A transition metal-based single-atom nanoscale enzyme with multiple enzyme activities and a preparation method and application thereof

Abstract

The present application relates to a kind of transition metal-based single atom nanoscale enzyme with multiple enzyme activities and its preparation method and application.The transition metal-based single atom nanoscale enzyme is metal-nitrogen carbon structure, and the formula is M-NC;Wherein, M is transition metal element, preferably selected from Zn, Fe, Cu, Mn, Ni or Co;And M is oxidation state, is coordinated with N atom to form M-N bond, and is distributed in nitrogen carbon skeleton in monodisperse form.

Description

Technical Field

[0001] This invention relates to a transition metal-based single-atom nanozyme with multi-enzyme activity, its preparation method, and its application, belonging to the field of nanomaterials technology. Background Technology

[0002] Enzymes, as highly efficient biocatalysts, can undergo specific catalytic reactions under mild conditions, exhibiting high catalytic activity and substrate specificity, and have been widely used in industry, medicine, and biology. As biocatalysts widely present in living organisms, natural enzymes possess good biocompatibility, rarely causing immune rejection reactions when applied in the biomedical field, making them potential diagnostic and therapeutic agents. Although natural enzymes show promising application prospects, their high preparation and purification costs, operational instability, susceptibility to environmental influences on catalytic activity, and difficulties in storage and recycling significantly limit their application in the biomedical field.

[0003] In 2007, scientists discovered that Fe3O4 nanoparticles possess intrinsic catalytic activity similar to horseradish peroxidase (HRP). In the presence of hydrogen peroxide, they can catalyze the oxidation of various HRP substrates, such as TMB, DAB, and OPD, producing the same color reaction as HRP catalysis. Scientists subsequently named these enzyme-like nanomaterials nanozymes. Nanozymes are nanomaterials capable of catalyzing natural enzyme substrates under physiological conditions and following reaction kinetics similar to those of natural enzymes. This catalytic activity originates from the unique nanostructure of the nanozyme itself, without the need for additional catalytic functional groups or natural enzymes. As a novel type of enzyme-like nanomaterial, nanozymes exhibit higher stability under harsh conditions, are easier to tune catalytic activity, and can be mass-produced compared to natural enzymes. Compared to ordinary nanomaterials, they possess superior catalytic activity and substrate specificity, showing great potential for application in the biomedical field. Even so, due to the lack of finely structured catalytic reactions, the catalytic activity of nanozymes under physiological conditions still lags behind that of natural enzymes, and further design of enzyme catalysts with high catalytic activity is needed.

[0004] In the field of catalysis, single-atom catalysts offer a significant potential pathway to significantly improve catalytic activity and selectivity due to their near 100% atomic dispersion and maximum metal utilization. Among these, single-atom nanozymes, using metal atoms as enzyme-like catalytic active sites, not only possess designable geometric structures and electronic coordination, effectively mimicking the metal active centers of natural enzymes at the atomic level, but also exhibit excellent structural stability, enabling highly efficient and sustained substrate catalysis. Applying single-atom catalysts to the biomedical field may bridge the gap between the activities of nanozymes and natural enzymes. In existing single-atom systems, the most typical approach is to chelate metals with carbon materials through high-content nitrogen doping, i.e., metal-nitrogen-carbon materials. However, current preparation processes for metal-nitrogen-carbon single-atom nanozymes often involve using organic ligands (2-methylimidazole, dicyandiamide, melamine, etc.) to chelate metal ions as organic precursors, followed by further pyrolysis and acid washing. This process is cumbersome and results in poor material dispersion, hindering its medical applications. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, the present invention aims to provide a transition metal-based single-atom nanozyme, its preparation method, and its applications. The single-atom nanozyme of this invention is prepared using a one-step solvothermal method, eliminating the need for pyrolysis and acid washing, making it convenient and rapid. Furthermore, this method is applicable to various metals, demonstrating its versatility.

[0006] In a first aspect, the present invention provides a transition metal-based single-atom nanozyme with multi-enzyme activity, wherein the transition metal-based single-atom nanozyme has a metal-nitrogen-carbon structure with the simplified formula M-NC; wherein M is a transition metal element, preferably selected from Zn, Fe, Cu, Mn, Ni, or Co; and M is in an oxidized state, coordinates with N atoms to form MN bonds, and is distributed in a monodisperse form in the nitrogen-carbon framework.

[0007] Preferably, N atoms exist mainly in the form of pyridine nitrogen and pyrrole nitrogen; C atoms exist mainly in the form of C=N, CC, and CN.

[0008] Preferably, the atomic content of each element in the transition metal-based single-atom nanozyme includes: M: 0.01%–5%; C: 60%–70%; N: 15%–25%; O: 10%–20%, with a total content of 100%.

[0009] Preferably, the morphology of the M-NC changes depending on the type of metal atom; wherein Zn-NC is strip-shaped, Fe-NC is plate-shaped, Cu-NC and Ni-NC are needle-shaped, Mn-NC is nanoflower-shaped, and Co-NC is dendritic.

