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Antibacterial activity of ZnO nanorods prepared by a hydrothermal method
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Thin Solid Films 516 (2008) 6167 – 6174 www.elsevier.com/locate/tsf
Antibacterial activity of ZnO nanorods prepared by a hydrothermal method K.H. Tam a , A.B. Djurišić a,⁎, C.M.N. Chan c , Y.Y. Xi a , C.W. Tse b , Y.H. Leung b , W.K. Chan b , F.C.C. Leung c , D.W.T. Au d a
Department of Physics, The University of Hong Kong, Pokfulam Road, Hong Kong Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong c Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong Department of Biology and Chemistry, City University of Hong Kong, Kowloon Tong, Hong Kong b
d
Received 9 February 2007; received in revised form 9 October 2007; accepted 16 November 2007 Available online 24 November 2007
Abstract We investigated antibacterial activity of ZnO nanorods prepared by a hydrothermal method against a gram-negative bacterium Escherichia coli and a gram-positive bacterium Bacillus atrophaeus. Antibacterial activity of ZnO nanorod coatings was studied on solid substrates covered with nutrient agar, as well as in liquid nutrient broth for different concentrations of ZnO nanorods, nanoparticles, and powder. ZnO exhibited antibacterial activity against both E. coli and B. atrophaeus, but it was considerably more effective in the latter case (at 15 mM vs. 5 mM concentration, respectively, showing zero viable cell count). For both organisms, damage of the cell membranes was found, and the effect was more pronounced for B. atrophaeus. Chemiluminescence analysis has been used to detect the release of hydrogen peroxide from ZnO structures, and the effect of H2O2 on the E. coli and B. atrophaeus was studied. Since significant differences were observed in the effect of ZnO nanostructures and H2O2 on B. atrophaeus, it can be concluded that there are other mechanisms contributing to the antibacterial activity of ZnO nanostructures. © 2007 Elsevier B.V. All rights reserved. Keywords: II–VI semiconductors; Nanomaterials; Antimicrobial activity
1. Introduction Microbial contamination is a serious issue in health care and food industry, so that development of antimicrobial agents and surface coatings has been attracting increasing attention in recent years. Due to the spread of antibiotic resistant infections, interest in alternative antimicrobial agents, such as inorganic materials, has been rising [1–12]. Antimicrobial properties have been demonstrated for metallic nanoparticles [1,9,10] and metal oxide powders and nanoparticles [2–7,11]. The inorganic materials can be used in different forms, such as powders [2,3,5,6,8,11,12], coated on cellulose fibers [4], or as a part of organic/inorganic nanocomposite coating [1,9,10]. Antimicrobial coatings are of great interest for protection of surfaces, since survival of microorganisms on surfaces in the ⁎ Corresponding author. E-mail address: [email protected] (A.B. Djurišić). 0040-6090/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.tsf.2007.11.081
environment can result in the spread of the diseases. In addition, coatings are expected to have better safety and stability, while nanoscale powders may pose a hazard upon inhalation [13]. It has been recognized that toxicity of nanoparticles is generally larger than in the case of larger particles of the same material, even for materials with relatively low toxicity [11]. Therefore, development of nanostructured coatings with antimicrobial properties is of considerable interest. In this work, we investigated antimicrobial properties of ZnO nanorods fabricated by a hydrothermal method. It has been demonstrated that ZnO powders and nanoparticles exhibit antimicrobial activity against Staphilococcus auerus [8] and Escherichia coli [8,11,12]. Antimicrobial properties of polymer coatings with ZnO tetrapods have also been studied [7], but there was no study of the ZnO nanorod arrays. ZnO nanorod arrays fabricated by a hydrothermal method have an advantage of low growth temperature (90 °C) [14–17] and variety of possible substrates, and the fabrication process is inexpensive and environmentally
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friendly. The low substrate temperature enables compatibility with temperature sensitive substrates such as plastics or textiles. With the use of a nanoparticle seeds, similar morphologies can be obtained on a variety of substrates (such as glass, quartz, Si etc.), including nonplanar substrates. Concerning the mechanism of the antibacterial activity of nanomaterials, several mechanisms have been proposed. In the case of metal nanoparticles, the antimicrobial properties are usually a result of release of metal ions over time [1,9,10]. Catalytic activity of TiO2 was proposed as a mechanism of its antibacterial activity [18], but other studies also reported bactericidal activity of TiO2 in the dark [4]. In the case of ZnO, release of H2O2 has been proposed as a mechanism responsible for antibacterial activity [8,12]. It has also been shown that metal oxide nanoparticles cause membrane damage [11,18,19], and in the case of ZnO nanoparticles and E. coli bacteria, cellular internalization of the nanoparticles has also been observed [11]. The membrane damage effect was larger for halogen doped MgO nanoparticles compared to the undoped ones [19], while in the case of TiO2 membrane damage was attributed to photocatalytic processes responsible for decomposition of the cell membrane [18]. Therefore, the mechanisms responsible for antibacterial activity of metal oxide nanostructures are still not fully clear. 2. Experimental details 2.1. Nanorod array preparation The nanorod arrays were prepared from equimolar aqueous solutions of zinc nitrate hydrate and hexamethylene tetramine [14–17]. Before the nanorod growth, the substrates were cleaned by sonication in acetone, ethanol, and deionized water, and then dried in an oven. Then, the seed layer was prepared either using nanoparticles [16,17] or zinc acetate solution [14], as described previously. For increasing the aspect ratio of the nanorods, polyethyleneimine can be added to the solution [15]. Zinc nitrate hydrate, hexamethylene tetramine, zinc acetate (for nanorod growth) and ZnO powder (for comparison of antibacterial properties, particle size 200–500 nm) were obtained from Aldrich, while ZnO nanoparticles (∼ 10 nm diameter) were obtained from Nanoscale Materials Inc. The morphology of the nanorods was examined by scanning electron microscopy (SEM) using a Leo 1530 field emission SEM, X-ray diffraction (XRD) using Bruker AXS SMART CCD diffractometer and transmission electron microscopy (TEM) using JEOL 2010F TEM and Philips Tecnai 20 TEM. BET surface area measurements were also performed. The samples were heated to 600 K under vacuum for out-gassing, and then the surface area was determined from nitrogen adsorption/desorption. Nanorods coated by silane surfactant or silane and Au nanoparticles were prepared by dipping the substrate into solution of 3-aminopropyltrimethoxysilane, followed by dipping in the suspension of gold nanoparticles. Gold nanoparticles (∼ 5 nm) were prepared by the reduction of HAuCl4 by sodium borohydride using polyacrylic acid as the surfactant. Due to the presence of carboxylic acid moieties on Au nanoparticle sur-
