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Titania and silvertitania composite films on glasspotent antimicrobialcoatings

Kristopher Page,a Robert G. Palgrave,a Ivan P. Parkin,*a Michael Wilson,b Shelley L. P. Savinc and

Alan V. Chadwickc

Received 15th August 2006, Accepted 19th October 2006First published as an Advance Article on the web 3rd November 2006

DOI: 10.1039/b611740f

Titania (anatase) and Ag-doped titania (anatase) coatings were prepared on glass microscope

slides by a solgel dip-coating method. The resultant coatings were characterised by X-ray

diffraction, X-ray absorption near edge structure (XANES), Raman, scanning electron

microscopy (SEM), wavelength dispersive X-ray (WDX) analysis, X-ray photoelectron

spectroscopy (XPS) and UV-vis techniques and shown to consist of anatase with ca. 0.21 atom%

Ag2O. Photocatalytic activity of the coatings was determined by photomineralisation of stearic

acid, monitored by FT-IR spectroscopy. Photocatalytically-active coatings were screened for their

antibacterial efficacy against Staphylococcus aureus (NCTC 6571), Escherichia coli(NCTC 10418)

and Bacillus cereus (CH70-2). Ag-doped titania coatings were found to be significantly more

photocatalytically and antimicrobially active than a titania coating. No antimicrobial activity was

observed in the darkindicating that silver ion diffusion was not the mechanism for antimicrobial

action. The mode of action was explained in terms of a charge separation model. The coatings

also demonstrated significantly higher activity against the Gram-positive organisms than against

the Gram-negative. The Ag2OTiO2 coating is a potentially useful coating for hard surfaces in a

hospital environment due to its robustness, stability to cleaning and reuse, and its excellent

antimicrobial response.

1. Introduction

Staphylococcus aureus is a Gram-positive bacterium which

colonises approximately 30% of individuals in developed

countries, mainly in the nose or on the skin.1,2 In a

colonisation of this type most people experience no symptoms

or any infection, however it is able to cause a variety of

diseases ranging from the trivial (e.g. boils) to the life-

threatening (e.g. toxic shock syndrome). Most S. aureus

infections can be treated with antibiotics1 as these are due to

infection by methicillin-sensitive S. aureus (MSSA). However,

some strains of the organism (known as methicillin-resistant S.

aureusMRSA) are resistant to a number of antibiotics, and

infections due to such strains are very difficult to treat.2

MRSA infections are more common in hospital environments

where the organism is usually passed on by direct contact,

usually by the hands of health care workers (nosocomial

infection).

24

S. aureus has achieved methicillin resistance byevolving both an efflux mechanism, which actively and non-

specifically expels antibiotics from the cell,5 and by the

production of an altered penicillin binding protein PBP2a

the product of the mecA gene which is insensitive to

methicillin.6 The spread of MRSA and other infections can

be controlled effectively through a rigorous hygiene regime.

Simple hand-washing is sufficient to help control the spread of

the organism,2,7 however this is of little use if the hospital

environment is heavily contaminated.3 Contamination of

surfaces touched by health care staff in the hospital environ-ment is obviously a potential reservoir for nosocomial

infection by MRSA3,4,8,9 and the organism can survive for

up to 9 weeks when it dries onto surfaces.4 An antimicrobial

coating that actively disinfects hard surfaces touched by

nursing staff will help to break the nosocomial infection loop.

Such a coating would be particularly useful as a means of

disinfection in high traffic communal areas and on items such

as door handles, taps and toilet flushes. An effective

antimicrobial coating would not necessarily be limited to these

areas, but could be employed in various roles across the

hospital in both surgical and communal areas.

Titanium dioxide (TiO2) is receiving considerable research

interest as a photocatalyst and consequently an antimicrobialcoating. TiO2 first came to the attention of the scientific

community when Fujishima and Honda demonstrated the

photolysis of water by a TiO2Pt electrochemical photocell in

1972.10,11 However it was not until 1985 that the efficacy of TiO2semiconductor particles as a means of microbial disinfection

was first realised by Matsunaga et al.12 It was found that

platinised TiO2, when irradiated with ultra band gap UV

radiation, acted as an antimicrobial agent, as a result of

photocatalytic processes taking place on the TiO2 surface. Mills

and LeHunte have written a key review in this area covering

photocatalytic and antimicrobial properties of titanium dioxide

and metal-doped titanium dioxide thin films.13

aDepartment of Chemistry, University College London, 20 GordonStreet, London, UK WC1H 0AJ. E-mail: [email protected] of Microbial Diseases, UCL Eastman Dental Institute,University College London, 256 Grays Inn Road, London, UKWC1X 8LDcSchool of Physical Sciences, Ingram Building, University of Kent,Canterbury, Kent, UK CT2 7NH

PAPER www.rsc.org/materials | Journal of Materials Chemistry

This journal is The Royal Society of Chemistry 2007 J. Mater. Chem., 2007, 17, 95104 | 95

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Anatase titanium dioxide has a band gap energy (Eg) of

3.2 eV.13 Irradiation of anatase TiO2 with UV radiation

greater than Eg causes promotion of an electron from the

valence band to the conduction band. This results in the

formation of an electronhole pair. This is a free electron in

the conduction band, and a hole in the valence band.1316

These reactive species then participate in oxidation and

reduction processes either within the TiO2 itself (electron andhole recombination), or with adsorbates at the surface.

Disinfection of a surface by photocatalysed reactions on

TiO2 is a popular possible alternative to using chemical

disinfectants such as chlorine bleach.

