Low-temperature-resistant iron monatomic nanoscale enzyme, preparation method thereof and application thereof in antiviral treatment

Abstract

The application belongs to the field of nano-enzyme and biomedical nanotechnology, and relates to a low-temperature-resistant iron single-atom nano-enzyme, a preparation method thereof and application thereof in antiviral treatment, the preparation method comprising the following steps: dissolving Fe(NO3)3.9H2O and Zn(NO3)2.6H2O in methanol, adding methanol containing dimethyl imidazole to obtain Fe-ZIF; adding the Fe-ZIF into an ethanol aqueous solution, and then adding ammonia water, resorcinol, formaldehyde, melamine and triphenylphosphine and stirring; centrifuging the mixed solution, discarding the supernatant and collecting the precipitate, drying and washing the precipitate to obtain Fe-ZIF@ARFMT, and pyrolyzing the Fe-ZIF@ARFMT in argon at 800-1000 DEG C to obtain FeN4P2- SAzymes. The low-temperature-resistant iron single-atom nano-enzyme has good safety and a broad-spectrum antiviral function at low temperature.

Description

Technical Field

[0001] This invention discloses a FeN4P2-SAzyme single-atom nanozyme for broad-spectrum antiviral use in cold chain environments, and provides a method for its application in cold chain outer packaging and protective clothing for anti-influenza virus, belonging to the field of nanozyme and biomedical nanotechnology. Background Technology

[0002] Natural enzymes are expensive, difficult to produce continuously, have poor stability, and are prone to denaturation and inactivation. Furthermore, natural enzymes generally only exert their catalytic activity at physiological temperatures, but in practical applications, especially in antimicrobial environments, low-temperature environments are unavoidable. Therefore, natural enzymes are difficult to use effectively on a large scale in practice. Since the discovery in 2007 that iron oxide nanozymes possess catalytic properties similar to horseradish peroxidase, the field of nanozyme research has rapidly emerged. Nanozymes with different morphologies, sizes, and materials have appeared one after another, and their catalytic mechanisms are gradually being understood. Unlike the catalytic characteristics of proteases, nanozymes, based on the properties of inorganic materials, do not require as stringent reaction conditions such as temperature and pH as proteases. Due to their high catalytic efficiency, stability, economy, and large-scale preparation capabilities, research on the application of nanozymes in medicine, chemical engineering, food, agriculture, and the environment has arisen. The discovery of nanozymes has not only advanced fundamental research in nanotechnology but also expanded their applications, such as tumor diagnosis and treatment, blood glucose and uric acid detection, immunoassay, label-free in vivo tracing, antibacterial activity, environmental monitoring and wastewater treatment, and pesticide and nerve agent surveillance. Although nanozymes have been extensively developed, their relatively low catalytic activity and poor kinetics severely limit their performance and further applications. In recent years, the emergence of single-atom nanocatalysts, with their unsaturated coordination environment, high atom utilization, unique metal-support interactions, and well-defined active sites, has led to higher catalytic activity in catalytic reactions. Simultaneously, their single active site can significantly improve catalytic selectivity and avoid side reactions. However, the improvement in enzyme activity remains limited. Phosphorus is a fundamental component of many natural enzymes and plays a crucial role in assisting electrons to tunnel from the substrate to the enzyme-like catalytic active site. Therefore, utilizing a phosphorus atom-driven strategy may, to some extent, improve the enzyme-like catalytic activity of nanomaterials. Therefore, this study will dope phosphorus atoms at the remote para-position of the Fe-NC center to explore its enzyme-like catalytic activity at low temperatures and its antiviral effects at low temperatures. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of traditional chemical disinfectants, such as poor disinfection performance at low temperatures, by providing a FeN4P2-SAzyme single-atom nanozyme for broad-spectrum antiviral activity, along with its activity evaluation method and application. This FeN4P2-SAzyme single-atom nanozyme exhibits improved safety and broad-spectrum antiviral activity at low temperatures. Furthermore, the invention provides a method for its application in cold chain packaging and protective clothing for anti-influenza virus applications, thereby enhancing the virus protection capabilities of high-risk groups such as those in live poultry markets, poultry farmers, and medical workers.

[0004] This invention prepares a FeN4P2-SAzyme single-atom nanozyme for broad-spectrum antiviral activity. The viruses are SARS-CoV-2, influenza A virus (IAV), transmissible gastroenteritis virus (TGEV), and porcine epidemic diarrhea virus (PEDV), wherein the IAVs are H1N1, H2N2, H3N2, H4N6, H5N1, H6N6, H7N9, H8N4, H9N2, H10N8, and H11N2.

[0005] This invention provides a method for preparing low-temperature resistant iron single-atom nanozymes, comprising the following steps: dissolving Fe(NO3)3·9H2O and Zn(NO3)2·6H2O in methanol, adding the solution to methanol containing dimethylimidazole to obtain Fe-ZIF; adding Fe-ZIF to an ethanol aqueous solution, then adding ammonia, resorcinol, formaldehyde, melamine and triphenylphosphine and stirring; centrifuging the mixture, discarding the supernatant and collecting the precipitate, drying and washing the precipitate to obtain Fe-ZIF@ARFMT; pyrolyzing Fe-ZIF@ARFMT in argon at 800-1000℃ to obtain low-temperature resistant iron single-atom nanozymes FeN4P2-SAzymes.