face, the particles can be attached on silane-treated ZnO surface. Excess silane was removed by rinsing in pure toluene. 2.2. Tests of antibacterial activity A gram-negative bacterium E. coli XL1-Blue and grampositive bacterium Bacillus atrophaeus ATCC 9372 were used for antibacterial activity testing. Culture broth (Luria–Bertani broth) and culture agar (Luria–Bertani agar) were used as culturing nutrient sources. E. coli was grown at 37 °C and B. atrophaeus was grown at 30 °C. The optical density of bacteria cell at 600 nm wavelength used for all testing was 0.3–0.4, in which cells are growing rapidly in the mid-log phase. The antibacterial activity of different ZnO nanorod coated plates (as described previously) was tested using a modified protocol from Ref. [18]. Twenty microlitres of bacteria was placed onto the coated plates and allowed to dry at 37 °C. Culture agar was then added on the top of the bacteria and kept at 37 °C (for E. coli) or 30 °C (for B. atrophaeus) overnight. Formation of colonies was determined by observation under an optical microscope. The minimum bactericidal concentrations (minimum concentration showing no viable cell growth) of the ZnO nanoparticles, ZnO nanorod and ZnO powder were determined. A serial dilution (1 to 100 mM) of ZnO nanoparticles, ZnO nanorod or ZnO powder with culture broth was prepared. One millilitre of bacteria cell in culture broth containing different concentrations of ZnO nanoparticles, ZnO nanorod or ZnO powder was incubated at appropriate temperature on a shaking platform at 250 rpm. After 24 h, 10 μl cell suspensions were collected from each sample tube, spread onto culture agar plate and incubated overnight (16 h). Colonies of bacteria present on culture agar plate indicated signs of bacterial growth. If no colonies are observed, the ZnO is considered as being bactericidal at that concentration. A control tube was prepared with no ZnO added and three replicate plates were prepared. The tests have been performed without the exposure to UV light. 2.3. Detection of hydrogen peroxide by chemiluminescence response Phosphate buffer solution (pH 7.4) was prepared by dissolving a tablet of phosphate buffered saline from Sigma in 250 mL deionized water. Different concentrations (1, 10, 100 mM) of ZnO nanorods, nanoparticles and powder in phosphate buffer solution were then prepared. Five microlitres of 5 mg/mL 2′,7′dichlorofluorescein diacetate in acetone, which initiated the chemiluminescence, was added into 1 mL of various concentration of ZnO in buffer solution. After 5 min incubation at room temperature in darkness, 200 μl of the solutions was pipetted into 96 well black microplate with two replicates. The chemiluminescence response was recorded by using fmax Fluorescene Microplate Reader (Molecular Devices Cooperation, Sunnyvale, California) with excitation at 485 nm and emission at 538 nm. The concentration of H2O2 was determined by comparison with chemiluminescence calibration curve obtained for known concentration of H2O2 in phosphate buffer solution. Experiments were carried out at room temperature.
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Fig. 1. SEM images of ZnO nanorods on a) Si, b) glass, c) concave glass surface, and d) convex glass surface.
2.4. Preparation of TEM samples The cell suspension was fixed in equal volume of 2.5% glutaraldehyde in cacodylate buffer (pH 7.4) overnight at 4 °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. One millimeter cube agar blocks were infiltrated and embedded in epoxy resin. Ultra-thin 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 subsequently examined under Philips Tecnai 12 TEM at 80 kV. 3. Results and discussion Fig. 1 shows the SEM images of the ZnO nanorods fabricated on different substrates with nanoparticle seeds, including
nonplanar glass substrates (both convex and concave surfaces). It can be observed that the nanorods can be grown on all substrates including nonplanar ones, although growth directions of the nanorods appear more random on nonplanar substrates. Nanorods are ∼ 250 nm long and have diameters in the range of 30–70 nm, and they are densely packed. In the case of a flexible substrate, such as Kapton foil, the nanorods have somewhat larger diameter and have more random growth directions compared to those fabricated on glass. The orientation of the nanorods can be improved when zinc acetate instead of ZnO nanoparticles is used to prepare the seed layer [14]. The difference between nanorods prepared with nanoparticle and zinc acetate derived seed layer is shown in Fig. 2. The use of acetate seed in combination with the addition of polyethyleneimine to the growth solution and repeating the growth process several times enables improved orientation of the nanorods perpendicular to the substrate, as well as increased the aspect ratio [15]. After repeating the growth process three times, nanorods with diameters in the range of 55–70 nm and
Fig. 2. SEM images of nanorods prepared with a) ZnO nanoparticle seed and b) acetate seed.
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Fig. 3. XRD spectrum of nanorods prepared with ZnO nanoparticle seed and acetate seed.
length of 800 nm can be fabricated. While the nanorod orientation is not critical for their antibacterial activity, longer nanorods would be preferable because they would be more difficult to be completely covered by surface contaminants and because
they have higher surface area. However, preparing the seed layer from zinc acetate solutions requires annealing at 350 °C, so that substrates requiring low processing temperature, such as polyester foils, in this case cannot be used. Fig. 3 shows the XRD spectrum of ZnO nanorods prepared with two different seed layers. It can be observed that ZnO nanorods have wurtzite structure in both cases, but the nanorods grown with acetate seed have better orientation since stronger signal from b0002N is obtained. In the case of nanoparticle seeds, strong peaks from other ZnO planes in addition to b0002N are obtained, indicating that ZnO nanorods in this case grow at different angles to the substrate, in agreement with SEM results. After the fabrication of nanorod arrays, they can be further modified by surface functionalization. By attaching different surfactants, polymer layers, or nanoparticles to the zinc oxide nanorod surface, their properties can be tailored to combine the properties of ZnO and material attached to the surface. Surface functionalization can be used to tailor various properties, from optical properties to antimicrobial activity. For example, it has been shown that vancomycin capped Au nanoparticles exhibit
Fig. 4. TEM images of a) ZnO nanorods, b) ZnO nanoparticles, c) ZnO powders, d) high resolution TEM image of a ZnO nanorod, e) ZnO nanorods with 3aminopropyltrimethoxysilane and f) ZnO nanorods with Au nanoparticles.
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Table 2 Minimum concentration of different forms of ZnO showing 100% antibacterial activity
Fig. 5. Optical microscope photos of E. coli on a) microscope slide, b) microscope slide with ZnO nanorods, c) microscope slide with ZnO nanorods with 3-aminopropyltrimethoxysilane and d) microscope slide with ZnO nanorods with Au nanoparticles.
