Graphene-silver nanocomposites and their use as antimicrobial compositions

Abstract

The present invention relates to an antimicrobial composition comprising a graphene-silver cation nanocomposite and its use as an antimicrobial agent. The antimicrobial agent is particularly useful for use as a disinfectant or as a coating to impart antimicrobial activity to a substrate. The antimicrobial agent can also be used to improve filtration efficiency in PPE, face masks, and filters in air filtration (HVAC) systems, as well as other airflow membranes and filters, to reduce the transmission of microbial pathogens.

Description

[Technical Field]

[0001] The present invention relates to the field of antimicrobial agents, and in particular to graphene-silver nanocomposites and compositions thereof for use in imparting antimicrobial activity to substrates. [Background technology]

[0002] Microorganisms (or microbes) are single cells, cell clusters, or multicellular microscopic (or macroscopic) organisms, including, but not limited to, bacteria, fungi, and viruses. Pathogenic microorganisms have the potential to cause numerous infectious diseases through various modes of transmission, including close contact, contact, or airborne transmission. For example, contamination of surfaces with one or more types of microorganisms, transfer of microorganisms between surfaces, and / or aerosol transfer of microorganisms through the air can result in the transmission of illness and disease.

[0003] Infectious diseases caused by pathogenic microorganisms continue to be a global problem, with recent outbreaks including swine flu, Ebola virus, Zika virus, norovirus, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and most recently coronavirus disease 19 (COVID-19), which has been declared a pandemic by the World Health Organization.

[0004] Pathogenic microorganisms are most commonly spread throughout a population by host-to-host transfer via respiratory droplets, close prolonged person-to-person contact, and touching an infected area followed by touching the mouth, nose, or eyes before washing the hands. Efforts to prevent transmission of infection have required the use of antimicrobial disinfectants, physical distancing from infected hosts, and physical protective barriers such as face masks, gloves, and protective clothing, also known as personal protective equipment (PPE). Pathogenic microorganisms have also been shown to spread as aerosols and can also be spread through airborne transmission. Studies of pathogen transmission in enclosed spaces have shown that air recirculation can also result in the spread of pathogenic microorganisms.

[0005] Current PPE is typically made of disposable or reusable materials designed to create a physical barrier to protect the wearer from exposure to pathogenic microorganisms. Similarly, current air filtration systems rely on materials that physically filter out dust, pollen, and other airborne fomites. Materials that filter out pathogenic microorganisms or physically block all exposure to pathogenic microorganisms have limited effectiveness and have raised new transmission challenges. For example, many disease-causing pathogenic microorganisms have been found to be smaller than the filtration size of most filter media used in face masks. In this regard, the virus that causes COVID-19 (SARS-CoV-2) has been identified as having a particle size of 0.125 μm, which is much smaller than the particle size filtered by most face mask materials, including N95 respirators. Further reductions in the filtration size of filter media are not possible without sacrificing the flow rate and exchange of breathable air for the wearer.

[0006] An additional risk of transmission also exists from contact with contaminated substrate surfaces and materials. Disease-causing microorganisms typically remain viable on filter media and other substrate materials for periods of time that can range from several hours to several days. For example, the SARS-CoV-2 virus has been shown to remain viable on materials for three days, and the H1N1 virus has been shown to persist on protective materials for six days. As a result, used and discarded substrate surfaces or protective materials can remain contaminated for relatively long periods of time, potentially posing a risk of secondary transmission of pathogens into the environment.

[0007] There is a need for compositions and / or materials that protect against pathogen transmission at multiple levels, i.e., compositions and / or materials that physically block, trap, or immobilize pathogens to prevent secondary transmission, and that can also destroy pathogens. Additionally, there is a need for antimicrobial compositions and / or materials that are suitable for application to substrate surfaces or fabrication into functional products, such as filter membranes for PPE and ventilation systems, to impart antimicrobial activity.

[0008] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should it be construed, that any of the above information constitutes prior art against the present invention. Summary of the Invention

[0009] The object of the present invention is to provide a graphene-silver nanocomposite and its use as an antimicrobial composition. According to one aspect of the present invention, a graphene-silver nanocomposite is prepared by combining graphene oxide (GO) and silver cations (Ag) bound to GO. + ) moieties are described.

[0010] According to another aspect of the present invention, GO and Ag bound to GO as a complex are prepared. + An antimicrobial nanocomposite is described comprising: According to another aspect of the present invention, GO-Ag + Antimicrobial formulations are described that include a nanocomposite and a solvent, carrier, diluent, and / or dispersant.

[0011] According to another aspect of the present invention, GO-Ag + The use of the nanocomposites as antimicrobial coatings to inactivate pathogenic microorganisms on a substrate is described. According to another aspect of the present invention, a method for imparting antimicrobial activity to a substrate is described, the method comprising: (a) preparing a substrate comprising graphene oxide (GO) and silver cations (Ag +(b) dispersing a nanocomposite comprising: (a) a nanocomposite comprising: (a) a cellulose acetate copolymer; (b ...

[0012] According to another aspect of the present invention, a personal protective equipment (PPE) is described having coated thereon an antimicrobial nanocomposite, the nanocomposite comprising graphene oxide (GO) and silver cations (Ag + ) is included.

[0013] According to another aspect of the present invention, a face mask is described having coated thereon an antimicrobial nanocomposite, the nanocomposite comprising graphene oxide (GO) and silver cations (Ag + ) is included.

[0014] According to another aspect of the present invention, an airflow membrane filter is described having coated thereon an antimicrobial nanocomposite, the nanocomposite comprising graphene oxide (GO) and silver cations (Ag + ) is included.

[0015] These and other features of the present invention will become more apparent in the following detailed description, in which reference is made to the accompanying drawings. [Brief explanation of the drawings]

[0016] [Figure 1] Figure 1 shows a schematic illustration of Ag complexation with oxygen-containing functional groups of GO and the formation of Ag nanoparticles on the surface of GO nanosheets. [Figure 2] 2A-2D are the results of XPS and AES analysis of a sample according to an embodiment of the present invention. [Figure 3] FIG. 3 is a chart showing a comparison of the chemical state of silver and the formed silver nanocomposite. [Figure 4-1] 4A-4F are SEM (SE) and SEM (backscattered electron imaging / Z count) images of GO before contact with silver cations. [Figure 4-2]4A-4F are SEM (SE) and SEM (backscattered electron imaging / Z count) images of GO before contact with silver cations. [Figure 5-1] 5A to 5C are EDS images and result charts of GO before contact with silver cations. [Figure 5-2] 5A to 5C are EDS images and result charts of GO before contact with silver cations. [Figure 5-3] 5A to 5C are EDS images and result charts of GO before contact with silver cations. [Figure 6-1] 6A-6F are SEM (SE) and SEM (Z count) images of GO after contact with silver cations but before purification. [Figure 6-2] 6A-6F are SEM (SE) and SEM (Z count) images of GO after contact with silver cations but before purification. [Figure 7-1] 7A and 7B are EDS images and elemental analysis results of GO after contact with silver cations but before purification. [Figure 7-2] 7A and 7B are EDS images and elemental analysis results of GO after contact with silver cations but before purification. [Figure 8] Figures 8A-8D are SEM (SE) and SEM (Z count) images of GO after contact with silver cations and after purification. [Figure 9-1] Figures 9A and 9B show EDS images and elemental analysis results of GO after contact with silver cations and after purification. [Figure 9-2] Figures 9A and 9B show EDS images and elemental analysis results of GO after contact with silver cations and after purification. [Figure 10] Figures 10A, 10B, and 10C are SEM images of both sides of a fabric sample coated with GO-Ag+ nanocomposite at a high loading concentration of 5 g / L. Figure 10A shows both the rough and fine surfaces, Figure 10B shows the fine surface, and Figure 10C shows the rough surface. [Figure 11]Figures 11A, 11B, 11C, 11D, 11E, and 11F show EDX analysis images of the microsurface of a fabric sample with GO-Ag nanocomposite at a high loading concentration of 5 g / L. Figure 11A shows the EDX image, Figure 11B shows the detection of C, Figure 11C shows the peak identification for the target elements (C, O, and Ag), Figure 11D shows the detection of O, Figure 11E shows the detection of Ag, and Figure 11F shows the peak identification for the target elements (C, O, and Ag). [Figure 12] Figures 12A, 12B, 12C, 12D, 12E, and 12F show EDX images of the rough surface of a fabric sample with a GO-Ag nanocomposite at a high loading concentration of 5 g / L. Figure 12A shows the electron image, Figure 12B shows the detection of C, Figure 12C shows the peak identification for the target elements (C, O, and Ag), Figure 12D shows the detection of O, Figure 12E shows the detection of Ag, and Figure 12F shows the peak identification for the target elements (C, O, and Ag). [Figure 13] Figure 13 is a table showing the elemental composition of both the coarse and fine surfaces of the fabric samples with GO-Ag+ nanocomposite at a high loading concentration of 5 g / L. [Figure 14] Figures 14A, 14B, 14C, and 14D are SEM images of both sides of a fabric sample coated with GO-Ag+ nanocomposite at a low loading concentration of 0.5 g / L. Figure 14A shows the rough surface at 500X, Figure 14B shows the fine surface at 500X, Figure 14C shows the rough surface at 2,000X, and Figure 14D shows the fine surface at 2,000X. [Figure 15] Figures 15A, 15B, 15C, 15D, and 15E show EDX images of the microsurface of a fabric sample with GO-Ag nanocomposite at a low loading concentration of 0.5 g / L. Figure 15A shows the EDX image, Figure 15B shows the detection of C, Figure 15C shows the detection of O, Figure 15D shows the detection of F, and Figure 15E shows the detection of Ag. [Figure 16]Figures 16A and 16B show the peak identifications for the target elements from Figure 15. Figure 16A shows the peak identifications for the target elements (C, O, F, Ag), and Figure 16B shows the peak identifications for the target elements (C, O, F, Ag). [Figure 17] Figures 17A, 17B, 17C, 17D, 17E, and 17F show EDX images of the rough surface of a fabric sample with GO-Ag nanocomposite at a low loading concentration of 0.5 g / L. Figure 17A shows the electron image, Figure 17B shows the detection of C, Figure 17C shows the peak identifications for the target elements (C, O, and Ag), Figure 17D shows the detection of O, and Figure 17E shows the detection of Ag. Figure 17F shows the peak identifications for the target elements (C, O, and Ag). [Figure 18] Figure 18 is a table showing the elemental composition of both the coarse and fine surfaces of the fabric samples with GO-Ag+ nanocomposite at a low loading concentration of 0.5 g / L. DETAILED DESCRIPTION OF THE INVENTION

[0017] Two-dimensional graphene oxide (GO) has shown promise as a nanomaterial for various applications, including biomedical applications, due to its lateral size and the colloidal properties of its nanosheets. The antibacterial effect of GO has also been reported, specifically as a support and stabilizer for antibacterial compounds, such as silver nanoparticles (AgNPs). More specifically, GO-AgNP nanocomposites are believed to improve the antibacterial effect of AgNPs by immobilizing them on GO and preventing nanoparticle migration.

