Preparation method of nano-enzyme and application thereof in detection of uranium ions in seawater

Abstract

This invention discloses a method for preparing nanozymes and their application in the detection of uranium ions in seawater, belonging to the field of uranium ion detection technology. The method for preparing the nanozyme involves synthesizing nitrogen-doped graphene (NG) using graphene and dicyandiamide, and then synthesizing FeN5-SA with hemin chloride, which is a single-atom nanozyme with a FeN5 structure. The nanozyme can be used for the detection of uranyl ions in seawater and has advantages such as high specific activity, acid and alkali resistance, high temperature resistance, and high activity.

Description

Technical Field

[0001] This invention belongs to the field of uranium ion detection technology, and particularly relates to a method for preparing nanozymes and their application in the detection of uranium ions in seawater. Background Technology

[0002] With social and industrial development, non-renewable resources are being gradually depleted, making it necessary to develop and utilize other energy sources. Nuclear energy is a clean energy source that provides 13% of the world's electricity without producing greenhouse gases or other environmental pollutants. Uranium, as a fundamental element of the nuclear industry, is receiving increasing attention from humankind. However, due to improper handling by humans, many problems have arisen, such as nuclear accidents and nuclear waste leaks, posing a serious threat to the ecological environment.

[0003] To date, analytical methods for uranium detection have been developed, such as X-ray fluorescence spectrometry, inductively coupled plasma mass spectrometry (ICP-MS), electrochemical methods, and atomic absorption spectrometry. However, most of these methods involve limitations due to time-consuming, complex, and expensive instruments, and poor portability, thus limiting their application to well-equipped laboratories. Colorimetric methods are among the most promising approaches for monitoring trace uranium in water systems because they enable simple, on-site monitoring without the need for additional advanced detection equipment.

[0004] In recent years, single-atom catalysts (SACs) with atomically dispersed metal sites have vividly mimicked the active sites of enzymes. Leveraging maximum atom utilization efficiency and strong metal-support interactions, the proposed SACs exhibit significantly higher catalytic activity and selectivity than traditional nanomaterials. Furthermore, the well-defined active metal sites contribute to a deeper understanding of structure-property relationships, providing potential guidance for the rational design of advanced enzyme-like catalysts at the atomic scale. Inspired by the active iron sites in natural HRP (horseradish peroxidase), Fe-NC single-atom catalysts with hemin-like structures have attracted widespread attention as mimics of POD (peroxidase). However, their intrinsic activity is still far from that of HRP, failing to meet the high sensitivity requirements of biosensing. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a method for preparing nanozymes and their application in the detection of uranium ions in seawater. The nanozyme is a highly active, high-loading single-atom nanozyme catalyst that can be used for the detection of uranium ions in seawater. It has advantages such as high specific activity, acid and alkali resistance, high temperature resistance, and high activity.

[0006] To achieve the above objectives, the present invention provides a method for preparing nanozymes, comprising the following steps:

[0007] Nitrogen-doped graphene (NG) is synthesized using graphene and dicyandiamide, and then NG is combined with heme chloride (Hemin) to synthesize a single-atom nanozyme (FeN5-SA) with an FeN5 structure, which is the nanozyme.

[0008] Five-coordinate heme iron active sites and unique three-dimensional structures can enhance the catalytic performance of catalysts. Axial ligands (such as histidine or cysteine) are considered to play a crucial role in controlling the electronic structure of active sites and even determining their catalytic performance. Specifically, the strong electron-donating groups of axial ligands on the metal active center can increase the electron density on the macrocycle by donating electrons to the unoccupied d orbitals of the metal ion. This interaction, named the "driving effect," significantly promotes the formation of protonated iron porphyrin (IV) reaction intermediates, enhancing the intrinsic catalytic activity. Inspired by the "driving effect," the introduction of axial ligands to construct five-coordinate environments in this invention may offer significant opportunities for vividly mimicking natural HRP and further developing high-performance enzyme-like SACs. Five-coordinate and four-coordinate Fe-N-CSACs were designed, and the effect of coordination structure on enzyme-like activity was investigated. Specifically, hemin was selected as a precursor for the FeN4 site and assembled on ultrathin nitrogen-doped graphene (NG) to form FeN5 SACs with additional axial ligands (FeN5-SA). Furthermore, as a comparison, FeN4 SACs (FeN4-SA) were prepared by assembling heme onto graphene. As expected, FeN5-SA exhibited better POD-like activity than FeN4-SA. Theoretical studies showed that the strong interaction between the active site and the intermediate, stemming from the driving effect of the added axial ligand, significantly enhanced the catalytic activity of FeN5-SA. FeN5-SA, with its good catalytic activity, was applied to the detection of uranyl ions in seawater. Experimental results showed that the sensing platform exhibited ideal sensitivity, selectivity, and recovery. This not only provides strong evidence for the crucial role of axial ligands in SACs but also demonstrates the superiority of designing enzyme-like catalysts at the atomic scale under the structural guidance of natural enzymes.

