Communication

DOI: 10.1002/cvde.200904285 Antimicrobial Activity in Thin Films of Pseudobrookite-Structured Titanium Oxynitride under UV Irradiation Observed for Escherichia coli**
By Zoie A. Aiken, Geoffrey Hyett, Charles W. Dunnill, Mike Wilson, Jonathan Pratten, and Ivan P. Parkin* For many years there has been an interest in the development of anti-microbial materials and coatings.[1,2] With the rise of antibiotic-resistant bacteria over the last decade, and with concern over the prominence of healthcare-associated infections (HCAIs), this interest is increasing. Anti-microbials can be used in a patient environment to reduce infection rates,[3,4] but also have applications in waste-water treatment and in the efcient production of safe drinking water through the enhancement of solar sterilization.[5] Many compounds have been utilized for their antimicrobial action including silver,[68] copper,[911] and poly(ethylene imine),[12,13] but the longevity of such materials may be hindered by leaching of the active components out of the coating. This leaching effect is also likely to cause rapid development of resistance in bacteria. One class of material that has been identied as being effective for anti-bacterial coatings is the light-activated photocatalysts. A photocatalyst is a semiconductor that can absorb ultra band-gap photons to produce electron-hole pairs which, at the surface of the semiconductor particles, generate radical species which then mineralize organic molecules, thus killing bacteria.[14] The most widely used photocatalyst is titania, TiO2, which is used in waterpurication and self-cleaning materials,[15,16] where it is activated by UV light. Although, due to a combination of efciency and low cost, TiO2 is the most commonly used photocatalyst for industrial purposes other semiconductors, including SrTiO3, Ta2O5, and In2O3 have been demonstrated as photocatalysts.[1719] More recently a new titanium oxynitride, Ti2.85O4N, has been discovered[20] and shown to be an effective UVactivated photocatalyst with a rate potentially exceeding that of TiO2,[21] and therefore worthwhile investigating as a
[*] Prof. I. P. Parkin, Dr. G. Hyett, Dr. C. W. Dunnill Christopher Ingold Laboratory, Department of Chemistry, University College London 20 Gordon Street, London, WC1H 0AJ (United Kingdom) E-mail: i.p.parkin@ucl.ac.uk Z. A. Aiken, Prof. M. Wilson, Dr. J. Pratten Division of Microbial Diseases, UCL Eastman Dental Institute 256 Grays Inn Road, London WC1X 8LD (United Kingdom) [**] This article is part of a special section on CVD of Biomaterials.

possible anti-bacterial coating. This was undertaken by inoculating samples of glass coated with TiO2 or with Ti2.85O4N with Escherichia coli and exposing these samples to a regime of UVC (254 nm) and UVA (365 nm) light. These tests found a 3.8 log10 (99.98%) bacterial reduction in the Ti2.85O4N-coated samples and a 3.9 log10 (99.99%) reduction in the TiO2 sample compared to the uncoated glass controls. This indicates that Ti2.85O4N is as effective a UV-activated anti-microbial as the commonly used phase TiO2. Atmospheric pressure (AP)CVD was used to synthesize a lm of Ti2.85O4N from titanium (IV) chloride, ethyl acetate, and ammonia on a 4 mm thick glass substrate. This coated substrate was then divided into seven equally sized sections of 32 mm 89 mm for analysis of chemical composition and anti-bacterial properties. A similar lm of TiO2 was also made using APCVD with the same synthetic conditions but without any ammonia present as a nitrogen source. The titania lm was also divided into seven sections for analysis. Uncoated glass was used as a control. The precursor ow rates and bubbler temperatures were chosen such that the titanium (IV) chloride and ethyl acetate, the metal and oxygen sources, were each delivered at similar rates of approximately 8 mmol min1. In the synthesis to produce titanium oxynitride, ammonia was supplied at 11 mmol min1. Such conditions allowed for the rapid deposition of a thin lm, which remained defect- and pinhole-free by eye. The use of such a large ow rate of nitrogen precursor ensured the formation of the oxynitride, rather than a nitrogen-doped titania phase.[22] Both lms were well adhered to the substrate and resistant to abrasion. In transmission the TiO2 lm was colorless, while the Ti2.85O4N lm was pale green/yellow. In reected light it was possible to observe interference fringes of color for both lms, indicating the lm thickness is of a similar order to the wavelengths of visible light. The lm on each section was characterized using powder X-ray diffraction (XRD). The patterns recorded on each of the sections were identical and could be matched to those previously recorded on samples of Ti2.85O4N, and also indexed using the Cmcm space group. This indexing gave the same lattice parameters for each of the lm sections of a 3.79(1) A, b 9.64(1) A, and c 9.89(1) A which , b 9.6486(1) A, compares with values of a 3.8040(1) A for the unit cell previously reported for and c 9.8688(1) A powder samples of Ti2.85O4N.[20] This conrmed that a lm of titanium (IV) oxynitride had been successfully synthesized. An example powder diffraction pattern of one of the lm sections, with indexing, is given in Figure 1. There was no evidence of the presence of TiO2 in any of the lm sections. XRD was also used to characterize the titania lm. This found that the only peaks present could be matched to 19

