A method for preparing gold magnetic nanozymes and a method for enhancing their catalytic activity

Abstract

This invention belongs to the field of nanoenzyme technology, specifically relating to a method for preparing gold magnetic nanoenzymes and a method for enhancing their catalytic activity. Citric acid is added to an aqueous dispersion of Fe3O4 and mixed to obtain citric acid-modified magnetic Fe3O4 nanoenzymes. The citric acid-modified magnetic Fe3O4 nanoenzymes are then added to a solution containing a reducing agent and mixed to obtain a dispersion. A gold-containing compound is added, and the mixture is heated to its boiling point until the solution changes from black to brownish-yellow. The solution is then centrifuged, washed, and magnetically separated. The resulting brownish-yellow substance is redispersed in water to obtain a gold magnetic cascade nanoenzyme. The prepared gold magnetic cascade nanoenzyme possesses cascade catalytic reaction activity.

Description

Technical Field

[0001] This invention belongs to the field of nanozyme technology, specifically relating to a method for preparing gold magnetic nanozymes and a method for enhancing their catalytic activity. Background Technology

[0002] Nanozymes are nanomaterials that possess catalytic activity similar to that of natural enzymes. Gold nanozymes (Au nanozymes) exhibit glucose oxidase activity, catalyzing the production of hydrogen peroxide and gluconic acid from glucose. Magnetic iron tetroxide nanozymes (Fe3O4 nanoozymes) possess peroxidase activity, catalyzing the production of reactive oxygen species from hydrogen peroxide. Currently, the catalytic activity of nanozymes is still lower than that of natural enzymes, and improving the catalytic activity of nanozymes is an important research direction in this field.

[0003] The essence of gold-magnetic cascade nanozymes is a composite material of gold-magnetic nanoparticles. Currently, the main methods for synthesizing gold-magnetic cascade nanoparticles include self-assembly, sonicochemical methods, wet chemical methods, chemical reduction methods, microemulsion methods, magnetron sputtering methods, and microemulsion methods. The self-assembly method involves first preparing Fe3O4 magnetic nanoparticles, then coating their surface with SiO2 and modifying it with amino molecules to form negatively charged amino-modified Fe3O4@SiO2 nanoparticles; next, using phosphorus tetrahydroxychloride to reduce chloroauric acid to synthesize small-diameter gold nanoparticles; then, mixing the dispersions of these two nanoparticles and synthesizing core-shell structured gold-magnetic nanoparticles through adsorption. However, this synthesis method is complex, time-consuming, and produces core-shell gold-magnetic nanoparticles with uneven particle size, easily forming individual magnetic nanoparticles or gold nanoparticles. The ultrasonic heating and microemulsion method involves uniformly mixing magnetic nanoparticles, glucose, and chloroauric acid reagents, followed by ultrasonic stirring and heating to synthesize gold-magnetic nanoparticles, but the gold content coated on the surface of the magnetic nanoparticles is relatively low. The wet synthesis process involves significant thermal diffusion during heating and temperature control, making it difficult to remove surfactants or ligands used as stabilizers. While excessive reducing or oxidizing agents can ensure the complete conversion of the precursor into nanoparticles, they also become byproducts, causing additional post-processing challenges. Chemical methods, which first synthesize oil-phase gold magnetic nanoparticles through high-temperature heating and then transfer them from the oil phase to the aqueous phase for subsequent biomedical applications, are more complex and can affect the efficiency and water dispersibility of the gold magnetic nanoparticles during the aqueous phase conversion.

[0004] Although methods for preparing gold magnetic nanoparticles have been explored, it is still difficult to prepare gold magnetic nanozymes with cascade catalytic activity. This is because traditional methods for preparing core-shell structured gold magnetic nanoparticles or excessive coating of ligands on the surface of nanoparticles can cause problems such as large mass transfer resistance in catalytic reactions and mismatch in cascade catalytic reaction rates, resulting in these gold magnetic nanoparticles not having cascade catalytic reaction activity. Summary of the Invention

[0005] To address the above problems, this invention provides a method for preparing gold magnetic nanozymes and a method for enhancing their catalytic activity.

