# Biocidal Silver and Silver/Titania Composite Films Grown by Chemical Vapour Deposition

Authors: D. W. Sheel; L. A. Brook; I. B. Ditta; P. Evans; H. A. Foster; A.  Steele; H. M. Yates
Journal: International Journal of Photoenergy
 (2008)

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## Abstract

This paper describes the growth and testing of highly active biocidal films based on  photocatalytically active films ofTiO2, grown by thermal CVD,  functionally and structurally modified by deposition of nanostructured silver via a novel flame assisted combination CVD process.  The resulting composite films are shown to be highly durable, highly photocatalytically active and are also shown to possess strong antibacterial behaviour. The deposition control, arising from the described approach, offers the potential to control the film nanostructure, which is proposed to be crucial in determining the photo and bioactivity of the combined film structure, and the transparency of the composite films.  Furthermore, we show that the resultant films are active to a range of organisms, including Gram-negative and Gram-positive bacteria, and viruses. The very high-biocidal activity is above that expected from the concentrations of silver present, and this is discussed in terms of nanostructure of the titania/silver surface. These properties are especially significant when combined with the well-known durability of CVD deposited thin films, offering new opportunities for enhanced application in areas where biocidal surface functionality is sought.


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## Body

## 1. Introduction

In recent years,TiO2 has been widely investigated for its interesting photoexcited properties, for
example, using photocatalysis can lead to the decomposition of organics into
harmless products under UV light irradiation [1]. The extent of the photoactivity depends on a
wide range of properties including morphology, crystallinity, and surface area.The
use ofTiO2 as a biocide was first demonstrated by Matsunaga et al.
(1985) [2].
Subsequently, there have been a number of reports of disinfection of bacteria, viruses, and other
micro-organisms. Most of this early work [3] used suspensions of TiO2 and planktonic organisms. More
recently, research has examined the interaction of organisms with biocidal thin
films of TiO2 anchored to solid surfaces [4–7]. Maness et al. have suggested that the mechanism by
which titania is able to kill bacteria involves the disruption of the cell
membrane following peroxidation of the membrane lipids by active oxygen species [3]. This is supported by the work of
Sunada et al. [8] who studied killing of Escherichia coli on thin Cu/TiO2 films
and showed that firstly, the outer
membrane was damaged followed by the cytoplamic membrane and that these
processes then allowed the complete degradation of the cells. Amézaga-Madrid et al. [9] studied the inactivation of Pseudomonas aeruginosa and showed cell
damage consistent with membrane and cell-wall damage.Bulk
Ag has been long used for coating many items including mirrors (for reflectance
properties) and electrical contacts, as it is the most conductive of all
metals. There is particular interest in nanoparticulate Ag due to its ability
to act as both an electron sink and as redox catalyst. The antimicrobial
properties of silver were well known to the ancient Egyptians and Greeks, for
example, Hippocrates mentions silver as a treatment for ulcers [10].
Since then silver has been widely used as an antimicrobial agent in
applications such as wound dressings and as surface coatings, for example,
catheters [11, 12]. Silver has also been incorporated into
bioglass [13]. Silver ions (Ag+) interact strongly with electron donors, and
the antimicrobial activity of Ag primarily involves interactions with
sulphydryl groups in proteins [14–17]. Silver also
reacts with other cellular components such as nucleic acids [18]. Silver has been
shown to inhibit energy production by inhibition of the respiratory chain of Escherichia
coli [19]. Indirect toxicity may also arise from salt formation
with silver ions that results in a chloride or anion limitation within the
cell.
Nanocrystalline silver [20] also releases Ag0 and has been shown to rapidly kill bacteria and
fungi [21].
Although Ag+ is rapidly inactivated by interaction with organic
matter, Ag0 is much more stable [22].The
combination of Ag andTiO2 for catalysis has been much studied for
mainly sol-gel produced materials [23–25] with some colloid production of mixed [26] and core-shell composite clusters [27]. Most of these papers conclude that Ag is
capable, under the correct conditions, of improving TiO2 photoactivity. The addition of Ag promotes the charge separation of the
electron-hole pairs from TiO2 after photon absorption by acting as
an electron sink. Also the plasmon
resonance in metallic Ag nanoparticles is considered to locally enhance the
electric field facilitating electron-hole production [28]. While most relate this improvement to
electronic effects, it has been pointed out that the addition of Ag can modify
the grain sizes of the TiO2, so increasing the surface area and
hence also the photoactivity [24].FACVD is a low-cost relatively
simple atmospheric pressure CVD technique that is compatible with small volume,
batch, and high volume continuous coating processes. A flame is used to provide the energy
required to crack the precursor species into fragments and subsequently forms
the film upon the substrate. Use of this method with low hazard aqueous
solutions of simple metal salts can yield thin films, which represents a major
advantage in terms of precursor cost and environmental impact compared to
alternative CVD methods. In a previous paper [29],
we also reported the use of aerosol delivery of aqueous solutions of silver
salts I combination with FACVD, overcoming the previous limitations that atmospheric
pressure CVD growth requires precursors with moderately high-vapour
pressures. In this publication, we
reported the first use of FACVD for silver deposition in combination with
titania. We showed that this composite had biocidal activity toward (gram
negative) Escherichia coli.In this paper, we have now
extended the work to explore a wider range of films structures, demonstrate
significantly enhanced activity levels, and widen the exploration of biocidal
activity to include gram positive bacteria (Staphylococcus
epidermidis), and a virus (bacteriophage).


## 2. Experimental

### 2.1. Growth

All films were grown on precoated (CVD) silica
coated barrier glass substrates. The barrier was a (60 nm) amorphous film of
SiO2 designed to prevent diffusion of impurity ions within the float
glass. These would all cause a reduction
in the quality and photoactivity of the films.
All TiO2 films used for biocidal testing were grown using an
atmospheric pressure CVD coater described previously [30].
The precursor used was titanium tetra-isopropoxide (7.79×10−4molmin−1) (TTIP from Sigma-Aldrich, Mo, USA), which was transported through the reactor by a carrier gas of nitrogen. The substrate temperature was 500C°
for TTIP.Additionally, some films were
grown usingTiCl4 and ethyl acetate at a substrate temperature of
650C°, for comparison purposes. The Ag films were grown using an
atmospheric pressure flame assisted CVD coater with a propane/oxygen flame,
described in detail previously [31]. The substrate temperature was set at 300C°.
An aqueous solution of 0.5 M AgNO3 was nebulised, into a carrier of N2, through the flame and onto the substrate.


### 2.2. Characterisation

Standard techniques of X-ray diffraction
(Siemens D5000, Surrey, UK), micro-Raman 514.5 nm Ar
line (Renishaw 1000, Gloucestershire, UK), UV/visible spectroscopy
(Hewlett Packard HP895A, Bracknell, UK), and SEM (Philips XL30, Surrey, UK, equipped with a Rontec Quantax silicon
drift diode detector 3rd generation (SDD3) energy dispersive X-ray spectrometer
(EDAX)) were used to characterise the samples.
Film thickness was estimated by relating the reflected colour to a
calibrated chart for thickness versus refractive index. X-ray photoelectron spectroscopy, XPS, (Kratos
AXIS Ultra, Calif, USA)
with an Al (monochromated)Kα radiation source was used to check the surface
characteristics of the films. It was
necessary to use a charge neutraliser as all the samples were insulating, due
mainly to the deposition on glass. This
tends to shift the peak positions up to 2 eV so the measurements are referenced
to the residual C 1s signal at 285 eV. Curve fitting used
CASA XP software using a mixture of Gaussian-Lorentzian functions to
deconvolute spectra.To test the functional
behaviour of the samples, both photoactivity and bioactivity were tested.


#### 2.2.1. Photocatalytic Behaviour

This was measured under UV (365 nm) at 20C°
under open laboratory conditions. The
emission spectrum of the UV tubes (Sylvania F15W/BLB-T8, Mass, USA) is shown in Figure 1. The degradation of stearic acid was
followed by FTIR (Bruker, Coventry, UK, Vector 22). A typical stearic acid layer
would have an integrated absorbance over this range of 1.0cm−1 corresponding to ca. 3.13×1015molecules cm−2 [32]. Stearic acid (100 μL of 10 mmol in methanol) was spun coated onto the sample.
After drying in an oven at 55C°, the sample was exposed to UV light
with an intensity of 3 mW/cm2.
The activity of the film was defined in cm−1min−1,
which indicated the rate of reduction in selected stearic acid peaks in the IR
region. The technique used was developed from work described
previously [30, 32, 33].Figure 1
Emission spectrum of UV lamps.


