Colloids and Surfaces A: Physicochem. Eng.

Aspects 374 (2011) 18

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Colloids and Surfaces A: Physicochemical and Engineering Aspects

journal homepage: www.elsevier.com/locate/colsurfa

Synthesis and characterization of zinc/iron oxide composite nanoparticles and their antibacterial properties
Tamar Gordon a , Benny Perlstein a , Or Houbara b , Israel Felner c , Ehud Banin b , Shlomo Margel a,
a b c

The Institute of Nanotechnology and Advanced Materials, Department of Chemistry, Bar Ilan University, 52900 Ramat Gan, Israel The Mina and Everard Goodman Faculty of Life Sciences, The Institute of Nanotechnology and Advanced Materials, Bar Ilan University, 52900 Ramat Gan, Israel Racah Institute of Physics, The Hebrew University, 91904 Jerusalem, Israel

a r t i c l e

i n f o

a b s t r a c t
Inorganic metal oxides may serve as effective disinfectants, due to their relatively non-toxic prole, chemical stability and efcient antibacterial activity. Among metal oxide nanoparticles, zinc oxide demonstrates signicant bacterial growth inhibition on a broad spectrum of bacteria, mainly by catalysis of reactive oxygen species (ROS) formation from water and oxygen. Aqueous suspensions of ZnO nanoparticles (ZnO nanouids) are the preferred formulation for using the antibacterial agent in liquid phases and for the incorporation of the nanoparticles in different commercial products. However, ZnO nanoparticles in aqueous media tend to aggregate into large occulates, due to their hydrophobic nature, and thus do not interact with microorganisms effectively. In this study, zinc oxide was combined with iron oxide to produce magnetic composite nanoparticles with improved colloidal aqueous stability, together with adequate antibacterial activity. For this purpose, the Zn/Fe oxide composite nanoparticles were synthesized by basic hydrolysis of Fe2+ and Zn2+ ions in aqueous continuous phase containing gelatin. The obtained composite nanoparticles were composed of iron oxide, zinc oxide and zinc ferrite phases. The effect of the weight ratio [Zn]/[Fe] of the composite nanoparticles on their properties (composition, size, magnetic behavior and colloidal stability) was elucidated. The antibacterial activity of these nanoparticles was tested against Staphylococcus aureus and Escherichia coli and was found to be dependent on the weight ratio [Zn]/[Fe], i.e., the higher the ratio, the higher the antibacterial activity. In addition, the activity against Staphylococcus aureus was signicantly higher than that observed against Escherichia coli. 2010 Elsevier B.V. All rights reserved.

Article history: Received 26 August 2010 Accepted 12 October 2010 Available online 29 October 2010 Keywords: Zinc oxide nanoparticles Iron oxide nanoparticles Zinc ferrite nanoparticles Antibacterial activity Magnetism

1. Introduction Antibacterial agents are of great importance in several industries, e.g., water disinfection, textiles, packaging, construction, medicine and food [1,2]. The organic compounds traditionally used for disinfection pose several disadvantages, including toxicity to the human body and sensitivity to high temperatures and pressures that are present in many industrial processes [13]. For these reasons, the interest in inorganic disinfectants such as metal oxides is increasing [1,35]. These inorganic compounds present strong antibacterial activity at low concentrations [6]. They are also much more stable in extreme conditions [3,4], considered as non-toxic, and some of them even contain mineral elements essential to the human body [79].

Corresponding author. Tel.: +972 3 5318861; fax: +972 3 6355208. E-mail address: shlomo.margel@mail.biu.ac.il (S. Margel). 0927-7757/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2010.10.015

Among metal oxide powders, ZnO demonstrates signicant growth inhibition of a broad spectrum of bacteria [10,11]. The suggested mechanism for the antibacterial activity of ZnO is based mainly on catalysis of formation of reactive oxygen species (ROS) from water and oxygen [1,3,5,12,13], that disrupt the integrity of the bacterial membrane, although additional mechanisms have also been suggested [6,10,1419]. Since the catalysis of radical formation occurs on the particle surface [5,20], particles with larger surface area demonstrate stronger antibacterial activity. Therefore, as the size of the ZnO particles decreases their antibacterial activity increases [1,10,12,21]. Aqueous suspensions of ZnO nanoparticles (ZnO nanouids) are the preferred formulation for using the antibacterial agent in liquid phases and for the incorporation of these nanoparticles in various commercial products such as building materials or water desalination systems [3,5,10]. The dispersion of metal oxide nanoparticles in physiological solutions is also important for biological in vitro and in vivo studies [22]. However, ZnO nanoparticles in aqueous media agglomerate into occulates ranging from several hundred nanometers to several microns and thus do not interact with