[0006] A method for preparing a gold magnetic cascade nanozyme includes the following steps:

[0007] Citric acid was added to an aqueous dispersion of Fe3O4 and mixed well to obtain a citric acid-modified magnetic Fe3O4 nanozyme. The molar ratio of citric acid to Fe3O4 in the aqueous dispersion of Fe3O4 was 1~5:1.

[0008] The citric acid-modified magnetic Fe3O4 nanozyme was added to a solution containing a reducing agent at a molar ratio of 0.1~0.2:1.5~3. The mixture was stirred to obtain a dispersion. A gold-containing compound was then added, and the reaction was continued until the solution changed from black to brownish-yellow. The mixture was then centrifuged, washed, and magnetically separated. The resulting brownish-yellow substance was redispersed in water to obtain the gold magnetic cascade nanozyme.

[0009] Preferably, the reducing agent is any one of sodium citrate, sodium citrate, tannic acid, phenylboronic acid, and sodium borohydride.

[0010] Preferably, the molar ratio of the citric acid-modified magnetic Fe3O4 nanozyme to the sodium citrate solution is 0.13:2.2.

[0011] Preferably, the gold-containing compound is chloroauric acid.

[0012] Preferably, the aqueous dispersion of Fe3O4 is obtained by dispersing Fe3O4 nanoparticles in water.

[0013] The gold magnetic cascade nanozyme prepared by the aforementioned preparation method.

[0014] A method for enhancing the catalytic activity of gold magnetic cascade nanozymes involves applying X-ray irradiation to the gold magnetic cascade nanozymes to enhance their catalytic activity.

[0015] Preferably, the tube voltage of the X-ray tube is 50 kV to 80 kV, and the tube current is 0.1 mA to 1 mA.

[0016] A method for enhancing the catalytic activity of gold magnetic cascade nanozymes involves applying a magnetic field to the gold magnetic cascade nanozymes to enhance their catalytic activity.

[0017] Preferably, the magnetic field stimulation conditions are a magnetic field frequency of 280 kHz to 500 kHz and an intensity of 180 Oe to 300 Oe.

[0018] In this invention, FeCl3·6H2O and FeCl2·4H2O provide divalent and trivalent iron ions as sources for the preparation of Fe3O4 nanoparticles. Citric acid modification increases the water dispersibility of the Fe3O4 nanoparticles and provides nucleation sites on the surface of the Fe3O4 nanoparticles for the synthesis of gold nanoparticles. Sodium citrate is fully dissolved and distributed on the surface of the Fe3O4 nanoparticles. Then, chloroauric acid (HAuCl4) is reduced, nucleated, and grown into gold nanoparticles. Chloroauric acid provides the gold source for the gold nanoparticles. HAuCl4 is reduced, then nucleates and grows into gold nanoparticles. As a gold precursor, chloroauric acid is readily soluble in water and undergoes thermal decomposition and reduction during solution heating to generate gold atoms, which further grow into gold nanoparticles.

[0019] To address the shortcomings of existing gold magnetic nanoparticles, such as core-shell gold magnetic nanoparticles or gold magnetic nanoparticles with excessive surface modification of ligands, which suffer from high mass transfer resistance of substrate and product during catalytic reactions and thus lack cascade catalytic activity, this invention proposes to first prepare a magnetic iron oxide nanozyme with citric acid surface modification, which has peroxidase catalytic activity; then, the magnetic nanozyme is mixed uniformly with sodium citrate and chloroauric acid reagents, and gold nanozyme particles are grown at multiple points on the surface of the magnetic nanozyme, i.e., gold magnetic cascade nanozymes.

[0020] This invention proposes a gold-magnetic cascade nanozyme with dual stimulus-responsiveness and a method for synergistically enhancing catalytic activity through magnetic field and X-ray stimulation. This method promises to achieve a synergistic enhancement of the catalytic activity of the gold-magnetic cascade nanozyme through a dual-effect mediated by magnetic field and X-ray stimulation, providing a simple, universal, and efficient new method for enhancing the catalytic activity of gold-magnetic cascade nanozymes for practical applications. Organically coupling two nanozymes can prepare nanozymes with cascade catalytic activity, thereby improving the overall catalytic activity of the nanozyme to a certain extent.

