Preparation and application of a Fe-Ag / CMP nanopolymer with catalase-like activity

Abstract

This invention provides a simple synthesis method for Fe-Ag / CMP nanopolymers with catalase-like activity and their application in hydrogen peroxide detection, belonging to the field of bioanalytical technology. The method of this invention includes the following steps: (1) preparing a 4-hydroxyethylpiperazine thiosulfonic acid (HEPES) standard buffer solution; (2) adding cytidine monophosphate (CMP) to the solution obtained in step (1) and stirring until the CMP is fully dissolved. Then, under vigorous stirring, ferric nitrate aqueous solution and silver nitrate aqueous solution are added sequentially, at which point the colorless solution turns pale yellow; (3) transferring the mixed solution obtained in step (2) to a centrifuge tube, centrifuging at 12000 rpm for 8 min to collect the precipitate, washing it three times with distilled water, and finally adding 4 mL of distilled water to obtain a 1 mg / mL Fe-Ag / CMP suspension. This invention utilizes CMP as a biological ligand and Ag... + Fe 3+ Simultaneous coordination, and the reducing amino groups on CMP can directly transfer Ag... + In situ reduction to silver nanoparticles (AgNPs) led to the one-step synthesis of a bimetallic Fe-Ag / CMP nanopolymer with hydrogen peroxide-like nanozyme activity, enabling the detection of hydrogen peroxide.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical technology, specifically relating to the simple synthesis of a bimetallic nanopolymer with hydrogen peroxide-like nanozyme activity and its application in hydrogen peroxide detection. Background Technology

[0002] Hydrogen peroxide (H2O2), as a reactive oxygen species, plays a crucial role in physiological processes such as biosynthesis, host defense, and cell signaling. The most widely used method for H2O2 detection is based on enzyme-catalyzed reactions; however, naturally occurring enzymes suffer from instability, easy inactivation, and high cost. Currently, researchers have synthesized a variety of nanozymes by mimicking natural enzymes, including gold nanoparticles, cerium dioxide, graphene oxide, and carbon nanotubes. However, existing nanozyme synthesis methods are complex and often exhibit poor biocompatibility and insufficient catalytic performance. Therefore, there is an urgent need to design and synthesize new nanozymes to replace existing ones.

[0003] Furthermore, to enhance the activity of nanozymes, studies have found that bimetallic nanozyme materials exhibit significantly improved catalytic performance compared to single-nanozyme materials, primarily due to the synergistic effect between bimetallic ions. It is well known that the regular arrangement of metal nodes and organic ligands in metal-organic frameworks (MOFs) provides abundant catalytic sites for MOF structures and endows MOF materials with nanozyme properties. Bimetallic nanozyme materials mainly include bimetallic MOF-based nanozymes and monometallic MOF complexes loaded with noble metal nanoparticles. However, MOF-based nanozymes often suffer from complex synthesis methods and poor biocompatibility, limiting their widespread application in biomedical analysis. Simultaneously, for MOF-based nanozymes loaded with noble metal nanoparticles, in-situ synthesis of nanoparticles via chemical reduction is commonly chosen. However, commonly used reducing agents such as ascorbic acid, sodium borohydride, and sodium citrate all possess certain chemical toxicity, hindering the low-cost, pollution-free, and low-toxicity green synthesis of nanoparticles.

[0004] Coordination polymers (CPs) are functional organic-inorganic hybrid materials formed by metal ions and bridging organic ligands. They have wide applications due to their mild polymerization conditions, high porosity, and high guest encapsulation efficiency. Biomolecules typically possess multiple metal-binding sites, and CPs constructed using these ligands exhibit good biocompatibility. Currently reported biomolecules, such as nucleotides, amino acids, peptides, and proteins, can be used to construct CPs. Among them, metal-nucleotide composites have attracted considerable attention due to their good biocompatibility and environmentally friendly preparation processes. Nucleotides have high metal-binding affinity and can self-assemble into various structures in the presence of metal ions. By selecting enzyme-like metal ions to coordinate with nucleotide analogs, nanozymes with tunable catalytic activity, high stability against harsh environments, flexible composition and structural design, and good biocompatibility can be constructed.