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microorganisms effectively [12,22,23]. Several research groups have applied different methods, e.g., ultrasonication, milling and use of stabilizing agents [3,16,22], in order to avoid nanoparticle aggregation, but this appears to have been only partially successful. By contrast, ferrouid (suspensions of magnetic iron oxide nanoparticles such as Fe3 O4 ) have good colloidal stability, and may be kept as an aqueous suspension without agglomeration for long periods of time. However, they are not known to have signicant antibacterial properties, in spite of the recent nding that Fe3 O4 magnetic nanoparticles possess catalytic activity toward the reduction of H2 O2 [24]. The iron oxide nanoparticles act as Fentons reagent (Fe2+ /Fe3+ in solutions) that reacts with hydrogen peroxide to produce hydroxyls and peroxide radicals. One may suggest that this radical formation acts synergistically with the ZnO nanoparticles, to enhance their antibacterial effect. In our laboratory, we have recently developed a new method for the preparation of stable non-toxic iron oxide magnetic nanoparticles of a narrow size distribution dispersed in an aqueous continuous phase [2527]. These nanoparticles are prepared by nucleation and then growth of thin magnetic iron oxide layers on gelatin/iron oxide nuclei. Combining the ZnO nanoparticles with magnetic iron oxide nanoparticles may improve the colloidal stability of the ZnO nanouid, and provide easier handling of the nanoparticles for concentrating and cleaning the nanouids from excess reagents by a magnetic column [2527]. In this study, several zinc/iron oxide composite nanoparticles were synthesized by basic hydrolysis of Fe2+ and Zn2+ ions of different weight ratios on gelatin/Zn/Fe nuclei. The stability of the different Zn/Fe oxide nanouids and their antibacterial activity were also investigated.

2.3. Nanoparticles characterization Quantitative analysis of Zn and Fe was performed by analyzing solutions of the nanoparticles in 6 N HCl, using inductively coupled plasma (ICP) spectrometer Ultima-2 (Jobin Yvon Horiba). ZnO was identied by X-ray diffraction (XRD) spectroscopy (Xray diffractometer, model D8 Advance, Bruker AXS, with Cu K radiation). Mssbauer studies (MS) of the iron oxide and the various Zn/Fe oxide composite nanoparticles were performed at room temperature (RT) using a conventional constant acceleration drive and 50 mCi 57 Co:Rh sources. The experimental magnetic spectra were analyzed by a least square t procedure, where a magnetic hyperne elds (Heff ) distribution was used. The 57 Fe isomer shifts (I.S.) are relative to -Fe, measured at RT. The dry diameter and size distribution of the various nanoparticles were measured by transmission electron microscopes (TEM) (Technai T12 BIO TWIN operating at 120 kV and JEOL-JEM2100 LaB6), using image analysis software AnalySIS Auto (Soft Imaging System GmbH, Germany). The hydrodynamic size and size distribution of the nanoparticles dispersed in aqueous continuous phase were measured by a submicron particle analyzer (N4MD, Coulter Electronics Ltd., Hialeah, FL, USA). Magnetic measurements were performed at room temperature, using a commercial (Quantum Design) superconducting quantum interference device (SQUID) magnetometer. 2.4. Antimicrobial studies The antimicrobial activity of the various nanoparticles was tested using two common bacterial pathogens: Escherichia coli (Gram negative) and Staphylococcus aureus (Gram positive). Briey, 105 cells/ml in LuriaBertani broth (LB) were shaken (250 rpm) in light with the various nanoparticles mixtures (0.3%) for 24 h at 37 C. The number of viable bacteria was determined by plating serial dilutions on LB agar plates and determining the number of colony forming units (CFU) [28]. 3. Results and discussion Iron oxide nanoparticles of 17.3 4.6 nm diameter were synthesized by nucleation, followed by controlled growth of iron oxide thin lms onto gelatin/iron oxide nuclei, as shown in Fig. 1. The nucleation step is based on the complexation of Fe2+ ions with chelating sites of the gelatin (probably primary amines and/or carboxylates), followed by partial oxidation with sodium nitrate (up to approximately 50%) of the chelated Fe2+ to Fe3+ , so that the water-soluble gelatin contains both Fe2+ and Fe3+ chelated ions. Gelatin/iron oxide nuclei were then formed by adding NaOH aqueous solution up to pH 9.5. The growth of the iron oxide lms onto the gelatin/iron oxide nuclei was accomplished by repeating the nucleation step four more times. Zn/Fe oxide composite nanoparticles were prepared similarly, substituting the Fe2+ ions by a mixture of Fe2+ and Zn2+ of different weight ratios. In this study ve Zn/Fe oxide composite nanoparticles of different [Zn]/[Fe] weight ratios (1:9, 3:7, 1:1, 8:2 and 9:1) were prepared. The [Zn]/[Fe] weight ratios of the produced composite nanoparticles were similar to the [Zn]/[Fe] ratios initially added. ZnO nanoparticles were also prepared similarly to the iron oxide nanoparticles, substituting the Fe2+ ions by Zn2+ ions and eliminating the NaNO3 oxidation step. Fig. 2 shows a picture of a similar concentration (0.4%) of the different nanoparticles dispersed in an aqueous continuous phase. The iron oxide nanoparticles and the Zn/Fe oxide composite nanopar-

