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Electrodeposition for antibacterial nickel-oxide-based coatings
Thin Solid Films 517 (2009) 6527–6530
Contents lists available at ScienceDirect
Thin Solid Films j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t s f
Electrodeposition for antibacterial nickel-oxide-based coatings Yan Yan Xi a, Bing Qiang Huang a, Aleksandra B. Djurišić a,⁎, Charis M.N. Chan b, Frederick C.C. Leung b, Wai Kin Chan c, Doris T.W. Au d a
Department of Physics, The University of Hong Kong, Pokfulam Road, Hong Kong School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong d Department of Biology and Chemistry, City University of Hong Kong, Kowloon Tong, Hong Kong b c
a r t i c l e
i n f o
Article history: Received 18 January 2009 Received in revised form 2 April 2009 Accepted 2 April 2009 Available online 8 April 2009 PACS: 81.07.Bc 81.15.Pq 87.19.xb
a b s t r a c t Nickel-oxide-based films exhibiting antibacterial activity against both Gram-negative (Escherichia coli) and Gram-positive bacteria (Bacillus atrophaeus) have been fabricated by electrodeposition from aqueous solutions. However, after annealing of the films, no antibacterial activity has been observed. As-deposited films were found to consist of a mixture of nickel-oxide hydroxide and nickel hydroxide, while annealing resulted in the conversion of the films into pure NiO. Also, annealed films exhibited no production of H2O2, unlike as-deposited films. Thus, antibacterial activity of as-deposited films is related to the presence of nickel-oxide hydroxide/nickel hydroxide which results in the production of reactive oxygen species and antibacterial activity. © 2009 Elsevier B.V. All rights reserved.
Keywords: Oxides Electrodeposition Bactericidal coatings X-ray diffraction Scanning electron microscopy
1. Introduction Nickel-oxide is a material of interest for a variety of practical applications, such as electrochromic material for smart windows [1,2], gas sensing [3], photovoltaics [4,5], catalysis [6], light-emitting devices [7], and electrochemical capacitors [8,9]. It can be fabricated by a variety of methods, such as magnetron sputtering [1,4], electrodeposition [2,7–9], hydrothermal synthesis [9], etc. Typically, a mixture of nickel-oxide-based materials (oxides, hydroxides, and oxyhydroxides) is obtained in the deposited films [1,9]. While the presence of nickel-oxide hydroxide (NiOOH) can be detrimental for some applications, such as light-emitting diodes [7], other applications, such as electrochromism, rely on transformation of different nickel-oxide [1], nickel hydroxide [1,10], and NiOOH [1,10] into one another. While many applications and properties of nickel-oxide-based materials have been investigated, antibacterial activity which has been observed in a variety of other metal oxide materials [11–19] has not been studied to date. Yet, antibacterial activity of inorganic materials is of significant interest due to the need for infection control and rising
⁎ Corresponding author. E-mail address: [email protected] (A.B. Djurišić). 0040-6090/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.tsf.2009.04.018
antibiotic resistance. For other metal oxides, different mechanisms have been proposed to explain antibacterial activity. Antibacterial activity of different metal oxides has been attributed to the photocatalytic production of reactive oxygen species, but this mechanism has so far been conclusively demonstrated for TiO2 [12]. In general, antibacterial activity has been demonstrated both in the dark and under illumination, but the difference in the growth inhibition in these two cases was dependent both on the type of metal oxide, as well as the type of bacteria (different responses were observed for the same material for Gram-positive and Gram-negative bacteria) [12]. For some metal oxides, such as ZnO, exposure to ambient light and UV light resulted in higher activity compared to dark conditions, but the optimal illumination conditions may be particle size dependent [14]. With or without illumination, production of reactive oxygen species (ROS), which would result in oxidative damage of the cell membrane or inside the cells, appears to be a likely mechanism of antibacterial activity of metal oxides [13,17]. These ROS can include hydroxyl groups, superoxide anions O− 2 , and hydrogen peroxide H2O2 [16]. Hydrogen peroxide release has been identified as a mechanism of antibacterial activity for some metal oxides, such as ZnO [17,18], while for others, such as MgO, antibacterial activity was attributed to O− 2 [19]. Damage of the cell membranes upon exposure to metal oxide nanoparticles is commonly
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Fig. 1. SEM image of nickel-oxide based nanostructured films electrodeposited on ITOcoated glass substrate at 50 °C: top view (left) and cross section (right) image.