[0005] Among them, cytidine monophosphate (CMP) is inexpensive and readily available, possesses multiple coordinating atoms, and can coordinate with various metal ions with enzyme-like activities to form coordination polymers with good biocompatibility and nanozyme activity. Furthermore, CMP is rich in reducing amino groups, allowing for the direct reduction of metal ions without the introduction of external reducing agents, thus achieving a green and simple synthesis of nanoparticles. Iron ions (Fe...) 3+ Fe is a common metal ion with catalase-like activity; for example, Fe3O4 and iron-based MOFs have been used as nanozymes. In addition, silver nanoparticles (AgNPs) also exhibit nanozyme activity. However, AgNPs existing in colloidal form are prone to aggregation and precipitation due to their high surface energy and density, thus losing enzyme activity. Therefore, researchers typically select a suitable biological template and grow silver nanoparticles in situ on it to improve the tendency of AgNPs to aggregate and become inactive. Using multiple ligand molecules with Ag+ and Fe... 3+ When metal ions coordinate separately, they easily form multi-component mixtures, and the symmetry of the resulting material structure decreases, leading to reduced stability. Summary of the Invention

[0006] To address the aforementioned problems, this invention discloses a simplified method for preparing bimetallic nanopolymers that synthesize hydrogen peroxide nanozymes. This invention utilizes the fact that CMP contains multiple coordinating atoms, enabling it to simultaneously coordinate with various metal ions to target Fe. 3+ With Ag + Then, the reducing property of the amino functional group is used to target Ag. + By reducing the enzyme, a hydrogen peroxide-like nanozyme with good biocompatibility—Fe-Ag / CMP nanopolymer—was synthesized in one step.

[0007] This invention first provides a method for synthesizing hybrid nanocomposites, the method comprising the following steps:

[0008] Step (1) Prepare HEPES standard buffer solution;

[0009] Step (2): Add CMP to the solution obtained in step (1) and stir until the CMP is fully dissolved. Then, add ferric nitrate aqueous solution and silver nitrate aqueous solution in sequence while stirring vigorously until the colorless solution turns pale yellow. The molar ratio of CMP, Fe(NO3)3 and AgNO3 obtained in step (2) is 1:1:(0.25~3).

[0010] (3) Transfer the mixed solution obtained in step (2) to a centrifuge tube, centrifuge at 12000 rpm for 8 min to collect the precipitate, wash it 3 times with distilled water, and finally resuspend it with 4 mL of distilled water to obtain a 1 mg / mL Fe-Ag / CMP suspension.

[0011] Furthermore, the HEPES standard buffer solution described in step (1) has a pH of 7.4 and a concentration of 0.1 mol / L.

[0012] Further, in step (2), the concentration of the silver nitrate solution is 5-60 mmol / L, the concentration of the ferric nitrate solution is 20 mmol / L, and the concentration of the CMP solution is 10 mmol / L.

[0013] Furthermore, the mixing conditions for the mixed solution described in step (2) are stirring at room temperature in the dark for 120 minutes.

[0014] The preparation method of Fe-Ag / CMP nanopolymer described in this invention is simple, easy to implement, and highly reproducible; the AgNPs reduced by CMP have a uniform size distribution and morphology.

[0015] Another important objective of this invention is to design the application of Fe-Ag / CMP nanopolymers in the detection of H2O2.

[0016] Furthermore, the method for detecting H2O2 using Fe-Ag / CMP is as follows: the synthesized Fe-Ag / CMP suspension is mixed with 6-22 mmol / L of 3,3',5,5'-tetramethylbenzidine solution (TMB), sodium acetate-acetic acid (NaAc-HAc) buffer solution with pH 3-11, and 30% H2O2 aqueous solution at a temperature of 15-65℃ for 10 min, and the absorption spectrum data is measured and recorded using a UV-Vis spectrophotometer.

