FIELD OF THE INVENTION

The present invention relates to an antimicrobial and/or antiviral nanoparticle covalently bonded to a substrate. The invention also relates to a coating comprising a plurality of nanoparticles wherein the coating is covalently bonded to a substrate. The invention also relates to a substrate having such nanoparticle or coating covalently bonded thereto. The invention further relates to a method of manufacturing nanoparticles and a coating of the invention. The invention also relates to the use of atomic layer deposition (ALD) and chemical vapour deposition (CVD) to deposit antimicrobial and/or antiviral material on a substrate. The invention further relates to uses of the nanoparticles, coatings and substrates of the invention.

BACKGROUND OF THE INVENTION

Germs such as viruses, bacteria and fungi can be readily transmitted from one host to another. Germs do not move themselves but rely on people, the environment, and/or medical equipment to move . Transmission can happen as a result of contact with a contaminated surface, sprays and splashes and inhalation of droplets carrying germs.

A large number of germs are harmless and even helpful to humans and animals. However, others are harmful and can cause infections. In such cases, the prevention of the spread of germs plays an important role in protecting vulnerable subjects.

The antimicrobial and antiviral properties of a number of metals such as silver, copper, zinc, gold, titanium, aluminium, tin, iron, arsenic, lanthanum, and molybdenum are known. Further, nanoparticles in the form of metal compounds such as titanium oxide, zinc oxide, aluminium oxide, copper oxide, silicon oxide and silver oxide, as well as other silver, copper and zinc compounds have been reported to elicit bactericidal properties through the generation of reactive oxygen species (ROS) that are able to target physical structures, metabolic pathways, and DNA synthesis of prokaryotic cells leading to cell death (Gold, K. et al. Antimicrobial Activity of Metal and Metal-Oxide Based Nanoparticles. Adv. Therap., 1(3) 1700033 (2018)). However, these nanoparticles can take hours to destroy microorganisms and their efficiency against viruses has not been established.

Therefore, there remains a need to provide new antiviral and antimicrobial agents that can efficiently kill microbes and viruses in a timely manner.

SUMMARY OF THE INVENTION

Here, presented for the first time, are nanoparticles that can efficiently kill microbes and viruses in a timely manner. The nanoparticles of the present invention can be used as an antimicrobial and/or antiviral surface coating. Using innate electrochemical activity to provide rapidly deployable, fast-acting and long-lasting protection, this technology is capable of inactivating viruses, such as coronavirus in seconds, which is a significant enhancement compared to current technologies such as silver and copper which produce the same effect in 24 to 48 hours.

The invention relates to a substrate having an antimicrobial and/or antiviral, ferroelectric nanoparticle covalently bonded thereto, wherein the nanoparticle comprises a metal and a dopant. Expressed in another way, the invention encompasses an antimicrobial and/or antiviral coating comprising a ferroelectric nanoparticle, wherein the nanoparticle comprises a metal and a dopant. Ideally the coating is covalently bonded to a substrate. In an embodiment, the nanoparticle or coating has an electric voltage in the range of 0.2 to 1.5 V, preferably in the range of 0.5 to 1 V, or in the range of 1 to 1.5 V. Alternatively or in addition, the nanoparticle or coating has an electric voltage in the range of from 0.5 to 8 V, preferable in the range of from 1 to 8 V or from 1.2 to 8 V. It will be appreciated that the present invention encompasses all the intermediate numbers, such as 0.7 V, 1.3 V, 1.4 V, 1.8 V, 3 V, 4.5 V, 6.3 V and the like.

In an alternative, the present invention resides in the use of ferroelectric nanoparticles as defined herein having a spontaneous electric polarisation that generates a potential difference or voltage of 0.5 V or greater, wherein use is as an antimicrobial and/or antiviral agent, optionally as a coating thereof. In one embodiment, the ferroelectric nanoparticles have an inherent voltage of between about 1 V to about 8 V. In a particular example, the nanoparticles have a voltage of between 1 V and 3.5 V, specifically about 1.5 V. Where the nanoparticles are used as a coating, the particles may be covalently bonded to a substrate. Expressed in another way, there is contemplated a method of reducing, minimising and/or preventing the spread of, or contamination by, microbial and/or viral particles comprising the use of ferroelectric nanoparticles as described herein.

In an embodiment, the metal is a metal oxide. In an embodiment, the metal oxide is selected from: zinc oxide, aluminium oxide, titanium oxide, silver oxide, copper oxide, iron oxide, manganese oxide, cerium oxide, molybdenum oxide, lanthanum oxide, arsenic trioxide, silicon dioxide, and any combination of these oxides as a metal alloy compound. Preferably, the metal oxide is selected from: TiCf. CuO, and ZnO.

In an embodiment, the substrate comprises a metal, textile, ceramic, plastic, wood, or glass layer. In an embodiment, the substrate comprises a polymer layer, optionally in combination with one or more additional layers, and the antimicrobial and/or antiviral nanoparticle is covalently bonded to the polymer layer. Preferably, the polymer is poly(3-hexylthiophene-2,5-diyl) (P3HT).

In an embodiment, the nanoparticle size is 1 nm to 100 nm. Preferably, the nanoparticle size is 1 nm to 10 nm.

In an embodiment, the dopant is a doping ion selected from: vanadium ion, lithium ion, niobium ion, tantalum ion, barium ion, titanium ion, zirconium ion, lanthanum ion, potassium ion, ammonium ion, silver ion, copper ion, magnesium ion, and any combination thereof. In an embodiment, the doping ion is a monovalent, divalent, trivalent, or tetravalent metal ion. In an embodiment, the doping ion is vanadium ion. In an embodiment, the doping ion is copper ion. In an embodiment, the ratio of metal to doping ion is in the range from 20: 1 to 4: 1. Preferably, the ratio of metal to doping ion is 10: 1.

The invention also relates to a substrate having an antimicrobial and/or antiviral coating applied thereon, the coating comprising a plurality of antimicrobial and/or antiviral nanoparticles as defined herein.

In an embodiment, the coating thickness is 0.5 nm to 5 pm. Preferably, the coating thickness is 30 nm to 100 nm.

In an embodiment, the substrate comprises a polymer layer, optionally in combination with one or more additional layers, and the antimicrobial and/or antiviral nanoparticles are covalently bonded to the polymer layer. Additional layers may be a backing or carrier layer, an adhesive layer and/or one or more release papers.

The invention also relates to a polymer layer wherein a coating is applied to at least a part of at least one surface of the polymer, and wherein the coating comprises a plurality of antimicrobial and/or antiviral nanoparticles as defined herein. In use, the polymer layer may be applied to a surface, such as metal, textile, ceramic, plastic, wood, or glass.

In an embodiment, the polymer is a semi-crystalline polymer. In an embodiment, the polymer is poly(3- hexylthiophene-2,5-diyl) (P3HT).

The invention also relates to a textile wherein a coating is applied to at least a part of at least one surface of the textile, and wherein the coating comprises a plurality of antimicrobial and/or antiviral nanoparticles as defined herein.

The invention also relates to an adhesive film comprising the substrate or coating as defined herein. In an embodiment, the film comprises an adhesive layer, a backing layer, and a release layer, wherein the substrate as defined herein is the backing layer. In an embodiment, the adhesive layer is protected with a removable protective layer or carrier sheet.

The invention further relates to the use of atomic layer deposition (ALD) to deposit antiviral and/or antimicrobial material on a substrate. The invention also relates to the use of chemical vapour deposition (CVD) to deposit antiviral and/or antimicrobial material on a substrate.

The invention also relates to the use of ALD in the manufacture of a substrate or coating as defined herein. The invention also relates to the use of CVD in the manufacture of a substrate or coating as defined herein. Expressed in another way, the invention encompasses the manufacture of a substrate or coating as defined herein using ALD or CVD.

The invention also relates to a method of making a substrate as defined herein, a polymer layer as defined herein, a textile as defined herein, or an adhesive film as defined herein.

The invention also relates to a method of making a substrate having an antimicrobial and/or antiviral coating, optionally the coating being as defined herein, applied thereon, wherein the method comprises the steps of: a) providing a precursor substrate having a reactive group comprising a leaving group (L) and a metal -containing precursor gas, b) performing a first cycle comprising a step of: diffusing the metal -containing precursor gas under conditions such that the precursor substrate and the metal -containing precursor gas can react together to displace the leaving group on the precursor substrate thereby forming a covalent bond between the substrate and the metal of the metalcontaining precursor gas; c) repeating step b).

In an embodiment, the metal-containing precursor comprises the metal and one or more alkyl or halide groups, wherein the alkyl or halide group is displaced during the reaction with the precursor substrate.

In an embodiment, said first cycle further includes the step of diffusing a second precursor gas, such as H2O(g), 02(g), Os(g), or H ₂ (g), wherein the second precursor gas can react with the metal covalently bonded to the substrate.

In an embodiment, the reactive group is a reactive oxygen-containing group. Preferably, the reactive oxygen-containing group is -OH and/or -COOH and the leaving group is H. The invention also relates to a method of making a substrate having an antimicrobial and/or antiviral coating, optionally the coating being as defined herein, applied thereon, wherein the method comprises the steps of: a) providing an OH-terminated polymer precursor substrate, a metal -containing precursor gas, and a second precursor gas, b) performing a first cycle including the steps of diffusing: i. the metal -containing precursor gas, and ii. a second precursor gas comprising ILC g), 02(g), 03(g), or TU g), wherein the metal-containing precursor gas can react with the OH-terminated polymer precursor substrate to form a covalent bond between the polymer and the metal, and wherein the second precursor gas can react with the metal covalently bonded to polymer to form metal oxide nanoparticles; c) repeating step b) for 30 to 120 cycles.