#### 2.2.2. Biocidal Activity Testing

The test used was a modification of the standard test
described by BS EN 13697:2001. Samples were cleaned by shaking gently
for 40 minutes in 40 mL of 100% methanol. Samples were removed aseptically,
placed in a UVA transparent plastic Petri dish, coated side uppermost, and
preirradiated under 15 W UVA lamps (Sylvania F15W/BLB-T8) with a 2.24
mWcm2 output for 24 hours.Escherichia coli B NCIMB 9482, E. coli ATCC 10536, and bacteriophage
T4 were obtained from the National Collection of Industrial and Marine
Bacteria, Aberdeen UK, and Staphylococcus
epidermidis NCTC11047 from the National Type Culture Collection, Colindale,
UK. Bacteria were subcultured into Nutrient Broth (Oxoid, Basingstoke,
UK), inoculated onto
Cryobank beads (Mast Diagnostics, Liverpool, UK) and stored at –70C°. Beads were
subcultured onto Nutrient Agar (Oxoid), incubated at 37C°for 24 hours and
stored at 5C°. A 50 μL loopful (a plastic loop calibrated to give a defined
volume) was inoculated in to 20 mL Nutrient broth (Oxoid) and incubated for 24
hours at 37C°. Cultures of E. coli ATCC 10536 and S. epidermidis were
centrifuged at 5000 x g for 10 minutes in a
bench centrifuge, and the cells washed in deionised water x 3 by centrifugation
and resuspension. Controls show that this process does not affect cell
viability over the test timescales. Cultures were resuspended in
water and adjusted to OD 0.5 at 600 nm (Camspec, M330, Cambridge, UK)
to give approximately 2×108 colony forming units (CFUs) mL−1.Stock bacteriophage suspension
was produced by addition of bacteriophage T4 to a 24-hour culture ofE. coli B and incubation at 37C° for a
further 24 hour. Remaining cells and cell debris were removed by centrifugation
at 5000 x g for 10 minutes. Bacteriophage were centrifuged by centrifugation at
20000 x g in a Sorvall RC6 centrifuge. The pellet was washed 3 x by
centrifugation and resuspension, plaque count determined and diluted
appropriately and stored at −20C°.Phage was assayed by the double
agar layer method [34]. Phage suspension
(0.1 mL) was mixed with 0.2 mL 24 hours E. coli B culture in nutrient broth (approx 2×108 CFU mL−1)
and 5 mL molten soft agar (Nutrient Broth +6gl−1 agar; Oxoid, UK)
and poured on to surface of a nutrient agar plate. When set, the plates were incubated at 37C° for 24 hours and plaques counted.A
suspension of 50μL of bacterial or phage was inoculated on to each test sample
and spread out using the edge of a flame sterilized microscope cover slip.The
samples were UV activated by exposure to three 15 W UVA lamps (the surface
incident irradiation was 2.25 mWcm−2 on each occasion). A sample
was removed immediately and the remaining samples removed at regular intervals.
Samples exposed to UVA but covered with a poly laminar UVA protection film
(Anglia Window Films UK) to block UVA but not infra-red, acted as controls.The
samples were immersed in 20 mL of sterile deionised water and vortexed for 60
seconds to resuspend the organisms. A viability count was performed by serial
dilution and plating onto nutrient agar in triplicate or by plaque count and
incubation at 37C° for 48 hours. Each experiment was performed in triplicate.


## 2.1. Growth

All films were grown on precoated (CVD) silica
coated barrier glass substrates. The barrier was a (60 nm) amorphous film of
SiO2 designed to prevent diffusion of impurity ions within the float
glass. These would all cause a reduction
in the quality and photoactivity of the films.
All TiO2 films used for biocidal testing were grown using an
atmospheric pressure CVD coater described previously [30].
The precursor used was titanium tetra-isopropoxide (7.79×10−4molmin−1) (TTIP from Sigma-Aldrich, Mo, USA), which was transported through the reactor by a carrier gas of nitrogen. The substrate temperature was 500C°
for TTIP.Additionally, some films were
grown usingTiCl4 and ethyl acetate at a substrate temperature of
650C°, for comparison purposes. The Ag films were grown using an
atmospheric pressure flame assisted CVD coater with a propane/oxygen flame,
described in detail previously [31]. The substrate temperature was set at 300C°.
An aqueous solution of 0.5 M AgNO3 was nebulised, into a carrier of N2, through the flame and onto the substrate.


## 2.2. Characterisation

Standard techniques of X-ray diffraction
(Siemens D5000, Surrey, UK), micro-Raman 514.5 nm Ar
line (Renishaw 1000, Gloucestershire, UK), UV/visible spectroscopy
(Hewlett Packard HP895A, Bracknell, UK), and SEM (Philips XL30, Surrey, UK, equipped with a Rontec Quantax silicon
drift diode detector 3rd generation (SDD3) energy dispersive X-ray spectrometer
(EDAX)) were used to characterise the samples.
Film thickness was estimated by relating the reflected colour to a
calibrated chart for thickness versus refractive index. X-ray photoelectron spectroscopy, XPS, (Kratos
AXIS Ultra, Calif, USA)
with an Al (monochromated)Kα radiation source was used to check the surface
characteristics of the films. It was
necessary to use a charge neutraliser as all the samples were insulating, due
mainly to the deposition on glass. This
tends to shift the peak positions up to 2 eV so the measurements are referenced
to the residual C 1s signal at 285 eV. Curve fitting used
CASA XP software using a mixture of Gaussian-Lorentzian functions to
deconvolute spectra.To test the functional
behaviour of the samples, both photoactivity and bioactivity were tested.


### 2.2.1. Photocatalytic Behaviour

This was measured under UV (365 nm) at 20C°
under open laboratory conditions. The
emission spectrum of the UV tubes (Sylvania F15W/BLB-T8, Mass, USA) is shown in Figure 1. The degradation of stearic acid was
followed by FTIR (Bruker, Coventry, UK, Vector 22). A typical stearic acid layer
would have an integrated absorbance over this range of 1.0cm−1 corresponding to ca. 3.13×1015molecules cm−2 [32]. Stearic acid (100 μL of 10 mmol in methanol) was spun coated onto the sample.
After drying in an oven at 55C°, the sample was exposed to UV light
with an intensity of 3 mW/cm2.
The activity of the film was defined in cm−1min−1,
which indicated the rate of reduction in selected stearic acid peaks in the IR
region. The technique used was developed from work described
previously [30, 32, 33].Figure 1
Emission spectrum of UV lamps.


### 2.2.2. Biocidal Activity Testing

The test used was a modification of the standard test
described by BS EN 13697:2001. Samples were cleaned by shaking gently
for 40 minutes in 40 mL of 100% methanol. Samples were removed aseptically,
placed in a UVA transparent plastic Petri dish, coated side uppermost, and
preirradiated under 15 W UVA lamps (Sylvania F15W/BLB-T8) with a 2.24
mWcm2 output for 24 hours.Escherichia coli B NCIMB 9482, E. coli ATCC 10536, and bacteriophage
T4 were obtained from the National Collection of Industrial and Marine
Bacteria, Aberdeen UK, and Staphylococcus
epidermidis NCTC11047 from the National Type Culture Collection, Colindale,
UK. Bacteria were subcultured into Nutrient Broth (Oxoid, Basingstoke,
UK), inoculated onto
Cryobank beads (Mast Diagnostics, Liverpool, UK) and stored at –70C°. Beads were
subcultured onto Nutrient Agar (Oxoid), incubated at 37C°for 24 hours and
stored at 5C°. A 50 μL loopful (a plastic loop calibrated to give a defined
volume) was inoculated in to 20 mL Nutrient broth (Oxoid) and incubated for 24
hours at 37C°. Cultures of E. coli ATCC 10536 and S. epidermidis were
centrifuged at 5000 x g for 10 minutes in a
bench centrifuge, and the cells washed in deionised water x 3 by centrifugation
and resuspension. Controls show that this process does not affect cell
viability over the test timescales. Cultures were resuspended in
water and adjusted to OD 0.5 at 600 nm (Camspec, M330, Cambridge, UK)
to give approximately 2×108 colony forming units (CFUs) mL−1.Stock bacteriophage suspension
was produced by addition of bacteriophage T4 to a 24-hour culture ofE. coli B and incubation at 37C° for a
further 24 hour. Remaining cells and cell debris were removed by centrifugation
at 5000 x g for 10 minutes. Bacteriophage were centrifuged by centrifugation at
20000 x g in a Sorvall RC6 centrifuge. The pellet was washed 3 x by
centrifugation and resuspension, plaque count determined and diluted
appropriately and stored at −20C°.Phage was assayed by the double
agar layer method [34]. Phage suspension
(0.1 mL) was mixed with 0.2 mL 24 hours E. coli B culture in nutrient broth (approx 2×108 CFU mL−1)
and 5 mL molten soft agar (Nutrient Broth +6gl−1 agar; Oxoid, UK)
and poured on to surface of a nutrient agar plate. When set, the plates were incubated at 37C° for 24 hours and plaques counted.A
suspension of 50μL of bacterial or phage was inoculated on to each test sample
and spread out using the edge of a flame sterilized microscope cover slip.The
samples were UV activated by exposure to three 15 W UVA lamps (the surface
incident irradiation was 2.25 mWcm−2 on each occasion). A sample
was removed immediately and the remaining samples removed at regular intervals.
Samples exposed to UVA but covered with a poly laminar UVA protection film
(Anglia Window Films UK) to block UVA but not infra-red, acted as controls.The
samples were immersed in 20 mL of sterile deionised water and vortexed for 60
seconds to resuspend the organisms. A viability count was performed by serial
dilution and plating onto nutrient agar in triplicate or by plaque count and
incubation at 37C° for 48 hours. Each experiment was performed in triplicate.


