Springer 2006
Journal of Nanoparticle Research (2007) 9:479–489 DOI 10.1007/s11051-006-9150-1
Technology and Applications
Investigation into the antibacterial behaviour of suspensions of ZnO nanoparticles (ZnO nanofluids) Lingling Zhang1, Yunhong Jiang1, Yulong Ding1,*, Malcolm Povey2 and David York3 1 Institute of Particle Science & Engineering, University of Leeds, Leeds, LS2 9JT, UK; 2Procter Department of Food Science, University of Leeds, Leeds, LS2 9JT, UK; 3Procter and Gamble Newcastle Technical Centre, Newcastle-upon-Tyne, NE12 9TS, UK; *Author for correspondence (Tel.: +44-113-343-2747; Fax: +44113-343-2405; E-mail:
[email protected]) Received 29 April 2006; accepted in revised form 31 July 2006
Key words: antibacterial activity, zinc oxide nanoparticles, nanofluids, mechanisms, E. coli, nanoengineering
Abstract The antibacterial behaviour of suspensions of zinc oxide nanoparticles (ZnO nanofluids) against E. Coli has been investigated. ZnO nanoparticles from two sources are used to formulate nanofluids. The effects of particle size, concentration and the use of dispersants on the antibacterial behaviour are examined. The results show that the ZnO nanofluids have bacteriostatic activity against E. coli. The antibacterial activity increases with increasing nanoparticle concentration and increases with decreasing particle size. Particle concentration is observed to be more important than particle size under the conditions of this work. The results also show that the use of two types of dispersants (Polyethylene Glycol (PEG) and Polyvinylpyrolidone (PVP)) does not affect much the antibacterial activity of ZnO nanofluids but enhances the stability of the suspensions. SEM analyses of the bacteria before and after treatment with ZnO nanofluids show that the presence of ZnO nanoparticles damages the membrane wall of the bacteria. Electrochemical measurements using a model DOPC monolayer suggest some direct interaction between ZnO nanoparticles and the bacteria membrane at high ZnO concentrations.
Introduction Antibacterial agents are of relevance to a number of industrial sectors including environmental, food, synthetic textiles, packaging, healthcare, medical care, as well as construction and decoration. They can be broadly classified into two types, organic and inorganic. Organic antibacterial materials are often less stable particularly at high temperatures and/or pressures compared to inorganic antibacterial agents (Sawai, 2003). This On visiting from the Tianjin University of Science & Technology, Tianjin, P.R. China.
presents a potential obstacle for the product formulation. As a consequence, inorganic materials such as metal and metal oxides have attracted lots of attention over the past decade due to their ability to withstand harsh process conditions (Wang et al. 1998; Hewitt et al. 2001; Fu et al., 2005; Makhluf et al., 2005). Of the inorganic materials, metal oxides such as TiO2, ZnO, MgO and CaO are of particular interest as they are not only stable under harsh process conditions but also generally regarded as safe materials to human beings and animals (Stoimenov et al., 2002; Fu et al., 2005). Some of the metal oxides e.g. MgO and CaO are essential minerals for human health
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(Yamamoto, 2001a, b, c; Roselli et al., 2003). Other metal oxides such as TiO2 and ZnO have been used extensively in the formulation of personal care products (Schumacher et al., 2004; Axtell et al., 2005; Li et al., 2006). This work is concerned about the antibacterial behaviour of ZnO particles for which there are only a small number of publications in the literature. These studies investigated the antibacterial activity of ZnO particles against Escherichia coli, Salmonella typhimurium, Bacillus subtilis, and Staphylococcus aureus etc. and the main conclusions of these studies can be summarised as: • ZnO particles are effective for inhibiting both Gram-positive and Gram-negative bacteria. They even have antibacterial activity against spores that are high-temperature and highpressure resistant (Sawai et al., 1995a, b, 1996a, b). • Smaller ZnO particles have a better antibacterial activity (Sawai et al., 1996a; Yamamoto, 2001a; Makhluf et al., 2005). • The antibacterial activity depends on the surface area and concentration, while the crystalline structure and particle shape have little effect. The higher the concentration and the larger the surface area, the better the antibacterial activity (Yamamoto et al., 1998). • High temperature treatment of ZnO particles has a significant effect on their antibacterial activity. Treatment at a higher temperature leads to a lower activity (Sawai et al., 1996a). • The mechanisms of the antibacterial activity of ZnO particles are not well understood although Sawai et al. (1996b, 1997, 1998) proposed that the generation of hydrogen peroxide be a main factor of the antibacterial activity, while Stoimenov et al. (2002) indicated that the binding of the particles on the bacteria surface due to the electrostatic forces could be a mechanism. These studies, however, have not addressed the effect of the formulation of ZnO suspensions on the antibacterial activity. This forms the first objective of this work. It is noted that the previous researchers only characterised ZnO powders in their dry form and paid little attention to the characterisation of suspensions. As the antibacterial tests are mostly done with suspensions, characterisation of the suspensions could provide more insight into the underlying antibacterial mecha-
nisms – the second objective of this work. From the practical application point of view, ZnO may be incorporated into liquid products in which case the use of a stabiliser may become necessary. In fact, Sawai et al. (1996b) have indicated that the stability of ZnO suspensions is important as the supernatant has no antibacterial activity. As a consequence, the third objective of this work is to investigate the effect of the use of polymeric dispersants on the antibacterial behaviour. Furthermore, as mentioned above, there is a disagreement in the dominant mechanism for the antibacterial behaviour of ZnO. The fourth objective of this work is therefore to provide more experimental evidence that could lead to a thorough understanding of the mechanisms.
Experimental Raw materials Dry zinc oxide nanoparticles from two suppliers, Nanophase Technologies and Nanostructured & Amorphous Materials (both of USA), were used in this work. The primary sizes of the nanoparticles given by the manufacturers were 24–71 nm for the Nanophase Technologies and 90–200 nm for the Nanostrutured & Amporphous Material products, respectively. Polyvinylpyrolidone (PVP) and Polyethylene Glycol 400 (PEG400) from Fluka were employed as dispersants. Luria-Bertani (LB) medium used for growing and maintaining bacterial cultures were purchased from Sigma-Aldrich (UK). Escherichia coli DH5a was provided by the Department of Biological Science of the University of Leeds. Potassium chloride and sodium chloride were purchased from Merck (USA). Formulation and characterisation of ZnO suspensions (ZnO nanofluids) ZnO nanoparticles from both manufacturers were used as received for producing suspensions. To gain more information of the shape, size distribution and morphology of the as-received nanoparticles, a scanning electron microscopy (SEM) was used. Figure 1(a) and (b) show the SEM images of the particles from the two suppliers, respectively. It can be seen that ZnO particles from both sources are in the form of agglomerates. Figure 1(b) also
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Figure 1. SEM images of ZnO nanoparticles as received from (a) Nanophase Technologies, (b) Nanostructured & Amorphous Materials.
shows the existence of particles that are much larger than 200 nm and some are even micron sized. To make ZnO nanofluids for the antibacterial tests, a preset amount of dry ZnO nanoparticles was mixed with distilled water in a glass beaker with the aid of a magnetic stirrer. Once particles were dispersed in water, the beaker was placed in an ultransoicator (Clifton, UK). The reason for the use of sonication was to break down the
agglomerates as seen in Figure 1(a) and (b). After 30 min of sonication, the so-called master ZnO nanofluid was produced, which had a concentration of 1 g/l. The master nanofluid was then diluted with distilled water to different concentrations for the antibacterial tests. As the ZnO nanoparticles from the Nanostructured & Amorphous Materials were very large as mentioned above, a Dyno-Mill (Willy A. Bachofen, Switzerland) was used to process the suspension for an hour at the
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room temperature. To enhance the stability of the suspension, two dispersants, PVP and PEG400, were used to produce suspensions of ZnO nanoparticles supplied by Nanophase Technologies and the amount of the dispersant was 10% of the amount of zinc oxide nanoparticles. Table 1 shows a list of nanofluids prepared for the antibacterial tests. Nanofluids prepared were autoclaved at 121C for 15 min and then characterised after cooling down to the room temperature by using an SEM for shape, size and morphology analyses and a Nano Sizer (Malvern Instruments) for size distribution and surface charge (Zeta potential) measurements.