[0018] Although GO-AgNP nanocomposites have been shown to have antibacterial effects, the use of GO-AgNP nanocomposites for broad-spectrum antimicrobial applications remains challenging due to, for example, their instability in water, which limits the availability of antimicrobially active forms of Ag. +It has been shown to have several limitations, such as the ability of GO-AgNPs to effectively release ions being affected. As a result, additional chemicals, solvents, and post-treatments are typically required to stabilize the GO-AgNP nanocomposite during use. According to certain embodiments of the present invention, silver cations (Ag) bound to GO are stable in water without the need for additional chemicals, solvents, or post-treatments. + GO-Ag containing GO nanosheets with ) moieties + Nanocomposites are described.

[0019] According to certain embodiments of the present invention, GO-Ag unexpectedly exhibits strong antibacterial, and even antifungal and / or antiviral effects. + Furthermore, in further embodiments, GO-Ag nanocomposites are described that unexpectedly exhibit potent antimicrobial effects against AMR and / or MDR pathogens. + Without being bound by theory, it is believed that GO is a nanocomposite of Ag. + providing a high surface area for cation binding, + Ag on nanocomposite + This is thought to further stabilize the cations. In this way, GO nanosheets can inhibit Ag, which causes oxidative stress to pathogens. + In addition, the functional oxygen groups on GO, combined with the physical shear effect of the sharp edges of graphene on pathogens, provide a stabilizing framework for the cations. + It is further believed that GO acts synergistically with the antimicrobial effects of cations. Specifically, GO encapsulates and traps pathogens due to its affinity for carbon, and then penetrates the cell membrane to bind Ag. + It is thought that this transfers the oxidative stress caused by the disease to the pathogen.

[0020] According to embodiments, unlike mixing preformed nanoparticles with GO, which often form aggregates, silver cations (Ag +The in-situ binding of Ag with GO promotes uniformly dispersed complexes. This increases the surface area of ​​the formed active material and allows Ag to create oxidative stress for pathogens. + According to an embodiment, GO-Ag + Nanocomposite GO and Ag + The components have a synergistic antimicrobial effect. In certain embodiments, GO-Ag + The nanocomposites exhibit broad-spectrum antimicrobial efficacy at concentrations of less than 0.1 μg / mL, less than 0.08 μg / mL, less than 0.06 μg / mL, less than 0.04 μg / mL, less than 0.02 μg / mL, less than 0.009 μg / mL, or less than 0.007 μg / mL.

[0021] In certain embodiments, GO-Ag + The nanocomposite consists of graphene oxide (GO) sheets and silver cations (Ag + In a further embodiment, the nanocomposite comprises a silver cation (Ag + In a further embodiment, the nanocomposite comprises GO sheets and Ag bound to GO by complex bonds. + The present invention also includes a combination of silver nanoparticles (AgNPs) chemically bound to GO and, in some cases, physically adsorbed thereto. According to certain embodiments, the majority of the bound silver is in cationic form (Ag + ) and bound to GO. According to some embodiments, the majority of the bound silver is in cationic form and bound to GO as a complex. In further embodiments, the majority of the bound silver is in cationic form and bound to GO as a complex, and also includes a small amount of silver nanoparticles. The silver nanoparticles are formed by the reduction of the cationic form followed by the oxidation of certain functional groups on GO. Such functional groups may include epoxy, hydroxyl, carboxyl, carbonyl, quinone, or any specific functional group introduced to the surface during GO fabrication. In certain embodiments, Ag +can be attached to the GO surface by adding functional groups, such as N or P functional groups, onto GO. According to certain embodiments described herein, GO-Ag + The nanocomposite is composed of complexed GO-Ag + In another embodiment, the GO-Ag + The nanocomposite is composed of complexed GO-Ag + and free Ag + According to further embodiments described herein, GO-Ag + The nanocomposites contain Ag in various chemical states, such as Ag(0) or Ag(1) salts, oxides, or metallic forms. In certain embodiments, 50-98% of the bound silver is bound to GO through complex bonding. According to other embodiments, 65-85% of the bound silver is bound to GO through complex bonding. In further embodiments, 70% of the bound silver is bound to GO through complex bonding. In other embodiments, 85-95% of the bound silver is bound to GO through complex bonding. In further embodiments, 90-95% of the bound silver is bound to GO through complex bonding.

[0022] In a further embodiment, the GO sheets of the present invention comprise Cu 2+ and / or Zn 2+ and / or Au 2+ GO-Ag of one or more other metals such as cations or combinations thereof + The nanocomposite is designed to have a sufficiently large surface area to accommodate additional antimicrobial agents, such as those added to the nanocomposite. + The nanocomposite further includes one or more other metals and metal-ligand complexes, such as copper (e.g., copper ions, copper nanoparticles), gold (gold ions, gold nanoparticles), and / or zinc (zinc ions, zinc nanoparticles), or any combination thereof.

[0023] According to an embodiment of the present invention, GO-Ag + The nanocomposite compositions provide broad-spectrum antimicrobial activity. In some embodiments, the described GO-Ag+ The nanocomposite composition has potent antimicrobial activity against bacterial, viral, or fungal pathogens, or any combination thereof. + The nanocomposite composition has antimicrobial activity against drug-resistant and / or multi-drug resistant strains of microorganisms.

[0024] In certain embodiments of the present invention, GO-Ag + Nanocomposite compositions are provided as formulations for inactivating a broad spectrum of pathogens on a variety of substrates. According to embodiments, formulations are provided that are useful for providing antimicrobial efficacy to a variety of surfaces. In some embodiments, the surfaces are solid substrates, including, but not limited to, countertops, door handles, faucets, telephones, cell phones, beds / bed frames, bedding, medical equipment, computers, automobile dashboards, chair components, various handheld devices and instruments, writing implements, surgical equipment, animal cages, kennels, and animal or livestock stables.

[0025] GO-Ag can be applied directly to substrate surfaces to impart antimicrobial effects + The nanocomposite formulations are provided in certain embodiments as solutions and / or suspensions and / or dispersions and / or emulsions. According to certain embodiments, the formulations are applied as disinfectants to high touchpoint substrate surfaces or enclosures. In certain embodiments, the substrate surface is a wall, or countertop, floor, bench, desk, or animal or livestock stable, kennel, or animal cage. In certain embodiments, the GO-Ag nanocomposite formulations described herein are also useful. +The nanocomposites can be attached to or coated (e.g., adsorbed or bound) onto the surface of an article of manufacture to impart antimicrobial properties to the article of manufacture. Certain embodiments of the present invention relate to formulations of the nanocomposites of the present invention provided as coatings that can be applied to the surface of materials to impart antimicrobial properties to various products. According to certain embodiments, these products include face masks, PPE, environmental cleaning wipes and other sanitation-related products, counters, door handles, walls, filters in air filtration (HVAC) systems, and other airflow membranes and filters to reduce the pathogenic activity of microbial pathogens.

[0026] According to certain embodiments, the formulations described herein impart antimicrobial properties to the treated surface by directly inactivating / killing microorganisms. In certain embodiments, the formulations also impart improved filtration efficiency to the treated substrate. According to such embodiments, GO-Ag + The hydrophilicity of the nanocomposite effectively attracts, retains, and / or immobilizes microorganisms on the treated substrate surface, thereby inhibiting their passage through the treated substrate. + The nanocomposite was able to attach microorganisms in saliva aerosol from infected individuals onto GO sheets, while simultaneously trapping Ag + causes oxidative stress to microorganisms. + This aspect of the nanocomposite offers particular applications in certain embodiments, such as face masks, air filters, and membranes.

[0027] According to various embodiments, the formulations described herein impart high antimicrobial properties to treated surfaces that remain stable over time. According to certain embodiments, the GO-Ag +Nanocomposite formulations kill (or otherwise inactivate) at least 75-100% of pathogens upon contact with the formulation. In further aspects, such formulations are stable for at least 3-6 weeks after application and kill (or otherwise inactivate) at least 90-100% of pathogens upon contact with the formulation. In other embodiments, efficacy persists for up to 3 months. According to further embodiments, efficacy persists for up to 2 months. In additional embodiments, efficacy persists for up to 1 month.

[0028] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0029] As used herein, the term "about" refers to approximately a + / - 10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to therein.

[0030] The use of the words "a" or "an" when used herein in conjunction with the term "comprising" can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more."

[0031] As used herein, the term "antimicrobial" refers to the destruction and / or inactivation of pathogenic microorganisms / microorganisms. The use of the terms "antiviral" and "viricidal" may be used interchangeably herein to refer to the destruction and / or inactivation of viruses.