[0017] Furthermore, the concentration of the Fe-Ag / CMP suspension used for detecting H2O2 is 0.1 mg / mL, the concentration of the TMB solution is 16 mmol / L, the concentration of the NaAc-HAc buffer solution is 0.1 mol / L, and the concentration of the H2O2 solution is 5–600 μmol / L.

[0018] Furthermore, the pH of the NaAc-HAc buffer solution is 4.

[0019] Furthermore, the reaction temperature is 45°C.

[0020] Furthermore, the volume ratio of the Fe-Ag / CMP suspension to the TMB solution, the NaAc-HAc buffer solution, and the aqueous solution of H2O2 is 1:2:33:4.

[0021] The beneficial effects of this invention are as follows:

[0022] 1. This invention utilizes CMP as a biological ligand to interact with Ag. + Fe 3+ Simultaneous coordination, and direct transfer of Ag using amino groups. + Fe-Ag / CMP nanopolymers with catalase-like activity were synthesized in one step by in-situ reduction to AgNPs.

[0023] 2. The method for synthesizing this nanopolymer is simple, with adjustable catalytic activity, high stability against harsh environments, and good biocompatibility. Compared with single-metal nanozymes, the enzyme activity is significantly improved. It successfully overcomes the shortcomings of natural enzymes, such as easy inactivation in harsh chemical environments, complex preparation, high purification and storage costs, and long processing time, and achieves highly sensitive and stable detection of hydrogen peroxide. Attached Figure Description

[0024] Figure 1 The images shown are transmission electron microscopy (TEM) images of the nanomaterials described in Example 1, where a is a TEM image of Fe / CMP and b is a TEM image of Fe-Ag / CMP.

[0025] Figure 2 The X-ray photoelectron spectra of the nanomaterials described in Example 1 are shown below, where a is the total elemental spectrum of the Fe-Ag / CMP nanopolymer, b is the X-ray photoelectron spectrum of Fe in Fe-Ag / CMP, and c is the X-ray photoelectron spectrum of Ag in Fe-Ag / CMP.

[0026] Figure 3 a is the ultraviolet absorption spectrum of the Fe-Ag / CMP nanopolymer described in Example 2 after being added to four different reaction systems, with the inset showing the color changes of the four systems during the reaction. Figure 3b is the UV-Vis absorption spectrum comparing the activities of Fe / CMP, Ag / CMP, and Fe-Ag / CMP catalases described in Example 3.

[0027] Figure 4 The diagram shows the optimized conditions for detecting Fe-Ag / CMP catalase activity as described in Example 4, where a, b, c, and d represent the effects of changes in pH, temperature, H2O2, and TMB concentrations on the activity of Fe-Ag / CMP catalase, respectively.

[0028] Figure 5 The graphs shown in Example 5 illustrate the detection of hydrogen peroxide using the Fe-Ag / CMP nanopolymer. In the graph, a is the UV-Vis absorption spectrum of the Fe-Ag / CMP nanopolymer for detecting hydrogen peroxide solutions ranging from 0 to 400 μmol / L, and b is a standard curve showing the concentration of hydrogen peroxide relative to the absorbance value. Detailed Implementation

[0029] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. It should be noted that the terms "front," "rear," "left," "right," "up," and "down" used in the following description refer to directions in the accompanying drawings, and the terms "inner" and "outer" refer to directions toward or away from the geometric center of a specific component, respectively.

[0030] Example 1:

[0031] Preparation and characterization of Fe-Ag / CMP nanopolymers

[0032] Testing instruments: JEM-2100 transmission electron microscope (JEOL) and Apreo 2S X-ray photoelectron spectrometer (APS).