In an alternative, the invention also relates a method as defined above where in step b) is repeated for between 1 and 10 cycles.

In an embodiment, the metal -containing precursor is selected from: Zn(Et)2, TiCfi. A1(CH3)3, Ag(fod)(PEt3)-Ci6H ₂₅ AgF ₇ O2P, (hfac)Cu-(I)(DMB), bis(N-isopropylketoiminate) iron(II), Mn2(CO)io , Ce(thd)4, or any combination thereof.

In an embodiment, step b) further includes performing a second cycle by diffusing a dopant gas; and wherein the first and second cycles are alternated in step c) such that ratios of metal to doping ion of 20: 1 to 4: 1 are produced. In an embodiment, the dopant gas is selected from (N,N-Di-i- propylacetamidinato)lithium, tetrakis(ethylmethylamido)vanadium(IV), cyclopentadienyl barium (cp-Ba), (tert-butylimido)bis(diethylamino)Niobium, Thallium(I) hexafluoroacetylacetonate, TiC14 (titanium tetrachloride), and copper(II) acetate [Cu(OAc)2].

In an embodiment, the substrate is selected from: wood, metal, glass, ceramic, plastic and textile.

In an embodiment, the method further includes priming the substrate to enable covalent bonding between the substrate and the antimicrobial and/or antiviral coating.

In an embodiment, the substrate comprises a polymer layer. In an embodiment, step a) further includes annealing the polymer. In an embodiment, the polymer is poly(3-hexylthiophene-2,5-diyl) (P3HT). In an embodiment, the method further includes step d) of applying the polymer layer on a product. In an embodiment, the polymer layer has a thickness of up to 1 pm.

The invention further relates to the use of the substrate as defined herein, a polymer layer as defined herein, a textile as defined herein, or an adhesive film as defined herein as an antimicrobial and/or antiviral agent.

The invention also relates to the use of the substrate as defined herein, a polymer layer as defined herein, a textile as defined herein, or an adhesive film as defined herein as an antiviral agent.

The invention also relates to the use of the substrate as defined herein, a polymer layer as defined herein, a textile as defined herein, or an adhesive film as defined herein for inactivating germs. In an embodiment, the germ is a virus. FIGURES:

Figure 1 : TEM image of iron oxide nanoparticles (100 nm) with low zeta potential value (less than 25 mV) aggregating to form large clusters (> 1pm).

Figure 2: Schematic diagram illustrating how a potential difference (voltage) occurs between two differently charged particles.

Figure 3: Cluster structure for ZnO. The schematic shows the wurtzite structure of the ZnO unit cell in a nanoparticle formed on a surface.

Figure 4: Schematic showing a release layer (1), an antimicrobial coating layer (2), a backing layer (3) and an adhesive layer (4).

Figure 5: Schematic showing an antimicrobial coating layer, an optional polymer layer and a substrate.

Figure 6: Schematic showing one- and two-step chemical vapour deposition method.

Figure 7: Schematic diagram of a substrate, based materials and coating of the invention.

Figure 8: Schematic diagram of ZnO deposition on -OH/-COOH surface via atomic layer deposition (ALD). Figure 9: Schematic diagram of ZnO deposition within/on a polymer substrate by ALD. A plan-view TEM image is shown in the top left comer.

Figure 10: UV-vis absorption comparison of polymer (P3HT), functionalized polymer with -COOH group (P3HT-COOH) and polymer/metal oxide deposited via ALD (P3HT-ZnO).

Figure 11: (a) GISAX and (b) GIWAX of polymer/metal oxide deposited via ALD.

Figure 12: Histogram of metal oxide nanoparticles sizes deposited by ALD within the polymer network. Figure 13: Hysteresis loop of doped metal oxide thin film deposited by ALD with ferroelectric properties. Figure 14: Antiviral effect of weak electric field (Sen, A. et al. Electroceutical Fabric Lowers Zeta Potential and Eradicates Coronavirus Infectivity upon contact. ChemRxiv (2020)).

DETAILED DESCRIPTION OF THE INVENTION

Nanoparticles

In one aspect, the invention relates to an antimicrobial and/or antiviral nanoparticle covalently bonded to a substrate, wherein the nanoparticle comprises a metal and a dopant. The invention therefore provides a substrate having an antimicrobial and/or antiviral nanoparticle covalently bonded thereto, wherein the nanoparticle comprises a metal and a dopant. It will be understood that the metal in the nanoparticle is doped with the dopant. The term “a” or “an” in the context of the present invention means at least one nanoparticle and encompasses one or more nanoparticles as well as a plurality of nanoparticles.

Generally, the zeta potential of nanoparticles has values that typically range from +100 mV to -100 mV. The zeta potential can be measured using dynamic light scattering. The magnitude of the zeta potential is predictive of the colloidal stability. Nanoparticles with zeta potential values greater than +25 mV or less than -25 mV typically have high degrees of stability as they can easily repel one another. However, in the -25 mV to +25 mV range, most nanoparticles aggregate in solution and become unstable, as shown in Figure 1. The zeta potential of a nanoparticle is dependent on the surface charge characteristics and the composition of the nanoparticle, but also on the pH of the solution. The pH value at which zeta potential is close to zero is known as the isoelectric point (IEP).

Viral particles, which are themselves nanoparticles, typically have an IEP of 3.5-7, which means that, at these pH values, viral particles can maintain less colloidal stability explaining why they generally remain in an unstable zeta potential range. The nanoparticles of the present invention aim, at least in part, to enhance the instability of viral nanoparticles, by creating an electric voltage. The antimicrobial and/or antiviral properties of the nanoparticles of the invention are derived, at least in part, from the action of the dopant, which induces a weak electric voltage (e.g. in the range of 0.2 to 1.5 V, preferably in the range of 0.5 to 1 V or 1 to 1.5 V) that is not present in the absence of the dopant. The presence of the dopant induces ferroelectric behaviour in the nanoparticle. The presence of an induced electric voltage and associated ferroelectric behaviour is detectable using ferroelectric hysteresis. Without wishing to be bound by theory, it is thought that the dopant induces the weak electric voltage by forming defect dipoles. Specifically, a small fraction of the metal atoms of the nanoparticle are replaced with dopant atoms of, e.g. another metal, thus forming defect dipoles. In the case of a doped metal oxide nanoparticle, each of the newly formed dipoles is composed of a replacement metal atom (dopant) paired with a neighbouring oxygen vacancy. The defect dipoles are too far apart to spontaneously align with one another. However, the defect dipoles can align under the effect of an external electric field. Dipoles that are aligned with an externally applied electric field (coercive field) will produce a non-zero net electric field. This induced electric field will remain in the material even after removal of the external electric field. The direction of a dipole moment changes upon application of an external electric field having a magnitude equal to the coercive field and a direction opposite to that of the dipole moment, thus flipping the direction of the aligned dipoles.

Ferroelectric materials are required by symmetry considerations also to be piezoelectric and pyroelectric. That is, the materials also have piezoelectric and pyroelectric properties. Piezoelectricity is an electric charge that accumulates in response to applied mechanical stress and, similarly, pyroelectricity is an electric charge that accumulates in response to a change in temperature, such as by heating or cooling.

For effective antimicrobial and/or antiviral properties, the desired minimum level of ferroelectric behaviour is one that induces a voltage in the range of 0.2 to 1.5 V, preferably in the range of 0.5 to 1 V or from 1 to 1.5 V. The desired level of innate electric behaviour may be ferroelectric or piezoelectric and induce a voltage of from 0.2 up to 8 V.

Typically, the radius of the metal ion in the nanoparticle is different from the radius of the dopant. This has the effect of inducing a defect dipole moment. The charge distribution within the nanoparticle is changed (see Figure 2).

Preferably, the nanoparticle further contains oxygen (O). Preferably, the nanoparticle comprises M-O- where M is the metal and O is covalently bound to the substrate:

M-O-Substrate.

Preferably, the nanoparticle comprises a metal oxide.

The nanoparticle may be covalently bound to one or more (e.g. 2 or 3) additional nanoparticle(s) depending on the valency of the metal. For example, in the case of a divalent metal oxide (e.g. ZnO), the following structure may be formed:

M-O-M-O-Substrate . In another example, in the case of a tetravalent metal oxide (e.g. TiCF). the following structure may be formed: z\

M Substrate

In an embodiment, the metal in the nanoparticle is selected from: zinc, aluminium, titanium, silver, copper, iron, manganese, cerium, molybdenum, lanthanum, arsenic, silicon and any combination of these as a metal alloy compound. Preferably, the metal in the nanoparticle is selected from: zinc, titanium, copper, aluminium, iron, and silicon. Preferably, the metal in the nanoparticle is zinc.

In an embodiment, the metal oxide is selected from: zinc oxide, aluminium oxide, titanium oxide, silver oxide, copper oxide, iron oxide, manganese oxide, cerium oxide, molybdenum oxide, lanthanum oxide, arsenic trioxide, silicon dioxide, and any combination of these oxides as a metal alloy compound. Preferably, the metal oxide is zinc oxide. Preferably, the metal oxide is titanium oxide. Preferably, the metal oxide is iron oxide. Preferably, the metal oxide is copper oxide. The metal oxide can be selected from: ZnO, AI2O3, TiO, TiCh, AgO, Ag ₂ O, CuO, CU2O, FeO, Fe2C>3, MnO, MnCF. CeCh, Ce2C>3, CesC , MoO2, AS2O3, HfCh, SiC>2, and any combination of these oxides as a metal alloy compound. Preferably, the metal oxide is selected from: TiCh, CuO, and ZnO.