[0010] On the other hand, the present invention provides a method for preparing a transition metal-based single-atom nanozyme with multi-enzyme activity, comprising:

[0011] (1) Add the transition metal salt to formamide or a mixture of formamide and other organic solvents, and dissolve by sonication to obtain solution A;

[0012] (2) After the obtained solution A is subjected to a solvothermal reaction at 170-240℃, it is cooled, washed and dried to obtain the transition metal-based single-atom nanozyme with multi-enzyme activity.

[0013] Preferably, the other organic solvents include commonly used organic solvents such as ethanol, ethylene glycol, glycerol, ethanolamine, N,N-dimethylformamide, and N,N-dimethylacetamide, wherein the volume ratio of formamide to organic solvent is not 0; the ratio of formamide or a mixture of formamide and other organic solvents to transition metal salt is (30-50) mL:(1-200) mM; the ultrasonic dissolution power is 40-320 W, and the time is 5-60 minutes.

[0014] Preferably, the transition metal salt is selected from at least one of zinc salt, iron salt, copper salt, manganese salt, nickel salt, and cobalt salt; the transition metal salt is at least one of the chloride salt, sulfate salt, nitrate salt, and acetate salt of Zn, Fe, Cu, Mn, Ni, and Co.

[0015] Preferably, the solvothermal reaction time is 10 to 48 hours.

[0016] Preferably, the cooling method is air cooling, water cooling, or furnace cooling; the washing method is centrifugation, with a centrifugation speed of 3000-12000 rpm, a centrifugation time of 5-30 min, and at least 4 washing cycles; the drying method is drying at 50-100℃ or freeze-drying at -50--5℃.

[0017] Thirdly, the present invention provides an application of a transition metal-based single-atom nanozyme, wherein the transition metal-based single-atom nanozyme M-NC simultaneously possesses multiple types of enzyme activities, including: oxidase-like enzyme (OXD), peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) activities, and the activities of each type of enzyme increase with the increase of transition metal loading.

[0018] Preferably, the enzyme-like activity of the transition metal-based single-atom nanozyme is pH and temperature dependent, exhibiting natural antioxidant enzyme activities such as CAT and SOD under physiological conditions, and achieving the effects of enzyme-like catalytic reactions on H2O2 and ·O2. - The removal.

[0019] Preferably, the enzyme-like activity of the transition metal-based single-atom nanozyme exhibits excellent stability; compared with the activity of natural enzymes, the enzyme-like activity of M-NC is stable under low temperature (-50~10℃), high temperature (50~300℃), acidic (pH=2~6.5), alkaline (pH=8~12) and long-term (more than one year) storage conditions; the transition metal-based single-atom nanozyme replaces natural antioxidant enzymes for antioxidant therapy.

[0020] Preferably, the transition metal-based single-atom nanozyme exhibits antioxidant effects in a cellular antioxidant model, and the higher the metal loading, the better the antioxidant effect, for use in cell protection.

[0021] Preferably, the cells include macrophages, fibroblasts, and endothelial cells.

[0022] The beneficial effects of this invention are as follows:

[0023] This invention utilizes a one-step solvothermal method to synthesize transition metal-based single-atom nanozymes (M-NC). The one-step solvothermal method described in this invention uses inexpensive and non-toxic formamide as a raw material, offering simplicity, low cost, and mild conditions. It eliminates the need for the pyrolysis and acid washing processes required in traditional single-atom nanozyme preparations, overcoming the drawbacks of complex processes and poor dispersibility in traditional single-atom nanozyme preparations, thus promoting the further application of single-atom nanozymes in the biomedical field. The preparation principle is further explained as follows: Formamide contains both amino and aldehyde groups. Under specific conditions, the nitrogen atom attacks the carbon atom, undergoing an intermolecular nucleophilic addition reaction—the Schiff base reaction—forming an unsaturated C=N bond. Simultaneously, the lone pair electrons of the nitrogen atom chelate with the metal atom (M) to form an MN coordinate bond. This one-step solvothermal method eliminates the need for deoxygenation treatments such as calcination, thereby preparing a metal nitrogen-carbon (M-NC) single-atom nanozyme while retaining a large number of oxygen-containing functional groups. The abundant oxygen-containing functional groups ensure the stability of this single-atom nanozyme under physiological conditions and can also act as electron donors to directly scavenge reactive oxygen species.

[0024] The transition metal-based single-atom nanozyme M-NC synthesized in this invention simultaneously possesses multiple enzyme activities, including OXD, POD, CAT, and SOD activities, and the activities of each enzyme increase with increasing metal loading. The prepared M-NC enzyme activity is pH and temperature dependent, exhibiting natural antioxidant enzyme activities such as CAT and SOD under physiological conditions, and can achieve the effects of enzyme-like catalytic reactions on H2O2 and ·O2. - It can clear [the virus]. At the same time, its enzyme-like activity exhibits excellent stability. Compared to natural enzyme activity, M-NC's enzyme-like activity remains stable under low temperature, high temperature, acidic, alkaline, and long-term storage conditions, making it a suitable alternative to natural antioxidant enzymes for antioxidant therapy.

