A dual action antimicrobial film

"A dual action antimicrobial film"

Introduction

The invention relates to a dual action antimicrobial film for coating a surface to provide a long term antimicrobial effect and prevent biofilm formation.

Silver and copper has been used for centuries in the treatment of burns and chronic wounds due to its ability to promote crust formation on the surface of the wound and provide anti-bacterial functionality. Its antimicrobial mechanism may be due to binding to proteins and enzymes inhibiting their function. The introduction of antibiotics in the 1940's led to a reduction in the use of silver in particular for bacterial infections. However, with the emergence of antibiotic resistant bacteria in the last number of decades silver has regained popularity in fighting infection and the spread of microbes.

Photocatalytic Ti0₂ has been shown to have, among other properties, anti-microbial activity due to its ability to breakdown organic material to C0₂ and H₂O.Ti0₂ can physically degrade the cell wall of the microbe leading to lysis and cell death. An increase in functionality of Ti0₂ can be accomplished by shifting the band onset into the visible light region via introduction of elemental dopants. However, in low lit or dark areas the photocatalytic effect is reduced or halted completed.

There is a need for improved antimicrobial films with enhanced properties.

Statements of Invention

According to the invention there is provided a dual action antimicrobial film comprising a visible transparency of greater than 75% transmission at visible light wavelengths of between 400 and 900nm, the film comprising a coating solution of soluble Ti0₂ and a metal species wherein the dual action antimicrobial activity comprises metal ion diffusion release and Ti0₂ photocatalytic activity. In one embodiment of the invention the film is a smooth film displaying regulated isotropic crystal growth and comprises a roughness factor (Ra) of less than 1.5nm.

In another embodiment of the invention the antimicrobial action of the film is active under both light and dark conditions.

Ina preferred embodiment of the invention the film prevents biofilm formation when the film is deposited on a substrate.

In one embodiment of the invention the antimicrobial film comprises an even concentration distribution of Ti and metal species comprising an average Ti0₂ to metal species concentration ratio of from 1:0.005 to 1:0.1.

In one embodiment of the invention the Ti0₂ comprises doped and/or undoped Ti0₂. Preferably the Ti0₂ is doped with sulphur and/or fluorine.

In one embodiment of the invention the metal species in is elemental or oxide form. Most preferably the metal is silver or copper.

In one embodiment of the invention the antimicrobial action of the film, metal ion diffusion release and Ti0₂ photocatalytic activity, provides a long term antimicrobial effect.

In one embodiment of the invention the coating solution comprises a homogenous solution of nano-particle sized soluble Ti0₂, metal species and/or a chelating agent and/or coordinating solvent. Preferably the particle size of the Ti0₂ and metal species in the coating solution is less than lOnm.

According to the invention there is provided a method of synthesising a dual action antimicrobial film as claimed in any preceding claim comprising the steps of; preparing and solubilising hydrous Ti0₂,

preparing a solution of Ag/TFA and/or Cu/TFA; combining the hydrous Ti0₂ with the Ag/TFA solution in the presence of a chelating agent and/or coordinating solvent to form a coating solution;

coating a substrate with the coating solution; and

annealing the substrate coated with the coating solution to form a film.

In one embodiment of the invention the substrate coated with the coating solution is annealed at a temperature between 500 and 1000°C.

According to the invention there is provided a substrate comprising a dual action antimicrobial film.

According to the invention there is provided a method of promoting antimicrobial activity comprising coating a substrate with a coating solution of nano-particle sized soluble Ti0₂, metal species and/or a chelating agent and/or coordinating solvent and annealing to form a transparent long acting dual action antimicrobial film.

