Preparation method of FeZn diatomic nano-enzyme with adjacent bimetal sites

Abstract

The invention specifically discloses a preparation method of a FeZn diatomic nano enzyme with adjacent double metal sites. The product is of a planar sheet structure, chitosan serves as a chelating support, and the product is prepared through in-situ doping of bimetallic precursors of Fe and Zn in a competitive coordination and high-temperature pyrolysis mode. The preparation method comprises the following steps: taking chitosan as a sacrificial template, taking ferric nitrate and zinc chloride as precursors, and carrying out in-situ conversion on the metal precursors into bimetallic atom sites by virtue of a high-temperature pyrolysis means, so as to prepare the FeZn diatomic nano-enzyme. FeZn diatoms exist in an adjacent state, the peroxidase-like activity of the FeZn diatoms is larger than that of monatomic Fe nano-enzyme and monatomic Zn nano-enzyme, the FeZn diatoms have excellent storage stability and high-temperature-resistant catalytic stability, and the preparation method has the advantages of being high in expandability, simple in process, green in raw material, stable in diatom structure and the like.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of nanoscience and biological catalysts, and particularly relates to a preparation method of FeZn bimetallic site adjacent FeZn diatomic nanoscale enzyme. BACKGROUND

[0002] Hydrogen peroxide (H2O2) as a key molecule of biological metabolism and environmental pollutants, its high sensitivity detection is essential in disease diagnosis (such as real-time monitoring of inflammation) and environmental analysis. Traditional H2O2 detection relies on natural enzymes such as horseradish peroxidase, but has defects such as poor stability, high cost, and harsh storage conditions. Single-atom nanoscale enzyme (SAzyme) has become an ideal alternative due to its high atom utilization, enzyme-like activity, and high stability. However, single-metal site nanoscale enzyme such as Fe-N-C has a bottleneck in H2O2 catalysis: it is difficult to simultaneously optimize H2O2 adsorption (requiring electrophilic sites) and ·OH radical generation (requiring low-valence metal reduction) with a single active center; the single reaction path limits the catalytic efficiency and the detection sensitivity. Heteronuclear diatomic nanoscale enzyme can realize electron coupling and synergistic catalysis through the synergistic effect of double metals, further improving its catalytic activity, substrate selectivity and reaction stability, thus breaking through the limitations of single-atom nanoscale enzyme.

[0003] At present, the main technical paths for preparing diatomic nanoscale enzyme include metal organic framework (MOFs) derivation method, impregnation annealing method, wet chemical self-assembly method, and gas phase migration method. MOFs are common single / diatomic catalyst precursors, and the MOFs derivation method utilizes MOFs to provide a skeleton template and uniformly distributed metal nodes; however, this method usually has problems such as complex synthesis process, easy collapse of pyrolysis structure, and limited content of active elements. The impregnation annealing method realizes the introduction of double metals through multi-step metal impregnation and heat treatment, but often faces problems such as uneven distribution of sites and poor batch stability. The wet chemical self-assembly method utilizes the co-assembly of double-metal macrocyclic compounds in solution and then carbonization. Although this method has a simple process, it has defects such as poor thermal stability, easy metal aggregation or non-uniform distribution. The gas phase migration method realizes the strategy of inducing another single atom to migrate and form a diatomic structure through metal vapor, which can form adjacent metal sites to a certain extent, but this method has strict requirements for reaction atmosphere and temperature window, and is difficult to realize universal regulation.

[0004] In summary, it is urgent to select appropriate templates and synthesis strategies to prepare diatomic nanoscale enzyme with uniform structure, stable site and high catalytic activity, so as to promote its sensing application and functional expansion in complex biological environment. SUMMARY

[0005] The present application aims at the deficiencies of the prior art, utilizes the natural multi-nitrogen and multi-hydroxyl structure of chitosan, good metal chelation ability, and the characteristics that carbonization can form a high N-doped carbon skeleton, limits with a chitosan template, regulates the metal coordination environment by adopting a competitive coordination method, and converts the metal precursor into a bimetallic atomic site in situ through high-temperature pyrolysis.