[0033] First, 6.464 mg of CMP was dissolved in 2 mL of 0.1 mol / L HEPES (pH = 7.4) standard buffer solution. Then, while stirring, 1 mL of 20 mmol / L AgNO3 aqueous solution and 1 mL of 20 mmol / L Fe(NO3)3 aqueous solution were added, and the mixture was stirred at room temperature in the dark for 120 min. After the reaction was completed, the yellow mixed solution was washed three times in a high-speed centrifuge and finally resuspended in 4 mL of ultrapure water to obtain a suspension of 1 mg / mL Fe-Ag / CMP nanopolymer. Fe / CMP was prepared without the addition of AgNO3 aqueous solution in the above steps; Ag / CMP was prepared without the addition of Fe(NO3)3 aqueous solution in the above steps. Figure 1 a shows that Fe / CMP is a nanopolymer with a porous structure. Upon addition of an aqueous solution of AgNO3, CMP reacts with Fe... 3+Ag + Simultaneous coordination, such as Figure 1 As shown in b, uniformly morphologically uniform and well-dispersed silver nanoparticles (AgNPs) appear on the surface and inside the pores of Fe / CMP. The AgNPs have a particle size of approximately 4 nm, thus confirming the successful preparation of Fe-Ag / CMP. The X-ray photoelectron spectroscopy (XPS) of the Fe-Ag / CMP nanopolymer prepared in Example 1 is shown below. Figure 2 As shown. Figure 2 As can be seen, the Fe-Ag / CMP nanopolymer is composed of five elements: carbon, nitrogen, oxygen, iron, and silver. Figure 2 The strong peaks in the binding energy of b at 717.6 eV, 725.1 eV, and 713.8 eV are attributed to Fe. 3+ The weak peaks at binding energies of 710.2 eV and 723.4 eV are attributed to Fe. 2+ Therefore, Fe mainly exists as Fe in Fe-Ag / CMP. 3+ Coordination occurs in the form of Fe, with a small portion of Fe... 3+ The amino group in CMP is reduced to Fe. 2+ .exist Figure 2 In c, the strong peaks at 374.2 eV and 368.3 eV can be observed to belong to Ag(0), proving that Ag + The amino groups in CMP were successfully reduced to AgNPs, further proving the successful preparation of Fe-Ag / CMP.

[0034] Example 2:

[0035] Enzyme activity assay of Fe-Ag / CMP nanopolymer

[0036] Test instrument: Agilent Cary-60 UV-Vis spectrophotometer.

[0037] Methods: To investigate the catalase-like activity of Fe-Ag / CMP nanopolymers, the same amount of Fe-Ag / CMP was added to four different reaction systems (including: (1) H2O2 + Fe-Ag / CMP, (2) TMB + Fe-Ag / CMP, (3) H2O2 + TMB, and (4) H2O2 + TMB + Fe-Ag / CMP) for enzymatic catalysis. After the experiment, the absorbance at 652 nm was recorded using a UV-Vis spectrophotometer. Each experiment was conducted three times under the same conditions. Figure 3As shown in the illustration, when H2O2 was absent in the reaction, TMB did not exhibit a significant color change regardless of the addition of Fe-Ag / CMP nanopolymer. When only TMB and H2O2 were present in the reaction, the mixed solution appeared light blue, indicating that H2O2 can slowly oxidize TMB without a catalyst. Furthermore, under the condition that H2O2, TMB, and Fe-Ag / CMP were present simultaneously, 3,3',5,5'-tetramethylbenzidine (TMB) was successfully oxidized to 3,3',5,5'-tetramethylbenzodiimide (TMBDI). Figure 3 The study also found the strongest absorption peak at 652 nm. This indicates that the Fe-Ag / CMP nanopolymer intensifies the oxidation reaction of H2O2 and TMB, verifying that the Fe-Ag / CMP nanopolymer possesses satisfactory catalase-like activity.

[0038] Example 3:

[0039] Comparison of catalase-like properties of three nanopolymers

[0040] Test instrument: Agilent Cary-60 UV-Vis spectrophotometer.