observed [11], consistent with the hypothesis of oxidative damage of the membrane by ROS. In some cases, penetration of metal oxide nanoparticles into the cells has also been observed [13]. However, that is likely not the dominant mechanism causing the damage of bacterial cells [11], and it is not relevant for nanocrystalline coatings, i.e. in the absence of free standing nanoparticles. In addition, electrostatic interactions as a mechanism of antibacterial activity have also been proposed, but this mechanism is inconsistent with the fact that antibacterial activity is still observed even with various molecules adsorbed onto the metal oxide surface [11,15]. In this work, we investigated the potential of electrodeposited nickel-oxide-based nanostructured film as an antibacterial material. These films can be of interest for number of potential applications, such as multifunctional coatings, as well as coating of nickel-plated tools, such as forceps, blades etc. For such applications, simple fabrication method, such as electrodeposition, is of significant interest. We have investigated both as-deposited films and annealed films to establish how the composition of the film affects the antibacterial activity. 2. Experimental details 2.1. Sample preparation
preparation of samples for TEM study, while LB culture agar was used for testing the antibacterial activity of the films. A single colony of bacteria was inoculated into 3 ml LB Broth and was grown to 108 colony-forming units/ml and 20 µl of bacteria was then placed onto the coated plates and incubated further for 4 h. LB culture agar was then added on the top of the bacteria and kept overnight. E. coli was grown at 37 °C and B. atrophaeus was grown at 30 °C in all experiments. The formation of colonies was determined by observation under an optical microscope. All the antibacterial tests have been performed without exposure to UV light. For examination of cell damage using TEM, the cell suspension was fixed in equal volume of 2.5% glutaraldehyde in cacodylate buffer (pH 7.4) over night at 4–8 °C. The specimens were then rinsed with 0.1 M cacodylate buffer followed by postfixation in 1% osmium tetroxide (OsO4) in cacodylate buffer. Fixed cell suspension was then washed with cacodylate buffer, centrifuged (2500 rpm for 10 min) and embedded in agar, followed by infiltration of 1 mm cube agar blocks which were then embedded in epoxy resin. Ultrathin sections (70 nm) were cut on Leica ULTRACUT UCT ultramicrotome. The sections were stained in Reynolds’ lead citrate and 2% uranyl acetate for 15 min and 20 min, respectively, and examined under Philips Tecnai 12 TEM at 80 kV. 2.3. Detection of hydrogen peroxide production Phosphate buffer solution (pH 7.4) was prepared by dissolving a tablet of phosphate buffered saline in 200 ml deionized water. Different concentrations (9.2, 46, and 92 mg/ml) of nanoparticle powders obtained from as-deposited and annealed films in phosphate buffer solution were then prepared. 5 ml of 5 mg/ml 2′, 7′-dichlorofluorescein diacetate in acetone was added into 1 ml of buffer solutions with different concentrations of the nanoparticle powder. After 5 min incubation at room temperature in darkness, 200 µl of the solution was pipetted into 96 well black microplate with 2 replicates. The chemiluminescence response (at room temperature) was recorded by using fmax Fluorescence Microplate Reader (Molecular Devices Corporation) with excitation at 485 nm and emission at 538 nm. The
The electrodeposition of nickel-oxide-based film was performed at 50 °C from a solution containing 0.2 M Ni(NO3)2 and 0.2 M hexamethylentetramine using a two electrode system. Indium tin oxide (ITO) coated glass served as a cathode, a Pt wire served as an anode, and the deposition voltage and time were 2.2 V and 1 min., respectively. After the deposition, the samples were dried in air. Properties and antibacterial activity of both as-grown and annealed (300 °C, in air) samples were studied. The morphology and structure of the deposited nanostructured films was examined by scanning electron microscopy (SEM) using a Leo 1530 field emission SEM at incident beam voltage of 5 kV, X-ray diffraction (XRD) using Bruker AXS SMART CCD diffractometer (using Cu-target Kα radiation at 40 kV and 40 mA at 0.05°/s scanning speed, and in Bragg–Brentano geometry for nanoparticle powder sample while for thin films masurement was performed at a low incident angle of 1°) and transmission electron microscopy (TEM) and selected area electron diffraction (SAED) using JEOL 2010F TEM and Philips Tecnai 20 TEM at incident beam voltage 200 kV. For TEM characterization, nanocrystallites from the films have been mechanically removed, dispersed in ethanol at a low concentration and then placed onto TEM grid. For XRD data analysis JCPDS-ICDD PDF2 reference database was used. 2.2. Tests of antibacterial activity A Gram-negative bacterium Escherichia coli XL1-Blue and Grampositive bacterium Bacillus atrophaeus ATCC 9372 were used for antibacterial activity testing. Luria–Bertani (LB) broth was used as culture medium providing nutrient source for culturing bacteria and the
Fig. 2. TEM images (left) and SAED pattern (right) for a), b) as-deposited films c),d) annealed films. The rings in SAED patterns correspond to nickel hydroxide (110) and nickel-oxide hydroxide (100) in b); while all the rings in d) can be identified as originating from different NiO planes.
Y.Y. Xi et al. / Thin Solid Films 517 (2009) 6527–6530
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Fig. 3. XRD patterns for as-deposited and annealed films. XRD of NiO nanoparticles is also shown for comparison. Open circles indicate peaks corresponding to nickel-oxide (JCPDS 711179), solid circles indicate peaks corresponding to nickel-oxide hydroxide (JCPDS 78-2343), solid squares indicate data corresponding to nickel hydroxide hydrate (JCPDS 38-0715), while open squares denote peaks corresponding to ITO (JCPDS 39-1058).
concentration of H2O2 was determined by comparison with chemiluminescence calibration curve obtained for known concentration of H2O2 in phosphate buffer solution. 3. Results and discussion
Fig. 5. TEM images of E. coli (a) control, (b) 9.2 mg/ml as-deposited nanoparticles and B. atrophaeus (c) control, (d) 9.2 mg/ml as-deposited nanoparticles.