[0025] The transition metal-based single-atom nanozyme M-NC synthesized in this invention exhibits excellent antioxidant effects and can be used for cell protection; the higher the metal loading, the better the cell protection effect. Therefore, the antioxidant effect of the material can be controlled by simply adjusting the metal loading, allowing it to be applied to different cell protection scenarios, such as sun protection, anti-photoaging, and inflammation treatment. Attached Figure Description

[0026] Figure 1 TEM images of the single-atom nanozymes Zn-NC, Fe-NC, Cu-NC, Co-NC, Mn-NC, Ni-NC, and NC obtained in Examples 1-6 and Comparative Examples (scale bar is 1 μm).

[0027] Figure 2 The XRD patterns of the single-atom nanozymes Zn-NC, Fe-NC, Cu-NC, Co-NC, Mn-NC, Ni-NC, and NC obtained in Examples 1-6 and the comparative examples are shown (the horizontal axis is 2θ (degrees), and the vertical axis is intensity (au)).

[0028] Figure 3 The simulated OXD and POD activities of the single-atom nanozymes Zn-NC, Fe-NC, Cu-NC, Co-NC, Mn-NC, Ni-NC, and NC obtained in Examples 1-6 and Comparative Examples are shown (the horizontal axis is wavelength (nm), and the vertical axis is absorbance (au)).

[0029] Figure 4 TEM images of the single-atom nanozymes Fe-NC-1, Fe-NC-2, Fe-NC-3, and NC obtained in Examples 7-9 and Comparative Examples;

[0030] Figure 5 The simulated activities of the single-atom nanozymes Fe-NC-1, Fe-NC-2, Fe-NC-3, and NC obtained in Examples 7-9 and the comparative examples are: OXD (wavelength (nm) on the x-axis, absorbance (au) on the y-axis), POD (wavelength (nm) on the x-axis, absorbance (au) on the y-axis), CAT (time (s) on the x-axis, dissolved oxygen (mg / L) on the y-axis), and SOD (wavelength (nm) on the x-axis, absorbance (au) on the y-axis).

[0031] Figure 6The simulated activities of the single-atom nanozyme Fe-NC-3 obtained in Example 9 at different pH values ​​are: OXD (wavelength (nm) on the x-axis, absorbance (au) on the y-axis), POD (wavelength (nm) on the x-axis, absorbance (au) on the y-axis), CAT (time (s) on the x-axis, dissolved oxygen (mg / L) on the y-axis), and SOD (wavelength (nm) on the x-axis, absorbance (au) on the y-axis). Among them, the POD activities at pH=3 and pH=4 are similar and the lines overlap, and the activities at pH=7 and pH=8 are similar and the lines overlap.

[0032] Figure 7 The cell viability of the single-atom nanozymes Fe-NC-1, Fe-NC-2, Fe-NC-3, and NC obtained in Examples 7-9 and Comparative Example 1 when co-cultured with endothelial cells HUVECs under normal and H2O2 conditions (x-axis: concentration (μg / mL), y-axis: cell viability (%)).

[0033] Figure 8 The XRD pattern of Cu-MIM obtained in Comparative Example 2 is shown (the horizontal axis is 2θ (degrees), and the vertical axis is intensity (au)). Detailed Implementation

[0034] The present invention will be further illustrated by the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0035] In this invention, the transition metal-based single-atom nanozyme possesses a variety of natural enzyme activities and exhibits antioxidant enzyme activity under physiological conditions, making it suitable for cell protection.

[0036] The transition metal-based single-atom nanozyme has a metal-nitrogen-carbon (M-NC) structure. The transition metal M includes, but is not limited to, transition metals such as Zn, Fe, Cu, Mn, Ni, and Co. M is in its oxidized state and coordinates with N atoms to form MN bonds, which are distributed in a monodisperse manner within the nitrogen-carbon framework. The N atoms mainly exist in the form of pyridine nitrogen and pyrrole nitrogen, and the C atoms mainly exist in the form of C=N, CC, and CN.

[0037] In an optional embodiment, the elemental percentage of metal M in the transition metal-based single-atom nanozyme is 0.01%–5%, C is 60%–70%, N is 15%–25%, and O is 10%–20%. The morphology of the M-NC varies depending on the type of metal atom; for example, Zn-NC is banded, Fe-NC is plate-like, Cu-NC and Ni-NC are needle-like, Mn-NC is nanoflower-like, and Co-NC is dendritic.

[0038] The following exemplifies a method for preparing transition metal-based single-atom nanozymes.

[0039] Add 1–200 mM of pyrotoxic metal salt to 30–50 mL of formamide and sonicate at 40–320 W for 5–60 min to obtain solution A.