[0009] Furthermore, the method for synthesizing NG using graphene and dicyandiamide is as follows:

[0010] Graphene was prepared into a graphene solution, dicyandiamide was added to the graphene solution, the mixture was stirred, freeze-dried, heated, washed, and dried to obtain NG.

[0011] Furthermore, the mass ratio of graphene to dicyandiamide is 1:5.

[0012] Furthermore, the stirring time is 24 hours; heating is carried out in a nitrogen atmosphere at a temperature of 700-900°C, preferably 800°C, for a heating time of 1-3 hours, preferably 3 hours; and the drying temperature is 80°C.

[0013] Furthermore, the method for synthesizing FeN5-SA from NG and Hemin is as follows:

[0014] NG is added to N,N-dimethylformamide (DMF) to prepare an NG solution;

[0015] Hemin was added to N,N-dimethylformamide to prepare a hemin solution;

[0016] The nanozyme is prepared by mixing the NG solution and the hemin solution, stirring at room temperature, centrifuging, washing the powder, and heating under nitrogen.

[0017] Furthermore, the powder obtained after centrifugation is washed with DMF and ultrapure water, and then heated at a temperature of 200-400℃, preferably 300℃, for a heating time of 1-3 hours, preferably 3 hours.

[0018] A nanozyme was prepared according to the above method.

[0019] Application of the nanozyme in the detection of uranium ions in seawater.

[0020] Furthermore, in the application of nanozymes for the detection of uranium ions in seawater, the specific method is as follows:

[0021] FeN5-SA, hydrogen peroxide, buffer solution, and tetramethylbenzidine (TMB) are mixed and reacted to generate OXTMB. This OXTMB is then added to seawater, where uranyl ions react with OXTMB to form a complex. The absorbance is measured. The minimum uranyl ion concentration that reduces absorbance is 3-5 ppb, preferably 3.67 ppb.

[0022] Furthermore, in the application of nanozymes for the detection of uranium ions in seawater, the amount of FeN5-SA is 3-5 μL, the hydrogen peroxide concentration is 90-120 mM, the TMB concentration is 1-3 mM, and the pH of the buffer solution is 2-4. Preferably, the amount of FeN5-SA is 5 μL, the hydrogen peroxide concentration is 100 mM, the TMB concentration is 3 mM, the pH of the buffer solution is 3, and the reaction temperature is 25℃.

[0023] Furthermore, as a comparison, FeN4-SA was synthesized as follows: Graphene was heated at 800℃ in a nitrogen atmosphere for 2 hours. 100 mg of graphene was added to 40 mL of DMF to prepare a solution. Then, 50 mg of hemin was added to 2 mg / mL of DMF. The two solutions were mixed and stirred at room temperature for 5 hours. After centrifugation, the powder was washed with DMF and ultrapure water and heated to 300℃ in a nitrogen atmosphere for 2 hours to obtain FeN4-SA.

[0024] Furthermore, as a comparison, FeN4-SA was used for the detection of uranium ions in seawater. The method is as follows: FeN4-SA reacts with hydrogen peroxide, a buffer solution, and TMB to generate OXTMB, and the absorbance is measured; uranyl ions react with the generated OXTMB to form a complex, and the absorbance is measured to detect the uranyl ions, thus realizing the detection of uranium ions in seawater. The amount of FeN4-SA is 3-5 μL, the concentration of hydrogen peroxide is 90-120 mM, the concentration of TMB is 1-3 mM, and the pH of the buffer solution is 2-4. Preferably, the amount of G-hemin is 5 μL, the concentration of hydrogen peroxide is 100 mM, the concentration of TMB is 3 mM, the pH of the buffer solution is 3, and the reaction temperature is 25℃.