enhanced antimicrobial activity [20]. It has also been shown that Au capped TiO2 nanoparticles exhibit somewhat improved antibacterial activity for E. coli [18]. ZnO nanorods, ZnO nanorods coated with 3-aminopropyltrimethoxysilane and ZnO nanorods coated with 3-aminopropyltrimethoxysilane and Au nanoparticles were prepared. The corresponding TEM images are shown in Fig. 4, together with TEM images of as-grown ZnO nanorods, nanoparticles, and powder. Small particle size of
ZnO
Escherichia coli (mM)
Bacillus atrophaeus (mM)
Nanorods Nanoparticles Powder
15 15 80
5 2 2
ZnO nanoparticles compared to ZnO powder can be clearly observed. The nanoparticles have the smallest particle size, followed by nanorods, and ZnO powder. The nanorods grow along [0001] direction (lattice spacing 5.2 Å) and have good crystallinity, as observed from high resolution TEM image. The coating of the nanorods with silane surfactant results in covering of the surface with a thin (2–3 nm) amorphous layer (Fig. 4e), in good agreement with the expected thickness of a monolayer of silane. After immersion into the suspension of Au nanoparticles, Au nanoparticles attached to nanorod surface can be clearly observed in Fig. 3f. We have investigated the antibacterial activity of ZnO nanorods on a substrate and in a liquid suspension. We found that the type of the seed layer (nanoparticles vs. zinc acetate derived seed) did not affect antibacterial activity as observed under optical microscope. No growth of E. coli bacteria was observed both in the case of nanoparticle seeds and seed obtained from zinc acetate solution. The substrate (Si, glass, or quartz) also did not affect biocidal activity. Annealing of the nanorods in different environments (air, forming gas) and at different temperatures (200 °C and 600 °C) affected the photoluminescence from ZnO but had no effect on antibacterial activity. Since no correlation has been observed between the antibacterial activity and photoluminescence features corresponding to surface defects (green emission) [17], we can
Table 1 Dependence of cell count on ZnO concentration for different forms of ZnO Concentration of ZnO (mM)
100 90 80 70 60 50 40 30 25 20 15 10 5 4 3 2 1 Control
Viable cell counts (106 cfu/mL) after incubation for 24 h Escherichia coli
Bacillus atrophaeus
NP
NR
Powder
NP
NR
Powder
0 0 0 0 0 0 0 0 0 0 0 8 233 433 467 567 627 1500
0 0 0 0 0 0 0 0 0 0 0 10 60 125 405 478 500 1500
0 0 0 0.18 3 7 10 11 33 148 270 599 733 800 987 1040 1070 1500
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 8000
0 0 0 0 0 0 0 0 0 0 0 0 0 0.68 7 10 17 8000
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 19 8000
The values are the average of 3 replication experiments. NP denotes nanoparticles, NR denotes nanorods.
Fig. 6. TEM images of E. coli a) control, b) ZnO powders, c) ZnO nanorods and d) ZnO nanoparticles.
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Fig. 7. TEM images of B. atrophaeus a) control, b) ZnO powders, c) ZnO nanorods and d) ZnO nanoparticles.
conclude that surface defects do not play a significant role in the antibacterial activity of ZnO. The effects of surface functionalization were also investigated. Comparison is shown in Fig. 5. Fig. 5a shows the bacteria growth on a microscope slide (no ZnO), while in the presence of ZnO (Fig. 5b–d) no growth was observed. The presence of surfactant or Au nanoparticles did not affect the antibacterial properties. The tests were repeated 3–5 times, and consistent results were obtained. In order to study the antibacterial activity of ZnO nanorods in more detail, the rods were detached from the substrate in ultrasonic bath in ethanol, centrifuged, re-suspended in methanol, and dried. Then, suspensions with different concentrations were prepared to test antibacterial activity in liquid nutrient broth. For comparison, antibacterial activity of ZnO nanoparticles and ZnO powder was also tested. The results are summarized in Tables 1 and 2. The tables give the average results of three replication experiments. The nanoparticles exhibit the best antibacterial activity in liquid phase for both organisms studied, in agreement with previous reports which found that antibacterial activity depends on the particle size [8]
and particle shape [7], with increase in antibacterial activity observed for decreasing size [8]. The performance of the nanorods is comparable to that of nanoparticles in the case of E. coli, and slightly worse in the case of B. atrophaeus. One possible reason for this is the tendency of the nanorods to aggregate together (as observed under TEM, see Fig. 4), so that in some cases they may be less likely to be in close contact with the bacteria in liquid phase compared to powder and nanoparticles at the same concentration. The difference in activity against these two types of bacteria can be attributed to different organization of the cell wall. Gram-positive bacteria typically have one cytoplasmic membrane and thick wall composed of multilayers of peptidoglycan [18]. On the other hand, gram-negative bacteria have more complex cell wall structure, with a layer of peptidoglycan between outer membrane and cytoplasmic membrane [11,18]. Thus, the cell membrane of gram-positive bacteria can be damaged more easily. In both cases, antibacterial activity of ZnO can be attributed to the damage of cell membranes, which leads to leakage of cell contents and cell death. However, exact cause of the membrane damage requires further study. To further clarify the mechanism of antibacterial activity and the difference in performance of various ZnO morphologies against E. coli and B. atrophaeus, detailed TEM studies have been performed. Figs. 6 and 7 show the TEM images of E. coli and B. atrophaeus, respectively, after treatment with different ZnO morphologies. ZnO concentration below the minimum bactericidal concentration has been used in all cases in order to be able to observe both intact and damaged cells. Control sample (no ZnO) is also shown. For all ZnO morphologies, similar types of cell damage are observed. In the case of E. coli, we can observe some intact cells and cells with obviously damaged membrane in addition to partially leaked out contents, similar to previously reported results [11]. Also, some of the cells exhibit shrinkage of cytoplasmatic material inside the cell wall (Fig. 6b and c). Although internalization of the ZnO rod or ZnO nanoparticles can be observed in some cases (see Fig. 8a), this is not a general occurrence. A large number of cells have been examined, and internalization has been observed in very few cases. Thus, mechanical damage of the cell membrane cannot be considered as the main mechanism of antibacterial activity of ZnO. In the case of B. atrophaeus, different ZnO morphologies cause similar types of cell damage. Cells with partially leaked out contents can be observed, and there are very
Fig. 8. a) TEM image showing internalization of a ZnO nanorod; TEM images of the cell damage induced by ZnO nanoparticles, b) E. coli and c) B. atrophaeus.
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Table 3 The concentration of H2O2 released from different ZnO structures ZnO type/concentration
1 mM
10 mM
100 mM
Nanoparticles Nanorods Powder
0 µg/L 0 µg/L 0 µg/L
0.123 µg/L 0.031 µg/L 0.023 µg/L
19.4 µg/L 0.365 µg/L 0.188 µg/L
few intact cells. Fig. 8b and c shows TEM images of a single cell to more clearly observe the cell damage in E. coli and B. atrophaeus cells exposed to ZnO nanoparticles. For B. atrophaeus cells, we can typically observe multiple breaches of the cell walls, while this is not the case for E. coli. The exact mechanism responsible for the observed cell wall damage in gram-positive bacteria requires further study. Since ZnO has a bandgap close to that of anatase TiO2 and can also be used in photocatalytic applications [11], it is possible that photocatalytic reactions cause the cell membrane damage similar to TiO2 nanoparticles [18], although membrane damage by released H2O2 [8,12] is also possible. It should be noted that antibacterial activity of ZnO and H2O2 release in our work has been studied without UV light, as in the Refs. [8,12]. Thus, we have tested the release of H2O2 from ZnO material with different morphologies, and the results are summarized in Table 3. The amount of released H2O2 is related to the particle size of ZnO, and it increased linearly with increasing concentration of ZnO. Due to the smallest particle size, geometric area is the highest for nanoparticles, followed by the nanorods, and then the powder, and the materials are not porous as observed from TEM images. Nevertheless, BET measurements have also been performed, and the highest surface area was obtained for nanoparticles, 40.6 m2/g. The powder and nanorods had comparable BET surface areas, 8.8 m2/g and 5.8 m2/g, respectively. In the case of nanorods, smaller area has been obtained than expected from the nanorod sizes, which is most likely due to the pronounced aggregation problems upon heating necessary for out-gassing to perform BET measurements. Nanorods were removed from the substrates, and collected after sonication and centrifugation, and then dried resulting in loose, fine powder. However, after BET measurements, nanorod powder was coarse and flaky. TEM imaging of the nanorods and powder has been repeated after BET measurements, and while sonication in ethanol
Fig. 9. TEM images after BET measurements of a) ZnO nanorods and b) ZnO powder.