[0041] Methods: To compare the catalase-like activities of the three nanopolymers, 5 μL of Fe / CMP, Ag / CMP, and Fe-Ag / CMP suspensions, 10 μL of TMB solution (10 mmol / L), 20 μL of H2O2 (10 mmol / L), and 165 μL of NaAc-HAc buffer solution (0.1 mol / L, pH = 4) were mixed thoroughly in a centrifuge tube. The mixture was then reacted at room temperature for 10 min, and the reaction was terminated by placing the tube in cold water. Finally, the absorbance of the three nanopolymers at 652 nm was measured using a spectrophotometer. Each experiment was performed three times under the same conditions. Figure 3 As shown in b, compared to Fe / CMP and Ag / CMP, the solution with added Fe-Ag / CMP nanopolymer exhibited the fastest color change rate and showed the strongest absorption peak in the UV-Vis spectrophotometer. This indicates that the bimetallic nanopolymer (Fe-Ag / CMP) possesses stronger catalase-like activity compared to single-metal nanopolymers. The order of catalase-like nanopolymer activity among the three nanopolymers is as follows: Fe-Ag / CMP > Fe / CMP > Ag / CMP. We speculate that the main reason for the high catalase activity of Fe-Ag / CMP nanopolymer is based on the fact that the larger specific surface area of ​​Fe / CMP can load a large number of AgNPs, providing more active sites for the catalytic reaction, and the synergistic effect between the two metals with enzyme-like activity.

[0042] Example 4:

[0043] Optimization of detection conditions for Fe-Ag / CMP nanopolymers

[0044] Testing instrument: Thermo Fisher Multiskan GO+ full-wavelength microplate reader.

[0045] Methods: To obtain optimal detection performance of Fe-Ag / CMP nanopolymers, we optimized several experimental conditions. The main factors affecting the catalytic performance of the nanozyme included: pH (3–11), temperature (15–65℃), H₂O₂ concentration (20–200 mmol / L), and TMB concentration (6–22 mmol / L). We recorded the corresponding absorbance values ​​at 652 nm using a full-wavelength microplate reader, and each experiment was performed three times under the same conditions. Figure 4 As shown in Figure a, when the pH of the NaAc-HAc buffer solution is 4, Fe-Ag / CMP exhibits the highest absorbance at 625 nm, indicating that it has the highest activity at pH 4. When the pH is higher than 5, the catalase-like activity of Fe-Ag / CMP decreases significantly, which we speculate may be due to the instability of H2O2 under alkaline conditions. Temperature also has a significant impact on the catalase-like activity of Fe-Ag / CMP, such as… Figure 4 As shown in b, the catalase-like activity of Fe-Ag / CMP significantly increased at 45℃, and then gradually decreased with increasing temperature. Therefore, 45℃ is the optimal temperature for Fe-Ag / CMP to maintain catalase-like activity. Furthermore, we investigated the effects of H2O2 and TMB concentrations on the catalase-like activity of Fe-Ag / CMP. Figure 4 c shows that when the H2O2 concentration is higher than 50 mmol / L, the catalase-like activity of Fe-Ag / CMP remains basically unchanged; while the optimal catalase-like activity can be observed when the TMB concentration is 16 mmol / L. Figure 4 d). We hypothesize that at higher concentrations of H2O2 and TMB, the enzyme activity tends to stabilize, and changing the concentrations of H2O2 and TMB no longer affects the activity of Fe-Ag / CMP catalases. Therefore, the optimal concentrations of H2O2 and TMB are 50 mmol / L and 16 mmol / L, respectively.

[0046] Example 5

[0047] Fe-Ag / CMP nanopolymers applied to the detection of hydrogen peroxide

[0048] Test instrument: Agilent Cary-60 UV-Vis spectrophotometer.