Preferably, the dopant is a doping ion. Preferably, the doping ion is a monovalent, divalent, trivalent, or tetravalent cation. Preferably, the doping ion is a monovalent, divalent, trivalent, or tetravalent metal ion. More preferably, the doping ion is a monovalent or divalent metal ion. The doping ion may be present in the nanoparticle structure as a metal oxide. In an embodiment, the doping ion is selected from a vanadium ion, lithium ion, niobium ion, tantalum ion, barium ion, titanium ion, zirconium ion, lanthanum ion, potassium ion, ammonium ion, silver ion, copper ion, and magnesium ion, and any combination thereof. Preferably, the doping ion is selected from a vanadium ion, lithium ion, niobium ion, tantalum ion, barium ion, copper ion, and titanium ion. Preferably, the doping ion is a vanadium ion. Preferably, the doping ion is a copper ion. Preferably, the nanoparticle comprises a metal oxide and a monovalent or divalent doping ion. More preferably, the nanoparticle is zinc oxide and the doping ion is vanadium ion.

In an embodiment, the ratio of metal in the nanoparticle to the doping ion is 20: 1 to 4: 1. Preferably, the ratio of metal in the nanoparticle to the doping ion is 10: 1 to 5: 1. More preferably, the ratio of metal in the nanoparticle to the doping ion is 10: 1. The skilled person will be able to optimise the level of doping in order to achieve the desired ferroelectric behaviour. The level of doping depends on the manufacture of the nanoparticles. It is determined based on a correlation of number of cycles carried out for the materials used to make the metal in the nanoparticle and the number of cycles carried out for the doping materials used to introduce the dopant.

In an embodiment, the substrate comprises a metal, textile, ceramic, plastic, glass, or wood layer. The antimicrobial and/or antiviral nanoparticle may be covalently bonded to the metal, textile, ceramic, plastic, glass, or wood layer. Alternatively, the layer may be treated or primed, e.g. with one or more of: paint, pigment, wax, varnish, carbon, or graphite, and the antimicrobial and/or antiviral nanoparticle is covalently bonded to the paint, pigment, wax, varnish, carbon, or graphite. In an embodiment, the substrate comprises a polymer layer, optionally in combination with one or more additional layers, and the antimicrobial and/or antiviral nanoparticle is covalently bonded to the polymer layer. The one or more additional layers may comprise a hydrophobic polymer, hydrophilic polymer, metal, textile, ceramic, plastic, and glass, or wood layer.

In an embodiment, the substrate consists of a polymer layer.

The polymer may be a hydrophobic or hydrophilic polymer. The polymer can be amorphous, semicrystalline, or crystalline. Preferably, the polymer is semi -crystalline. Preferably, the polymer is poly(3- hexylthiophene-2,5-diyl) (P3HT). Preferably, the polymer has a high thermal stability, mechanical strength and high chemical stability.

In one embodiment the invention relates to an antimicrobial and/or antiviral metal oxide nanoparticle covalently bonded to a polymer substrate, wherein the nanoparticle is doped with a monovalent, divalent, trivalent, or tetravalent doping ion. Preferably the metal oxide is zinc oxide and the doping ion is vanadium. Preferably, the polymer is poly(3-hexylthiophene-2,5-diyl) (P3HT).

Preferably, a plurality of antimicrobial and/or antiviral nanoparticles are covalently bonded to the substrate.

In an embodiment, the nanoparticle size is 1 nm to 100 nm. Preferably, the nanoparticle size is 1 nm to 75 nm. Preferably, the nanoparticle size is 1 nm to 50 nm. Preferably, the nanoparticle size is 1 nm to 25 nm. Preferably, the nanoparticle size is 1 nm to 10 nm. For example, a zinc oxide nanoparticle size is 1 nm to 10 nm. Preferably, the zinc oxide nanoparticle size is 2.3 nm to 6.5 nm. Preferably, the zinc oxide nanoparticle has an average size of 3.5 nm.

The size of a nanoparticle can be measured by transmission electron microscopy (TEM) or scanning electron microscopy (SEM). For nanoparticles of less than 20 nm in size, TEM gives a precise size estimate and a high-resolution shape. The TEM method with plan-view mode can be used to capture the image of nanoparticles coated on a conductive substrate.

In an embodiment, the nanostructure of the nanoparticle has high temperature stability. Preferably, the nanostructure is stable up to 500 °C. Preferably, the nanostructure is stable up to 200 °C.

Preferably, the nanoparticle has no environmental impact. In particular, the materials forming the nanoparticle are used in low concentration and are not toxic to humans and the environment. Many of these materials are already being used in commercial products. In addition, the nanoparticles are bound to the substrate surface and hence are not released into the environment.

Nanoparticle coatings

The antimicrobial and/or antiviral nanoparticle as described above and anywhere herein can be formulated as a coating on a substrate or a surface. Thus, in another aspect, the invention relates to an antimicrobial and/or antiviral coating applied on a substrate, the coating comprising a plurality of antimicrobial and/or antiviral nanoparticles covalently bonded to the substrate. It will be understood that the coating will be covalently bonded to the substrate by virtue of the antimicrobial and/or antiviral nanoparticles that are covalently bonded to the substrate . The coating as defined herein may be a continuous layer of nanoparticles or may be a sporadic layering or film of nanoparticles. In one embodiment, the coating is a continuous layer of nanoparticles on the substrate. In another embodiment, the coating is a sporadic layering or film of nanoparticles on the substrate. The invention therefore provides a substrate having an antimicrobial and/or antiviral coating applied thereon, the coating comprising a plurality of antimicrobial and/or antiviral nanoparticles as defined anywhere herein.

The nanoparticles may be as defined anywhere herein.

In an embodiment, the coating comprises a cluster of aggregated nanoparticles in which the cluster comprises at least one nanoparticle that is covalently bonded (i) to the substrate and (ii) to at least one additional nanoparticle in the cluster. A cluster structure for ZnO is shown in Figure 3. In addition to nanoparticles being covalently bound to other nanoparticles, additional ionic interactions between nanoparticles may be present in the cluster.

In an embodiment, the substrate comprises one or more layers. Preferably, the coating is applied to substantially the entirety of the surface of the substrate (or, in the case of a substrate comprising more than one layer, to the entirety of the upper surface of the upper layer of the substrate) to form a coating layer.

The coating may comprise nanoparticles doped to a level of 5% to 20% by weight, preferably around 10% by weight.

In an embodiment, the substrate comprises a polymer layer, optionally in combination with one or more additional layers, and the antimicrobial and/or antiviral nanoparticle is covalently bonded to the polymer layer. In this embodiment, it will be understood that the polymer layer is located between the coating and any additional layers of the substrate. In an embodiment, the substrate consists of a polymer layer. The polymer may be semi-crystalline or crystalline. Preferably, the polymer is a semi-crystalline polymer. Preferably, the polymer is poly(3-hexylthiophene-2,5-diyl) (P3HT). Suitable polymers for use in the polymer layer are as described anywhere herein.

Where the substrate comprises a permeable layer, the nanoparticles can be formed within and/or on the surface of the substrate. Particularly, where the substrate comprises a polymer layer, the nanoparticles may be present within the polymer layer, as well as in the coating that is on the polymer layer. As such, at least a part of one or more clusters of nanoparticles may be present within the polymer layer, as well as in the coating that is located on the polymer layer. The polymer layer may comprise 20% to 50% by weight, preferably around 30% by weight of the nanoparticles.

In one embodiment, the coating thickness is 0.5 nm to 5 pm. Preferably, the coating thickness is 30 nm to 100 nm. Preferably, the coating thickness is 50 to 80 nm.

In one embodiment the invention relates to an antimicrobial and/or antiviral coating applied on a polymer substrate, the coating comprising a plurality of antimicrobial and/or antiviral metal oxide nanoparticles including a dopant, wherein the dopant is a monovalent, divalent, trivalent, or tetravalent doping ion. Preferably the metal oxide in zinc oxide and the doping ion is vanadium. Preferably, the polymer is poly(3- hexylthiophene-2,5-diyl) (P3HT).

The versatility and ease of use of the coating comprising the nanoparticles makes it desirable, especially with respect to its ability to retroactively treat substrate surfaces that are already in use in order to render them antiviral and/or antimicrobial.

In one aspect, a polymer layer is provided, wherein a coating is applied to at least a part of at least one surface of the polymer, and wherein the coating comprises a plurality of antimicrobial and/or antiviral nanoparticles as defined herein, each covalently bonded to the polymer layer. Preferably, the polymer is a semi-crystalline polymer. Preferably, the polymer is poly(3-hexylthiophene-2,5-diyl) (P3HT).

The polymer may be a polymer functionalised with -COOH groups or a polymer functionalised with -OH groups. Polymers functionalised with -COOH groups include carboxylic acid-poly(ethylene glycol)-b- poly(8-caprolactone), poly(l-(2-carboxyethyl)pyrrole) (PPyCOOH), poly(dimethylsiloxane) functionalised with COOH group (PDMS-COOH), and poly(3 -hexylthiophene) with COOH group (P3HT- COOH). Polymers functionalised with -OH groups include polyester with functional OH group, PIM- 6FDA-OH, poly(ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNIPAM), poly(N,N- dimethylacrylamide) (PDMAM), and poly(tert-butyl acrylate) (PtBA). Polymers that do not comprise -OH and/or -COOH groups can be functionalised (e.g. with reactive -OH groups) via treatments such as oxygen plasma treatment or illumination. Such polymers include hydrophobic polymers, such as polystyrene, PVC, polypropylene and derivatives thereof such as polypropylene derivatives e.g. biaxially oriented polypropylene (BOPP).