Fig. 10. TEM images of bacteria treated with H2O2 a) E. coli (low magnification), b) E. coli (high magnification), c) B. atrophaeus (low magnification) and d) B. atrophaeus (high magnification).
resulted in separation of powder particles, nanorods were still clumped together, as observed in Fig. 9. Thus, the differences observed in surface area measurements and antibacterial activity of nanorods in the solution are likely due to their tendency to aggregate. However, this tendency does not affect antibacterial activity on the substrate, since nanorod position is fixed and aggregation is not possible. However, TEM investigation of the effects of H2O2 (30 mg/ mL) on the E. coli and B. atrophaeus reveals that no damage of B. atrophaeus cells is observed at concentrations causing damage to E. coli, as shown in Fig. 10. This is different from the behavior observed with all ZnO morphologies, indicating existence of a different mechanism of antibacterial activity. While the release of H2O2 from ZnO may contribute to the cell damage of E. coli, cell damage of B. atrophaeus is caused by a different mechanism since no effect of H2O2 is observed in this case. Thus, there is a correlation between ZnO particle size and the production of H2O2 and antibacterial activity, but there is no
Fig. 11. Optical microscope photos of E. coli on a microscope slide with ZnO nanorods after repeating the incubation of the bacteria on the same slide a) two times and b) three times.
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correlation between the antibacterial activity and H2O2 (especially for B. atrophaeus). Another possible mechanism is that the release of Zn2+ ions is responsible for the observed antibacterial activity. It is well known that ZnO can be unstable in the solution, and that under illumination H2O2 is produced and Zn2+ ions concentration is increased as a result of ZnO decomposition [21]. It should be noted that experiments on H2O2 production in Ref. [21] have been done under different conditions, i.e. presence of halogen anions in the solution and UV illumination. However, ZnO is in general unstable in acidic solution even without illumination, and the release of Zn2+ ions is a possibility which merits further investigation. We have also explored the implications of the coating stability and durability, by repeating the tests of bacterial growth on the same substrate. Washing the substrates in ethanol, exposure to UV light, and cleaning with UV light and ozone were investigated. In the case of ethanol washing in between applying the bacteria (same procedure as described previously), obvious bacteria growth was observed after third repeated test. Similar results were obtained after exposure to UV light (5 min, 1200 mJ/cm2), and further improvement in growth inhibition in subsequent trials was observed after exposure to UV light and ozone, but some variation from one trial to another is observed. An example of bacteria growth on second and third trial is shown in Fig. 11. The fact that repeated incubation results in eventual loss of antibacterial activity is likely due to the fact that it is necessary for nanorods to be in contact with the cells to observe antibacterial activity. Thus, no antibacterial activity will be observed once surface contamination, dead bacteria residue etc. has covered the nanorods. It is expected that this can be improved by using longer nanorods, or improved method of removing organic residue from the surface. 4. Conclusion We have demonstrated antibacterial activity of ZnO nanorod array fabricated by a hydrothermal method. The nanorod coating exhibited good inhibition of growth of bacteria on a variety of substrates, and antibacterial activity was not affected by surface modifications such as silane surfactant or gold nanoparticle attachment. The study of antibacterial activity in the liquid phase revealed that cell death occurred due to cell membrane damage. Consequently, ZnO is more effective for gram-positive than gram-negative bacteria because they have simpler cell membrane structure. While the release of H2O2 from ZnO is proportional to ZnO concentration and surface area and it may contribute to the cell damage of E. coli, it is not the main mechanism of antibacterial activity of ZnO. Further study is in progress on exact mechanism of membrane damage and long term stability of the coatings.
Acknowledgements This work is partly supported by the University Development Fund grant and Outstanding Young Researcher Award of the University of Hong Kong, Faculty Development Fund grant of the Science Faculty, the University of Hong Kong, 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, and Prof. K. Y. Chan from the University of Hong Kong and the Department of Chemistry, Hong Kong Baptist University for BET measurements. References [1] F. Furno, K.S. Morley, B. Wong, B.L. Sharp, P.L. Arnold, S.M. Howdle, R. Bayston, P.D. Brown, P.D. Winship, H.J. Reid, J. Antimicrob. Chemother. 54 (2004) 1019. [2] J. Sawai, T. Yoshikawa, J. Appl. Microbiol. 96 (2004) 803. [3] L. Huang, D.Q. Li, Y.J. Lin, M. Wei, D.G. Evans, X. Duan, J. Inorg. Biochem. 99 (2005) 986. [4] W.A. Daoud, J.H. Xin, Y.H. Zhang, Surf. Sci. 599 (2005) 69. [5] O.B. Koper, J.S. Klabunde, G.L. Marchin, K.J. Klabunde, P. Stoimenov, L. Bohra, Curr. Microbiol. 44 (2002) 49. [6] J. Sawai, J. Microbiol. Methods 54 (2003) 177. [7] T. Xu, C.S. Xie, Prog. Org. Coat. 46 (2003) 297. [8] O. Yamamoto, Int. J. Inorg. Mater. 3 (2001) 642. [9] N. Ciofi, L. Torsi, N. Ditaranto, L. Sabatini, P.G. Zambonin, G. Tantillo, L. Ghibelli, M. D'Alessio, T. Bleve-Zacheo, E. Traversa, Appl. Phys. Lett. 85 (2004) 2417. [10] N. Ciofi, L. Torsi, N. Ditaranto, G. Tantillo, L. Ghibelli, L. Sabatini, T. Bleve-Zacheo, M. D'Alessio, P.G. Zambonin, E. Traversa, Chem. Mater. 17 (2005) 5255. [11] R. Brayner, R. Ferrari-Iliou, N. Brivois, S. Djediat, M.F. Benedetti, F. Fiévet, Nano Lett. 6 (2006) 866. [12] O. Yamamoto, M. Komatsu, J. Sawai, Z.E. Nakagawa, J. Mater. Sci., Mater. Med. 15 (2004) 847. [13] P.J.A. Borm, W. Kreyling, J. Nanosci. Nanotechnol. 4 (2004) 521. [14] L.E. Greene, M. Law, D.H. Tan, M. Montano, J. Goldberger, G. Somorjai, P. Yang, Nano Lett. 5 (2005) 1231. [15] M. Law, L.E. Greene, J.C. Johnson, R. Saykally, P. Yang, Nature Mater. 4 (2005) 455. [16] L.E. Greene, M. Law, J. Goldberger, F. Kim, J.C. Johnson, Y. Zhang, R.J. Saykally, P. Yang, Angew. Chem., Int. Ed. Engl. 42 (2003) 3031. [17] D. Li, Y.H. Leung, A.B. Djurišić, Z.T. Liu, M.H. Xie, S.L. Shi, S.J. Xu, W.K. Chan, Appl. Phys. Lett. 85 (2004) 1601. [18] G. Fu, P.S. Vary, C.T. Lin, J. Phys. Chem., B 109 (2005) 8889. [19] P.K. Stoimenov, R.L. Klinger, G.L. Marchin, K.J. Klabunde, Langmuir 18 (2002) 6679. [20] H. Gu, P.L. Ho, E. Tong, L. Wang, B. Xu, Nano Lett. 3 (2003) 1261. [21] J. Doménech, A. Prieto, J. Phys. Chem. 90 (1986) 1123.