The effectiveness of the TiO2 as a photocatalyst is in part

dependent upon the rate of production of hydroxyl radicals at

the surface of the semiconductor. This is in turn dependent

upon other factors. These include the energy of the light

illuminating the surface and the competition between electron

hole recombination and the redox processes occurring on the

surface.17

Titanium dioxide thin films have been formed on glass, steel

and other surfaces by a wide range of techniques, especially bysolgel and chemical vapour deposition.18,19 Furthermore they

have been looked at as antimicrobial coatings and shown to be

efficient especially under sunlight or black light irradiation.13

Commercial products making use of TiO2 photocatalyst

include self cleaning glasses such as Pilkington Activ2 and

Saint Gobain Bioclean, self cleaning tiles (TOTO Inc.) and in

air purifiers.11 The formation of silver-doped titania thin films

has received less attention.20 Silver is incorporated into the

titania film by first forming the film, often using a paste

method using Degussa P-25, followed by impregnation with an

aqueous solution that contains silver ions.21,22 Reduction of

this film by photolysis forms nanoparticulate silver nuggets

within a host titania matrix. These films have shown to be bothmore and less active than the parent titania host matrix in the

photomineralisation of organic molecules.21,22 The destruction

of a particular pollutant has been related to the sensitivity of

its radical and the ability of the silvertitania film to stabilise

photo-produced electrons and holes. The ability of silver

titania thin films to act as antimicrobial coatings has received

scant attention, although one report on preliminary antimi-

crobial tests showed that the coating halts E. coli colony

formation.20 The use of silver as a microbicide is well known

and a host of commercial products exist for use in wound

dressings, ear-pieces, face masks, catheters, plasters and even

for deodorisation of socks.23 A number of commercial

antimicrobial surface treatments also exist which rely on themicrobicidal activity of the Ag+ ionthese include AgION2

(AgION Technologies Inc.)24 and SilvaGard2 (AcryMed

Inc.).25 In all of these instances the silver is impregnated in

the products in its nanoparticulate form or as a silver salt such

as silver nitrate. The mode of action has been shown to

correlate directly with the diffusion of Ag+ into solution. This

mode of action works equally well in the dark as in the light as

it is not directly related to the photocatalytic mechanism

associated with the host titania.

In this paper we report the synthesis of titania and silver-

doped titania nanoparticulate thin films from a solgel route.

We demonstrate that the silver-doped titania thin films are

significantly more active than titania films both as a

photocatalyst and as an antimicrobial agent when illuminated

with 365 nm light. We show that the silver is present in the

films as Ag2O by XPS and X-ray absorption spectroscopy

(XAS). The silver-doped titania films are rugged and have

survived multiple reuses and cleaning with no depletion in

antimicrobial effect. We provide a comparison of the

antimicrobial efficiencies of the films for Gram-positive,Gram-negative and spore-forming bacteria. Furthermore we

observe no antimicrobial activity from these films in the dark,

indicating that the mode of action is not, unlike previous

studies, due to silver ion diffusion. We conclude that the mode

of action of these films is related to the ease of stabilisation of

the photo-generated electronhole pair. These new films are

easy to apply at the point of manufacture and have the

potential to be used in a clinical environment for reducing

bacterial loads and hence nosocomial infections.

Experimental

The chemicals used in this investigation were all purchasedfrom Sigma-Aldrich Chemical Co; propan-2-ol; butan-1-ol;

pentane-2,4-dione (acetylacetone); silver nitrate; titanium (IV)

n-butoxide and acetonitrile. The thin films were prepared on

standard low iron microscope slides (BDH). These were

supplied cleaned and polished, but were nonetheless washed

with distilled water, dried and rinsed with propan-2-ol and left

to air dry before use (2 h).

Solgel synthesis

Ag-doped TiO2 film. The procedure was carried out in air.

Titanium n-butoxide (17.02 g, 0.05 mol) was chelated with a

mixture of pentane-2,4-dione (2.503 g, 0.025 mol) in butan-1-ol(32 cm3, 0.35 mol). A clear, straw yellow solution was

produced, with no precipitation. This was covered with a

watch glass and stirred for an hour. Distilled water (3.6 g,

0.2 mol) was dissolved in propan-2-ol (9.04 g, 0.15 mol)

and added to hydrolyse the titanium precursor. The solution

remained a clear straw yellow colour, with no precipitate.

The solution was stirred for a further hour. Silver nitrate

(0.8510 g, 0.005 mol) was dissolved in acetonitrile (1.645 g,

0.04 mol). This was added to the pale yellow titanium

solution, which was stirred for a final hour. After the final

stirring, the resultant sol was a slightly deeper yellow in

colour, but remained clear and without precipitate. The sol

was used within 30 min for dip-coating. The TiO2 filmcontrols were made in a similar manner and to the same

thickness/crystallinity.

Dip-coating. For dip-coating the glass microscope slides, the

sols were transferred to a tall and narrow 50 cm3 beaker. This

ensured that most of the slide could be immersed in the sol. A

dip-coating apparatus was used to withdraw the slide from the

sol at a steady rate of 120 cm min21. If more than one coat was

required, the previous coat was allowed to dry before repeating

the process. Alternative substrate materials were also coated.

These included martensitic stainless steel; aluminium; brass;

galvanised steel and Pilkington float glass.

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Calcination/annealing. All films were annealed in a furnace

at 500 uC for one hour, with a rate of heating of 5 uC min21.