[0040] In optional embodiments, the transition metal salt includes, but is not limited to, transition metal salts such as zinc salts, iron salts, copper salts, manganese salts, nickel salts, and cobalt salts. The metal salt includes, but is not limited to, one or a mixture of two of the chloride, sulfate, nitrate, and acetate salts of the same metal element.

[0041] Solution A was transferred to a stainless steel hydrothermal reactor, placed in an oven, and the solvothermal reaction was initiated by setting the temperature. The reaction temperature and time for the solvothermal reaction were 170–240°C and 10–24 h, respectively. For the M-NC, the higher the content of metal M and the lower the temperature and shorter the time within the preparation conditions, the smaller the size.

[0042] After the reaction is complete, the mixture is cooled to room temperature, the reaction vessel is opened, and the reaction liquid is transferred to a centrifuge tube. The resulting reactants are washed with a mixture of ultrapure water and ethanol and dried to obtain the transition metal-based single-atom nanozyme M-NC. The cooling methods include, but are not limited to, air cooling, water cooling, and furnace cooling; the washing method is centrifugation, with a centrifugation speed of 3000–12000 rpm and a centrifugation time of 5–30 min, and at least 4 washes; the drying methods include, but are not limited to, drying at 50–100℃ or freeze-drying.

[0043] In this invention, the transition metal-based single-atom nanozyme M-NC simultaneously possesses multiple types of enzyme activities, including oxidase-like (OXD), peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) activities, and the activities of each type of enzyme increase with the increase of metal loading.

[0044] Among them, the enzyme-like activity of transition metal-based single-atom nanozymes is pH and temperature dependent, exhibiting the activity of natural antioxidant enzymes such as CAT and SOD under physiological conditions. They can achieve the catalytic reaction of H2O2 and ·O2 through enzyme-like catalytic reactions. - The removal.

[0045] Among them, transition metal-based single-atom nanozymes exhibit excellent stability in their enzyme-like activity. Compared with the activity of natural enzymes, the enzyme-like activity of M-NC remains stable under low temperature, high temperature, acidic, alkaline, and long-term storage conditions, and can replace natural antioxidant enzymes for antioxidant therapy. Furthermore, transition metal-based single-atom nanozymes have shown antioxidant effects in cellular antioxidant models, with higher metal loading resulting in better antioxidant effects.

[0046] Currently, there are no reports of directly synthesizing metal nitrogen-carbon single-atom nanoenzyme materials with multi-enzyme activity using a one-step solvothermal method. Furthermore, the method in this invention is applicable to a variety of metals and has universality. The materials prepared all exhibit excellent antioxidant activity.

[0047] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0048] Example 1

[0049] Dissolve 20.43 mg of zinc chloride hexahydrate in 30 mL of formamide and sonicate for 30 min until completely dissolved to prepare Zn 2+ A 5 mM solution was prepared and then transferred to a stainless steel hydrothermal reactor. The reactor was then dried at 180°C for 12 h in a forced-air drying oven. After the reaction was complete, the mixture was air-cooled to room temperature. The reactor was then opened, and the reaction liquid was transferred to centrifuge tubes. The centrifuge was set to 12000 rpm and centrifuged for 5 min. The precipitate obtained by centrifugation was washed three times with a mixture of ultrapure water and ethanol, and then dried in a 60°C oven for 6 h. The prepared single-atom nanozyme is designated Zn-NC. The atomic contents of the obtained transition metal-based single-atom nanozyme are as follows: Zn: 0.12%; C: 62.08%; N: 24.61%; O: 13.19%, with a total content of 100%.

[0050] Example 2

[0051] 24.33 mg of anhydrous ferric chloride was dissolved in 30 mL of formamide and sonicated for 30 min until completely dissolved, to prepare Fe 3+A 5 mM solution was prepared and then transferred to a stainless steel hydrothermal reactor. The reactor was then dried at 180°C for 12 hours in a forced-air drying oven. After the reaction was complete, the mixture was air-cooled to room temperature. The reactor was then opened, and the reaction liquid was transferred to centrifuge tubes. The centrifuge was set to 12000 rpm and centrifuged for 5 minutes. The precipitate obtained by centrifugation was washed three times with a mixture of ultrapure water and ethanol, and then dried in a 60°C oven for 6 hours. The prepared single-atom nanozyme is designated Fe-NC. The atomic contents of the obtained transition metal-based single-atom nanozyme are as follows: Fe: 0.22%; C: 62.86%; N: 23.50%; O: 13.42%, with a total content of 100%.

[0052] Example 3

[0053] Dissolve 25.57 mg of copper chloride dihydrate in 30 mL of formamide, and sonicate for 30 min until completely dissolved to prepare Cu 2+ A 5 mM solution was prepared and then transferred to a stainless steel hydrothermal reactor. The reactor was then dried at 180°C for 12 hours in a forced-air drying oven. After the reaction was complete, the mixture was air-cooled to room temperature. The reactor was then opened, and the reaction liquid was transferred to centrifuge tubes. The centrifuge was set to 12000 rpm and centrifuged for 5 minutes. The precipitate obtained by centrifugation was washed three times with a mixture of ultrapure water and ethanol and dried in a 60°C oven for 6 hours. The prepared single-atom nanozyme is designated Cu-NC. The atomic contents of the obtained transition metal-based single-atom nanozyme are as follows: Cu: 0.27%; C: 63.55%; N: 22.91%; O: 13.27%, with a total content of 100%.