2. Experimental 2.1. Materials Gelatin (from porcine skin), ferric chloride tetrahydrate, zinc chloride and sodium nitrate were all purchased from Sigma Aldrich (Rehovot, Israel). Water was puried by passing deionized water through an Elgastat Spectrum reverse osmosis system (Elga Ltd., High Wycombe, UK).

2.2. Preparation of the Fe3 O4 , the ZnO and the Zn/Fe oxide composite nanoparticles Magnetic iron oxide (Fe3 O4 ) nanoparticles were prepared as described previously [25,26]. Briey, 240 l FeCl2 4H2 O solution (10 mmol/5 ml 0.01 N HCl) were added to 80 ml aqueous solution containing 240 mg porcine gelatin, followed by 86 l NaNO3 solution (6 mmol/5 ml H2 O). After a reaction time of 10 min, the pH was raised to 9.5 by adding NaOH aqueous solution (1 N). This procedure was repeated four more times. Excess reagents were removed by extensive dialysis against water, which also neutralized the pH of the nanoparticle dispersions. Zn/Fe oxide composite nanoparticles were prepared in a similar manner, substituting the Fe2+ ions for a mixture of Fe2+ and Zn2+ of different weight ratios. The mixtures containing weight ratio [Zn]/[Fe] of 1:9, 3:7, 1:1, 8:2 and 9:1 were prepared by mixing different volumes of FeCl2 4H2 O solution (10 mmol/5 ml 0.01 N HCl) with appropriate volumes of ZnCl2 solution (10 mmol/5 ml 0.01 N HCl). 240 l of each of the [Zn]/[Fe] mixtures were then added to the gelatin solution. ZnO nanoparticles were also prepared similarly, substituting the Fe2+ ions by Zn2+ ions and eliminating the NaNO3 oxidation step.

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Fig. 1. A scheme describing the formation of the magnetic iron oxide nanoparticles.

ticles containing a relatively high percentage of iron have a typical black color, which changes to lighter shades of brown and then yellow with increasing percentage of Zn. The ZnO nanoparticle aqueous suspension has a typical white color. No precipitation was detected in the suspensions for at least 2 weeks after the completion of the synthesis. All nanoparticles aqueous suspensions were translucent due to their small particle size, except for the ZnO suspension, which already contains agglomerated particles as will be illustrated later. Mssbauer spectra of the iron oxide nanoparticles and the [Zn]/[Fe] oxide composite nanoparticles of the weight ratio 1:9 demonstrate magnetism at RT and smeared broad magnetic sextets without any distinct peaks. The distance between the extreme lines indicates that the blocking temperature (TB ) of these samples is well