General

Characterisation of the synthesised coatings was carried out by

field emission scanning electron microscopy (SEM) (Jeol JSM-

6301F), wavelength dispersive X-ray (WDX) analysis (Philips

ESEM) and by Raman techniques (Renishaw 1000). Powder

X-ray diffraction (XRD) was carried out at glancing angles

with a 0.5 mm collimator using an AXS D8 Discover

instrument equipped with a general area detection diffraction

system (GADDS). The TiO2 coating was examined with an

angle of incidence of 5u over an angular range of 1066u for a

15 min period. The AgTiO2 coatings were examined with an

angle of incidence of 1.5u over an angular range of 1062.5u for

a 30 min period. X-Ray absorption near edge structure

(XANES) measurements were made on station 9.3 at the

CCLRC Daresbury Synchrotron Radiation Source. The

synchrotron has an electron energy of 2 GeV and the average

current during the measurements was 150 mA. Ag K-edge

extended X-ray absorption fine structure (EXAFS) spectra for

the films were collected at room temperature in fluorescence

mode using ten films added together to give effectively 20 layers

of the sample. Ag2O, AgO, and Ag metal powder were used as

standards, along with a Ag metal foil reference, these were

collected in standard transmission mode. The standards were

prepared by thoroughly mixing the ground material with

powdered polyvinylpyrrolidine diluent and pressing into

pellets in a 13 mm IR press. Spectra were typically collected

to k = 1 6 A21 (k is the wave vector associated with the

photoelectron) and several scans were taken to improve the

signal-to-noise ratio. For these measurements the amount of

sample in the pellet was adjusted to give an absorption of

about md= 1 (where m is the absorption coefficient and dis the

sample thickness). The data were processed in the conventional

manner using the Daresbury suite of EXAFS programmes:

EXCALIB and EXBACK.26,27 UV-vis spectra were obtained

using a Thermo Spectronic Helios Alpha single beam

instrument. WDX (Philips ESEM) was performed on car-

bon-coated samples, and SEM imaging (JEOL JSM-6301F)

was performed on gold-coated samples. X-Ray photoelectron

spectroscopy (XPS) measurements were carried out on a VG

ESALAB 220i XL instrument using focussed (300 mm spot)

monochromatic Al-Ka X-ray radiation at a pass energy of

20 eV. Scans were acquired with steps of 50 meV. A flood gun

was used to control charging and the binding energies were

referenced to surface elemental carbon at 284.6 eV. Depth

profile analysis was undertaken using argon sputtering.

Photocatalytic activity

The photocatalytic activity of the films was monitored by

Fourier transform infrared (FTIR) spectroscopy (Perkin

Elmer Paragon 1000). The films were firstly activated by

30 min exposure to UV radiation from a 254 nm germicidal

lamp (Vilber Lourmat VL-208G; 8WBDH/VWR Ltd). The

IR spectrum of each stearic acid over-layer was then recorded

over the range 30002700 cm21 and the areas of the peaks

between 29502875 and 28632830 cm21 (the CH stretching

regions of stearic acid) were integrated. Monitoring the

integrated area is directly analogous to measuring the

concentration of stearic acid on the surface, and so can be

used to monitor the degree of photomineralisation after UV

irradiation. Slides were irradiated for a set period and then the

IR measured after each irradiation. The stearic acid over-layer

was applied by dip-coating the sample slides in a 0.02 mol dm23

solution of stearic acid in methanol. To compare thephotocatalytic ability between samples it was ensured that

the initial peak areas were as close in value as possible. At the

end of the experiments the peak areas were normalised to the

initial starting value, such that comparison could be made.

Rates of photocatalysis (in molecules cm22 min21) were also

calculated when the stearic acid decay profile could be fitted to

an appropriate rate law.28,29

Water droplet contact angle

Photoactive films often demonstrate photoinduced super-

hydrophilicity (PSH). The degree of PSH can be gauged by

observing the change in contact angle of a water droplet on the

film surface after UV illumination. The samples were pre-

irradiated for 30 min under a 254 nm germicidal lamp (Vilber

Lourmat VL-208GBDH/VWR Ltd), and then a 4 ml droplet

of distilled water was placed on the surface. The diameter of

the drop was then measured after it had settled. The volume

diameter data were then entered into a computer programme

to calculate the contact angle of the water droplet. If a coating

demonstrates PSH after UV illumination, the water droplet

will be seen to spread out and have a very low contact angle

with the coating surface. Droplets were added and measured

after every consecutive 30 min of illumination time for 2 h.

Antibacterial activity

The antibacterial activity of the films was assessed against

Staphylococcus aureus (NCTC 6571), Escherichia coli (NCTC

10418) and Bacillus cereus (CH70-2; mixed vegetative and

endospore). Samples were tested in duplicate against a suite of

controls (detailed below). Sample coatings and the controls

were irradiated under a 254 nm germicidal UV lamp (Vilber

Lourmat VL-208G from VWR Ltd; 8 W) for 30 min to both

activate and disinfect the films. The sample slides were then

transferred to individual moisture chambers (made from Petri

dishes with moist filter paper in the base). An overnight culture

in nutrient broth (Oxoid Ltd, Basingstoke UK) was then

vortexed and 25 ml aliquots of the culture pipetted onto each

film in duplicate. The samples were then irradiated by a black-light UV lamp, 365 nm (Vilber Lourmat VL-208BLB; 8W

from VWR Ltd) for the desired length of time. The irradiance

of the 365 nm lamp was measured at 1.4 mW cm22 using a

Solarmeter Model 5.0 Total UV (A + B) hand held meter

(Solartech Inc., Michigan USA). After the desired irradiation

period, the bacterial droplets were swabbed from the surface

using sterile calcium alginate swabs (Technical Service

Consultants Ltd). The swabs were transferred aseptically to

4 ml Calgon Ringer solution (Oxoid Ltd, Basingstoke UK) in

a glass bijoux containing 57 small glass beads. The bijoux was

then vortexed until the entire swab had dissolved. For all

bijoux, serial 10-fold dilutions of the bacterial suspension were


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prepared down to 1 6 1026 in phosphate buffered saline