[0032] The use of the terms "antimicrobial" and "bactericidal" may be used interchangeably herein to refer to the destruction and / or inactivation of bacteria. The use of the terms "antifungal" and "fungicidal" may be used interchangeably herein to mean the destruction and / or inactivation of fungi.

[0033] As used herein, the words "comprising" (and its grammatical variations, such as "comprise" and "comprises"), "having" (and its grammatical variations, such as "have" and "has"), "including" (and its grammatical variations, such as "includes" and "include"), or "containing" (and its grammatical variations, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrequited elements or method steps.

[0034] "Attenuate," "inhibit," "prevent," "treat," and grammatical variations thereof, as used herein, refer to a measurable decrease in a given parameter or event.

[0035] As used herein, the term "pathogen" means an organism capable of causing disease or injury in a host, and includes, but is not limited to, bacteria, viruses, protozoa, fungi, and parasites.

[0036] As used herein, the term "minimum inhibitory concentration" or "MIC" refers to the lowest concentration of an antimicrobial compound / agent that reduces the viability of the initial inoculum of microorganisms by ≧99.9%. As used herein, the terms "drug resistance" or "drug-resistant" refer to a pathogen that is resistant to one or more antimicrobial agents.

[0037] As used herein, the term "multidrug resistant" or "MDR" refers to a pathogen that is resistant to two or more antimicrobial agents, drugs, or medications. As used herein, the term "complex bond," also known as acid-base Lewis interactions, coordinate bonds, chelate bonds, or coordinate covalent bonds, refers to the formation of a coordination compound in which silver ions are bound to GO via coordinate covalent bonds. The complex or chelate bond occurs because the oxygen groups of GO have at least two unshared electron pairs, and both electrons involved in the bond come from the ligand. These unshared electron pairs are regions of negative charge to which silver cations are attracted. When only two unshared electron pairs form a complex with silver, this is known as a bidentate configuration. When three unshared electron pairs form a complex with silver, this is known as a tridentate configuration.

[0038] It is contemplated that any embodiment discussed herein may be implemented with respect to any method of the composition of the invention, and vice versa. Additionally, the compositions of the invention can be used to practice the methods of the invention.

[0039] Fabrication of graphene oxide / silver cation nanocomposites Graphene oxide (GO) is a two-dimensional form of carbonaceous material whose edges and basal planes are adorned with oxygen-containing groups, including epoxide, carboxyl, and hydroxyl groups. GO is known to have a high surface area and is non-corrosive. GO can be synthesized by standard methods known in the art, such as the Staudenmaier method, the Hofmann method, the Brodie method, the Hummers method, and electrochemical exfoliation, which include mechanical or thermal exfoliation, chemical vapor deposition (CVD), and epitaxial growth.

[0040] According to certain embodiments, GO may be synthesized by any standard method known in the art. In certain embodiments, GO may be synthesized by electrochemical exfoliation. In further embodiments, GO may be synthesized by the Hummers method.

[0041] According to further embodiments, GO may be synthesized by a modified Hummers method. In one particular embodiment, GO is synthesized by a modified Hummers method that eliminates phosphoric acid from the process steps. Unexpectedly, it has been discovered that eliminating the use of phosphoric acid from the synthesis process reduces the number of chemicals required, making the synthesis more efficient and reducing the number of steps.

[0042] In a preferred embodiment, GO is synthesized to maximize the surface area available for binding silver ions. According to such an embodiment, the method is adapted to synthesize smaller graphene flakes to produce GO with increased surface area. In certain embodiments, for example, the size of the GO flakes is reduced through ultrasonic treatment. In certain embodiments, the graphene flakes have a particle size in the range of 500 nm to 5 μm. In further embodiments, the graphene flakes have a particle size in the range of 750 nm to 4 μm. In additional embodiments, the graphene flakes have a particle size in the range of 1 to 3 μm.

[0043] In various embodiments, the GO has an oxygen content of 5-40%. In further embodiments, the GO has an oxygen content of 10-35%. In additional embodiments, the GO has an oxygen content of 15-25%. In further embodiments, the GO has an oxygen content of 20-25%. In other embodiments, the GO has an oxygen content of 20-35%. In additional embodiments, the GO has an oxygen content of 28-35%.

[0044] In various embodiments, the GO has 1-10 layers with an interlayer distance in the range of 0.3 nm to 1 nm. In certain embodiments, the GO has 1-10 layers with an interlayer distance in the range of 0.3-0.8 nm. In further embodiments, the GO has 1-10 layers with an interlayer distance of at least about 0.8 nm. In additional embodiments, the GO has 1-10 layers with an interlayer distance of at least about 0.4 nm.

[0045] In certain embodiments, the GO has at least 3 layers. In further embodiments, the GO has at least 4 layers. In additional embodiments, the GO has at least 5 layers. In other embodiments, the GO has at least 7 layers.

[0046] Silver nanoparticles (AgNPs) have been widely studied as antimicrobial agents, but the effectiveness of AgNPs in inactivating various types of bacteria and viruses is limited by the ability of AgNPs to release silver ions. AgNPs can also be released from AgNPs to directly interact with phosphorus- or sulfur-containing biomolecules, including DNA, RNA, and proteins. + The release rate of AgNPs influences the antimicrobial efficacy of AgNPs. In particular, the size, shape, and concentration of AgNPs have been identified as limiting factors affecting their antimicrobial capacity.

[0047] Therefore, various embodiments described herein may be used to prepare silver cations (Ag + ) to GO. In certain embodiments, the GO contains 3-25 wt.% silver in its cationic form. In other embodiments, the concentration of silver cations bound to GO is 5-15 wt.%. In further embodiments, the concentration of silver cations bound to GO is 10-20 wt.%. According to further embodiments, the concentration of silver cations bound to GO is 3-10 wt.%. In other embodiments, the concentration of silver cations bound to GO is 4-8 wt.%.

[0048] According to a further embodiment, GO-Ag + The nanocomposite also contains colloidal silver, i.e., AgNPs. In such an embodiment, GO-Ag + The Ag / AgNP nanocomposites were prepared by mixing these two forms of silver in the ratios of 500:1, 400:1, 300:1, 200:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, and 5:1 Ag. + In certain embodiments, the Ag bound to the GO nanocomposite may comprise AgNPs in a ratio of 0.1:0.2. +According to a further embodiment, the ratio of Ag to AgNPs bound to the GO nanocomposite is 50:1. + The ratio of AgNPs to AgNPs is 40:1. According to another embodiment, the Ag NPs bound to the GO nanocomposite + The ratio of AgNPs to AgNPs is 20:1. According to one particular embodiment, the Ag bound to the GO nanocomposite + In a further embodiment, the ratio of Ag to AgNPs bound to the GO nanocomposite is 15:1. + The ratio of AgNPs to AgNPs is 10:1. According to one particular embodiment, the Ag bound to the GO nanocomposite + In a further embodiment, the ratio of Ag NPs bound to the GO nanocomposite is 11:1. + The ratio of AgNPs to AgNPs is 10:1 to 15:1.

[0049] FIG. 1 shows the Ag + The non-limiting reaction of ions and AgNPs to adhere to GO is shown. GO-Ag as described herein + It is further contemplated that the nanocomposite may include additional metals. Exemplary, but not limiting, nanocomposites described herein include GO-Ag + The nanocomposite and one or more other metals. Exemplary other metals include, but are not limited to, copper (e.g., copper ions, copper nanoparticles), gold (gold ions, gold nanoparticles), and zinc (zinc ions, zinc nanoparticles).

[0050] In certain embodiments, the GO-Ag + The nanocomposite is mixed with additives such as cations and nanomaterials or combinations thereof. Cations include, but are not limited to, Ag + , Cu 2+ , Zn 2+ Metal nanomaterials include, but are not limited to, Ag, Cu, and Zn. In further embodiments, these additives are chemically bonded to GO and, in some cases, physically adsorbed to GO.

[0051] According to one non-limiting embodiment of the present invention, the antimicrobial nanocomposite comprises: (a) synthesizing graphene oxide (GO); (i) mixing graphite powder with a 98% sulfuric acid solution in a volume to mass ratio of sulfuric acid solution to graphite powder of about 30 mL:1 g to form a suspension; (ii) sonicating the suspension three times at 6 hour intervals for 30 minutes at 50°C; (iii) transferring the suspension to an ice-water bath and adding potassium permanganate to the suspension in portions in a mass ratio of said potassium permanganate to graphite powder of about 4:1; (iv) stirring the mixture at 35°C for up to 12 hours with intermittent sonication for 15-30 minutes after 8 hours; (v) reducing the temperature of the mixture to below 5°C in an ice bath and adding distilled water at a temperature of 2-5°C to the mixture in a volume to mass ratio of about 100 mL:1 g of water to graphite; (vi) adding 30% hydrogen peroxide dropwise to the mixture until the color of the mixture changes from dark brown to yellow; (vii) sonicating the mixture for 30 minutes to promote separation of the GO nanosheets; and (viii) purifying the GO nanosheets by washing three times with a 1 M hydrochloric acid solution, followed by three times with a mixture of water and ethanol (8:2 v / v), the mixture being sonicated for 30 min between each wash, and the pH of the mixture being adjusted to 3-4 by adding a 1 M potassium hydroxide solution; and (b) immobilizing silver cations on GO nanosheets, (i) sonicating GO nanosheets in deionized water at a volume to mass ratio of about 0.1 g:30 mL GO nanosheets to water for 30 minutes to form a suspension, and adjusting the pH of the suspension to 10 using 0.1 M sodium hydroxide solution; (ii) adding 10 M silver nitrate solution to the suspension in a volume-to-mass ratio of silver nitrate solution to GO nanosheets of about 0.1 g:2 mL; (iii) adding deionized water to the suspension in a volume-to-mass ratio of deionized water to GO nanosheets of about 0.1 g:20 mL and stirring at 60° C. for 20 hours to reduce the viscosity of the solution; and (iv) centrifugal washing of the suspension in deionized water three times to recover the graphene oxide-silver cation nanocomposite; and It is produced by a method comprising:

[0052] Without being limited to any particular theory, it is believed that the GO-silver cation nanocomposite forms in part by an initial monolayer of silver cations being deposited and strongly bound to the GO sheet, and subsequent layers of silver cations being weakly bound, primarily by physisorption.