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Fig. 1. XRD pattern recorded for the Ti2.85O4N-coated glass using Cu Ka radiation. The pattern could be indexed in the Cmcm space group with a 3.79 A, b 9.64 A, and c 9.89 A, as shown. Fig. 2. Concentration of E. coli remaining on the thin lms after exposure to 1 h 254 nm light and 4 h 365 nm light (C AL) or just the latter light dose (C AL). Thin lms were also exposed to the activation step only (C AL) or incubated in the dark throughout (C AL). Uncoated glass slides were either exposed to both light doses (G AL) or incubated in the absence of light (G AL). Horizontal lines indicate median values, bars indicate the inter-quartile range, and circles indicate outliers.

the database pattern for the anatase phase of TiO2. The production of an anatase lm by APCVD under the investigated condition, is expected, based on previous work.[23] The XRD patterns could also be used to determine the average particle size by use of the Scherrer method.[24] This analysis of the peak broadening gave a particle size of 40 nm for the titanium oxynitride lm and 60 nm for the titanium dioxide lm. Thickness of the lms was determined by spectroscopic analysis of the interference fringes apparent on the surface of each lm. Some variation in lm thickness was found as a function of position, but the sections of lm analyzed in the microbiology experiments were found to be in the range of 8001000 nm for both lms. Microbiological testing was carried out on the seven sections of each of the thin lms and also on sections of uncoated glass as a control. Samples were denoted C for the Ti2.85O4N-coated samples, T for the TiO2 thin lms, and G for the uncoated glass. The experiments then proceeded with three steps; activation of the sample with 1 h of 254 nm light (A), inoculation with E. coli, and then, nally, exposure to 4 h of 365 nm light (labeled L). For control purposes both light exposure periods were also carried out in the dark, A- and L-, respectively, in separate experiments. When the titanium (IV) oxynitride lm was pre-exposed to 1 h of 254 nm light, inoculated with E. coli, and then exposed to 4 h of 365 nm light (C AL), a 3.8 log10 (99.98%) reduction in bacterial count was observed, compared with the uncoated control exposed to the same light conditions (G AL). This difference was highly statistically signicant ( p < 0.01), and is shown graphically, along with the bacterial counts for a number of the other conditions, in Figure 2. The reduction observed for the TiO2 lm (T AL) was slightly higher at 3.9 log10 (99.99%), but comparable. Exposing the uncoated slides to both light incubation steps (G AL) resulted in a 0.5 log10 reduction of E. coli compared with the slides incubated in the absence of light 20
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(G AL); therefore the G AL slide was used as the negative control throughout. This also demonstrates that E. coli is resistant to UV light alone, without the presence of a photocatalytic coating. For the uncoated test group with the activation step, but no exposure to 365 nm light (G AL) there was no difference in the number of bacteria recovered from the sample compared to that without any UV exposure (G A-L). These comparisons show that regardless of the lighting exposure, there was no appreciable reduction in the number of bacteria for experiments with uncoated glass samples. For the Ti2.85O4N-coated lms the pre-inoculation, activation step (A) was found to enhance the activity of the thin lms when they were subsequently exposed to the 365 nm light. The oxynitride thin lms which were not activated but exposed to only 365 nm light for four hours produced a 1.2 log10 (93.2%) reduction in the number of E. coli observed ( p < 0.01), compared with the 3.8 log10 kill with the activation step (both relative to the negative control). The activation step alone, however, was not enough to exert a signicant killing effect on E. coli and there was no signicant decrease in the number of bacteria recovered from thin lms which were exposed to just the activation step (C AL). In addition, no signicant decrease in the number of recoverable E. coli was observed from the thin lms which were incubated in the absence of light throughout (C AL). As has already been noted, the titania lm when exposed to 365 nm light with an activation step (T AL) had a 3.9 log10 reduction in bacterial count. It was also found, however, that for the titania samples the activation step was unnecessary and that exposure to 365 nm light alone
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(T AL) will lead to a 3.9 log10 reduction after four hours of exposure. These results show that in the Ti2.85O4N-coated samples no anti-bacterial activity was observed without UV exposure after the inoculation (L), therefore the mode of action is unlikely to be related to the diffusion of ions onto the surface and is genuinely photo-activated. This is benecial because ion diffusion as an anti-bacterial mechanism can lead to the rapid development of bacterial resistance. In conclusion, we have demonstrated that a novel titanium oxynitride thin lm is capable of killing surfaceassociated E. coli. The thin lms are activated by exposure to 254 nm UV light for 1 h before the bacterial inoculum is added and the thin lms are irradiated with 365 nm UV light for 4 h. This is a rate of bacterial kill as effective as TiO2. Given the wide use that titania nds in anti-bacterial materials this is a signicant result, indicating that with further optimization, lms of Ti2.85o4N might nd use in anti-microbial applications.