(Oxoid Ltd, Basingstoke UK) in a sterile 96 well plate. Each

dilution was then plated in duplicate onto agar. Mannitol salt

agar (Oxoid Ltd, Basingstoke UK) was used for S. aureus,

MacConkey agar (Oxoid Ltd, Basingstoke UK) was used for

E. coli and nutrient agar (Oxoid Ltd, Basingstoke UK) was

used for B. cereus. Inoculated plates were then incubated

overnight at 37 uC. After incubation a colony count was

performed for the dilution with the optimal countable number

of colonies (30 to 300 colonies). The data were then processed,

taking into account the dilution factor and the mean values of

duplicate experiments. The end result is a direct comparison of

the number of viable bacteria per ml on the samples to that on

a glass control. Experiments were repeated at least twice,

giving four data points for each sample tested. Experiments of

2, 4 and 6 h irradiation were conducted for S. aureus;

experiments of 6 h were carried out for E. coliand experiments

of 2 and 4 h were carried out for B. cereus.

Appropriate use of controls is essential in determining

whether the coating by itself, UV exposure by itself, or a

combination of the two is the cause of any observedmicrobicidal effect. For each coating under test the following

system of positive and negative controls is required: (1) L+S+

(in UV light with an active substrate); (2) L+S2 (in UV light

with an inactive substrate); (3) L2S+ (in the dark with an

active substrate); (4) L2S2 (in the dark with an inactive

substrate). By using a system of controls as shown it is possible

to deduce from the results which conditions result in the

antibacterial effect. Photocatalytic coatings should not be

antimicrobially active without the activation by UV light, and

so only the L+S+ sample should show antibacterial activity. A

comparison of L+S+ and L2S2 enables kill levels to be

calculated. (Note: depending upon the bacterium being

investigated, exposure to UV light by itself may have amicrobicidal effect. That is to say that the L+S2 sample may

in some cases demonstrate a measurable kill.)

Results

Synthesis

A simple solgel method was used to produce both the TiO 2 and

silver-dopedTiO2 films.The generalprinciple behindthis method

is the hydrolysis of a titanium precursor and its subsequent

polymerisation into a TiOTi network. By dip-coating the

microscope slides, a thin film of titanium precursor is deposited

and the gelation of the sol is substantially accelerated.17

Annealing of the samples in a furnace drives off the last tracesof solvent, removes carbon and further enhances the polymerisa-

tion of the precursor into a crystalline anatase network.

The synthetic technique for doping Ag nanoparticles into a

titania film was similar to that of the pure titania film. Key to a

successful synthesis is the chelation of the metal sites involved;

this prevents agglomeration of nanoparticulate Ag and also

stops the instant gelation that occurs upon addition of AgNO3to an acidified titanium precursor. This effect was observed in

preliminary experiments without the use of stabilising solvents.

Acetylacetone (pentane-2,4-dione) in butan-1-ol was used to

stabilise the Ti centre and acetonitrile was used as a

coordinating solvent to stabilise the Ag.

Physical characterisation

The TiO2 and Ag-doped TiO2 films had a multicoloured hue,

dependent upon the angle from which they are viewed. The

appearance of the coatings is due to refringence effects

resulting from a small variation in the coating thickness.

Films of different thickness were made by varying the number

of dip-coats appliedhowever all films had a uniform

appearance, and were smooth. All of the AgTiO2 had abluishpurple hue (possibly due to nanoparticulate silver),

with a distinct yelloworange tinge in certain lighting

conditions. Notably TiO2 films without silver did not show

the distinct bluishpurple hue or the yelloworange tinge.

Under an optical microscope the surface of the one-coat film

TiO2Ag was featureless, however in the four dip-coat film,

cracking of the surface was visible. All thicknesses of the

coating were resistant to standard scratch tests with a stainless

steel spatula, could not be removed by Scotch1 tape and were

generally rugged. Indeed the film could only be removed by

chipping the glass substrate. Repeated dipping of the coatings

into distilled water had no effect on the coatings surface,

which could not be wiped off. Depositing coatings ontoalternative substrates (brass, aluminium, SnO2, silica and

stainless steel) produced films of identical appearance to those

made on glass microscope slides. In particular, films deposited

onto stainless steel had excellent uniformity and retained the

ruggedness and adherence of the films coated on glass. This

robust physical behaviour is significantly better than paste,21,22

traditional solgel and physical vapour deposition (PVD)

prepared titania films and is most akin to those made by

chemical vapour deposition (CVD)18,19 such as the commercial

products Pilkington Activ and Saint Gobain Bioclean (ca.

2550 nm thick anatase TiO2, deposited by on-line CVD at

650 uC).

Characterisation

Powder X-ray diffractograms of the TiO2 films were indexed

as anatase (I41/amdz, a = 3.776 A, c = 9.486 A). The AgTiO2diffractograms were slightly less well defined than the TiO2diffractogram but did show peaks attributed to anatase TiO2(Fig. 1). Furthermore, the AgTiO2 patterns exhibited one

other significant peak at 31.5u 2h which was absent in the TiO2pattern and must therefore be due to the difference in

compositionpossibly due to the incorporation of a Ag

compound rather than crystalline Ag. Database patterns for

crystalline Ag do not correlate with this observed peak. The

best pattern match for this peak and the remainder of thediffractogram is for the silver oxides AgO and Ag2O. Both

silver oxide species correspond well with their most intense

peaks aligning with the additional peak observed in the

experimental pattern.