[0053] antimicrobial activity In various embodiments, the GO-Ag + The nanocomposites, and / or compositions or formulations comprising these nanocomposites, exhibit antimicrobial activity against a spectrum of microbial targets. In particular, and unexpectedly, the GO-Ag nanocomposites described herein + The nanocomposite was found to exhibit minimum inhibitory concentrations (MICs) of <1 μg / mL against a spectrum of microbial targets. The unexpectedly low MIC values ​​across this spectrum were due to the GO-Ag + These results suggest that the nanocomposites are effective antimicrobial agents with minimal risk of toxicity in animals and humans.

[0054] According to certain embodiments, the GO-Ag + The nanocomposite exhibits an MIC of <1 μg / mL against microbial pathogens. In a further embodiment, the GO-Ag nanocomposite described herein +The nanocomposite exhibits an MIC of <0.5 μg / mL against microbial pathogens. In other embodiments, the GO-Ag nanocomposite described herein + The nanocomposite exhibits an MIC of <0.25 μg / mL against microbial pathogens. In further embodiments, the GO-Ag nanocomposite described herein + The nanocomposite exhibits an MIC of <0.125 μg / mL against microbial pathogens. In other embodiments, the GO-Ag nanocomposite described herein + The nanocomposite exhibits an MIC of <0.0625 μg / mL against microbial pathogens. In a further embodiment, the GO-Ag nanocomposite described herein + The nanocomposite exhibits an MIC of <0.031 μg / mL against microbial pathogens. In other embodiments, the GO-Ag nanocomposite described herein + The nanocomposite exhibits an MIC of <0.0156 μg / mL against microbial pathogens. In further embodiments, the GO-Ag nanocomposite described herein + The nanocomposite exhibits an MIC of <0.008 μg / mL against microbial pathogens.

[0055] In certain embodiments, the GO-Ag + The nanocomposite exhibits antimicrobial effects against viral, bacterial, and / or fungal pathogens. In further embodiments, the GO-Ag nanocomposite described herein + The nanocomposites and / or compositions or formulations comprising these nanocomposites exhibit virucidal and / or antiviral effects against viral targets, including, but not limited to, enveloped viruses such as herpesviruses, poxviruses, hepadnaviruses, asfarviruses, flaviviruses, alphaviruses, togaviruses, coronaviruses, hepatitis D virus, orthomyxoviruses, paramyxoviruses, rhabdoviruses, bunyaviruses, filoviruses, and retroviruses. According to further embodiments, the GO-Ag +The nanocomposite compositions may be prepared from a wide variety of viruses, including those from the Adenoviridae, Arenaviridae (e.g., Ippivirus and Lassa virus), Birnaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae (e.g., yellow fever virus, dengue virus, hepatitis C virus), Hepadnaviridae (e.g., hepatitis B virus), Herpesviradae (e.g., human herpes simplex virus type 1), Orthomyxoviridae (e.g., For example, they exhibit virucidal and / or antiviral effects against viral pathogens including viruses from the families Influenza A, B, and C), Paramyxoviridae (e.g., mumps virus, measles virus, and respiratory syncytial virus), Picornaviridae (e.g., poliovirus and hepatitis A virus), Poxviridae, Reoviridae, Retroviradae (e.g., BLV-HTLV retrovirus, HIV-1, HIV-2, bovine immunodeficiency virus, and feline immunodeficiency virus), Rhabodoviridae (e.g., rabies virus), and Togaviridae (e.g., rubella virus).Non-limiting examples of relevant pathogenic viruses include, but are not limited to, various strains of influenza virus, cytomegalovirus, various strains of respiratory syncytial virus (such as human respiratory syncytial virus and certain animal strains), various strains of parainfluenza virus (such as human parainfluenza virus and certain animal strains), coronavirus (such as human coronavirus, SARS-CoV, MERS-CoV, and Covid-19 coronavirus), rhinovirus (such as human rhinovirus), enterovirus (such as human enterovirus), adenovirus (such as human adenovirus), bocavirus (such as human bocavirus), metapneumovirus (such as human metapneumovirus), dengue virus, various hepatitis viruses, human immunodeficiency virus (HIV), West Nile virus, rabies virus, human papillomavirus (HPV), Epstein-Barr virus (EBV), and polyomavirus. In certain embodiments of the present invention, GO-Ag is used. + The nanocomposite exhibits virucidal and / or antiviral effects against influenza virus, flavivirus (such as dengue virus or yellow fever virus), parainfluenza virus, human metapneumovirus, respiratory syncytial virus, coronavirus (such as Covid-19 coronavirus, SARS coronavirus, MERS coronavirus), rhinovirus, or adenovirus.

[0056] In a further embodiment, the GO-Ag +The nanocomposites and / or compositions or formulations comprising these nanocomposites exhibit antimicrobial effects against bacterial pathogens. According to certain embodiments, the bacterial pathogens include gram-positive bacteria. In other embodiments, the bacterial pathogens include gram-negative bacteria. Bacterial pathogens include, for example, various bacterial species of the genera Bacillus, Yersinia, Francisella, Haemophilus, Streptococcus, Staphylococcus, Pseudomonas, Mycobacterium, and Burkholderia.Non-limiting examples of relevant pathogenic bacterial species include, but are not limited to, Bacillus anthracis, Yersinia pestis, Francisella tularensis, Streptococcus pnemoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia, Corynebacterium diphtheriae, Legionella pneumophila, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Mycobacterium tuberculosis, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Escherichia coli, Coxiella burnetii, Clostridium spp., and Shigella spp.

[0057] In certain embodiments, the GO-Ag +The nanocomposites and / or compositions or formulations containing these nanocomposites exhibit antifungal effects against fungal pathogens, such as Histoplasma capsulatum, Coccidiodes immitis, Blastomyces dermatitidis, Cryptococcus neoformans, Aspergillus fumigatus, Candida albicans, and Pneumocystis carinii.

[0058] In a further embodiment, the GO-Ag + The nanocomposite exhibits antimicrobial efficacy against antimicrobial-resistant (AMR) and / or multidrug-resistant (MDR) microbial pathogens. In certain embodiments, AMR and / or MDR pathogens include Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Acinetobacter baumannii, Stenotrophomonas maltophilia, Streptococcus pneumoniae, Staphylococcus aureus, Candida auris, influenza virus, extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli, ESBL-producing Klebsiella pneumoniae, carbapenem-resistant organisms (CROs), Enterobacter species, and the like. spp.), penicillin-resistant Streptococcus pneumoniae, CA-MRSA, HA-MRSA, and Acinetobacter baumannii species complex. +The nanocomposites exhibit antimicrobial efficacy against antimicrobial-resistant (AMR) and / or multidrug-resistant (MDR) microbial pathogens known as ESKAPE pathogens. According to such embodiments, the GO-Ag nanocomposite described herein + The nanocomposite exhibits antimicrobial efficacy against any one or more of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species.

[0059] antimicrobial agents The present invention is directed to GO-Ag + An antimicrobial formulation comprising a nanocomposite is provided. The formulation can be formulated for application by various methods. For example, the formulation can be formulated as a powder, spray, gel, foam, film, or coating for application to a substrate. In certain embodiments, the formulation can include one or more suitable agents, carriers, diluents, and / or dispersants, as would be known to those skilled in the art. If desired, other active ingredients, such as additional antimicrobial agents, can be included in the formulation.

[0060] According to various embodiments, the GO-Ag described herein + The nanocomposite formulation was GO-Ag + To facilitate application of the nanocomposite to a substrate surface, it may be provided as a solution and / or suspension and / or dispersion and / or emulsion. For example, the GO-Ag nanocomposite described herein may be provided as a solution and / or suspension and / or dispersion and / or emulsion. + The nanocomposite is GO-Ag + The nanocomposite is formulated as an aqueous suspension by suspending it in an industry-accepted solvent, carrier, and / or diluent. In certain embodiments, the formulation comprises the GO-Ag nanocomposite as described herein. +The nanocomposite may be combined with ethanol, deionized water, both ethanol and deionized water, isopropyl alcohol, isopropyl alcohol and deionized water, isopropyl alcohol, ethanol, and deionized water, isopropyl alcohol, hydrogen peroxide, and deionized water, hydrogen peroxide, ethanol, isopropyl alcohol, and water, or any combination thereof. In certain embodiments, the antimicrobial formulations described herein contain between about 40 mg / L and 5 g / L of GO-Ag. + In another embodiment, the formulation has a nanocomposite concentration of GO-Ag at a concentration of about 2-20 g / L. + In a further embodiment, the formulation comprises a GO-Ag nanocomposite at a concentration of about 3-5 g / L. + Includes nanocomposites.

[0061] In certain embodiments, the antimicrobial formulation may be formulated as a dispersible powder or granules, which can then be used to prepare an aqueous suspension by adding a suitable solvent, carrier, and / or diluent, such as water. Such dispersible powders or granules may be prepared by dispersing GO-Ag in admixture with one or more dispersing or wetting agents and / or suspending agents. + Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional additives, such as colorants and / or fragrances, may also be included in these formulations.