## 2.2.1. Photocatalytic Behaviour

This was measured under UV (365 nm) at 20C°
under open laboratory conditions. The
emission spectrum of the UV tubes (Sylvania F15W/BLB-T8, Mass, USA) is shown in Figure 1. The degradation of stearic acid was
followed by FTIR (Bruker, Coventry, UK, Vector 22). A typical stearic acid layer
would have an integrated absorbance over this range of 1.0cm−1 corresponding to ca. 3.13×1015molecules cm−2 [32]. Stearic acid (100 μL of 10 mmol in methanol) was spun coated onto the sample.
After drying in an oven at 55C°, the sample was exposed to UV light
with an intensity of 3 mW/cm2.
The activity of the film was defined in cm−1min−1,
which indicated the rate of reduction in selected stearic acid peaks in the IR
region. The technique used was developed from work described
previously [30, 32, 33].Figure 1
Emission spectrum of UV lamps.


## 2.2.2. Biocidal Activity Testing

The test used was a modification of the standard test
described by BS EN 13697:2001. Samples were cleaned by shaking gently
for 40 minutes in 40 mL of 100% methanol. Samples were removed aseptically,
placed in a UVA transparent plastic Petri dish, coated side uppermost, and
preirradiated under 15 W UVA lamps (Sylvania F15W/BLB-T8) with a 2.24
mWcm2 output for 24 hours.Escherichia coli B NCIMB 9482, E. coli ATCC 10536, and bacteriophage
T4 were obtained from the National Collection of Industrial and Marine
Bacteria, Aberdeen UK, and Staphylococcus
epidermidis NCTC11047 from the National Type Culture Collection, Colindale,
UK. Bacteria were subcultured into Nutrient Broth (Oxoid, Basingstoke,
UK), inoculated onto
Cryobank beads (Mast Diagnostics, Liverpool, UK) and stored at –70C°. Beads were
subcultured onto Nutrient Agar (Oxoid), incubated at 37C°for 24 hours and
stored at 5C°. A 50 μL loopful (a plastic loop calibrated to give a defined
volume) was inoculated in to 20 mL Nutrient broth (Oxoid) and incubated for 24
hours at 37C°. Cultures of E. coli ATCC 10536 and S. epidermidis were
centrifuged at 5000 x g for 10 minutes in a
bench centrifuge, and the cells washed in deionised water x 3 by centrifugation
and resuspension. Controls show that this process does not affect cell
viability over the test timescales. Cultures were resuspended in
water and adjusted to OD 0.5 at 600 nm (Camspec, M330, Cambridge, UK)
to give approximately 2×108 colony forming units (CFUs) mL−1.Stock bacteriophage suspension
was produced by addition of bacteriophage T4 to a 24-hour culture ofE. coli B and incubation at 37C° for a
further 24 hour. Remaining cells and cell debris were removed by centrifugation
at 5000 x g for 10 minutes. Bacteriophage were centrifuged by centrifugation at
20000 x g in a Sorvall RC6 centrifuge. The pellet was washed 3 x by
centrifugation and resuspension, plaque count determined and diluted
appropriately and stored at −20C°.Phage was assayed by the double
agar layer method [34]. Phage suspension
(0.1 mL) was mixed with 0.2 mL 24 hours E. coli B culture in nutrient broth (approx 2×108 CFU mL−1)
and 5 mL molten soft agar (Nutrient Broth +6gl−1 agar; Oxoid, UK)
and poured on to surface of a nutrient agar plate. When set, the plates were incubated at 37C° for 24 hours and plaques counted.A
suspension of 50μL of bacterial or phage was inoculated on to each test sample
and spread out using the edge of a flame sterilized microscope cover slip.The
samples were UV activated by exposure to three 15 W UVA lamps (the surface
incident irradiation was 2.25 mWcm−2 on each occasion). A sample
was removed immediately and the remaining samples removed at regular intervals.
Samples exposed to UVA but covered with a poly laminar UVA protection film
(Anglia Window Films UK) to block UVA but not infra-red, acted as controls.The
samples were immersed in 20 mL of sterile deionised water and vortexed for 60
seconds to resuspend the organisms. A viability count was performed by serial
dilution and plating onto nutrient agar in triplicate or by plaque count and
incubation at 37C° for 48 hours. Each experiment was performed in triplicate.


## 3. Results and Discussion

In order to understand the
influence of the various layer structures, four types of film were produced and
characterised. That of just Ag,TiO2/Ag
deposited sequentially to form layers of Ag both over and under TiO2 and TiO2 for reference. The
two multilayered structures were deposited, so that the effect of Ag either
above or below the TiO2 layer could be assessed. The Ag films were produced with a range of
thickness (60–90 nm), by increasing
the number of passes of the moving substrate under the flame (see [35] for a full description of the FACVD system). The number of passes is approximately linearly related to the thickness.


### 3.1. Optical Properties

TheTiO2 films
(single and combined) were all transparent, showing interference fringes of
varying colour depending on their thickness (40–120 nm). All of these films were very strongly adhered
to the substrate (measured by tape pull test).
Visually all the Ag films were reflective, with a pale pink tinge, which
darkened to purple for thicker coatings, and eventually developed a silver
appearance. The multilayered films were
again reflective, those with TiO2 grown over the Ag exhibiting
accentuation of the reflected colour relating to the TiO2 thickness. There was an increased
hardness of TiO2/Ag over Ag, which was relatively soft. (Tested by
scratch resistance with a variable load traversing needle.)


### 3.2. Crystallinity

#### 3.2.1. XRD

All Ag films were crystalline
showing metallic cubic Ag only (JCPDS 04-0783).Those films of
Ag/TiO2 showed no mixed species only those of Ag and TiO2. TiO2 was
grown directly onto the glass substrate, as expected anatase only, as discussed
in a previous publication [36].Use of Scherrer’s formula [37] allows the calculation of crystallite size. The calculation is ideally for a powder not for a thin film, so will
contain line width broadening from strain as well as crystallite size. Despite
these reservations, the values obtained will give an idea of the changes
occurring.Considering one of the thicker
Ag samples, there was an apparent increase in Ag crystallite size from 21 nm to
59 nm on addition of theTiO2 layer. Calculations, on Ag layers
covered by over-layers of TiO2, suggest crystallite size values of 33 nm and 51 nm for anatase. The anatase value was similar to that calculated from
other TiO2 films (of similar thickness) grown under identical
conditions (average 30 nm). There was no obvious change in crystallite size in TiO2 when it is the lower layer, as expected since relatively low
temperatures (300C°) were used to deposit the Ag. In previous work [35], we have
shown that at higher substrate temperatures, the Ag crystallite size increases
when TiO2 overgrowth is undertaken. In this case, this most likely
arises due to the high TiO2 growth temperature (650C°),
leading to annealing and growth of the Ag crystallites. As an independent check, an Ag sample was
heated (650C°, 100min−1) without the addition
of more Ag. Although some vaporisation
of the silver film appeared to have occurred, it was possible to calculate that
the crystallite size still increased significantly (21 nm to 37 nm) on heating
under these conditions.Similarly, the crystallite
size for Ag growth either onTiO2 or directly on the barrier glass
appeared to be equivalent in all cases, indicating that the FACVD deposition
process was controlling this property.Growth ofTiO2 layer, over the thin layer of Ag produced no changes in the Ag crystallite
size. This is attributed to the relatively low-growth temperature (500C°)
used for TTIP, which was probably not high enough to alter the Ag crystallite
size. The anatase crystallite size was 30 nm.