[0054] Example 4

[0055] Dissolve 35.69 mg of cobalt chloride hexahydrate in 30 mL of formamide and sonicate for 30 min until completely dissolved to prepare Co 2+ A 5 mM solution was prepared and then transferred to a stainless steel hydrothermal reactor. The reactor was then dried at 180°C for 12 h in a forced-air drying oven. After the reaction was complete, the mixture was air-cooled to room temperature. The reactor was then opened, and the reaction liquid was transferred to centrifuge tubes. The centrifuge was set to 12000 rpm and centrifuged for 5 min. The precipitate obtained by centrifugation was washed three times with a mixture of ultrapure water and ethanol and dried in a 60°C oven for 6 h. The prepared single-atom nanozyme is designated Co-NC. The atomic contents of the obtained transition metal-based single-atom nanozyme are as follows: Co: 0.18%; C: 62.73%; N: 24.12%; O: 12.97%, with a total content of 100%.

[0056] Example 5

[0057] Dissolve 27.685 mg of manganese chloride tetrahydrate in 30 mL of formamide, and sonicate for 30 min until completely dissolved to prepare Mn 2+A 5 mM solution was prepared and then transferred to a stainless steel hydrothermal reactor. The reactor was then dried at 180°C for 12 hours in a forced-air drying oven. After the reaction was complete, the mixture was air-cooled to room temperature. The reactor was then opened, and the reaction liquid was transferred to centrifuge tubes. The centrifuge was set to 12000 rpm and centrifuged for 5 minutes. The precipitate obtained by centrifugation was washed three times with a mixture of ultrapure water and ethanol and dried in a 60°C oven for 6 hours. The prepared single-atom nanozyme is designated Mn-NC. The atomic contents of the obtained transition metal-based single-atom nanozyme are as follows: Mn: 0.25%; C: 63.17%; N: 25.81%; O: 10.77%, with a total content of 100%.

[0058] Example 6

[0059] 43.62 mg of nickel nitrate hexahydrate was dissolved in 30 mL of formamide and sonicated for 30 min until completely dissolved, to prepare Ni 2+ A 5 mM solution was prepared and then transferred to a stainless steel hydrothermal reactor. The reactor was then dried at 180°C for 12 h in a forced-air drying oven. After the reaction was complete, the mixture was air-cooled to room temperature. The reactor was then opened, and the reaction liquid was transferred to centrifuge tubes. The centrifuge was set to 12000 rpm and centrifuged for 5 min. The precipitate obtained by centrifugation was washed three times with a mixture of ultrapure water and ethanol, and then dried in a 60°C oven for 6 h. The prepared single-atom nanozyme is designated Ni-NC. The atomic contents of the obtained transition metal-based single-atom nanozyme are as follows: Ni: 0.17%; C: 61.29%; N: 24.34%; O: 14.2%, with a total content of 100%.

[0060] Example 7

[0061] 4.866 mg of anhydrous ferric chloride was dissolved in 30 mL of formamide and sonicated for 30 min until completely dissolved, to prepare Fe 3+ A 1 mM solution was prepared. This solution was then transferred to a stainless steel hydrothermal reactor and reacted at 220°C for 12 h in a forced-air drying oven. After the reaction was complete, the reactor was air-cooled to room temperature. The reactor was then opened, and the reaction liquid was transferred to centrifuge tubes. The centrifuge was set to 12000 rpm and centrifuged for 5 min. The precipitate obtained by centrifugation was washed three times with a mixture of ultrapure water and ethanol and dried in a 60°C oven for 6 h. The prepared single-atom nanozyme was designated Fe-NC-1. The atomic contents of the obtained transition metal-based single-atom nanozyme were as follows: Fe: 0.05%; C: 67.13%; N: 17.80%; O: 15%, with a total content of 100%.

[0062] Example 8

[0063] 24.33 mg of anhydrous ferric chloride was dissolved in 30 mL of formamide and sonicated for 30 min until completely dissolved, to prepare Fe 3+A 5 mM solution was prepared and then transferred to a stainless steel hydrothermal reactor. The reactor was then dried at 220°C for 12 hours in a forced-air drying oven. After the reaction was complete, the mixture was air-cooled to room temperature. The reactor was then opened, and the reaction liquid was transferred to centrifuge tubes. The centrifuge was set to 12000 rpm and centrifuged for 5 minutes. The precipitate obtained by centrifugation was washed three times with a mixture of ultrapure water and ethanol, and then dried in a 60°C oven for 6 hours. The prepared single-atom nanozyme was designated Fe-NC-2. The atomic contents of the obtained transition metal-based single-atom nanozyme were as follows: Fe: 0.14%; C: 69.82%; N: 16.40%; O: 13.64%, with a total content of 100%.