Brief Description of the Invention

The invention will be more clearly understood from the following description thereof, given by way of example only, with reference to the accompanying drawings, in which:

Fig. 1 is a schematic illustration of the formation of biofilms on a silver release surface;

Fig. 2 is a schematic illustration of the prevention of biofilm formation using the dual action antimicrobial film according to the invention;

Fig. 3 is a schematic illustration of the dual action of the antimicrobial film via (a) photocatalytic Ti0₂ and (b) Ag⁺ release according to the invention; Fig. 4 shows X-ray diffraction (XRD) analysis of the Ti0₂ and silver components in a deposited matrix film according to the invention;

Fig. 5 is a photograph illustrating the hybrid nature of the films coated onto (a) ceramic and (b) steel coupons;

Fig. 6 is an Energy Dispersive X-ray Spectroscopy (EDS) spectra of

Ag-S/F/Ti0₂ coated steel samples;

Fig. 7 is a table of the elemental concentration of Ti and Ag on coated steel films;

Fig. 8 is an Energy Dispersive X-ray Spectroscopy (EDS) spectra of

Ag-S/F/Ti0₂ coated ceramic samples;

Fig. 9 is a table of the elemental concentration of ceramic coated films;

Fig. 10 is a graph showing the % transmission between uncoated and Ag- S/F/Ti0₂ coated glass samples;

Fig. 11 is a graph showing the % transmission between uncoated and Cu- F/Ti0₂ coated glass samples;

Fig. 12 is a table showing silver ion release under different conditions;

Fig. 13 is a table showing silver ion release sustained over time;

Fig. 14 is a table showing silver ion release sustained over time when placed in a new saline solution;

Fig. 15 is a table showing the ion diffusion of copper and silver over time;

Fig. 16 is an atomic force microscopy measurement showing the difference between (a) Uncoated glass and (b) Ag-S/F/Ti0₂ coated glass; Fig. 17 is a schematic illustration of test and control conditions prior to spiking the samples with a bacterial load; and

Fig. 18 is a table of results of the test and controls outlined in Fig. 17 subjected to Escherichia coli and Staphylococcus aureus survival testing.

Detailed description

Antimicrobials containing titanium dioxide and silver are known. US2009/0220600 (Parkin et al) describes a film consisting of a titanium dioxide host matrix comprising silver oxide particles. The film is prepared from a colloidal precursor solution. Silver oxide nanoparticles are deposited in or upon the titanium dioxide film. The antimicrobial effect of the films is activated by exposure to a light source. The silver nanoparticle solutions are quite unstable.

Surprisingly it was found in the present invention that combining photocatalytic Ti0₂ and anti-bacterial Ag and/or Cu species into a single composite film provides a dual action coating for surfaces having enhanced properties. The dual action coating has surprisingly been found to provide a sustained long term antimicrobial effect and prevent biofilm formation. The antimicrobial film of the invention has been shown to maintain a long term antimicrobial action under both light and dark conditions. The antimicrobial film of the invention has been shown to have a very high microbial kill rate under dark conditions.

The solutions of Ti0₂ and metal species are homogenous in nature with approximately less than 5nm size particles resulting in an even distribution of Ag/Cu ions among the Ti0₂ particles. As the particle size begins at a very low level the Ag is well distributed among the Ti0₂ particles. In comparison to other known films comprising silver and Ti0₂, the films of the present invention show no particle agglomeration. With the films of the present invention the Ag/Cu species are evenly integrated into the Ti0₂ matrix during film formation on a 'nano-level'. This results in an increase in the anti-microbial activity of the film as there is a greater distribution of metal species on the surface leading to greater diffusion of silver/copper ions with the ability to attack microbes. Conventional silver ion release films are susceptible to biofilm formation. Silver ion release can initially slow down the formation of biofilms but eventually viable biofilms will propagate. It has been reported that in less than 10 days a mature biofilm layer will establish on silver ion releasing surfaces. Once the surface has been covered with a biofilm layer its antimicrobial action is lost and microbial propagation on the surface will occur as shown in Fig. 1.

A biofilm layer is a combination of biomolecules that can adhere onto an artificial surface. It provides a means of survival for microbes outside of a host organism and a vehicle for re-infection of a population. It acts as an anchor surrounding the microbial cells protecting them against unfavourable conditions such as temperature or chemical attack through cleaning. Human pathogens have shown resistance to antibiotic attack through biofilm formation.

