Preparation method and application of difunctional nano-enzyme Eu (at) Ce-MOF

Abstract

The invention belongs to the technical field of nano composite materials, and relates to a preparation method and application of a difunctional nano enzyme Eu (at) Ce-MOF. According to the invention, ceric ammonium nitrate is used as an inorganic metal source, terephthalic acid is used as an organic ligand, and Ce-MOF is synthesized in one step by using a hydrothermal method; and then introducing europium nitrate hexahydrate and sodium citrate to prepare the Eu (at) Ce-MOF composite material through one-step in-situ functionalization. Ce-MOF is functionalized by using europium nitrate hexahydrate, and second metal activity is introduced, so that the bi-metal synergistic effect is realized to enhance the peroxidase-like activity of the composite material; meanwhile, the introduced Eu < 3 + > is used as a recognition element and has specific recognition capability on Pi, specific recognition sites of the Eu-coated Ce-MOF composite material are increased, and the difunctional nano-enzyme with strong enzyme-like activity and specific recognition capability is prepared.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanocomposite materials, and specifically relates to a preparation method and application of a Eu@Ce-MOF nanozyme with strong peroxidase-like properties and specific recognition ability. Background Art

[0002] Metal-organic frameworks (MOFs) are a class of highly ordered porous materials, which are a stable framework structure formed by the coordination of inorganic metal ions and organic ligands. Among the many MOFs materials, Ce-MOF has been widely studied due to its excellent crystallinity and controllable porosity. The Ce metal site can form a coordinated and stable framework structure with a variety of organic ligands, and the Ce in the framework 3+ / Ce 4+ The cycle can provide electrons to catalyze hydrogen peroxide to produce hydroxyl radicals, which then oxidize the chromogenic substrate to exhibit peroxidase-like activity. In addition, Ce-MOF can be widely used for the detection and adsorption of Pi by forming a Ce-OP bond with phosphate (Pi). However, there are two problems with the single Ce metal site in Ce-MOF: first, the peroxidase activity of Ce-MOF materials is low; second, it lacks the specific recognition ability for Pi, which limits its application in biosensing.

[0003] Therefore, it is necessary to develop a novel preparation method of bifunctional nanozyme Eu@Ce-MOF composite material, which has strong peroxidase-like activity and the ability to specifically recognize Pi. Summary of the invention

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

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

[0006] Introducing the second metal site Eu 3+ It has two functions:

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

[0008] (2)Introduced Eu 3+Eu-OP bonds can be formed to specifically recognize Pi, increase the specific recognition sites of Eu@Ce-MOF composite materials, and improve their specific recognition performance.

[0009] There are carboxyl (-COOH) functional groups on the surface of Ce-MOF, and -COOH and Eu 3+ There is a coordination interaction, so Ce-MOF can interact with europium nitrate hexahydrate through the surface -COOH 3+ Coordinate binding.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

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

[0012] (2) placing the mixed solution c in a vacuum drying oven for hydrothermal reaction to obtain a light yellow mixed solution;

[0013] (3) dialyzing the light yellow mixed solution obtained in step (2), centrifuging, drying, and grinding the dialyzed mixed solution to obtain a light yellow solid powder, namely Ce-MOF, and storing it at room temperature;

[0014] (4) dissolving the Ce-MOF obtained in step (3) in deionized water to obtain a Ce-MOF solution, and adding a europium nitrate hexahydrate solution and a sodium citrate solution to the Ce-MOF solution, stirring at room temperature, and then centrifuging, collecting the centrifugal precipitate, and washing it with N,N-dimethylformamide and deionized water respectively; after washing, separating the precipitate and drying it to obtain a product, which is Eu@Ce-MOF.

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

[0016] Preferably, the mixed solution c in step (2) is placed in a vacuum drying oven for hydrothermal reaction at 100° C. for 1 h.

[0017] Preferably, the molecular weight cutoff of the dialysis bag in step (3) is 14,000, and the dialysis time is 12-48 hours.

[0018] Preferably, the centrifugal conditions in step (3) are: centrifugal speed is 10000 rpm, centrifugal time is 10-15 min; drying temperature is 50-60° C., and drying time is 6-8 h.

[0019] Preferably, the volume ratio of the Ce-MOF solution, europium nitrate hexahydrate solution and 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] Furthermore, the concentration of the Ce-MOF solution was 1 mg / mL, the concentration of the europium nitrate hexahydrate solution was 30 mM, and the concentration of the sodium citrate solution was 20 mM.

[0021] Preferably, the stirring time at room temperature in step (4) is 10-60 min; the centrifugal conditions are: the centrifugal rate is 10000 rpm, the centrifugal time is 10-15 min; the drying temperature is 55-60°C, and the time is 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] Beneficial effects of the present invention

[0024] (1) The present invention prepares a novel bifunctional nanozyme Eu@Ce-MOF composite material, which can enhance the peroxidase-like properties while increasing the Pi-specific recognition sites.