#### 3.2.2. Raman

Titania and titania underlayer
samples confirmed the presence of anatase (398, 515, 636cm−1) (see Figure 2). There was an
additional signal at 972cm−1 which was the only signal seen for the
reference thick Ag sample. This is tentatively assigned to a plasmon resonance,
which is generally defined as a coupled oscillation of conductance electrons
when interacting with an external electromagnetic wave of specific
wavelength. The position of the plasmon
relates to the size (shape or distribution) of the Ag particles [38, 39].Figure 2
(a) Raman spectra for silver and (b)TiO2 over silver film.


### 3.3. Chemical Composition

XPS of all the samples
containing Ag confirmed this to be metallic Ag with the3d5/2 peak
appearing at 368.7 eV and only an O 1s signal at 533 eV relating to absorbed
water and no signal for an oxide (528.2–531 eV) [40] (see Figure 2). XPS of all the samples
(Ag, TiO2, TiO2/Ag, Ag/TiO2) showed no major
impurities in the wide scan, save the expected presence of C (standard
calibration reference) and small amounts of Cl in the case of TiO2 grown from TiCl4 and ethyl acetate.
Of major importance was the fact that both multilayer samples show
signals from both TiO2 and Ag. As XPS only samples about 5 nm of the
surface, this established that the surface consists of both Ag and TiO2.A high resolution scan of the
Ag 3d region (see Figure3(a)) for the reference Ag film (30 passes) showed a 3d5/2 peak at 368.7 eV and a 3d3/2 peak at 374.7 eV, which were
both shifted to lower binding energies when TiO2 was grown on the
same Ag film (3d5/2=367.9eV and 3d3/2=373.9eV).XPS high-resolution scan of (a) Ag 3dTiO2 over Ag(30) (including sample Ag(30), (b) Ti 2p, (c) XPS high-resolution scan for O 1s.
(a)(b)(c)The titania over Ag samples
showed a slightly lower intensity of Ag (when spectra were overlaid). This is
as expected, as it is partially masked by theTiO2 layer, and in
order to be located at the exposed surface of the sample the Ag atoms would
have had to diffuse through the TiO2 layer.High resolution Ti 2p spectra
(see Figure3(b)) showing the 2p1/2 and 2p3/2 signals
(464.7 eV, 459.0 eV) displayed no differences in position and width from that
of a standard CVD deposited TiO2 sample.The O 1s (see Figure3(c))
showed signals assigned to O bound to Ti4+ (530.2 eV) and O bound
to H (532.8 eV) from absorbed water, on the lower trace. The upper trace for a thick layer of Ag (30
passes) showed only the O 1s signal for absorbed water.From this, it can be confirmed
that only Ag andTiO2 were present. There was no shift in the positions
of the Ti 2p and O 1s peaks when Ag was present, establishing that there was no
significant chemical interaction of the Ag with the Ti and O. Quantitative calculation of the elements gave
1:1:2.2 ratio for Ag:Ti:O, that is, consistent with the presence of Ag metal and an oxide with the stoichiometry, TiO2.2.XPS results for Ag overTiO2 gave very similar results to those above, confirming the presence of TiO2 and Ag. As expected, the Ag signals were stronger as more Ag was expected to be
present at the surface layer.For Ag on top, it can be seen that the top
surface contains bothTiO2 and Ag, despite the fact that they were
grown as independent layers. The XPS
characteristics for the multilayer of Ag on TiO2 are readily
explained on the basis that the Ag grows as nanocrystallites rather than as a
continuous film. There are two possible explanations for the presence of Ag on
the surface when it was originally formed as the lower layer. Either there was
preferential growth of the TiO2 on the barrier glass rather than the
Ag (inhibited on Ag or faster growth on glass) or the Ag may diffuse to the
surface due to the growth temperature required for TiO2 growth.


### 3.4. UV/Visible Spectroscopic Characteristics

Deposition ofTiO2 on top of silver (see Figure 4(b)) showed a UV/vis absorption signal at 310 nm
which relates to the TiO2 (see trace of commercial TiO2 on glass, Figure 4(f)). There is also a
signal at 357 nm, which may be of the same origin as the shoulder seen on the
Ag film with a long broad tail (see Figure 4(a)). The spectra from a sample
prepared by growing a thin over-layer of Ag (2 passes) (since the silver films
are not continuous, the number of passes-giving relative
amount of silver deposited—is considered
more valuable than a nominal approximated thickness) on TiO2 showed only a signal
relating to the TiO2 and no sign of any Ag plasmon (see Figure 4(e)). Due to the small crystallite size (11 nm) and
the lower Ag concentration, this is possibly hidden by the broad strong TiO2 absorption band.Figure 4
UV/Vis spectra of (a) thick layer of Ag (30 passes), (b)TiO2 on Ag (30), (c) TiO2 (TTIP) over Ag (4 passes), (d) Annealed TiO2 (TTIP) over Ag (4 passes), (e) thin layer of Ag (2 passes) over TiO2, (f) commercial TiO2 on glass.Spectra,
for a sample prepared using TTIP as a reactant in order to deposit only anatase
over Ag (4 passes), again gave absorption bands relating to bothTiO2 and an Ag plasmon (see Figure 4(c)).
Interestingly on annealing this sample at 650C°, the surface
plasmon shifted from 564 nm to 602 nm (see Figure 4(d)). This is in line with
the expected change of wavelength as the particle size increases.


### 3.5. Surface Structure

The FACVD process used leads
to the growth of a nanostructured surface rather than a continuous Ag
film. The density, size, and spacing of
the nanostructured surface will depend critically on the growth conditions
chosen. The image in Figure5(a) shows growth of Ag (seen as bright particles)
on top of TiO2. Separated Ag particles between TiO2 crystallites are readily observed.SEM images of (a) Ag(2) on top ofTiO2, (b) TiO2 on barrier glass, (c) TiO2 (TTIP) over Ag (4 passes).
(a)(b)(c)The form of theTiO2 round and under the Ag is similar to that expected for TiO2 grown
directly on a glass substrate. The example shown in Figure 5(b) for comparison
(as this is the most commonly used alternative process for CVD of titania) is for a TiO2 layer grown using
TiCl4 and ethyl acetate with a thickness of ca. 120 nm.The sample (see Figure5(c))
with an over-layer of TiO2 on Ag shows similar images, of the TiO2 to that of TiO2 grown direct on barrier glass. This is to be expected as both are only of
anatase. In this instance, the SEM images look very similar, although with
slightly smaller features resulting from growth on the Ag underlayer.SEM images forTiO2 over-layers on Ag do not show any obvious signs of Ag nanoparticles on the
surface. However, use of EDAX at a series of reducing accelerating voltages
(i.e., sampling closer to the surface) clearly showed that the Ag signal became
stronger nearer the surface, supporting our contention that at least some Ag is
likely to be either exposed or very close to the surface of the sample.
Unfortunately, it is not possible to accurately determine the penetration depth
as this will depend on many variables including the accelerating voltage,
sample matrix, and chemical identity (extent of absorption). The depth will generally vary from a few
hundreds of nm to a couple of μm.


### 3.6. Surface Ag Elution

The rate of elution of silver
was tested by aqueous solution extraction-simulating the biocidal testing
procedure. The sample was treated as if undergoing a bacterial
test, but water and no bacteria added. After set times additional water was
added, and then withdrawn, to nitric acid and the Ag content checked by ICPMS.
The intention was to explore if the levels of silver eluted were sufficient to
explain the biocidal activity (see Figure6). A further objective was to assess
the longevity of the bacteriocidal activity (by repeated extraction cycles). The Ag elution results are
in the order silver > titania > titania under silver > titania over
silver > titania only.Figure 6
▴ titania under, □ titania over, ▪ silver only, ∘ titania over with UV.The concentrations of silver in the bacteria sampling
solutions, for titania/silver combinations, are~100 to 1000 ppb. For silver alone, this rises to ~10000ppb.
It is interesting to note that silver under titania gives higher elution rates.
This is compatible with the biocidal activity results.