In one example, the substrate comprises a banknote. Current banknotes are made from a derivative of polypropylene (biaxially oriented polypropylene (BOPP)) and this provides a suitable substrate or surface for deposition of the antimicrobial and/or antiviral coating of the invention thereon.

In one aspect, an adhesive film is provided (Figure 4). In the adhesive film, the substrate may comprise an adhesive layer (4) and a backing layer (3), wherein the antimicrobial and/or antiviral coating (2) is applied on at least a part of the backing layer (3). Preferably, the coating is applied to substantially the entirety of the backing layer (3) to form a coating layer (2). Preferably, the substrate further comprises a removable release layer (1) located over the coating layer (2). The release layer can be made of any suitable material, for example paper liners, siliconized paper, siliconized film, polyester liner, and poly-coated papers. Preferably, the release layer is made of a paper liner. Preferably, the backing layer (3) is an acetate sheet. The acetate sheet may have any suitable thickness. Preferably, the acetate sheet has a thickness in the range of 0.2 to 0.3 mm. Adhesive materials include silicone adhesives, polyurethane and isocyanate adhesives, acrylic adhesives, epoxy resins, and rubber-based adhesives (e.g. based on butadiene-styrene, butyl, polyisobutylene or nitrile compounds). In an embodiment, the adhesive film comprises a polymer layer located between the coating layer (2) and the backing layer (3), wherein the antimicrobial and/or antiviral nanoparticle is covalently bonded to the polymer layer (Figure 5). In this embodiment, the polymer layer is over the backing layer (3). Therefore, in an embodiment, the adhesive film comprises five layers: a release layer (1), an antiviral and/or antimicrobial coating layer (2), an optional polymer layer, a backing film layer (3), and an adhesive layer (4). The adhesive film may optionally include one or more additional layers. For example, the adhesion of the adhesive layer may be improved by a primer layer. The adhesive layer (4) may be protected with a removable protective layer. The protective layer and the release layer (1) are removed during use.

The adhesive film can be easily applied on surfaces e.g. high tactile surfaces such as, walls, floors, doors, light switches, pens, telephones, worktops, keyboards, handles (e.g. drawer handles, trolley handles), knobs, buttons (e.g. lift button and ATM machine buttons).

In one aspect, the substrate is a textile, wherein the coating is applied to at least a part of at least one surface of the textile. Preferably, the coating is applied to one surface of the textile. Alternatively, the coating is applied to both surfaces of the textile. Preferably, the coating is applied to substantially the entirety of one surface of the textile. Alternatively, the coating is applied to the entirety of both surfaces of the textile.

Suitable textiles include silk, wool, leather, linen, cotton, flax, jute, bamboo, rayon, nylon, polyester, acrylic, and some inorganic fibres, such as cloth of gold, glass fibre, and asbestos cloth. The coated textile can be used, for example, as face masks, surgical gowns and other medical textiles as well as for general clothing and accessories.

Other substrates can be coated with the coating of the invention, including polyethylene terephthalate (PET)Zpolyester, polyimide, polyvinyl chloride (PVC)Zvinyl, rubber, silicone, acrylic, glass/fiberglass, fluoropolymer, metal foil (aluminium, aluminium-reinforced), cloth, paper, polytetrafluoroethylene (PTFE), and polyvinylidene fluoride (PVDF).

Precursor substrates having available leaving groups prior to deposition of the coating are particularly desirable. Such leaving groups can be utilised during deposition of the coating to react with a precursor to the nanoparticle thereby forming the nanoparticle covalently bonded to the substrate. Leaving groups will be known to a person skilled in the art and include, but are not limited to, a hydrogen in an -OH group or -COOH group. Leaving groups may be naturally present on certain precursor substrates. For other precursor substrates, leaving groups may be introduced by functionalising the surface of the precursor substrate.

The coating of the invention can be integrated into many products such as mobile phone screens, shopping trolleys, buttons used in lifts, cash/card machines, bank notes and coins, door handles, water taps, textiles such as surgical gowns and apparel, mattresses, furniture, reading glasses or sunglasses, kitchenware and tableware, office desks and chairs, computer keyboards, products in the automotive and transport industry, handrails in public transport systems, personal protective equipment (PPE) such as face masks, medical devices, and bedrails in hospitals.

Method of manufacture

In addition to antimicrobial and antiviral properties of the nanoparticles and coating of the invention, an important parameter to produce a reliable surface coating is the chemical process used to deposit the materials forming the nanoparticles and ultimately the coating on the surface of a substrate. As such, high- precision coating processes such as chemical vapour deposition (CVD), and in particular atomic layer deposition (ALD), can be employed to coat materials in a highly controllable and uniform manner.

In an aspect, the invention relates to the use of chemical vapour deposition (CVD) to deposit antiviral and/or antimicrobial material on a substrate. In an aspect, the invention relates to the use of chemical vapour deposition (CVD) to produce antimicrobial and/or antiviral nanoparticles covalently bound to a substrate. The features of the antimicrobial and/or antiviral nanoparticles covalently bound to a substrate may be as described anywhere herein.

In an aspect, the invention relates to the use of atomic layer deposition (ALD) to deposit antiviral and/or antimicrobial material on a substrate. In an aspect, the invention relates to the use of atomic layer deposition (ALD) to produce antimicrobial and/or antiviral nanoparticles covalently bound to a substrate. The features of the antimicrobial and/or antiviral nanoparticles covalently bound to a substrate may be as described anywhere herein.

The substrate can be any suitable material such as anywhere described herein, for example polymer, metal, glass, ceramic, plastic, wood, and textile. The features of antimicrobial and/or antiviral nanoparticles covalently bound to a substrate may be as described anywhere herein.

In one embodiment, the invention relates to a method of making a coating according to the invention, wherein the method comprises depositing nanoparticles (preferably doped metal oxide nanoparticles) on a substrate using CVD (preferably ALD). The dopant can be co-deposited with the metal of the nanoparticle (1-step deposition). Alternatively, the dopant can be deposited in a separate step to the metal of the nanoparticle (2-step deposition). Examples of 1-step deposition and 2-step deposition are shown in Figure 6.

In one embodiment, the invention relates to a method of making a coating according to the invention, wherein the method comprises depositing nanoparticles (preferably doped metal oxide nanoparticles) on an OH-terminated precursor polymer substrate using CVD (preferably ALD). See Figure 7.

CVD and ALD are techniques using vapours as precursors which can be utilised to adhere nanoparticles of the invention on surfaces, thus enabling production of a nanoparticle coating on a surface.

CVD can generally be described as follows:

CVD is parent to a family of processes whereby a solid material is deposited from a vapour by a chemical reaction occurring on or in the vicinity of a heated substrate surface. By varying experimental conditions, including substrate material, substrate temperature, composition of the reaction gas mixture, and total pressure gas flows, materials with a wide range of physical, tribological, and chemical properties can be grown. CVD is a technique that relies on the formation of a gaseous species containing the coating element within a coating retort or chamber. Alternatively, the gaseous species may be generated external to the coating retort and introduced via a delivery system. In this technique, precursor gases (often diluted in carrier gases) are delivered into the reaction chamber at approximately ambient temperatures. As the precursor gases pass over or come into contact with a heated substrate (above room temperature) in the presence of a reducing atmosphere, they react or decompose forming a solid phase which is deposited onto the substrate. The substrate temperature is critical and can influence what reactions will take place.

Using the CVD method, a wide variety of coatings may be formed, ranging from soft, ductile coatings to those with hard, ceramic like properties. Coating thicknesses can vary from 100 nm to over 200 pm.

ALD can generally be described as follows:

ALD is a deposition method which can use two precursors to deposit a metal oxide layer on a substrate with atomic scale precision. The substrate is exposed sequentially to (i) a metal-organic precursor in the form of a low-pressure vapour gas and (ii) gaseous water to form the desired metal oxide on the substrate. ALD uses low vacuum (in the range of 0.1 mbar -5 mbar) with adjustable temperature (25 °C to 350 °C) which allows to deposit metal oxide with different ranges of thicknesses.

ALD systems that can be used are widely available. ALD systems include Beneq ALD system, Oxford Instrument ALD system, LotusAT ALD system, and Forge Nano ALD system.

ALD is a reliable and simple technique to deposit a wide range of metals on a variety of substrate materials, including metal, glass, ceramic, plastic, wood, and textile. ALD is a method of choice for the following reasons:

1. ALD can dope nanostructures in the atomic scale with wide range of elements;

2. Nanostructures size is easily controllable with the number of ALD cycles;

3. The nanostructure can be grown at low temperature (e.g. < 100 °C);

4. A strong covalent bond is formed between the nanoparticle and the substrate, which produces a long-lasting coating and thus long-lasting antimicrobial and/or antiviral activity.

