Preparation method and application of bifunctional nano-enzyme Eu@Ce-MOF

Abstract

The application belongs to the technical field of nanocomposites, and relates to a preparation method and application of a bifunctional nano-enzyme Eu@Ce-MOF; the application selects cerium ammonium nitrate as an inorganic metal source, terephthalic acid as an organic ligand, and uses a hydrothermal method to synthesize Ce-MOF in one step; then, europium nitrate hexahydrate and sodium citrate are introduced to prepare the Eu@Ce-MOF composite material in one step in situ functionalization. The europium nitrate hexahydrate is used to functionalize the Ce-MOF, a second metal activity is introduced, the synergistic effect of the double metals is enhanced, and the peroxidase activity of the composite material is increased; meanwhile, the Eu 3+ As a recognition element, the Eu@Ce-MOF composite material has specific recognition ability for Pi, the specific recognition sites of the Eu@Ce-MOF composite material are increased, and a bifunctional nano-enzyme with strong enzyme-like activity and specific recognition ability is prepared.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanocomposites, and particularly relates to a preparation method of a strong peroxidase and a Eu@Ce-MOF nanoenzyme with specific recognition capability and application thereof. BACKGROUND

[0002] Metal-organic frameworks (MOFs) are a class of highly ordered porous materials formed by inorganic metal ions and organic ligands through coordination to form a stable framework structure. Among the numerous MOFs materials, Ce-MOF has been widely studied due to its excellent crystallinity and controllable porosity. Ce metal sites can form a coordination stable framework structure with a variety of organic ligands, and the Ce 3+ / Ce 4+ Cycles can provide electron catalytic hydrogen peroxide to generate hydroxyl radicals to further oxidize the color developing substrate, thereby exhibiting peroxidase-like activity. In addition, Ce-MOF can form a Ce-O-P bond with phosphate (Pi) and is widely used for the detection and adsorption of Pi. However, there are two problems with the single Ce metal site in Ce-MOF: first, the peroxidase-like activity of Ce-MOF material is low; and second, the lack of specific recognition ability for Pi limits its application in biological sensing.

[0003] Therefore, it is necessary to develop a preparation method of a new type of dual-functional nanoenzyme Eu@Ce-MOF composite material, which has strong peroxidase-like activity and specific recognition ability for Pi. SUMMARY

[0004] In view of the problems in the prior art, the application aims to solve the problems of low peroxidase-like activity of Ce-MOF material and lack of specific recognition Pi site.

[0005] Based on this, the application proposes a method for preparing Eu@Ce-MOF by introducing a second metal active site by in-situ functionalization. The method selects ammonium cerium nitrate as an inorganic metal source and terephthalic acid as an organic ligand, and uses a hydrothermal method to synthesize Ce-MOF in one step; and then introduces europium nitrate hexahydrate and sodium citrate to prepare Eu@Ce-MOF composite material by in-situ functionalization in one step.

[0006] The introduction of the second metal site Eu 3+ has two effects:

[0007] (1) The second metal active site is introduced into the Ce-MOF framework, and the prepared composite material has stronger peroxidase-like activity through the synergistic effect of the two metals;

[0008] (2) The introduced Eu 3+The Eu-O-P bond can specifically recognize Pi, increase the specific recognition sites of the Eu@Ce-MOF composite material, and improve the specific recognition performance thereof.

[0009] The carboxyl (-COOH) functional group exists on the surface of the Ce-MOF, and the -COOH can be combined with Eu 3+ There is a coordination interaction, so the Ce-MOF can be combined with the europium nitrate hexahydrate through the surface -COOH and Eu 3+ Coordination binding.