### 3.7. Functional Properties

#### 3.7.1. Photoactivity

Photocatalytic activity
assessment was undertaken via degradation of stearic acid under UV light (365 nm). The rate at which the stearic acid
was decomposed is shown by the integrated area under the IR signals from the
stearic acid (2957.5, 2922.8, and 2853.4cm−1), which are directly
proportional to the concentration. The
rates were obtained from the gradient of a linear (first order) fit to the raw
data. As an indication of the fit error, the variance has also been given. This would give a value of 1 for a perfect
fit. All Ag samples gave broadly similar
results irrespective of the Ag deposition thickness. For example, a thick layer
gave a stearic acid decomposition rate of 0.0017cm−1min−1 (variance 0.946) while that for a thin layer was 0.0020cm−1min−1(variance 0.961).
The multilayered films were UV active, although to varying
degrees, depending on a range of factors.(1) Titania over Silver
TiO2 over a thin
layer of Ag (2 passes) (0.015, 0.010cm−1min−1, variance
0.968) was more active than Ag and generally higher than TiO2 alone
(see Figure 7).
The comparison values forTiO2 on barrier glass are for pure anatase. Growth of TiO2 over Ag (4 passes) gave an activity of 0.0024cm−1min−1(variance 0.974) for the combined layer, which is similar to that of just the
Ag, but less than that for a single layer of TiO2 (0.006cm−1min−1,
variance 0.963) of similar thickness.
The conditions of growth ofTiO2 have been shown to alter some of the physical properties of the
underlying Ag (e.g., crystallite size) and so this in turn may effect the activity of
the multilayer, along with the rutile/anatase ratio.Figure 7
Photoactivity for the degradation of stearic acid (normalised) for▪ Ag(2), □TiO2 on Ag(2), ▴TiO2 on Ag repeat.(2) Silver over Titania
A thin layer of Ag (2 passes) was deposited
on laboratory thermally grownTiO2. Ag(2) on TiO2 activity (0.0082cm−1min−1,
variance 0.981) (sample A in Figure 8) is referenced against TiO2 chosen from the
thickest area of the substrate before the Ag was grown. This reference should
indicate the maximum activity available from anywhere on the CVD coated plate,
(average 0.007cm−1min−1). To illustrate the effect of thickness, a
second point chosen from a thinner area gives a lower activity of 0.006cm−1min−1 and variance of 0.940 (see Figure 8, sample B).
For comparison with
commercially grown CVD titania, Ag(2)
was deposited on a (uniform) commercially available (Saint-Gobain Bioclean) CVDTiO2 coated glass giving photoactivity of 0.0054cm−1min−1 and variance of 0.958 (over 60 minutes) against the commercial TiO2 coating of average 0.003cm−1min−1 (variance 0.976) (see Figure 8).Figure 8
Photoactivity for the degradation of stearic acid (normalised) for Ag overTiO2 (sample A ▴, sample B ▪), reference TiO2⋄ and Ag on a commercial TiO2 coating□.


#### 3.7.2. Biocidal Activity

The combined thermal and FACVD
grown multilayer films were investigated for biocidal activity using the
bacteriaEscherichia coli (Gram-negative), Staphylococcus epidermidis (Gram-positive), and
bacteriophage T4.The extension of the biocidal
work to Gram-positive bacteria is important because the different cell wall
structure from the Gram-negativeE. coli has been shown to decrease susceptibility to photocatalytic disinfection. The
ability to disinfect viruses is also important, and bacteriophage is used as
models for inactivation of mammalian viruses. There is also a large size
difference between bacteria and viruses, and surface morphology may play a
significant role. Plain glass was used
as a control. The error bars represent
the standard deviation from three separate biocidal tests. In some cases, the
SD is very low and error bars are smaller than the data points. Comparisons were made between FACVD Ag layers
alone, photoactive TiO2 films, and FACVD Ag over-coated with TiO2. All the Ag films used were about 60 nm thick
(4 passes), while the comparison TiO2 reference samples were
approximately 80 nm thick.The biocidal activity was
measured by the technique outlined in Section2. Example results (E. coli) are shown in Figure 9(a) for a TiO2 layer only;
see Figure 9(b) for Ag on glass and TiO2 over Ag (see Figure 9(c)).(a) Killing ofE. coli on a TiO2 film on glass
• with control sample ∘. (b) Killing of E. coli on Ag layer on glass (note: minimum test time 20 minutes) • with control sample ∘. (c) Killing of E. coli on TiO2 layer over Ag on glass • with control sample ∘.
(a)(b)(c)It can be seen that the
thermally grownTiO2 film was biocidally active for the Gram-negative E. coli, with a >5 log reduction
in 180–240 minutes. All the Ag films tested were highly
bacteriocidal, and most gave effectively 100% kill in under the minimum test
time of 20 minutes. Although the rate of killing was slower on TiO2 over Ag, the rate was much higher than on plain TiO2 and these films
also showed a significant enhancement of durability.Gram-positive
bacteriumStaphylococcus epidermidis on Ag over TiO2 was also completely killed (see Figure 10) but at a slower rate taking approx
60 minutes.Figure 10
Killing ofS. epidermidis on Ag over TiO2.It took 80 minutes to achieve
equivalent deactivation of Bacteriophage T4 (see Figure11) on Ag over TiO2.Figure 11
Killing of Bacteriophage T4 on Ag overTiO2• with control sample ∘.


## 3.1. Optical Properties

TheTiO2 films
(single and combined) were all transparent, showing interference fringes of
varying colour depending on their thickness (40–120 nm). All of these films were very strongly adhered
to the substrate (measured by tape pull test).
Visually all the Ag films were reflective, with a pale pink tinge, which
darkened to purple for thicker coatings, and eventually developed a silver
appearance. The multilayered films were
again reflective, those with TiO2 grown over the Ag exhibiting
accentuation of the reflected colour relating to the TiO2 thickness. There was an increased
hardness of TiO2/Ag over Ag, which was relatively soft. (Tested by
scratch resistance with a variable load traversing needle.)


## 3.2. Crystallinity

### 3.2.1. XRD

All Ag films were crystalline
showing metallic cubic Ag only (JCPDS 04-0783).Those films of
Ag/TiO2 showed no mixed species only those of Ag and TiO2. TiO2 was
grown directly onto the glass substrate, as expected anatase only, as discussed
in a previous publication [36].Use of Scherrer’s formula [37] allows the calculation of crystallite size. The calculation is ideally for a powder not for a thin film, so will
contain line width broadening from strain as well as crystallite size. Despite
these reservations, the values obtained will give an idea of the changes
occurring.Considering one of the thicker
Ag samples, there was an apparent increase in Ag crystallite size from 21 nm to
59 nm on addition of theTiO2 layer. Calculations, on Ag layers
covered by over-layers of TiO2, suggest crystallite size values of 33 nm and 51 nm for anatase. The anatase value was similar to that calculated from
other TiO2 films (of similar thickness) grown under identical
conditions (average 30 nm). There was no obvious change in crystallite size in TiO2 when it is the lower layer, as expected since relatively low
temperatures (300C°) were used to deposit the Ag. In previous work [35], we have
shown that at higher substrate temperatures, the Ag crystallite size increases
when TiO2 overgrowth is undertaken. In this case, this most likely
arises due to the high TiO2 growth temperature (650C°),
leading to annealing and growth of the Ag crystallites. As an independent check, an Ag sample was
heated (650C°, 100min−1) without the addition
of more Ag. Although some vaporisation
of the silver film appeared to have occurred, it was possible to calculate that
the crystallite size still increased significantly (21 nm to 37 nm) on heating
under these conditions.Similarly, the crystallite
size for Ag growth either onTiO2 or directly on the barrier glass
appeared to be equivalent in all cases, indicating that the FACVD deposition
process was controlling this property.Growth ofTiO2 layer, over the thin layer of Ag produced no changes in the Ag crystallite
size. This is attributed to the relatively low-growth temperature (500C°)
used for TTIP, which was probably not high enough to alter the Ag crystallite
size. The anatase crystallite size was 30 nm.


### 3.2.2. Raman

Titania and titania underlayer
samples confirmed the presence of anatase (398, 515, 636cm−1) (see Figure 2). There was an
additional signal at 972cm−1 which was the only signal seen for the
reference thick Ag sample. This is tentatively assigned to a plasmon resonance,
which is generally defined as a coupled oscillation of conductance electrons
when interacting with an external electromagnetic wave of specific
wavelength. The position of the plasmon
relates to the size (shape or distribution) of the Ag particles [38, 39].Figure 2
(a) Raman spectra for silver and (b)TiO2 over silver film.