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

[0026] (3) The Eu@Ce-MOF prepared by the present invention has stronger peroxidase-like activity due to the bimetallic synergistic effect. It can be clearly seen from the normalized comparison that the peroxidase-like activities of Ce-MOF and Eu(NO3)3 are 59.3% and 5.3% of that of Eu@Ce-MOF. Eu@Ce-MOF achieves a synergistic effect of 1+1>2, proving that Eu@Ce-MOF has stronger peroxidase-like activity.

[0027] (4) The Eu@Ce-MOF prepared by the present invention introduces the second recognition site Eu 3+ , increase the specific recognition site of Pi, through the selective test (such as attached Figure 6) This indicates that due to the introduction of specific recognition sites, interfering ions in the surface water environment cannot affect the performance of Eu@Ce-MOF in specific detection of Pi, proving that Eu@Ce-MOF successfully realizes specific recognition of Pi. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the preparation process of the bifunctional nanozyme Eu@Ce-MOF of the present invention.

[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 the X-ray diffraction pattern of Eu@Ce-MOF and Ce-MOF, and Figure B is the X-ray photoelectron spectrum of Eu@Ce-MOF and Ce-MOF.

[0031] Figure 4 Figure 3 is the UV-visible absorption spectra of different solutions. The inset is the normalized concentric arc diagram comparing the peroxidase activities of Ce-MOF, Eu(NO3)3 and Eu@Ce-MOF. In the figure, a, b, c and d are H2O2+TMB, Eu(NO3)3+H2O2+TMB, Ce-MOF+H2O2+TMB and Eu@Ce-MOF+H2O2+TMB, respectively.

[0032] Figure 5 UV-visible absorption spectra of (a) Eu(NO3)3, (b) Ce-MOF and (c) Eu@Ce-MOF before and after Pi recognition, where the solid line represents before the addition of Pi and the dotted line represents after the addition of Pi; the inset is a comparison of the absorbance difference before and after the addition of Pi.

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

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art of the present invention generally understand. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and / or materials related to the documents. In the event of conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0037] Embodiment 1:

[0038] Step synthesis method Figure 1 As shown, the specific operations are 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 ammonium cerium nitrate was dissolved in 8 mL of deionized water to obtain a mixed solution b; then the mixed solution b was added to the mixed solution a and stirred for 10 min to obtain solution c.

[0040] (2) placing solution c in a vacuum drying oven for hydrothermal reaction at 100° C. for 1 h, and obtaining a light yellow mixed solution after the reaction;

[0041] (3) The obtained light yellow mixed solution was transferred to a dialysis bag with a molecular weight cutoff of 14,000 and dialyzed for 24 hours, with water replaced every 6 hours to remove unreacted small molecule impurities. Finally, the dialyzed mixed solution was centrifuged (centrifugal speed of 10,000 rpm, time of 10 minutes) and dried (60°C, 8 hours) into a light yellow powder to obtain 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 Ce-MOF solution (1 mg / mL), 2 mL of europium nitrate hexahydrate solution (30 mM), and 0.2 mL of 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, and the centrifugal precipitate was collected and washed three times with N,N-dimethylformamide and deionized water respectively. After washing, the precipitate was separated and dried at 60°C for 8 h to obtain a dry product, namely Eu@Ce-MOF.

[0043] Characterization of Eu@Ce-MOF:

[0044] Figure 2 Figure A is a transmission electron microscope image of Eu@Ce-MOF. It can be seen from the figure that Eu@Ce-MOF presents a uniform spherical shape. Figure B is a high-magnification transmission electron microscope image of Eu@Ce-MOF, showing that its lattice spacing is 0.33nm, which matches the (111) crystal plane of cerium. Figure C is an element distribution diagram of Eu@Ce-MOF, which contains four elements: carbon, oxygen, cerium, and europium. This preliminarily proves that Eu 3+ Successfully functionalized Ce-MOF.

[0045] Figure 3 Figure A in the middle shows the X-ray diffraction patterns of Eu@Ce-MOF and Ce-MOF, which successfully proves that Eu 3+ Functionalization of Ce-MOF will not destroy the crystal structure of Ce-MOF; Figure B is the X-ray photoelectron spectrum 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] UV-visible absorption spectroscopy was used to verify the peroxidase-like activity of europium nitrate hexahydrate, Ce-MOF, and Eu@Ce-MOF. Figure 4 As shown in the figure, in the presence of H2O2, europium nitrate hexahydrate can catalyze a small amount of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to generate blue oxidized TMB (ox-TMB), with a weak absorption peak at 652nm, while Ce-MOF exhibits good catalytic activity (line c), and the absorption peak at 652nm is stronger than that of europium nitrate hexahydrate. The absorption peak of Eu@Ce-MOF at 652nm is further enhanced, proving that it has the strongest peroxidase-like activity (line d). By normalized comparison, the enzyme-like activities of europium nitrate hexahydrate and Ce-MOF are 5.3% and 59.3% of that of Eu@Ce-MOF, indicating that the use of Eu 3+Functionalized Ce-MOF successfully achieved the effect of bimetallic synergistic enhancement of enzyme-like activity.