Thin Solid Films 516 (2008) 6167 – 6174 www.elsevier.com/locate/tsf
Antibacterial activity of ZnO nanorods prepared by a hydrothermal method K.H. Tam a , A.B. Djurišić a,⁎, C.M.N. Chan c , Y.Y. Xi a , C.W. Tse b , Y.H. Leung b , W.K. Chan b , F.C.C. Leung c , D.W.T. Au d a
Department of Physics, The University of Hong Kong, Pokfulam Road, Hong Kong Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong c Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong Department of Biology and Chemistry, City University of Hong Kong, Kowloon Tong, Hong Kong b
d
Received 9 February 2007; received in revised form 9 October 2007; accepted 16 November 2007 Available online 24 November 2007
Abstract We investigated antibacterial activity of ZnO nanorods prepared by a hydrothermal method against a gram-negative bacterium Escherichia coli and a gram-positive bacterium Bacillus atrophaeus. Antibacterial activity of ZnO nanorod coatings was studied on solid substrates covered with nutrient agar, as well as in liquid nutrient broth for different concentrations of ZnO nanorods, nanoparticles, and powder. ZnO exhibited antibacterial activity against both E. coli and B. atrophaeus, but it was considerably more effective in the latter case (at 15 mM vs. 5 mM concentration, respectively, showing zero viable cell count). For both organisms, damage of the cell membranes was found, and the effect was more pronounced for B. atrophaeus. Chemiluminescence analysis has been used to detect the release of hydrogen peroxide from ZnO structures, and the effect of H2O2 on the E. coli and B. atrophaeus was studied. Since significant differences were observed in the effect of ZnO nanostructures and H2O2 on B. atrophaeus, it can be concluded that there are other mechanisms contributing to the antibacterial activity of ZnO nanostructures. © 2007 Elsevier B.V. All rights reserved. Keywords: II–VI semiconductors; Nanomaterials; Antimicrobial activity
1. Introduction Microbial contamination is a serious issue in health care and food industry, so that development of antimicrobial agents and surface coatings has been attracting increasing attention in recent years. Due to the spread of antibiotic resistant infections, interest in alternative antimicrobial agents, such as inorganic materials, has been rising [1–12]. Antimicrobial properties have been demonstrated for metallic nanoparticles [1,9,10] and metal oxide powders and nanoparticles [2–7,11]. The inorganic materials can be used in different forms, such as powders [2,3,5,6,8,11,12], coated on cellulose fibers [4], or as a part of organic/inorganic nanocomposite coating [1,9,10]. Antimicrobial coatings are of great interest for protection of surfaces, since survival of microorganisms on surfaces in the ⁎ Corresponding author. E-mail address: [email protected] (A.B. Djurišić). 0040-6090/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.tsf.2007.11.081
environment can result in the spread of the diseases. In addition, coatings are expected to have better safety and stability, while nanoscale powders may pose a hazard upon inhalation [13]. It has been recognized that toxicity of nanoparticles is generally larger than in the case of larger particles of the same material, even for materials with relatively low toxicity [11]. Therefore, development of nanostructured coatings with antimicrobial properties is of considerable interest. In this work, we investigated antimicrobial properties of ZnO nanorods fabricated by a hydrothermal method. It has been demonstrated that ZnO powders and nanoparticles exhibit antimicrobial activity against Staphilococcus auerus [8] and Escherichia coli [8,11,12]. Antimicrobial properties of polymer coatings with ZnO tetrapods have also been studied [7], but there was no study of the ZnO nanorod arrays. ZnO nanorod arrays fabricated by a hydrothermal method have an advantage of low growth temperature (90 °C) [14–17] and variety of possible substrates, and the fabrication process is inexpensive and environmentally
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friendly. The low substrate temperature enables compatibility with temperature sensitive substrates such as plastics or textiles. With the use of a nanoparticle seeds, similar morphologies can be obtained on a variety of substrates (such as glass, quartz, Si etc.), including nonplanar substrates. Concerning the mechanism of the antibacterial activity of nanomaterials, several mechanisms have been proposed. In the case of metal nanoparticles, the antimicrobial properties are usually a result of release of metal ions over time [1,9,10]. Catalytic activity of TiO2 was proposed as a mechanism of its antibacterial activity [18], but other studies also reported bactericidal activity of TiO2 in the dark [4]. In the case of ZnO, release of H2O2 has been proposed as a mechanism responsible for antibacterial activity [8,12]. It has also been shown that metal oxide nanoparticles cause membrane damage [11,18,19], and in the case of ZnO nanoparticles and E. coli bacteria, cellular internalization of the nanoparticles has also been observed [11]. The membrane damage effect was larger for halogen doped MgO nanoparticles compared to the undoped ones [19], while in the case of TiO2 membrane damage was attributed to photocatalytic processes responsible for decomposition of the cell membrane [18]. Therefore, the mechanisms responsible for antibacterial activity of metal oxide nanostructures are still not fully clear. 2. Experimental details 2.1. Nanorod array preparation The nanorod arrays were prepared from equimolar aqueous solutions of zinc nitrate hydrate and hexamethylene tetramine [14–17]. Before the nanorod growth, the substrates were cleaned by sonication in acetone, ethanol, and deionized water, and then dried in an oven. Then, the seed layer was prepared either using nanoparticles [16,17] or zinc acetate solution [14], as described previously. For increasing the aspect ratio of the nanorods, polyethyleneimine can be added to the solution [15]. Zinc nitrate hydrate, hexamethylene tetramine, zinc acetate (for nanorod growth) and ZnO powder (for comparison of antibacterial properties, particle size 200–500 nm) were obtained from Aldrich, while ZnO nanoparticles (∼ 10 nm diameter) were obtained from Nanoscale Materials Inc. The morphology of the nanorods was examined by scanning electron microscopy (SEM) using a Leo 1530 field emission SEM, X-ray diffraction (XRD) using Bruker AXS SMART CCD diffractometer and transmission electron microscopy (TEM) using JEOL 2010F TEM and Philips Tecnai 20 TEM. BET surface area measurements were also performed. The samples were heated to 600 K under vacuum for out-gassing, and then the surface area was determined from nitrogen adsorption/desorption. Nanorods coated by silane surfactant or silane and Au nanoparticles were prepared by dipping the substrate into solution of 3-aminopropyltrimethoxysilane, followed by dipping in the suspension of gold nanoparticles. Gold nanoparticles (∼ 5 nm) were prepared by the reduction of HAuCl4 by sodium borohydride using polyacrylic acid as the surfactant. Due to the presence of carboxylic acid moieties on Au nanoparticle sur-
face, the particles can be attached on silane-treated ZnO surface. Excess silane was removed by rinsing in pure toluene. 2.2. Tests of antibacterial activity A gram-negative bacterium E. coli XL1-Blue and grampositive bacterium Bacillus atrophaeus ATCC 9372 were used for antibacterial activity testing. Culture broth (Luria–Bertani broth) and culture agar (Luria–Bertani agar) were used as culturing nutrient sources. E. coli was grown at 37 °C and B. atrophaeus was grown at 30 °C. The optical density of bacteria cell at 600 nm wavelength used for all testing was 0.3–0.4, in which cells are growing rapidly in the mid-log phase. The antibacterial activity of different ZnO nanorod coated plates (as described previously) was tested using a modified protocol from Ref. [18]. Twenty microlitres of bacteria was placed onto the coated plates and allowed to dry at 37 °C. Culture agar was then added on the top of the bacteria and kept at 37 °C (for E. coli) or 30 °C (for B. atrophaeus) overnight. Formation of colonies was determined by observation under an optical microscope. The minimum bactericidal concentrations (minimum concentration showing no viable cell growth) of the ZnO nanoparticles, ZnO nanorod and ZnO powder were determined. A serial dilution (1 to 100 mM) of ZnO nanoparticles, ZnO nanorod or ZnO powder with culture broth was prepared. One millilitre of bacteria cell in culture broth containing different concentrations of ZnO nanoparticles, ZnO nanorod or ZnO powder was incubated at appropriate temperature on a shaking platform at 250 rpm. After 24 h, 10 μl cell suspensions were collected from each sample tube, spread onto culture agar plate and incubated overnight (16 h). Colonies of bacteria present on culture agar plate indicated signs of bacterial growth. If no colonies are observed, the ZnO is considered as being bactericidal at that concentration. A control tube was prepared with no ZnO added and three replicate plates were prepared. The tests have been performed without the exposure to UV light. 