity. This invention also provides two methods for detecting profenofos residues in vegetables using the dual-enzyme active nanozyme UiO-66(Ce)-Cu: (1) Colorimetric detection method: which has the advantages of simple operation, efficient process, and intuitive and clear results; (2) Fluorescence detection method: which has the advantages of short time consumption and fast detection speed, and can also meet the needs of rapid on-site screening.

Claims

1. A method for preparing a dual-enzyme active nanozyme, wherein the dual-enzyme active nanozyme is copper-modified UiO-66(Ce), and the dual enzymes are a phosphorylase and a laccase, characterized in that, Includes the following steps: Step 1: UiO-66(Ce) powder is prepared using terephthalic acid and cerium ammonium nitrate as precursors and N,N-diethylformamide as solvent. Step 2: Disperse the UiO-66(Ce) obtained in Step 1 in N,N-diethylformamide, add copper acetate to the solution, and the ratio of UiO-66(Ce), N,N-diethylformamide to copper acetate is 8-11 mg:3 mL:8-11 mg; after ultrasonic treatment, heat the mixed solution; place the obtained solid in N,N-diethylformamide and acetone for immersion treatment in sequence; vacuum dry to obtain nanoenzyme UiO-66(Ce)-Cu powder, which is the dual-enzyme active nanoenzyme.

2. The preparation method according to claim 1, characterized in that, In step two, the heating temperature is 90-120℃.

3. The preparation method according to claim 1, characterized in that, In step two, the soaking treatment solution is changed every 10-12 hours and repeated 2-3 times.

4. The preparation method according to claim 1, characterized in that, In step two, the vacuum drying temperature is 60-80℃.

5. The application of the dual-enzyme active nanozyme prepared by the method of claim 1 in the detection of propargite.

6. The application according to claim 5, characterized in that, The concentration range for the detection of profenofos is 0.05-50 μg / mL.

7. The application according to claim 5, characterized in that, The method for detecting propargite is either colorimetric or fluorescence detection.

8. The application according to claim 7, characterized in that, The colorimetric detection method is as follows: A dual-enzyme active nanozyme at a concentration of 4-6 mg / mL and the sample to be tested are added to a Tris-HCl buffer solution with a concentration of 0.15-0.2 mol / L and a pH of 8.5-9.

0. The dual-enzyme active nanozyme is dispersed in an aqueous solution, and the sample to be tested is a solution obtained by extraction with an organic solvent. The volume ratio of Tris-HCl buffer solution, dual-enzyme active nanozyme, and sample to be tested in the mixed solution is 35-40:2-3:2-3. After maintaining the mixed solution at 40-50℃ for 10-30 minutes, a phosphate buffer solution with a concentration of 1.0-1.5 mol / L and a pH of 2.0-2.5 and a 4-AP solution with a concentration of 40-60 mmol / L are added, wherein the volume ratio of the mixed solution, phosphate buffer solution, and 4-AP solution is 39-46:4-5:2-3. After reacting for 15-25 min, the colorimetric product was filtered through a 0.22 μm filter membrane; the absorbance of the filtrate was measured at 510 nm, and the content of profenofos in the sample was calculated using a standard curve.

9. The application according to claim 7, characterized in that, The fluorescence detection method is as follows: The sample to be tested is added to a Tris-HCl buffer solution with a concentration of 0.15-0.2 mol / L and a pH of 8.5-9.

0. The sample to be tested is a solution obtained by extraction with an organic solvent. The volume ratio of the sample to the Tris-HCl buffer solution is 2-3:40-45. Then, 4-MUP with a concentration of 0.8-1.2 mol / L and a dual-enzyme active nanozyme with a concentration of 4-6 mg / mL are added to the mixed solution. The dual-enzyme active nanozyme is dispersed in an aqueous solution. The volume ratio of the mixed solution, 4-MUP, and dual-enzyme active nanozyme is 84-96:2-5:1-3. After reacting for 4-5 min, the fluorescence emission intensity at 448 nm under a 360 nm excitation wavelength is recorded. The content of profenofos in the sample is calculated using a standard curve.

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