[0025] To ensure more accurate absorbance measurements, hydrogen peroxide and buffer solution can be added to FeN5-SA / FeN4-SA and allowed to react for 1-2 minutes before adding TMB and measuring the absorbance. Alternatively, after TMB is oxidized to OXTMB, wait 3 minutes before adding uranyl ions.

[0026] The recyclability of FeN5-SA and HRP was tested and compared. The specific method is as follows: Hydrogen peroxide, buffer solution, and TMB were added to each material separately, and the absorbance was measured. The original solution was discarded, and then hydrogen peroxide, buffer solution, and TMB were added again, and the absorbance was measured again. This process was repeated 3-5 times, and the changes in absorbance were compared. Preferably, the absorbance was measured after adding hydrogen peroxide, buffer solution, and TMB to each material separately, discarding the original solution, adding hydrogen peroxide, buffer solution, and TMB again, and the absorbance was measured again. This process was repeated 5 times, and the changes in absorbance were compared.

[0027] A sensor for detecting uranium ions in seawater was prepared using the aforementioned nanozyme.

[0028] Compared with the prior art, the present invention has the following advantages and technical effects:

[0029] (1) When using the nanozyme of the present invention to detect uranium ions in seawater, the minimum concentration of uranyl ions that can reduce absorbance is 3-5 ppd.

[0030] (2) The nanozyme FeN5-SA of the present invention has higher activity, higher degree of color change and higher specific activity than FeN4-SA under the same reaction conditions.

[0031] (3) The nanozyme FeN5-SA of the present invention is more resistant to high temperature and acid and alkali than HRP, and is more stable and can be reused. Attached Figure Description

[0032] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0033] Figure 1 SEM, TEM, SAED, and HRTEM images of FeN5-SA;

[0034] Figure 2 SEM, TEM, SAED, and HRTEM images of FeN4-SA;

[0035] Figure 3 pH dependence of FeN5-SA and HRP;

[0036] Figure 4 Temperature dependence of FeN5-SA and HRP;

[0037] Figure 5 The absorbance results for FeN5-SA, FeN4-SA, and the control group with different substrates are shown.

[0038] Figure 6 The specific activities of FeN5-SA and FeN4-SA;

[0039] Figure 7 For the recyclability of FeN5-SA and HRP;

[0040] Figure 8 This is a detection graph for uranyl ions. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] The room temperature in this invention refers to 25±2℃.

[0047] Example 1: Synthesis of NG (Introduction of a nitrogen source into graphene)

[0048] 100 mg of graphene was added to 50 mL of ultrapure water to make a solution, then 500 mg of dicyandiamide was added, stirred for 24 h, freeze-dried, heated at 800 °C in a nitrogen atmosphere for 2 h, then washed three times with ultrapure water, and dried at 80 °C to obtain NG.

[0049] Example 2: Synthesis of FeN4-SA

[0050] Graphene was added to 40 mL of DMF to prepare a graphene solution; then 50 mg of hemin was added to 2 mg / mL of DMF to prepare a hemin solution; the two solutions were mixed and stirred at room temperature for 5 h, centrifuged, and the powder obtained was washed with DMF and ultrapure water. FeN4-SA was obtained by heating at 300 °C for 2 h under nitrogen.

[0051] Example 3: Synthesis of FeN5-SA

[0052] The NG synthesized in Example 1 was added to 40 mL of DMF to prepare an NG solution; then 50 mg of hemin was added to 2 mg / mL of DMF to prepare a hemin solution; the two solutions were mixed and stirred at room temperature for 5 h, centrifuged, and the powder obtained was washed with DMF and ultrapure water. The powder was then heated at 300 °C for 2 h under nitrogen to obtain FeN5-SA.

