A kind of oxidation mimetic enzyme and its preparation method and application

Abstract

The present invention belongs to the technical field of organic pollutant detection, and specifically discloses an oxidative mimetic enzyme and a preparation method and application. The present invention mixes copper salt, adenine and water to react, obtains a reaction solution, and obtains a gram-level oxidative mimetic enzyme after drying. The present invention also uses the oxidative mimetic enzyme and combines three sensing units to construct a colorimetric sensor array, which not only realizes high-throughput rapid detection and identification of five aromatic amines as low as 50 μM, but also can quantitatively detect single aromatic amines, such as o-phenylenediamine and p-phenylenediamine, and even accurately identify multi-component mixed systems, and finally successfully applies to the identification and differentiation of five aromatic amines in actual water samples. The present invention has the characteristics of simple preparation method and environmental friendliness, provides a simple and efficient method for high-throughput rapid detection and identification of aromatic amines in actual water samples, and also provides very important theoretical and experimental guidance for promoting the application of nanoenzymes in actual systems.

Description

Technical Field

[0001] The present invention relates to the technical field of organic pollutant detection, and in particular to an oxidation mimicking enzyme and a preparation method and application thereof. Background Art

[0002] Nanozymes are a new type of nanomaterial with enzyme-like activity. They tend to gradually replace natural enzymes because they can make up for the shortcomings of natural enzymes, such as low stability, high cost, difficulty in modification, and storage. At present, nanozymes have been widely used in the fields of biosensing, bioimaging and disease diagnosis, as well as environmental monitoring and protection, especially in the detection and degradation of phenolic substances, showing great potential application value.

[0003] In environmental testing, aromatic amines are a class of pollutants with high mutagenicity, teratogenicity and carcinogenicity. At present, the detection of aromatic amines is limited to the detection of a single pollutant, which makes it difficult to achieve high-throughput rapid detection. On the other hand, the detection performance in the actual system cannot meet the actual needs. When multiple aromatic amines coexist, it is not only difficult to detect, but also impossible to identify. Therefore, how to disclose a sensing strategy based on high-stability oxidative mimetic enzymes to achieve high-throughput rapid detection and identification of multiple aromatic amines is an urgent problem to be solved. Summary of the invention

[0004] In view of this, the present invention provides an oxidative mimetic enzyme and a gram-scale preparation method and application to solve the problem that existing detection methods cannot simultaneously perform high-throughput rapid detection and identification of multiple aromatic amines.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A method for preparing an oxidative mimetic enzyme comprises the following steps:

[0007] Copper salt, adenine and water are mixed for reaction to obtain a reaction solution, and the reaction solution is dried to obtain an oxidative mimetic enzyme.

[0008] Preferably, the mass volume ratio of the copper salt, adenine and water is 0.2-0.25 g: 0.1-0.15 g: 10 mL.

[0009] Preferably, the reaction temperature is 65-80° C., and the reaction time is 10-30 min.

[0010] Preferably, the copper salt includes one or more of copper nitrate, copper chloride, copper sulfate and copper acetate.

[0011] Preferably, a freezing operation is also included before drying, the freezing time is 10 to 14 hours, and the freezing temperature is 0 to 5°C.

[0012] Another object of the present invention is to provide a method for preparing oxidative mimetic enzymes at gram scale.

[0013] Another object of the present invention is to provide a method for rapidly detecting and identifying a variety of aromatic amines using an oxidative mimicking enzyme in high throughput.

[0014] Preferably, a colorimetric sensor array is constructed by using oxidative mimicking enzymes to achieve simultaneous detection and identification of multiple aromatic amines in actual water bodies.