above RT. The t of both spectra indicates a maximum Heff value of 455 (4) kOe. The RT Heff values for Fe2 O3 and Fe2 O3 are around 515 and 495 kOe, respectively, whereas for Fe3 O4 the two Heff values are 491 and 453 kOe [29]. These results, therefore, may indicate that the smeared magnetic spectra of these two samples (iron oxide and [Zn]/[Fe] of weight ratio of 1:9) correspond to nano-size Fe3 O4 which are magnetically ordered at RT. The Zn phase (ZnFe2 O4 as discussed later) in the 1:9 [Zn]/[Fe] oxide nanoparticles could not be detected, and probably was hidden within the smeared spectrum. The RT Mssbauer spectra obtained for the remainder of the Zn/Fe oxide samples are very similar to each other and show one doublet only with an I.S. of 0.340.36 mm/s and a quadrupole splitting (1/2e2 Qq) in the range of 0.560.64 mm/s. Therefore, our interpretation is that the Mssbauer spectra of these Zn/Fe oxide

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Fig. 2. A picture showing the visibility of the iron oxide, the zinc oxide and the various Zn/Fe oxide composite nanoparticles dispersed in an aqueous continuous phase (0.4%), from iron oxide only (left) to zinc oxide only (right).

nanoparticles belong to nano-size Fe3 O4 particles with TB values lower than RT. Indeed, typical study of the magnetic behavior of the [Zn]/[Fe] sample of weight ratio 3:7 as a function of temperature at 14 Oe indicates clearly that its TB is around 100 K. It is quite obvious that for lower Fe concentrations TB should be at lower temperatures. Alternatively, Mssbauer measurements of nano-size ZnFe2 O4 particles performed recently at RT [30], also exhibit a pure quadrupole splitting of about 0.42 (2) mm/s. Thus, the determination made above is not conclusive, and these Zn/Fe oxide composite nanoparticles may contain ZnFe2 O4 phase and/or Fe3 O4 phase. XRD studies were performed in order to identify the ZnO phase of the various nanoparticles. Due to the similarity in the structure of Fe3 O4 and ZnFe2 O4 the two phases are indistinguishable by XRD. However, from the XRD spectra it can be concluded that the composite nanoparticles with the higher [Zn]/[Fe] weight ratios, i.e., 8:2 and 9:1, contain, in addition to the iron oxide or zinc ferrite phases, a hexagonal phase of wurtzite ZnO (PDF 01-089-1397). XRD studies of the ZnO nanoparticles exhibit that they are composed of the same wurtzite ZnO phase. ZnO phase was not identied in the XRD spectra of the composite samples of [Zn]/[Fe] of weight ratios 1:9, 3:7 and 1:1, either due to its low concentration (below the detection limit) and/or low crystallinity, or because of its absence. The fast Fourier transform (FFT) analysis of the high resolution TEM (HRTEM) images of the nanoparticles provides further support for the conclusions made regarding the identication of each phase. Typical analyses of the 3:7 [Zn]/[Fe] oxide composite nanoparticles and the ZnO nanoparticles are presented in Figs. 3 and 4, respectively. The micrograph shown in Fig. 3 illustrates a single crystalline of the 3:7 [Zn]/[Fe] oxide composite nanoparticle (center of the image). The nanoparticle displays lattice-fringe contrast. The distances measured between these lattice fringes are 0.298 nm, matching the interplanar spacing d0 2 2 of both the cubic FCC structure of Fe3 O4 and ZnFe2 O4 . The inset at the top right corner (A) represents the computed Fourier transform pattern of the portion of the image marked by the white square and looks like a diffraction pattern identical to one that would be recorded in a diffraction experiment. Analysis of this pattern reveals sets of reections that can readily be referred to the FCC structure of both Fe3 O4 (PDF 03 and ZnFe2 O4 (PDF 01-082-1042, a = 8.44 A). 065-3107, a = 8.39 A) Because these oxides are structurally similar they could not be distinguished in the high resolution image. But this image analyzing technique suggests that these 3:7 [Zn]/[Fe] oxide composite nanoparticles might be composed of one or both of the above mentioned oxides (Fe3 O4 or ZnFe2 O4 ). This observation is also in good agreement with the Mssbauer ndings that also show the possibility of these two oxides. The inset in the left top corner of Fig. 3(B) represents the ltered and magnied portion of the image outlined by the white square. The distances measured between lattice fringes are 0.29 nm, 0.26 nm and 0.26 nm matching respectively,