## 3.2.1. XRD

All Ag films were crystalline
showing metallic cubic Ag only (JCPDS 04-0783).Those films of
Ag/TiO2 showed no mixed species only those of Ag and TiO2. TiO2 was
grown directly onto the glass substrate, as expected anatase only, as discussed
in a previous publication [36].Use of Scherrer’s formula [37] allows the calculation of crystallite size. The calculation is ideally for a powder not for a thin film, so will
contain line width broadening from strain as well as crystallite size. Despite
these reservations, the values obtained will give an idea of the changes
occurring.Considering one of the thicker
Ag samples, there was an apparent increase in Ag crystallite size from 21 nm to
59 nm on addition of theTiO2 layer. Calculations, on Ag layers
covered by over-layers of TiO2, suggest crystallite size values of 33 nm and 51 nm for anatase. The anatase value was similar to that calculated from
other TiO2 films (of similar thickness) grown under identical
conditions (average 30 nm). There was no obvious change in crystallite size in TiO2 when it is the lower layer, as expected since relatively low
temperatures (300C°) were used to deposit the Ag. In previous work [35], we have
shown that at higher substrate temperatures, the Ag crystallite size increases
when TiO2 overgrowth is undertaken. In this case, this most likely
arises due to the high TiO2 growth temperature (650C°),
leading to annealing and growth of the Ag crystallites. As an independent check, an Ag sample was
heated (650C°, 100min−1) without the addition
of more Ag. Although some vaporisation
of the silver film appeared to have occurred, it was possible to calculate that
the crystallite size still increased significantly (21 nm to 37 nm) on heating
under these conditions.Similarly, the crystallite
size for Ag growth either onTiO2 or directly on the barrier glass
appeared to be equivalent in all cases, indicating that the FACVD deposition
process was controlling this property.Growth ofTiO2 layer, over the thin layer of Ag produced no changes in the Ag crystallite
size. This is attributed to the relatively low-growth temperature (500C°)
used for TTIP, which was probably not high enough to alter the Ag crystallite
size. The anatase crystallite size was 30 nm.


## 3.2.2. Raman

Titania and titania underlayer
samples confirmed the presence of anatase (398, 515, 636cm−1) (see Figure 2). There was an
additional signal at 972cm−1 which was the only signal seen for the
reference thick Ag sample. This is tentatively assigned to a plasmon resonance,
which is generally defined as a coupled oscillation of conductance electrons
when interacting with an external electromagnetic wave of specific
wavelength. The position of the plasmon
relates to the size (shape or distribution) of the Ag particles [38, 39].Figure 2
(a) Raman spectra for silver and (b)TiO2 over silver film.


## 3.3. Chemical Composition

XPS of all the samples
containing Ag confirmed this to be metallic Ag with the3d5/2 peak
appearing at 368.7 eV and only an O 1s signal at 533 eV relating to absorbed
water and no signal for an oxide (528.2–531 eV) [40] (see Figure 2). XPS of all the samples
(Ag, TiO2, TiO2/Ag, Ag/TiO2) showed no major
impurities in the wide scan, save the expected presence of C (standard
calibration reference) and small amounts of Cl in the case of TiO2 grown from TiCl4 and ethyl acetate.
Of major importance was the fact that both multilayer samples show
signals from both TiO2 and Ag. As XPS only samples about 5 nm of the
surface, this established that the surface consists of both Ag and TiO2.A high resolution scan of the
Ag 3d region (see Figure3(a)) for the reference Ag film (30 passes) showed a 3d5/2 peak at 368.7 eV and a 3d3/2 peak at 374.7 eV, which were
both shifted to lower binding energies when TiO2 was grown on the
same Ag film (3d5/2=367.9eV and 3d3/2=373.9eV).XPS high-resolution scan of (a) Ag 3dTiO2 over Ag(30) (including sample Ag(30), (b) Ti 2p, (c) XPS high-resolution scan for O 1s.
(a)(b)(c)The titania over Ag samples
showed a slightly lower intensity of Ag (when spectra were overlaid). This is
as expected, as it is partially masked by theTiO2 layer, and in
order to be located at the exposed surface of the sample the Ag atoms would
have had to diffuse through the TiO2 layer.High resolution Ti 2p spectra
(see Figure3(b)) showing the 2p1/2 and 2p3/2 signals
(464.7 eV, 459.0 eV) displayed no differences in position and width from that
of a standard CVD deposited TiO2 sample.The O 1s (see Figure3(c))
showed signals assigned to O bound to Ti4+ (530.2 eV) and O bound
to H (532.8 eV) from absorbed water, on the lower trace. The upper trace for a thick layer of Ag (30
passes) showed only the O 1s signal for absorbed water.From this, it can be confirmed
that only Ag andTiO2 were present. There was no shift in the positions
of the Ti 2p and O 1s peaks when Ag was present, establishing that there was no
significant chemical interaction of the Ag with the Ti and O. Quantitative calculation of the elements gave
1:1:2.2 ratio for Ag:Ti:O, that is, consistent with the presence of Ag metal and an oxide with the stoichiometry, TiO2.2.XPS results for Ag overTiO2 gave very similar results to those above, confirming the presence of TiO2 and Ag. As expected, the Ag signals were stronger as more Ag was expected to be
present at the surface layer.For Ag on top, it can be seen that the top
surface contains bothTiO2 and Ag, despite the fact that they were
grown as independent layers. The XPS
characteristics for the multilayer of Ag on TiO2 are readily
explained on the basis that the Ag grows as nanocrystallites rather than as a
continuous film. There are two possible explanations for the presence of Ag on
the surface when it was originally formed as the lower layer. Either there was
preferential growth of the TiO2 on the barrier glass rather than the
Ag (inhibited on Ag or faster growth on glass) or the Ag may diffuse to the
surface due to the growth temperature required for TiO2 growth.


## 3.4. UV/Visible Spectroscopic Characteristics

Deposition ofTiO2 on top of silver (see Figure 4(b)) showed a UV/vis absorption signal at 310 nm
which relates to the TiO2 (see trace of commercial TiO2 on glass, Figure 4(f)). There is also a
signal at 357 nm, which may be of the same origin as the shoulder seen on the
Ag film with a long broad tail (see Figure 4(a)). The spectra from a sample
prepared by growing a thin over-layer of Ag (2 passes) (since the silver films
are not continuous, the number of passes-giving relative
amount of silver deposited—is considered
more valuable than a nominal approximated thickness) on TiO2 showed only a signal
relating to the TiO2 and no sign of any Ag plasmon (see Figure 4(e)). Due to the small crystallite size (11 nm) and
the lower Ag concentration, this is possibly hidden by the broad strong TiO2 absorption band.Figure 4
UV/Vis spectra of (a) thick layer of Ag (30 passes), (b)TiO2 on Ag (30), (c) TiO2 (TTIP) over Ag (4 passes), (d) Annealed TiO2 (TTIP) over Ag (4 passes), (e) thin layer of Ag (2 passes) over TiO2, (f) commercial TiO2 on glass.Spectra,
for a sample prepared using TTIP as a reactant in order to deposit only anatase
over Ag (4 passes), again gave absorption bands relating to bothTiO2 and an Ag plasmon (see Figure 4(c)).
Interestingly on annealing this sample at 650C°, the surface
plasmon shifted from 564 nm to 602 nm (see Figure 4(d)). This is in line with
the expected change of wavelength as the particle size increases.


## 3.5. Surface Structure

The FACVD process used leads
to the growth of a nanostructured surface rather than a continuous Ag
film. The density, size, and spacing of
the nanostructured surface will depend critically on the growth conditions
chosen. The image in Figure5(a) shows growth of Ag (seen as bright particles)
on top of TiO2. Separated Ag particles between TiO2 crystallites are readily observed.SEM images of (a) Ag(2) on top ofTiO2, (b) TiO2 on barrier glass, (c) TiO2 (TTIP) over Ag (4 passes).
(a)(b)(c)The form of theTiO2 round and under the Ag is similar to that expected for TiO2 grown
directly on a glass substrate. The example shown in Figure 5(b) for comparison
(as this is the most commonly used alternative process for CVD of titania) is for a TiO2 layer grown using
TiCl4 and ethyl acetate with a thickness of ca. 120 nm.The sample (see Figure5(c))
with an over-layer of TiO2 on Ag shows similar images, of the TiO2 to that of TiO2 grown direct on barrier glass. This is to be expected as both are only of
anatase. In this instance, the SEM images look very similar, although with
slightly smaller features resulting from growth on the Ag underlayer.SEM images forTiO2 over-layers on Ag do not show any obvious signs of Ag nanoparticles on the
surface. However, use of EDAX at a series of reducing accelerating voltages
(i.e., sampling closer to the surface) clearly showed that the Ag signal became
stronger nearer the surface, supporting our contention that at least some Ag is
likely to be either exposed or very close to the surface of the sample.
Unfortunately, it is not possible to accurately determine the penetration depth
as this will depend on many variables including the accelerating voltage,
sample matrix, and chemical identity (extent of absorption). The depth will generally vary from a few
hundreds of nm to a couple of μm.