[0015] Preferably, the method for preparing the colorimetric sensor array comprises the following steps:

[0016] Sensing unit 1: mixed oxidative mimetic enzyme, NaCl solution and BR buffer solution;

[0017] Sensing unit 2: mixed oxidative mimetic enzyme, NaBr solution and BR buffer solution;

[0018] Sensing unit 3: mixed oxidative mimetic enzyme, NaI solution and BR buffer solution;

[0019] 50 μM to 10 mM aromatic amines were added to a final volume of 1 mL of the sensor unit at room temperature for 5 to 15 minutes, UV–vis spectra were recorded, and absorbance values ​​were used to distinguish aromatic amines; this was repeated 5 times to generate a sensor array of 5 aromatic amines × 3 sensor units × 5 parallel experiments; origin and linear discriminant analysis of the data was performed using IBM SPSS Statistics;

[0020] When preparing the sensor units 1 to 3, the concentration of the oxidative mimic enzyme is independently 0.05 to 0.2 mg / mL; the concentration of the NaCl solution, the NaBr solution and the NaI solution is independently 0.05 to 10 mmol / L;

[0021] The volume ratio of the oxidative mimetic enzyme, NaCl solution, and BR buffer solution was 1:1:7;

[0022] The volume ratio of the oxidative mimetic enzyme, NaBr solution, and BR buffer solution was 1:1:7;

[0023] The volume ratio of the oxidative mimetic enzyme, NaI solution, and BR buffer solution was 1:1:7;

[0024] The pH value of the BR buffer solution is 7.

[0025] Preferably, the aromatic amine includes one or more of o-phenylenediamine (OPD), p-phenylenediamine (PPD), 1,8-naphthalenediamine (1,8-NDA), 1,5-naphthalenediamine (1,5-NDA), and 2-aminoanthracene (2-AA).

[0026] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The gram-scale oxidative mimetic enzyme prepared by the present invention has good water solubility, high stability and high activity, and exhibits the property of salt (NaCl / NaBr / NaI) regulating and enhancing catalytic activity. Based on this, a colorimetric sensor array with the above three sodium salts as sensing channels was designed, which simultaneously detected o-phenylenediamine (OPD), p-phenylenediamine (PPD), 1,8-naphthalenediamine (1,8-NDA), 1,5-naphthalenediamine (1,5-NDA), and 2-aminoanthracene (2-AA) as low as 50 μM, and can accurately identify multi-component mixed systems, and has been successfully applied to identify and distinguish various aromatic amines in various actual water samples.

[0028] (2) The preparation method of the present invention is simple, easy to realize mass production, and environmentally friendly. It provides a simple and feasible method for the simultaneous high-throughput rapid detection and identification of aromatic amines in actual water samples, and at the same time provides very important theoretical and experimental guidance for promoting the application of nanozymes in actual systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the linear discriminant analysis (3D-LDA) diagram of the colorimetric sensor array 3D prepared in Example 4. A corresponds to the discrimination of aromatic amines with a final concentration of 50 μM, B corresponds to the discrimination of aromatic amines with a final concentration of 100 μM, C corresponds to the discrimination of aromatic amines with a final concentration of 1 mM, and D corresponds to the discrimination of aromatic amines with a final concentration of 10 mM. E and F are 2D-LDA diagrams for the discrimination and attribution of 20 unknown samples with a final concentration of 1 mM aromatic amines, and F is an enlarged portion of the selected portion of E;

[0030] Figure 2 This is the linear discriminant analysis diagram (3D-LDA) of the colorimetric sensor array 3D prepared in Example 4. A1 is the discrimination and linearity of o-phenylenediamine (OPD) with a concentration of 50 μM-10 mM, A2 is the discrimination and linearity of p-phenylenediamine (PPD) with a concentration of 50 μM-10 mM, and B1-B2 are the linear responses of concentration and absorbance. B1 is the discrimination and linearity of o-phenylenediamine with a concentration of 50 μM-10 mM, and B2 is the discrimination and linearity of p-phenylenediamine with a concentration of 50 μM-10 mM;