The anti-microbial activity of the Ag-S/F/Ti0₂ films of the present invention utilises and combines two mechanisms of action, silver and/or copper ion release and photocatalytic Ti0₂. Microbes are attacked by silver ion release as soon as they come into contact with the film killing them. The silver/copper species work by diffusing from the film as metal ions that are absorbed by the microbe and attack the cellular functions via a number of methods, e.g. inhibition of protein assembly, redox cycling, membrane lipid peroxidation etc. The second component of the film is Ti0₂ which is a light activated catalyst which continues to fully breakdown the organic matter of the dead microbe. In this way the dual action of the film of the present invention prevents any formation of a biofilm and enables continued and sustained metal ion release activity. Fig. 2 is a schematic showing the dual action of the silver and Ti0₂.

The anti-microbial mechanism of silver and copper is based on binding and inhibition of protein function, inhibiting protein assembly, redox cycling, membrane peroxidation etc. Ti0₂ employs a different mode of action by physically breaking down the structure of the cell. It attacks the organic material of the cell wall and cell contents. Ti0₂ is a light activated semi-conductor material, a light activated catalyst that generates reactive oxygen species that attack the microbe reacting with it to form C0₂ and H₂0. Photocatalytic Ti0₂ is used as an effective tool against biofilm formation. Photons of light are absorbed generating electron-hole pairs that migrate to the surface of the Ti0₂ particle. There they react with surface water to produce a number of reactive oxygen species (ROSs) via a series of oxidation and reduction reactions. The principle reactive species responsible for the anti-microbial action is the hydroxyl radical which attacks organic material in a highly oxidative, nonspecific fashion. The hydroxyl radical causes the breakdown of the cell wall and cytoplasmic membrane where leakage of cytoplasm, including critical ions such as K⁺, results in cell death. The breakdown of organic matter by Ti0₂ eradicates the presence of biofilms. Activated Ti0₂ breaks down the biomolecules that make up the biofilms as well as the attach threads that anchor the biofilm to the surface. Photocatalytic Ti0₂ requires light for activation. In areas that are not illuminated consistently or areas that are dimly lit the Ag and Cu ion diffusion provides effective antibacterial activity to boost the efficiency of the film.

The Ag-S/F/Ti0₂ films of the present invention provide a dual action coating for surfaces. The composite Ag-S/F/Ti0₂ films according to the present invention provide a hybrid dual action coating as shown in Fig. 3. It has been found that silver and copper bind and inhibit protein function while the Ti0₂ breaks down the structure of the organic material. In areas not illuminated consistently the silver or copper ion release is utilised to boost the anti-microbial efficiency of the film. In addition the breakdown of organic matter by Ti0₂ eradicates the issue of biofilms.

The presence of both species, Ti0₂ and metal species, in the deposited film is shown in Fig, 4 which shows the results of X-ray diffraction (XRD) analysis carried out. Each species has its own characteristic crystal form and XRD has the ability to differentiate between them. The XRD pattern contains all the characteristic peaks of anatase Ti0₂ as well as additional peaks that can be attributed to silver. The XRD analysis indicates the presence of elemental silver in the deposited film.

Originally, the silver species was believed to be in oxide form due to annealing of the films at high temperature in an oxygen environment leading to thermal oxidation of silver in the matrix film. XRD analysis confirms the presence of elemental silver in films, however, this does not rule out the possibility of smaller quantities of silver species in the oxide form present in the matrix film. This is also true for copper species present in a matrix film.

A common issue with silver ion containing solutions is their inherent instability in solution particularly in mixtures with other materials such as anions or material particles etc.

In the present invention the dual action antimicrobial film comprises a coating solution of Ti0₂ and a metal species containing silver or copper. A coating solution comprising both silver and copper species in combination with Ti0₂ may also be used. The coating solution is deposited onto a substrate and annealed at high temperatures to form a dual action antimicrobial film of the invention.

The coating solution of the invention is very stable over long periods of time. The coating solution has been found to be stable after several months of storage. The solution comprises very low nano-meter particles, approximately in the range of less than 5nm, so the metal species distribution in solution is very homogenous. The smaller particle size results in a strong electrostatic repulsion between particles. A chelating agent and/or a coordinating solvent is also added to the coating solution. The combination of small particle size and chelating agent and/or coordinating solvent result in a coating solution which can be stored and used over long periods of time and in which there are high concentrations of Ti0₂ and metal species present. The coating solutions are used to produce permanent films with long-term functionality.

