Antimicrobial Mechanism Based on H2O2 ... - ACS Publications

3 downloads 1660 Views 5MB Size Report
Apr 9, 2013 - China Academy of Space Technology, Beijing 100094, China. § ... Oxygen vacancy (VO) in the surface layer of ZnO ..... Supporting Information.
Article pubs.acs.org/Langmuir

Antimicrobial Mechanism Based on H2O2 Generation at Oxygen Vacancies in ZnO Crystals Xiaoling Xu,† Dan Chen,† Zhigang Yi,† Man Jiang,† Li Wang,‡ Zuowan Zhou,*,† Ximei Fan,† Yong Wang,† and David Hui§ †

Key Laboratory of Advanced Technologies of Materials (Ministry of Education), School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu, Sichuan 610031, China ‡ China Academy of Space Technology, Beijing 100094, China § Department of Mechanical Engineering, University of New Orleans, New Orleans, Louisiana 70148, United States S Supporting Information *

ABSTRACT: The production of H2O2 has been taken for a crucial reason for antimicrobial activity of ZnO without light irradiation. However, how the H2O2 generates in ZnO suspension is not clear. In the present work, the comparatively detections on three kinds of ZnO, tetrapod-like ZnO whiskers (t-ZnO), nanosized ZnO particles (n-ZnO), and microsized ZnO particles (m-ZnO), showed that the antimicrobial activity of ZnO was correlated with its production of H2O2. Oxygen vacancy (VO) in the surface layer of ZnO crystals determined by XPS indicated that it was quite probably involved in the production of H2O2. To validate the role of VO, the concentration of VO in t-ZnO was adjusted by heat-treatment under the atmospheres of H2, vacuum, and O2, respectively, and the H2O2 production and antimicrobial effect were detected. Consistently, the t-ZnO treated in H2, which possessed the most VO in its crystal, produced the most H2O2 and displayed the best antimicrobial activity. These results provide the basis for developing a more detailed mechanism for H2O2 generation catalyzed by ZnO and for taking greater advantage of this type of antimicrobial agent. size,18 orientation,19 and lattice constant20 have been found to affect the antimicrobial activity of ZnO. However, the detailed mechanism for the activity of ZnO is still under debate. Through detecting the production of reactive oxygen species (ROS) by electron spin resonance (ESR) technique, Applerot9 and Lipovsky21 et al. from Bar-Ilan University suggested that the antimicrobial activity of ZnO was due to the production of hydroxyl radicals (•OH). Yet, they did not clearly specify the light conditions for the measurements. Because ZnO is a kind of typical photocatalyst, it can produce •OH under relevant light excitation. So, it is difficult to know if the production of • OH originates from the light irradiation or not. It is not clear if the antimicrobial activity of ZnO with light irradiation may be due to the production of •OH to some extent. Yet, the mechanism for the antimicrobial activity of ZnO in the dark is still not that clear. Some studies suggested that the released zinc ions from ZnO were responsible for the nanotoxicity.22,23 Yet, there were still many studies that showed that the concentrations of released Zn2+ ions were not high enough to give rise to the corresponding activity of antimicrobial.9,18,24,25 Generation of H2O2 has been proposed to be one of the principal elements for displaying antimicrobial activity, and the

1. INTRODUCTION The emergence of infectious diseases and the continuous development of antibiotic resistance among a variety of pathogenic bacteria pose a serious threat to public health worldwide.1,2 It is of great importance to develop appropriate new strategies for controlling bacterial infections. Development of antimicrobial agents that are not liable to cause drugresistance is a proper method. Antimicrobial agents can be broadly classified into two types, organic compounds and inorganic materials. Organic antimicrobial agents are quickacting relatively to inorganic antimicrobial materials. However, the broad use of them will make more and more bacteria drugresistant.3 On the other hand, inorganic antimicrobial materials can achieve appropriate disinfection without forming harmful byproducts and are more stable than organic antimicrobial agents.4,5 As a consequence, the interest in inorganic antimicrobial materials such as metal and metal oxides has been rising in recent years.6−9 Of the inorganic antimicrobial materials, ZnO is a remarkable one due to the good thermal stability, marked antimicrobial activity, and low cost.9−12 Therefore, the fundamental study on antimicrobial activity of ZnO has become a hot topic in recent years.9,13,14 ZnO exhibits antimicrobial activity to both grampositive bacteria like Staphyloccocus aureus and gram-negative bacteria like Escherichia coli, and even has antimicrobial effect on spores like the Bacillus subtilis spores.15−17 The particle © 2013 American Chemical Society