[0006] The competitive coordination of the Fe and Zn bimetallic precursor with the amino and hydroxyl groups in the chitosan can regulate the anchoring distance and coordination configuration of the metal, inhibit metal agglomeration during pyrolysis, carbonize the chitosan skeleton to form an N-doped carbon base carrier, and anchor the metal atoms on the surface or pore structure of the carrier, so as to realize the stable anchoring and collaborative arrangement of the Fe-Zn bimetallic atom on the atomic scale.

[0007] One of the purposes of the present application is to provide a new strategy for synthesizing a diatomic nanoscale enzyme with strong metal site anchoring ability, simple process, and green raw materials, so as to provide industrialization possibilities for developing high-performance diatomic nanoscale enzymes.

[0008] The second purpose of the present application is to develop a diatomic nanoscale enzyme with high catalytic activity and catalytic stability, which can replace natural enzymes or existing nanoscale enzymes for the development of H2O2 detection sensors, and provide ideas for solving the application bottleneck of poor stability and high cost of H2O2 sensor sensing elements.

[0009] The technical scheme of the present application is as follows:

[0010] One, a FeZn diatomic nanoscale enzyme with adjacent bimetallic sites:

[0011] The FeZn diatomic nanoscale enzyme with adjacent bimetallic sites is prepared by using chitosan as a chelation support, adopting competitive coordination and high-temperature pyrolysis, and doping Fe and Zn bimetallic precursors in situ.

[0012] The FeZn diatomic nanoscale enzyme with adjacent bimetallic sites has a planar sheet structure, and Fe and Zn are doped in a diatomic adjacent state on the nitrogen heterocarbon carrier formed by chitosan;

[0013] The FeZn diatomic nanoscale enzyme with adjacent bimetallic sites has peroxidase-like catalytic activity, room temperature storage stability, and high-temperature catalytic stability, that is, the peroxidase-like activity remains unchanged at room temperature storage and high temperature.

[0014] Two, a preparation method of a FeZn diatomic nanoscale enzyme with adjacent bimetallic sites, comprising the following steps:

[0015] (1) First, prepare an aqueous solution containing chitosan and zinc chloride and stir uniformly at a specific temperature as an A solution; then prepare an aqueous solution containing iron nitrate and stir uniformly as a B solution;

[0016] (2) then the B solution is added dropwise to the A solution at a certain temperature under stirring to form a mixed solution, and then the temperature is adjusted and stirred to dryness to obtain a solid;

[0017] (3) the stirred solid is placed in a vacuum box for overnight drying, and then is ground into powder; then is placed in a tube furnace and calcined at a lower temperature under a flowing nitrogen atmosphere;

[0018] (4) the calcined solid is ground into powder, washed with a nitric acid solution overnight, and then the washed powder is filtered to be neutral, and then is dried overnight;

[0019] (5) is placed in a tube furnace again, and is calcined at a higher temperature under a flowing nitrogen atmosphere, and finally is cooled to room temperature to obtain a black powder FeZn diatomic nanoscale enzyme material.

[0020] In the step (1), the mass ratio of chitosan and zinc chloride used is 1:2, and the aqueous solution containing chitosan and zinc chloride is stirred and uniformly mixed at 60℃ as the A solution.

[0021] In the step (2), the B solution is added dropwise to the A solution at 60℃ under continuous stirring for 3 hours, and then the temperature is adjusted to 80℃ until the mixed solution is stirred to dryness to obtain a solid, and the mass ratio of chitosan in the A solution and iron nitrate in the B solution is 10:1.

[0022] The heating condition in the step (3) is that the heating speed is 5℃ / min from room temperature to 750℃ under a nitrogen flow of 100mL / min, and the heating lasts for 2 hours.

[0023] In the step (4), the calcined solid is ground into powder in a proportion of 1g:100mL, washed with a 1M nitric acid solution overnight, and then dried at 90℃ after being neutralized.

[0024] The heating condition in the step (5) is that the heating speed is 5℃ / min from room temperature to 900℃ under a nitrogen flow of 100mL / min, and the heating lasts for 2 hours.

[0025] In the method, only the A solution is used to prepare a Zn monatomic nanoscale enzyme, and only the B solution is used to prepare a Fe monatomic nanoscale enzyme.