Morphology of the bacteria SEM analyses were performed to investigate the morphological changes of the E. coli. For doing so, the E. coli culture was fixed with 2.5% glutaraldehyde for at least 2 h. The sample was then washed twice with de-ionised water each for 20 min. The washed sample was then put into 1% osmium tetroxide for 4 h, followed by washing twice with de-ionised water each 20 min. The washed sample was then put onto a stub for air drying, coating with 3 nm platinum, and SEM analyses using a LEO Gemini 1530 field emission SEM at a voltage of 5 kV. ZnO nanoparticle–bacteria wall interaction
Antibacterial tests Antibacterial tests were performed by measuring the growth curve of E. coli DH5a incubated in the LB broth medium in the presence of nanofluids containing different concentrations of ZnO nanoparticles. All experiments were performed in the dark. In a typical experiment for construction of the growth curves, 50 ll of the E. coli culture with an approximately concentration of 106–107 colony forming units per millilitre (CFU/ml) were inoculated in a 5 ml solution mixer, which contained autoclaved LB broth medium and autoclaved ZnO nanofluid. Cultures were grown at 37C under an agitation condition. The growth curve was determined by measuring the time evolution of the optical density (OD) of the sample contained in a 10 mm optical path length quartz cuvette. The measurements were done at 600 nm with a spectrophotometer (Thermo Electron Corporation, USA) at a frequency of once an hour. A blank LB broth medium cultured under the same conditions was used as a control.
Table 1. List of master nanofluids prepared for the antibacterial tests Sample Nanoparticle Processing code suppliers methods
Stabiliser Average size, nm
ZnO-1 ZnO-2 ZnO-3 ZnO-4 ZnO-5
– PEG400 PVP – –
Nanophase Nanophase Nanophase N&A, US N&A, US
Ultrasonication Ultrasonication Ultrasonication Ultrasonication Nano-grinding
249 293 314 2417 230
To investigate potential interactions between ZnO nanoparticles and the bacteria membrane wall, a three-electrode system was used, which consisted of a platinum counterelectrode, an Ag/AgCl (versus 3.5 mol/dm3 KCl) reference electrode and a hanging mercury drop electrode as the working electrode; see details of the technique in the literature (Leermakers & Nelson, 1990; Nelson et al., 2001; Neville et al., 2004). A Langmuir monolayer composed of dioleoyl-phophatidylcholine (DOPC) molecules was used to model the external layer of the bacterial membrane. The DOPC monolayer was deposited on the mercury drop electrode, which had an area of 0.0088 cm2 and submerged in the ZnO nanofluid with 0.1 mol/dm3 NaCl. The liquid phase was fully deaerated with special grade argon before each experiment and a blanket of argon was maintained above the liquid during the experiment to avoid any introduction of oxygen. Application of an electrical potential across the electrode system generated electrical current. The relation between the applied potential and the resultant current in the presence and absence of ZnO nanoparticles provided information of the interaction between the DOPC monolayer and the particles.