[0006] Furthermore, the pyrolysis step is as follows: Fe-ZIF@ARFMT is pyrolyzed in argon at 900℃ for 2 hours.

[0007] Furthermore, the mass ratio of Fe(NO3)3·9H2O, Zn(NO3)2·6H2O and dimethylimidazole is 1:40~50:45~55.

[0008] Furthermore, the mass-to-volume ratio of Fe-ZIF, ammonia, resorcinol, formaldehyde, melamine, and triphenylphosphine is: 6–7 mg: 4–6 μL: 5–6 mg: 7–8 μL: 4–5 mg: 7–8 mg.

[0009] Furthermore, in the methanol containing dimethylimidazole, the concentration of dimethylimidazole is 0.08–0.12 g / mL.

[0010] Further, the mixture was centrifuged, the supernatant was discarded, and the precipitate was collected. The precipitate was dried in an oven at 60-70℃ for 7-10 hours and washed three times each with water and ethanol to obtain Fe-ZIF@ARFMT.

[0011] The present invention also provides a low-temperature resistant iron single-atom nanozyme, which is prepared by the above method.

[0012] The present invention also provides the application of the above-mentioned low-temperature resistant iron single-atom nanozyme in antiviral applications.

[0013] Furthermore, the virus is one or more of the following: novel coronavirus, influenza A virus, transmissible gastroenteritis virus, and swine epidemic diarrhea virus; the influenza A virus is one or more of the following: H1N1, H2N2, H3N2, H4N6, H5N1, H6N6, H7N9, H8N4, H9N2, H10N8, and H11N2.

[0014] Furthermore, the low-temperature resistant iron single-atom nanozyme can be used in the outer packaging of cold chain transportation or in antiviral protective clothing.

[0015] The present invention also provides the application of the above-mentioned low-temperature resistant iron single-atom nanozyme in catalyzing the redox reaction of O2 and TMB.

[0016] Beneficial effects

[0017] This invention relates to a scientifically sound, simple, low-cost, and stable preparation process for FeN4P2-SAzyme single-atom nanozymes, which is also safe and non-toxic. FeN4P2-SAzyme single-atom nanozymes can significantly reduce or kill various viruses at low temperatures, including SARS-CoV-2-Fluc, IAV, PEDV, and TGEV. FeN4P2-SAzyme single-atom nanozymes can be used as a broad-spectrum antiviral agent in the outer packaging of cold chain transportation. Furthermore, the application of FeN4P2-SAzyme single-atom nanozymes in low-temperature anti-influenza virus protective clothing has shown significant protective effects, which is of great significance for improving the influenza protection capabilities of high-risk groups such as live poultry market workers, poultry farmers, and medical workers.

[0018] The FeN4P2-SAzyme single-atom nanozyme of the present invention can be prepared in large quantities, has low cost, good stability at room temperature, and good safety. Therefore, the application of this broad-spectrum antiviral agent has certain economic value and application prospects.

[0019] Based on the emerging single-atom nanocatalyst technology, this study developed FeN4P2-SAzyme single-atom nanozyme and discovered its novel biological function—broad-spectrum antiviral activity under low-temperature conditions. This is expected to solve the key bottleneck problem of traditional disinfectants' antiviral activity at low temperatures, and will help block the spread of imported pathogens and major infectious disease pathogens such as the novel coronavirus and influenza virus, as well as improve the protection against viruses for high-risk groups such as medical workers. Attached Figure Description

[0020] Figure 1 The OXD-like activity of FeN4P2-SAzyme single-atom nanozyme at low temperature; Figure 1 A represents the enzyme-like catalytic activity of FeN4P2-SAzyme single-atom nanozyme, FeN4 nanozyme, and Fe3O4 nanozyme; Figure 1 B represents the enzyme-like catalytic activity of FeN4P2-SAzyme single-atom nanozyme under conditions of 25℃, 4℃ and -20℃; Figure 1 C is a visual diagram of enzyme catalytic activity;

[0021] Figure 2 FeN4P2-SAzyme single-atom nanozymes exhibit antiviral activity against various viruses at low temperatures, among which... Figure 2 A represents SARS-CoV-2-Fluc; Figure 2 B represents IAV (H1N1); Figure 2 C stands for TGEV; Figure 2 D stands for PEDV; Figure 2 E represents the antiviral activity of various natural enzymes against IAV at low temperatures. * P < 0.05, the difference was significant; ** P < 0.01 (the difference is highly significant);

[0022] Figure 3 FeN4P2-SAzyme single-atom nanozymes exhibit antiviral activity against multiple subtypes of influenza viruses, including H2N2, at low temperatures. Figure 3 A) H3N2 ( Figure 3 B), H4N6 Figure 3 C), H5N1 Figure 3 D), H6N6 Figure 3 E), H7N9 ( Figure 3 F), H8N4 Figure 3 G), H9N2 Figure 3 H), H10N8 ( Figure 3 I) and H11N2 subtype IAV ( Figure 3 J)( * P < 0.05, the difference was significant; ** P < 0.01 (the difference is highly significant);