[0010] To achieve the above object, the present application adopts the following technical scheme:

[0011] (1) Dissolve terephthalic acid in N,N-dimethylformamide and stir to obtain a mixed solution a; dissolve ammonium cerium nitrate in deionized water and stir to obtain a mixed solution b; then add the mixed solution b to the mixed solution a and stir to obtain a mixed solution c;

[0012] (2) Put the mixed solution c into a vacuum drying box for hydrothermal reaction, and obtain a light yellow mixed solution after reaction;

[0013] (3) The light yellow mixed solution obtained in step (2) is subjected to dialysis treatment, and the mixed solution after dialysis treatment is centrifuged, dried and ground to obtain a light yellow solid powder, which is Ce-MOF, and is stored at room temperature;

[0014] (4) Dissolve the Ce-MOF obtained in step (3) in deionized water to obtain a Ce-MOF solution, and add a europium nitrate hexahydrate solution and a sodium citrate solution to the Ce-MOF solution under normal temperature conditions and stir, then centrifuge, collect the centrifugal precipitate and wash with N,N-dimethylformamide and deionized water respectively; the precipitate after washing is separated and dried to obtain the product, which is Eu@Ce-MOF.

[0015] Preferably, in step (1), the amount of terephthalic acid, ammonium cerium nitrate, N,N-dimethylformamide and deionized water is 0.177g:0.558g:24mL:8mL; the stirring time of the mixed solution b added to the mixed solution a is 5-30min.

[0016] Preferably, in step (2), the hydrothermal reaction of the mixed solution c in the vacuum drying box is carried out at 100℃ for 1h.

[0017] Preferably, in step (3), the molecular weight cut-off of the dialysis bag is 14000, and the dialysis time is 12-48h.

[0018] Preferably, the centrifugation in step (3) is carried out at a speed of 10000 rpm for 10-15 min, and the drying is carried out at a temperature of 50-60℃ for 6-8 h.

[0019] Preferably, the volume ratio of the Ce-MOF solution, the europium nitrate hexahydrate solution and the sodium citrate solution used in step (4) is 20:10:1, wherein the concentration of the Ce-MOF solution is 1-10 mg / mL, the concentration of the europium nitrate hexahydrate solution is 30-300 mM, and the concentration of the sodium citrate solution is 20-200 mM.

[0020] Further, the concentration of the Ce-MOF solution is 1 mg / mL, the concentration of the europium nitrate hexahydrate solution is 30 mM, and the concentration of the sodium citrate solution is 20 mM.

[0021] Preferably, the stirring in step (4) is carried out at room temperature for 10-60 min, the centrifugation is carried out at a speed of 10000 rpm for 10-15 min, and the drying is carried out at a temperature of 55-60℃ for 6-8 h.

[0022] The Eu@Ce-MOF prepared based on the above method has good peroxidase-like activity and can be used for phosphate (Pi) detection.

[0023] Advantages of the present application

[0024] (1) The present application prepares a novel bifunctional nanoscale enzyme Eu@Ce-MOF composite material, which can enhance the peroxidase-like activity and increase the specific recognition site of Pi.

[0025] (2) The present application uses a one-step in-situ functionalization method to prepare the composite material from the Ce-MOF solution, the europium nitrate hexahydrate solution and the sodium citrate solution, and the synthesis temperature is room temperature, the preparation conditions are mild, and the required time is short.

[0026] (3) The Eu@Ce-MOF prepared by the present application has stronger peroxidase-like activity due to the synergistic effect of the double metals, and through normalized comparison, it can be obviously seen that the peroxidase-like activity of Ce-MOF and Eu(NO3)3 is 59.3% and 5.3% of that of Eu@Ce-MOF, the Eu@Ce-MOF realizes the synergistic effect of 1+1>2, and it is proved that the Eu@Ce-MOF has stronger peroxidase-like activity.

[0027] (4) The Eu@Ce-MOF prepared by the present application has a second recognition site Eu 3+ , which increases the specific recognition site of Pi, and through selective test (such as the attached Figure 6It is illustrated that the interference ions in the surface water environment cannot affect the performance of the Eu@Ce-MOF in the specific detection of Pi due to the introduction of the specific recognition site, and it is proved that the Eu@Ce-MOF successfully realizes the specific recognition of Pi. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Figure is a schematic diagram of the preparation process of the bifunctional nanoscale enzyme Eu@Ce-MOF.