2.3. Detection of hydrogen peroxide by chemiluminescence response Phosphate buffer solution (pH 7.4) was prepared by dissolving a tablet of phosphate buffered saline from Sigma in 250 mL deionized water. Different concentrations (1, 10, 100 mM) of ZnO nanorods, nanoparticles and powder in phosphate buffer solution were then prepared. Five microlitres of 5 mg/mL 2′,7′dichlorofluorescein diacetate in acetone, which initiated the chemiluminescence, was added into 1 mL of various concentration of ZnO in buffer solution. After 5 min incubation at room temperature in darkness, 200 μl of the solutions was pipetted into 96 well black microplate with two replicates. The chemiluminescence response was recorded by using fmax Fluorescene Microplate Reader (Molecular Devices Cooperation, Sunnyvale, California) with excitation at 485 nm and emission at 538 nm. The concentration of H2O2 was determined by comparison with chemiluminescence calibration curve obtained for known concentration of H2O2 in phosphate buffer solution. Experiments were carried out at room temperature.
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Fig. 1. SEM images of ZnO nanorods on a) Si, b) glass, c) concave glass surface, and d) convex glass surface.
2.4. Preparation of TEM samples The cell suspension was fixed in equal volume of 2.5% glutaraldehyde in cacodylate buffer (pH 7.4) overnight at 4 °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. One millimeter cube agar blocks were infiltrated and embedded in epoxy resin. Ultra-thin 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 subsequently examined under Philips Tecnai 12 TEM at 80 kV. 3. Results and discussion Fig. 1 shows the SEM images of the ZnO nanorods fabricated on different substrates with nanoparticle seeds, including
nonplanar glass substrates (both convex and concave surfaces). It can be observed that the nanorods can be grown on all substrates including nonplanar ones, although growth directions of the nanorods appear more random on nonplanar substrates. Nanorods are ∼ 250 nm long and have diameters in the range of 30–70 nm, and they are densely packed. In the case of a flexible substrate, such as Kapton foil, the nanorods have somewhat larger diameter and have more random growth directions compared to those fabricated on glass. The orientation of the nanorods can be improved when zinc acetate instead of ZnO nanoparticles is used to prepare the seed layer [14]. The difference between nanorods prepared with nanoparticle and zinc acetate derived seed layer is shown in Fig. 2. The use of acetate seed in combination with the addition of polyethyleneimine to the growth solution and repeating the growth process several times enables improved orientation of the nanorods perpendicular to the substrate, as well as increased the aspect ratio [15]. After repeating the growth process three times, nanorods with diameters in the range of 55–70 nm and
Fig. 2. SEM images of nanorods prepared with a) ZnO nanoparticle seed and b) acetate seed.
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Fig. 3. XRD spectrum of nanorods prepared with ZnO nanoparticle seed and acetate seed.
length of 800 nm can be fabricated. While the nanorod orientation is not critical for their antibacterial activity, longer nanorods would be preferable because they would be more difficult to be completely covered by surface contaminants and because
they have higher surface area. However, preparing the seed layer from zinc acetate solutions requires annealing at 350 °C, so that substrates requiring low processing temperature, such as polyester foils, in this case cannot be used. Fig. 3 shows the XRD spectrum of ZnO nanorods prepared with two different seed layers. It can be observed that ZnO nanorods have wurtzite structure in both cases, but the nanorods grown with acetate seed have better orientation since stronger signal from b0002N is obtained. In the case of nanoparticle seeds, strong peaks from other ZnO planes in addition to b0002N are obtained, indicating that ZnO nanorods in this case grow at different angles to the substrate, in agreement with SEM results. After the fabrication of nanorod arrays, they can be further modified by surface functionalization. By attaching different surfactants, polymer layers, or nanoparticles to the zinc oxide nanorod surface, their properties can be tailored to combine the properties of ZnO and material attached to the surface. Surface functionalization can be used to tailor various properties, from optical properties to antimicrobial activity. For example, it has been shown that vancomycin capped Au nanoparticles exhibit
Fig. 4. TEM images of a) ZnO nanorods, b) ZnO nanoparticles, c) ZnO powders, d) high resolution TEM image of a ZnO nanorod, e) ZnO nanorods with 3aminopropyltrimethoxysilane and f) ZnO nanorods with Au nanoparticles.
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Table 2 Minimum concentration of different forms of ZnO showing 100% antibacterial activity
Fig. 5. Optical microscope photos of E. coli on a) microscope slide, b) microscope slide with ZnO nanorods, c) microscope slide with ZnO nanorods with 3-aminopropyltrimethoxysilane and d) microscope slide with ZnO nanorods with Au nanoparticles.
enhanced antimicrobial activity [20]. It has also been shown that Au capped TiO2 nanoparticles exhibit somewhat improved antibacterial activity for E. coli [18]. ZnO nanorods, ZnO nanorods coated with 3-aminopropyltrimethoxysilane and ZnO nanorods coated with 3-aminopropyltrimethoxysilane and Au nanoparticles were prepared. The corresponding TEM images are shown in Fig. 4, together with TEM images of as-grown ZnO nanorods, nanoparticles, and powder. Small particle size of
ZnO
Escherichia coli (mM)
Bacillus atrophaeus (mM)
Nanorods Nanoparticles Powder
15 15 80
5 2 2
ZnO nanoparticles compared to ZnO powder can be clearly observed. The nanoparticles have the smallest particle size, followed by nanorods, and ZnO powder. The nanorods grow along [0001] direction (lattice spacing 5.2 Å) and have good crystallinity, as observed from high resolution TEM image. The coating of the nanorods with silane surfactant results in covering of the surface with a thin (2–3 nm) amorphous layer (Fig. 4e), in good agreement with the expected thickness of a monolayer of silane. After immersion into the suspension of Au nanoparticles, Au nanoparticles attached to nanorod surface can be clearly observed in Fig. 3f. We have investigated the antibacterial activity of ZnO nanorods on a substrate and in a liquid suspension. We found that the type of the seed layer (nanoparticles vs. zinc acetate derived seed) did not affect antibacterial activity as observed under optical microscope. No growth of E. coli bacteria was observed both in the case of nanoparticle seeds and seed obtained from zinc acetate solution. The substrate (Si, glass, or quartz) also did not affect biocidal activity. Annealing of the nanorods in different environments (air, forming gas) and at different temperatures (200 °C and 600 °C) affected the photoluminescence from ZnO but had no effect on antibacterial activity. Since no correlation has been observed between the antibacterial activity and photoluminescence features corresponding to surface defects (green emission) [17], we can
Table 1 Dependence of cell count on ZnO concentration for different forms of ZnO Concentration of ZnO (mM)
100 90 80 70 60 50 40 30 25 20 15 10 5 4 3 2 1 Control
Viable cell counts (106 cfu/mL) after incubation for 24 h Escherichia coli
Bacillus atrophaeus
NP
NR
Powder
NP
NR
Powder
0 0 0 0 0 0 0 0 0 0 0 8 233 433 467 567 627 1500
0 0 0 0 0 0 0 0 0 0 0 10 60 125 405 478 500 1500
0 0 0 0.18 3 7 10 11 33 148 270 599 733 800 987 1040 1070 1500
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 8000
0 0 0 0 0 0 0 0 0 0 0 0 0 0.68 7 10 17 8000
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 19 8000
The values are the average of 3 replication experiments. NP denotes nanoparticles, NR denotes nanorods.