[0053] Taking FeN4-SA synthesized in Example 2 and FeN5-SA synthesized in Example 3 as examples, the following experiments were conducted:

[0054] SEM, TEM, SAED, and HRTEM were performed on FeN5-SA and FeN4-SA, respectively. The results are shown in [Figure number missing]. Figure 1 and Figure 2 .Depend on Figure 1 and Figure 2It can be seen that both FeN5-SA and FeN4-SA exhibit a wrinkled nanosheet structure and no nanoparticle aggregation, proving the successful synthesis of single-atom nanozymes.

[0055] Determination of acid and alkali resistance: Equal amounts of FeN5-SA and HRP were placed in strong acid and strong alkali environments respectively, and then reacted with H2O2 solution.

[0056] The pH-dependent results for FeN5-SA and HRP are shown in [the table]. Figure 3 ,Depend on Figure 3 It can be seen that FeN5-SA has better environmental tolerance and can still maintain relatively good enzyme activity under extreme pH conditions.

[0057] Temperature dependence determination: FeN5-SA and HRP were placed at 4℃, 15℃, 25℃, 40℃, 50℃, 60℃, 70℃, and 80℃, respectively. Kinetics were measured by reacting 5 μL of FeN5-SA with 100 μL of 100 mM hydrogen peroxide and 100 μL of pH 3 buffer solution, followed by the addition of 100 μL of 3 mM TMB solution. Similarly, 5 μL of HRP was reacted with 100 μL of 100 mM hydrogen peroxide and 100 μL of pH 3 buffer solution, followed by the addition of 100 μL of 3 mM TMB solution, and kinetics were measured again.

[0058] The temperature dependence of FeN5-SA and HRP is shown in the figure. Figure 4 ,Depend on Figure 4 It can be seen that FeN5-SA has a higher tolerance to temperature compared with HRP, and its enzyme activity can still be well maintained over a relatively wide temperature range.

[0059] FeN5-SA reacts with hydrogen peroxide, buffer solution, and TMB to generate OXTMB; absorbance is measured.

[0060] kinetics of FeN5-SA:

[0061] Add 0, 1.5, 3, 4.5, 6, and 7.5 μL of material to each of the six wells of the microplate, followed by 200 μL of NaAc-HAc buffer solution at pH 3, 50 μL of 100 mM hydrogen peroxide solution, and 50 μL of 1 mM TMB solution, and measure the kinetics.

[0062] Measurement of hydrogen peroxide kinetics:

[0063] Add 100 μL of hydrogen peroxide solution with concentrations of 0, 3, 9, 15, 30, 60, 100, and 150 mM to each of the eight wells of the microplate (no hydrogen peroxide is added for the 0 concentration well), then add 5 μL of FeN5-SA material, 100 μL of pH 3 buffer solution (200 μL of buffer solution is added for the 0 concentration well), and finally add 100 μL of 1 mM TMB solution. Measure the kinetics.

[0064] Measuring TMB dynamics:

[0065] Add 5 μL of FeN5-SA material, 100 μL of 100 mM hydrogen peroxide solution, and 100 mM pH 3 buffer solution to each of the eight wells of the microplate. Finally, add TMB solution with concentrations of 0, 0.3, 1, 2.7, 3.6, 4.5, and 5.4 mM (add 200 mM TMB solution to the wells with a TMB concentration of 0 when adding the buffer solution) and measure the kinetics.

[0066] Analyze the kinetic data: React 5 μL FeN5-SA with 100 μL of 100 mM hydrogen peroxide and 100 μL of pH 3 buffer solution, then add 100 μL of 3 mM TMB solution to obtain OXTMB, and measure the absorbance.

[0067] FeN4-SA reacts with hydrogen peroxide, buffer solution, and TMB to generate OXTMB. The absorbance is measured: 5 μL of FeN4-SA is reacted with 100 μL of 100 mM hydrogen peroxide and 100 μL of pH 3 buffer solution. Then, 100 μL of 3 mM TMB solution is added to obtain OXTMB. The absorbance is measured and compared with the absorbance obtained with FeN5-SA as the catalyst.