Results and discussion Characterization of ZnO nanofluids ZnO nanofluids prepared with the method described in the Experimental section have a pH value of
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7.2 ± 0.1 at which the zeta-potential is approximately +24 mV. It is also found that the isoelectric point (IEP) of ZnO is 9.4, consistent with the reported data in the literature (Sheng & Liu, 2004). SEM analyses of the ZnO nanofluids reveal little changes to the morphology of nanoparticles due to ultrasonication and the use of dispersants except for disintegration of some nanoparticle agglomerates. However, the use of Dyno-Mill to process suspensions made with ZnO particles from Nanostructured & Amorphous Materials changes the morphology of ZnO particles significantly. Figure 2 shows the results. It can be seen that large ZnO crystals as shown in Figure 1(b) have been milled down to below 100 nm. Figure 2 also indicates that the milled particles are also in the form of agglomerates. All nanofluids as shown in Table 1 are analysed by using the Malvern Nano-Sizer for particle size distribution and average size. Two peaks are generally observed for all samples, which is likely due to the presence of agglomerates as shown in Figures 1 and 2. The average sizes of the samples are shown in the last column of Table 1. It can be seen that the use of ultrasonication does not seem to be effective in breaking down nanoparticle agglomerates and the use of dispersants does not enhance the size reduction. However, it is observed that the
use of dispersant enhances considerably the stability of the suspensions thus brings in benefit. Note that particle sizes measured by the Malvern Nano-Sizer are hydrodynamic diameter based on the Stokes–Einstein equation, which is expected to be larger than the actual particle size. A comparison between the sizes of ZnO-4 and ZnO-5 in Table 1 suggests that the use of DynoMill reduces the particle size significantly, which agrees with the SEM analysis; see Figures 1(b) and 2. Antibacterial test results Antibacterial tests were carried out with nanofluids formulated under different conditions as shown in Table 1. These master nanofluids are diluted to different concentrations for the tests. The results are presented in this section. Effect of nanoparticle concentration Autoclaved Sample ZnO-1 (Table 1) was mixed with autoclaved LB medium to make nanofluids with ZnO concentrations of 0.1 and 0.25 g/l. Figure 3 shows the growth curves of the two nanofluids together with the negative control. As the value of the optical density (OD) at 600 nm represents the absorbance of the bacteria, an increase
Figure 2. An SEM image of ZnO nanoparticles after grinding with the Dyno-Mill for 1 h(Nanostructured & Amorphous Materials).
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OD at 600nm
OD at 600nm
2.5
Negative control 2417nm ZnO 0.1 g/l 230nm ZnO 0.1 g/l
4
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Time, hour Figure 3. Growth curves of E. coli in LB medium inoculated with 107 CFU of bacteria in the presence of two different concentrations of ZnO (ZnO-1).
in the number of bacteria implies more light being absorbed by the bacteria. As a consequence, Figure 3 shows clearly a bacteriostatic action of ZnO nanoparticles against E. coli. In particular, little bacteria growth is seen for 8 h with nanofluid containing 0.25 g/l ZnO made from the ZnO-1 Sample (Table 1). Similar behaviour has been observed by Sawai et al. (1995a), Sawai (2003) and Yamamoto (2001a) although a different method called conductance method was used by them. With such a method, the growth inhibitory effect is studied by measuring the so-called Detection Time (DT) at which the bacteria concentration reaches approximately 107 viable organisms per ml. Effect of the particle (agglomerate) size Samples ZnO-4 and ZnO-5 as shown in Table 1 were used to make nanofluids for investigating the effect of particle (agglomerate) size. The particle concentration used in these tests is 0.1 g/l. Figure 4 shows the results. It can be seen that smaller particles (agglomerates) have a much better bacteriostatic activity. Particles in Sample ZnO-4 have a very large average particle size (2417 nm) and there is little or no antibacterial activity with the growth curve almost overlapping the negative control curve. A comparison of the data with 0.1 g/l nanofluids shown in Figures 3 and 4 indicates that the antibacterial performance of ZnO particles from the two suppliers is similar (the average particle sizes in the two cases are similar). An inspection of Figures 3 and 4 also indicates that the effect of particle concentration seems to be
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Time, hour Figure 4. Growth curves of E. coli in LB medium inoculated with 107 CFU of bacteria in the presence of ZnO suspensions of different particle sizes.
