CN111990409B

CN111990409B - Broad-spectrum antiviral material and preparation method and application thereof

Info

Publication number: CN111990409B
Application number: CN202010651081.7A
Authority: CN
Inventors: 王世珍; 段凌暄; 刘凯泷
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2020-07-08
Filing date: 2020-07-08
Publication date: 2021-06-22
Anticipated expiration: 2040-07-08
Also published as: CN111990409A; BE1028412A1; BE1028412B1

Abstract

The invention discloses a broad-spectrum antiviral material, a preparation method and application thereof, and mainly aims to research the broad-spectrum antiviral performance of a modified graphene oxide-polyethyleneimine composite material and application thereof in protective articles. The alkylated graphene oxide-polyethyleneimine serving as a broad-spectrum antiviral material provided by the invention has the advantages of simple preparation process and low cost, has good inactivation effect on various viruses under low concentration, can enable virus particles adsorbed on the surface of a protective material to be broken and lose the infectious activity, and can be sprayed on surfaces of non-woven fabrics, paper, furniture, in-vehicle spaces and the like, such as a mask and a protective clothing, so that the alkylated graphene oxide-polyethyleneimine can be used for disinfecting living spaces and moving spaces for a long time, preventing infection and diffusion and improving the protection strength of medical care personnel and the broad masses.

Description

Broad-spectrum antiviral material and preparation method and application thereof

Technical Field

The invention relates to the technical field of materials, in particular to a preparation method and application of a graphene and polyethyleneimine composite antiviral material.

Background

The spread and dissemination of pathogenic microorganisms has become a global focus of general attention, with regard to human health, economic development and social stability. Where the losses caused by the virus are extremely large. Viruses can be classified into DNA viruses and RNA viruses, etc. The virus of the genus coronavirus is a positive-strand single-stranded RNA virus having an envelope (also called an overcoat) with a spinous process thereon, and has a diameter of about 80 to 120 nm. Coronaviruses are a large family of viruses known to cause the common cold and more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). In order to prevent various pathogenic microorganisms in the living environment from invading human health and avoid various infectious diseases caused by the invasion, the development of antiviral materials has become one of the hot topics in the scientific research and production fields. Polyethyleneimine (PEI) is a high molecular polymer, and due to its excellent conductivity and biocompatibility, it becomes an effective electron conductor between immobilized enzyme and electrode. PEI can be divided into two main classes of branched polyethyleneimine (also called branched polyethyleneimine) and linear polyethyleneimine (also called linear polyethyleneimine), the molecular weight is from 1000Da to 1600KDa, and the structures of the PEI are long chains with larger molecular weights. The functionalized PEI material has broad-spectrum antiviral performance, can damage the mantle of the virus and lead to the splitting of virus particles, thereby inhibiting the replication and the transmission of the virus, and is safe and nontoxic to human cells. Most of the currently reported functionalized PEI with antiviral activity is linked on the surface of a substrate such as glass, and the like, and cannot be directly applied to various materials such as fabrics, plastics, resins and the like, so that the wide application of the PEI is limited.

Graphene Oxide (GO) is an important derivative of Graphene, and has a wide application prospect in the fields of biological medicines, drug sustained release, packaging materials and the like. The graphene oxide has a two-dimensional lamellar structure similar to graphene, and the specific surface area is large. GO has a large number of oxygen-containing groups on the two-dimensional plane of the carbon skeleton to be modified on the surface and the edge, and the oxygen-containing groups comprise epoxy groups, carboxyl groups, hydroxyl groups and the like. The existence of the oxygen-containing groups also enables GO to have better dispersibility and stability in an aqueous solution, and has certain antibacterial and antiviral effects due to the fact that the GO has negative charges. However, there is no report on an antiviral material combining graphene oxide and polyethyleneimine.

Disclosure of Invention

The invention provides a broad-spectrum antiviral material and a preparation method and application thereof.

One of the technical schemes adopted by the invention for solving the technical problems is as follows:

a broad-spectrum antiviral material is an alkylated graphene oxide-polyethyleneimine composite (GO-N-PEI).

The alkylated graphene oxide-polyethyleneimine composite (GO-N-PEI) has a plurality of alkylated polyethyleneimine units with the same or different carbon chain lengths, wherein the alkyl groups in the alkylated polyethyleneimine units comprise at least one of C1-C20 alkyl (preferably C4-C20 alkyl), C1-C20 hydroxyalkyl (preferably C4-C20 hydroxyalkyl) or C7-C20 aralkyl.

In the alkylated graphene oxide-polyethyleneimine composite material (GO-N-PEI), polyethyleneimine is at least one of linear polyethyleneimine (preferably 22KDa, 87KDa, 217KDa or 500KDa) with the molecular weight of 20-500 KDa or branched polyethyleneimine (preferably 70KDa, 87KDa or 1600KDa) with the molecular weight of 20-1600 KDa. The antiviral effect of the linear polyethyleneimine is superior to that of the branched polyethyleneimine.