Raman analysis of both TiO2 and AgTiO2 types was

attempted in the range 100 to 1000 cm21. A characteristic

anatase TiO2 scattering pattern was produced (Fig. 2), with a

sharp and intense peak at 143 cm21, and further peaks at 197,

396, 519 and 639 cm21 in the undoped TiO2 pattern. The less

well defined Raman pattern for the Ag-doped samples is most

probably due to the lower level of crystallinity in the samples

as observed by the comparatively weak anatase peaks in the


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XRD. No Raman patterns for silver oxides were apparent.

This is most likely due to the low concentration of Ag2O in the

films and the poor Raman scattering power of Ag2O compared

with the TiO2 matrix.

Ag K-edge XAS spectra were collected for the three Ag-

doped TiO2 films made from sols with Ag concentrations of

5%, 10% and 20%, Ag metal foil, Ag metal powder, Ag 2O and

AgO powders. The Ag K-edge XANES data for the dopedsamples are shown in Fig. 3(a) along with the corresponding

data for Ag metal powder, Ag2O and AgO. The energy scales

of all the spectra have been consistently normalised to the Ag

K-edge at 25 518 eV and the spectra shifted on the y-axis for

ease of viewing. Fig. 3(a) shows that the local environment of

the Ag atoms has a distinct effect on the shape of the XANES

spectra. This can be used to identify the local environment of

the Ag atoms in the Ag-doped TiO2 films. In each case, the

shape of the XANES spectra for the doped films matches that

of the Ag2O standard, indicating that the silver is present in the

Fig. 1 Powder XRD patterns for four coat TiO2 (lower trace) and two and four coat AgTiO2 coatings (upper and middle traces respectively).

The Ag-oxide peak is marked with an asterisk (*).

Fig. 2 Raman pattern for four coat TiO2 film.

Fig. 3 (a) The Ag K-edge XANES for Ag2O, AgO, Ag powder and

Ag-doped TiO2 films. No pre edge features were observed; (b) the Ag

K-edge XANES for Ag2O and Ag-doped TiO2 films.


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films as Ag2O [Fig. 3(b)]. The pattern for silver metalas also

shown in Fig. 3(a) is very different to that observed and cant

be detected in the samples measured. No bands were observed

before the edge in any of the XANES experiments.

Furthermore as the XAS gave such a good match to Ag2O

[see Fig. 3(b)] it is unlikely that the silver is present within the

titania lattice as a discrete solid solution AgxTi22xO2 because

this would give a different edge shape pattern. Hence the filmsare best described as composites of anatase titania with small

amounts of homogeneously distributed silver (I) oxide.

SEM and WDX techniques were used to study the

composition and morphology of the coated surfaces. WDX

analysis confirmed the presence of Ag in the AgTiO2 with

ratios of 1 part Ag to 100 parts Ti (or less). This was

significantly lower than the silver : titania ratio in the starting

sol (1 : 10). SEM imaging showed minor shrink cracking in the

single or double dip-coated films. The severity of shrink

cracking increased with increasing film thickness. At higher

magnifications, both coating types had similar morphologies,

consisting of granular structures. A high magnification

(6

160 000) image of the two coat Ag-doped coating (Fig. 4)displayed the granular and uneven nature of the coating. In the

top left quarter of the image, a high electron density artefact

can be observed. This indicates the presence of an agglomer-

ated island that contains Ag since this has a higher electron

density than the TiO2 matrixsuch islands were seen

randomly dispersed across the surface of the film. Also, during

the course of the SEM studies, the nanocrystalline nature of

the TiO2 coating was observedparticles of 30 nm size on

average can be seen in a 6 400 000 image (Fig. 5).

Observation of particles of this size correlates well with the

crystallite sizes calculated by the Scherrer equation from the

XRD line broadeningwhich corresponds best to nanocrys-

talline titania, rather than a fully crystalline phase. End onSEM studies were also carried out to measure the thickness of

the films. The two coat materials had a thickness of

approximately 150 nm and a four coat material was

approximately twice this thickness, at ca. 300 nm.

X-Ray photoelectron spectroscopy was undertaken on two

sets of four coat AgTiO2 films, one on a set exposed to UV

light and one on the films as made. Both gave the same XPS

profile. The titanium to oxygen atomic ratio was as expected

2 : 1, no other elements were detected other than carbon andsilicon at a few atom%. The percentage of the carbon

decreased dramatically on etching indicating that it was

residual carbon from within the XPS chamber. The Si

abundance was constant with etching and probably a result

of breakthrough to the underlying glass on regions where there

was a small crack in the titania coating, notably it was only

seen in one of the four samples analysed. Silver was detected

both at the surface and throughout the film and its abundance

was invariant with sputter depth. The silver was typically

detected at below 1 atom%significantly lower than that in

the initial sol but comparable to that observed by WDX

analysis (values ranged around 0.2 atom%, however accurate

quantification was difficult at such low levels). The detectionlimit of the instrument is approximately 0.1 atom% and for

quantification it is 0.2 atom%. XPS spectra were collected and

referenced to elemental standards. The Ti 2p3/2 and O 1s

binding energy shifts of 458.6 eV and 530.1 eV match exactly

literature values for TiO2.30 In the sample exposed to UV light

just prior to measurement there was a small shoulder to both

the Ti and O peaks that correspond to Ti2O3. Interestingly the

silver 3d5/2 XPS showed a single environment centred at

367.8 eV which gave a best match for Ag2O (literature reports

at 367.7367.9 eV) rather than for silver metal 368.3 eV

(Fig. 6).30 Hence the XPS is consistent with the silver being

oxidised as Ag(I) rather than a metallic form in the thin films.

Furthermore sputtering studies showed no change in the silverenvironment with sputter depth. This indicates that the silver is

present as Ag2O and not a Ag2O coated Ag particle; as

otherwise an asymmetry to the peak shape would have

occurred.