Fig. 1 shows the SEM images of the top view and side view of the electrodeposited films on ITO substrates. To closely examine morphology and structure, we have performed TEM, SAED, and XRD measurements for as-deposited and annealed films. Fig. 2 shows the TEM images and the corresponding SAED patterns for the asdeposited and annealed films, while Fig. 3 shows the XRD patterns of as-deposited and annealed films, with the XRD pattern of NiO nanoparticles shown for comparison. It can be observed that both asdeposited and annealed films consist of small nanoscale particles. Larger grains observed in top-view SEM (Fig. 1a) likely represent aggregates of smaller particles since much smaller size can be observed in TEM (Fig. 2). Based on XRD and SAED results, we can conclude that the annealed films consist entirely of NiO (JCPDS 71-
1179), while for as-deposited films we can identify a mixture of different phases such as nickel-oxide hydroxide (JCPDS 78-2343) and nickel hydroxide hydrate (JCPDS 38-0715). A mixture of nickel hydroxide hydrate and nickel-oxide hydroxide, which was converted to NiO after annealing at 500 °C, was also obtained previously by chemical bath deposition [20]. In general, for solution based deposition of nickel-oxide based films, the appearance (dark vs. greenish) and the composition of the deposited films are strongly dependent on the deposition conditions [20]. The annealing temperature required for conversion of the electrochemically deposited films to NiO was found to be at or above 250 °C [8].
Fig. 4. Optical microscope photos of E. coli on (a) ITO glass, (b) ITO glass with as-deposited film, (c) ITO glass with annealed film and B. atrophaeus on (d) ITO glass, (e) ITO glass with as-deposited film, (f) ITO glass with annealed film.
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To examine the potential of the prepared films as an antibacterial coating, we have performed tests on both as-deposited and annealed films. Fig. 4 shows the optical microscope photos of E. coli and B. atrophaeus on different substrates. Bare ITO glass (Fig. 4a and d) was used as a control sample. Bacteria growth can be clearly observed that both control samples and annealed films, while no growth was found for as-deposited films. Thus, while pure NiO did not exhibit any antibacterial activity, as-deposited films exhibited antibacterial activity against both Gram-positive and Gram-negative bacteria. To examine the mechanism of antibacterial activity, we have prepared samples with and without the powder obtained from the asdeposited films at a low concentration (9.2 mg/ml, to obtain some undamaged and some damaged cells), and examined the cells using TEM. Fig. 5 shows the TEM images of the bacteria with (right side) and without (left side) exposure to nanoparticle dispersions obtained from as-deposited films. Both in the case of E. coli and B. atrophaeus, clear cell damage (damaged cell walls and/or leaked out cell content) can be observed in samples exposed to nanoparticle dispersions. It has been shown previously that electrodeposited nickel-oxide based films contain water, as well as two protons associated with a nickel vacancy–oxygen atom complex [8]. In addition, various nickeloxide based phases (NiO, NiOOH and Ni(OH)2) can be converted to one another by dehydration/oxidation reactions [1], and such reactions are expected to occur in aqueous solutions. These processes could potentially contribute to the creation of ROS in aqueous media and thus antibacterial activity. To examine this hypothesis, we determined the release of H2O2 for different concentrations of nanoparticle powders obtained from as-deposited and annealed films. In the case of annealed NiO films, no release of hydrogen peroxide has been detected. For nanoparticle powders obtained from as-deposited films, the H2O2 release increased with increased particle concentration. For 9.2 mg/ml solution of nanoparticle powders, H2O2 concentration of 3.4 µg/L was obtained, while for 46 and 92 mg/ml, obtained H2O2 concentrations were 6.3 µg/L and 10.8 µg/L, respectively. It should be noted that H2O2 concentration released for low particle concentration is significantly higher than that released from ZnO nanoparticles and nanorods [11]. However, we have also previously found that H2O2 concentrations as high as 30 µg/L did not cause significant damage of B. atrophaeus cells [11]. This indicates that other ROS in addition to H2O2 likely contribute to the antibacterial activity. In addition, the exact mechanism of the cell damage of Gram-positive and Gram-negative bacteria could be different. These types of bacteria have significantly different cell wall structure, and thus can exhibit different reactions to various chemical agents, with Gram-negative bacteria often exhibiting higher resistance due to more complex cell wall structure [21]. Several works on antibacterial activity