[0023] Figure 4 To evaluate the anti-influenza virus activity of FeN4P2-SAzyme single-atom nanozyme under cold chain conditions; Figure 4 (A) is a schematic diagram of a model for anti-IAV based FeN4P2-SAzyme single-atom nanozyme on cold chain outer packaging; Figure 4 (B) To evaluate the effectiveness of HA titer against IAV on cold chain outer packaging; * P < 0.05, the difference was significant; ** P < 0.01 (the difference is highly significant); Figure 4 (C) To utilize TCID 50 Titer evaluation of the effectiveness of anti-IAV on cold chain outer packaging ( * P < 0.05, the difference was significant; ** P < 0.01 (the difference is highly significant);

[0024] Figure 5 (A) is a schematic diagram of the structure of a low-temperature anti-influenza protective suit based on FeN4P2-SAzyme single-atom nanozyme; * P < 0.05, the difference was significant; ** P < 0.01 (the difference is highly significant); Figure 5 (BD) To evaluate the effectiveness of anti-influenza protective clothing against influenza virus using HA titer; * P < 0.05, the difference was significant; ** P < 0.01 (the difference is highly significant); Figure 5 (EG) for utilizing TCID 50 Titer evaluation of the effectiveness of anti-influenza protective clothing in protecting against influenza virus ( * P < 0.05, the difference was significant; ** P < 0.01 (the difference was highly significant). Detailed Implementation

[0025] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0026] A method for evaluating the activity of FeN4P2-SAzyme single-atom nanozymes with broad-spectrum antiviral activity at low temperatures, comprising the following steps:

[0027] 1) The preparation process of FeN4P2-SAzyme single-atom nanozyme is as follows: First, 0.08g Fe(NO3)3·9H2O and 3.4g Zn(NO3)2·6H2O are dissolved in 40mL methanol, and then 40mL methanol containing 4g dimethylimidazole is added to obtain Fe-ZIF. Fe-ZIF was added to a mixed solution of deionized water (50 mL) and ethanol (20 mL), followed by the addition of ammonia (200 μL), resorcinol (220 mg), formaldehyde (295 μL), melamine (180 mg), and triphenylphosphine (300 mg). The mixture was stirred at 70 °C and 700 rpm for 22–26 h. The reaction solution was then transferred to a reaction vessel and maintained at 115–125 °C for 24 h. The resulting liquid was centrifuged, the supernatant was discarded, and the precipitate was collected. The precipitate was dried in an oven at 60–70 °C for 7–10 h and washed three times each with water and ethanol to obtain Fe-ZIF@ARFMT. Finally, Fe-ZIF@ARFMT was pyrolyzed in argon at 900 °C for 2 h to obtain FeN4P2-SAzymes. Prepare MEM and DMEM cell culture media, SAR-CoV-2-Fluc, IAV (H1N1, H2N2, H3N2, H4N6, H5N1, H6N6, H7N9, H8N4, H9N2, H10N8 and H11N2 subtypes), TGEV and PEDV, HEK293T cells overexpressing angiotensin-converting enzyme 2 (HEK293T-ACE2), canine kidney cells (MDCK cells), porcine kidney cells (PK-15 cells) and porcine testicular cells (ST cells), 1% chicken erythrocytes, and PEDV antibody.

[0028] 2) Weigh 40 mg of FeN4P2-SAzyme single-atom nanozyme into a 15 mL centrifuge tube, add 10 mL of anhydrous ethanol, and then sonicate in an ultrasonic cleaner for 15 min. Then centrifuge the FeN4P2-SAzyme single-atom nanozyme to the bottom of the centrifuge tube, discard the supernatant (upper clear liquid), and then add 5 mL of PBS buffer (phosphate buffer solution) to wash three times, discarding the supernatant (washing three times means adding PBS, centrifuging the FeN4P2-SAzyme single-atom nanozyme to the bottom of the centrifuge tube, discarding the supernatant, and repeating the operation three times) to remove any remaining anhydrous ethanol.

[0029] 3) Resuspend the FeN4P2-SAzyme single-atom nanozyme centrifuged to the bottom of the tube in 4 mL of PBS buffer, and adjust the concentration of FeN4P2-SAzyme single-atom nanozyme to 10 mg / mL. This concentration is the storage concentration of FeN4P2-SAzyme single-atom nanozyme. Prepare FeN4P2-SAzyme single-atom nanozyme suspensions with concentrations of 8 mg / mL, 4 mg / mL, 2 mg / mL, 1 mg / mL, and 0.5 mg / mL, respectively.