[0062] According to certain embodiments, the formulations may be prepared for application by spin coating, dip coating, bath dip coating, spray coating, solvent evaporation. + The nanocomposite coating is fixed by air drying, heat drying, thermal drying, sun drying, vacuum drying, or drying under ambient conditions, or a combination of these methods. + The nanocomposite is coated onto the substrate surface to a thickness ranging from about 5 nm to about 5 μm. +The nanocomposite is coated onto the substrate surface to a thickness ranging from about 100 nm to about 3 μm. + The nanocomposite is coated onto the substrate surface to a thickness ranging from about 200 nm to about 2 μm. + The nanocomposite is coated onto the substrate surface to a thickness ranging from about 300 nm to about 1.5 μm. In another embodiment, the GO-Ag + The nanocomposite is coated onto the surface of the substrate to a thickness in the range of about 500 nm to about 1.0 μm.

[0063] When applied to a substrate surface, the GO-Ag + The nanocomposite formulations are stable and maintain the ability to kill (or otherwise inactivate) at least 75-100% of pathogens upon contact with the formulation after application to the treated surface. In a further aspect, such formulations are stable for at least 3-6 weeks after application and kill (or otherwise inactivate) at least 90-100% of pathogens upon contact with the formulation on the treated surface. In other embodiments, the GO-Ag formulations described herein are also suitable for use in treating microbial infections. + The antimicrobial efficacy of the nanocomposite formulations persists for up to three months. According to further embodiments, the GO-Ag nanocomposite formulations described herein + The antimicrobial efficacy of the nanocomposite formulations persists for up to two months. In additional embodiments, the GO-Ag nanocomposite formulations described herein + The antimicrobial efficacy of the nanocomposite formulations lasts for up to one month. According to various embodiments, the formulations described herein impart high antimicrobial properties to treated surfaces that remain stable over time.

[0064] In some embodiments, the above-referenced formulation components may be used in various non-limiting combinations to provide formulations optimized for specific purposes (e.g., selectively targeting one or more specific types of microorganisms and killing or inhibiting them at a particular level, etc.). Additionally, in some embodiments, the above-referenced components comprising any one of the formulation embodiments may be recognized by those of skill in the art by trade name or trademark or formula chemical nomenclature. Other formulations and methods of making antimicrobial formulations are known in the art and within the capabilities of one of skill in the art.

[0065] Methods and Uses GO-Ag as described herein + The nanocomposites and / or compositions or formulations comprising these nanocomposites are useful as broad-spectrum antimicrobial agents, including antiviral, antibacterial, and / or antifungal agents in a wide variety of applications. For example, GO-Ag + The nanocomposites can be used to disinfect a variety of materials, including equipment, textiles, enclosure walls, countertop surfaces, or high-touch surfaces of any metal, plastic, stone, and / or wood. + The nanocomposites may also be used as surface coatings to impart broad-spectrum antimicrobial, antiviral, antibacterial, and / or antifungal properties to reduce or eliminate the activity of microbial pathogens on various materials and articles of manufacture, including, but not limited to, PPE, face masks, counters, door handles, walls, air filtration (HVAC) system filters, and other airflow membranes and filters.

[0066] The disinfectant formulation of the present invention can be applied to the surface to be disinfected (i.e., cleaned) by various spraying techniques. In one embodiment, the disinfectant formulation of the present invention is applied using a spray nozzle, a diffuser, or a misting machine. Alternatively, the disinfectant formulation of the present invention can be formulated into an aerosol formulation. Further means of applying the disinfectant solution of the present invention are within the capabilities of those skilled in the art. The disinfectant formulation of the present invention can be applied as is or diluted before application.

[0067] In certain embodiments, the GO-Ag + The nanocomposites are particularly useful as virucides and / or antivirals against viral pathogens, such as enveloped viruses. According to further embodiments, the GO-Ag nanocomposites described herein are + The nanocomposites are useful as virucides and / or antivirals against coronaviruses. In further embodiments, the GO-Ag nanocomposites described herein are + The nanocomposites are useful as virucidal and / or antiviral agents against the SARS-CoV-2 virus.

[0068] In various embodiments, the GO-Ag + The nanocomposites may be provided in compositions / formulations and used directly or may be attached to the surface of an article of manufacture. Thus, for example, the GO-Ag nanocomposites described herein may be used in compositions / formulations and applied directly to an article of manufacture. + The nanocomposite may be provided in a solution and / or suspension and / or dispersion and / or emulsion for direct use. In certain embodiments, the GO-Ag nanocomposite described herein may be prepared in a solution and / or suspension and / or dispersion and / or emulsion for direct use. + The nanocomposites can be attached to or coated (e.g., adsorbed or bonded) to the surface of an article of manufacture to impart antimicrobial, antiviral, antibacterial, and / or antifungal properties to the article of manufacture. Thus, for example, the GO-Ag nanocomposites described herein can be used in conjunction with other nanocomposites. + The nanocomposites can be attached to high-contact surfaces of metals, plastics, stone, and wood.

[0069] In addition to reducing microbial transmission via transfer from high-touch surfaces, the GO-Ag + The nanocomposites can also be attached to certain surfaces to reduce the transmission of microorganisms via airborne aerosols. For example, the GO-Ag nanocomposite described herein + The nanocomposites may be attached to face masks, protective clothing, respirators, and other PPE, filters in HVAC systems, and other airflow membranes and filters to capture, reduce the activity of, or destroy microbial pathogens. In certain embodiments, the GO-Ag nanocomposites described herein may be used in various applications. + The nanocomposites can be attached to or coated on the surfaces of PPE, face masks, counters, door handles, walls, filters in air filtration (HVAC) systems, and other airflow membranes and filters. In certain embodiments, the GO-Ag nanocomposites described herein + The nanocomposites can be used to improve the filtration efficiency of substrates such as medical masks, PPE, airflow membranes, and filters. In certain embodiments, the GO-Ag nanocomposites described herein + The nanocomposites may increase the bacterial and viral filtration efficiency of face masks and filters in air filtration (HVAC) systems. According to further embodiments, the GO-Ag nanocomposite described herein + The nanocomposites may be used to improve the filtration efficiency of water filtration membranes, including, but not limited to, reverse osmosis, dialysis, and ion-selective membranes and filters, including filter papers, fabrics, and substrate filter beds. + Application of the nanocomposite improves the filtration efficiency of the treated substrate by at least 85%, 90%, 95%, 97%, 98%, or 99% over the uncoated substrate.

[0070] In various embodiments, the GO-Ag +The nanocomposites and / or compositions or formulations comprising these nanocomposites are used to kill, capture, immobilize, and / or otherwise inactivate any of a wide variety of pathogen targets and / or to prevent the spread of pathogens and associated diseases in animals and humans.

[0071] In order to gain a better understanding of the invention described herein, the following examples are set forth. It is understood that these examples are intended to describe exemplary embodiments of the invention and are not intended to limit the scope of the invention in any way.

[0072] Example Example 1: Fabrication of graphene-silver cation nanocomposite GO-Ag + The nanocomposites were prepared according to the following method, based on 1 g of graphite powder.

[0073] Synthesis of GO One gram of graphite powder was immersed in 30 mL of H2SO4 (98%) solution in an Erlenmeyer flask under a fume hood with stirring at 50 °C and 300 rpm for 18 hours. The suspension was sonicated three times for 30 minutes every 6 hours using a sonication bath. 4 g of KMnO4 was then added portionwise to the mixture. During the exothermic oxidation reaction, the temperature of the mixture was reduced using an ice / water bath. The mixture was kept under continuous stirring at 35 °C for up to 12 hours. After 8 hours of stirring, three ultrasonic bath treatments (15–30 minutes each) were used. Next, 100 mL of cold distilled water (2–5 °C) was added to the mixture. The temperature of the mixture was maintained below 5 °C using an ice bath. H2O2 (30%) was then added dropwise to the diluted mixture until the color of the mixture changed from dark brown to yellow. Prior to purification, the mixture was treated in an ultrasonic bath for 30 min to facilitate the separation of the exfoliated GO nanosheets from each other. The final suspension was washed three times with HCl (1 M) and three times with a water / ethanol mixture (8:2 v / v). Between purification steps, ultrasonic bath treatment (30 min) was used to facilitate the removal of impurities intercalated between the GO layers. The pH of the sample was adjusted to 3–4 with 1 M KOH solution during centrifugation for better precipitation. The final GO nanosheets were dispersed in distilled water and stored for further use.

[0074] Silver + ) Synthesis of doped GO Graphene oxide (GO) nanosheets were synthesized as a platform for silver cations. To synthesize Ag-doped GO nanosheets, 1 g of dried, untreated GO powder was dispersed in 100 mL of DI water in a 250 mL Erlenmeyer flask using a sonication bath for 30 min. The pH of the GO suspension was adjusted to 10 using a 0.1 M NaOH solution. Subsequently, 2 mL of 10 M AgNO3 solution was added to the suspension under stirring (400 rpm). The mixture was stirred at 60 °C for 20 h. Finally, the Ag-doped GO nanosheets were collected using a centrifuge (4,000 rpm), washed three times with DI water, and dried overnight in an oven at approximately 60 °C.

[0075] Example 2: Complex binding of silver cations to GO nanosheets The GO surface is known to act as a reducing agent. + It is also possible to reduce some of the cations to metallic Ag(0). + This reduction of cations may be accompanied by the simultaneous oxidation of other graphene functional groups (such as quinone to hydroquinone). C and O-containing species (phenolic, carbonyl, etc.) have also been observed to oxidize during this process. On the other hand, Ag + Ag(0) particles may exist on the surface in the +1 oxidation state, or may simply coordinate with oxygen or other functional groups on the GO surface. If Ag(0) particles are present, they can contact microorganisms (pathogens) and directly affect them by interfering with DNA / RNA replication or denaturing other proteins in the cell (ribosomes, cell membranes, etc.); alternatively, Ag can contact the fluid (surrounding the pathogen) and oxidize Ag(0) to Ag(+), which will disrupt cell membranes or denature proteins.