of different nanomaterials have shown that these materials exhibit higher activity against Gram-positive bacteria [12], while in others the opposite effect has been observed [13]. This can possibly be due to the kind of reactive oxygen species produced, and the bacterial species used for testing. For example, E. coli can produce superoxide dismutase which could transform O− 2 into H2O2, while S. aureus can produce catalase which would decompose H2O2 into water and oxygen [13]. These differences, together with the differences in cell wall structure, could contribute to the different behavior of Gram-positive and Gram-negative bacteria when exposed to the same chemical agent. Thus, we can conclude that although we have observed a clear correlation between H2O2 release and antibacterial activity of nickeloxide based coatings, it is likely that other reactive oxygen species such as oxy-hydroxyl groups and superoxide ions also contribute to the antibacterial activity. It has been proposed that there are additional
undetermined mechanisms of antibacterial activity in addition to the production of reactive oxygen species under illumination [12] and the production of H2O2 [11]. Since annealed films do not exhibit any antibacterial activity, activity due to Ni ions is unlikely, since Ni is present in both as-deposited and annealed films. On the other hand, annealed films also do not produce any H2O2, which indicates that antibacterial activity is likely related to the production of ROS, including but not necessarily limited to H2O2. Considering the fact that the as-deposited films consist of a mixture of nickel hydroxide hydrate and nickel-oxide hydroxide, possible ion exchange reactions could result in release of protons or electrons [1], which in turn could result in formation of a variety of ROS. 4. Conclusions Films exhibiting antibacterial activity against both Gram-positive and Gram-negative bacteria have been prepared by simple electrodeposition method. As-deposited films consisted of a mixture of nickeloxide hydroxide and nickel hydroxide hydrate. Annealing of the films at 300 °C resulted in conversion to NiO and disappearance of antibacterial activity. Antibacterial activity of as-deposited films was attributed to the release of reactive oxygen species which caused cell membrane damage of the bacteria. Acknowledgements This work is partly supported by the University Development Fund grant and Outstanding Young Researcher Award of the University of Hong Kong, Hung Hing Ying Physical Sciences research Fund, and Research Fund for the Control of Infectious Diseases grant (Ref. No. 07060602) funded by the Health, Welfare, and Food Bureau, Hong Kong Government. The authors would like to thank Mr. M. W. L. Chiang from City University of Hong Kong for his help in obtaining the TEM images. References [1] G.A. Niklasson, C.G. Granqvist, J. Mater. Chem. 17 (2007) 127. [2] M.S. Wu, C.-H. Yang, Appl. Phys. Lett. 91 (2007) 033109. [3] K.I. Arshak, L.M. Cavanagh, I. Gaidan, E.G. Moore, S.A. Clifford, IEEE Sens. J. 7 (2007) 925. [4] S.-S. Kim, K.-W. Park, J.-H. Yum, Y.-E. Sung, Sol. Energy Mater. Sol. Cells 90 (2006) 283. [5] J. He, H. Lindström, A. Hagfeldt, S.-E. Lindquist, J. Phys. Chem. B 103 (1999) 8940. [6] J. Li, R. Yan, B. Xiao, D.T. Liang, D.H. Lee, Energy Fuels 22 (2008) 16. [7] Y.Y. Xi, Y.F. Hsu, A.B. Djurišić, A.M.C. Ng, W.K. Chan, H.L. Tam, K.W. Cheah, Appl. Phys. Lett. 92 (2008) 113505. [8] D.-D. Zhao, M.W. Xu, W.-J. Zhou, J. Zhang, H.L. Li, Electrochim. Acta 53 (2008) 2699. [9] Y.Y. Xi, D. Li, A.B. Djurišić, M.H. Xie, K.Y.K. Man, W.K. Chan, Electrochem. Solid-State Lett. 11 (2008) D56. [10] M. Vidotti, C. van Greco, E.A. Ponzio, S. Córdoba de Torresi, Electrochem. Commun. 8 (2006) 554. [11] K.H. Tam, A.B. Djurišić, C.M.N. Chan, Y.Y. Xi, C.W. Tse, Y.H. Leung, W.K. Chan, F.C.C. Leung, D.T.W. Au, Thin Solid Films 516 (2008) 6167. [12] L.K. Adams, D.Y. Lyon, P.J.J. Alvarez, Water Res. 40 (2006) 3527. [13] S. Makhluf, R. Dror, Y. Nitzan, Y. Abramovich, R. Jelinek, A. Gedanken, Adv. Funct. Mater. 15 (2005) 1708. [14] N. Jones, B. Ray, K.T. Ranjit, A.C. Manna, FEMS Microbiol. Lett. 279 (2008) 71. [15] L. Zhang, Y. Jiang, Y. Ding, M. Povey, D. York, J. Nanopart. Res. 9 (2007) 479. [16] M.S. Wong, W.-C. Chu, D.-S. Sun, H.-S. Huang, J.-H. Chen, P.-J. Tsai, N.T. Lin, M.-S. Yu, S.-F. Hsu, S.-L. Wang, H.-H. Chang, Appl. Environ. Microbiol. 72 (2006) 6111. [17] J. Sawai, J. Microbiol. Methods 54 (2003) 177. [18] O. Yamamoto, K. Nakakoshi, T. Sasamoto, H. Nakagawa, K. Miura, Carbon 39 (2001) 1643. [19] J. Sawai, H. Kojima, H. Igarashi, A. Hashimoto, S. Shoji, T. Sawaki, A. Hakoda, E. Kawada, T. Kokugan, M. Shimizu, World J. Microbiol. Biotechnol. 16 (2000) 187. [20] S.-Y. Han, D.-H. Lee, Y.-J. Chang, S.-O. Ryu, T.-J. Lee, C.-H. Chang, J. Electrochem. Soc. 153 (2006) C382. [21] G. Fu, P.S. Vary, C.T. Lin, J. Phys. Chem. B 109 (2005) 8889.