The antimicrobial films of the invention are homogenous on a 'nano-level' scale and show regulated isotropic crystal growth. The solutions of Ti0₂ and Ag or Cu ions are homogenous in nature with approximately less than 5nm size particles resulting in an even distribution of Ag/Cu ions among the Ti0₂ particles. The coating solution contains preformed Ti0₂ particles which during film formation result in isotropic crystal growth. Isotropic crystal growth has no preferred orientation and instead grow in an even fashion in each direction producing very smooth films. As the particle size begins at a very low level the Ag and Cu species are well distributed among the Ti0₂ particles, without any agglomeration as can be found in other films comprising silver and Ti0₂. In comparison, during film formation in the present invention, the Ag/Cu species are evenly integrated into the Ti0₂ matrix on a 'nano-level' . This results in an increase in the anti-microbial activity of the film as there is a greater distribution of metal species on the surface leading to greater diffusion of silver/copper ions with the ability to attack microbes.

The small particle size also provides an antimicrobial film with superior adherence to a substrate. During film formation the small particle size of the components in the coating solution ensure that more particles are adhered per surface area of a substrate. The films of the present invention show no cracking. The films are very smooth and show enhanced transparency.

The antimicrobial films of the present invention may have a thickness as low as lOnm. Any thickness of film may be made depending on the application using the antimicrobial films of the invention.

The antimicrobial properties of the films and the films ability to prevent biofilm formation ensure that the films of the present invention have application in many different areas where microbiological cleanliness is necessary. The films may be applied to any suitable surface in order to provide antimicrobial properties, for example metal surfaces such as taps and metal work surfaces, ceramic surfaces, such as wash basins, baths and toilets or glass surfaces, such as doors and windows. The films may also be applied to furniture, such as beds, or medical equipment and instruments. Other applications for the films include surfaces for use in a medical environment, such as tiles, work surfaces, door handles, taps and beds. The films may be used in hospitals and other places, for example in food processing facilities, dining areas, play areas, swimming pools, communal areas and nursing homes.

While the metal species described in the examples are silver and copper other suitable metal species having antimicrobial properties such as zinc may be used.

While the Ti0₂ described in the examples is doped the Ti0₂ in the dual action antimicrobial films of the invention may be doped or undoped. Both doped and undoped Ti0₂ exhibit photocatalysis and the ion diffusion from the metal species allows the dual action film to produce an anti-microbial effect in light and dark conditions. The present invention will be more fully understood from the following examples.

The synthesis method of doped Ti0₂ was carried out essentially as described in International Patent Application No. PCT/EP2011/62010 the relevant contents of which are incorporated herein by reference.

The production of Ag/TFA-S/Ti0₂/TFA involves two separate synthesis pathways for a) Ag/TFA or Cu/TFA and b) S/Ti0₂/TFA. Formulation of both species is required with a chelating agent to generate the coating solution. The coating solution is then deposited onto a substrate and annealed at a high temperature to produce the hybrid Ag-S/F/Ti0₂ film of the invention. The following describes the synthesis process, formulation and film generation of the material.

Synthesis of Ag/TFA

The silver species to be introduced is produced from the reaction of TFA and Ag₂0 in a polar solvent (see below). The Ag/TFA species is not isolated and is formulated with soluble Ti0₂and/or chelating agent and/or coordination solvent to the appropriate concentration.

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Synthesis of Cu/TFA

The copper species to be introduced is produced in a similar fashion via the reaction of TFA and Cu(N0₃)₂ in a polar solvent

Synthesis of S/TiO^TFA

Titanium isopropoxide (Ti(OPr)₄) is added to water with some hydrous Ti0₂ seed and ethyl acetoacetate. The addition of Ti(OPr)₄ to H₂0 in itself produces amorphous Ti0₂ rather than hydrous Ti0₂, see below.