The second technical scheme adopted by the invention for solving the technical problems is as follows:

a preparation method of a broad-spectrum antiviral material comprises the step of carrying out alkylation treatment on a graphene oxide-polyethyleneimine composite material (GO-PEI) to obtain the alkylated graphene oxide-polyethyleneimine composite material (GO-N-PEI).

Specifically, the alkylation treatment method comprises the following steps: performing graphene oxide-polyethyleneimine composite (GO-PEI) functionalization by using an alkylating reagent, adding the alkylating reagent with the final concentration of 0.01-4M into chloroform or tert-amyl alcohol serving as a solvent, and reacting for 1-48 h at the temperature of 0-80 ℃.

The alkylating agent comprises at least one of halogenated alkanes with alkyl groups with different carbon chain lengths (C1-C20) or benzoyl alkylating agent.

Preferably, the alkylating agent is an alkyl halide represented by a formula RX, wherein R is selected from C1-C20 alkyl (preferably C4-C20 alkyl), C1-C20 hydroxyalkyl (preferably C4-C20 hydroxyalkyl) or C7-C20 aralkyl; x is Cl, Br or I.

In one embodiment: the benzoyl alkylating agent includes aromatic amides and the like.

Further, the alkylation treatment method comprises the steps of firstly treating with alkyl bromide or alkyl fluoride and then treating with alkyl iodide.

In one embodiment: the preparation method of the graphene oxide-polyethyleneimine composite material (GO-PEI) comprises the following steps: dropwise adding a Polyethyleneimine (PEI) aqueous solution (10-200 mg/mL) into a Graphene Oxide (GO) aqueous solution (1-20 mg/mL), carrying out ultrasonic treatment for 5-30 min, then adding carbodiimide (EDC), stirring at room temperature for 30-180 min, then adding the carbodiimide (EDC) for reacting overnight, finally adding sodium chloride and urea into the obtained solution, centrifuging to remove precipitates, collecting a suspension, and carrying out ultrafiltration to completely remove unreacted Polyethyleneimine (PEI) to obtain a stable graphene oxide-polyethyleneimine composite material (GO-PEI); wherein the formula proportion of the 10-200 mg/mL Polyethyleneimine (PEI) aqueous solution, the 1-20 mg/mL Graphene Oxide (GO) aqueous solution, the carbodiimide (EDC), the sodium chloride and the urea is 9-11 mL: 10-100 mL: 200-800 mg: 2-10 g: 2-20 g.

Further, the mass ratio of the carbodiimide (EDC) added for the first time to the carbodiimide (EDC) added for the second time is 1: 1.5-5.

In one embodiment: the preparation method of the Graphene Oxide (GO) aqueous solution comprises the following steps: preparing graphene oxide by a Hummer method, dissolving graphene oxide in pure water, performing ultrasonic dispersion to obtain a uniformly dispersed graphene oxide suspension, centrifuging, and taking a supernatant, namely a 1-20 mg/mL graphene oxide solution.

The third technical scheme adopted by the invention for solving the technical problems is as follows:

a using method of a broad-spectrum antiviral material is characterized in that 0.1-500 mg/mL of a dispersion liquid of an alkylated graphene oxide-polyethyleneimine composite material (GO-N-PEI) is attached to a material to be treated.

The material to be treated comprises at least one of fabric, plastic or leather. Such as a nonwoven fabric, a meltblown fabric, a cell phone housing, or a vehicle interior, etc.

The dispersion is applied by dipping, coating or spraying to achieve adhesion to the material to be treated.

The solvent of the dispersion liquid is an organic solvent or a mixed liquid of organic solvents and water in different proportions.

The organic solvent comprises at least one of methanol, ethanol, isopropanol, n-butanol, tetrahydrofuran, dimethyl sulfoxide, ethyl acetate, methyl tert-butyl ether, diethyl ether, toluene, dioxane, petroleum ether, n-pentane, cyclopentane, n-hexane, cyclohexane or n-heptane.

The alkylated graphene oxide-polyethyleneimine composite (GO-N-PEI) can play an antiviral role by destroying virus particles, so that viruses lose infectivity.

The virus includes at least one of dengue virus, coronavirus, enterovirus, influenza virus, or herpes simplex virus.

Wherein the coronavirus includes SARS coronavirus, novel coronavirus COVID-19, porcine enteropathogenic coronavirus, etc.; such enteroviruses include, for example, norovirus and the like; examples of the influenza virus include H1N1 influenza virus and the like.

In addition, the alkylated graphene oxide-polyethyleneimine composite (GO-N-PEI) also has antibacterial activity, and bacteria comprise at least one of escherichia coli or staphylococcus aureus.

The alkylated graphene oxide-polyethyleneimine composite material (GO-N-PEI) is safe and nontoxic in contact with human skin.

The physical properties, antiviral properties, etc. of the broad-spectrum antiviral material of the present invention are measured by the following methods:

and (3) physical property measurement: dispersing the alkylated graphene oxide-polyethyleneimine composite material (GO-N-PEI) in an organic solvent or a mixed solution of the organic solvent and water in a certain proportion, and performing ultrasonic treatment for 0.1-1 h to obtain a dispersion liquid of 0.1-500 mg/mL. The charging properties of the material particles were measured using Zeta potential. And (4) obtaining the material morphology characteristics by using a scanning electron microscope (TEM). And (3) uniformly dripping the solution on a glass sheet, drying, and measuring a contact angle by using a contact angle meter to judge the hydrophobicity of the antiviral composite material. Particle size was determined using dynamic light scattering.