Fig. 6. TEM images of E. coli a) control, b) ZnO powders, c) ZnO nanorods and d) ZnO nanoparticles.
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Fig. 7. TEM images of B. atrophaeus a) control, b) ZnO powders, c) ZnO nanorods and d) ZnO nanoparticles.
conclude that surface defects do not play a significant role in the antibacterial activity of ZnO. The effects of surface functionalization were also investigated. Comparison is shown in Fig. 5. Fig. 5a shows the bacteria growth on a microscope slide (no ZnO), while in the presence of ZnO (Fig. 5b–d) no growth was observed. The presence of surfactant or Au nanoparticles did not affect the antibacterial properties. The tests were repeated 3–5 times, and consistent results were obtained. In order to study the antibacterial activity of ZnO nanorods in more detail, the rods were detached from the substrate in ultrasonic bath in ethanol, centrifuged, re-suspended in methanol, and dried. Then, suspensions with different concentrations were prepared to test antibacterial activity in liquid nutrient broth. For comparison, antibacterial activity of ZnO nanoparticles and ZnO powder was also tested. The results are summarized in Tables 1 and 2. The tables give the average results of three replication experiments. The nanoparticles exhibit the best antibacterial activity in liquid phase for both organisms studied, in agreement with previous reports which found that antibacterial activity depends on the particle size [8]
and particle shape [7], with increase in antibacterial activity observed for decreasing size [8]. The performance of the nanorods is comparable to that of nanoparticles in the case of E. coli, and slightly worse in the case of B. atrophaeus. One possible reason for this is the tendency of the nanorods to aggregate together (as observed under TEM, see Fig. 4), so that in some cases they may be less likely to be in close contact with the bacteria in liquid phase compared to powder and nanoparticles at the same concentration. The difference in activity against these two types of bacteria can be attributed to different organization of the cell wall. Gram-positive bacteria typically have one cytoplasmic membrane and thick wall composed of multilayers of peptidoglycan [18]. On the other hand, gram-negative bacteria have more complex cell wall structure, with a layer of peptidoglycan between outer membrane and cytoplasmic membrane [11,18]. Thus, the cell membrane of gram-positive bacteria can be damaged more easily. In both cases, antibacterial activity of ZnO can be attributed to the damage of cell membranes, which leads to leakage of cell contents and cell death. However, exact cause of the membrane damage requires further study. To further clarify the mechanism of antibacterial activity and the difference in performance of various ZnO morphologies against E. coli and B. atrophaeus, detailed TEM studies have been performed. Figs. 6 and 7 show the TEM images of E. coli and B. atrophaeus, respectively, after treatment with different ZnO morphologies. ZnO concentration below the minimum bactericidal concentration has been used in all cases in order to be able to observe both intact and damaged cells. Control sample (no ZnO) is also shown. For all ZnO morphologies, similar types of cell damage are observed. In the case of E. coli, we can observe some intact cells and cells with obviously damaged membrane in addition to partially leaked out contents, similar to previously reported results [11]. Also, some of the cells exhibit shrinkage of cytoplasmatic material inside the cell wall (Fig. 6b and c). Although internalization of the ZnO rod or ZnO nanoparticles can be observed in some cases (see Fig. 8a), this is not a general occurrence. A large number of cells have been examined, and internalization has been observed in very few cases. Thus, mechanical damage of the cell membrane cannot be considered as the main mechanism of antibacterial activity of ZnO. In the case of B. atrophaeus, different ZnO morphologies cause similar types of cell damage. Cells with partially leaked out contents can be observed, and there are very
Fig. 8. a) TEM image showing internalization of a ZnO nanorod; TEM images of the cell damage induced by ZnO nanoparticles, b) E. coli and c) B. atrophaeus.
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Table 3 The concentration of H2O2 released from different ZnO structures ZnO type/concentration
1 mM
10 mM
100 mM
Nanoparticles Nanorods Powder
0 µg/L 0 µg/L 0 µg/L
0.123 µg/L 0.031 µg/L 0.023 µg/L
19.4 µg/L 0.365 µg/L 0.188 µg/L
few intact cells. Fig. 8b and c shows TEM images of a single cell to more clearly observe the cell damage in E. coli and B. atrophaeus cells exposed to ZnO nanoparticles. For B. atrophaeus cells, we can typically observe multiple breaches of the cell walls, while this is not the case for E. coli. The exact mechanism responsible for the observed cell wall damage in gram-positive bacteria requires further study. Since ZnO has a bandgap close to that of anatase TiO2 and can also be used in photocatalytic applications [11], it is possible that photocatalytic reactions cause the cell membrane damage similar to TiO2 nanoparticles [18], although membrane damage by released H2O2 [8,12] is also possible. It should be noted that antibacterial activity of ZnO and H2O2 release in our work has been studied without UV light, as in the Refs. [8,12]. Thus, we have tested the release of H2O2 from ZnO material with different morphologies, and the results are summarized in Table 3. The amount of released H2O2 is related to the particle size of ZnO, and it increased linearly with increasing concentration of ZnO. Due to the smallest particle size, geometric area is the highest for nanoparticles, followed by the nanorods, and then the powder, and the materials are not porous as observed from TEM images. Nevertheless, BET measurements have also been performed, and the highest surface area was obtained for nanoparticles, 40.6 m2/g. The powder and nanorods had comparable BET surface areas, 8.8 m2/g and 5.8 m2/g, respectively. In the case of nanorods, smaller area has been obtained than expected from the nanorod sizes, which is most likely due to the pronounced aggregation problems upon heating necessary for out-gassing to perform BET measurements. Nanorods were removed from the substrates, and collected after sonication and centrifugation, and then dried resulting in loose, fine powder. However, after BET measurements, nanorod powder was coarse and flaky. TEM imaging of the nanorods and powder has been repeated after BET measurements, and while sonication in ethanol
Fig. 9. TEM images after BET measurements of a) ZnO nanorods and b) ZnO powder.