[0064] Example 9

[0065] 48.66 mg of anhydrous ferric chloride was dissolved in 30 mL of formamide and sonicated for 30 min until completely dissolved, to prepare Fe 3+ A 10 mM solution was prepared and then transferred to a stainless steel hydrothermal reactor. The reactor was then dried at 220°C for 12 hours in a forced-air drying oven. After the reaction was complete, the mixture was air-cooled to room temperature. The reactor was then opened, and the reaction liquid was transferred to centrifuge tubes. The centrifuge was set to 12000 rpm and centrifuged for 5 minutes. The precipitate obtained by centrifugation was washed three times with a mixture of ultrapure water and ethanol and dried in a 60°C oven for 6 hours. The prepared single-atom nanozyme was designated Fe-NC-3. The atomic contents of the obtained transition metal-based single-atom nanozyme were as follows: Fe: 0.29%; C: 68.24%; N: 15.24%; O: 16.23%, with a total content of 100%.

[0066] Comparative Example 1

[0067] 30 mL of formamide was directly transferred to a stainless steel hydrothermal reactor and reacted at 180 °C for 12 h in a forced-air drying oven. After the reaction was complete, the reactor was air-cooled to room temperature, the reactor was opened, and the reaction liquid was transferred to a centrifuge tube. The centrifuge was set to 12000 rpm and centrifuged for 5 min. The precipitate obtained by centrifugation was washed three times with a mixture of ultrapure water and ethanol and dried in a 60 °C oven for 6 h. The prepared material is denoted as NC.

[0068] Comparative Example 2

[0069] 25.57 mg of copper chloride dihydrate and 2-methylimidazole (2-MIM) were dissolved in 30 mL of ultrapure water and sonicated for 30 min until completely dissolved to prepare a Cu 2+Solutions of Cu-MIM and 2-MIM were prepared at 5 mM and 50 mM, respectively. These solutions were then transferred to a stainless steel hydrothermal reactor and reacted at 180°C for 12 h in a forced-air drying oven. After the reaction was complete, the reactor was air-cooled to room temperature, the reactor was opened, and the reaction liquid was transferred to centrifuge tubes. The centrifuge was set to 12000 rpm and centrifuged for 5 min. The precipitate obtained after centrifugation was washed three times with a mixture of ultrapure water and ethanol and dried in a 60°C oven for 6 h. The prepared single-atom nanozyme is designated Cu-MIM.

[0070] TEM images were taken of the single-atom nanozymes Zn-NC, Fe-NC, Cu-NC, Co-NC, Mn-NC, Ni-NC, and NC obtained in Examples 1-6 and Comparative Example 1, as shown. Figure 1 As shown, the morphology of M-NC changes with the type of metal atom. NC and Zn-NC are banded, Fe-NC is plate-like, Cu-NC and Ni-NC are needle-like, Mn-NC is nanoflower-like, and Co-NC is dendritic. This is related to the different polymerization modes of formamide self-polymerization reaction after different metal ions are coordinated with high N content. Figure 2 The XRD results of M-NC showed that all M-NCs had diffraction peaks only around 27°, consistent with NC, indicating that the incorporation of transition metal atoms did not affect the phase composition of formamide self-polymers, suggesting that the transition metal atoms were dispersed in the nitrogen-carbon framework in a monodisperse form.

[0071] Using 2-methylimidazolium, an organic ligand commonly used in the preparation of single-atom nanozymes, as Comparative Example 2, we verified the unique role of formamide ligand in the preparation of single-atom nanozymes. Cu... 2+ Cu-MIM was prepared by one-step solvothermal reaction by mixing with 2-methylimidazole. Its optical images and XRD diffraction patterns are shown below. Figure 8 As shown in the figure, the XRD diffraction pattern reveals that Cu-MIM prepared by the one-step solvothermal method is CuO, and Cu-NC (XRD characteristic peak at 27°) cannot be prepared. This is attributed to the uniqueness of the functional groups in the formamide ligand. Formamide contains both amino and aldehyde groups. Under specific conditions, the nitrogen atom attacks the carbon atom, undergoing an intermolecular nucleophilic addition reaction—the Schiff base reaction—to form an unsaturated C=N bond. Simultaneously, the lone pair electrons of the nitrogen atom chelate with the metal atom (M) to form an MN coordinate bond. This preparation method is a one-step solvothermal method, eliminating the need for deoxygenation treatments such as calcination, thus preparing a metal nitrogen-carbon (M-NC) single-atom nanozyme while retaining a large number of oxygen-containing functional groups. The abundant oxygen-containing functional groups ensure the stability of this single-atom nanozyme under physiological conditions and can also act as electron donors to directly scavenge reactive oxygen species.