[0026] III. The FeZn diatomic nanoscale enzyme with adjacent bimetallic sites is applied to catalytic decomposition of hydrogen peroxide.

[0027] The FeZn diatomic nanoscale enzyme with adjacent bimetallic sites is applied to catalytic decomposition of hydrogen peroxide.

[0028] The FeZn bimetallic site adjacent to the FeZn bimetallic nanoscale enzyme exhibits higher specific activity and excellent Michaelis constant than the Fe monatomic nanoscale enzyme and the Zn monatomic nanoscale enzyme, and has peroxidase-like catalytic activity, room temperature storage stability and high-temperature-resistant catalytic stability.

[0029] The preparation method comprises the following steps: using chitosan as a sacrifice template, using iron nitrate and zinc chloride as precursors, and converting the metal precursors into bimetallic atomic sites in situ through high-temperature pyrolysis, so as to prepare the FeZn bimetallic nanoscale enzyme. The mass ratio of the chitosan and the zinc chloride is 1:2, and the mass ratio of the chitosan and the iron nitrate is 10:1.

[0030] The present application has the following beneficial effects:

[0031] (1) The Fe-Zn bimetallic site with controllable structure and stable adjacent structure is constructed:

[0032] The effective regulation of the atomic spacing and anchoring environment between Fe and Zn atoms is realized in the precursor stage through the competitive coordination between chitosan and metal precursors. After high-temperature pyrolysis, the Fe-Zn bimetallic site with a clear directional coordination structure is formed, which significantly improves the synergistic effect and electronic regulation ability of the catalytic center.

[0033] (2) The high-activity and high-selectivity hydrogen peroxide catalytic ability is realized:

[0034] Compared with the monatomic nanoscale enzyme, the prepared FeZn bimetallic nanoscale enzyme has stronger substrate affinity and catalytic reaction rate in the peroxidase-like reaction, and is especially suitable for high-sensitivity identification and continuous monitoring of H2O2.

[0035] (3) A green, low-cost and scalable preparation path is adopted:

[0036] The natural chitosan is used as a template, and a one-step pyrolysis process is combined, so that the raw material source is extensive, the environment is friendly, and the process is simple and easy to control, and is suitable for batch production. DETAILED DESCRIPTION

[0037] Figure 1 It is a preparation flowchart of the FeZn bimetallic site adjacent to the FeZn bimetallic nanoscale enzyme;

[0038] Figure 2 It is a high-resolution transmission electron microscopy diagram of the FeZn bimetallic nanoscale enzyme (Fig. a), the Fe monatomic nanoscale enzyme (Fig. b) and the Zn monatomic nanoscale enzyme (Fig. c);

[0039] Figure 3 It is a spherical aberration correction transmission electron microscopy (Fig. a) and a Fe-Zn atomic pair spacing statistical diagram (Fig. b) of the FeZn bimetallic nanoscale enzyme;

[0040] Figure 4 Figures for the synchrotron radiation characterization of FeZn diatomic nanoszyme; a represents the Fe K-edge X-ray absorption near-edge structure spectra of diatomic nanoszyme and standard, b represents the Fe K-edge X-ray fine structure absorption spectra of diatomic nanoszyme and standard, c represents the fitting figure of Fe K-edge X-ray fine structure absorption spectra of diatomic nanoszyme and theoretical calculation value, d represents the Zn K-edge X-ray absorption near-edge structure spectra of diatomic nanoszyme and standard, e represents the Zn K-edge X-ray fine structure absorption spectra of diatomic nanoszyme and standard, and f represents the fitting figure of Zn K-edge X-ray fine structure absorption spectra of diatomic nanoszyme and theoretical calculation value;

[0041] Figure 5 Figures for the comparison of kinetic constants of FeZn diatomic nanoszyme, Fe monatomic nanoszyme and Zn monatomic nanoszyme; a and b respectively represent the Michalis-Menten curve and Lineweaver-Burk figure of FeZn diatomic nanoszyme to different concentrations of H2O2, c and d respectively represent the Michalis-Menten curve and Lineweaver-Burk figure of FeZn diatomic nanoszyme to different concentrations of TMB, e and f respectively represent the Michalis-Menten curve of Fe monatomic nanoszyme to different concentrations of H2O2 and TMB, g and h respectively represent the Michalis-Menten curve of Zn monatomic nanoszyme to different concentrations of H2O2 and TMB.