UV-vis spectroscopy of the TiO2 and AgTiO2 thin films on

glass was carried out in the range 300800 nm. A band edge for

the O22 to Ti4+ transition in anatase TiO217 was observed in all

of the types of coating at approximately 380 nm. This coupled

with XRD and Raman evidence showed that the anatase form

of TiO2 was present in all films. An approximate value of the

optical band gap for the coatings was obtained by extrapola-

tion on a plot of (ahn)1/2 versus hn, where a is the absorbance of

Fig. 4 SEM image of two coat AgTiO2 coating6 160 000, scale bar

100 nm.

Fig. 5 SEM image of TiO2 coating6 400 000, scale bar 10 nm.


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the film (a = 2log T/T0; T, sample optical transmission; T0,

substrate optical transmission) and hn the photon energy. The

band gap for the TiO2 coating was in the region of 3.0 eV

which is to be expected for the anatase form of TiO2 (3.2 eV).13

Band gap plots for the Ag-doped coatings were not aseasy to interpret as that of TiO2, giving a band gap range of

2.83.4 eV. Ag metal nanoparticles could not be detected by

the observation of a plasmon band31,32 in the UV visible

spectra of the AgTiO2 films. However, nanoparticulate silver

was detected in the initial starting sol by this method,

exhibiting a broad plasmon band at 430 nm. There was,

therefore, considering the UV, XAS and XPS spectra, little

evidence for the incorporation of these Ag metal nanoparticles

into the coatings intact without transformation into an oxide.

Functional properties I: photocatalysis and water droplet contact

angle

All of the films showed photocatalytic activity with 254 nmgermicidal lamp illumination over a period of eight hours. The

reason for choosing the 254 nm (4.88 eV) lamp was to make

sure that the radiation was of greater energy than the TiO2band gap (3.2 eV). The degree of photocatalysis observed

varied between the different coatings, as shown in Fig. 7. It can

be clearly seen that the Ag-doped coatings were significantly

more photocatalytically active than the undoped TiO2 coating

of the same thickness. Amongst the different thickness

Ag-doped coatings there was also a difference in the

photocatalytic activity. The two-coat AgTiO2 film had the

highest initial rate of photocatalysis. The zero order rate

constants for the degradation of stearic acid were calculated at

4.05 6 1012 molecules cm22 min21 for TiO2 and 5.85 6

1012 molecules cm22 min21 for a Ag2OTiO2 coating of the

same thickness. The photoactivity of the TiO2 films generated

in this study to photomineralise stearic acid was slightly lowerthan our previous work using CVD and solgel prepared

films.17 In previous work depositions had been conducted on

barrier glass which has a diffusion layer to stop sodium ion

diffusion from the glass substrate into the film. It has been

noted previously that sodium diffusion during calcinations can

reduce the photocatalytic ability of titania films.28,33 However

our XPS and WDX studies did not detect any sodium in the

titania films so if present it must be less than the 0.1 atom%

detection limit of these techniques.

Initial water contact angle measurements showed that all of

the samples were hydrophilic as they made ca. 15u water

contact angles and they became superhydrophilic upon

exposure to UV radiation. The AgTiO2 samples had contactangles of around 1u after only the initial 30 min of irradiation

with 254 nm. These angles decreased further upon subsequent

exposure to the germicidal lamp (254 nm)but as they were so

low they were difficult to quantify. However, it showed that

the 2-coat AgTiO2 film had a very high degree of

photoinduced superhydrophilicity, as did the three and four

coat versions of the same coating. Photoinduced super-

hydrophilicity was not observed in the coatings deposited

onto metal substrate materials, with initial contact angles

being significantly higher (ca. 20u) than for equivalent coatings

on glass. This may be due to metal ions diffusing into the

coating during the annealing step.

Functional properties II: microbicidal activity

The antimicrobial activity of the coatings was assessed against

three different micro-organisms; Staphylococcus aureus (NCTC

6571), Escherichia coli (NCTC 10418) and Bacillus cereus

(CH70-2). These organisms represent a spectrum of different

classes of bacterium. S. aureus is perhaps the most important

target for this investigation, because of its direct link withMRSA

and hospital acquired infections. S. aureus is also a fairly typical

example of a Gram-positive organism, so it serves as a useful

indicator of the behaviour of a sample coating towards this class

of micro-organism. In the interests of completeness and

experimental rigour, the coatings were also tested against E.coli, a Gram-negative organism and with B. cereus, a Gram-

positive spore-forming organism. It should be noted that the

same coatings were reused for all antimicrobial testing and that

all experiments were carried out in duplicate and repeated twice.

The samples were cleaned between uses by wiping with

isopropanol wipes (as commonly used to clean hard surfaces in

hospitals). The Ag-doped coatings performed very well under

conditions of reuse, maintaining a constant level of effectiveness

despite being handled, cleaned and reused.

Staphylococcus aureus (NCTC 6571). Experiments with

S. aureus were carried out on timescales of two hours, four

Fig. 6 XPS Ag 3d profile for a four coat AgTiO2 coating.

Fig. 7 Relative photocatalytic abilities of all coatings.


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hours and six hours. Both the Ag-doped and un-doped TiO2coatings displayed antibacterial activity towards S. aureus,

although to varying degrees (Fig. 8). The two-coat AgTiO2coating proved to be extremely effective against S. aureus.

After six hours of illumination under 365 nm UV radiation the

two coat AgTiO2 coating proved to be 99.997% effective

against an inoculum of approximately 2.15 6 109 cfu ml21

(colony-forming units ml21) S. aureus. As a point of reference,

the analogous TiO2 coating displayed an effectiveness of

49.925% against the same inoculum. Supplementary studies

carried out at four and two hours of illumination enabled

elucidation of relative antimicrobial activity between coating

types, and also of the relationship between UV light dose and

antimicrobial activity.