more important than that of particle size under the conditions of this work. The influence of particle size of on the antibacterial activity of metal oxides has been reported in the literature; see for example Yamamoto (2001a) and Makhluf (2005). Yamamoto (2001a) examined the effect of primary particle size of ZnO over a range of 100 and 800 nm obtained by milling in a planetary ball mill. He used electrical conductivity as the parameter to evaluate the antibacterial activity and showed that the antibacterial activity increased with decreasing particle size. Makhluf et al. (2005) produced MgO nanoparticles with different particle sizes by the microwave method and tested their antibacterial behaviour. By adjusting the concentration of the reactant Mg(Ac)2, they were able to produce MgO particles with the primary particle size between 8 and 23 nm. Their antibacterial tests with 1 g/l MgO particles showed that the antibacterial activity increased with decreasing primary particle size. These observations seem to be in broad agreement with that of this work. It should be noted that the benchmark of this work is based on the actual agglomerate size rather than the primary particle size as used by Yamamoto (2001a) and Makhluf et al. (2005). As mentioned in the Introduction section, the dominant mechanism of the antibacterial behaviour of ZnO particles is still unclear; there is insufficient information to judge which benchmark should be used. More discussion on the antibacterial mechanisms is presented later in this paper.
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Effect of the presence of a stabiliser ZnO suspensions in the absence of a stabiliser can have stability issue, which bears significance to the shelf-life. This could be overcome by using a dispersant, which, however, could have impact on the antibacterial behaviour. In this work, PEG400 and PVP are considered which are shown to be good stabilising agents for ZnO nanofluids. Their effects on the antibacterial behaviour are shown in Figure 5 for Samples ZnO-1, ZnO-2, and ZnO-3 (Table 1). These tests were done with a ZnO concentration of 0.1 g/l. It can be seen that the use of PEG has little effect on the antibacterial performance of ZnO, whereas the use of PVP gives a slightly negative effect. Given the experimental uncertainty and small size difference of particles between the PVP and PEG stabilised nanofluids, the effects of both PEG400 and PVP on the antibacterial activity are very small. The effect of stabilisers on the antibacterial behaviour has also been studied by Cho et al. (2005) with silver and platinum nanoparticle suspensions. In their study, poly-(N-vinyl-2-pyrrolidone) (PVP) and sodium dodecylsulfate (SDS) were used as the stabilisers, and the antibacterial tests were on S. aureus (KCTC 1928) and E. coli (KCTC 1041) with the cup diffusion method. Their results showed that both silver and platinum nanoparticles had strong antibacterial activity. However, the silver and platinum nanoparticles stabilised by the SDS did not show antibacterial activity. These observations disagree with that of this work as described above where little effect of the dispersants is observed. The exact reasons for 3.5
Negative control ZnO 0.1g/l ZnO + PEG 0.1g/l ZnO + PVP 0.1 g/l
OD at 600nm
3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 0
2
4
6
8
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Time, hour Figure 5. Growth curves of E. coli in LB medium inoculated with 107 CFU of bacteria in the presence of different dispersants.