[0042] Figure 6 Figures for the room temperature storage stability of FeZn diatomic nanoszyme and horseradish peroxidase (Figure a) and relative activity at different temperatures (Figure b). DETAILED DESCRIPTION

[0043] The present application will be further explained in conjunction with specific embodiments, but the embodiments do not limit the present application in any form.

[0044] Embodiments and comparisons of the present application are as follows:

[0045] Example 1: Preparation method of FeZn diatomic nanoszyme with adjacent bimetallic sites

[0046] Figure 1 The figure shows the general preparation process of FeZn diatomic nanoszyme with adjacent bimetallic sites. 4.8 g of chitosan and 9.6 g of zinc chloride were mixed in a 150 mL aqueous solution and stirred at 60°C for 2 h. Then, 10 mL of an aqueous solution containing 0.48 g of ferric nitrate was added dropwise to the above solution at 60°C, and stirring was continued for 3 h.

[0047] The temperature was then adjusted to 80 °C until the solution was stirred dry. The stirred dry solid was dried in a vacuum oven at 60 °C overnight, ground to a powder, then placed in a tube furnace and calcined to 750 °C at a heating rate of 5 °C / min under flowing nitrogen (100 mL / min) for 2 h.

[0048] The calcined solid was ground to a powder and washed with 1 M nitric acid solution overnight at a ratio of 1 : 100 (g:mL). The washed powder was filtered to neutrality, dried at 90 °C overnight, then placed in a tube furnace and heated to 900 °C at a heating rate of 5 °C / min under flowing nitrogen (100 mL / min) for 2 h, cooled to room temperature to obtain FeZn biatomic black powder.

[0049] Comparative Example 1 : Monoatomic Fe nanoszyme preparation method;

[0050] The temperature was then adjusted to 80 °C until the solution was stirred dry. The stirred dry solid was dried in a vacuum oven at 60 °C overnight, ground to a powder, then placed in a tube furnace and calcined to 750 °C at a heating rate of 5 °C / min under flowing nitrogen (100 mL / min) for 2 h.

[0051] Comparative Example 2: Monoatomic Zn nanoszyme preparation method;

[0052] The temperature was then adjusted to 80 °C until the solution was stirred dry. The stirred dry solid was dried in a vacuum oven at 60 °C overnight, ground to a powder, then placed in a tube furnace and calcined to 750 °C at a heating rate of 5 °C / min under flowing nitrogen (100 mL / min) for 2 h.

[0053] 1) Characterization by electron microscopy, etc.

[0054] Figure 2 High-resolution transmission electron microscopy images of the FeZn biatomic nanoszyme, Fe monatomic nanoszyme, and Zn monatomic nanoszyme of the present application. It can be seen from the images that the Fe monatomic nanoszyme appears as obvious aggregated particles; the FeZn biatomic nanoszyme and the Zn monatomic nanoszyme present a sheet structure without obvious crystalline structure, and have clear carbon lines. The introduction of the Zn precursor is conducive to the formation of dispersed Fe monatomic atoms.

[0055] Figure 3 STEM and Fe-Zn atomic pair spacing statistical diagram of the FeZn biatomic nanoszyme of the present application. The circled part in the diagram is the Fe-Zn biatomic pair. The bright spots in the diagram are atoms dispersed on the substrate. The spacing of 150 pairs of Fe-Zn atomic pairs was counted, and it was found that the proportion of Fe-Zn biatomic pairs was greater than 70%, and the atomic pair spacing was 0.21 nm.

[0056] Figure 4 Synchronous radiation characterization diagram of the FeZn biatomic nanoszyme. The results can confirm that Fe-Zn in the FeZn biatomic nanoszyme exists in an adjacent state, and Fe and Zn are connected with 4 N respectively; and the valence of Fe is +2 to +3 valence, and the valence of Zn is +2 valence. The structure is similar to the active center of hematin.