Coating technologies such as CVD, and particularly ALD, can be seamlessly integrated into manufacturing processes, which facilitates rapid product development. A textile and tape compatible version of the ALD machine known as roll-to-roll ALD is already available. The CVD machine is also widely utilised in different manufacturing lines. These coating technologies have been reported to coat substrates with speeds of over one meter per second for the preparation of polymer films with high barrier properties. Web speeds of over 10 meters per second have been reported for food packaging applications (Atomic Layer Deposition for Continuous Roll-to-Roll Processing, S.M. George et al., Society of Vacuum Coaters, 54th Annual Technical Conference Proceedings, Chicago, IL April 16-21, 2011 ISSN 0737-5921). The overall coating process is very cost-effective.

In an aspect, the invention relates to a method of making a coating according to the invention, wherein the method comprises the steps of: a) providing a precursor substrate having a reactive group comprising a leaving group (L) and a metalcontaining precursor gas, b) performing a first cycle comprising a step of: diffusing the metal -containing precursor gas under conditions such that the precursor substrate and the metal-containing precursor gas can react together to displace the leaving group on the precursor substrate thereby forming a covalent bond between the substrate and the metal of the metal -containing precursor gas; c) repeating step b).

The precursor substrate is optionally cleaned before growing the nanoparticles thereon. The substrate may be cleaned by any suitable method known in the art, for example using deionised water, acetone, and isopropanol in an ultrasonic bath, followed by treatment with UV-ozone or oxygen plasma.

In one embodiment, the first cycle further includes the step of diffusing a second precursor gas, such as H ₂ O(g). 02(g), Os(g), or H ₂ (g), wherein the metal-containing precursor gas can react with the substrate to form a covalent bond between the substrate and the metal of the metal-containing precursor gas, and the second precursor gas can react with the covalently bonded metal. The continued diffusion of the metal - containing precursor gas and the second precursor gas leads to the formation of nanoparticles covalently bonded to the substrate. Nanoparticles covalently bonded to the substrate are capable of further reacting with the metal-containing precursor gas and the second precursor gas to produce more nanoparticles, thereby growing a nanoparticle structure.

Where the substrate comprises a permeable layer, the nanoparticles can be formed within and/or on the surface of the substrate.

In one embodiment, step b) may be repeated for 30 to 120 cycles. Preferably, 60 to 120 cycles are carried out in step c) of the method. More preferably, 80 to 100 cycles are carried out in step c) of the method. In an alternative embodiment, step b) may be repeated for up to 10 cycles.

The size of the nanoparticles of the coating formed with the present method depends on the processing temperature and any post-thermal treatment (e.g. heating the coated substrate at 120 °C to 140 °C for 20 to 30 minutes). When the number of cycles increases, more nanoparticles form within and/or on top of the substrate and result in agglomerated nanoparticles (clusters). Preferably, clusters with nanoparticles having an average size of 5 nm to 10 nm are formed. The thickness of the deposited coating can be measured, for example, using atomic force microscopy.

In one embodiment, the reactive group is a reactive oxygen-containing group. In one embodiment, the leaving group is hydrogen. Preferably, the reactive oxygen-containing group is -OH and/or -COOH. Most preferably, the reactive oxygen-containing group is -OH. The presence of oxygen-containing groups such as -COOH and/or -OH on the substrate initiates the formation of metal compounds, such as metal oxides, by reacting with a metal -containing precursor gas. The nanoparticles are formed within and/or on the surface of the substrate containing reactive groups (e.g. -OH and/or -COOH) via a chemical bond between the metal-containing precursor gas and these reactive groups (e.g. -OH and/or -COOH). Any substrate with suitable reactive groups (e.g. -OH and/or -COOH) can be used to form chemically bonded nanoparticles (e.g. metal oxides). The substrate can be a hydrophobic polymer, a hydrophilic polymer, a metal, a textile, ceramic, wood or glass. In the present method, nanoparticles then continue to be formed using the two precursor gases comprising the metalcontaining precursor compound and H2O(g), 02(g), O4g)- or H2(g). When a polymer is used as the substrate, the nanoparticles start forming via a chemical bond to the -OH and/or -COOH group. These then form clusters of nanoparticles mainly in the amorphous region of the polymer when the number of cycles increases.

In one embodiment, the substrate is selected from: metal, glass, ceramic, plastic, wood and textile. Preferably, the substrate is glass. Preferably, the substrate is plastic. Preferably, the substrate is textile.

In one embodiment, the substrate is an OH and/or COOH- terminated surface. In one embodiment, the substrate is a polymer. Polymers functionalised with -COOH groups include carboxylic acid-poly(ethylene glycol)-b-poly(8-caprolactone), poly( 1 -(2-carboxyethyl)pyrrole) (PPyCOOH), poly(dimethylsiloxane) functionalised with COOH group (PDMS-COOH), and poly(3 -hexylthiophene) with COOH group (P3HT- COOH). Polymers functionalised with -OH groups include polyester with functional OH group, PIM- 6FDA-OH, polyethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNIPAM), poly(N,N- dimethylacrylamide) (PDMAM), and poly(tert-butyl acrylate) (PtBA). Alternatively, polymers that do not comprise -OH and/or -COOH groups can be functionalised (e.g. with reactive -OH groups) via treatments such as oxygen plasma treatment or illumination. Such polymers include hydrophobic polymers, such as polystyrene, PVC, polypropylene and derivatives thereof such as polypropylene derivatives e.g. biaxially oriented polypropylene (BOPP).

Preferably, the polymer is functionalised with -OH groups. Preferably, the polymer is functionalised with -OH end groups and/or -OH side-chain groups.

Preferably, the polymer is poly(3-hexylthiophene-2,5-diyl) (P3HT).

Preferably, step a) further includes annealing the polymer to enhance its crystallinity.

In one embodiment, when the substrate is a polymer, the method further includes step d) applying the polymer as a polymer layer on a product. Preferably, the polymer layer has a thickness of up to 1 pm, preferably up to 500 nm . More preferably, the polymer layer has a thickness of 30 nm to 100 nm . Generally, the thickness of the coating on the polymer layer is in the range of 0.5 nm to 5 pm. The polymer layer can be applied on a product via coating techniques such as spin-coating, dip-coating, printing or drop-casting.

In an aspect, the invention relates to a method of making a substrate having an antimicrobial and/or antiviral coating applied thereon, wherein the method comprises the steps of: a) providing an OH-terminated polymer precursor substrate, a metal -containing precursor gas, and a second precursor gas, b) performing a first cycle including the steps of diffusing: i. the metal -containing precursor gas, and ii. a second precursor gas comprising H2O(g), 02(g), Os(g), or H2(g), wherein the metal-containing precursor gas can react with the OH-terminated polymer precursor substrate to form a covalent bond between the polymer and the metal, and wherein the second precursor gas can react with the metal covalently bonded to polymer to form metal oxide nanoparticles; c) repeating step b) for 30 to 120 cycles.

Alternatively, step b) of the method may only be repeated for up to 10 cycles.

The metal-containing precursor may comprise one or more alkyl or halide, particularly chloride, groups where the alkyl or halide group is displaced during the reaction with the precursor substrate.

Metal-containing precursors include diethyl zinc (DEZ) for Zn, titanium tetrachloride for Ti, trimethylaluminum (TMA) for Al, triethylphosphine(6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5 - octanedionate)silver(I) [Ag(fod)(PEt3)-Ci6H2sAgF7O2P] for Ag, hexafluoroacetyl-acetonateCu(I)(3,3- Dimethyl-1 -butene) ((hfac)Cu-(I)(DMB)) for Cu, bis(N-isopropylketoiminate) iron(II) for Fe, Mn2(CO)io for Mn, tetrakis(2,2,6,6-tetramethyl-3,5-heptanedionato)cerium [Ce(thd)4] for Ce. In one embodiment, the metal-containing precursor gas is selected from: Zn(Et)2, TiC’E. A1(CH3)3, Ag(fod)(PEt3)-Ci6H25AgF7O2P, (hfac)Cu-(I)(DMB), bis(N-isopropylketoiminate) iron(II), Mn2(CO)io , Ce(thd)4, or any combination thereof. When two or more metal-containing precursor gases are combined, complex metal nanoparticles such as alloys can be formed.

In an embodiment, multiple metal -containing precursor gases can be used. When multiple metal -containing precursor gases are used, each metal-containing precursor gas is pulsed in the deposition chamber sequentially to avoid any cross-reaction between each gas and/or by-product formed in the chamber.

Preferably, the following combinations of metal-containing precursor and second precursor can be used: i) triethylphosphine(6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5 -octanedionate)silver(I) [Ag(fod)(PEt3)-Ci6H ₂₅ AgF ₇ O2P] as the Ag precursor and EE as the reducing agent, to form silver oxide; ii) hexafluoroacetyl-acetonateCu(I)(3,3-Dimethyl-l-butene) ((hfac)Cu-(I)(DMB)) and O3, to form copper oxide; iii) [bis(N-isopropylketoiminate) iron(II)] and H2O, to form iron oxide; iv) Mn2(C0) 10 and O3, to form manganese oxide; v) tetrakis(2,2,6,6-tetramethyl-3,5-heptanedionato)cerium [Ce(thd)4] and O3, to form cerium oxide.

Two-step deposition:

A two-step deposition can be carried out using ALD.

In one embodiment, step b) further includes performing a second cycle after the first cycle, the second cycle comprising diffusing a dopant precursor gas. Preferably the first and second cycles are alternated as nonoverlapping pulses, with a purge or evacuation step between each pulse.