[0030] 4) The prepared 200 μL of FeN4P2-SAzyme single-atom nanozyme suspensions at different concentrations were respectively incubated with 200 μL of SAR-CoV-2-Fluc, IAV (H1N1, H2N2, H3N2, H4N6, H5N1, H6N6, H7N9, H8N4, H9N2, H10N8, and H11N2 subtypes), TGEV, and PEDV at 4℃ for 2 h. The inhibition rate of SAR-CoV-2-Fluc was detected using the Bio-Lite Luciferase Assay System kit, and the hemagglutination (HA) titer and tissue half-maximal infectious dose (TCID) of IAV were detected. 50 Changes in TCID detected by TGEV and PEDV 50 Changes in HA titer of IAV require detection using 1% chicken red blood cells, and TCID detection is also required. 50 MDCK cells are required, and the culture medium for MDCK cells is MEM; TCID of TGEVs is detected. 50 ST cells are required; the culture medium for TGEV is DMEM; TCID of PEDV is used for detection. 50 PK-15 cells are required. The culture medium for PK-15 is DMEM, and the antibody required for immunofluorescence is PEDV monoclonal antibody.

[0031] 5) Determination of OXD-like activity

[0032] Using TMB as the enzyme substrate, the oxidase-like (OXD) activities of FeN4P2-SAzyme single-atom nanozymes, FeN4 nanozymes, and Fe3O4 nanozymes were investigated. OXD catalyzes the redox reaction of O2 with TMB to generate a blue product (oxidized TMB, TMBox). The OXD-like activity of the FeN4P2-SAzyme single-atom nanozyme was determined by the change in absorbance of TMBox at 652 nm. The reaction was carried out at pH 4.55 (sodium acetate aqueous solution), and the effects of 10 μg / mL FeN4P2-SAzyme single-atom nanozyme and different concentrations of TMB were measured.

[0033] 6) Determination of inhibition rate:

[0034] Follow the manufacturer's instructions for the Bio-Lite Luciferase Assay system to perform the assay, as follows: Remove the cell culture plate from the incubator and allow it to equilibrate to room temperature for 30 minutes. Add an equal volume of Bio-Lite assay reagent, also equilibrated to room temperature. Allow the plate to lyse at room temperature for at least 3 minutes before assaying.

[0035] 7) Evaluation of the inactivation effect of natural enzymes on IAV

[0036] 4 mg of natural lipoxygenase, horseradish peroxidase (HRP), and catalase (CAT) were dissolved in 1 mL of deionized water. The hemagglutination titer of 4 mg / mL natural lipoxygenase, HRP, and CAT with IAV was evaluated after 2 h at 4 °C and pH = 7.

[0037] 8) Determination of hemagglutination (HA) titer:

[0038] ① Preparation of 1% chicken red blood cell suspension: Collect fresh chicken blood into an Erlenmeyer flask containing anticoagulant (sodium citrate solution); dispense the anticoagulant chicken blood into 10mL centrifuge tubes, balance, and centrifuge at 1200rpm for 10min; aspirate the supernatant and discard the uppermost layer of white, viscous homogeneous material (the part where white blood cells and platelets are concentrated); add 10mL PBS and gently resuspend the red blood cells with a dropper; repeat the above steps twice, and if necessary, wash four times until the supernatant is clear and transparent. For the last wash, centrifuge at 1200rpm for 15min, remove and measure the red blood cell volume, discard the supernatant, and add PBS at a volume ratio to prepare a 1% red blood cell solution. Store at 4℃ for later use. Shake the red blood cells well before use.

[0039] ② Add 25 μL of PBS to each well of a 96-well hemagglutination plate; add 25 μL of virus to the first column of wells in the 96-well hemagglutination plate, and serially dilute from left to right up to well 11, discarding the 25 μL. Well 12 is the negative control. Add 25 μL of PBS to each well; add 25 μL of 1% red blood cells to each well, and gently shake the hemagglutination plate to mix the liquid in the wells; place the hemagglutination plate in a 37°C incubator for 10 min; after the specified time, tilt the V-shaped hemagglutination plate so that the red blood cells in the negative control wells are attached. Then observe the other experimental wells, and the dilution at which the red blood cells are completely attached is taken as the HA titer of the virus.

[0040] 9) Median Infectious Dose (TCID) in Tissue Cultures 50 The determination of )

[0041] For IAV, MDCK cells were seeded into 96-well cell culture plates. After the cells formed a monolayer, the culture supernatant was removed and the cells were washed twice with sterile PBS. Then, serially diluted 10-fold virus solution was seeded onto the cell surface. The infected cells were cultured at 37°C and 5% CO2. After 72 hours of infection, the number of positive wells was counted, and TCID was calculated according to the Reed-Muench method (Reed LML. Am J Hyg, 27:493-497(1938)). 50 For TGEV, PK-15 cells are required; for PEDV, Vero cells are required.

[0042] 10) The evaluation results of the activity of FeN4P2-SAzyme single-atom nanozymes at low temperature are as follows:

[0043] A. FeN4P2-SAzyme single-atom nanozymes at final concentrations of 0.5 mg / mL-4 mg / mL can significantly inhibit SARS-CoV-2-Fluc, and the inhibition rate of SARS-CoV-2-Fluc by 0.5-4 mg / mL FeN4P2-SAzymes can reach more than 94.4%, and the inhibition rate of SARS-CoV-2-Fluc after treatment with 4 mg / mL FeN4P2-SAzymes is as high as 99.91%.