[0029] Figure 2 Figure A is a transmission electron microscope image of Eu@Ce-MOF; Figure B is a high-magnification transmission electron microscope image of Eu@Ce-MOF; Figure C is an element distribution map of Eu@Ce-MOF.

[0030] Figure 3 Figure A is an X-ray diffraction pattern of Eu@Ce-MOF and Ce-MOF, and Figure B is an X-ray photoelectron spectroscopy pattern of Eu@Ce-MOF and Ce-MOF.

[0031] Figure 4 Figure is the ultraviolet-visible absorption spectrum of different solutions, and the insert is a normalized concentric circle arc diagram comparing the peroxidase activity of Ce-MOF, Eu(NO3)3 and Eu@Ce-MOF; a, b, c and d in the figure respectively represent H2O2+TMB, Eu(NO3)3+H2O2+TMB, Ce-MOF+H2O2+TMB and Eu@Ce-MOF+H2O2+TMB.

[0032] Figure 5 Figure is the ultraviolet-visible absorption spectrum of Eu(NO3)3, Ce-MOF and Eu@Ce-MOF before and after recognizing Pi, wherein the solid line represents before adding Pi, and the dotted line represents after adding Pi; the insert is a comparison of absorbance difference before and after adding Pi.

[0033] Figure 6 Figure is the influence of common interference ions in the surface water environment on the specific detection performance of Eu@Ce-MOF for Pi. DETAILED DESCRIPTION

[0034] Now, various exemplary embodiments of the present application will be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0035] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended as an admission that the reference is prior art, but rather that the reference is part of the technical literature that is relevant to A person of ordinary skill in the art.

[0036] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Additional implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The examples in the specification are exemplary only and are not intended to be limiting.

[0037] Example 1:

[0038] The synthesis method is shown in Figure 1 The specific operation is as follows:

[0039] (1) 0.177 g of terephthalic acid was dissolved in 24 mL of N,N-dimethylformamide to obtain solution a; 0.558 g of cerium nitrate was dissolved in 8 mL of deionized water to obtain mixed solution b; then mixed solution b was added to mixed solution a and stirred for 10 min to obtain solution c,

[0040] (2) Solution c was placed in a vacuum drying box for hydrothermal reaction, the reaction condition was 100°C, and the time was 1 h, and a light yellow mixed solution was obtained after reaction;

[0041] (3) The obtained light yellow mixed solution was transferred into a dialysis bag with a molecular weight cut-off of 14000 and dialyzed for 24 h, and the water was changed every 6 h to remove unreacted small molecular impurities. Finally, the dialyzed mixed solution was centrifuged (centrifugal speed was 10000 rpm, time was 10 min), dried (60°C, 8h) into a light yellow powder, which was Ce-MOF.

[0042] (4) The Ce-MOF obtained in step (3) was dissolved in deionized water to obtain a Ce-MOF solution; then 4 mL of the Ce-MOF solution (1 mg / mL), 2 mL of an europium nitrate hexahydrate solution (30 mM), and 0.2 mL of a sodium citrate solution (20 mM) were mixed and stirred at room temperature for 20 min, the obtained mixed solution was centrifuged at 10,000 rpm for 15 min, the centrifugal precipitate was washed with N,N-dimethylformamide and deionized water for 3 times respectively, and the precipitate was separated after washing and dried at 60°C for 8 h to obtain a dried product, which was Eu@Ce-MOF.

[0043] Characterization of Eu@Ce-MOF:

[0044] Figure 2 Figure A is a transmission electron microscope image of Eu@Ce-MOF, which shows that Eu@Ce-MOF is uniformly spherical; Figure B is a high-magnification transmission electron microscope image of Eu@Ce-MOF, which shows that the lattice spacing of Eu@Ce-MOF is 0.33 nm, which matches the (111) crystal plane of cerium; and Figure C is an element distribution map of Eu@Ce-MOF, which contains four elements of carbon, oxygen, cerium, and europium, which preliminarily proves that Eu 3+ Ce-MOF is successfully functionalized.