## 3.6. Surface Ag Elution

The rate of elution of silver
was tested by aqueous solution extraction-simulating the biocidal testing
procedure. The sample was treated as if undergoing a bacterial
test, but water and no bacteria added. After set times additional water was
added, and then withdrawn, to nitric acid and the Ag content checked by ICPMS.
The intention was to explore if the levels of silver eluted were sufficient to
explain the biocidal activity (see Figure6). A further objective was to assess
the longevity of the bacteriocidal activity (by repeated extraction cycles). The Ag elution results are
in the order silver > titania > titania under silver > titania over
silver > titania only.Figure 6
▴ titania under, □ titania over, ▪ silver only, ∘ titania over with UV.The concentrations of silver in the bacteria sampling
solutions, for titania/silver combinations, are~100 to 1000 ppb. For silver alone, this rises to ~10000ppb.
It is interesting to note that silver under titania gives higher elution rates.
This is compatible with the biocidal activity results.


## 3.7. Functional Properties

### 3.7.1. Photoactivity

Photocatalytic activity
assessment was undertaken via degradation of stearic acid under UV light (365 nm). The rate at which the stearic acid
was decomposed is shown by the integrated area under the IR signals from the
stearic acid (2957.5, 2922.8, and 2853.4cm−1), which are directly
proportional to the concentration. The
rates were obtained from the gradient of a linear (first order) fit to the raw
data. As an indication of the fit error, the variance has also been given. This would give a value of 1 for a perfect
fit. All Ag samples gave broadly similar
results irrespective of the Ag deposition thickness. For example, a thick layer
gave a stearic acid decomposition rate of 0.0017cm−1min−1 (variance 0.946) while that for a thin layer was 0.0020cm−1min−1(variance 0.961).
The multilayered films were UV active, although to varying
degrees, depending on a range of factors.(1) Titania over Silver
TiO2 over a thin
layer of Ag (2 passes) (0.015, 0.010cm−1min−1, variance
0.968) was more active than Ag and generally higher than TiO2 alone
(see Figure 7).
The comparison values forTiO2 on barrier glass are for pure anatase. Growth of TiO2 over Ag (4 passes) gave an activity of 0.0024cm−1min−1(variance 0.974) for the combined layer, which is similar to that of just the
Ag, but less than that for a single layer of TiO2 (0.006cm−1min−1,
variance 0.963) of similar thickness.
The conditions of growth ofTiO2 have been shown to alter some of the physical properties of the
underlying Ag (e.g., crystallite size) and so this in turn may effect the activity of
the multilayer, along with the rutile/anatase ratio.Figure 7
Photoactivity for the degradation of stearic acid (normalised) for▪ Ag(2), □TiO2 on Ag(2), ▴TiO2 on Ag repeat.(2) Silver over Titania
A thin layer of Ag (2 passes) was deposited
on laboratory thermally grownTiO2. Ag(2) on TiO2 activity (0.0082cm−1min−1,
variance 0.981) (sample A in Figure 8) is referenced against TiO2 chosen from the
thickest area of the substrate before the Ag was grown. This reference should
indicate the maximum activity available from anywhere on the CVD coated plate,
(average 0.007cm−1min−1). To illustrate the effect of thickness, a
second point chosen from a thinner area gives a lower activity of 0.006cm−1min−1 and variance of 0.940 (see Figure 8, sample B).
For comparison with
commercially grown CVD titania, Ag(2)
was deposited on a (uniform) commercially available (Saint-Gobain Bioclean) CVDTiO2 coated glass giving photoactivity of 0.0054cm−1min−1 and variance of 0.958 (over 60 minutes) against the commercial TiO2 coating of average 0.003cm−1min−1 (variance 0.976) (see Figure 8).Figure 8
Photoactivity for the degradation of stearic acid (normalised) for Ag overTiO2 (sample A ▴, sample B ▪), reference TiO2⋄ and Ag on a commercial TiO2 coating□.


### 3.7.2. Biocidal Activity

The combined thermal and FACVD
grown multilayer films were investigated for biocidal activity using the
bacteriaEscherichia coli (Gram-negative), Staphylococcus epidermidis (Gram-positive), and
bacteriophage T4.The extension of the biocidal
work to Gram-positive bacteria is important because the different cell wall
structure from the Gram-negativeE. coli has been shown to decrease susceptibility to photocatalytic disinfection. The
ability to disinfect viruses is also important, and bacteriophage is used as
models for inactivation of mammalian viruses. There is also a large size
difference between bacteria and viruses, and surface morphology may play a
significant role. Plain glass was used
as a control. The error bars represent
the standard deviation from three separate biocidal tests. In some cases, the
SD is very low and error bars are smaller than the data points. Comparisons were made between FACVD Ag layers
alone, photoactive TiO2 films, and FACVD Ag over-coated with TiO2. All the Ag films used were about 60 nm thick
(4 passes), while the comparison TiO2 reference samples were
approximately 80 nm thick.The biocidal activity was
measured by the technique outlined in Section2. Example results (E. coli) are shown in Figure 9(a) for a TiO2 layer only;
see Figure 9(b) for Ag on glass and TiO2 over Ag (see Figure 9(c)).(a) Killing ofE. coli on a TiO2 film on glass
• with control sample ∘. (b) Killing of E. coli on Ag layer on glass (note: minimum test time 20 minutes) • with control sample ∘. (c) Killing of E. coli on TiO2 layer over Ag on glass • with control sample ∘.
(a)(b)(c)It can be seen that the
thermally grownTiO2 film was biocidally active for the Gram-negative E. coli, with a >5 log reduction
in 180–240 minutes. All the Ag films tested were highly
bacteriocidal, and most gave effectively 100% kill in under the minimum test
time of 20 minutes. Although the rate of killing was slower on TiO2 over Ag, the rate was much higher than on plain TiO2 and these films
also showed a significant enhancement of durability.Gram-positive
bacteriumStaphylococcus epidermidis on Ag over TiO2 was also completely killed (see Figure 10) but at a slower rate taking approx
60 minutes.Figure 10
Killing ofS. epidermidis on Ag over TiO2.It took 80 minutes to achieve
equivalent deactivation of Bacteriophage T4 (see Figure11) on Ag over TiO2.Figure 11
Killing of Bacteriophage T4 on Ag overTiO2• with control sample ∘.