The two-step deposition method of making a coating according to the invention may comprise the steps of: a) providing a precursor substrate having a reactive group comprising a leaving group (L), a metal containing precursor, a second precursor gas, and a dopant precursor gas, b) (i) performing a first cycle comprising: diffusing the metal -containing precursor gas under conditions such that the precursor substrate and the metal-containing precursor gas react together to displace the leaving group on the precursor substrate thereby forming a covalent bond between the substrate and the metal of the metalcontaining precursor gas; and diffusing the second precursor gas, such as H2O(g), 02(g), Odg)- or H ₂ (g). wherein the second precursor gas reacts with the covalently bonded metal to form additional reactive groups, (ii) performing a second cycle comprising: diffusing the dopant precursor gas under conditions such that the additional reactive group on the metal covalently bonded to the substrate in the first cycle and the dopant precursor gas react together to form a covalent bond; and diffusing the second precursor gas, such as H2O(g), 02(g), Odg)- or H2(g), wherein the second precursor gas reacts with the covalently bonded dopant to form additional reactive groups, c) repeating the first and second cycles of step b) such that nanoparticles comprising the metal and the dopant are formed.

The additional reactive groups formed when the second precursor gas reacts with the covalently bonded metal, in addition of being capable of reacting with the dopant precursor gas, are capable of reacting with additional metal of the metal-containing precursor gas in order to covalently bond additional metal to the substrate.

One-step deposition:

A one-step deposition can be carried out using CVD.

In one embodiment, the first cycle further includes diffusing a dopant precursor gas.

The one-step deposition method of making a coating according to the invention may comprise the steps of: a) providing a precursor substrate having a reactive group comprising a leaving group (L), a metalcontaining precursor, a second precursor gas, and a precursor dopant gas, b) performing a cycle comprising: diffusing the metal -containing precursor gas, the second precursor gas, such as TBC g), 02(g), Os(g), or H ₂ (g), and the dopant precursor gas under conditions such that:

(i) the precursor substrate and the metal-containing precursor gas react together to displace the leaving group on the precursor substrate thereby forming a covalent bond between the substrate and the metal of the metal -containing precursor gas,

(ii) the precursor substrate and the dopant precursor gas react together to displace the leaving group on the precursor substrate thereby forming a covalent bond between the substrate and the doping ion of the dopant precursor gas, and

(iii) the second precursor gas reacts with the covalently bonded metal and the covalently bonded doping ion to form additional reactive groups, which then react with additional metal of the metalcontaining precursor gas and additional doping ions of the dopant precursor gas in order to covalently bond additional metal and additional doping ions to the substrate, c) repeating step b) such that nanoparticles comprising the metal and the dopant are formed.

In an embodiment, multiple dopant precursor gases can be used. When multiple dopant gases are used, each dopant gas is pulsed in the deposition chamber sequentially to avoid any cross-reaction between each gas and/or by-product formed in the chamber.

The dopant precursor gas can be selected from: (N,N-Di-i-propylacetamidinato)lithium, tetrakis(ethylmethylamido)vanadium(IV), cyclopentadienyl barium (cp-Ba), (tert- butylimido)bis(diethylamino)Niobium, TiC14 (titanium tetrachloride), Thallium(I) hexafluoroacetylacetonate and copper(II) acetate [Cu(OAc)2] .

In an embodiment, the metal-containing precursor gas comprises an alkyl and the second precursor gas is an oxygen-based precursor gas. Preferably, ratios of metal in the nanoparticles to doping ion of 20: 1 to 4: 1 are produced. Preferably, ratios of metal in the nanoparticles to doping ion of 10: 1 to 5: 1 are produced. More preferably, a ratio of metal in the nanoparticles to doping ion of 10: 1 is produced. The amount of dopant can be controlled easily in the process of deposition. Metal -containing precursor gas(es) and precursor dopant gas(es) are deposited using a number of cycles proportional with the desired ratio of metal to doping ion desired.

Nanoparticles as defined herein can be obtained by co-deposition of different metal oxides such as ZnO/TiO ₂ , ZnO/Al ₂ O ₃ , ZnO/VO ₂ , ZnO/ CuO, CuO/ TiO ₂ , CuO/ A1 ₂ O ₃ , SiO ₂ /CuO, SiO ₂ /TiO ₂ , and SiO ₂ /ZnO.

The deposition time varies depending on the type of substrate. Preferably, the deposition time for the metalcontaining precursor gas is in the range of 0. 1 second to 0.5 second. Preferably, the deposition time for the second precursor gas is in the range of 0.1 second to 0.5 second. Preferably, the deposition time for the dopant precursor gas is in the range of 0.1 second to 0.5 second. Preferably, the waiting time between each gas deposition is 0.5 second to 1 second. The wait time allows for purging out any by-product formed as a result of each precursor reaction with the substrate and/or the nanoparticle structure.

The deposition is preferably carried out at a temperature below the thermal stability of the substrate. In one embodiment, steps b) and c) are carried out at a temperature below 150 °C. Preferably, steps b) and c) are carried out at a temperature below 100 °C. Preferably, steps b) and c) are carried out at a temperature between 70 °C and 100 °C. More preferably, steps b) and c) are carried out at a temperature between 80 °C and 90 °C.

Uses

The nanoparticles and coating according to the present invention exhibit an antimicrobial and/or antiviral activity. In particular, the present nanoparticles and coating produce a rapid (within seconds or minutes) inactivation of viruses, including coronavirus. This is achieved, at least in part, by producing inherent electrical properties (e.g. ferroelectric properties) to inactivate viral particles, which, unlike the prior art, produces an electric field without the need for electrically conductive medium. In particular, Sen et al (Electroceutical Fabric Lowers Zeta Potential and Eradicates Coronavirus Infectivity upon contact. ChemRxiv (2020)) discloses the use of a very weak (0.5 V) electric voltage that is set up between alternating dots of Ag and Zn printed on a polyester fabric. However, it is believed that this voltage is too weak to provide either sufficient electrolysis of the environment surrounding the microbe/virus and/or to destabilise the electrochemical properties of the microbial/viral membrane sufficiently to render the microbe/virus non-viable.

Specifically, the doping of the nanoparticles induces ferroelectric properties. For example, the doping of zinc oxide with vanadium confers ferroelectric properties. The nanoparticles produce a weak electric voltage of about 0.2 to 1.5 V, more preferably of about 0.5 to 1 V or 1 to 1.5 V, regardless of environment conditions, which destabilises the electrokinetic properties of viruses in seconds by changing the zeta potential of the viral particles. A piezoelectric voltage of 0.5 to 8 V is also possible and encompassed within the scope of the present invention

The present nanoparticles and coating may combine additional multiple mechanisms of action to inactivate microbes and/or viruses:

- Oxygen containing nanoparticles may release reactive oxygen species (ROS) which damage the membrane and RNA/DNA proteins of viruses and microbes. ROS can be generated not only from metal oxides but from any redox-active metal (such as some of the transition metals) which can reduce oxygen in the environment. The sequential reduction of oxygen through the addition of electrons leads to the formation of a number of ROS including superoxide anions (O ^2- ), hydrogen peroxide (H2O2), hydroxyl radicals, and hydroxyl ion (HO-). The release of ROS can be measured using electron paramagnetic resonance (EPR) spectroscopy.

- Metal ions released by the nanoparticles may change the structure of the nucleic acid and affect viral genetic information. Metal ion concentrations are known to efficiently impair the replication of a number of RNA viruses, e.g. by interfering with correct proteolytic processing of viral polyproteins. For example, it has been shown that coronavirus and arterivirus replication can be inhibited by increased Zn ²⁺ levels (te Velthuis, A. J. et al. Zn(2+) inhibits coronavirus and arterivirus RNA polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture. (PLoS Pathog., 6(1 l):el001176 (2010)). The release of metal ions can be measured using fluorescent sensors.

- The nanoparticles may damage lipids and proteins in viral envelopes (e.g. glycoprotein spikes) and hinder the virus from binding to the host cells (Akhtar, S. et al. Antibacterial and antiviral potential of colloidal Titanium dioxide (TiO2) nanoparticles suitable for biological applications. Materials Research Express, 6(10) (2019)).

Standard tests to measure antiviral and antimicrobial properties include cytopathic effect (CPE) inhibition assay, plaque assay, qPCR assay and flow cytometry.

A cytopathic effect (CPE) inhibition assay can be used to measure the antiviral property of the nanoparticles of the invention. Many combinations of cells and viruses can be used to measure viral infectivity via CPE assay. MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) and ATP luminescence assays are both efficient and simple ways of quantifying the CPE. A range of viruses and host cell lines are used to quantify the cytopathic effect of a particular virus to a particular cell line after exposure to the coated substrates of the invention.

In summary the cells are seeded in cell culture plates and incubated for 18-24h. Aqueous suspension of viral particle are then placed on a small size of the coated substrate material (e.g. 2 cm x 2 cm sample) and also uncoated substrate (uncoated control) for a specified contact time (i.e. this is the time under investigation which specifies the speed of action of the coated substrate). The viral particles are then recovered from the substrate by rinsing serum- free medium on the substrate and the efficiency of this viral recovery is quantified using nanoparticle tracking analysis. The recovered viruses are used to infect the seeded host cells while using parallel sets of cells infected with untreated viruses as positive control and uninfected cells as negative control. Cell viability test of these cell sets is then carried out using the MTT assay kit which is purchased from a manufacturer (MTT Assay Kit (Cell Proliferation) (ab211091 )) or ATP luminescence (commercial kit ViralToxGlo (Promega)).