And (3) investigating damage conditions of GO, GO-PEI and GO-N-PEI materials to virus particles, researching influences of structures and physical properties of the GO, GO-PEI and GO-N-PEI materials on antiviral capacity, and investigating material properties including PEI length and an antiviral mechanism of damage action of alkylation modified hydrophobicity to a virus envelope. And respectively and uniformly coating GO, GO-PEI and GO-N-PEI materials on different glass slides and then drying. And (3) transferring 10 mu L of virus liquid by using a liquid transfer gun, uniformly dripping the virus liquid on a glass slide, covering a cover glass Polyethylene (PE) sheet, pressing a heavy object to uniformly distribute the virus liquid, and placing for 5-30 min to ensure that the virus is fully contacted with GO, GO-PEI and GO-N-PEI materials respectively. And repeatedly washing the surface of the PE sheet of the cover glass with 1mL of PBS for a plurality of times, then washing the surface of the glass slide for a plurality of times, respectively eluting and respectively collecting viruses which are contacted with GO, GO-PEI and GO-N-PEI materials, and uniformly mixing the obtained buffer solution containing the viruses and then diluting to obtain six dilutions.

Referring to a plaque formation experiment, MDCK cell culture (Madin Darby Canine Kidney) plates are divided into four groups of blank samples (virus solution not contacted with an antiviral material), a GO group (virus solution contacted with the GO material), a GO-PEI group (virus solution contacted with the GO-PEI material) and a GO-N-PEI group (virus solution contacted with the GO-N-PEI material), wherein each hole of the four groups is respectively added with different dilutions of virus solution not contacted with the antiviral material, virus solution contacted with the GO-PEI material and virus solution contacted with the GO-N-PEI material, and the layers are respectively coated by 25 muL. After coating, the cells were cultured for 2 to 3 days, and the number of plaques produced after death of the cells in each group was counted under a microscope (x 10). The log reduction value LRV (Log reduction value) of the virus concentration in each group of virus solutions exposed to GO, GO-PEI and GO-N-PEI materials was calculated compared to the blank (virus solution not exposed to the antiviral material).

The preparation method comprises the steps of carrying out dip coating and spray coating of melt-blown cloth by using a dispersion liquid of an alkylated graphene oxide-polyethyleneimine composite material (GO-N-PEI), and carrying out surface spraying on materials such as mobile phone shells and vehicle interiors. And (3) inspecting the antiviral performance of the dipped and sprayed melt-blown cloth materials after drying, and comparing the effect difference of the two coating modes.

Immersing the non-woven fabric into the GO-N-PEI dispersion liquid, carrying out non-woven fabric dipping coating and spraying, researching influence factors in the coating process, carrying out 100-1000W power microwave treatment for 10min, and turning over to obtain a more uniform sample. And (3) inspecting the antiviral performance of the coated non-woven fabric material, and comparing the effect difference of the two coating modes.

Effect of contact time on virucidal effect: and respectively measuring the antiviral ability under the contact time of 5-60 minutes. And detecting the difference of the shape damage degree of the virus particles by using an electron microscope according to different contact time. The virus solution contacted with the antiviral material was spread on MDCK cell culture (Madin Darby Canine Kidney) plates, different dilutions of the virus were added to each well from low to high, and the log reduction value LRV (Log reduction value) of the virus concentration was measured separately.

The antibacterial activity of the cationic antibacterial polymer is influenced by the hydrophilicity and hydrophobicity, the charge density, the alkyl chain length and other factors. The method combines the advantages of graphene oxide and a polyethyleneimine cationic polymer, the graphene oxide is combined with high-molecular polyethyleneimine by utilizing electrostatic binding force to prepare graphene oxide grafted polyethyleneimine, and further molecular design and functional modification are carried out to obtain a novel efficient broad-spectrum antiviral material alkylated graphene oxide-polyethyleneimine composite material (GO-N-PEI). Compared with GO, the antiviral performance of the PEI material is improved, compared with the traditional functional PEI material which can not be sprayed, the PEI material has improved antiviral performance, and the PEI material can be applied to various materials and environments in the modes of spraying, dipping, coating and the like.

The equipment, reagents, processes, parameters and the like related to the invention are conventional equipment, reagents, processes, parameters and the like except for special description, and no embodiment is needed.

All ranges recited herein include all point values within the range.

In the present invention,% is mass% unless otherwise specified or indicated in a general sense.

In the invention, the room temperature, namely the normal environment temperature, can be 10-30 ℃.

The 'overnight reaction' in the invention means that the reaction time is 10-16 h.