[0045] Figure 3 Figure A is an X-ray diffraction pattern of Eu@Ce-MOF and Ce-MOF, which successfully proves the preparation of Eu 3+ Functionalization of Ce-MOF does not destroy the crystal structure of Ce-MOF; and Figure B is an X-ray photoelectron spectroscopy pattern of Eu@Ce-MOF and Ce-MOF, which further proves the successful preparation of Eu@Ce-MOF.

[0046] Enzyme-like catalytic activity of Eu@Ce-MOF:

[0047] The ultraviolet-visible absorption spectrum was used to verify the peroxidase-like activity of europium nitrate hexahydrate, Ce-MOF, and Eu@Ce-MOF. As shown in Figure Figure 4 In the presence of H2O2, europium nitrate hexahydrate can catalyze a small amount of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to generate blue ox-TMB, and the absorption peak at 652 nm is weak, while Ce-MOF exhibits good catalytic activity (line c), and the absorption peak at 652 nm is stronger than that of europium nitrate hexahydrate. The absorption peak at 652 nm of Eu@Ce-MOF is further enhanced, which proves that it has the strongest peroxidase-like activity (line d). Through normalized comparison, the enzyme-like activity of europium nitrate hexahydrate and Ce-MOF is 5.3% and 59.3% of that of Eu@Ce-MOF, which indicates that Eu 3+The functionalized Ce-MOF successfully realizes the synergistic effect of double metals to enhance the enzyme-like activity.

[0048] Specific recognition of Pi by Eu@Ce-MOF:

[0049] (1) The specific recognition of Pi by Eu(NO3)3.6H2O, Ce-MOF and Eu@Ce-MOF was compared by UV-Vis absorption spectrum. As shown in FIG. 1, after the addition of Pi to the solutions of Eu(NO3)3.6H2O, Ce-MOF and Eu@Ce-MOF, the UV absorption peak at 652 nm was obviously decreased, because Pi can inhibit the peroxidase-like activity thereof. It can be seen from the UV-Vis absorption spectrum that the decrease trend of Eu@Ce-MOF after the addition of Pi is the most obvious. By comparing the difference in the absorption peak intensity at 652 nm before and after the addition of Pi, as shown in FIG. 2, the difference of Eu@Ce-MOF is the largest, that is, the specific recognition of Pi by Eu@Ce-MOF is the best. Figure 5 Figure 5 The inset shows that the difference of Eu@Ce-MOF is the largest, that is, the specific recognition of Pi by Eu@Ce-MOF is the best. 3+ The functionalized Ce-MOF successfully improves the ability of specific recognition of Pi.

[0050] (2) The influence of common interfering ions in the surface water environment on the specific detection of Pi by Eu@Ce-MOF was investigated, so as to prove that the bifunctional nanoscale enzyme Eu@Ce-MOF has the specific recognition function of Pi. As shown in FIG. 3, the influence of the addition of K+, Na+, Mg2+, Ca2+, Zn2+, Cd2+, Co2+, Pb2+, Cu2+, Cl-, NO3-, Br-, SO42-, CO32-, SO32-, HCO3- and other interfering ions on the peroxidase-like activity of Eu@Ce-MOF was investigated (wherein Mix represents the total of all interfering ions except Pi, and the concentration of the interfering ions is 100 μM, and the concentration of Pi is 50 μM); it can be seen from the figure that only when Pi exists, there is an obvious signal change, which further proves that the bifunctional nanoscale enzyme has the ability of specific detection of Pi, and can be applied to the detection of Pi. Figure 6 + ,Na + ,Mg 2+ ,Ca 2+ ,Zn 2+ ,Cd 2+ ,Co 2+ ,Pb 2+ ,Cu 2+ ,Cl - ,NO3 - ,Br - ,SO4 2- ,CO3 2- ,SO3 - ,HCO3 - and other interfering ions; it can be seen from the figure that only when Pi exists, there is an obvious signal change, which further proves that the bifunctional nanoscale enzyme has the ability of specific detection of Pi, and can be applied to the detection of Pi.

[0051] ​​In conclusion, the second metal active site is introduced by one-step in-situ functionalization, and a bifunctional nanoscale enzyme Eu@Ce-MOF is prepared, which can have strong peroxidase-like activity and the ability of specific recognition of Pi.