[0044] When the final concentration of B.FeN4P2-SAzyme single-atom nanozyme is 0.25 mg / mL-4 mg / mL, it can reduce the TCID of H1 subtype IAV. 50 The titer decreased significantly, especially at 4 mg / mL for single-atom nanozymes, which reduced the viral TCID. 50 The titer dropped to 0; different concentrations of FeN4P2-SAzyme single-atom nanozymes also significantly induced the HA titer and TCID of IAV subtypes H1N1, H2N2, H3N2, H4N6, H5N1, H6N6, H7N9, H8N4, H9N2, H10N8, and H11N2. 50 The titer decreased to varying degrees.

[0045] When the final concentration of C.FeN4P2-SAzyme single-atom nanozyme is 0.25 mg / mL-4 mg / mL, it can reduce the TCID of TGEV. 50 The titer decreased significantly, especially when the final concentration of the single-atom nanozyme was 4 mg / mL, indicating a significant decrease in viral TCID50. 50 The titer dropped to 0;

[0046] When the final concentration of D.FeN4P2-SAzyme single-atom nanozyme is 0.5 mg / mL-4 mg / mL, it can reduce the TCID of PEDV. 50The titer decreased significantly, especially when the final concentration of the nanozyme was 2 mg / mL and 4 mg / mL, indicating a decrease in viral TCID. 50 The titer dropped to 0;

[0047] E. 4 mg / mL natural lipoxygenase, HRP, CAT and IAV were reacted at 4℃ and pH=7 for 2 h and the IAV hemagglutination titer remained unchanged at 4 log2. Natural enzymes cannot exert antiviral effects at low temperature.

[0048] Example 1: Evaluation of OXD activity of FeN4P2-SAzyme single-atom nanozymes at low temperature

[0049] Experimental material: FeN4P2-SAzyme single-atom nanozyme was synthesized in our laboratory;

[0050] Methods: 1) Synthesis of FeN4P2-SAzyme single-atom nanozymes

[0051] First, 0.08 g of Fe(NO3)3·9H2O and 3.4 g of Zn(NO3)2·6H2O were dissolved in 40 mL of methanol solution. Then, 40 mL of methanol containing 4 g of dimethylimidazole was added to obtain Fe-ZIF. Fe-ZIF was added to a mixed solution of deionized water (50 mL) and ethanol (20 mL), followed by ammonia (200 μL), resorcinol (220 mg), formaldehyde (295 μL), melamine (180 mg), and triphenylphosphine (300 mg). The mixture was stirred at 70 °C and 700 rpm for 22–26 h. The reaction solution was then transferred to a reaction vessel and kept at 115–125 °C for 24 h. The supernatant was discarded and the precipitate was collected by centrifugation. The precipitate was dried in an oven at 60–70 °C for 7–10 h and washed three times each with water and ethanol to obtain Fe-ZIF@ARFMT. Finally, Fe-ZIF@ARFMT was pyrolyzed in argon at 900℃ for 2 h to obtain FeN4P2-SAzymes.

[0052] 2) Determination of OXD activity of FeN4P2-SAzyme single-atom nanozymes

[0053] Using 3,3',5,5'-Tetramethylbenzidine (TMB) as the enzyme substrate, the oxidase-like (OXD) activities of FeN4P2-SAzyme single-atom nanozymes, FeN4 nanozymes, and Fe3O4 nanozymes were investigated. OXD catalyzes the redox reaction of O2 with TMB to generate a blue product (oxidized TMB, TMBox). The OXD-like activity of FeN4P2-SAzyme single-atom nanozymes was determined by the change in absorbance of TMBox at 652 nm. The reaction was carried out at pH 4.55 (sodium acetate aqueous solution), and the effects of 10 μg / mL FeN4P2-SAzyme single-atom nanozymes and different concentrations of TMB were measured.

[0054] Test results: such as Figure 1 As shown in (A), the FeN4P2-SAzyme single-atom nanozyme exhibits higher enzyme-like catalytic activity than FeN4 and Fe3O4 nanozymes. This is because FeN4P2-SAzyme, being a single-atom nanozyme, reduces the catalytic active center to the atomic level, thereby improving the intrinsic catalytic efficiency of each metal atom. Furthermore, the addition of phosphorus atoms during preparation, a fundamental component of many natural enzymes, plays a crucial role in the tunneling of auxiliary electrons from the substrate to the enzyme-like catalytic active center, contributing to enhanced nanozyme catalytic activity. This demonstrates that reducing the catalytic activity of nanozymes to the atomic level and adding phosphorus atoms can yield better catalytic activity. Figure 1 As shown in (BC), FeN4P2-SAzyme single-atom nanozymes exhibit high enzyme-like catalytic activity at 25℃, 4℃, and -20℃, with no significant differences between the different temperatures. The enzyme-like catalytic activity visualization also shows that the intensity of the blue product formed by FeN4P2-SAzyme single-atom nanozymes catalyzing the substrate TMB does not differ significantly between different temperature conditions.