Compared with the background technology, the technical scheme has the following advantages:

the invention prepares a novel efficient broad-spectrum antiviral material, namely alkylated graphene oxide-polyethyleneimine composite material (GO-N-PEI), by preparing a graphene oxide-polyethyleneimine composite functionalized material and further carrying out molecular modification and functional modification. The antiviral material has simple preparation process and low cost, has good inactivation effect on broad-spectrum viruses under low concentration, can be attached to the surfaces of various materials such as fabrics, plastics, leather and the like by spraying, dipping and the like, can be used for biochemical protection of protective materials such as masks, protective clothing and the like, can be sprayed on the surfaces of non-woven fabrics, paper, furniture, in-vehicle spaces and the like, and can be used for long-term disinfection of living spaces and moving spaces to prevent infection and diffusion.

Drawings

The invention is further illustrated by the following figures and examples.

Fig. 1 is a surface Scanning Electron Microscope (SEM) image of a graphene oxide-polyethyleneimine composite material (GO-PEI) prepared in example 1 of the present invention.

Fig. 2 is an atomic force microscope image of the alkylated graphene oxide-polyethyleneimine composite (GO-N-PEI) prepared in example 2 of the present invention.

FIG. 3 is a graph of the effect of contact time on antiviral performance studied in example 4 of the present invention, wherein the contact time is plotted on the abscissa and the virus kill rate is plotted on the ordinate.

Detailed Description

The present invention will be described in detail with reference to the following examples:

example 1

1) Preparing a graphene oxide solution: preparing graphene oxide by a Hummer method, dissolving graphene oxide with corresponding mass in pure water, performing ultrasonic dispersion to obtain a uniformly dispersed graphene oxide suspension, centrifuging, and taking a supernatant, namely a graphene oxide solution with the concentration of 20 mg/mL;

2) preparing a graphene oxide-polyethyleneimine composite material (GO-PEI): 10mL of 217kDa linear PEI in water (200mg/mL) was added dropwise to 100mL of GO in water (20mg/mL), sonicated for 30min, and 300mg of carbodiimide (EDC) was added. After stirring at room temperature for 180min, 500mg of EDC was added and the reaction was allowed to proceed overnight. Finally, 10g of sodium chloride and 20g of urea are added into the obtained solution, and then the solution is centrifuged at 10000rpm for 15min to remove precipitates. The suspension was collected, ultra pure water was added and ultrafiltration was repeated with 300kDa ultrafiltration tube until complete removal of unreacted PEI to obtain a stable GO-PEI solution which was stored at 4 ℃. When in use, the dried GO-PEI composite material is obtained by freeze drying and other modes.

3) Preparation of alkylated graphene oxide-polyethyleneimine (GO-N-PEI) composite: performing GO-PEI composite material functionalization by using an alkylating reagent, adding 1-bromododecane with the final concentration of 1M into chloroform serving as a solvent, reacting for 36 hours at 60 ℃, adding 2M iodomethane with the final concentration of 2M into the mixture, reacting for 12 hours at 40 ℃, and obtaining the antiviral material GO-N-PEI by centrifugal separation and other modes.

4) Determination of physical properties of the materials: dispersing the antiviral composite material GO-N-PEI in an aqueous solution containing 10% ethanol, and performing ultrasonic treatment for 0.1-1 h until the dispersion is uniform to obtain a 500mg/mL dispersion liquid. The particle size was determined to be 500nm by dynamic light scattering. And (4) obtaining the material morphology characteristics by a scanning electron microscope (TEM). The solution was uniformly dropped on a glass plate, and after drying, the contact angle was measured to be 73.5 degrees.

5) Virus transfection experiments were performed: and respectively inspecting the damage condition of GO, GO-PEI and GO-N-PEI materials to the porcine intestinal pathogenic coronavirus. 20 mu L of GO, GO-PEI and GO-N-PEI materials are evenly coated on a glass slide and then dried. Transferring 10 μ L of virus liquid with a pipette, uniformly dripping on a glass slide, covering with a cover glass (PE sheet), pressing with a heavy object to uniformly distribute the virus liquid, and standing for 5min to make the virus fully contact with GO, GO-PEI and GO-N-PEI materials. Repeatedly washing the surface of the PE sheet of the cover glass by 1mL of PBS buffer solution for 10 times, then washing the surface of the glass slide for 10 times, respectively eluting and collecting viruses contacted with GO, GO-PEI and GO-N-PEI materials, and uniformly mixing the obtained buffer solution containing the viruses and then diluting to obtain six dilutions.

6) 25. mu.L of the virus solution obtained in step 5) was added to each well of MDCK cell culture (Madin Darbey Canine Kidney) plates and coated. Culturing for 2-3 days after coating, calculating the number of plaques generated after cell death under a microscope (multiplied by 10), taking virus liquid which is not contacted with an antiviral material as 2 parallel blank samples, comparing the blank samples with the virus liquid which is not contacted with the antiviral material, and calculating logarithmic reduction values (LRV (log reduction value) of virus concentrations in each group of virus liquids which are contacted with GO, GO-PEI and GO-N-PEI materials, wherein the values are GO 0.6, GO-PEI 0.9 and GO-N-PEI 1.2 respectively.