[0072] Application Example 1: Enzyme-like Activity of Transition Metal-Based Single-Atom Nanozymes

[0073] The OXD and POD-like activities of M-NC were evaluated using the 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric method.

[0074] Evaluation of OXD-like activity: 100 μL of 1 mg / mL M-NC dispersion and 100 μL of 20 mM TMB-HCl aqueous solution were added to 800 μL of acetate-sodium acetate buffer solution (HAc-NaAc) at different pH values ​​(pH = 3, 4, 5, 6, 7, 8). After thorough mixing, the mixture was reacted at 37 °C for 30 min. 100 μL of the solution was then used to measure the full spectrum absorbance at 500-800 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0075] POD-like activity evaluation: 100 μL of 1 mg / mL M-NC dispersion, 100 μL of 20 mM TMB-DMSO solution, 100 μL of 1 mM H2O2 solution, and 700 μL of acetate-sodium acetate buffer solution (HAc-NaAc) with different pH values ​​(pH = 3, 4, 5, 6, 7, 8) were added to the solution. After thorough mixing, the mixture was reacted at 37℃ for 30 min. 100 μL of the solution was then used to measure the full spectrum absorbance at 500-800 nm using a microplate reader.

[0076] The ability of M-NC to catalyze the production of O2 from H2O2 was determined using a dissolved oxygen analyzer to evaluate its CAT-like activity.

[0077] CAT-like activity evaluation: 200 μL of M-NC dispersions at concentrations of 0 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, and 800 μg / mL were added to 1600 μL of PBS buffer solution (pH = 3, 4, 5, 6, 7, 8, 20 mM) at different pH values. Then, 200 μL of 100 mM H2O2 solution was added. After thorough mixing, the change in O2 content in the reaction system was immediately measured using a dissolved oxygen meter. The data was recorded every 5 seconds for a total of 300 seconds.

[0078] The SOD-like activity of M-NC was evaluated using a total SOD activity assay kit (WST-8 method).

[0079] SOD-like activity evaluation: The SOD-like activity of different concentrations of M-NC was evaluated strictly in accordance with the kit instructions. After incubating the reaction system at 37℃ for 30 min, the full spectrum absorption of 410-550 nm was measured using an ELISA reader. The lower the absorption peak, the higher the SOD-like activity.

[0080] like Figure 3As shown, Zn-NC, Fe-NC, Cu-NC, Co-NC, Mn-NC, Ni-NC, and NC exhibit absorption peaks at 652 nm to varying degrees in the presence or absence of H2O2, indicating that single-atom nanozymes of different metals possess different enzyme activities. For example, NC, Cu-NC, and Ni-NC only exhibit POD-like activity, while Co-NC, Mn-NC, and Cu-NC possess both OXD and POD-like activities, and Zn-NC lacks both OXD and POD-like activities. To further investigate the enzyme-like activity of this type of single-atom nanozyme, Fe-NC with different Fe atom loadings were prepared, namely the materials prepared in Examples 7-8, namely Fe-NC-1, Fe-NC-2, and Fe-NC-3, respectively. The main focus was on studying the influence of metal loading on enzyme-like activity.

[0081] like Figure 4 As shown, the morphology of the material gradually changes from strip-like to amorphous after different Fe atom loading, and is more inclined to a plate-like structure. The material size gradually decreases, indicating that the Fe atom loading significantly affects the formamide polymerization process, thereby controlling the material morphology and size.

[0082] Figure 5 The study showed the effect of Fe atom loading on the enzyme activity of the material. The results indicated that Fe-NC simultaneously possesses four natural enzyme-mimicking activities: OXD, POD, CAT, and SOD. Moreover, the higher the Fe atom loading, the stronger the activity of each enzyme. This suggests that the enzyme activity of this material mainly originates from the Fe-N coordinated active sites.

[0083] like Figure 6 As shown, the enzyme-like activity of Fe-NC is also pH-dependent. Under acidic conditions, it mainly exhibits OXD and POD mimicry activities, while under neutral or alkaline conditions, it mainly exhibits CAT and SOD mimicry activities, and can achieve H2O2 and ·O2 - The removal of these substances shows great application potential in the field of biomedical antioxidants.

[0084] Application Example 2: Cytotoxicity and Antioxidant Effects of Transition Metal-Based Single Atom Nanozymes. Cell viability after co-incubation of Fe-NC with cells was tested using alamar Blue™ (Thermo Fisher Scientific Inc., USA) in both the presence and absence of H2O2, to evaluate the cytotoxicity and antioxidant effects of Fe-NC. The steps are as follows:

[0085] Add 100 μL of cell suspension (1×10⁴ cells / mL) to a 96-well white plate and incubate for 24 h.

[0086] Remove the original culture medium, add 100 μL of a culture medium dispersion containing different concentrations of Fe-NC and H2O2 (final concentration 800 μM), and incubate for 24 h;

[0087] Remove the original culture medium, add 110 μL of culture medium containing 10% Alamar Blue™ staining solution, and incubate in the dark for 2 hours.