[0057] 2) Enzyme activity test

[0058] Comparison of the peroxidase activity of the FeZn biatomic nanoszyme, Fe monatomic nanoszyme, and Zn monatomic nanoszyme.

[0059] For 3,3',5,5'-tetramethylbenzidine (TMB) kinetic determination, 10 μL of nanoszyme (0.1 mg / mL), 20 μL of H2O2 (100 mM), and 10 μL of different concentrations of TMB stock solution (final concentration of 0.1 to 1 mM) were added to 0.1 M sodium acetate-acetic acid buffer (pH 4.5), and the final volume was 200 μL. For H2O2 kinetic determination, different concentrations of H2O2 (final concentration of 1 to 80 mM) were added to 200 μL of 0.1 M HAc-NaAc buffer (pH 4.5), and TMB (1 mM) and nanoszyme loading (5 μg / mL) were kept constant. All the above reactions were determined by measuring the absorbance of oxidized TMB (Δε = 39,000 M-1cm-1) at a wavelength of 652 nm and a frequency of once every 7.0 seconds by a microplate spectrophotometer. The kinetic constant (Vmax) of the FeZn biatomic nanoszyme was 0.25 U / mg, and the Michaelis constant (Km) was 0.25 mM. max and K m) by fitting the reaction rate values and substrate concentrations to the typical Michaelis-Menten equation.

[0060] Figure 5 Figure 2. Comparison of kinetic constants of FeZn diatomic nanoszyme, Fe monatomic nanoszyme and Zn monatomic nanoszyme. It can be seen that the maximum reaction rate (V max ) and Michaelis constant (K m ) of FeZn diatomic nanoszyme are greater than those of Fe monatomic nanoszyme and Zn monatomic nanoszyme, indicating that the peroxidase-like activity of FeZn diatomic nanoszyme is higher than that of Fe or Zn monatomic nanoszyme.

[0061] 3) Comparison of stability of FeZn diatomic nanoszyme and horseradish peroxidase.

[0062] (1) FeZn diatomic nanoszyme and horseradish peroxidase were stored at room temperature (25℃), and the activity was detected at 1, 3, 5, 10, 20, 30, 40, 50, 60 days, respectively, and the relative activity of FeZn diatomic nanoszyme or horseradish peroxidase under different storage days was compared.

[0063] (2) FeZn diatomic nanoszyme and horseradish peroxidase were incubated at 30℃, 60℃, 90℃ for 5 minutes, respectively, and then the activity was detected, and the relative activity of FeZn diatomic nanoszyme or horseradish peroxidase under different treatment temperatures was compared.

[0064] (3) The activity detection method of FeZn diatomic nanoszyme is as follows: 1 mM chromogenic substrate TMB is added to 0.1 M sodium acetate-sodium acetate buffer (pH 4.5) containing 10 mM H2O2 and 5 μg / mL nanoszyme. After reaction at 25℃ for 5 minutes, the oxidized TMB was quantified by spectrophotometry at λ = 652 nm. The relative activity is the absorbance ratio of the treated sample to the untreated sample at 652 nm.

[0065] (4) The activity detection method of horseradish peroxidase is as follows: 1 mM chromogenic substrate TMB is added to 0.1 M sodium acetate-sodium acetate buffer (pH 4.5) containing 10 mM H2O2 and 5 μg / mL HRP. After reaction at 25℃ for 5 minutes, the oxidized TMB was quantified by spectrophotometry at λ = 652 nm. The relative activity is the absorbance ratio of the treated sample to the untreated sample at 652 nm.

[0066] Figure 6The figure shows the room temperature storage stability graph and the relative activity graph at different temperatures of FeZn diatomic nanoscale enzyme and horseradish peroxidase. It can be seen from the figure that after being stored at room temperature for 60 days, the activity of FeZn diatomic nanoscale enzyme is basically not affected; the activity of HRP enzyme is reduced by more than 90%; in addition, the relative activity of FeZn diatomic nanoscale enzyme at 60 DEG C and 90 DEG C is basically consistent with that at 30 DEG C, while the relative activity of HRP enzyme is reduced by more than 90% after being treated at 90 DEG C. The above results show that FeZn diatomic nanoscale enzyme with adjacent bimetallic sites has excellent room temperature storage stability and high-temperature resistant stability.