7) Coating non-woven fabrics: and (3) carrying out dip coating and spray coating on the non-woven fabric by using the dispersion liquid (containing 10% ethanol aqueous solution) of the GO-N-PEI material, inspecting the antiviral performance of the coated non-woven fabric material after drying, comparing the antiviral performance with that of the non-woven fabric which is not treated by using the GO-N-PEI material, and comparing the effect difference of the two coating modes. The LRV was 1.1 by spray method and 1.2 by dip method.

Example 2

1) Preparing a graphene oxide solution: preparing graphene oxide by a Hummer method, dissolving graphene oxide with corresponding mass in pure water, performing ultrasonic dispersion to obtain a uniformly dispersed graphene oxide suspension, centrifuging, and taking a supernatant, namely a graphene oxide solution with the concentration of 2 mg/mL;

2) preparing a graphene oxide-polyethyleneimine composite material (GO-PEI): 10mL of 1600kDa aqueous solution of branched polyethyleneimine PEI (10mg/mL) was added dropwise to 10mL of aqueous GO (2mg/mL), sonicated for 5min, and 50mg EDC was added. After stirring at room temperature for 30min, 150mg of EDC was added and the reaction was allowed to proceed overnight. Finally, 2g of sodium chloride and 2g of urea are added into the obtained solution, and then the solution is centrifuged at 10000rpm for 15min to remove the precipitate. The suspension was collected, ultra pure water was added and ultrafiltration was repeated with 2000KDa ultrafiltration tube until complete removal of unreacted PEI to give a stable GO-PEI solution which was stored at 4 ℃. When in use, the dried GO-PEI composite material is obtained by freeze drying and other modes.

3) Preparing a GO-N-PEI composite material: performing GO-PEI composite material functionalization by using an alkylating reagent, adding 1-bromohexadecane with the final concentration of 0.5M into tertiary amyl alcohol serving as a solvent, and reacting for 24 hours at the temperature of 80 ℃. And adding 1M methyl iodide to react for 12h at 40 ℃, and obtaining the antiviral material GO-N-PEI by centrifugal separation and other modes.

4) Determination of physical properties of the materials: dispersing the antiviral composite material GO-N-PEI in an aqueous solution containing 20% N-butyl alcohol, and performing ultrasonic treatment for 0.1h to obtain a dispersion liquid of 0.1 mg/mL. The particle size was determined to be 100nm by dynamic light scattering. The solution was uniformly dropped on a glass plate, and after drying, the contact angle was measured to be 69.4 degrees.

5) Virus transfection experiments were performed: and respectively inspecting the damage condition of GO, GO-PEI and GO-N-PEI to the H1N1 influenza virus. 20 mu L of GO, GO-PEI and GO-N-PEI materials are evenly coated on a glass slide and then dried. Transferring 10 μ L of virus liquid with a pipette, uniformly dripping on a glass slide, covering with a cover glass PE sheet, pressing with a heavy object to uniformly distribute the virus liquid, and standing for 30min to make the virus fully contact with GO, GO-PEI and GO-N-PEI materials. Repeatedly washing the surface of a PE sheet of the cover glass with 1mL of PBS for 10 times, then washing the surface of the glass slide for 10 times, respectively eluting and collecting viruses which are contacted with GO, GO-PEI and GO-N-PEI materials, and uniformly mixing the obtained buffer solution containing the viruses and then diluting to obtain six dilutions.

6) 25 μ L of virus solution obtained in step 5) was added to each well of MDCK cell culture plates at different dilutions for plating. Culturing for 2-3 days after coating, calculating the number of plaques generated after cell death under a microscope (multiplied by 10), taking virus liquid which is not contacted with an antiviral material as 2 parallel blank samples, and comparing the blank samples (the virus liquid which is not contacted with the antiviral material), calculating logarithmic reduction values LRV of virus concentrations in each group of virus liquids which are contacted with GO, GO-PEI and GO-N-PEI materials, wherein the logarithmic reduction values LRV are respectively GO 0.7, GO-PEI 1.1 and GO-N-PEI 3.9.

7) Melt-blown fabric coating: and carrying out dip coating and spray coating on a melt-blown fabric by using the dispersion (containing 20% of N-butyl alcohol aqueous solution) of the GO-N-PEI material. And (3) inspecting the antiviral performance of the coated melt-blown fabric material after drying, comparing the antiviral performance with the melt-blown fabric which is not treated by the GO-N-PEI material, and comparing the effect difference of the two coating modes. The LRV was 2.1 by spray method and 2.8 by dip method.

Example 3

1) Preparing a graphene oxide solution: preparing graphene oxide by a Hummer method, dissolving graphene oxide with corresponding mass in pure water, performing ultrasonic dispersion to obtain a uniformly dispersed graphene oxide suspension, centrifuging, and taking supernatant fluid, namely a graphene oxide solution with the concentration of 10 mg/mL;

2) preparing a graphene oxide-polyethyleneimine composite material (GO-PEI): 10mL of an aqueous solution of 87kDa branched PEI (100mg/mL) was added dropwise to 60mL of an aqueous GO solution (10mg/mL), sonicated for 15min, and 150mg EDC was added. After stirring at room temperature for 120min, 300mg of EDC was added and the reaction was allowed to proceed overnight. Finally, 5g of sodium chloride and 10g of urea are added into the obtained solution, and then the solution is centrifuged at 10000rpm for 15min to remove the precipitate. The suspension was collected, ultra pure water was added and ultra filtration was repeated using a 100kDa ultra filtration tube until the unreacted PEI was completely removed, resulting in a stable GO-PEI solution which was stored at 4 ℃. The GO-PEI composite material is obtained by freeze drying and other modes when in use.