Ti(OPr)₄ + H₂0 → (am)Ti0₂ + IPA The amorphous Ti0₂ is required to be converted into nano-particulate hydrous-Ti0₂ due to its high absorbance to both cationic and anionic species. The ability to attract donor species is crucial to the coordination of organic acetate groups during the solubilisation step.

The conversion of amorphous Ti0₂ to hydrous nano-particle Ti0₂ is carried out at elevated temperatures, see equation below. The high temperature encourages particle growth from amorphous Ti0₂ to more crystalline nano-particulate hydrous Ti0₂.

Ti(OPr)₄ + H₂0 → H₂O.Ti0₂ + IPA

The nano-particulate hydrous Ti0₂ is dried and the solubilisation of the material involves refluxing in TFA before removal of excess TFA and isolation of Ti0₂/TFA.

H2O.T1O2 + TFA → Ti0₂/TFA + H₂0

The doping step is carried out using sulphur to produce the S/Ti0₂/TFA.

T1O₂/TFA + S → S/Ti0₂/TFA

Coating Solution Formulation

Example 1

There are four constituents of the coating solution to produce Ag-S/F/Ti0₂ films.

• S/Ti0₂/TFA solution

• Ag/TFA solution

• Chelating agent

• Coordinating solvent

For a functioning coating solution all constituents must exhibit long term stability and remain in solution. Ionic Ag⁺ (from Ag/TFA) is known for its inherent instability in solution. It easily precipitates out of solution under many conditions. Unfortunately the addition with S/Ti0₂/TFA alone to a solution leads to a breakdown in the stability of Ag⁺ and precipitation. To insure the stability of the coating solution a chelating agent can be added which coordinates around the Ag⁺ ion and maintains its stability in solution. The chelating agent may be a homopolymeric polyethylenimine containing a combination of primary, secondary and tertiary amine groups with a general formula:

Chelating agent formula: -(CH₂-CH₂-NH_n)_m-

Where n = 0, 1, 2

m = 10-10⁵

The addition of a coordinating solvent such as acetonitrile also contributes the stability of the material in solution. The addition of a chelating agent and/or a coordination solvent allows the formulated coating solutions to be stored and used when required.

Example 2

There are two constituents of the coating solution to produce Cu-/F/Ti0₂ films.

• Ti0₂/TFA

• Cu/TFA

For solutions containing copper (substituting silver) no stability issues are witnessed and there is no requirement for a chelating agent and/or a coordinating solvent.

Film Formation

The coating solution [e.g. Ag/TFA-S/Ti0₂/TFA] may be applied to a surface such as the surface of a ceramic tile, steel, a polymeric element, glass, a silica element or a zeolite element or any other suitable surface.

Deposition of the coating solution [e.g. Ag/TFA-S/Ti0₂/TFA] according to the present invention may be carried out using conventional wet chemistry methods such as dip coating, spin coating or spray coating. Preferably dip and spray techniques are used on high temperature substrates.

The general procedure to deposit the coating solution of the invention onto substrates was carried out as described essentially in International Patent Application No. PCT/EP2011/62010 the relevant contents of which are incorporated herein by reference.

Annealing Example 1

The substrate with the deposited Ag/TFA-S/Ti0₂/TFA coating solution is placed in a furnace at a high temperature. Depending on the method of deposition and the substrate involved the annealing conditions are amended. For example ceramic substrates have a higher annealing temperature while glass has a longer anneal duration etc.

During the high temperature anneal the film is formed, while the nano-particulate S/Ti0₂/TFA particles sinter together to form a dense film of crystalline anatase Ti0₂. Sulfur and fluorine doping of the Ti0₂ occurs during the annealing step (see below). The Ag species is integrated with the Ti0₂ particles and evenly distributed throughout the film producing a matrix coating. The chelating agent and/or coordination solvent, which is organic based, is removed through decomposition at high temperature.