the interplanar spacings d2 2 0 , d3 1 1 and d1 3 1 in the cubic FCC structure of Fe3 O4 or that of ZnFe2 O4 . This is sufcient proof that the 3:7 [Zn]/[Fe] oxide composite nanoparticles contains Fe3 O4 and/or ZnFe2 O4 . Fig. 4 represents a HRTEM micrograph of the ZnO nanoparticles, displaying typical lattice-fringe contrast of closely-packed ZnO nanoparticles (5 nm diameter). Fourier analysis of the lattice images was readily available to provide identication of the small ZnO nanoparticles. The Fourier transform pattern taken from the image is illustrated in the inset in Fig. 4. Analysis of this pattern revealed ring pattern like reections that can be referred and indexed in terms of the hexagonal structure describing ZnO c = 5.21 A, PDF 01-089-1397). From (cell parameters: a = 3.25 A, these reections the following distances were calculated: 0.28 nm, 0.266 nm and 0.248 nm, corresponding to the interplanar spacing d1 0 1 0 , d0 0 0 2 and d1 0 1 1 planes, respectively. The dry diameter of the iron oxide, the zinc oxide and the Zn/Fe oxide composite nanoparticles, as measured by TEM, was found to be less than 20 nm. Table 1 shows that the dry diameter of the iron oxide nanoparticles is signicantly larger than that of the zinc oxide: 17.3 4.6 and 3.9 0.5, respectively. In addition, the particle

Fig. 3. High resolution electron micrograph (FFT processed) of the 3:7 [Zn]/[Fe] oxide composite nanoparticle showing a single crystalline nanoparticle (center of the image). The nanoparticle displays lattice-fringe contrast. The distances measured between these lattice fringes are 0.298 nm, matching the interplanar spacing d0 2 2 of both the cubic FCC structure of Fe3 O4 and ZnFe2 O4 . Inset (A) is the computed Fourier transform taken from the marked area (white square), showing sets of reections corresponding to the interplanar spacing, d2 2 0 , d3 1 1 and d1 3 1 planes. Insert (B) is the processed and magnied image of the marked area (white square) showing the lattice fringes of the d2 2 0 , d3 1 1 and d1 3 1 planes with the marked interplanar spacings.

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Table 1 Mean diameter of the iron oxide, the zinc oxide and the zinc/iron oxide composite nanoparticles. [Zn]/[Fe] (w/w) Particle diameter [nm] Dry Iron oxide 1:9 3:7 1:1 8:2 9:1 Zinc oxide 17.3 14.9 11.3 4.2 3.5 3.4 3.9 4.6 4.0 2.5 0.5 0.5 0.7 0.5 Hydrodynamic 100 111 125 138 138 161 296 36 34 59 62 37 48 47

Fig. 4. High resolution electron micrograph of ZnO nanoparticles showing closely packed agglomerate-like ZnO crystalline nanoparticles (5 nm). The inset is the computed Fourier transform pattern of the image showing sets of reections corresponding to the interplanar spacing d1 0 1 0 , d0 0 0 2 and d1 0 1 1 planes.

size decreases as the weight ratio of [Zn]/[Fe] increases, as demonstrated in Table 1 and in the TEM (Fig. 5) and the HRTEM images shown in Figs. 3 and 4. Table 1 also demonstrates that the hydrodynamic diameter of the different oxide nanoparticles, as measured by light scattering, is signicantly higher than that of the dry diameter, e.g., the hydrodynamic diameter of the iron oxide nanoparticles, the 3:7 [Zn]/[Fe] oxide composite nanoparticles and the zinc oxide nanoparticles are 100 36, 125 59 and 296 47 nm, respectively while that of the dry diameter are 17.3 4.6, 11.3 2.5 and 3.9 0.5 nm, respectively. The main reason for this difference is due to the fact that the hydrodynamic diameter measurements are performed in aqueous continuous phase and also include the water

Fig. 5. TEM images of the iron oxide nanoparticles (A), the 3:7 [Zn]/[Fe] composite nanoparticles (B), the 8:2 [Zn]/[Fe] composite nanoparticles (C) and the ZnO nanoparticles (D).