[0067] It can be seen from the implementation that the FeZn diatomic in the diatomic nanoscale enzyme of the product of the application exists in an adjacent state, and the peroxidase-like activity thereof is greater than that of monatomic Fe nanoscale enzyme and monatomic Zn nanoscale enzyme, and has excellent storage stability and high-temperature resistant catalytic stability. Moreover, the preparation method has the advantages of strong scalability, simple process, green raw materials and stable diatomic structure, and provides an important way for constructing novel high-performance diatomic nanoscale enzyme.

[0068] The above examples are used to explain and illustrate the application, but not to limit the application, and any modification and change made to the application within the spirit and protection scope of the claims of the application all fall into the protection scope of the application.

Claims

1. A FeZn diatomic nanozyme with adjacent bimetallic sites, characterized in that: The FeZn biatomic nanozyme adjacent to the bimetallic site is prepared by in-situ doping of bimetallic precursors of Fe and Zn using chitosan as a chelating scaffold and competitive coordination and high-temperature pyrolysis.

2. The FeZn diatomic nanozyme with adjacent bimetallic sites according to claim 1, characterized in that: The FeZn diatomic nanozyme adjacent to the bimetallic site has a planar sheet structure, and Fe and Zn are doped in a diatomic adjacent state on a nitrogen-containing carbon support formed by chitosan.

3. A method for preparing FeZn diatomic nanozymes with adjacent bimetallic sites, characterized in that, Includes the following steps: (1) First, prepare an aqueous solution containing chitosan and zinc chloride and stir it at a specific temperature to obtain solution A; then prepare an aqueous solution containing ferric nitrate and stir it to obtain solution B. (2) Then, at a specific temperature, solution B is added dropwise to solution A and stirred to form a mixed solution. Then, the temperature is adjusted and the mixture is stirred until dry to obtain a solid. (3) After the solid was dried, it was placed in a vacuum box and dried overnight, then ground into powder; then it was placed in a tube furnace and heated and calcined at a low temperature under flowing nitrogen. (4) The solid after heating and calcination is ground into powder, washed with nitric acid solution overnight, filtered until neutral, and then dried overnight; (5) It is placed back into the tube furnace and heated and calcined at a high temperature under flowing nitrogen, and finally cooled to room temperature to obtain black powder FeZn diatomic nanoenzyme material.

4. The method for preparing FeZn diatomic nanozymes with adjacent bimetallic sites according to claim 3, characterized in that: In step (1), the mass ratio of chitosan to zinc chloride is 1:2, and the aqueous solution containing chitosan and zinc chloride is stirred and mixed at 60°C to form solution A.

5. The method for preparing FeZn diatomic nanozymes with adjacent bimetallic sites according to claim 3, characterized in that: In step (2), solution B is added dropwise to solution A at 60°C and stirred for 3 hours. Then the temperature is adjusted to 80°C until the mixed solution is dried to obtain a solid. The mass ratio of chitosan used in solution A to ferric nitrate in solution B is 10:

1.

6. The method for preparing FeZn diatomic nanozymes with adjacent bimetallic sites according to claim 3, characterized in that: The heating conditions in step (3) are as follows: heating from room temperature to 750°C at a heating rate of 5°C / min under nitrogen gas with a flow rate of 100 mL / min, for 2 hours.

7. The method for preparing FeZn diatomic nanozymes with adjacent bimetallic sites according to claim 3, characterized in that: In step (4), the solid after heating and calcination is ground into powder according to the ratio of 1g:100mL, washed overnight with 1M nitric acid solution, and then dried overnight at 90°C after neutralization.

8. The method for preparing FeZn diatomic nanozymes with adjacent bimetallic sites according to claim 3, characterized in that: The heating conditions in step (5) are as follows: heating from room temperature to 900°C at a heating rate of 5°C / min under nitrogen gas with a flow rate of 100 mL / min, for 2 hours.

9. The application of a FeZn diatomic nanozyme with adjacent bimetallic sites prepared by the preparation method according to any one of claims 1-8, characterized in that: Application in the catalytic decomposition of hydrogen peroxide.

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