Experimental
Films of Ti2.85O4N and TiO2 were synthesized using APCVD on glass substrates of dimensions 220 mm 85 mm 4 mm using titanium (IV) chloride (TiCl4, Aldrich 99.9%), ethyl acetate (Fisher, reagent grade), and ammonia (anhydrous, BOC used only in oxynitride synthesis) as precursors. A cold-wall reactor was used to deposit the lms, with the substrates placed in the reactor on a graphite block containing three Whatman heater cartridges. The substrates were made of standard commercial oat glass (Pilkington NSG Group) coated with a silicon dioxide barrier layer to prevent the diffusion of metal ions from the glass into the deposited thin lms. During the deposition, a substrate temperature of 550 8C was used with a reaction time of 60 s. The mixing chambers were kept at 250 8C, and the gas delivery lines at 200 8C, to prevent condensation of the reagent vapors. Figure 3 shows a schematic diagram of the reactor. Both the TiCl4 and ethyl acetate precursors were heated in brass bubblers through which nitrogen gas (N2, BOC, oxygen free) was passed to transport the vapors to the reactor. The TiCl4 bubbler was heated to 61 8C and the content transported using a N2 gas ow of 2 L min1 equivalent to 8 mmol min1 calculated using the method described by Betsch [25]. The ethyl acetate bubbler was heated to 44 8C, and the oxygen source transported with a ow rate of 0.5 L min-1 giving a ow rate of 8 mmol min1. Both the

TiCl4 and ethyl acetate vapor ows were combined in the same mixing chamber with an additional plain line N2 ow of 12 L min1. The nal reagent, ammonia, used only in the synthesis of the oxynitride, was introduced through a second mixing chamber, and into a separate channel in the bafe manifold. This prevented the pre-reaction of TiCl4 and NH3 which can form an undesirable solid adduct. NH3 was introduced into the reactor under its own vapor pressure without carrier gas at a ow rate of 0.26 L min1 giving a molar ow of 11 mmol min1. The thin lms were characterized using powder XRD, conducted using a Bruker D8 Discover, tted with a GADDS area X-ray detector, and utilizing Cu Ka12 radiation. The patterns were interpreted, in the case of the titania lm, by comparison with known database patterns, and for the Ti2.85O4N by Le Bail renement using the GSAS and EXPGUI software packages, and comparison with previously published data [26,27]. For anti-microbial testing the lms were cut into seven equal pieces of 85 mm 30 mm 4 mm in size. E. coli ATCC 25922 was stored in 10% glycerol at 80 8C and subcultured weekly onto 5% Columbia blood agar plates (Oxoid Ltd., Basingstoke, UK). An overnight culture was prepared by inoculating a single colony into 20 mL nutrient broth and incubating in an orbital incubator (Sanyo BV, Loughborough, UK) at a speed of 200 rpm for 18 h at 37 8C. A 1 mL aliquot of the culture was centrifuged at 12,000 rpm before the pellet was re-suspended in 1 mL phosphate-buffered saline (PBS). An optical density of 0.05 Absorbance units on a spectrophotometer (GE Healthcare, Buckinghamshire, UK) at 600 nm was achieved by adding around 300 mL of this suspension to 10 mL PBS. This equates to approximately 107 cfu per mL or 2.5 105 cfu per 25 mL. The test group of thin lms were exposed to two light exposure steps activation under the UVC (254 nm) light for 1 h, then irradiation under the UVA (365 nm) light for 4 h, designated AL. The thin lms were also subjected to control conditions by exposure to the UVA step only (AL), the UVC step only (AL) or incubation in the dark throughout (AL). Uncoated controls were also tested and exposed to either both light steps (G AL) or neither of the light incubation steps (G AL). During activation, thin lms were placed in square petri dishes (24 cm2) with the lid removed. For the L/L conditions, the thin lms were placed in custom-made moisture chambers, with the lid in place. The moisture chamber was assembled by lining a square 24 cm 24 cm petri dish with lter paper saturated with sterile distilled water and placing wooden sticks, on which to rest the thin lms, on top of the lter paper. The bacterial inoculum was added after the activating step and the moisture chamber prevented evaporation of the bacterial droplet. A 25 mL aliquot of the bacterial suspension was inoculated onto a clean lm and was sampled for 20 s using a cotton-tipped swab in a standardized manner, with continual rotation of the swab head. The swab was then inoculated into 1 mL phosphate-buffer solution, vortexed for 2 min before being serially diluted ten-fold. A 20 mL aliquot of each dilution was spread onto MacConkey agar plates and enumeration was performed after 48 h incubation at 37 8C, using the viable count technique. The experiment was performed in duplicate, and repeated three times to demonstrate reproducibility. The Mann-Whitney U test was used to determine the statistical signicance of any differences observed between the test and control groups.

Received: November 5, 2009 Revised: January 18, 2010

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