[0055] Example 2: Evaluation of the anti-influenza virus activity of FeN4P2-SAzyme single-atom nanozyme at low temperature

[0056] Experimental materials: FeN4P2-SAzyme single-atom nanozyme was synthesized in our laboratory; IAV (H1N1) was isolated, identified, and preserved by the Key Laboratory of Animal Infectious Diseases of the Ministry of Agriculture. 1% chicken red blood cells were required to detect the HA titer of IAV, and TCID was also tested. 50All required MDCK cells were prepared or preserved in this laboratory. MEM cell culture medium was purchased from Hyclone. Lipoxygenase was purchased from Sigma-Aldrich; horseradish peroxidase (HRP) was purchased from Salarbio; catalase (CAT) was also used.

[0057] Methods: 1) Synthesis of FeN4P2-SAzyme single-atom nanozymes

[0058] First, 0.08 g of Fe(NO3)3·9H2O and 3.4 g of Zn(NO3)2·6H2O were dissolved in 40 mL of methanol solution. Then, 40 mL of methanol containing 4 g of dimethylimidazole was added to obtain Fe-ZIF. Fe-ZIF was added to a mixed solution of deionized water (50 mL) and ethanol (20 mL), followed by ammonia (200 μL), resorcinol (220 mg), formaldehyde (295 μL), melamine (180 mg), and triphenylphosphine (300 mg). The mixture was stirred at 70 °C and 700 rpm for 22–26 h. The reaction solution was then transferred to a reaction vessel and kept at 115–125 °C for 24 h. The supernatant was discarded and the precipitate was collected by centrifugation. The precipitate was dried in an oven at 60–70 °C for 7–10 h and washed three times each with water and ethanol to obtain Fe-ZIF@ARFMT. Finally, Fe-ZIF@ARFMT was pyrolyzed in argon at 900℃ for 2 h to obtain FeN4P2-SAzymes.

[0059] Weigh 40 mg of FeN4P2-SAzyme single-atom nanozyme into a 15 mL centrifuge tube, add 10 mL of anhydrous ethanol, and then sonicate in an ultrasonic cleaner for 15 min. Centrifuge the FeN4P2-SAzyme single-atom nanozyme to the bottom of the tube, discard the supernatant, and wash three times with 5 mL of PBS buffer, discarding the supernatant again. Resuspend the FeN4P2-SAzyme single-atom nanozyme at the bottom of the tube with 1 mL of PBS buffer, adjusting the concentration of FeN4P2-SAzyme single-atom nanozyme to 40 mg / mL. This concentration is the storage concentration of FeN4P2-SAzyme single-atom nanozyme. Prepare FeN4P2-SAzyme single-atom nanozyme suspensions with concentrations of 5 mg / mL to 80 mg / mL as needed.

[0060] 2) Preparation of cold chain outer packaging

[0061] like Figure 4 As shown in (A), a selected area (5cm) with flat and concave surfaces was created using a block of ice simulating cold chain packaging and a heated iron block as a base. 2 Seal with plastic film, freeze and shape.

[0062] 3) Interaction of FeN4P2-SAzyme single-atom nanozyme with influenza virus under cold chain conditions

[0063] In the selected area (5cm) 2 The surface coating concentration is 0.8 mg / cm³. 2 FeN4P2-SAzyme was frozen at -20°C for 30 min. IAV was sprayed onto selected areas and incubated at -20°C for different times. After incubation, the FeN4P2-SAzyme areas were placed in tubes and centrifuged immediately, then analyzed using HA and TCID. 50 Methods for measuring viral titers.

[0064] 4) Determination of hemagglutination (HA) titer

[0065] ① Preparation of 1% chicken red blood cell suspension: Collect fresh chicken blood into an Erlenmeyer flask containing anticoagulant (sodium citrate solution); dispense the anticoagulant chicken blood into 10mL centrifuge tubes, balance, and centrifuge at 1200rpm for 10min; aspirate the supernatant and discard the uppermost layer of white, viscous homogeneous material (the part where white blood cells and platelets are concentrated); add 10mL PBS and gently resuspend the red blood cells with a dropper; repeat the above steps twice, and if necessary, wash four times until the supernatant is clear and transparent. For the last wash, centrifuge at 1200rpm for 15min, remove and measure the red blood cell volume, discard the supernatant, and add PBS at a volume ratio to prepare a 1% red blood cell solution. Store at 4℃ for later use. Shake the red blood cells well before use.