[0076] GO-Ag + Peak fitting was performed to investigate the silver species bound to the nanocomposite and to account for the inclusion of Ag(1)-complexes as well as (Ag(0)) nanoparticles on GO. X-ray photoelectron spectroscopy (XPS) and Auger electron spectroscopy (AES) of one exemplary product according to the present invention (defined in Table 1) were performed (see Figures 2A and 2B, Table 1).

[0077] [Table 1]

[0078] [Table 2]

[0079] [Table 3]

[0080] The calculated Auger parameter value for the exemplary product was 723.2 eV, confirming that the primary composition was not Ag or AgO (see Figures 2C and 2D). Peak fitting of the Ag 3d5 / 2 data (Table 2) indicates that approximately 92% of the silver in the exemplary product was in the form of Ag(I) complexes, and the remaining 8% could be attributed to the form of clustered Ag(O) nanoparticles. No form of nitrogen was detected, thus confirming the removal of nitrate from the final product. The downward shift of the Ag 3d5 / 2 peak of the exemplary product compared to Ag(O) clearly indicates that the chemical state of the former is Ag(I) (Figure 3). The elemental composition of C and O in the exemplary product was maintained similar to that of GO, except for the addition of approximately 4.5 atomic % Ag (Tables 1 and 3). This suggests that Ag(I) ions are complexed with functional groups present on GO. The positively charged Ag(I) ions, originally introduced into the system by the addition of AgNO3, are electrostatically attracted toward the negatively charged functional groups with unshared electron pairs on the GO surface, forming complex bonds such as coordinate covalent bonds (both electrons shared in the bond come from the functional groups on the GO sheet, which in this case are the ligands).

[0081] Example 3: Morphological characterization of graphene-silver cation nanocomposites GO-Ag + The surface morphology of the nanocomposite was examined (a) before contact with silver cations and (b) after contact but before purification (purification involves the removal of Ag from silver nitrate). +The surfaces of the GO samples were examined by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) after (a) the application of silver cations (nitrate removal after application), and (b) the purification of the GO samples. SEM analysis was performed in two different modes: SE (scanning electron) and backscattered electron (BSE or Z-count). In the second mode, different components with different electron scattering levels produce different contrasts, providing a more detailed view of the surface (e.g., areas with more silver are brighter). Measurements were performed on samples diluted with ethanol. Note that negative results should be read as zero. Figures 4A–4F show SEM (SE) and SEM (Z-count) images of GO before contact with silver cations, and Figures 5A–5C show EDS images and charts of the results for GO before contact with silver cations. Figures 6A-6F are SEM (SE) and SEM (Z-count) images of GO after contact with silver cations but before purification, Figures 7A-7B are EDS images and charts of the results for GO after contact with silver cations but before purification, Figures 8A-8D are SEM (SE) and SEM (Z-count) images of GO after contact with silver cations and after purification, and Figures 9A-9B are EDS images and charts of the results for GO after contact with silver cations and after purification.

[0082] Example 4: Broad-spectrum antimicrobial activity of graphene-silver cation nanocomposites GO-Ag was assayed against seven representative bacteria and one yeast strain using the macrobroth dilution method according to Clinical Laboratory Standards Institute (CLSI) documents M7, M11, and M60. + The MICs of the nanocomposites were determined. The following microorganisms were tested: 1. Streptococcus pneumoniae (ATCC® 33400) 2. Haemophilus influenzae (ATCC® 51907D-5) 3. Streptococcus pyogenes (Group A hemolytic streptococcus) (ATCC® 12344D-5) 4. Moraxella catarrhalis (ATCC® 19606D-5) 5. Staphylococcus aureus (ATCC® 12600) 6. Escherichia coli (ATCC® 10798) 7. Fusobacterium nucleatum (ATCC® 25586D-5) 8. Candida albicans (ATCC® 14053) GO-Ag + The nanocomposite (sample number: 09-002-501; October 22, 2020) was first dissolved in sterile water to a concentration of 2 mg / mL. Mueller-Hinton broth was used for S. pneumoniae, S. pyogenes, S. aureus, M. catarrhalis, and E. coli. Haemophilus test broth was used for H. influenzae, and fastidious anaerobic broth was used for Fusobacterium nucleatum. The concentration ranges tested were (µg / mL): 1, 0.5, 0.25, 0.125, 0.0625, 0.031, 0.0156, 0.008, and 0.004 (a total of nine target concentrations). Each concentration was tested in quintuplicate.

[0083] Each microorganism was prepared into a 0.5 McFarland turbidity standard (equivalent to 105 cfu / mL) and then diluted in ascending order of GO-Ag concentration. + Each tube containing the nanocomposite was inoculated and incubated at 37°C in 5% CO2 for 18–24 hours, except for Fusobacterium nucleatum, which was incubated anaerobically. After incubation, the tubes were examined for turbidity. The lowest concentration at which no turbidity was observed in all quintuplicate samples was considered the MIC. A growth control (GO-Ag) was used for each microorganism and for each test set. +Sterility and growth controls were used for each microorganism to verify the absence of contamination and the GO-Ag + It was confirmed that growth occurred in broth medium in the absence of .

[0084] result Table 4 shows the GO-Ag values ​​reflected by the MICs against each of the microorganisms tested: Streptococcus pyogenes (Group A hemolytic streptococcus) (ATCC® 12344D-5), Staphylococcus aureus (ATCC® 12600), Streptococcus pneumoniae (ATCC® 33400), Moraxella catarrhalis (ATCC® 19606D-5), Haemophilus influenzae (ATCC® 51907D-5), Escherichia coli (ATCC® 10798), Candida albicans (ATCC® 14053), and Fusobacterium nucleatum (ATCC® 25586D-5). + Results showing the antimicrobial activity of the nanocomposites are presented. MICs are based on complete inhibition in all quintuple replicates, and an "X" in the table indicates that no visible turbidity was observed in GO-Ag nanocomposites. + Represents the minimum concentration of the nanocomposite.

[0085] [Table 4]

[0086] Consideration The results of this evaluation are + This shows that the nanocomposite can inhibit common aerobic bacteria and yeasts at very low concentrations. However, the anaerobic bacteria (Fusobacterium nucleatum) used in this evaluation was not inhibited over the entire concentration range tested. However, this is due to the fact that the agar used as the medium is Ag-rich. + This was due to the negation of the activity of

[0087] GO-Ag + The nanocomposite appears to be active at extremely low concentrations against both Gram-positive bacteria (e.g., Streptococcus and Staphylococcus) and Gram-negative bacteria (e.g., E. coli, H. influenzae, M. catarrhalis), as well as common yeasts (e.g., Candida albicans).

[0088] GO-Ag required to achieve antimicrobial efficacy + The relatively low concentration of the nanocomposite is much lower than the concentration required for commonly used antibiotics to have a similar effect. Overall, the results of this evaluation suggest that GO-Ag + The nanocomposite is shown to be a novel compound with broad-spectrum antibacterial, antiviral, and antifungal activity at extremely low concentrations of 0.008–0.0625 μg / mL.

[0089] Example 5: Antimicrobial activity of graphene-silver cation nanocomposites against drug-resistant and multidrug-resistant pathogens GO-Ag + The efficacy of the nanocomposite was further tested against a wide range of exemplary, but not limited to, antimicrobial-resistant (AMR) and multidrug-resistant (MDR) microorganisms, including: 1. Gram-negative bacteria: Pseudomonas aeruginosa (two separate isolates) Escherichia coli (extended-spectrum beta-lactamase-producing bacteria [ESBL]) E. coli (carbapenem-resistant [CRO]) Klebsiella pneumoniae (ESBL) Klebsiella pneumoniae (CRO) Enterobacter aerogenes Stenotrophomonas maltophilia 2. Gram-positive bacteria: Nosocomial methicillin-resistant Staphylococcus aureus (HA-MRSA) Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) Vancomycin-resistant Enterococcus faecium (VRE) Penicillin-resistant Streptococcus pneumoniae The same method as described in Example 4 was used and the results are presented in Table 5. In particular, GO-Ag + The nanocomposite (sample number: 09-002-501; October 22, 2020) was first dissolved in sterile water to a concentration of 2 mg / mL. Mueller-Hinton (MH) broth was used for all microorganisms. The concentrations tested ranged from 1, 0.5, 0.25, 0.125, 0.0625, 0.031, 0.0156, 0.008, and 0.004 μg / mL (a total of nine target concentrations). Each concentration was tested in quintuplicate.

[0090] Each organism was analyzed using a 0.5 McFarland turbidity standard (10 5 cfu / mL), and the GO-Ag + Each tube containing 100 mg of 10 ... + Sterile and growth controls were used for each microorganism to verify the absence of contamination and GO-Ag, respectively. + It was confirmed that growth occurred in broth medium in the absence of .

[0091] Table 5 shows the GO-Ag as reflected by the MIC against each of the AMR microorganisms tested, namely, Serratia marcescens (S. mar), Pseudomonas aeruginosa (P. aer1), Pseudomonas aeruginosa (P. aer2), Escherichia coli (ESBL), Escherichia coli (CRO), Klebsiella pneumoniae (ESBL), Klebsiella pneumoniae (CRO), Enterobacter aerogenes, Stenotrophomonas maltophilia, methicillin-resistant Staphylococcus aureus (MRSA), CA-methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and penicillin-resistant Streptococcus pneumoniae. + Results showing the antimicrobial activity of the nanocomposite are presented. GO-Ag against exemplary microorganisms tested. + The average MIC values ​​(measured in μg / mL) of the nanocomposites are presented. The MIC was based on complete inhibition in all quintuplicate samples, and an "X" in the table indicates that no visible turbidity was observed in GO-Ag. + Represents the minimum concentration of the nanocomposite.