A fluorescent cell viability assay can be used to measure the antimicrobial properties of the nanoparticles of the invention. To measure anti-microbial activity, fluorescent dyes are used throughout different techniques as a rapid way to distinguish between populations and determine the viability of cells. An example is the bacterial viability assay kit from abeam (ab 189818), which utilises two highly specific, ultrasensitive fluorescent reagents to assess quickly and easily the percentage of live and dead cells within a bacterial culture. The total cell stain is permeant to all cells and thus all bacteria within the culture will be stained allowing the total number to be calculated. The dead cell stain is impermeant to living cells and as such will only be able to enter and stain dead cells; this allows the number of dead cells to be calculated. The ratio of dead to live cells can then be quickly and easily calculated. In this method, an aqueous suspension of the bacterial cells is placed on a small size of the coated substrate material (e.g. 2 cm x 2 cm sample) and also uncoated substrate (uncoated control) for a specified contact time (i.e. this is the time under investigation which specifies the speed of action of the coated substrate). The suspension is then recovered from the substrate by rinsing serum free medium on the substrate. Cells are then harvested by spinning at 10,000 g for 10 min and resuspended in a buffer. Cells are incubated for 1 hr and spun at 10,000 g for 10 min. Cells are resuspended and the procedure is repeated. Total cell stain and dead cell stain are then added. Cells are either incubated for 1 hr and analysed with a microplate reader or incubated for 15 min and analysed with fluorescence microscope.

In one aspect, the invention relates to the use of: i. the coating of the invention, or ii. the substrate according to the invention, or iii. the textile of the invention, or iv. the polymer layer of the invention, or v. the adhesive fdm of the invention as an anti-microbial agent.

In another aspect, the invention relates to the use of: i. the coating of the invention, or ii. the substrate according to the invention, or iii. the textile of the invention, or iv. the polymer layer of the invention, or v. the adhesive fdm of the invention as an anti-viral agent.

In another aspect, the invention relates to the use of: i. the coating of the invention, or ii. the substrate according to the invention, or iii. the textile of the invention, or iv. the polymer layer of the invention, or v. the adhesive fdm of the invention for inactivating germs. In one embodiment, the germ is a virus. Preferably, the virus is coronavirus, influenza, or norovirus. More preferably, the virus is coronavirus.

In one embodiment, inactivation ofthe germs occurs within 1 second to 10 minutes. Preferably, inactivation of the germs occurs within 1 to 60 seconds. More preferably, inactivation of the germs occurs within 30 seconds. More preferably, inactivation ofthe germs occurs within 15 seconds.

EXAMPLES

Example 1 - Preparation of the polymer substrate

A semi-crystalline polymer such as poly (3 -hexylthiophene) (P3HT) with a low molecular weight of 5.5 kDa and carboxylic acid ending group (-COOH) was used as the substrate top layer within/on which the nanoparticles were grown. The polymer in a form of powder was dissolved at a concentration of 5 mg/ml in a low boiling point solvent such as chloroform and was spin coated on top of a glass substrate. The spin coating was performed in an inert environment (nitrogen glovebox) and the polymer thickness was in the range of 35 - 40 nm. Following the formation of a thin layer of polymer, a controllable hotplate was used to anneal the polymer up to its melting point (185 °C). The polymer was then cooled down slowly (with a rate of 5 °C/min) to the room temperature. This process was performed in a nitrogen glove box to avoid polymer exposure to moisture. When the polymer reached room temperature, the atomic layer deposition (ALD) process was started as described by Moghaddam et al (Nano Lett. (2013) 13(9), 4499-4504).

Example 2: Preparation of a ZnO coating on a polymer substrate using ALD

As shown in Figures 8 and 9, the polymer substrate was loaded inside the ALD chamber and a low vacuum of about 0.1-5 mbar was applied to the chamber for ALD reaction. To form and grow the ZnO within the polymer network, two ZnO ALD precursors which are diethylzinc (DEZ) and H2O were introduced in a sequential way in the chamber. The set temperature for the reaction was 80 °C and the two gases were conducted in the chamber in a sequential way with a pulse time of 0.1 second and purge time of 0.5 second. The number of cycles was typically varied between 30 to 120 cycles, but sometimes lower cycle numbers were found to be sufficient. The number of cycles determines the load of ZnO within/on the polymer substrate.

Example 3 : Preparation of ZnO coating on a glass substrate using ALD

A glass substrate has abundant -OH groups on its surface. These -OH groups are reactive sites to the metal precursor (DEZ in the case of ZnO deposition). In the first cycle, diethylzinc (DEZ) reacted with the -OH groups on the surface of the glass substrate and formed O-Zn-Et in the first half cycle. The second half of the cycle introduced water vapour to the chamber to react with O-Zn-OH at the end of the first cycle. 60 cycles were run at 80 °C with a pulse time of 0.1 second and purge time of 0.5 second. The thickness of the ZnO layer formed on the glass substrate was around 0.5 nm per cycle as measured by atomic force microscopy (AFM) and this thickness increased to 1 nm per cycle upon increase of the temperature to around 200 °C.

Interestingly, without the inclusion of a polymer coating and depending on the number of deposition cycles, nanoparticles were deposited either in the form of island growth on the surface or formation of thin film, the latter being seen above twenty cycles.

Example 4: Preparation of a TiO coating on a glass substrate using ALD

For the deposition of TiCL. a glass substrate was put at 220 °C for 10 min prior to the deposition. The substrate was exposed to titanium tetrachloride (Ti Cl 4) and water in a sequential way to form a TiCL layer. TiCL was introduced in the chamber with a pulse duration of 0. 1 second and purge duration of 0.5 second. TiCL reacted with the -OH group on the surface of the glass substrate to form the O-Ti-O-C L and HC1 as a by-product which was purged out of the chamber. The second precursor, H2O as the second half of the first cycle was pulsed in and completed the reaction to make O-Ti-O-OH and HC1 as a by-product which was purged out at the end of the second cycle. Each deposition cycle made a layer of TiO2 that was around 0.2 nm thick, as estimated by the ratio of thickness and number of cycles (Thickness/number of cycles). The final thickness of the TiCF coating was 25-30 nm as measured by AFM.

Example 5: Preparation of a doped ZnO coating using ALD

To obtain a ZnO layer containing 10% of vanadium, a combination of tetrakis [ethylmethylamino] vanadium (V(NC ₂ H ₅ CH ₃ )4 or TEMAV) and H2O was used at the ratio of 10:90. After 9 cycles of ZnO deposited at 80 °C, 1 cycle of vanadium oxide was deposited at 80 °C. Vanadium doped ZnO was formed. The nanostructure obtained showed ferroelectric properties as determined by measuring the capacitance of doped ZnO in a capacitor structure versus external applied voltage. Ferroelectric properties are illustrated as a hysteresis loop (see Figure 14, for example). To obtain copper doped ZnO, the same procedure used for vanadium is applicable for Cu doped ZnO. The precursors used for CuO in ALD were copper(II) acetate [Cu(OAc)2] and water vapor. The nanostructure obtained had ferroelectric properties due to the presence of copper within the ZnO thin film.

Example 6: Characterisation of the coating - UV-visible absorption

The samples were tested with UV-visible absorption in which UV-visible spectroscopy was used to send light at different wavelengths, from 200 nm to 900 nm, and the transmission of light from the film was measured. The absorbance of the film at different wavelengths from UV to near infrared region was then calculated using software associated with the spectrophotometer. (Hewlett-Packard 8453 UV-vis spectrometer with a 280-1100 nm spectral range) The polymer without carboxylic acid was chosen as a reference and P3HT with COOH groups and without/with AED treatment was chosen for comparison. Referring to Figure 10, P3HT without -COOH acid groups showed higher crystallinity due to the better resolved peaks at 2 eV and 2.2 eV as a result of 0-0 and 0-1 vibronic absorption. These peaks were less resolved upon chemical modification of P3HT with -COOH functional ending group. This can be due to the change of the crystallinity of the polymer as a result of introducing the functional group in the polymer. Upon treatment of P3HT-COOH with ALD and formation of (un-doped) ZnO, the absorption peaks were much less resolved. This is due to two reasons: the formation of ZnO within the polymer can change the crystalline domain configuration and size of the polymer; in addition, the presence of ZnO nanoparticles within the polymer network can increase the light scattering which results in less absorption of the polymer/ZnO structure. The P3HT/ZnO structure showed an extra absorption starting at 3.3 eV which is for ZnO nanoparticles.

Example 7: Characterisation of the coating - Crystallinity (GIWAX and GISAX)

Referring to Figure 11, grazing incidence small angle x-ray diffraction (GISAX) (Figure 1 la) as described by Moghaddan et al (Nano Lett. (2013) supra) was used to measure the distance between the ZnO nanoparticles within the polymer network and it was shown to be in the range of 12.5 nm, which is consistent with the polymer chain length (in the range of 12-14 nm). Without the polymer, the crystalline properties of ZnO did not change because the measurement used shows the structure of the metal oxide which was highly related to the processing condition of ZnO.

Grazing incidence wide angle x-ray diffraction (GIWAX) (Figure 1 lb) as described by Moghaddan et al (Nano Lett. (2013) supra) was used to find out the crystallinity of any ultrathin/ thin film structure. The ZnO nanoparticle and P3HT structure were assessed by GIWAX. ZnO deposited by 60 cycles of ALD at low temperature (80 °C), has shown poly-crystalline structure at [1 0 0], [0 02] and [1 0 1] directions. P3HT has only shown weak crystallinity at [1 0 0] direction.