Fig. 10. TEM images of bacteria treated with H2O2 a) E. coli (low magnification), b) E. coli (high magnification), c) B. atrophaeus (low magnification) and d) B. atrophaeus (high magnification).
resulted in separation of powder particles, nanorods were still clumped together, as observed in Fig. 9. Thus, the differences observed in surface area measurements and antibacterial activity of nanorods in the solution are likely due to their tendency to aggregate. However, this tendency does not affect antibacterial activity on the substrate, since nanorod position is fixed and aggregation is not possible. However, TEM investigation of the effects of H2O2 (30 mg/ mL) on the E. coli and B. atrophaeus reveals that no damage of B. atrophaeus cells is observed at concentrations causing damage to E. coli, as shown in Fig. 10. This is different from the behavior observed with all ZnO morphologies, indicating existence of a different mechanism of antibacterial activity. While the release of H2O2 from ZnO may contribute to the cell damage of E. coli, cell damage of B. atrophaeus is caused by a different mechanism since no effect of H2O2 is observed in this case. Thus, there is a correlation between ZnO particle size and the production of H2O2 and antibacterial activity, but there is no
Fig. 11. Optical microscope photos of E. coli on a microscope slide with ZnO nanorods after repeating the incubation of the bacteria on the same slide a) two times and b) three times.
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correlation between the antibacterial activity and H2O2 (especially for B. atrophaeus). Another possible mechanism is that the release of Zn2+ ions is responsible for the observed antibacterial activity. It is well known that ZnO can be unstable in the solution, and that under illumination H2O2 is produced and Zn2+ ions concentration is increased as a result of ZnO decomposition [21]. It should be noted that experiments on H2O2 production in Ref. [21] have been done under different conditions, i.e. presence of halogen anions in the solution and UV illumination. However, ZnO is in general unstable in acidic solution even without illumination, and the release of Zn2+ ions is a possibility which merits further investigation. We have also explored the implications of the coating stability and durability, by repeating the tests of bacterial growth on the same substrate. Washing the substrates in ethanol, exposure to UV light, and cleaning with UV light and ozone were investigated. In the case of ethanol washing in between applying the bacteria (same procedure as described previously), obvious bacteria growth was observed after third repeated test. Similar results were obtained after exposure to UV light (5 min, 1200 mJ/cm2), and further improvement in growth inhibition in subsequent trials was observed after exposure to UV light and ozone, but some variation from one trial to another is observed. An example of bacteria growth on second and third trial is shown in Fig. 11. The fact that repeated incubation results in eventual loss of antibacterial activity is likely due to the fact that it is necessary for nanorods to be in contact with the cells to observe antibacterial activity. Thus, no antibacterial activity will be observed once surface contamination, dead bacteria residue etc. has covered the nanorods. It is expected that this can be improved by using longer nanorods, or improved method of removing organic residue from the surface. 4. Conclusion We have demonstrated antibacterial activity of ZnO nanorod array fabricated by a hydrothermal method. The nanorod coating exhibited good inhibition of growth of bacteria on a variety of substrates, and antibacterial activity was not affected by surface modifications such as silane surfactant or gold nanoparticle attachment. The study of antibacterial activity in the liquid phase revealed that cell death occurred due to cell membrane damage. Consequently, ZnO is more effective for gram-positive than gram-negative bacteria because they have simpler cell membrane structure. While the release of H2O2 from ZnO is proportional to ZnO concentration and surface area and it may contribute to the cell damage of E. coli, it is not the main mechanism of antibacterial activity of ZnO. Further study is in progress on exact mechanism of membrane damage and long term stability of the coatings.
Acknowledgements This work is partly supported by the University Development Fund grant and Outstanding Young Researcher Award of the University of Hong Kong, Faculty Development Fund grant of the Science Faculty, the University of Hong Kong, 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, and Prof. K. Y. Chan from the University of Hong Kong and the Department of Chemistry, Hong Kong Baptist University for BET measurements. References [1] F. Furno, K.S. Morley, B. Wong, B.L. Sharp, P.L. Arnold, S.M. Howdle, R. Bayston, P.D. Brown, P.D. Winship, H.J. Reid, J. Antimicrob. Chemother. 54 (2004) 1019. [2] J. Sawai, T. Yoshikawa, J. Appl. Microbiol. 96 (2004) 803. [3] L. Huang, D.Q. Li, Y.J. Lin, M. Wei, D.G. Evans, X. Duan, J. Inorg. Biochem. 99 (2005) 986. [4] W.A. Daoud, J.H. Xin, Y.H. Zhang, Surf. Sci. 599 (2005) 69. [5] O.B. Koper, J.S. Klabunde, G.L. Marchin, K.J. Klabunde, P. Stoimenov, L. Bohra, Curr. Microbiol. 44 (2002) 49. [6] J. Sawai, J. Microbiol. Methods 54 (2003) 177. [7] T. Xu, C.S. Xie, Prog. Org. Coat. 46 (2003) 297. [8] O. Yamamoto, Int. J. Inorg. Mater. 3 (2001) 642. [9] N. Ciofi, L. Torsi, N. Ditaranto, L. Sabatini, P.G. Zambonin, G. Tantillo, L. Ghibelli, M. D'Alessio, T. Bleve-Zacheo, E. Traversa, Appl. Phys. Lett. 85 (2004) 2417. [10] N. Ciofi, L. Torsi, N. Ditaranto, G. Tantillo, L. Ghibelli, L. Sabatini, T. Bleve-Zacheo, M. D'Alessio, P.G. Zambonin, E. Traversa, Chem. Mater. 17 (2005) 5255. [11] R. Brayner, R. Ferrari-Iliou, N. Brivois, S. Djediat, M.F. Benedetti, F. Fiévet, Nano Lett. 6 (2006) 866. [12] O. Yamamoto, M. Komatsu, J. Sawai, Z.E. Nakagawa, J. Mater. Sci., Mater. Med. 15 (2004) 847. [13] P.J.A. Borm, W. Kreyling, J. Nanosci. Nanotechnol. 4 (2004) 521. [14] L.E. Greene, M. Law, D.H. Tan, M. Montano, J. Goldberger, G. Somorjai, P. Yang, Nano Lett. 5 (2005) 1231. [15] M. Law, L.E. Greene, J.C. Johnson, R. Saykally, P. Yang, Nature Mater. 4 (2005) 455. [16] L.E. Greene, M. Law, J. Goldberger, F. Kim, J.C. Johnson, Y. Zhang, R.J. Saykally, P. Yang, Angew. Chem., Int. Ed. Engl. 42 (2003) 3031. [17] D. Li, Y.H. Leung, A.B. Djurišić, Z.T. Liu, M.H. Xie, S.L. Shi, S.J. Xu, W.K. Chan, Appl. Phys. Lett. 85 (2004) 1601. [18] G. Fu, P.S. Vary, C.T. Lin, J. Phys. Chem., B 109 (2005) 8889. [19] P.K. Stoimenov, R.L. Klinger, G.L. Marchin, K.J. Klabunde, Langmuir 18 (2002) 6679. [20] H. Gu, P.L. Ho, E. Tong, L. Wang, B. Xu, Nano Lett. 3 (2003) 1261. [21] J. Doménech, A. Prieto, J. Phys. Chem. 90 (1986) 1123.