[0092] The results of this evaluation demonstrate the efficacy of GO-Ag against many difficult-to-treat clinical infections, including those involving the respiratory tract, urinary tract, skin, and soft tissues, as well as exemplary known AMR and MDR pathogens, including ESKAPE pathogens with bacteremia at very low concentrations. + The unexpectedly low MIC values ​​were in the range of 0.008–0.031 μg / mL (Table 5). The GO-Ag nanocomposite showed excellent efficacy against fully susceptible microorganisms (i.e., E. coli, S. aureus, and Streptococcus pneumoniae). + Based on previous evaluation of (see Example 4), the MICs against AMR strains were either equivalent or one dilution higher.

[0093] [Table 5-1]

[0094] [Table 5-2]

[0095] Example 6: Comparison of antimicrobial and synergistic effects GO-Ag according to embodiments disclosed herein + The antimicrobial efficacy of the nanocomposite was compared with that of previously reported nanocomposites. As shown in Table 6, the MICs of the nanocomposites reported so far were significantly higher for the GO-Ag nanocomposite than for the previously reported nanocomposites. + The unexpectedly improved performance of the nanocomposite is demonstrated.

[0096] Table 6 shows the antimicrobial efficacy of GO-Ag against various pathogens compared to known metal and graphene composites reported in the following literature: +(a) Zhong, L. and Yun, K. “Graphene oxide-modified Zn particle synthesis: synthesis characterization: antibacterial properties.” International Journal of Nanomedicine Spec. Iss. 10, 79-92;(b)Matar, Susan A. et al. “The antibacterial biofilm activity of metal-doped mullite ceramics against pathogenic bacteria.”African Journal of Microbiology Research 7 (23), June 2013, 2939-2947;(c)Salman, Halah Dawood, “Evaluation and Comparison the Antibacterial Activity of Silica Particles and AginoNPs Silver Nitrate (AgNO3) on Some Pathogenic Bacteria.”Journal of Global Pharma Technology, December 2016;(d)Panacek et al.“Silver colloid nanoparticles: Synthesis, characterization and their antibacterial activity.” Journal of Physical Chemistry ( B3 ), 1101 16248-16253;(e)Anni, Feng et al.“Facile Synthesis of Silver nanoparticles with High Antibacterial Activity.”Materials 11 (12), Dec 2018;(f)Ulkuseven, Bahri et al.“Synthesis, Characterization and antimicrobial Activity of d8-10 Metal Complexes of nine 2-substituted-1H-Benzimidazoles.”Metal-Based Drugs 6 (3), 1999;(g)Mazarin de Moraes, AC et al. “Graphene oxide-silver nanocomposite as a promising biocidal agent against methicillin-resistant Staphylococcus aureus.” Int J Nanomedicine 10:6847-6861, 2015. With reference to the comparative data presented in Table 6, these data are consistent with the GO-Ag + The results show that nanocomposites significantly outperform graphene oxide (GO), graphene oxide-silver nanoparticles (AgNPGO), silver nitrate (AgNO), silver nanoparticles (AgNP), and zinc oxide-graphene oxide (ZnO.GO) nanocomposites. The GO-Ag nanocomposites according to embodiments disclosed herein, as reflected in the MICs against representative pathogens, are significantly superior to those of graphene oxide (GO), graphene oxide-silver nanoparticles (AgNPGO), silver nitrate (AgNO), silver nanoparticles (AgNP), and zinc oxide-graphene oxide (ZnO.GO). + The MICs of the nanocomposites are at least 100×–10,000× lower than the MICs of previously reported nanocomposites.

[0097] Furthermore, these data suggest that when considering broad-spectrum antimicrobial effects, graphene oxide with silver cations (GO-Ag + The unexpected synergistic effect of the complex is shown.

[0098] [Table 6-1]

[0099] [Table 6-2]

[0100] Example 7: Virucidal activity of graphene-silver NP nanocomposites The virucidal activity of GO-AgNP nanocomposite was evaluated for comparative purposes. Fabrication of GO-AgNP nanocomposite To synthesize Ag-doped GO nanosheets, 0.1 g of dried raw GO powder was dispersed in 30 mL of DI water in a 100 mL Erlenmeyer flask using a sonication bath for 30 min. The pH of the GO suspension was adjusted to 10 using a 0.1 M NaOH solution. Subsequently, 2 mL of 0.25 M AgNO3 solution was added to the suspension under stirring (400 rpm). Next, 20 mL of DI water was added to the suspension to reduce the viscosity of the solution. The mixture was stirred at 60 °C for 20 h.

[0101] Methods for SARS-CoV-2 Replication / Inhibition Testing The virucidal activity of the GO-AgNP nanocomposite was tested at the ImPaKt Facility at Western University. The GO-AgNP nanocomposite was in the form of a high-viscosity paste, which was applied to the bottom of a 12-well dish using a flat-edged measuring spoon.

[0102] SARS-CoV-2 Wuhan strain virus was serially diluted four times to approximately 200,000 infectious units (IU), 20,000 IU, 2,000 IU, and 200 IU per 20 μL. 200 μL of SARS-CoV-2 dilutions were applied to GO-AgNP-treated and untreated surfaces. After 1 and 12 hours of incubation of the virus with the treated surfaces, 20 μL of the suspension from each well was added to wells of a new 96-well plate containing approximately 20,000 Vero cells in DMEM medium. The final multiplicities of infection were 2.0, 0.2, 0.02, and 0.002 infectious units per cell for each virus dilution. Viral cytopathic effect (vCPE) in cells was observed on day 1, and vCPE was measured on day 3.

[0103] SARS-CoV-2 replication / inhibition test results The results of the replication / inhibition studies are presented in Table 7. At 2,000 IU, the GO-AgNP nanocomposite provided approximately 20-30% protection against SARS-CoV-2 infection of VERO E6 cells. At 200 IU and 20 IU, approximately 10-20% protection was observed against SARS-CoV-2 infection of VERO E6 cells. No protection was observed at 20,000 IU of virus. At 12 hours, there was a significant decrease in apparent viral activity in both treated and untreated conditions. No differences were observed between treated and untreated surfaces.

[0104] [Table 7]

[0105] Example 8: Graphene-silver cation nanocomposite formulation for surface coating GO-Ag + To apply the nanocomposite to the surface, GO-Ag + The nanocomposites were further formulated as suspensions using a mixture of ethanol and deionized water (DI). In a 100 mL suspension, 60–70 mL of ethanol and 30–40 mL of DI water were used as diluents. + The nanocomposites were suspended in a diluent at concentrations of 0.1–5 g / L to form dispersions. + The resulting formulation containing the nanocomposite was applied to surfaces and filter media by either dip coating or spray coating, followed by air or heat drying to form the GO-Ag + The nanocomposite was fixed on the medium.

[0106] Example 9: Morphological characterization of surface-coated graphene-silver cation nanocomposites GO-Ag by applying it by dip coating +The surface morphology of nanocomposite-coated fabric samples was investigated by scanning electron microscopy (SEM), field emission SEM (FE-SEM), coupled with energy dispersive X-ray (EDX) spectroscopy.

[0107] GO-Ag prepared by the method described in Example 8 + After dip-coating with the nanocomposite formulation, EDX elemental analysis was performed to evaluate the amount of carbon, oxygen, silver, etc. on the fabric (polyurethane) surface. The formulation was applied at two different concentrations: low and high loading (0.5 g / L and 5 g / L).

[0108] For FE-SEM-EDX analysis, a sample of the coated fabric (1 cm × 1 cm) was cut and coated with a thin layer of gold using metal evaporation to make the fabric surface conductive. Subsequently, the sample was analyzed. After selecting an appropriate area of ​​the fabric at an appropriate magnification, elemental mapping was performed using EDX.

[0109] As shown in Figures 10A, 10B, and 10C, each fabric sample has two distinct surfaces: a rough surface and a fine surface. The rough surface appears to be more hydrophobic in the thicker fabrics, while the fine surface appears to be hydrophilic in the thinner fabrics. This explains the higher concentration of Ag element on the fine surface, as the coating is an aqueous dispersion and adheres to the hydrophilic surface. The surface Ag maps also show a relatively uniform distribution, indicating that the coating is well dispersed in the water / ethanol medium and that dip coating is an effective method for coating mask materials.

[0110] The elemental composition of each coated fabric sample surface was also determined by EDX analysis, as shown in Figures 11-18. Each color in the EDX maps corresponds to a specific element, as shown in Figures 11, 12, 15, and 17. Because EDX analyzes a sample depth of approximately 1-3 μm, the black areas lacking Ag represent wavy or textured areas of the fabric.

[0111] Example 10: Virucidal activity of graphene-silver cation nanocomposites A viral titer reduction test using SARS-CoV-2 was performed using GO-Ag + This was performed on N-95 mask materials coated with the nanocomposite GO-Ag. + The nanocomposite coating was found to effectively reduce viral activity by 99% after two hours of exposure.

[0112] Preparation of materials The coated N-95 mask material was prepared according to the methods described herein in Examples 1 and 8. The material was prepared 5 weeks prior to virucidal testing.

[0113] The coated N-95 mask material had a dark gray appearance. Uncoated N-95 material served as a control. Prior to testing, the material was sterilized by autoclaving at 121°C for 30 minutes before analysis. After sterilization, the material was cut into approximately 0.5 x 0.5 cm squares in a biosafety cabinet (BSC) and placed in sterile 1.5 mL tubes.