Example 8: Characterisation of the coating - Atomic force microscopy (AFM)

As described by Moghaddan et al (Nano Lett. (2013) supra), AFM was used to determine the nanoparticle coating thickness. The coverage of the polymer on the substrate could be seen as well as whether well- ordered nanoparticles with a specific range of nanoparticle sizes were deposited on the surface of the substrate.

Also as described by Moghaddan et al (Nano Lett. (2013) supra), in order to measure the thickness of the coating, the coated substrate was slightly scratched with a sharp needle point in the glass surface and the AFM tip was put at the vicinity of the scratch to measure and capture the image both on the polymer substrate and on the glass substrate. Example 9: Characterisation of the coating - Transmission electron microscopy ITEM)

As described by Moghaddan et al (Nano Lett. (2013) supra), TEM measurements have been carried out both in plan-view and cross-sectional TEM.

The plan view showed different lines formed on the surface of the polymer and these lines were from a few nanometers up to 0.5 micron. The size of these nanoparticles as agglomerated structures varied from 2.3 nm to 6.5 nm with an average size of 3.7 nm (Figure 12).

The cross-sectional showed how the ZnO-P3HT was formed using ALD. The structure showed a comblike structure of ZnO within the polymer network with length scale of 10-12 nm in agreement with GISAX measurement.

Example 10: Characterisation of the coating - P-V measurement

Polakowski and Muller (Appl. Phys. Lett. (2015) 106. 232905) have measured the polarisation versus voltage with the use of an analyser, function/waveform generator, power amplifier and probe station (see Yoon et al (2019) Journal of Vacuum Science & Technology B37, 050601). For a ferroelectric material in a format of ferroelectric capacitor, the polarisation versus voltage upon voltage sweep above the coercive field of the ferroelectric materials showed hysteresis loops. An example of this measurement for a doped metal oxide (Al -doped HfCE) deposited by ALD is illustrated in Figure 13.

Example 11 : Anti-viral effect of weak electric field

Sen, A. et al (Electroceutical Fabric Lowers Zeta Potential and Eradicates Coronavirus Infectivity upon contact. ChemRxiv (2020)) have shown that weak electric voltage eradicates the infectivity of coronavirus (ATCC® VR-2384TM) upon contact in less than 1 minute by affecting the electrokinetic properties of the viral particles. When exposed to a weak electric field, the zeta-potential of viral particles is moved close to zero, which affects surface charge interaction between individual particles producing large sizes (in the micron range) of viral aggregates. As described by Mohseni M. et al (Materials and Design (2020) 191, 108610) the rapid aggregation (i.e. in less than 10 seconds) of iron-oxide nanoparticles with similar size (100 nm) to Sars-CoV-2 viral particles was observed at the zeta-potential value of less than 25 mV, which affected the colloidal stability of these particles in the solution. Figure 1 shows a TEM image of the aggregated 100 nm iron-oxide nanoparticles suspended in ddFEO.

This effect has been validated in a recent study by Sen, A. et al. (2020) supra (https://chemrxiv.org/engage/chemrxiv/article-details/60c74b 33bb8cla34d03dbl70) where exposure to 0.5 V for durations of 1 and 5 min has resulted in inactivation of coronavirus. The MTT assay carried on the infected cells indicated that cells infected with the virus which was exposed to the electric field generating substrate were viable at levels comparable to those with no coronavirus exposure at all. Infection of cells with coronavirus caused marked loss of cell viability, however, such cytopathic effect was completely absent once the virus was exposed to the electric field generating substrate (Figure 14) for both 1 min and 5 min contact times. These results were further validated using a calcein/PI fluorescence assay in the same study.

Example 12: Anti-viral effect of ZnO nanoparticles

Zinc oxide films grown by atomic layer deposition (ALD) have shown a considerable amount of zinc -ion. For example, a ZnO coated layer of 50 nm thickness at 120 °°C has shown to produce a Zn ²⁺ concentration of 9.5 mg/L (Kaariainen M. L. et al. Zinc release from atomic layer deposited zinc oxide thin fdms and its antibacterial effect on Escherichia coli. Applied Surface Science, 287(15), 375-380 (2013)). Zn ²⁺ concentrations are known to efficiently impair replication of a number of RNA viruses, e.g. by interfering with correct proteolytic processing of viral polyproteins. It has also been shown that corona- and arterivirus replication can be inhibited by increased Zn ²⁺ levels (te Velthuis, A. J. W. et al. Zn ²⁺ Inhibits Coronavirus and Arterivirus RNA Polymerase Activity In Vitro and Zinc Ionophores Block the Replication of These Viruses in Cell Culture. PLoS Pathog., 11(6), el001176 (2010)).

Example 13: Anti-viral effect of TiO? nanoparticles

Published studies have demonstrated the inactivation of influenza virus through TiCE photocatalysis using TiCE nanoparticles immobilised on a glass plate (Nakano R. et al. Photocatalytic inactivation of influenza virus by titanium dioxide thin fdm. Photochem. Photobiol. Sci., 11, 1293-1298 (2012)). In this study, the viral titers were dramatically reduced by the photocatalytic reaction. The inactivation of the influenza A virus (H1N1) with TiCE was dependent on the UV-A light intensity. However, even with a low intensity of UV-A (0.01 mW cm ² ). a viral reduction of approximately 4-loglO was observed within 2h exposure time. In a separate study, 61og reduction of the influenza A virus (H3N2) was observed even in darkness when TiCE nanoparticles were in direct contact with viral particles for less than Ih at high concentration of nanoparticles (7 mg/ml) (Mazurkova N. A., et al. Interaction of titanium dioxide nanoparticles with influenza virus. Nanotechnol Russia, 5, 417-420 (2010)).

The main mechanism of anti-viral activity of TiCE nanoparticles is attributed to damaging lipids in viral envelopes, due to which the glycoprotein spikes are damaged, and their attachment was blocked that could not initiate infection (Akhtar, S. at al. Antibacterial and antiviral potential of colloidal Titanium dioxide (TiO2) nanoparticles suitable for biological applications. 2019 Mater. Res. Express 6 105409). Similar to Coronavirus, Influenza A is also an enveloped virus, with a genome of a single strand RNA, and relies on its surface spike proteins to enter cells and infect them (Jiang C. et al. Comparative review of respiratory diseases caused by coronaviruses and influenza A viruses during epidemic season. Microbes and Infection, 22, 236e244 (2020)).

Example 14: Anti-microbial effect of the coating

Pasquet et al (Science Direct (2014); 457(5) p.263-274 https://doi.Org/10.1016/j.colsurfa.2014.05.057) investigated the antimicrobial activity of ZnO on microbial cultures in broth medium. In this study, an aqueous suspension of the bacterial cells was placed on a small size of the coated substrate material (2 cm x 2 cm sample) and also uncoated substrate (control) for a specified contact time (i.e. this is the time under investigation which specifies the speed of action of the coated substrate). The suspension was then recovered from the substrate by rinsing serum free medium on the substrate. Cells were then harvested by spinning at 10,000 g for 10 min and resuspended in a buffer. Cells were incubated for 1 hr and spun at 10,000 g for 10 min. Cells were resuspended and the procedure repeated. Total cell stain and dead cell stain were then added. Cells were either incubated for 1 hr and analysed with a microplate reader or incubated for 15 min and analysed with fluorescence microscope.

Example 15: Ferroelectric modelling

The invention relies on the non-uniform size distribution of ferroelectric and piezoelectric nanoparticles and harnesses the ferro- and piezoelectric properties of these nanoparticles as an antiviral and/or antimicrobial solution. Nanoparticles of different sizes develop different polarization compared to the neighbouring nanoparticles under an external stimulus, such as voltage or pressure. A nanoscale potential difference is created between the nanoparticles coated on a surface. When a germ lands between the nanoparticles the potential difference between the nanoparticles disturbs the electrical charge on the surface of the germ to the extent that the germ is then unable to function.

In the case of piezoelectric particles, the voltage created across the particles is proportional to the thickness of the particle. Considering the area and the external force are the same, the voltage will be smaller for the thinner particles, causing a potential gradient between the neighbouring nanoparticles which can be more than 1 volt.

The voltage across a piezoelectric nanoparticle can be estimated using the relation:

Voltage = (d33 x Force x Thickness) / (s x a _r x Area)

When Force (F) is applied on a piezoelectric material with a Thickness of (d) and on an Area of (A) with piezoelectric coefficient of d33, it produces a voltage of V is produced. Epsilon(o) is permittivity of free space and Epsilon(r) is relative permittivity.

Using typical values for doped ZnO and the force on the particle to be 1 micro Newton, the voltage across lOOnm and 50nm thick nanoparticle of area lOOnm x lOOnm, will be about 6V and 3 V respectively, giving a potential difference of 3V which is sufficient to cause viruses and germs to be deactivated or killed.

Similarly, the remnant field in polarised ferroelectric particles also depends on their size that creates a field gradient between the non-similar ferroelectric nanoparticles. If QI and Q2 are the charges on the surface of neighbouring particles, as shown in Figure 2, the potential difference can be estimated using the following electrostatic equation: dV = 9 x 10 ⁹ (Q ₂ - Qi) / d

If QI and Q2 are equivalent to 100 and 200 electron charges respectively and are separated by about lOOnm, a potential difference between them can be about 1.5V. The local potential gradient disturbs the zetapotential of the germs and enhances their instability. A local potential difference of more than 1.3V can also cause water electrolysis which also kills the germ.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Those skilled in the art will recognise, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

All patents and publications referenced herein are hereby incorporated by reference in their entireties.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.