the observed difference require further investigation; however, different physical and chemical properties of silver and platinum from that of ZnO are likely to a reason. Different physical and chemical properties of the antibacterial materials could imply different mechanisms of the antibacterial behaviour, and the presence of a dispersant may affect the mechanisms in different ways. SEM analysis of the E. coli To gain direct evidence of the antibacterial behaviour of ZnO nanoparticles, SEM analyses were performed on some E. coli samples before and after antibacterial tests. Figure 6(a) and (b) show respectively the bacteria images before and after treatment for 5 h with 0.2 g/l ZnO nanofluids made with Sample ZnO-5. One can clearly see that treatment of the bacteria with ZnO nanoparticles has led to considerable damage to some E. coli and the damage has caused the breakdown of the membrane of the bacteria. Interaction between ZnO nanoparticles and bacteria membrane wall SEM analyses as described above indicate damages to the bacterial membrane. One of the possible reasons for the damage could be direct interaction between ZnO nanoparticles and the external membrane surface. To investigate this, a monolayer of dioleyl phosphatidylcholine (DOPC) molecules was used to simulate the bacteria membrane wall. Figure 7 shows the results in the form of current as a function of the applied potential across the electrode system (called AC Voltammogram) under various conditions. Shown in Figure 7(a) is the current–voltage relation in the absence of ZnO nanoparticles. Two sharp pseudocapacitive peaks are observed, which correspond to two consecutive phase transitions (Leermakers et al., 1990). In the presence of ZnO nanoparticles, an extra peak forms in the AC Voltammograms; see Figure 7(b)–(d). This peak is mainly caused by the cations of Zn. The height of the Zn peak is seen to increase with increasing ZnO concentration. An inspection of the Voltammograms indicates little changes in the shape and size of the two peaks formed by the DOPC monolayer at ZnO concentrations lower than 0.4 g/l, suggesting little direct interactions between ZnO nanoparticles and the
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Figure 6. SEM images of E. coli: (a) before antibacterial tests; (b) after treatment with 0.2% ZnO nanofluids for 5 h.
DOPC lipids at these concentrations. At high concentrations, particularly 2 g/l ZnO, small interaction seems to exist; see Figure 8 which overlays the data shown in Figure 7(a) and (d). More work is therefore needed to confirm these observations. Discussion on the antibacterial mechanisms A number of mechanisms have been proposed to interpret the antibacterial behaviour of metal
oxides. Makhluf et al. (2005) investigated the antibacterial behaviour of MgO and attributed the behaviour to the following mechanisms: production of active oxygen species due to the presence of MgO, interaction between MgO particles and membrane cell wall, penetration of individual MgO particles into cell and reformation of MgO within the cell. Sawai et al. (1996b) studied the antibacterial behaviour of ZnO particles by using a chemiluminescence and oxygen electrode analysis. They reported that H2O2 was produced in ZnO
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Figure 7. Current–applied potential relationships across the electrode system in absence (a) and presence (b–d) of ZnO nanofluids with 0.1 mol/dm NaCl electrolytes.
slurry and the concentration of H2O2 produced was linearly proportional to the ZnO particle concentration of the slurry. H2O2 was also detected by Yamamoto et al. (2000) with a spectrophotofluorometer. Stoimenov et al. (2002), on the other hand, suggested that the electrostatic interactions between the bacteria surface and nanoparticles be a reason. The results obtained in this work clearly show that the presence of ZnO nanoparticles leads to damages to the membrane wall of E. Coli. Such damages may be partly due to direct interactions between ZnO nanoparticles and bacteria membrane surface as shown in Figure 8. ZnO nanoparticles used in this work are in the form of agglomerates with an average size greater than 200 nm. These large agglomerates are less likely to penetrate into the cell wall to damage the bacteria from the interior. This is supported by the observation of little effect due to the use of
dispersant (PVP and PEG), which are long-chain polymers and are likely to be adsorbed onto the ZnO particle surface. Adsorption of the long-chain polymer onto the ZnO particle surface implies a barrier between the bacteria cell wall and ZnO nanoparticles. The little effect due to the use of dispersant also seems to exclude the electrostatic interaction as a possible mechanism as proposed by Stoimenov et al. (2002). The small effect of the ZnO particle (agglomerate) size suggests that the penetration mechanism proposed by Makhluf et al. (2005) for MgO particles be less unlikely a major mechanism for the observed antibacterial behaviour. It seems that active oxygen species generated by ZnO particles could be a mechanism although there is no direct evidence from the results of this study. The presence of active oxygen species has been detected by Sawai et al. (1996b) and Yamamoto et al. (2000). However, it is less clear
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membrane damage may be partly due to the direct interaction between ZnO nanoparticles and bacteria membrane wall. The mechanisms of the antibacterial behaviour are discussed. The results suggest that both the direct nanoparticle-cell membrane wall interaction and generation of active oxygen species be mechanisms but there are a number of questions remained to be answered before a firm conclusion could be drawn.