Contents lists available at ScienceDirect
Thin Solid Films j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t s f
Electrodeposition for antibacterial nickel-oxide-based coatings Yan Yan Xi a, Bing Qiang Huang a, Aleksandra B. Djurišić a,⁎, Charis M.N. Chan b, Frederick C.C. Leung b, Wai Kin Chan c, Doris T.W. Au d a
Department of Physics, The University of Hong Kong, Pokfulam Road, Hong Kong School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong d Department of Biology and Chemistry, City University of Hong Kong, Kowloon Tong, Hong Kong b c
a r t i c l e
i n f o
Article history: Received 18 January 2009 Received in revised form 2 April 2009 Accepted 2 April 2009 Available online 8 April 2009 PACS: 81.07.Bc 81.15.Pq 87.19.xb
a b s t r a c t Nickel-oxide-based films exhibiting antibacterial activity against both Gram-negative (Escherichia coli) and Gram-positive bacteria (Bacillus atrophaeus) have been fabricated by electrodeposition from aqueous solutions. However, after annealing of the films, no antibacterial activity has been observed. As-deposited films were found to consist of a mixture of nickel-oxide hydroxide and nickel hydroxide, while annealing resulted in the conversion of the films into pure NiO. Also, annealed films exhibited no production of H2O2, unlike as-deposited films. Thus, antibacterial activity of as-deposited films is related to the presence of nickel-oxide hydroxide/nickel hydroxide which results in the production of reactive oxygen species and antibacterial activity. © 2009 Elsevier B.V. All rights reserved.
Keywords: Oxides Electrodeposition Bactericidal coatings X-ray diffraction Scanning electron microscopy
1. Introduction Nickel-oxide is a material of interest for a variety of practical applications, such as electrochromic material for smart windows [1,2], gas sensing [3], photovoltaics [4,5], catalysis [6], light-emitting devices [7], and electrochemical capacitors [8,9]. It can be fabricated by a variety of methods, such as magnetron sputtering [1,4], electrodeposition [2,7–9], hydrothermal synthesis [9], etc. Typically, a mixture of nickel-oxide-based materials (oxides, hydroxides, and oxyhydroxides) is obtained in the deposited films [1,9]. While the presence of nickel-oxide hydroxide (NiOOH) can be detrimental for some applications, such as light-emitting diodes [7], other applications, such as electrochromism, rely on transformation of different nickel-oxide [1], nickel hydroxide [1,10], and NiOOH [1,10] into one another. While many applications and properties of nickel-oxide-based materials have been investigated, antibacterial activity which has been observed in a variety of other metal oxide materials [11–19] has not been studied to date. Yet, antibacterial activity of inorganic materials is of significant interest due to the need for infection control and rising
⁎ Corresponding author. E-mail address: [email protected] (A.B. Djurišić). 0040-6090/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.tsf.2009.04.018
antibiotic resistance. For other metal oxides, different mechanisms have been proposed to explain antibacterial activity. Antibacterial activity of different metal oxides has been attributed to the photocatalytic production of reactive oxygen species, but this mechanism has so far been conclusively demonstrated for TiO2 [12]. In general, antibacterial activity has been demonstrated both in the dark and under illumination, but the difference in the growth inhibition in these two cases was dependent both on the type of metal oxide, as well as the type of bacteria (different responses were observed for the same material for Gram-positive and Gram-negative bacteria) [12]. For some metal oxides, such as ZnO, exposure to ambient light and UV light resulted in higher activity compared to dark conditions, but the optimal illumination conditions may be particle size dependent [14]. With or without illumination, production of reactive oxygen species (ROS), which would result in oxidative damage of the cell membrane or inside the cells, appears to be a likely mechanism of antibacterial activity of metal oxides [13,17]. These ROS can include hydroxyl groups, superoxide anions O− 2 , and hydrogen peroxide H2O2 [16]. Hydrogen peroxide release has been identified as a mechanism of antibacterial activity for some metal oxides, such as ZnO [17,18], while for others, such as MgO, antibacterial activity was attributed to O− 2 [19]. Damage of the cell membranes upon exposure to metal oxide nanoparticles is commonly
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Fig. 1. SEM image of nickel-oxide based nanostructured films electrodeposited on ITOcoated glass substrate at 50 °C: top view (left) and cross section (right) image.