3) Preparing a GO-N-PEI composite material: performing GO-PEI composite material functionalization by using an alkylating reagent, adding 1-bromon-hexane with the final concentration of 0.5M into tertiary amyl alcohol serving as a solvent, and reacting for 12 hours at 50 ℃. And adding iodomethane with the final concentration of 2M, reacting for 12h at 40 ℃, and obtaining the antiviral material GO-N-PEI by means of centrifugal separation and the like.

4) Determination of physical properties of the materials: dispersing the antiviral composite material GO-N-PEI in an aqueous solution containing 10% of methyl tert-butyl ether, and performing ultrasonic treatment for 0.2h to obtain a dispersion liquid of 25 mg/mL. The particle size was determined to be 300nm by dynamic light scattering. And (4) obtaining the material morphology characteristics by a scanning electron microscope (TEM). The solution was uniformly dropped on a glass plate, and the contact angle measured after drying was 78.3 degrees.

5) Virus transfection experiments were performed: and respectively investigating the damage conditions of GO, GO-PEI and GO-N-PEI to norovirus. 20 mu L of GO, GO-PEI and GO-N-PEI materials are evenly coated on a glass slide and then dried. Transferring 10 μ L of virus liquid with a liquid transfer gun, uniformly dripping on a glass slide, covering with a cover glass PE sheet, pressing with a heavy object to uniformly distribute the virus liquid, and standing for 15min to make the virus fully contact with GO, GO-PEI and GO-N-PEI materials. Repeatedly washing the surface of a PE sheet of the cover glass with 1mL of PBS for 10 times, then washing the surface of the glass slide for 10 times, respectively eluting and collecting viruses which are contacted with GO, GO-PEI and GO-N-PEI materials, and uniformly mixing the obtained buffer solution containing the viruses and then diluting to obtain six dilutions.

6) 25 μ L of virus solution obtained in step 5) was added to each well of MDCK cell culture plates at different dilutions for plating. Culturing for 2-3 days after coating, calculating the number of plaques generated after cell death under a microscope (multiplied by 10), taking virus liquid which is not contacted with an antiviral material as 2 parallel blank samples, comparing the blank samples (the virus liquid which is not contacted with the antiviral material), and calculating logarithmic reduction values LRV of virus concentrations in each group of virus liquids which are contacted with GO, GO-PEI and GO-N-PEI materials, wherein the logarithmic reduction values LRV are respectively GO 0.9, GO-PEI 1.2 and GO-N-PEI 3.6.

7) Coating non-woven fabrics: dip coating and spray coating of the nonwoven fabric were performed using the dispersion of GO-N-PEI material described above (aqueous solution containing 15% methyl t-butyl ether). And (3) inspecting the antiviral performance of the coated non-woven fabric material after drying, comparing the antiviral performance with that of the non-woven fabric which is not treated by the GO-N-PEI material, and comparing the effect difference of the two coating modes. The LRV was 2.1 by spray method and 3.3 by dip method.

Example 4

1) Preparing a graphene oxide solution: preparing graphene oxide by a Hummer method, dissolving graphene oxide with corresponding mass in pure water, performing ultrasonic dispersion to obtain a uniformly dispersed graphene oxide suspension, centrifuging, and taking supernatant, namely a graphene oxide solution with the concentration of 5 mg/mL;

2) preparing a graphene oxide-polyethyleneimine composite material (GO-PEI): 10mL of a 500kDa linear PEI aqueous solution (50mg/mL) was added dropwise to 100mL of GO aqueous solution (5mg/mL), sonicated for 10min, and 100mg EDC was added. After stirring at room temperature for 180min, 400mg of EDC was added and the reaction was allowed to proceed overnight. Finally, 8g of sodium chloride and 15g of urea are added into the obtained solution, and then the solution is centrifuged at 10000rpm for 15min to remove the precipitate. The suspension was collected, ultra pure water was added and ultra filtration was repeated using an ultra filtration tube of 1000KDa until complete removal of unreacted PEI to give a stable GO-PEI solution which was stored at 4 ℃. The GO-PEI composite material is obtained by freeze drying and other modes when in use.

3) Preparing a GO-N-PEI composite material: performing GO-PEI composite material functionalization by using an alkylating reagent, adding 4-fluorodiphenylmethane with the final concentration of 1M as the alkylating reagent by using tertiary amyl alcohol as a solvent, and reacting for 48 hours at 80 ℃. And adding iodomethane with the final concentration of 2.5M, reacting for 12h at 40 ℃, and obtaining the antiviral material GO-N-PEI by means of centrifugal separation and the like.