[0052] It should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; therefore, although the present application has been described in detail with reference to the above examples, those skilled in the art should understand that the present application can still be modified or replaced equivalently; and all technical solutions and improvements without departing from the spirit and scope of the present application should be covered in the scope of claims of the present application.

Claims

1. A preparation method of a bifunctional nanoscale enzyme Eu@Ce-MOF, characterized in that, The steps are as follows: (1) terephthalic acid is dissolved in N,N-dimethylformamide and stirred to obtain a mixed solution a; cerium ammonium nitrate is dissolved in deionized water and stirred to obtain a mixed solution b; then the mixed solution b is added to the mixed solution a and stirred to obtain a mixed solution c; (2) the mixed solution c is placed in a vacuum drying box for hydrothermal reaction, and a light yellow mixed solution is obtained after reaction; (3) the light yellow mixed solution obtained in step (2) is subjected to dialysis treatment, and the mixed solution after dialysis treatment is centrifuged, dried and ground to obtain a light yellow solid powder, which is Ce-MOF, and the Ce-MOF is stored at room temperature; (4) the Ce-MOF obtained in step (3) is dissolved in deionized water to obtain a Ce-MOF solution, and europium nitrate hexahydrate solution and sodium citrate solution are added to the Ce-MOF solution under normal temperature conditions and stirred, followed by centrifugation, collection of the centrifugal precipitate, washing with N,N-dimethylformamide and deionized water respectively; the precipitate after washing is separated and dried to obtain the product, which is Eu@Ce-MOF.

2. The method for preparing bifunctional nanoscale enzyme Eu@Ce-MOF according to claim 1, characterized in that, In step (1), the amount of terephthalic acid, cerium ammonium nitrate, N,N-dimethylformamide and deionized water is 0.177 g: 0.558 g: 24 mL: 8 mL; the stirring time of the mixed solution b added to the mixed solution a is 5-30 min.

3. The method for preparing bifunctional nanoscale enzyme Eu@Ce-MOF according to claim 1, characterized in that, In step (2), the hydrothermal reaction of the mixed solution c in the vacuum drying box is carried out at 100℃ for 1 h.

4. The method for preparing bifunctional nanoscale enzyme Eu@Ce-MOF according to claim 1, characterized in that, In step (3), the dialysis bag used for dialysis has a molecular weight cut-off of 14000, and the dialysis time is 12-48 h.

5. The method for preparing bifunctional nanoscale enzyme Eu@Ce-MOF according to claim 1, characterized in that, In step (3), the centrifugation conditions are: centrifugal speed is 10000 rpm, centrifugation time is 10-15 min; drying temperature is 50-60℃, time is 6-8h.

6. The method for preparing bifunctional nanoscale enzyme Eu@Ce-MOF according to claim 1, characterized in that, In step (4), the volume ratio of the Ce-MOF solution, the europium nitrate hexahydrate solution and the sodium citrate solution used is 20:10:1; wherein the concentration of the Ce-MOF solution is 1-10 mg / mL, the concentration of the europium nitrate hexahydrate solution is 30-300 mM, and the concentration of the sodium citrate solution is 20-200 mM.

7. The method for preparing bifunctional nanoscale enzyme Eu@Ce-MOF according to claim 6, characterized in that, The concentration of the Ce-MOF solution is 1 mg / mL, the concentration of the europium nitrate hexahydrate solution is 30 mM, and the concentration of the sodium citrate solution is 20 mM.

8. The method for preparing bifunctional nanoscale enzyme Eu@Ce-MOF according to claim 1, characterized in that, In step (4), the normal temperature stirring time is 10-60 min; the centrifugation conditions are: centrifugal speed is 10000 rpm, centrifugation time is 10-15 min; the drying temperature is 55-60℃, and the time is 6-8h.

9. The bifunctional nanoscale enzyme Eu@Ce-MOF prepared by the method of any one of claims 1-8.

10. The use of the bifunctional nanoscale enzyme Eu@Ce-MOF of claim 9 for phosphate detection.

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