T. Gordon et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 374 (2011) 18

Fig. 6. Magnetization curves at room temperature of the iron oxide, ZnO and [Zn]/[Fe] oxide composite nanoparticles of weight ratio 3:7, 1:1 and 8:2: ferromagnetic hysteresis loop curves (A) paramagnetic and diamagnetic curves (B).

Fig. 7. Means and standard errors of CFU per ml of E. coli (A) and S. aureus (B) incubated for 24 h in LB media in the presence of the iron oxide, the zinc oxide and the various Zn/Fe oxide composites (0.3%). The control refers to bacteria incubated without nanoparticles. *No CFU count.

molecules adsorbed on the surface of these nanoparticles. Table 1 also exhibits that the ZnO nanoparticles possess the highest hydrodynamic diameter, whereas the iron oxide nanoparticles show the smallest hydrodynamic diameterabout 3 times smaller than the ZnO nanoparticles (100 36 nm vs. 296 47 nm, respectively), contrary to the dry diameter ratio of those nanoparticles (17.3 4.6 for the iron oxide and 3.9 0.5 nm for the ZnO nanoparticles). This relatively high hydrodynamic diameter of the ZnO nanoparticles may be due to partial agglomeration of these nanoparticles, due to their relatively hydrophobic nature. Indeed, Fig. 5 illustrates that the iron oxide nanoparticles and the Zn/Fe oxide composite nanoparticles are composed of single non-agglomerated particles, while the ZnO nanoparticles (Fig. 5D) are composed of closely packed agglomerated nanoparticles. Magnetization curves at room temperature of the iron oxide, the ZnO and the Zn/Fe oxide composite nanoparticles are illustrated in Fig. 6. Ferromagnetic behavior was found only for the iron oxide nanoparticles and the [Zn]/[Fe] oxide composite nanoparticles of weight ratios 1:9 and 3:7. Fig. 6A exhibits the typical hysteresis loop at room temperature of the iron oxide and the 3:7 [Zn]/[Fe] oxide nanoparticles. The ferromagnetism is attributed to the magnetite and to the zinc ferrite phases [31]. As expected, the magnetite nanoparticles show a higher magnetic saturation moment than the Zn/Fe oxide composite nanoparticles, due to the lower Fe quantity of the latter (the values are stated in units of emu/g nanoparticles). For the [Zn]/[Fe] oxide composite nanoparticles of 3:7 weight ratio, the temperature dependence of the magnetization curve measured at 14 Oe indicates that its TB is below room temperature (around 100 K). However, isothermal eld dependence of the magnetization at RT shows a ferromagnetic-like behavior, which may be related to a ferromagnetic residue at RT. Fig. 6B exhibits that the Zn/Fe oxide

composite nanoparticles with [Zn]/[Fe] weight ratio of 1:1 and 8:2 show paramagnetic behavior, whereas zinc/iron oxide composite nanoparticles with [Zn]/[Fe] weight ratio of 9:1 and ZnO nanoparticles demonstrate diamagnetic behavior [32]. The antimicrobial activity of the iron oxide, the zinc oxide and the various Zn/Fe oxide composite nanoparticles has been examined on two common bacterial pathogens: the Gram negative bacterium E. coli and the Gram positive bacterium S. aureus. The results are summarized in Fig. 7. The ZnO nanoparticles exhibit the highest bactericidal activity and completely eradicated both bacterial species. The Zn/Fe oxide composite nanoparticles show variable activity on the two species. Overall it is clear that the S. aureus is more sensitive to some of the zinc/iron oxide composite nanoparticles than E. coli. For example, a bactericidal affect against S. aureus is evident in all particles with a [Zn]/[Fe] weight ratio higher than 1:1 (i.e., [Zn]/[Fe] weight ratio 8:2, 9:1 and ZnO nanoparticles), while complete killing of E. coli was caused only by particles containing only ZnO. Furthermore, a bacteriostatic affect on S. aureus is evident in nanoparticles in which the [Zn]/[Fe] weight ratio was 1:1, whereas those particles had no affect on E. coli. These ndings are in good agreement with a previous report that also demonstrated that S. aureus is more sensitive to ZnO than E. coli [28]. One explanation for the higher resistance observed in E. coli compared to S. aureus is due to differences in the polarity of their cell membrane. Sonohara et al. [33] have reported that the S. aureus membrane has a smaller negative charge than E. coli. This would allow a higher level of penetration of negatively charged free radicals such as superoxide radical anions and peroxide ions, causing damage and cell death to S. aureus at lower concentrations than those required to damage E. coli [21,28,33].