[0049] Implementation Method: Based on the excellent catalase activity of Fe-Ag / CMP nanopolymers, we designed a simple and sensitive H2O2 colorimetric sensor by utilizing the characteristic that Fe-Ag / CMP can catalyze the oxidation of TMB by H2O2 to produce a significant color change. 5 μL of 0.1 mg / mL Fe-Ag / CMP nanopolymer, 10 μL of TMB solution (16 mmol / L), 165 μL of NaAc-HAc buffer solution (0.1 mol / L, pH = 4), and 20 μL of H2O2 at different concentrations (5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 400, 500, 600 μmol / L) were mixed thoroughly in a centrifuge tube. The mixture was then reacted at 45 °C for 10 min, and the reaction was terminated by placing the tube in cold water. Finally, the absorbance was measured at 652 nm using a spectrophotometer. Each experiment was performed three times under the same conditions. Figure 4 As shown in Figure a, it is clearly visible that the absorbance measured at 652 nm increases with increasing H₂O₂ concentration (0–400 μmol / L). Based on the known hydrogen peroxide concentration and the absorbance values ​​of the solution at this concentration obtained experimentally, we established a concentration-absorbance standard curve. Figure 4 In sample b, a good linear relationship was observed between the concentration of H₂O₂ and the absorbance of TMBDI in the range of 5–400 μmol / L (R). 2 =0.999), and the linear regression equation is y = 0.00257x + 0.14499 (where x is the concentration of H2O2 and y is the corresponding absorbance value). The detection limit of this colorimetric sensor for H2O2 is 1.35 μmol / L. The absorbance of a prepared hydrogen peroxide solution of unknown concentration can be measured using the above method. Substituting the absorbance value into the linear regression equation will yield the concentration of the unknown hydrogen peroxide solution, thus enabling the detection of hydrogen peroxide concentration.

[0050] The technical means disclosed in this invention are not limited to those disclosed in the above embodiments, but also include technical solutions composed of any combination of the above technical features.

Claims

1. A method for preparing Fe-Ag / CMP nanopolymer having catalase-like activity, characterized in that, The method comprises the following steps: Step (1): preparing a HEPES standard buffer solution; Step (2): adding cytidine monophosphate (CMP) into the solution obtained in step (1) and stirring until the cytidine monophosphate is fully dissolved; then adding an aqueous ferric nitrate solution and an aqueous silver nitrate solution under vigorous stirring until the colorless solution turns light yellow; wherein the molar ratio of cytidine monophosphate, ferric nitrate and silver nitrate is 1:1:(0.25-3); Step (3): transferring the mixed solution obtained in step (2) into a centrifuge tube, centrifuging at a speed of 12000 rpm for 8 min to collect the precipitate, washing with distilled water for 3 times, and finally resuspending with 4 mL of distilled water to obtain a suspension of Fe-Ag / CMP nanopolymers at a concentration of 1 mg / mL.

2. The method for preparing Fe-Ag / CMP nanopolymers having a catalase-like activity according to claim 1, characterized in that, The pH of the HEPES standard buffer solution in step (1) is 7.4 and the concentration is 0.1 mol / L.

3. The method for preparing Fe-Ag / CMP nanopolymers having a catalase-like activity according to claim 1, characterized in that, In step (2), the concentration of the aqueous silver nitrate solution is 5-60 mmol / L, the concentration of the aqueous ferric nitrate solution is 20 mmol / L, and the concentration of the CMP solution is 10 mmol / L.

4. Fe-Ag / CMP nanopolymers prepared by the method according to any one of claims 1-3.

5. The use of Fe-Ag / CMP nanopolymer according to claim 4 for the detection of hydrogen peroxide. The method for detecting H2O2 using Fe-Ag / CMP is as follows: The synthesized Fe-Ag / CMP suspension is mixed with 6-22 mmol / L of a 3,3',5,5'-tetramethylbenzidine solution (TMB), a sodium acetate-acetic acid (NaAc-HAc) buffer solution with a pH of 3-11, and a 30% H2O2 aqueous solution at a temperature of 15-65℃ for 10 min, and the absorption spectrum data is measured and recorded using an ultraviolet-visible spectrophotometer.

6. The use of Fe-Ag / CMP nanopolymer according to claim 5 in the detection of hydrogen peroxide, characterized by: The concentration of the Fe-Ag / CMP suspension for detecting H2O2 is 0.1 mg / mL, the concentration of the TMB solution is 16 mmol / L, and the concentration of the NaAc-HAc buffer solution is 0.1 mol / L.

7. The use of Fe-Ag / CMP nanopolymer according to claim 5 in the detection of hydrogen peroxide, characterized by: The pH of the NaAc-HAc buffer solution is 4.

8. The use of Fe-Ag / CMP nanopolymer according to claim 5 in the detection of hydrogen peroxide, characterized by: The reaction temperature for detecting H2O2 is 45℃.

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