## 3.7.1. Photoactivity

Photocatalytic activity
assessment was undertaken via degradation of stearic acid under UV light (365 nm). The rate at which the stearic acid
was decomposed is shown by the integrated area under the IR signals from the
stearic acid (2957.5, 2922.8, and 2853.4cm−1), which are directly
proportional to the concentration. The
rates were obtained from the gradient of a linear (first order) fit to the raw
data. As an indication of the fit error, the variance has also been given. This would give a value of 1 for a perfect
fit. All Ag samples gave broadly similar
results irrespective of the Ag deposition thickness. For example, a thick layer
gave a stearic acid decomposition rate of 0.0017cm−1min−1 (variance 0.946) while that for a thin layer was 0.0020cm−1min−1(variance 0.961).
The multilayered films were UV active, although to varying
degrees, depending on a range of factors.(1) Titania over Silver
TiO2 over a thin
layer of Ag (2 passes) (0.015, 0.010cm−1min−1, variance
0.968) was more active than Ag and generally higher than TiO2 alone
(see Figure 7).
The comparison values forTiO2 on barrier glass are for pure anatase. Growth of TiO2 over Ag (4 passes) gave an activity of 0.0024cm−1min−1(variance 0.974) for the combined layer, which is similar to that of just the
Ag, but less than that for a single layer of TiO2 (0.006cm−1min−1,
variance 0.963) of similar thickness.
The conditions of growth ofTiO2 have been shown to alter some of the physical properties of the
underlying Ag (e.g., crystallite size) and so this in turn may effect the activity of
the multilayer, along with the rutile/anatase ratio.Figure 7
Photoactivity for the degradation of stearic acid (normalised) for▪ Ag(2), □TiO2 on Ag(2), ▴TiO2 on Ag repeat.(2) Silver over Titania
A thin layer of Ag (2 passes) was deposited
on laboratory thermally grownTiO2. Ag(2) on TiO2 activity (0.0082cm−1min−1,
variance 0.981) (sample A in Figure 8) is referenced against TiO2 chosen from the
thickest area of the substrate before the Ag was grown. This reference should
indicate the maximum activity available from anywhere on the CVD coated plate,
(average 0.007cm−1min−1). To illustrate the effect of thickness, a
second point chosen from a thinner area gives a lower activity of 0.006cm−1min−1 and variance of 0.940 (see Figure 8, sample B).
For comparison with
commercially grown CVD titania, Ag(2)
was deposited on a (uniform) commercially available (Saint-Gobain Bioclean) CVDTiO2 coated glass giving photoactivity of 0.0054cm−1min−1 and variance of 0.958 (over 60 minutes) against the commercial TiO2 coating of average 0.003cm−1min−1 (variance 0.976) (see Figure 8).Figure 8
Photoactivity for the degradation of stearic acid (normalised) for Ag overTiO2 (sample A ▴, sample B ▪), reference TiO2⋄ and Ag on a commercial TiO2 coating□.


## 3.7.2. Biocidal Activity

The combined thermal and FACVD
grown multilayer films were investigated for biocidal activity using the
bacteriaEscherichia coli (Gram-negative), Staphylococcus epidermidis (Gram-positive), and
bacteriophage T4.The extension of the biocidal
work to Gram-positive bacteria is important because the different cell wall
structure from the Gram-negativeE. coli has been shown to decrease susceptibility to photocatalytic disinfection. The
ability to disinfect viruses is also important, and bacteriophage is used as
models for inactivation of mammalian viruses. There is also a large size
difference between bacteria and viruses, and surface morphology may play a
significant role. Plain glass was used
as a control. The error bars represent
the standard deviation from three separate biocidal tests. In some cases, the
SD is very low and error bars are smaller than the data points. Comparisons were made between FACVD Ag layers
alone, photoactive TiO2 films, and FACVD Ag over-coated with TiO2. All the Ag films used were about 60 nm thick
(4 passes), while the comparison TiO2 reference samples were
approximately 80 nm thick.The biocidal activity was
measured by the technique outlined in Section2. Example results (E. coli) are shown in Figure 9(a) for a TiO2 layer only;
see Figure 9(b) for Ag on glass and TiO2 over Ag (see Figure 9(c)).(a) Killing ofE. coli on a TiO2 film on glass
• with control sample ∘. (b) Killing of E. coli on Ag layer on glass (note: minimum test time 20 minutes) • with control sample ∘. (c) Killing of E. coli on TiO2 layer over Ag on glass • with control sample ∘.
(a)(b)(c)It can be seen that the
thermally grownTiO2 film was biocidally active for the Gram-negative E. coli, with a >5 log reduction
in 180–240 minutes. All the Ag films tested were highly
bacteriocidal, and most gave effectively 100% kill in under the minimum test
time of 20 minutes. Although the rate of killing was slower on TiO2 over Ag, the rate was much higher than on plain TiO2 and these films
also showed a significant enhancement of durability.Gram-positive
bacteriumStaphylococcus epidermidis on Ag over TiO2 was also completely killed (see Figure 10) but at a slower rate taking approx
60 minutes.Figure 10
Killing ofS. epidermidis on Ag over TiO2.It took 80 minutes to achieve
equivalent deactivation of Bacteriophage T4 (see Figure11) on Ag over TiO2.Figure 11
Killing of Bacteriophage T4 on Ag overTiO2• with control sample ∘.


## 4. Discussion

All grown films were
polycrystalline, consisting of cubic Ag andTiO2. The TiO2 is stoichiometric anatase when the bottom layer, and a mixture of anatase and rutile when over silver. XPS confirms that there is no chemical interaction between the Ag and
the TiO2. One of the interesting features of this work is that
although the Ag and TiO2 layers were grown sequentially the overall
result shows that the surface consists of both Ag and TiO2, for TiO2 samples if the substrate temperature during growth (or after annealing
post growth) is sufficiently high (>~600C).All the samples had some UV photoactivity,
which is of differing values depending on the exact arrangement of the
layers. The addition of Ag layers gives
comparable or improved photoactivity of the multilayers, particularly in the
case of Ag on TiO2, over that of single layer of Ag or TiO2 of comparable thickness.The biocidal results show a
high degree of activity for both Ag and Ag/TiO2 films and
interestingly some of the most active results are from Ag under TiO2.Sökmen et al. [41] used TiO2 loaded with 1% Ag but in
suspension and got 100% kill in 15 minutes probably due to the presence of
silver ions. On TiO2 alone, Amézaga-Madrid [9] only got a 70% reduction after 40 minutes whereas Sunada et al. [42] saw a 6 log kill after 90 minutes with similar bacteria loadings to our
own, but kill time was
longer with larger doses. Kikuchi et al. [43] reported a 4-log reduction in 1 hour.
Kühn et al. [44] reported a 6-log reduction of E. coli on P25 coated plexiglass in 1 hour.
Direct comparison of biocidal activity to other data available in the
literature is complicated by the different test methods used. However, it appears clear that, for thin
films-based biocidal activity, the films grown in this work are highly active.The demonstration of high
activity across organisms is particularly encouraging, as Gram-positive
bacteria have been shown to be more resistant [45] to photocatalytic disinfection than Gram-negative bacteria, both in suspension
and on air filters [46, 47] probably due to their different cell wall structure.Similarly, the observed high
activity against bacteriophage shows that nonenveloped DNA viruses can also be
inactivated and suggests that the films can be active across a wide range of
organism dimensions.The proposed mechanism for
this activity is one that is strongly influenced by the thickness of theTiO2 (80 nm). Our data suggest that this should be sufficiently thick so as to
develop critical level crystallinity, but thin enough to allow silver to
diffuse through (and into) the TiO2 film as it grows. This is supported by the reported XPS
results.It should be noted that the UV
levels employed in this work are of similar intensity to that found in full
sunlight.The silver elution results
show that for titania/silver combinations, the level of silver in the bacteria
sampling solutions was
~100 to 1000 ppb. A silver level of
around 1 ppm is normally considered the minimum for significant biocidal
activity. The very high levels of
bactericidal activity seen with the films grown by the combination CVD process
seem unlikely to be due to silver alone.
Furthermore, it was observed that no killing occurred in the dark, at
times up to 80 minutes, demonstrating that photocatalytic activity was crucial
for high-level biocidal activity.We can speculate that the
nanostructure of Ag deposited by FACVD may be crucial in determining biocidal
activity. Ag grown on glass or Ag
over-coated withTiO2, both benefit from the high-structure control
capability of the FACVD approach. When deposited onto TiO2, the
structure flexibility will be, at least, partially predefined by this
underlayer. Silver molecules can be oxidised at the silver/titania interface,
and thus we have designed the multilayer system to incorporate a diffusion-based
replenishment capability, giving the potential for extended activity. It is interesting to note that atomic
absorption measurements of the bacteria solutions above the films showed
concentrations below 1 ppm (Atomic Abs. detection limit).CVD films of titania are
highly durable. Indeed photocatalytic
titania films made by CVD are used in commercial window “easy clean” coatings
which are used on external surfaces. We have, additionally, tested our films on
simulated (humidity, temperature, and exposure) and compared them to commercial
samples, demonstrating close to similar performance. The titania films thus (as well as being
active) also act as a host for silver.The
combination of Ag by FACVD andTiO2, by CVD, offers unique
advantages in that the CVD TiO2 coating imparts a major enhancement
to durability and as we previously have shown [29] the
activity was retained along with chemical and abrasion resistance
compatible with potential application of the technology. In addition, it is
noteworthy that the combined Ag/TiO2 films are thin and as such
impart only moderate changes to visual appearance.This combined flexible process
along with the associated transparency and durability offers opportunity for
application in the increasing number of areas where bio-active surface
functionality is sought. The demonstrated capability of the films to show
highly efficient biocidal activity against a range of organisms reinforces this
potential.



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*Source: 168185-2008-08-21.xml*