[0048] Eu@Ce-MOF specific recognition of Pi performance:

[0049] (1) The specific recognition performance of europium nitrate hexahydrate, Ce-MOF and Eu@Ce-MOF for Pi was compared using UV-visible absorption spectroscopy. Figure 5 As shown in the figure, after adding Pi to europium nitrate hexahydrate, Ce-MOF and Eu@Ce-MOF solutions, the ultraviolet absorption peak at 652nm has a significant decrease. This is because Pi can inhibit their peroxidase-like activity. From the UV-visible absorption spectrum, it can be seen that the decrease trend of Eu@Ce-MOF after adding Pi is the most obvious. By comparing the difference in the absorption peak intensity at 652nm before and after adding Pi, as shown in the figure, Figure 5 As shown in the inset, Eu@Ce-MOF has the largest difference, that is, using Eu 3+ Functionalized Ce-MOF successfully improved the ability to specifically recognize Pi.

[0050] (2) The effects of common interfering ions in surface water environments on the specific detection performance of Eu@Ce-MOF for Pi were investigated to prove that the bifunctional nanozyme Eu@Ce-MOF has a specific recognition function for Pi. Figure 6 As shown in the figure, the K + ,Na + Mg 2+ ,Ca 2+ ,Zn 2+ ,Cd 2+ ,Co 2+ ,Pb 2+ ,Cu 2+ ,Cl - ,NO3 - ,Br - ,SO4 2- ,CO3 2- ,SO3 - ,HCO3 - Whether the addition of interfering ions affects the peroxidase-like activity of Eu@Ce-MOF (where Mix represents the sum of all interfering ions except Pi, where the interfering ion concentration is 100 μM and the Pi concentration is 50 μM); it can be seen from the figure that there is an obvious signal change only when Pi is present, which further proves that the bifunctional nanozyme has the ability to specifically detect Pi and can be used for the detection of Pi.

[0051] In summary, the present invention introduces a second metal active site through one-step in situ functionalization to prepare a bifunctional nanozyme Eu@Ce-MOF, which has both strong peroxidase-like activity and the ability to specifically recognize Pi.

[0052] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although this specification has described the present invention in detail with reference to the above embodiments, a person skilled in the art should understand that the present invention can still be modified or replaced by equivalents; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a bifunctional nanozyme Eu@Ce-MOF, characterized in that: Here are the steps: (1) dissolving terephthalic acid in N,N-dimethylformamide and stirring to obtain a mixed solution a; dissolving ammonium cerium nitrate in deionized water and stirring to obtain a mixed solution b; then adding the mixed solution b to the mixed solution a and stirring to obtain a mixed solution c; (2) placing the mixed solution c in a vacuum drying oven for hydrothermal reaction to obtain a light yellow mixed solution; (3) dialyzing the light yellow mixed solution obtained in step (2), centrifuging, drying, and grinding the dialyzed mixed solution to obtain a light yellow solid powder, namely Ce-MOF, and storing the powder at room temperature; (4) dissolving the Ce-MOF obtained in step (3) in deionized water to obtain a Ce-MOF solution, and adding a europium nitrate hexahydrate solution and a sodium citrate solution to the Ce-MOF solution, stirring at room temperature, and then centrifuging, collecting the centrifugal precipitate, and washing it with N,N-dimethylformamide and deionized water respectively; after washing, separating the precipitate and drying it to obtain a product, which is Eu@Ce-MOF.

2. The method for preparing the bifunctional nanozyme Eu@Ce-MOF according to claim 1, characterized in that: In step (1), the dosage 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 adding mixed solution b to mixed solution a is 5-30 min.

3. The method for preparing the bifunctional nanozyme Eu@Ce-MOF according to claim 1, characterized in that: The mixed solution c in step (2) is placed in a vacuum drying oven for hydrothermal reaction at 100° C. for 1 h.

4. The method for preparing the bifunctional nanozyme Eu@Ce-MOF according to claim 1, characterized in that: The molecular weight cut-off of the dialysis bag in step (3) is 14,000, and the dialysis time is 12-48 hours.

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

6. The method for preparing the bifunctional nanozyme Eu@Ce-MOF according to claim 1, characterized in that: The volume ratio of the Ce-MOF solution, europium nitrate hexahydrate solution and 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.

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

8. The method for preparing the bifunctional nanozyme Eu@Ce-MOF according to claim 1, characterized in that: The stirring time at room temperature in step (4) is 10-60 min; the centrifugal conditions are: the centrifugal rate is 10000 rpm, the centrifugal time is 10-15 min; the drying temperature is 55-60°C, and the time is 6-8 h.

9. The bifunctional nanozyme Eu@Ce-MOF prepared according to any one of the methods of claims 1-8.

10. Use of the bifunctional nanozyme Eu@Ce-MOF according to claim 9 for phosphate detection.

Images