observed [11], consistent with the hypothesis of oxidative damage of the membrane by ROS. In some cases, penetration of metal oxide nanoparticles into the cells has also been observed [13]. However, that is likely not the dominant mechanism causing the damage of bacterial cells [11], and it is not relevant for nanocrystalline coatings, i.e. in the absence of free standing nanoparticles. In addition, electrostatic interactions as a mechanism of antibacterial activity have also been proposed, but this mechanism is inconsistent with the fact that antibacterial activity is still observed even with various molecules adsorbed onto the metal oxide surface [11,15]. In this work, we investigated the potential of electrodeposited nickel-oxide-based nanostructured film as an antibacterial material. These films can be of interest for number of potential applications, such as multifunctional coatings, as well as coating of nickel-plated tools, such as forceps, blades etc. For such applications, simple fabrication method, such as electrodeposition, is of significant interest. We have investigated both as-deposited films and annealed films to establish how the composition of the film affects the antibacterial activity. 2. Experimental details 2.1. Sample preparation
preparation of samples for TEM study, while LB culture agar was used for testing the antibacterial activity of the films. A single colony of bacteria was inoculated into 3 ml LB Broth and was grown to 108 colony-forming units/ml and 20 µl of bacteria was then placed onto the coated plates and incubated further for 4 h. LB culture agar was then added on the top of the bacteria and kept overnight. E. coli was grown at 37 °C and B. atrophaeus was grown at 30 °C in all experiments. The formation of colonies was determined by observation under an optical microscope. All the antibacterial tests have been performed without exposure to UV light. For examination of cell damage using TEM, the cell suspension was fixed in equal volume of 2.5% glutaraldehyde in cacodylate buffer (pH 7.4) over night at 4–8 °C. The specimens were then rinsed with 0.1 M cacodylate buffer followed by postfixation in 1% osmium tetroxide (OsO4) in cacodylate buffer. Fixed cell suspension was then washed with cacodylate buffer, centrifuged (2500 rpm for 10 min) and embedded in agar, followed by infiltration of 1 mm cube agar blocks which were then embedded in epoxy resin. Ultrathin sections (70 nm) were cut on Leica ULTRACUT UCT ultramicrotome. The sections were stained in Reynolds’ lead citrate and 2% uranyl acetate for 15 min and 20 min, respectively, and examined under Philips Tecnai 12 TEM at 80 kV. 2.3. Detection of hydrogen peroxide production Phosphate buffer solution (pH 7.4) was prepared by dissolving a tablet of phosphate buffered saline in 200 ml deionized water. Different concentrations (9.2, 46, and 92 mg/ml) of nanoparticle powders obtained from as-deposited and annealed films in phosphate buffer solution were then prepared. 5 ml of 5 mg/ml 2′, 7′-dichlorofluorescein diacetate in acetone was added into 1 ml of buffer solutions with different concentrations of the nanoparticle powder. After 5 min incubation at room temperature in darkness, 200 µl of the solution was pipetted into 96 well black microplate with 2 replicates. The chemiluminescence response (at room temperature) was recorded by using fmax Fluorescence Microplate Reader (Molecular Devices Corporation) with excitation at 485 nm and emission at 538 nm. The
The electrodeposition of nickel-oxide-based film was performed at 50 °C from a solution containing 0.2 M Ni(NO3)2 and 0.2 M hexamethylentetramine using a two electrode system. Indium tin oxide (ITO) coated glass served as a cathode, a Pt wire served as an anode, and the deposition voltage and time were 2.2 V and 1 min., respectively. After the deposition, the samples were dried in air. Properties and antibacterial activity of both as-grown and annealed (300 °C, in air) samples were studied. The morphology and structure of the deposited nanostructured films was examined by scanning electron microscopy (SEM) using a Leo 1530 field emission SEM at incident beam voltage of 5 kV, X-ray diffraction (XRD) using Bruker AXS SMART CCD diffractometer (using Cu-target Kα radiation at 40 kV and 40 mA at 0.05°/s scanning speed, and in Bragg–Brentano geometry for nanoparticle powder sample while for thin films masurement was performed at a low incident angle of 1°) and transmission electron microscopy (TEM) and selected area electron diffraction (SAED) using JEOL 2010F TEM and Philips Tecnai 20 TEM at incident beam voltage 200 kV. For TEM characterization, nanocrystallites from the films have been mechanically removed, dispersed in ethanol at a low concentration and then placed onto TEM grid. For XRD data analysis JCPDS-ICDD PDF2 reference database was used. 2.2. Tests of antibacterial activity A Gram-negative bacterium Escherichia coli XL1-Blue and Grampositive bacterium Bacillus atrophaeus ATCC 9372 were used for antibacterial activity testing. Luria–Bertani (LB) broth was used as culture medium providing nutrient source for culturing bacteria and the
Fig. 2. TEM images (left) and SAED pattern (right) for a), b) as-deposited films c),d) annealed films. The rings in SAED patterns correspond to nickel hydroxide (110) and nickel-oxide hydroxide (100) in b); while all the rings in d) can be identified as originating from different NiO planes.
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Fig. 3. XRD patterns for as-deposited and annealed films. XRD of NiO nanoparticles is also shown for comparison. Open circles indicate peaks corresponding to nickel-oxide (JCPDS 711179), solid circles indicate peaks corresponding to nickel-oxide hydroxide (JCPDS 78-2343), solid squares indicate data corresponding to nickel hydroxide hydrate (JCPDS 38-0715), while open squares denote peaks corresponding to ITO (JCPDS 39-1058).
concentration of H2O2 was determined by comparison with chemiluminescence calibration curve obtained for known concentration of H2O2 in phosphate buffer solution. 3. Results and discussion
Fig. 5. TEM images of E. coli (a) control, (b) 9.2 mg/ml as-deposited nanoparticles and B. atrophaeus (c) control, (d) 9.2 mg/ml as-deposited nanoparticles.