[0031] Figure 3This is a linear discriminant analysis diagram (3D-LDA) of the colorimetric sensor array 3D prepared in Example 4. A is the discrimination of aromatic amines with a final concentration of 500 μM in the tap water application environment, B is the discrimination of aromatic amines with a final concentration of 500 μM in the river water application environment, C is the discrimination of aromatic amines with a final concentration of 500 μM in the sewage application environment, and D is the discrimination of aromatic amines with a final concentration of 500 μM in the seawater application environment. DETAILED DESCRIPTION

[0032] A method for preparing an oxidative mimetic enzyme comprises the following steps:

[0033] Copper salt, adenine and water are mixed for reaction to obtain a reaction solution, and the reaction solution is dried to obtain an oxidative mimetic enzyme.

[0034] In the present invention, the mass volume ratio of the copper salt, adenine and water is 0.2-0.25 g: 0.1-0.15 g: 10 mL, preferably 0.23-0.25 g: 0.12-0.15 g: 10 mL, and more preferably 0.24 g: 0.14 g: 10 mL.

[0035] In the present invention, the reaction temperature is 65-80°C, specifically 68°C, 70°C, 72°C, 75°C, 78°C; the reaction time is 10-30min, specifically 12min, 14min, 15min, 16min, 18min, 20min, 22min, 24min, 25min, 26min, 28min.

[0036] In the present invention, the copper salt includes one or more of copper nitrate, copper chloride, copper sulfate and copper acetate.

[0037] In the present invention, a freezing operation is also included before drying, and the freezing time is 10 to 14 hours, specifically 10.5 hours, 11 hours, 11.5 hours, 12 hours, 12.5 hours, 13 hours, and 13.5 hours; the freezing temperature is 0 to 5°C, specifically 1°C, 2°C, 3°C, and 4°C.

[0038] The invention also provides a gram-level oxidative mimic enzyme prepared by the preparation method.

[0039] The invention also provides an application of the oxidation mimicking enzyme in the detection of aromatic amines.

[0040] In the present invention, a colorimetric sensor array is constructed by using an oxidative mimicking enzyme to achieve high-throughput rapid detection and identification of a variety of aromatic amines.

[0041] In the present invention, the method for preparing the colorimetric sensor array comprises the following steps:

[0042] Sensing unit 1: mixed oxidative mimetic enzyme, NaCl solution and BR buffer solution;

[0043] Sensing unit 2: mixed oxidative mimetic enzyme, NaBr solution and BR buffer solution;

[0044] Sensing unit 3: mixed oxidative mimetic enzyme, NaI solution and BR buffer solution;

[0045] At room temperature, add 50μM to 10mM, specifically 100μM, 500μM, 1mM, 2mM, 4mM, 5mM, 6mM, 8mM of aromatic amines to a final volume of 1mL of the sensor unit for 5 to 15min, specifically 6min, 7min, 8min, 9min, 10min, 12min, 14min; record UV-vis spectra, and use absorbance values ​​to distinguish aromatic amines; repeat 5 times to generate a sensor array of 5 aromatic amines × 3 sensor units × 5 parallel experiments; use IBM SPSS Statistics to perform origin and linear discriminant analysis on the data.

[0046] When preparing sensor units 1 to 3, the concentration of the oxidative mimetic enzyme is independently 0.05 to 0.2 mg / mL, specifically 0.08 mg / mL, 0.1 mg / mL, 0.12 mg / mL, 0.14 mg / mL, 0.15 mg / mL, 0.16 mg / mL, 0.18 mg / mL; the concentrations of the NaCl solution, the NaBr solution, and the NaI solution are independently 0.05 to 10 mmol / L, specifically 0.1 mmol / L, 0.5 mmol / L, 1 mmol / L, 2 mmol / L, 3 mmol / L, 5 mmol / L, 6 mmol / L, 8 mmol / L;

[0047] The volume ratio of the oxidative mimetic enzyme, NaCl solution, and BR buffer solution was 1:1:7;

[0048] The volume ratio of the oxidative mimetic enzyme, NaBr solution, and BR buffer solution was 1:1:7;

[0049] The volume ratio of the oxidative mimetic enzyme, NaI solution, and BR buffer solution was 1:1:7;

[0050] The pH value of the BR buffer solution is 7.