Escherichia coli (NCTC 10418). Six hour experiments were

carried out with the two coat AgTiO2 coating against E. coli.

The results were not as striking as with S. aureus, but

nonetheless revealed that the coatings exerted an antimicrobial

effect. The coating averaged an effectiveness of 69% against an

inoculum of ca. 2.61 6 109 cfu ml21 E. coli. The coating was

noticeably less effective against E. coli than S. aureus, even

though the size of the inoculum was similar.

Bacillus cereus (CH70-2). The two-coat AgTiO2 coating

was also tested against B. cereus, another Gram-positive

organism, but one that forms spores under adverse environ-mental conditions. Four and two hour experiments were

carried out against this organism using the two coat AgTiO2coating only. The coating achieved greater than 99.9% kills of

this organism at both 2 h and 4 h exposure periods with 365 nm

UV. It should be noted however, that this was from an initial

concentration ofca. 1.06 108 cfu ml21 B. cereus. Further, the

UV light control L+S2 showed no measurable kill at 2 h and a

64% kill at 4 h of exposure. This demonstrates that the coating

is extremely effective after just 2 h against an inoculum in the

region of one hundred million cfu ml21. This level of

contamination is still significantly greater than what would

be found on a contaminated surface. For example, S. aureus

contamination of a surface was shown typically to be between

4 and 7 cfu cm22.34

Discussion

There are a number of avenues that can be followed in an

attempt to provide an explanation for the enhanced activity of

the Ag-doped TiO2 coating over that of an un-doped TiO2film. In reality, there is likely no one single reason for the

increased activity, rather the observation results from a

combination of effects. The simplest explanation is one of

surface microstructure. The Ag-doped films displayed islands

with a high silver density. This in itself is a good explanation

for the difference in activities, but it does not take into account

other evidence from the characterisation of the coatings. XRD,

XPS and XANES analysis elucidated the presence of the silver

oxide Ag2O. It is possible that these species act as a source of

electrons and as charge separators because of their high

electron density relative to the TiO2 matrix. These factors

would enhance the overall photoactivity of the coating by

firstly donating extra electrons to the conduction band whichin turn are able to produce more reactive species at the catalyst

surface, and secondly by blocking electronhole recombination

which stops the production of radicals at the surface. Indeed,

this explanation is supported by the photocatalysis results.

There have also been reports in the literature of some silver

oxides exhibiting semiconductor behaviour35,36 and Ag2O is

quoted in the literature as having a band gap of 2.25 eV

(550 nm).37 This may go some way to explaining the apparent

change in the optical band gap of the AgTiO2 films over the

TiO2 coating.

It is difficult to compare photocatalysis results with the

literature since there is not as yet an agreed universal reference

against which photocatalysis can be measured. However, theuse of Pilkington Activ2 glass (which is TiO2 coated) as a

reference photocatalyst has been proposed, since this would

make a reliable standard.38 Preliminary photocatalytic results

in our laboratory indicate the AgTiO2 films are considerably

more active. It is equally difficult to compare the micro-

biological results of this investigation with other work in the

literature because of the great diversity in techniques used, and

in the precise details of the experiments performed. The vast

majority of studies of TiO2 antimicrobials are carried out in

solution using a suspension of Degussa P25 TiO2.14,15,38 This is

fundamentally different from the thin film coatings prepared in

this study because the surface area of active catalyst in

suspension would be significantly greater than that availableon a thin film surface (perhaps up to 10 000 times greater).

Furthermore, titania particles in suspension can be ingested by

cells via phagocytosisthis has been shown to cause rapid

cellular damage in addition to that caused by photocataly-

sis.39,40 Consequently, literature results from this method differ

greatly from those obtained in this study. Most studies also

examined only E. coli. However, the efficacy against E. coli

when using a suspended powder is variable from study to

study. One study used an inoculum of 16 106 cfu ml21 E. coli

in a P25 suspension and observed 85% effectiveness after 20 min

exposure to UV (peak wavelength 356 nm), and 100%

effectiveness after an hour.39 This compares with 69%

Fig. 8 Bacterial kills for the two coat AgTiO2 solgel prepared

coating against Staphylococcus aureus after 2, 4 and 6 h illumination

times with 365 nm radiation. The viable counts are expressed as

colony-forming units ml21. L+S+ refers to the exposure of an active

coating (identity in brackets) to UV light. L+S2 refers to the exposure

of an uncoated slide to UV light. L2S+ refers to an active coating

(identity in brackets) kept in the dark and L2S2 refers to an uncoated

slide kept in the dark.


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effectiveness against 1 6 109 cfu ml21 E. coli after 6 h UV

illumination (365 nm) for the two-coat AgTiO2 coating

prepared in this study.

The antimicrobial effect of titania coatings is derived from

the production of hydroxyl radicals,15,16 hence a rationalisa-

tion for the relative effectiveness of the coating against Gram-

positive and Gram-negative organisms can be offered.