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The ability of ZnO to inhibit bacterial growth by generation of radical oxygen species is well documented [1,3,5,12,13]. ZnO is a semiconductor with a wide band gap. As with other semiconductors, radiation of ZnO with higher photon energy than its band gap causes movement of electrons from the valence band (vb) to the conduction band (cb) of the particle. The result of this process is the formation of a positive area that can be described as a hole (h+) in the valence band and a free electron in the conduction band. On the surface of the ZnO particles, these holes react with hydroxyl groups and absorb water to create a hydroxyl radical. In the presence of oxygen, the lone electron in the conduction band creates a superoxide ion, which can also become a hydroxyl radical, and so forth. Derivatives of this active oxygen damage the bacterial cell [5]. It could be hypothesized that zinc ferrite, also a semiconductor, would act in a similar manner, and would catalyze the formation of free radicals [34]. This, however, is not the case. The composite nanoparticles containing zinc ferrite (i.e., those with the [Zn]/[Fe] weight ratio of 1:9 and 3:7) did not show signicant antibacterial properties, proving that zinc ferrite has no signicant antibacterial activity. It can therefore be assumed, that the antibacterial activity of the zinc/iron oxide composite nanoparticles characterized in our study are mediated by the ZnO phase. In support of this hypothesis, iron oxide nanoparticles and composite nanoparticles with the [Zn]/[Fe] weight ratio of 1:9 and 3:7, that show either zero or very limited antimicrobial activity, did not contain any ZnO according to our XRD measurements. This may explain the lack of antibacterial activity in those particles. 4. Conclusions The present study demonstrates the unique synthesis of iron oxide, zinc oxide and Zn/Fe oxide composite nanoparticles of sizes ranging between 3 and 17 nm, by nucleation followed by controlled growth of Zn/Fe oxide lms onto gelatin/Zn/Fe oxide nuclei. The iron oxide nanoparticles are composed of magnetite. The composite nanoparticles with the [Zn]/[Fe] weight ratios of 1:9, 3:7 and 1:1 are composed of magnetite and zinc ferrite (ZnO phase was not detected). The composite nanoparticles with the [Zn]/[Fe] ratios of 8:2 and 9:1 are composed of zinc oxide and either zinc ferrite and/or magnetite phases. This study illustrates that the integration of magnetite and/or zinc ferrite phases into the Zn/Fe oxide composite nanoparticles stabilizes these nanoparticles against agglomeration: the lower the [Zn]/[Fe] ratio, the higher the stabilization effect, and thus the magnetization of the nanoparticles. This study also exhibits that the higher the [Zn]/[Fe] weight ratio the higher the antibacterial activity of the nanoparticles against E. coli and Staphylococcus aureus, while the latter was found to be more sensitive. Although the most efcient antibacterial activity against E. coli was demonstrated for ZnO particles, the Zn/Fe oxide composite nanoparticles with [Zn]/[Fe] weight ratio higher than 1:1 have shown good bacteriostatic activity on Staphylococcus aureus, and to a lesser extent against E. coli. Therefore, these composite nanoparticles, applied at higher concentrations, may also be used as antibacterial agents, when good colloidal stability is required (as ZnO nanoparticles agglomerate immediately after synthesis). In the future, the study of the antibacterial activity of the ZnO and the Zn/Fe oxide composite nanoparticles will be extended to include other bacterial strains, and the utilization of these nanoparticles for water purication from organic waste as well as microbial pollutants will also be investigated. Acknowledgements This study was partially supported by a Minerva Grant (Microscale & Nanoscale Particles and Films). The authors also thank to Dr. Judith Grinblat for her help in the HRTEM analysis.

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