[0021] The gold magnetic cascade nanozyme preparation method proposed in this invention has the following advantages: (1) The entire preparation process is carried out in an aqueous environment, which can ensure that the gold magnetic nanozyme has good water dispersibility. At the end of the preparation, the gold magnetic cascade nanozyme can be easily obtained by magnetic separation method, while removing byproducts such as gold nanoparticles, excess oxidants and surfactants; (2) The gold magnetic nanozyme presents a structure in which gold nanoparticles are attached to the surface of magnetic nanoparticles at multiple points, which greatly eliminates the large mass transfer resistance defect of traditional core-shell coated or excessive ligand modified structures of gold magnetic nanoparticles.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] By growing gold nanoparticles on the surface of citric acid-modified magnetic Fe3O4 nanoparticles, the prepared gold magnetic nanozymes no longer have a core-shell structure and the surface ligand modification is not excessive. This greatly reduces the mass transfer resistance in the cascade catalytic reaction process, thus facilitating the cascade catalytic reaction activity.

[0024] To address the current lack of advanced technologies for enhancing the catalytic activity of gold magnetic nanoparticles, this invention proposes for the first time a dual-effect synergistic enhancement of the cascade catalytic activity of gold magnetic nanozymes. This synergistic effect is achieved by combining the magnetocaloric effect generated by the induction of alternating magnetic fields by magnetic nanoparticles with the photoelectron effect generated by the absorption of X-rays by gold nanoparticles. This method of enhancing the cascade catalytic activity of gold magnetic nanozymes through dual stimulation by magnetic fields and X-rays is not limited by penetration depth. It utilizes thermal and photoelectron effects to enhance both magnetic and gold nanozymes separately, ultimately synergistically enhancing the overall catalytic activity of the gold magnetic nanozymes. Compared to traditional methods, this method shows a more significant enhancement of enzyme activity.

[0025] Compared with existing methods for enhancing nanozyme activity, this invention employs an alternating magnetic field and X-rays, with a deep penetration depth, to maximize the number of catalytic active sites on gold magnetic nanozymes. Utilizing the magnetocaloric and photoelectron effects, it directly enhances the redox cascade reaction of nanozymes, achieving highly efficient enhancement of the catalytic activity of gold magnetic nanozymes. This is because X-ray photon energies, between 124 eV and 1.24 MeV, possess advantages such as collimation, no energy attenuation, and no penetration depth limitations. X-ray irradiation can maximize the probability of increasing the number of catalytic active sites on the surface of gold nanoparticles and electron transfer in the catalytic reaction, thereby accelerating the first reaction process of the cascade catalysis. Furthermore, the magnetocaloric heating effect induced by the alternating magnetic field in the magnetic nanoparticles can increase the number of catalytic active sites on the surface of the magnetic nanoparticles and increase the redox reaction rate, thereby accelerating the second reaction process of the cascade catalysis, ultimately significantly enhancing the overall cascade catalytic activity. This method, based on the synergistic enhancement of the cascade catalytic activity of gold magnetic nanozymes using magnetic fields and X-rays, holds promise for the application of gold magnetic nanozymes in rapid in vitro biomarker detection and efficient catalytic therapy for malignant tumors, with significant potential socio-economic benefits. Attached Figure Description

[0026] Figure 1 This is a schematic diagram illustrating the preparation of the gold magnetic cascade nanozyme of the present invention.

[0027] Figure 2 This is a schematic diagram illustrating the concept of the present invention, which utilizes magnetic fields and X-rays to synergistically enhance the catalytic activity of gold magnetic cascade nanozymes.

[0028] Figure 3 This is a TEM image of the gold magnetic cascade nanozyme prepared in this invention.

[0029] Figure 4UV-Vis absorption spectrum for X-ray-enhanced catalytic activity of gold magnetic cascade nanozymes.

[0030] Figure 5 UV-Vis absorption spectrum of gold magnetic cascade nanozymes enhanced by alternating magnetic field. Detailed Implementation

[0031] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0032] Example 1

[0033] A method for preparing gold magnetic nanozymes includes the following steps:

[0034] FeCl3·6H2O (37.5 mg) and FeCl2·4H2O (12.5 mg) were dissolved in deionized water (50 mL). NaOH solution (0.1 mol / L) was slowly added while stirring vigorously until the solution color changed from orange to black. Fe3O4 nanoparticles were collected using a magnetic separation method. Specifically, a magnet was brought close to a three-necked flask, and when the black Fe3O4 nanoparticles were attracted to the magnet, the solution in the three-necked flask was removed, and the black substance was collected. After centrifugation and washing multiple times, Fe3O4 nanoparticles were obtained. The Fe3O4 nanoparticles were redispersed in deionized water to obtain an aqueous dispersion of Fe3O4. 38.4 mg of citric acid was added to the Fe3O4 aqueous dispersion with a concentration of 0.13 mmol / L. At this point, the molar ratio of citric acid to Fe3O4 in the aqueous dispersion was 2:1. The mixture was subjected to intermittent ultrasonic oscillation for 4 hours, centrifuged and washed multiple times, and the citric acid-modified magnetic Fe3O4 nanozyme was collected by magnetic separation.

[0035] 0.13 mmol / L citric acid-modified magnetic Fe3O4 nanozyme was added to 50 mL of sodium citrate (2.2 mM) solution and stirred for 2 hours to obtain a dispersion. 0.6 mL of chloroauric acid HAuCl4 (25 mM) was added to the dispersion, and the mixture was stirred and intermittently sonicated. The mixture was heated to the boiling point until the solution changed from black to brownish-yellow. The solution was then processed using the magnetic separation method described above, and centrifuged and washed to obtain a brownish-yellow substance. The brownish-yellow substance was redispersed in deionized water to obtain the gold magnetic cascade nanozyme.

[0036] TEM image of the gold magnetic cascade nanozyme prepared in this invention is shown below. Figure 3As shown, the white arrows represent Fe3O4 nanoparticles, and the red arrows represent Au nanoparticles, proving that the gold magnetic cascade nanozyme was successfully prepared.

[0037] Example 2

[0038] A method for enhancing the catalytic activity of gold magnetic cascade nanozymes using X-rays includes the following steps:

[0039] 3 mL of gold magnetic nanozyme was dispersed in PBS buffer to obtain a gold magnetic nanozyme dispersion, which was then placed in a cuvette and irradiated with X-rays (80 kV, 1 mA).

[0040] The absorbance of the solution at 450 nm was recorded in real time using ultraviolet-visible absorption spectroscopy to determine the catalytic activity of the gold magnetic nanozyme. Specifically, gold nanoparticles catalyze the reaction of glucose, oxygen, and water to produce gluconic acid and hydrogen peroxide. Magnetic Fe3O4 nanoparticles further catalyze the generation of reactive oxygen species from hydrogen peroxide and catalyze the color reaction of the substrate o-phenylenediamine. The catalytic activity of the gold magnetic nanozyme can be characterized by real-time recording of the absorbance of the dispersion (at a wavelength of 450 nm).

[0041] Experimental results showed that dispersing 3 mL of gold magnetic cascade nanozyme in PBS buffer to obtain a gold magnetic nanozyme dispersion, and placing it in a cuvette, followed by X-ray irradiation (80 kV, 1 mA), caused the gold nanoparticles in the gold magnetic nanozyme to absorb X-ray energy, exciting photoelectrons and promoting the catalytic conversion of glucose, oxygen, and water into gluconic acid and hydrogen peroxide. This accelerated the color reaction of the substrate o-phenylenediamine, specifically a significant increase in absorbance at 450 nm. The absorbance of the dispersion at 450 nm was recorded in real time using UV-Vis absorption spectroscopy, thus verifying that X-ray irradiation can enhance the catalytic activity of the gold magnetic nanozyme cascade (e.g., ...). Figure 4 ).

[0042] Example 3

[0043] A method for enhancing the catalytic activity of gold magnetic cascade nanozymes using a magnetic field includes the following steps:

[0044] 3 mL of gold magnetic cascade nanozyme was dispersed in PBS buffer to obtain a gold magnetic nanozyme dispersion, which was then placed in a cuvette and stimulated by applying a magnetic field (435 kHz, 250 Oe).

[0045] The absorbance of the solution at 450 nm was recorded in real time using ultraviolet-visible absorption spectroscopy to determine the catalytic activity of the gold magnetic nanozyme. Specifically, gold nanoparticles catalyze the reaction of glucose, oxygen, and water to produce gluconic acid and hydrogen peroxide. Magnetic Fe3O4 nanoparticles further catalyze the generation of reactive oxygen species from hydrogen peroxide and catalyze the color reaction of the substrate o-phenylenediamine. The catalytic activity of the gold magnetic nanozyme can be characterized by real-time recording of the absorbance of the dispersion (at a wavelength of 450 nm).