[0051] In the present invention, the aromatic amine includes one or more of o-phenylenediamine (OPD), p-phenylenediamine (PPD), 1,8-naphthalenediamine (1,8-NDA), 1,5-naphthalenediamine (1,5-NDA), and 2-aminoanthracene (2-AA).

[0052] Example 1

[0053] Dissolve 0.24g of copper nitrate and 0.14g of adenine in 10ml of hot water and heat in a water bath (70°C) for 20 minutes to obtain a dark blue solution. The resulting solution was cooled to room temperature in an ice bath and filtered, and finally transferred to a dialysis bag for further purification. The final solution was transferred to a clean petri dish, sealed and frozen in a refrigerator for 12h (0°C). Then, take out the sample and vacuum dry it in a freeze dryer for 12 hours to finally obtain the oxidative mimetic enzyme.

[0054] Example 2

[0055] Dissolve 0.25g of copper nitrate and 0.15g of adenine in 10ml of hot water and heat in a water bath (80°C) for 30 minutes to obtain a dark blue solution. The resulting solution was cooled to room temperature in an ice bath and filtered, and finally transferred to a dialysis bag for further purification. The final solution was transferred to a clean petri dish, sealed and frozen in a refrigerator for 10h (0°C). Then, take out the sample and vacuum dry it in a freeze dryer for 12 hours to finally obtain the oxidative mimetic enzyme.

[0056] Example 3

[0057] Dissolve 0.24g of copper nitrate and 0.14g of adenine in 10ml of hot water and heat in a water bath (70°C) for 20 minutes to obtain a dark blue solution. The resulting solution was cooled to room temperature in an ice bath and filtered, and finally transferred to a dialysis bag for further purification. The final solution was transferred to a clean culture dish, sealed and frozen in a refrigerator for 14h (4°C). Then, take out the sample and vacuum dry it in a freeze dryer for 12 hours to finally obtain the oxidative mimetic enzyme.

[0058] Example 4

[0059] A colorimetric sensor array was constructed using three sensing units (NaCl, NaBr, NaI) to recognize five representative aromatic amines (o-phenylenediamine (OPD), p-phenylenediamine (PPD), 1,5-naphthalenediamine (1,5-NDA), 1,8-naphthalenediamine (1,8-NDA), and 2-aminoanthracene (2-AA)).

[0060] Separately mix 100 μL of the oxidative mimetic enzyme prepared in Example 1 (1 mg / mL), 100 μL of NaCl, NaBr, NaI (1 mM), and 700 μL of BR buffer solution (pH = 7). Subsequently, add 50 μM to 10 mM aromatic amine (100 μL) to the mixture to a final volume of 1 mL and continue at room temperature for 5 minutes. Record the UV-visible spectrum and use the absorbance values ​​to distinguish aromatic amines. Repeat this process 5 times each time to generate a training data matrix of 5 aromatic amines × 3 sensing units × 5 repeated experiments. Use SYSTATV13.0 to perform origin and linear discriminant analysis (LDA) on the data. The results show that, Figure 1 As shown in AD, the five aromatic amine compounds independently formed tight clusters without any cross-overlapping between each other, indicating that the sensor array distinguished and detected the five aromatic amine compounds with 100% accuracy, proving that the three-channel sensor array can identify the five aromatic amines at 50μM to 10mM.

[0061] In the same way, 100 μL Cu-A (1 mg / mL), 100 μL NaCl, NaBr, NaI (1 mM) and 700 μL BR buffer solution (pH = 7) were mixed and reacted for 5 minutes. Subsequently, 10 mM unknown sample (100 μL) was added to the mixture, and the unknown sample could be accurately classified by the size of the Mahalanobis distance in LDA ( Figure 1 EF).