Fig. 1 shows the SEM images of the top view and side view of the electrodeposited films on ITO substrates. To closely examine morphology and structure, we have performed TEM, SAED, and XRD measurements for as-deposited and annealed films. Fig. 2 shows the TEM images and the corresponding SAED patterns for the asdeposited and annealed films, while Fig. 3 shows the XRD patterns of as-deposited and annealed films, with the XRD pattern of NiO nanoparticles shown for comparison. It can be observed that both asdeposited and annealed films consist of small nanoscale particles. Larger grains observed in top-view SEM (Fig. 1a) likely represent aggregates of smaller particles since much smaller size can be observed in TEM (Fig. 2). Based on XRD and SAED results, we can conclude that the annealed films consist entirely of NiO (JCPDS 71-
1179), while for as-deposited films we can identify a mixture of different phases such as nickel-oxide hydroxide (JCPDS 78-2343) and nickel hydroxide hydrate (JCPDS 38-0715). A mixture of nickel hydroxide hydrate and nickel-oxide hydroxide, which was converted to NiO after annealing at 500 °C, was also obtained previously by chemical bath deposition [20]. In general, for solution based deposition of nickel-oxide based films, the appearance (dark vs. greenish) and the composition of the deposited films are strongly dependent on the deposition conditions [20]. The annealing temperature required for conversion of the electrochemically deposited films to NiO was found to be at or above 250 °C [8].
Fig. 4. Optical microscope photos of E. coli on (a) ITO glass, (b) ITO glass with as-deposited film, (c) ITO glass with annealed film and B. atrophaeus on (d) ITO glass, (e) ITO glass with as-deposited film, (f) ITO glass with annealed film.
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To examine the potential of the prepared films as an antibacterial coating, we have performed tests on both as-deposited and annealed films. Fig. 4 shows the optical microscope photos of E. coli and B. atrophaeus on different substrates. Bare ITO glass (Fig. 4a and d) was used as a control sample. Bacteria growth can be clearly observed that both control samples and annealed films, while no growth was found for as-deposited films. Thus, while pure NiO did not exhibit any antibacterial activity, as-deposited films exhibited antibacterial activity against both Gram-positive and Gram-negative bacteria. To examine the mechanism of antibacterial activity, we have prepared samples with and without the powder obtained from the asdeposited films at a low concentration (9.2 mg/ml, to obtain some undamaged and some damaged cells), and examined the cells using TEM. Fig. 5 shows the TEM images of the bacteria with (right side) and without (left side) exposure to nanoparticle dispersions obtained from as-deposited films. Both in the case of E. coli and B. atrophaeus, clear cell damage (damaged cell walls and/or leaked out cell content) can be observed in samples exposed to nanoparticle dispersions. It has been shown previously that electrodeposited nickel-oxide based films contain water, as well as two protons associated with a nickel vacancy–oxygen atom complex [8]. In addition, various nickeloxide based phases (NiO, NiOOH and Ni(OH)2) can be converted to one another by dehydration/oxidation reactions [1], and such reactions are expected to occur in aqueous solutions. These processes could potentially contribute to the creation of ROS in aqueous media and thus antibacterial activity. To examine this hypothesis, we determined the release of H2O2 for different concentrations of nanoparticle powders obtained from as-deposited and annealed films. In the case of annealed NiO films, no release of hydrogen peroxide has been detected. For nanoparticle powders obtained from as-deposited films, the H2O2 release increased with increased particle concentration. For 9.2 mg/ml solution of nanoparticle powders, H2O2 concentration of 3.4 µg/L was obtained, while for 46 and 92 mg/ml, obtained H2O2 concentrations were 6.3 µg/L and 10.8 µg/L, respectively. It should be noted that H2O2 concentration released for low particle concentration is significantly higher than that released from ZnO nanoparticles and nanorods [11]. However, we have also previously found that H2O2 concentrations as high as 30 µg/L did not cause significant damage of B. atrophaeus cells [11]. This indicates that other ROS in addition to H2O2 likely contribute to the antibacterial activity. In addition, the exact mechanism of the cell damage of Gram-positive and Gram-negative bacteria could be different. These types of bacteria have significantly different cell wall structure, and thus can exhibit different reactions to various chemical agents, with Gram-negative bacteria often exhibiting higher resistance due to more complex cell wall structure [21]. Several works on antibacterial