[0068] The absorbance results of FeN5-SA, FeN4-SA, and the control group (where only H2O2 and TMB are present) with different substrates are shown in Figure 5. Figure 5 It can be seen that FeN5-SA exhibits higher peroxidase activity when H2O2 and TMB are present simultaneously.

[0069] The specific activities of FeN5-SA and FeN4-SA are shown in the figure. Figure 6 ,Depend on Figure 6 It can be seen that FeN5-SA has a higher specific activity than FeN4-SA.

[0070] S1, HRP is used to test the recyclability of materials:

[0071] Add 50 μL of HRP solution, 1 mL of pH 3 buffer solution, and 1 mL of 100 mM hydrogen peroxide solution to a test tube. Pipette 200 μL into each of the six wells. Finally, add 100 μL of 1 mM TMB solution to each well and spot-swipe. Discard the remaining liquid in the test tube. Repeat this process 5 times.

[0072] S2, FeN5-SA are materials whose recyclability is tested.

[0073] Add 50 μL of FeN5-SA solution, 1 mL of pH 3 buffer solution, and 1 mL of 100 mM hydrogen peroxide solution to a test tube. Pipette 200 μL into each of the six wells. Finally, add 100 μL of 1 mM TMB solution to each well and spot-swipe. Discard the remaining liquid in the test tube. Repeat this process 5 times.

[0074] S3, compare the data of the two materials, the results are shown in... Figure 7 The results showed that FeN5-SA has good recyclability.

[0075] Different concentrations of uranyl ions react with OXTMB generated from FeN5-SA to form complexes. The absorbance is measured to detect the uranyl ions. Figure 8 ,Depend on Figure 8 It is known that the minimum concentration of uranyl ions that can reduce absorbance is 3-5 ppb.

[0076] In summary, the nanozyme of the present invention can be used for the detection of uranium ions in seawater, and has advantages such as high specific activity, acid and alkali resistance, high temperature resistance, and high activity.

[0077] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. Application of a nano-enzyme in detection of uranium ions in seawater, characterized in that, The specific method is as follows: a single-atom nanozyme with FeN5 structure, hydrogen peroxide, buffer solution and tetramethylbenzidine are mixed and reacted to generate OXTMB. The mixture is then added to seawater, where uranyl ions react with OXTMB to form a complex, and the absorbance is measured. The method for preparing the nanozyme includes the following steps: synthesizing nitrogen-doped graphene using graphene and dicyandiamide, mixing the nitrogen-doped graphene solution and heme chloride solution, stirring at room temperature, centrifuging, washing the powder, and heating under nitrogen to obtain a single-atom nanozyme with an FeN5 structure, which is the nanozyme.

2. Use according to claim 1, characterized in that, The method for synthesizing nitrogen-doped graphene using graphene and dicyandiamide is as follows: Graphene was prepared into a graphene solution, dicyandiamide was added to the graphene solution, the mixture was stirred, freeze-dried, heated, washed, and dried to obtain nitrogen-doped graphene.

3. The application according to claim 2, characterized in that, The mass ratio of graphene to dicyandiamide is 1:

5.

4. The application according to claim 2, characterized in that, When synthesizing nitrogen-doped graphene, the stirring time is 24 hours; when synthesizing nitrogen-doped graphene, it is heated in a nitrogen atmosphere at a temperature of 700-900℃ for 1-3 hours; the drying temperature after washing is 80℃.

5. The application according to claim 1, characterized in that, The method for synthesizing single-atom nanozymes with an FeN5 structure from nitrogen-doped graphene and heme chloride is as follows: Nitrogen-doped graphene was added to N,N-dimethylformamide to prepare a nitrogen-doped graphene solution; Heme chloride was added to N,N-dimethylformamide to prepare a heme chloride solution; The nanozyme is prepared by mixing the nitrogen-doped graphene solution and the heme chloride solution, stirring at room temperature, centrifuging, washing the powder, and heating under nitrogen.

6. The application according to claim 5, characterized in that, The powder obtained after centrifugation was washed with N,N-dimethylformamide and ultrapure water, and then heated at 200-400℃ for 1-3 hours.

7. An application of a sensor in detecting uranium ions in seawater, characterized in that, The sensor is prepared from the nanozyme used in the application of claim 1.

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