4) Determination of physical properties of the materials: dispersing the antiviral composite material GO-N-PEI in an aqueous solution containing 20% dimethyl sulfoxide, and performing ultrasonic treatment for 0.2h to obtain a 25mg/mL dispersion. The particle size was determined to be 300nm by dynamic light scattering. And (4) obtaining the material morphology characteristics by a scanning electron microscope (TEM). The solution was uniformly dropped on a glass plate, and the contact angle measured after drying was 108.3 degrees.

5) Virus transfection experiments were performed: and respectively investigating the damage conditions of GO, GO-PEI and GO-N-PEI to the herpes simplex virus. 20 mu L of GO, GO-PEI and GO-N-PEI materials are evenly coated on a glass slide and then dried. Transferring 10 μ L of virus liquid with a liquid transfer gun, uniformly dripping on a glass slide, covering with a cover glass PE sheet, pressing with a heavy object to uniformly distribute the virus liquid, and standing for 15min to make the virus fully contact with GO, GO-PEI and GO-N-PEI materials. Repeatedly washing the surface of a PE sheet of the cover glass with 1mL of PBS for 10 times, then washing the surface of the glass slide for 10 times, respectively eluting and collecting viruses which are contacted with GO, GO-PEI and GO-N-PEI materials, and uniformly mixing the obtained buffer solution containing the viruses and then diluting to obtain six dilutions.

6) 25 μ L of virus solution obtained in step 5) was added to each well of MDCK cell culture plates at different dilutions for plating. Culturing for 2-3 days after coating, calculating the number of plaques generated after cell death under a microscope (multiplied by 10), taking virus liquid which is not contacted with an antiviral material as 2 parallel blank samples, and comparing the blank samples (the virus liquid which is not contacted with the antiviral material), calculating logarithmic reduction values LRV of virus concentrations in each group of virus liquids which are contacted with GO, GO-PEI and GO-N-PEI materials, wherein the logarithmic reduction values LRV are respectively GO 0.5, GO-PEI 0.6 and GO-N-PEI 1.3. The influence of the contact time of the virus liquid and the GO-N-PEI material on virus killing activity is considered, as shown in figure 3, compared with the virus liquid without the contact of the virus liquid with the antiviral material, the virus killing rate can reach about 90% after the GO-N-PEI material is contacted for 5min, and can be continuously maintained after the GO-N-PEI material is contacted for 10min, until the virus killing rate can reach over 90% after the GO-N-PEI material is contacted for 20 min.

7) Coating the surface of the mobile phone shell: and (3) spraying the surface of the mobile phone shell by using the dispersion liquid (aqueous solution containing 20% of dimethyl sulfoxide) of the GO-N-PEI material. LRV was determined to be 0.8 compared to cell phone shells not treated with GO-N-PEI material.

Example 5

2) preparing a graphene oxide-polyethyleneimine composite material (GO-PEI): 10mL of a 70kDa branched PEI aqueous solution (100mg/mL) was added dropwise to 60mL of GO aqueous solution (10mg/mL), sonicated for 15min, and 150mg EDC was added. After stirring at room temperature for 120min, 300mg of EDC was added and the reaction was allowed to proceed overnight. Finally, 5g of sodium chloride and 10g of urea are added into the obtained solution, and then the solution is centrifuged at 10000rpm for 15min to remove the precipitate. The suspension was collected, ultra pure water was added and ultra filtration was repeated using a 100kDa ultra filtration tube until the unreacted PEI was completely removed, resulting in a stable GO-PEI solution which was stored at 4 ℃. When in use, the dried GO-PEI composite material is obtained by freeze drying and other modes.

3) Preparing a GO-N-PEI composite material: performing GO-PEI composite material functionalization by using an alkylating reagent, adding 1-bromohexadecane with the final concentration of 0.5M into tertiary amyl alcohol serving as a solvent, and reacting for 12 hours at 50 ℃. Then adding 1-iodine-2-methylbutane with the final concentration of 2.5M, reacting for 1h at 80 ℃, and obtaining the antiviral material GO-N-PEI by means of centrifugal separation and the like.

5) Virus transfection experiments were performed: and respectively inspecting the damage conditions of GO, GO-PEI and GO-N-PEI to the avian infectious bronchitis virus. 20 mu L of GO, GO-PEI and GO-N-PEI materials are evenly coated on a glass slide and then dried. Transferring 10 μ L of virus liquid with a liquid transfer gun, uniformly dripping on a glass slide, covering with a cover glass PE sheet, pressing with a heavy object to uniformly distribute the virus liquid, and standing for 15min to make the virus fully contact with GO, GO-PEI and GO-N-PEI materials. Repeatedly washing the surface of a PE sheet of the cover glass with 1mL of PBS for 10 times, then washing the surface of the glass slide for 10 times, respectively eluting and collecting viruses which are contacted with GO, GO-PEI and GO-N-PEI materials, and uniformly mixing the obtained buffer solution containing the viruses and then diluting to obtain six dilutions.

6) 25 μ L of virus solution obtained in step 5) was added to each well of MDCK cell culture plates at different dilutions for plating. Culturing for 2-3 days after coating, calculating the number of plaques generated after cell death under a microscope (multiplied by 10), taking virus liquid without contacting with an antiviral material as 2 parallel blank samples, and comparing the blank samples (the virus liquid without contacting with the antiviral material), and calculating the logarithmic reduction values LRV of the virus concentrations in each group of virus liquids with contacting with GO, GO-PEI and GO-N-PEI materials as GO 0.3, GO-PEI 0.4 and GO-N-PEI 3.6 respectively.