Sintering and doping of Ti0₂: S/Ti0₂/TFA → S/F/Ti0₂

Matrix film formation: Ag/TFA + S/Ti0₂/TFA → Ag-S/F/Ti0₂

Example 2

The substrate with the deposited Cu/TFA-Ti0₂/TFA material is placed in a furnace at a high temperature. Nano-particulate Ti0₂/TFA particles sinter together to form a dense film of crystalline anatase Ti0₂ at high temperature. Fluorine doping of the Ti0₂ occurs during this annealing step. The Cu species is integrated with the Ti0₂ particles and evenly distributed throughout the film producing a matrix coating.

Sintering and doping of Ti0₂: Ti0₂/TFA → F/Ti0₂

Matrix film formation: Cu/TFA + Ti0₂/TFA → Cu-F/Ti0₂ Silver ion concentration

The silver species of the Ag-S/F/Ti0₂ film is fully integrated into the coating with an even concentration distribution. The film exhibits silver ion diffusion properties giving it its anti-microbial activity.

Analysis of the deposited Ag-S/F/Ti0₂ films confirms the hybrid nature of the film. The films produced are robust and durable displaying no reduction in film quality with the addition of Ag in comparison to pure Ti0₂ films. No substrate affects were found with both steel (a) and ceramic (b) substrates successfully coated and tested as shown in Fig 5.

The elemental analysis of the Ag-S/F/Ti0₂ films on their respective substrates was carried out using energy dispersive x-ray spectroscopy (EDS). The EDS analysis of coated steel samples confirms the presence and concentration distribution of Ag as shown in Fig.6.

The elemental concentration of the films on steel was found to be evenly distributed between coated samples as shown in the table in Fig. 7. Samples (a) and (b) show two different areas of coupon composition mapping. An average Ti:Ag concentration ratio of 1:0.083 was found.

Coated ceramic samples were also analysed using EDS as shown in Fig. 8.

The concentration ratio of Ag is equivalent to Ti for samples on both substrates averaging a ratio of 1:0.078 for the ceramic samples as shown in the table in Fig. 9.

The transparency of the films was very high compared to uncoated substrate. Fig. 10 is UV-vis spectrum showing the % transmission between uncoated and Ag-S/F/Ti0₂ coated glass samples.

Fig. 11 is UV-vis spectrum showing the transparency data of matrix film containing copper with Ti0₂. The films show a transmission greater than 75% over the visible light wavelengths (400 to 900nm). The high transmission is an indication of the high physical qualities of the dual action film of the present invention.

Ion Diffusion Studies

Steel and ceramic samples were coated and annealed at high temperature to produce durable Ag-S/F/Ti0₂ films. The coated substrates were placed in saline to replicate biological systems. The saline was removed and analysed using Atomic Absorption to determine the silver ion concentration that has diffused from the film. Sustained release over a period of time was demonstrated showing the potential long term use of the product.

Different Ti:Ag concentrations of the Ag-S/F/Ti0₂ deposited films, the number of coats as well as the effect of varying test conditions were investigated. However no significant difference was found between samples and conditions- different compositions of Ag-S/F/Ti0₂ films did not contribute any changes in ion release (silver concentration, parts per billion (ppb)), double coated samples did not exhibit greater release over single coats, varying the annealing temperature of the films also had no effect while more concentrated saline solutions did not promote more diffusion as shown in the table in Fig. 12.

Continual and sustained ion release over time was examined to show the long term use of the product. Interestingly, the concentration of the silver remained static past a certain level when left in the same saline solution as shown in the table in Fig. 13.

However, when the same test sample was removed from the saline solution and placed in a new saline solution the concentration of silver ions reached similar or higher levels again. This indicates that an equilibrium is reached in ion concentration before release from the film stops. This property may regulate the release of silver from the Ag-S/F/Ti0₂ film where the silver is only released as silver is removed from the surface. This ensures the maintenance of a continuous concentration of silver over time showing its potential for long term use as shown in the table in Fig. 14.