[0088] 100 μL was taken from each well and placed into a black 96-well plate. The fluorescence intensity of each well was measured using a microplate reader (excitation / emission wavelength: 560 / 590 nm). Three replicates were set up for each group of samples, and the mean ± standard deviation was taken as the cell viability result.

[0089] like Figure 7 As shown, the addition of Fe-NC had no significant effect on cell activity in the absence of H2O2, indicating that the material has excellent biocompatibility and can be used in the biomedical field. Under stimulation with 800 μM H2O2, cell activity decreased significantly by about 40%, indicating that the cells underwent oxidative stress. The addition of Fe-NC with different Fe atom loadings of 200 μg / mL significantly alleviated cellular oxidative stress and restored cell activity. Moreover, the higher the Fe atom loading, the stronger the cell activity, indicating that the Fe atom loading significantly improved the antioxidant activity of Fe-NC, thereby affecting the antioxidant behavior of cells and improving cell activity.

Claims

1. A transition metal-based single-atom nanozyme with multi-enzyme activity, characterized in that, The transition metal-based single-atom nanozyme has a metal-nitrogen-carbon structure with the abbreviated formula M-NC; wherein M is a transition metal element, selected from Fe, Cu, Mn, or Co, and M is in an oxidized state, coordinating with N atoms to form MN bonds, and is distributed in a monodisperse form in the nitrogen-carbon framework; N atoms exist in the form of pyridine nitrogen and pyrrole nitrogen; C atoms exist in the form of C=N, CC, or CN; the transition metal-based single-atom nanozyme simultaneously possesses multiple enzyme activities, including: oxidase-like enzyme (OXD), peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) activities; The preparation method of the transition metal-based single-atom nanozyme with multi-enzyme activity includes: (1) Add the transition metal salt to formamide or a mixture of formamide and other organic solvents, and dissolve by ultrasonication to obtain solution A; wherein: the ratio of the formamide or the mixture of formamide and other organic solvents to the transition metal salt is (30~50) mL:(1~200) mM; the ultrasonic dissolution power is 40~320W, and the time is 5~60 minutes; (2) The obtained solution A is subjected to a solvothermal reaction at 170~240°C for 10~48 hours, followed by cooling, washing and drying to obtain the transition metal-based single-atom nanozyme with multi-enzyme activity.

2. The transition metal-based single-atom nanozyme according to claim 1, characterized in that, The atomic content of each element in the transition metal-based single-atom nanozyme includes: M: 0.01%–5%; C: 60%–70%; N: 15%–25%; O: 10%–20%, with a total content of 100%.

3. The transition metal-based single-atom nanozyme according to claim 1, characterized in that, The morphology of the M-NC varies depending on the type of metal atom; Fe-NC is plate-like, Cu-NC is needle-like, Mn-NC is nanoflower-like, and Co-NC is dendritic.

4. The transition metal-based single-atom nanozyme according to claim 1, characterized in that, The other organic solvents include at least one of ethanol, ethylene glycol, glycerol, ethanolamine, N,N-dimethylformamide, and N,N-dimethylacetamide.

5. The transition metal-based single-atom nanozyme according to claim 1, characterized in that, The transition metal salt is selected from at least one of iron salts, copper salts, manganese salts, and cobalt salts; the transition metal salt is at least one of the chloride salts, sulfate salts, nitrate salts, and acetate salts of Fe, Cu, Mn, and Co.

6. The transition metal-based single-atom nanozyme according to claim 1, characterized in that, The cooling method is air cooling, water cooling, or furnace cooling; the washing method is centrifugation, with a centrifugation speed of 3000~12000 rpm, a centrifugation time of 5~30 min, and washing at least 4 times; the drying method is drying at 50~100°C or freeze-drying at -50~-5°C.

7. The application of the transition metal-based single-atom nanozyme of claim 1 in the field of enzymes for non-disease diagnosis and treatment, characterized in that, The activities of these various enzymes increase with increasing transition metal loading.

8. The application according to claim 7, characterized in that, The enzyme-like activity of the transition metal-based single-atom nanozyme is pH and temperature dependent. Under physiological conditions, it exhibits natural antioxidant enzyme activity of CAT and SOD, and achieves the effects of enzyme-like catalytic reactions on H2O2 and •O2. - The removal.

9. The application according to claim 7, characterized in that, The enzyme-like activity of the transition metal-based single-atom nanozyme is stable; compared with the activity of natural enzymes, the enzyme-like activity of M-NC is stable at low temperatures of -50 to 10°C, high temperatures of 50 to 300°C, acidic pH of 2 to 6.5, alkaline pH of 8 to 12, and when stored for more than one year.

10. The application according to claim 7, characterized in that, The transition metal-based single-atom nanozymes exhibit antioxidant effects in cellular antioxidant models, with higher metal loading resulting in better antioxidant effects, thus enabling their use in cell protection.

11. The application according to claim 10, characterized in that, The cells include macrophages, fibroblasts, and endothelial cells.

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