[0114] Preparation and testing of SARS-CoV-2 10 5.8 SARS-CoV-2 virus stock with a titer of infectious units (IU) / ml was prepared by 2.9(IU) / ml. A 500 ml volume of diluted virus stock was added to the 1.5 ml tube containing the square coated material. The tube containing virus and coated material was placed on a tube rotator for 2 hours at 22°C. Untreated material (0-001-011) exposed to virus supernatant served as a control. The collected supernatant was diluted 100-fold to dilute any chemicals / materials that may have been released from the coated material during incubation. The collected supernatant (100-fold diluted) was further serially diluted from 1:100 to 1:100,000 and subsequently added to 20,000 Vero E6 cells in a 96-well flat-bottom plate.

[0115] A 1:100 dilution of the virus stock to infect 20,000 cells represents a multiplicity of infection (MOI) of 0.02. Infection of Vero E6 cells was monitored by viral cytotoxicity. Cytotoxicity of supernatant-derived material (1:100 dilution) in the absence of virus was measured visually.

[0116] result As shown in Table 8, the viral titer was + After two hours of exposure to the nanocomposite-coated N-95 respirator material, there was a two-log reduction in infectious virus, corresponding to a 99% reduction. Exposure to the control mask material, i.e., untreated N-95 material, resulted in no reduction in viral titer. All experiments were performed in triplicate. Results varied by less than 5% and are reported as a 99% reduction in infectious virus (or 99% virucidal activity).

[0117] [Table 8]

[0118] Example 11: Improving microbial filtration efficiency According to certain embodiments described herein, GO-Ag +The nanocomposites can be formulated for use in PPE, air filtration (HVAC) systems, and other airflow membranes and filters to reduce or eliminate the activity of microbial pathogens. The effectiveness of PPE and other airflow membrane / filter systems depends on their filtration efficiency (FE) against microbial pathogens.

[0119] Without being bound by theory, GO-Ag + The hydrophilic nature of the nanocomposite is believed to attract, retain, and immobilize microorganisms on the treated surface, inhibiting their passage through the filtration device. Improving the filtration efficiency of substrates such as medical masks, combined with the inactivation / killing of microorganisms, results in improved performance of the filtration device.

[0120] GO-Ag + The ability of the nanocomposites to improve the filtration efficiency of 3- or 4-ply medical masks was tested against representative bacterial and viral pathogens using standard industry test methods.

[0121] Preparation of materials The coated N-95 mask material was prepared according to the methods described herein in Examples 1 and 6. The material was prepared 5 weeks prior to virucidal testing.

[0122] The coated N-95 mask material had a dark gray appearance. Uncoated N-95 material served as a control. Prior to testing, the material was sterilized by autoclaving at 121°C for 30 minutes, followed by test analysis.

[0123] Bacterial Filtration Efficiency Test Bacterial filtration efficiency (BFE) testing was performed at increasing exposure levels to allow for measurements of filtration efficiencies greater than those measurable using standard BFE procedures. Testing was performed according to standard ASTM F2101 (Standard Test Method for Evaluating the Bacterial Filtration Efficiency (BFE) of Medical Face Mask Materials, Using a Biological Aerosol of Staphylococcus aureus, ASTM International, West Conshohocken, PA, 2001), in which a bacterial challenge suspension of S. aureus (Staphylococcus aureus ATCC 6538) was aerosolized using a nebulizer to a concentration of 5 × 10 6 More than 100 colony-forming units (CFU) were delivered to the target. The aerosol suspension was drawn through the user side of the test article into a glass impinger (AGI) at a constant flow rate of 28.3 LPM to collect live microorganisms. The test area was approximately 38.5 cm. 2 The exposure fluid was delivered for 5 minutes, and sampling was performed for a total of 6 minutes to ensure the aerosol chamber was emptied. Viable microorganisms collected during the AGI were enumerated using a membrane filtration procedure. S. aureus exposure fluid was collected at 28.3 LPM using a six-stage Andersen sampler, and mean particle size (MPS) was determined using the procedure outlined in ASTM F2101. An uncoated control was performed to ensure reproducibility and reliability.

[0124] The exposure level in these studies was determined to be 6.9667 x 10^6 CFU of S. aureus with a mean particle size of 2.83 μm. Viral filtration efficiency test Viral filtration efficiency (VFE) testing using MS2 bacteriophage was performed at increasing exposure levels to allow for measurements of filtration efficiencies greater than those measurable using standard VFE procedures. Testing was performed according to standard ASTM F2101 (Standard Test Method for Evaluating the Bacterial Filtration Efficiency (BFE) of Medical Face Mask Materials, Using a Biological Aerosol of Staphylococcus aureus, ASTM International, West Conshohocken, PA, 2001). In this test, an MS2 bacteriophage (MS2 bacteriophage ATCC 15597-B1) exposure suspension was aerosolized using a nebulizer to a concentration of 1 × 10 7 More than 100 plaque-forming units (PFU) were delivered to the target. The aerosol suspension was drawn through the user-facing side of the test article into an aerosol glass impinger (AGI) at a constant flow rate of 28.3 LPM to collect viable microorganisms. The exposure fluid was delivered for 5 minutes, with sampling for a total of 6 minutes to ensure the aerosol chamber was emptied. Viable microorganisms collected during the AGI were enumerated by visualizing cleared zones (plaques) representing viable MS2 bacteriophage that had penetrated the mask using a single agar layer procedure with an E. coli host. A six-stage Andersen sampler was used to collect MS2 bacteriophage exposure fluid at 28.3 LPM, and mean particle size (MPS) was determined using the procedure outlined in ASTM F2101. Controls were performed to ensure reproducibility and reliability.

[0125] The exposure level in these studies was determined to be 2.2933 x 10^7 CFU of MS2 bacteriophage, which has a mean particle size of 2.76 μm. result The BFE (%) and VFE (%) were calculated as follows.

[0126]

number

[0127] Where C = exposure level T = total CFU recovered downstream of the test article MPS was identified as follows.

[0128]

number

[0129] During the ceremony, Px = 50% effective cutoff diameter at stage X as specified by the manufacturer Cx = uncorrected counts in stage X (stages 1 and 2) or "probable hit" counts (stages 3 through 6) determined using the positive hold conversion chart from the Cascade Impactor manual. As presented in Table 9, GO-Ag + The nanocomposite-coated masks removed 98.9% more bacteria and 97.8% more viruses than the uncoated masks, resulting in masks with viral and bacterial filtration efficiencies of over 99.99%.

[0130] [Table 9]

[0131] [Table 10]

[0132] The disclosures of all patents, patent applications, publications, and database entries referenced herein are expressly incorporated herein by reference in their entirety, to the same extent as if each individual patent, patent application, publication, and database entry was specifically and individually indicated to be incorporated by reference.

[0133] While the present invention has been described with reference to certain specific embodiments, various modifications will be apparent to those skilled in the art without departing from the spirit and scope of the invention. All such modifications apparent to those skilled in the art are intended to be included within the scope of the following claims.

Claims

1. Graphene oxide (GO) and silver cations (Ag) bound to the GO as Ag(I) complexes. ＋ ), wherein the silver cations (Ag + ) are bound to the GO by complex bonds.

2. The nanocomposite of claim 1 , wherein the complex bond is a chelating bond or a coordinate covalent bond.

3. 10. The nanocomposite of claim 1, comprising 3-80 wt / wt%, 10-20 wt / wt%, or 4-8 wt / wt% silver cations bound to the GO.

4. 10. The nanocomposite of claim 1, further comprising silver nanoparticles covalently bonded to the GO of the nanocomposite.

5. 5. The nanocomposite of claim 4, wherein the ratio of silver cations to silver particles is within the range of a ratio of 10:1 to 15:

1.

6. The silver bound to the GO is approximately 90-99% silver cations in the form of Ag(I) complexes (Ag ＋ 5. The nanocomposite of claim 4, comprising about 1 to 10% silver nanoparticles in the form of clustered Ag(0) nanoparticles.

7. Copper cation (Cu ２＋ ) or zinc cation (Zn ２＋ 10. The nanocomposite of claim 1, further comprising:

8. 10. The nanocomposite of claim 1 having a particle size in the range of 2 to 10 μm.

9. 10. The nanocomposite of claim 1, which is effective in killing microbial pathogens.

10. 10. The nanocomposite of claim 9, wherein the microbial pathogen is the SARS-CoV-2 virus.

11. 10. The nanocomposite of claim 9, wherein the microbial pathogen is a drug-resistant strain.

12. 10. An antimicrobial formulation comprising the nanocomposite of claim 1 and a solvent, carrier, diluent, and / or dispersant.

13. 13. The antimicrobial formulation of claim 12 having a nanocomposite concentration of 40 mg / L to 5 g / L.

14. 13. The antimicrobial formulation of claim 12, formulated for application to a substrate as a liquid spray, mist, foam, dip coat bath, wipe, or coating.

15. 13. The antimicrobial formulation of claim 12, which is an antimicrobial coating applied to face masks, personal protective equipment (PPE), environmental cleaning wipes, counters, door handles, walls, or airflow membranes and filters.

16. 16. The antimicrobial formulation of claim 15, wherein the coating has a thickness in the range of about 5 nm to about 5 μm, about 100 nm to about 3 μm, about 200 nm to about 2 μm, about 300 nm to about 1.5 μm, or about 500 nm to about 1.0 μm.

17. 1. A method for imparting antimicrobial activity to a substrate, comprising: a. Dispersing the nanocomposite of claim 1 in deionized water, ethanol, or deionized water and ethanol; b. applying the nanocomposite dispersion to the substrate; and c) fixing said nanocomposite to a surface by air drying or heat drying.

18. The method of claim 17 , wherein the nanocomposite is applied to the substrate by spray coating or dip coating.

19. 20. The method of claim 17, wherein the nanocomposite is applied to the substrate as a coating having a thickness in the range of about 5 nm to about 5 μm, about 100 nm to about 3 μm, about 200 nm to about 2 μm, about 300 nm to about 1.5 μm, or about 500 nm to about 1.0 μm.

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