[0066] ② Add 25 μL of PBS to each well of a 96-well hemagglutination plate; add 25 μL of virus to the first column of wells in the 96-well hemagglutination plate, and serially dilute from left to right up to well 11, discarding 25 μL. Well 12 is the negative control. Add 25 μL of PBS to each well; add 25 μL of 1% red blood cells to each well, and gently shake the hemagglutination plate to mix the liquid in the wells; place the hemagglutination plate in a 37°C incubator for 10 min; after the specified time, tilt the V-shaped hemagglutination plate so that the red blood cells in the negative control wells are attached to the plate. Then observe the other experimental wells, and take the dilution at which the red blood cells are completely attached as the HA titer of the virus;

[0067] 5) Median Infectious Dose (TCID) in Tissue Cultures 50 The determination of )

[0068] For IAV, MDCK cells were seeded into 96-well cell culture plates. After the cells formed a monolayer, the culture supernatant was removed and the cells were washed twice with sterile PBS. Then, serially diluted 10-fold virus solution was seeded onto the cell surface. The infected cells were cultured at 37°C and 5% CO2. After 72 hours of infection, the number of positive wells was counted, and TCID was calculated according to the Reed-Muench method (Reed LML. Am J Hyg, 27:493-497(1938)). 50 .

[0069] Test results: such as Figure 4 As shown in (BC), when the concentration of FeN4P2-SAzyme single-atom nanozyme was 4 mg / mL, there was no significant difference in the inactivation of IAV at 4℃ and 25℃. It reduced the HA titer of IAV to 0 at 15 min and 5 min, respectively, and reduced the TCID of IAV to 0 at 1 h. 50 The potency dropped to 0. Although the FeN4P2-SAzyme single-atom nanozyme killed IAV slowly at -20℃, it could still reduce the HA and TCID of IAV within 2 hours. 50 The valence dropped to 0.

[0070] The above results demonstrate that FeN4P2-SAzyme single-atom nanozymes retain significant anti-influenza virus activity even under cold chain conditions. Therefore, FeN4P2-SAzyme single-atom nanozymes can be used as a novel anti-influenza virus material in cold chain transportation, providing a new perspective for virus control in cold chain environments.

[0071] Example 3: Application of FeN4P2-SAzyme single-atom nanozyme in low-temperature anti-influenza virus protective clothing

[0072] Experimental materials: FeN4P2-SAzyme single-atom nanozymes were synthesized in our laboratory; IAVs (H1N1, H3N2, and H7N9) were isolated, identified, and preserved by the Key Laboratory of Animal Infectious Diseases of the Ministry of Agriculture. 1% chicken erythrocytes were required to detect the HA titer of IAVs, and TCID was also tested. 50 All required MDCK cells were prepared or preserved in this laboratory. MEM cell culture medium was purchased from Hyclone.

[0073] Methods: 1) Synthesis of FeN4P2-SAzyme single-atom nanozymes

[0074] First, 0.08 g of Fe(NO3)3·9H2O and 3.4 g of Zn(NO3)2·6H2O were dissolved in 40 mL of methanol solution. Then, 40 mL of methanol containing 4 g of dimethylimidazole was added to obtain Fe-ZIF. Fe-ZIF was added to a mixed solution of deionized water (50 mL) and ethanol (20 mL), followed by ammonia (200 μL), resorcinol (220 mg), formaldehyde (295 μL), melamine (180 mg), and triphenylphosphine (300 mg). The mixture was stirred at 70 °C and 700 rpm for 22–26 h. The reaction solution was then transferred to a reaction vessel and kept at 115–125 °C for 24 h. The supernatant was discarded and the precipitate was collected by centrifugation. The precipitate was dried in an oven at 60–70 °C for 7–10 h and washed three times each with water and ethanol to obtain Fe-ZIF@ARFMT. Finally, Fe-ZIF@ARFMT was pyrolyzed in argon at 900℃ for 2 h to obtain FeN4P2-SAzymes.

[0075] Weigh 40 mg of FeN4P2-SAzyme single-atom nanozyme into a 15 mL centrifuge tube, add 10 mL of anhydrous ethanol, and then sonicate in an ultrasonic cleaner for 15 min. Centrifuge the FeN4P2-SAzyme single-atom nanozyme to the bottom of the tube, discard the supernatant, and wash three times with 5 mL of PBS buffer, discarding the supernatant again. Resuspend the FeN4P2-SAzyme single-atom nanozyme at the bottom of the tube with 1 mL of PBS buffer, adjusting the concentration of FeN4P2-SAzyme single-atom nanozyme to 40 mg / mL. This concentration is the storage concentration of FeN4P2-SAzyme single-atom nanozyme. Prepare FeN4P2-SAzyme single-atom nanozyme suspensions with concentrations of 5 mg / mL to 80 mg / mL as needed.

[0076] 2) Preparation method of anti-influenza virus protective clothing based on FeN4P2-SAzyme single-atom nanozyme.

[0077] As attached Figure 5 As shown in (A), a randomly selected area (5cm) on the protective suit. 2 50 μL of FeN4P2-SAzyme single-atom nanozymes of different concentrations were coated, dried by airflow for 30 min, and then sprayed with IAV and incubated for 5 min, 10 min and 15 min.

[0078] 3) Place the protective suit in a 1.5 mL finger tube containing 200 μL PBS. After washing and squeezing, harvest the viral suspension. Then, use the hemagglutination (HA) titer and tissue half-maximal infectious dose (TCID) to determine the viral suspension. 50 The method measures the number of live viruses.