Further Ion diffusion work was carried out to confirm the long term use of the film. Films containing either silver or copper with Ti0₂ were deposited on ceramic and glass samples. The samples were left in saline solutions at room temperature for specified periods of time and at each time point the saline is removed and retained. The same coated samples were then placed in fresh saline and the cycle continued until testing reached 100 days duration. The saline solutions were tested for elemental determination by inductively coupled plasma mass spectroscopy. Diffusion of ions (copper and silver) is witnessed over the course of the test period for on both glass and ceramic samples indicating the versatility of the films.

These set of tests involved films with F doped Ti0₂ with both copper and silver on ceramic and glass substrates. The films were shown to have a long term antimicrobial effect. The results are shown in the Table in Fig. 15.

Film smoothness: Atomic Force Microscopy

The smoothness of the films is an indication of the uniformity of the film on the substrate surface and provides evidence for the isotropic growth of the particles during sintering to form a dense even film. Film roughness can affect the adhesion of the film to the substrate as it is important that as much of the film is in contact with the substrate interface. Smooth films can also enhance the film's physical properties by reducing light diffraction in comparison to rougher films resulting in increased transparency and clarity of the film with reduced haziness and negligible impact on the aesthetic of the underlying substrate. Atomic force measurements were carried out on both coated and uncoated samples and the smoothness of the film was found to be very similar. The smoothness is calculated by using a Ra value or roughness factor, the uncoated sample was found to have an Ra value of 0.99nm while the coated sample was marginally higher at 1.15nm. (Fig. 16).

Combating Biofilm Formation

While the antimicrobial activity of the Ag-S/F/Ti0₂ films can be clearly seen on colony microbial cells they also play a role in reducing and eradicating the formation of biofilms.

The photocatalytic activity of the Ag-S/F/Ti0₂ films according to the present invention was quantified using indicator dyes such as methylene blue, resazurin or silver nitrate. The colour of the dye changes in proportion to the activity of the photocatalytic film. The rate of colour change, using the Lab colour space scheme (L.a.b.), was analysed to identify any changes in the activity of the film due to adjustments in formulation, film thickness as well as from chemical and physical resistance testing.

Anti-microbial Functionality of Ag-S/F/Ti0₂ films

The anti-microbial activity of the Ag-S/F/Ti0₂ films was assessed via microbiological survival studies. The results demonstrate their exceptionally potency at killing both gram negative Escherichia coli ATCC 25922 and gram positive Staphylococcus aureus ATCC 25923 under fluorescent illumination and in dark conditions. Test conditions and procedure were executed using aseptic techniques and all testing was carried out in triplicate. To ensure accuracy two parallel serial dilutions and inoculated Petri dishes were carried out where an average was taken. Samples were divided into a number of control and test categories as set out in Fig. 17. Both coated and uncoated aseptic samples were spiked with a bacterial load, incubated for 15 hours at 30-35°C under fluorescent light irradiation as well as in the dark. 'Pre- activated' samples were irradiated with fluorescent lighting prior to testing.

The microbiological results were successful in displaying the anti-bacterial properties of Ag-S/F/Ti0₂, as shown in the table in Fig. 18. Results reveal that environmental stresses or lack of resources cannot be attributed to the testing conditions as no significant losses (&lt;log 1) were encountered for any of the controls. The hybrid films produce a biocidal surface that totally reduces microbial density, not a single colony forming unit was present for the coated samples for both gram negative and gram positive test regimes.

All coated samples recorded a total loss of bacteria density below detectable levels. Significantly this included samples left in the dark. The Ag-S/F/Ti0₂ films according to the present invention are durable, functional coats formed by conventional coating/annealing methods and can be easily applied to common surfaces. Functional testing has demonstrated that the films according to the present invention have superior antimicrobial activity under both light and dark conditions. There is huge potential in the films ability to reduce infection transmission on surfaces of internal environments. The antimicrobial action of the films of the present invention, metal ion diffusion release and Ti0₂ photocatalytic activity, provide a long term antimicrobial effect. Long term may be several months if not years as the Ti0₂ is a catalyst and is not used up during the photocatalytic process. It should therefore work for the lifetime of the substrate. The silver ion release is at low levels and should also continue for years.

The invention is not limited to the embodiment hereinbefore described, with reference to the accompanying drawings, which may be varied in construction and detail.