[0046] 3 mL of gold magnetic cascade nanozyme was dispersed in PBS buffer to obtain a gold magnetic nanozyme dispersion. The dispersion was placed in a cuvette, and a magnetic field (435 kHz, 250 Oe) was applied to stimulate the Fe3O4 nanoparticles in the gold magnetic nanozyme, causing a magnetothermal heating effect. This thermal effect accelerated the catalytic conversion of hydrogen peroxide to reactive oxygen species (ROS) by the Fe3O4 nanoparticles, thereby accelerating the color reaction of the substrate o-phenylenediamine, specifically a significant increase in absorbance at 450 nm. The absorbance of the dispersion at 450 nm was recorded in real-time using UV-Vis absorption spectroscopy to verify that the alternating magnetic field can enhance the catalytic activity of the gold magnetic nanozyme cascade (e.g., ...). Figure 5 ).

[0047] Ultimately, experiments confirmed that the magnetocaloric effect or photoelectronic effect can efficiently enhance the catalytic activity of gold magnetic nanozyme cascade reactions.

[0048] The closest existing technology to the present invention is the following enhanced gold magnetic nanozyme technology: (1) In a magnetic nanoparticle surface modified with natural glucose oxidase material, the magnetic nanoparticles are heated by an external magnetic field, which can enhance the enzyme catalytic activity, but the efficiency is low, and the magnetothermal heating effect can easily cause protein denaturation and loss of catalytic activity of natural glucose oxidase. (2) Although gold magnetic nanoparticles have been studied extensively, these gold magnetic nanoparticles have not been reported to have peroxidase-glucose oxidase cascade catalytic activity, and there is no method to enhance the catalytic activity of such cascade nanozymes. Since the gold magnetic nanozyme prepared in this invention is an inorganic material composite system, it has higher stability than natural enzyme proteins and has magnetothermal and photoelectronic effects under the action of external magnetic fields and X-rays. Therefore, we propose for the first time to use magnetic fields or X-rays to achieve a simple and efficient enhancement of the catalytic activity of gold magnetic cascade nanozymes.

[0049] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0050] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0051] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing a gold magnetic cascade nanozyme, characterized in that, Including the following methods: Citric acid was added to an aqueous dispersion of Fe3O4 and mixed well to obtain a citric acid-modified magnetic Fe3O4 nanozyme. The molar ratio of citric acid to Fe3O4 in the aqueous dispersion of Fe3O4 was 1~5:

1. The citric acid-modified magnetic Fe3O4 nanozyme was added to a solution containing a reducing agent at a molar concentration ratio of 0.1~0.2:1.5~3. The mixture was stirred to obtain a dispersion. A gold-containing compound was added, and the reaction was continued until the solution changed from black to brownish-yellow. The solution was then centrifuged, washed, and magnetically separated. The brownish-yellow substance was redispersed in water to obtain the gold magnetic cascade nanozyme. The reducing agent is any one of sodium citrate, tannic acid, phenylboronic acid, and sodium borohydride.

2. The preparation method according to claim 1, characterized in that, The molar ratio of the citric acid-modified magnetic Fe3O4 nanozyme to the sodium citrate solution is 0.13:2.

2.

3. The preparation method according to claim 1, characterized in that, The gold-containing compound is chloroauric acid.

4. The preparation method according to claim 1, characterized in that, The aqueous dispersion of Fe3O4 is obtained by dispersing Fe3O4 nanoparticles in water.

5. The gold magnetic cascade nanozyme prepared by the preparation method according to any one of claims 1 to 4.

6. A method for enhancing the catalytic activity of gold magnetic cascade nanozymes, characterized in that, X-ray irradiation and magnetic field stimulation are applied to the gold magnetic cascade nanozyme according to claim 5 to enhance its catalytic activity.

7. The method according to claim 6, characterized in that, The X-ray tube voltage is 50 kV to 80 kV, and the tube current is 0.1 mA to 1 mA.

8. The method according to claim 6, characterized in that, The conditions for magnetic field stimulation are a magnetic field frequency of 280kHz to 500kHz and an intensity of 180 Oe to 300 Oe.

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