Previous research examining the toxicity mechanism of TiO2against micro-organisms showed that the lethal action

involved breach of the cytoplasmic membrane and the

resultant leakage of intracellular components.39,41 For this to

occur, the hydroxyl radicals produced at the coating surface

must be able to directly attack the cytoplasmic membrane. The

differing morphologies of Gram-positive and Gram-negative

cell envelopes means that the passage of hydroxyl radicals

from coating surface to cytoplasmic membrane is hindered to

differing extents. For S. aureus, the only barrier is the

peptidoglycan layer and the periplasmic space. Despite having

a thick layer of peptidoglycan, S. aureus is likely afforded little

protection from the hydroxyl radicals. This is because the

peptidoglycan is composed of a fairly open network polymerof N-acetylmuramic acid and N-acetylglucosamine polysac-

charide chains, with peptide bridges. In contrast, the passage

of hydroxyl radicals towards the cytoplasmic membrane of

E. coli is significantly hindered by the morphology of the cell

envelope. In Gram-negative organisms, such as E. coli, the

cytoplasmic membrane is protected by a thin layer of

peptidoglycan, followed by an outer membrane. The outer

membrane presents a significant barrier to hydroxyl radical

passage since it is comprised of a complex layer of lipids,

lipopolysaccharides and proteins. The outer membrane layer

presents an attractive target for approaching hydroxyl radicals

because of this composition. Although the outer membrane is

semi-permeable, many of the hydroxyl radicals will react withthe lipid constituents of the membrane rather than pass

through it. Once the membrane is breached, however, there are

no further significant obstacles blocking the approach of the

radicals to disrupt the cytoplasmic membrane and cell death

can be observed.39 This interpretation is supported by a recent

study of the photokilling of E. coli by TiO2 thin films.40 The

bactericidal action was found to be a two step process in which

the outer membrane is compromised first, followed by the

cytoplasmic membrane. Hence the Gram-negative envelope

affords better protection against the hydroxyl radical as a

cytotoxic agent. This rationale would therefore account for the

higher antimicrobial activity of the AgTiO2 towards Gram-

positive organisms than Gram-negative.In the films prepared here, no antibacterial activity was

observed from the AgTiO2 films in the absence of lightthis

implies that the silver has no direct role in promoting increased

bacterial kills. The presence of silver as an oxide within the film

enhanced the antimicrobial and photocatalytic properties.

Solutions of silver sols have applications as antibacterial

agents where the active component of these solutions is the

Ag+ ions which disrupt bacterial metabolism.42 The silver sols

display a large surface area and are known to be partially

oxidised by atmospheric oxygen to give Ag2O. While this is

only sparingly soluble in water it is sufficient to provide

antibacterial effects. These antibacterial effects are manifested

independently of whether a light source is used or not. In the

films made in this study no bacterial kill was observed in the

dark. This is strong evidence that the films are not functioning

as microbicides due to the presence of silver ions, as we would

have observed some kill in the absence of light. Silver metal is

normally quite resistant to oxidation in air and requires

stronger oxidising agents such as ozone to convert to the oxide.

The fact that the silver is present as Ag2O in these films is aconsequence of the high temperature anneal and the fact that

the silver is embedded in a titanium dioxide matrix. Although

the silver is present as the oxide, UV illumination of titania can

in principle convert this to the native metal in the presence of

titanium dioxide. However XPS studies of the Ag2OTiO2 film

both before and after UV irradiation did not show any change

in the silver environmentthe binding energy shifts match well

for Ag2O and no lower energy peak was seen as would be

characteristic of silver formation. Hence this combined with

the lack of any antibacterial activity in the dark seems, even

after 12 cycles of UV irradiation, to indicate that any possible

formation of silver metal in this system occurs below the

detection limits of the experiments used. Recent work hasshown that at elevated temperature in the presence of oxygen

the most stable thermodynamic form is Ag2O.43 This correlates

nicely with what was observed in this study. However, the

presence of the silver oxide Ag2O in conjunction with titania

did show a marked enhancement over a pure titania film as a

photocatalyst. This is most likely due to stabilisation of

photogenerated electronhole pairs at the titania surface by

localisation of the photogenerated electron onto the silver

oxide.

Conclusion

Photocatalytically-active and antimicrobially-active coatingswere synthesised by a simple solgel dip-coating technique.

The resultant coatings were characterised by glancing angle

X-ray diffraction, XPS, XANES, Raman spectroscopy, SEM,

WDX and UV-vis spectroscopy and shown to consist of

anatase titania with embedded Ag2O particles. Photocatalytic

activity of the coatings was determined by photomineralisation

of stearic acid and monitored by FT-IR spectroscopy.

Coatings demonstrating high photocatalytic activity against

stearic acid were then screened for antibacterial efficacy

against Staphylococcus aureus (NCTC 6571), Escherichia coli

(NCTC 10418) and Bacillus cereus (CH70-2). Ag-doped

coatings were found to be significantly more photocatalytically

and antimicrobially active than a regular TiO2 coating. Thiswas explained in terms of a charge separation model. Notably

the coatings showed no activity against bacteria in the dark

indicating that their efficacy is not due to silver ions acting as a

microbicide. The coatings also demonstrated significantly

higher activity against the Gram-positive organisms than

against the Gram-negative. This was explained in terms of

the comparative morphologies of the cell envelopes and the

permeability of these envelopes to the likely toxic agent, the

hydroxyl radical. The two coat AgTiO2 coating would appear

to be a potentially useful coating for hard surfaces in a hospital

environment due to its robustness, stability to cleaning and

reuse, and its excellent antimicrobial response to all organisms


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tested thus far. Such a coating would need to be applied at the

point of manufacture of a particular itemand could not be

retrofitted to existing surfaces because of the heat treatment

required to generate the active coatings. However on new

products it could create a very potent antimicrobial coating.

Acknowledgements

The Horshall fund is thanked for financial support. Professor

Parkin is a Royal Society Wolfson Trust merit holder. K.P.

would like to thank Ms Valerie Decraene for her help and

advice during the antimicrobial testing. Mr Kevin Reeves is

thanked for his assistance with SEM imaging and WDX

analysis. CCLRC Daresbury is thanked for provision of

XANES time.

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