[0079] 4) Determination of hemagglutination (HA) titer

[0080] ① Preparation of 1% chicken red blood cell suspension: Collect fresh chicken blood into an Erlenmeyer flask containing anticoagulant (sodium citrate solution); dispense the anticoagulant chicken blood into 10mL centrifuge tubes, balance, and centrifuge at 1200rpm for 10min; aspirate the supernatant and discard the uppermost layer of white, viscous homogeneous material (the part where white blood cells and platelets are concentrated); add 10mL PBS and gently resuspend the red blood cells with a dropper; repeat the above steps twice, and if necessary, wash four times until the supernatant is clear and transparent. For the last centrifugation, centrifuge at 1200rpm for 15min, remove and measure the red blood cell volume, discard the supernatant, and add PBS at a volume ratio to prepare a 1% red blood cell solution. Store at 4℃ for later use. Shake the red blood cells well before use.

[0081] ② Add 25 μL of PBS to each well of a 96-well hemagglutination plate; add 25 μL of virus to the first column of wells in the 96-well hemagglutination plate, and serially dilute from left to right up to well 11, discarding 25 μL. Well 12 is the negative control. Add 25 μL of PBS to each well; add 25 μL of 1% red blood cells to each well, and gently shake the hemagglutination plate to mix the liquid in the wells; place the hemagglutination plate in a 37°C incubator for 10 min; after the specified time, tilt the V-shaped hemagglutination plate so that the red blood cells in the negative control wells are attached to the plate. Then observe the other experimental wells, and take the dilution at which the red blood cells are completely attached as the HA titer of the virus;

[0082] 5) Median Infectious Dose (TCID) in Tissue Cultures 50 The determination of )

[0083] For IAV, MDCK cells were seeded into 96-well cell culture plates. After the cells formed a monolayer, the culture supernatant was removed and the cells were washed twice with sterile PBS. Then, serially diluted 10-fold virus solution was seeded onto the cell surface. Infected cells were cultured at 37°C and 5% CO2. After 72 hours of infection, the number of positive wells was counted, and TCID was calculated according to the Reed-Muench method (Reed LML. Am J Hyg, 27:493-497(1938)). 50 .

[0084] Test results: such as Figure 5 As shown in (BG), compared with the control group, the addition of FeN4P2-SAzyme coated with single-atom nanozyme to the protective clothing reduced the viral HA and TCID levels. 50 The titer can be significantly reduced, especially at 0.8 mg / cm³. 2 Under the action of high concentration FeN4P2-SAzyme single-atom nanozymes, H1N1, H3N2 and H7N9 IAV can be completely inactivated within 15 min.

[0085] The above results indicate that the anti-influenza protective clothing prepared based on FeN4P2-SAzyme single-atom nanozyme has a significant protective effect against influenza.

Claims

1. The application of low-temperature resistant iron single-atom nanozymes in antiviral treatment at low temperatures, characterized in that, The preparation method of the low-temperature resistant iron single-atom nanozyme includes the following steps: Fe(NO3)3⋅9 H2O and Zn(NO3)2⋅6 H2O are dissolved in methanol, and then added to methanol containing dimethylimidazole to obtain Fe-ZIF; Fe-ZIF is added to an ethanol aqueous solution, and then ammonia, resorcinol, formaldehyde, melamine and triphenylphosphine are added and stirred; the mixture is centrifuged, the supernatant is discarded and the precipitate is collected, the precipitate is dried and washed to obtain Fe-ZIF@ARFMT, and Fe-ZIF@ARFMT is pyrolyzed in argon at 800~1000℃ to obtain the low-temperature resistant iron single-atom nanozyme FeN4P2-SAzymes.

2. The application according to claim 1, characterized in that, The pyrolysis step is as follows: Fe-ZIF@ARFMT is pyrolyzed in argon at 900℃ for 2 h.

3. The application according to claim 1, characterized in that, The mass ratio of Fe(NO3)3⋅9 H2O, Zn(NO3)2⋅6 H2O and dimethylimidazole is 1:40~50:45~55.

4. The application according to claim 1, characterized in that, The mass-to-volume ratio of Fe-ZIF, ammonia, resorcinol, formaldehyde, melamine, and triphenylphosphine is: 6~7 mg: 4~6 μL: 5~6 mg: 7~8 μL: 4~5 mg: 7~8 mg.

5. The application according to claim 1, characterized in that, Centrifuge the mixture, discard the supernatant, and collect the precipitate. Dry the precipitate in an oven at 60-70℃ for 7-10 h, and wash it three times each with water and ethanol to obtain Fe-ZIF@ARFMT.

6. The application according to claim 1, characterized in that, The virus is one or more of the following: novel coronavirus, influenza A virus, transmissible gastroenteritis virus, and porcine epidemic diarrhea virus. The influenza A virus is one or more of the following: H1N1, H2N2, H3N2, H4N6, H5N1, H6N6, H7N9, H8N4, H9N2, H10N8, and H11N2.

7. The application according to claim 1, characterized in that, The low-temperature resistant iron single-atom nanozyme is used in the outer packaging of cold chain transportation or in antiviral protective clothing.

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