7) Coating the surface of the plastic toy: dip coating and spray coating were performed using the dispersion of GO-N-PEI material described above (aqueous solution containing 15% methyl t-butyl ether). The antiviral performance of the coated plastic toy is examined after drying, and compared with the plastic toy which is not treated by the GO-N-PEI material, the effect difference of the two coating modes is compared. The LRV was 2.1 by spray method and 3.3 by dip method.

8) And (3) testing antibacterial performance: performing antibacterial performance test according to national standard GBT 20944.3-2008, and putting the sterilized GO-N-PEI sample solution into 10⁵Shaking the bacterium solution of escherichia coli and staphylococcus aureus with the concentration for 18h, absorbing the test solution after shaking, diluting the test solution by a 10-fold dilution method, coating a plate, putting the plate into a constant-temperature constant-humidity incubator for culturing for 24h, taking out the plate, and calculating the bacteriostatic rate of a bacterial colony, wherein the LRV is 3.2.

The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.

Claims

1. A broad spectrum antiviral material, characterized by: the antiviral material is an alkylated graphene oxide-polyethyleneimine composite material; carrying out alkylation treatment on the graphene oxide-polyethyleneimine composite material to obtain the alkylated graphene oxide-polyethyleneimine composite material; the preparation method of the graphene oxide-polyethyleneimine composite material comprises the following steps: dripping 10-200 mg/mL of a polyethyleneimine water solution into 1-20 mg/mL of a graphene oxide water solution, ultrasonically dispersing for 5-30 min, then adding carbodiimide, stirring at room temperature for 30-180 min, then adding the carbodiimide, reacting overnight, adding sodium chloride and urea into the obtained solution, removing precipitates, collecting a suspension, and removing unreacted polyethyleneimine by ultrafiltration to obtain a graphene oxide-polyethyleneimine composite material; wherein the formula proportion of the 10-200 mg/mL polyethyleneimine water solution, the 1-20 mg/mL graphene oxide water solution, the carbodiimide, the sodium chloride and the urea is 9-11 mL: 10-100 mL: 200-800 mg: 2-10 g: 2-20 g; the mass ratio of the carbodiimide added for the first time to the carbodiimide added for the second time is 1: 1.5-5.

2. The broad spectrum antiviral material of claim 1, wherein: the alkylated graphene oxide-polyethyleneimine composite material has a plurality of alkylated polyethyleneimine units with the same or different carbon chain lengths, wherein the alkyl group in the alkylated polyethyleneimine units comprises at least one of C1-C20 alkyl, C1-C20 hydroxyalkyl or C7-C20 aralkyl; the polyethyleneimine comprises at least one of linear polyethyleneimine with the molecular weight of 20-500 KDa or branched polyethyleneimine with the molecular weight of 20-1600 KDa.

3. The broad spectrum antiviral material of claim 1, wherein: the alkylation treatment method comprises the following steps: chloroform or tert-amyl alcohol is used as a solvent, 0.01-2M of an alkylating reagent is added, and the reaction is carried out for 1-48 h at the temperature of 0-80 ℃.

4. The broad spectrum antiviral material of claim 3, wherein: the alkylating reagent is alkyl halide shown as a formula RX, wherein R is C1-C20 alkyl, C1-C20 hydroxyalkyl or C7-C20 aralkyl; x is Cl, Br or I.

5. The broad spectrum antiviral material of claim 1, wherein: the preparation method of the graphene oxide aqueous solution comprises the following steps: preparing graphene oxide by a Hummer method, dissolving graphene oxide in pure water, performing ultrasonic dispersion to obtain a uniformly dispersed graphene oxide suspension, and centrifuging to obtain a supernatant, namely a graphene oxide aqueous solution.

6. A method of using the broad spectrum antiviral material of claim 1 or 2, wherein: attaching 0.1-500 mg/mL of a dispersion of the alkylated graphene oxide-polyethyleneimine composite material to a material to be treated; the material to be treated comprises at least one of fabric, plastic or leather; the dispersion is applied by dipping, coating or spraying to achieve adhesion to the material to be treated.

7. The method of using broad spectrum antiviral material of claim 6, wherein: the solvent of the dispersion liquid is an organic solvent or a mixed liquid of the organic solvent and water; the organic solvent comprises at least one of methanol, ethanol, isopropanol, n-butanol, tetrahydrofuran, dimethyl sulfoxide, ethyl acetate, methyl tert-butyl ether, diethyl ether, toluene, dioxane, petroleum ether, n-pentane, cyclopentane, n-hexane, cyclohexane or n-heptane.

8. The method of using broad spectrum antiviral material of claim 6, wherein: the material has antibacterial and antiviral activity, the bacteria including at least one of Escherichia coli or Staphylococcus aureus; the virus includes at least one of dengue virus, coronavirus, enterovirus, influenza virus, or herpes simplex virus.