[0062] According to the method, 100 μL OPD / PPD (50 μM-10 mM), 100 μL Cu-A (1 mg / mL), 100 μL NaCl, NaBr, NaI (1 mM) and 700 μL BR buffer solution (pH = 7) were mixed, and the absorption value at the UV characteristic peak was recorded and processed and calculated. Figure 2 As shown, the sensor array designed by this method can accurately quantify OPD ( Figure 2 A1, B1) and PPD( Figure 2 A2, B2).

[0063] The sensor array designed by the present invention is designed with an actual system of 100 μL aromatic amine (dissolved in the actual water system, 500 μM), 100 μL Cu-A (dissolved in the actual water system, 1 mg / mL), 100 μL NaCl, NaBr, NaI (dissolved in the actual water system, 1 mM) and 700 μL BR buffer solution (pH = 7). The sensor array designed in this embodiment is used to detect actual water systems (respectively: tap water, river water, sea water, and sewage). The detection results are as follows: Figure 3 As shown, it has good sensing performance and can identify and detect aromatic amine compounds with an accuracy of 100%.

[0064] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0065] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An application of an oxidative mimetic enzyme in the detection of aromatic amines, characterized in that: A colorimetric sensor array was constructed by combining oxidative mimetic enzymes with three sensing units to detect aromatic amines. The method for preparing the oxidation mimicking enzyme comprises the following steps: The copper salt, adenine and water are mixed for reaction to obtain a reaction solution, which is dried to obtain an oxidative mimic enzyme; The method for preparing the colorimetric sensor array comprises the following steps: Sensing unit 1: mixed oxidative mimetic enzyme, NaCl solution and BR buffer solution; Sensing unit 2: mixed oxidative mimetic enzyme, NaBr solution and BR buffer solution; Sensing unit 3: mixed oxidative mimetic enzyme, NaI solution and BR buffer solution; 50 μM to 10 mM aromatic amines were added to a final volume of 1 mL of sensor unit at room temperature for 5 to 15 minutes, UV–vis spectra were recorded, and absorbance values ​​were used to distinguish aromatic amines; this was repeated 5 times to generate a sensor array of 5 aromatic amines × 3 sensor units × 5 parallel experiments; origin and linear discriminant analysis of the data was performed using IBM SPSS Statistics; When preparing the sensor units 1 to 3, the concentration of the oxidative mimic enzyme is independently 0.05 to 0.2 mg / mL; the concentration of the NaCl solution, the NaBr solution and the NaI solution is independently 0.05 to 10 mmol / L; The volume ratio of the oxidative mimetic enzyme, NaCl solution, and BR buffer solution was 1:1:7; The volume ratio of the oxidative mimetic enzyme, NaBr solution, and BR buffer solution was 1:1:7; The volume ratio of the oxidative mimetic enzyme, NaI solution, and BR buffer solution was 1:1:7; The pH value of the BR buffer solution is 7.

2. The use according to claim 1, characterized in that: The mass volume ratio of the copper salt, adenine and water is 0.2-0.25 g: 0.1-0.15 g: 10 mL.

3. The use according to claim 1, characterized in that: The reaction temperature is 65-80° C., and the reaction time is 10-30 min.

4. The use according to claim 1, characterized in that: The copper salt includes one or more of copper nitrate, copper chloride, copper sulfate and copper acetate.

5. The use according to claim 1, characterized in that: Before drying, a freezing operation is also included, the freezing time is 10 to 14 hours, and the freezing temperature is 0 to 5°C.

6. The use according to claim 1, characterized in that: The five aromatic amines are o-phenylenediamine, p-phenylenediamine, 1,8-naphthalenediamine, 1,5-naphthalenediamine and 2-aminoanthracene.

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