activity of different nanomaterials have shown that these materials exhibit higher activity against Gram-positive bacteria [12], while in others the opposite effect has been observed [13]. This can possibly be due to the kind of reactive oxygen species produced, and the bacterial species used for testing. For example, E. coli can produce superoxide dismutase which could transform O− 2 into H2O2, while S. aureus can produce catalase which would decompose H2O2 into water and oxygen [13]. These differences, together with the differences in cell wall structure, could contribute to the different behavior of Gram-positive and Gram-negative bacteria when exposed to the same chemical agent. Thus, we can conclude that although we have observed a clear correlation between H2O2 release and antibacterial activity of nickeloxide based coatings, it is likely that other reactive oxygen species such as oxy-hydroxyl groups and superoxide ions also contribute to the antibacterial activity. It has been proposed that there are additional
undetermined mechanisms of antibacterial activity in addition to the production of reactive oxygen species under illumination [12] and the production of H2O2 [11]. Since annealed films do not exhibit any antibacterial activity, activity due to Ni ions is unlikely, since Ni is present in both as-deposited and annealed films. On the other hand, annealed films also do not produce any H2O2, which indicates that antibacterial activity is likely related to the production of ROS, including but not necessarily limited to H2O2. Considering the fact that the as-deposited films consist of a mixture of nickel hydroxide hydrate and nickel-oxide hydroxide, possible ion exchange reactions could result in release of protons or electrons [1], which in turn could result in formation of a variety of ROS. 4. Conclusions Films exhibiting antibacterial activity against both Gram-positive and Gram-negative bacteria have been prepared by simple electrodeposition method. As-deposited films consisted of a mixture of nickeloxide hydroxide and nickel hydroxide hydrate. Annealing of the films at 300 °C resulted in conversion to NiO and disappearance of antibacterial activity. Antibacterial activity of as-deposited films was attributed to the release of reactive oxygen species which caused cell membrane damage of the bacteria. Acknowledgements This work is partly supported by the University Development Fund grant and Outstanding Young Researcher Award of the University of Hong Kong, Hung Hing Ying Physical Sciences research Fund, and Research Fund for the Control of Infectious Diseases grant (Ref. No. 07060602) funded by the Health, Welfare, and Food Bureau, Hong Kong Government. The authors would like to thank Mr. M. W. L. Chiang from City University of Hong Kong for his help in obtaining the TEM images. References [1] G.A. Niklasson, C.G. Granqvist, J. Mater. Chem. 17 (2007) 127. [2] M.S. Wu, C.-H. Yang, Appl. Phys. Lett. 91 (2007) 033109. [3] K.I. Arshak, L.M. Cavanagh, I. Gaidan, E.G. Moore, S.A. Clifford, IEEE Sens. J. 7 (2007) 925. [4] S.-S. Kim, K.-W. Park, J.-H. Yum, Y.-E. Sung, Sol. Energy Mater. Sol. Cells 90 (2006) 283. [5] J. He, H. Lindström, A. Hagfeldt, S.-E. Lindquist, J. Phys. Chem. B 103 (1999) 8940. [6] J. Li, R. Yan, B. Xiao, D.T. Liang, D.H. Lee, Energy Fuels 22 (2008) 16. [7] Y.Y. Xi, Y.F. Hsu, A.B. Djurišić, A.M.C. Ng, W.K. Chan, H.L. Tam, K.W. Cheah, Appl. Phys. Lett. 92 (2008) 113505. [8] D.-D. Zhao, M.W. Xu, W.-J. Zhou, J. Zhang, H.L. Li, Electrochim. Acta 53 (2008) 2699. [9] Y.Y. Xi, D. Li, A.B. Djurišić, M.H. Xie, K.Y.K. Man, W.K. Chan, Electrochem. Solid-State Lett. 11 (2008) D56. [10] M. Vidotti, C. van Greco, E.A. Ponzio, S. Córdoba de Torresi, Electrochem. Commun. 8 (2006) 554. [11] K.H. Tam, A.B. Djurišić, C.M.N. Chan, Y.Y. Xi, C.W. Tse, Y.H. Leung, W.K. Chan, F.C.C. Leung, D.T.W. Au, Thin Solid Films 516 (2008) 6167. [12] L.K. Adams, D.Y. Lyon, P.J.J. Alvarez, Water Res. 40 (2006) 3527. [13] S. Makhluf, R. Dror, Y. Nitzan, Y. Abramovich, R. Jelinek, A. Gedanken, Adv. Funct. Mater. 15 (2005) 1708. [14] N. Jones, B. Ray, K.T. Ranjit, A.C. Manna, FEMS Microbiol. Lett. 279 (2008) 71. [15] L. Zhang, Y. Jiang, Y. Ding, M. Povey, D. York, J. Nanopart. Res. 9 (2007) 479. [16] M.S. Wong, W.-C. Chu, D.-S. Sun, H.-S. Huang, J.-H. Chen, P.-J. Tsai, N.T. Lin, M.-S. Yu, S.-F. Hsu, S.-L. Wang, H.-H. Chang, Appl. Environ. Microbiol. 72 (2006) 6111. [17] J. Sawai, J. Microbiol. Methods 54 (2003) 177. [18] O. Yamamoto, K. Nakakoshi, T. Sasamoto, H. Nakagawa, K. Miura, Carbon 39 (2001) 1643. [19] J. Sawai, H. Kojima, H. Igarashi, A. Hashimoto, S. Shoji, T. Sawaki, A. Hakoda, E. Kawada, T. Kokugan, M. Shimizu, World J. Microbiol. Biotechnol. 16 (2000) 187. [20] S.-Y. Han, D.-H. Lee, Y.-J. Chang, S.-O. Ryu, T.-J. Lee, C.-H. Chang, J. Electrochem. Soc. 153 (2006) C382. [21] G. Fu, P.S. Vary, C.T. Lin, J. Phys. Chem. B 109 (2005) 8889.