Resultant Current, mA
0.04 0.03 0.02 0.01 0.00 -0.01 -0.02 No ZnO 2g/l ZnO
-0.03 -0.04 -1.2
-1.0
-0.8
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Applied Potential, V Figure 8. Comparison of Figures 7(a) (no ZnO) and 7(d) (2 g/l ZnO).
how the active oxygen species are produced and how to improve the active oxygen production if they were the main mechanism. It is known that ZnO can be photo-catalytic under the UV light, which could be a reason for the production of the active oxygen species. However, all the antibacterial tests of this work were done under the dark conditions. A further question is therefore how photoactive the ZnO nanoparticles are in suspensions under the dark conditions. Further work is underway aiming at answering these questions, which will be reported in the future.
Concluding remarks This paper is concerned about the use of suspensions of ZnO nanoparticles (ZnO nanofluid) as antibacterial agents. Various ZnO nanofluids are formulated, characterised and tested for their antibacterial behaviour. The results show that ZnO nanofluids have the bacteriostatic activity against E. coli. The antibacterial activity increases with increasing particle concentration and decreasing particle size. Particle concentration is observed to more important than particle size under the conditions of this work. The use of PEG and PVP dispersants shows little effect on the antibacterial activity but enhances the stability of the suspensions. SEM analysis of the bacteria before and after treatment with ZnO nanofluids shows that the presence of ZnO particles causes damages to the membrane wall of the bacteria. Electrochemical analyses indicate that the
Acknowledgements The work is financially supported by the Nanomanufacturing Institute of the University of Leeds and Procter and Gamble Company. The authors would like to extend their thanks to Dr Jianjun Fang of the Biological Science Department at the University of Leeds for providing the E. coli strain. Sincere thanks are due to Mr. Adrian Hick of Faculty of Biological Sciences for assisting in the SEM analysis, and Dr. Andrew Nelson and Mr. Zachary Coldrick of School of Chemistry for advices and performing experiments on the electrochemical analyses. References Axtell H.C, S.M. Hartley & R.A. Sallavanti, 2005. Multifunctional protective fiber and methods for use. United States Patent, US2005026778. Cho K.H., J.E. Park, T. Osaka & S.G. Park, 2005. The study of antimicrobial activity and preservative effects of nanosilver ingredient. Electrochim. Acta 51, 956–960. Fu G., P.S. Vary & C.T. Lin, 2005. Anatase TiO2 nanocomposites for antimicrobial coating. J. Phys. Chem. B 109, 8889–8898. Hewitt C.J., S.T. Bellara, A. Andreani, G. Nebe-von-Caron & C.M. Mcfarlane, 2001. An evaluation of the antibacterial action of ceramic powder slurries using multiparameter flow cytometry. Biotechnol. Lett. 23, 667–675. Leermakers F.A.M. & A. Nelson, 1990. Substrate-induced structural changes in electrode-adsorbed lipid layers. J. Electroanal. Chem. 278, 53–72. Li Y., P. Leung, L. Yao, Q.W. Song & E. Newton, 2006. Antimicrobial effect of surgical masks coated with nanoparticles. J. Hosp. Infect. 62, 58–63. Makhluf S., R. Dror, Y. Nitzan, Y. Abramovich, R. Jelinek & A. Gedanken, 2005. Microwave-assisted synthesis of nanocrystalline MgO and its use as Bacteriocide. Adv. Funct. Mater. 15, 1708–1715. Nelson A., N. Geddes & J. Tattersall, 2001. Interaction of channel-blocking bispyridinium compounds with supported phospholipid layers. Cell. Mol. Biol. Lett. 6, 319–326.
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