Received: January 28, 2013 Revised: April 7, 2013 Published: April 9, 2013 5573

dx.doi.org/10.1021/la400378t | Langmuir 2013, 29, 5573−5580

Langmuir

Article

Zinc remaining in the supernatants was determined using atomic absorption spectrometry (AAS, SpectrAA 220FS, VARIAN). 2.4. Detection of •OH. ESR method with 5,5-dimethy-1pyrroline-N-oxide (DMPO) and fluorescence method with terephthalate were both employed to analyze the production of •OH in ZnO suspensions. ESR method for detecting •OH in ZnO suspensions in the dark or under light irradiation was performed by ESR spectrometer (EMX-8/2.7, Bruker) at room temperature with DMPO as the radical trap. Before being detected on the ESR spectrometer, aqueous suspension with 2 g/L of ZnO and 0.05 mol/L of DMPO was prepared in darkroom. Next, 25 μL of the mixed suspension was injected into a quartz capillary tube under weak red light. The ESR spectra were obtained after kepting the tube in a sample chamber under no illumination or under simulated solar light illumination. Data acquisition parameters were as follows: magnetic field, 351 mT; microwave power, 2 mW; modulation frequency, 100 kHz; modulation amplitude, 0.1 mT; microwave frequency, 9.86 GHz; sweep time, 41.943 s. Generation of •OH in terephthalate solution yields fluorescent 2hydroxy-terephthalate. The reaction product is stable for hours and can readily be assessed using standard fluorimeters. The production of • OH can be determined from the fluorescent intensity at 425 nm in the fluorescence spectra at 312 nm excitation. Reactions for detecting • OH by fluorescence method were carried out in beakers containing suspensions of ZnO with a concentration of 2 g/L, terephthalate with a concentration of 2 × 10−3 mol/L. Before the reactions, the suspensions were treated with ultrasonator for 10 min in the dark. During the reactions, the suspensions were kept in the dark or under the simulated solar irradiation with a 150 W xenon arc lamp (XBO 150W/CR OFR, OSRAM) as the light source. Magnetic stirrer was involved to ensure the homogenization of the suspensions. The reactions were stopped by removing the ZnO particles from the suspensions through centrifuging and filtering with Millipore membrane. The fluorescence spectra of the filtrates containing 2hydroxy-terephthalate were detected with a fluorescence spectrophotometer (F-7000, Hitachi, Japan). 2.5. Detection of H2O2. H2O2 generated from ZnO suspension was determined by UV−vis spectrophotometry with KI and starch.28 Reactions for detecting the generation of H2O2 in ZnO suspensions were conducted without any light irritation in beakers containing aqueous suspensions of the ZnO with a concentration of 5 g/L. Before reactions, the suspensions were treated with ultrasonator for 10 min in the dark. Magnetic stirrer was involved to ensure homogenization of the suspensions. The reactions were stopped by removing the ZnO particles from the suspensions through centrifuging (8000 rpm, 5 min) and filtering with Millipore membranes. Five milliliters of filtrate was added to a 10 mL volumetric flask, and then 0.5 mL of NaCl solution (200 g/L), 0.2 mL of HCl solution (3.6 vol %), 0.3 mL of KI solution (10 g/L), and 0.2 mL of soluble starch solution (10 g/L) were successively dropped into the volumetric flask, and finally the flask was brought up to volume with water. The absorbance at 580 nm of the final solution was detected by UV−vis spectrophotometer (UV-2550, Shimadzu, Japan). 2.6. Detection of VO in ZnO. The concentration of VO in the surface layer of ZnO was determined by the deviation from the stoichiometry ratio, which was obtained from the molar ratio of oxygen and zinc determined by X-ray photoelectron spectroscopy (XPS). The XPS spectra were detected on a Thermo Scientific ESCALAB 250 spectrometer with monochromatic 150 W Al Kα radiation. The base pressure was about 6.5 × 10−10 mbar, and the binding energies were referenced to the C1s line at 284.8 eV from alkyl or adventitious carbon. The fittings of XPS curves were analyzed with the software XPSPEAK. The detailed calculation process for the atomic percentages of lattice Zn and lattice O was given in the Supporting Information.29 ESR method was also employed to analyze the VO in ZnO samples. To compare the amount of VO in ZnO, an equivalent amount of each ZnO sample was put into the same position in the sample chamber of ESR spectrometer. The ESR spectra were recorded at 77 K. X-band data acquisition parameters were as follows: magnetic field, 336 mT;

H2O2 in ZnO suspension was detected by many methods such as chemiluminescence,26 fluorescence,20 and oxygen electrode method.13 However, the factors affecting the generation of H2O2 in ZnO suspension and the control of antimicrobial activity of ZnO have not been intensively studied, and the mechanism for the generation of H2O2 is unclear, although it is very important for developing more effective antimicrobial materials. In this Article, the effects that influence the antimicrobial activity of ZnO without light irradiation were studied using ZnO samples with different morphologies, including tetrapodlike ZnO whiskers (t-ZnO), nanosized ZnO particles (n-ZnO), and microsized ZnO particles (m-ZnO). It was preliminarily found that oxygen vacancies (VO) in surface layer of ZnO crystals have a distinct effect on the generation of H2O2 in ZnO suspension. Accordingly, a novel and effective approach to adjust the antimicrobial activity of ZnO by controlling the VO in it was proposed.

2. EXPERIMENTAL SECTION 2.1. Materials. The t-ZnO was prepared by the gas expanding method using metallic zinc as the raw material as our earlier report.27 The n-ZnO was provided by Meidilin (Gansu, China), and the m-ZnO was provided by Aushin (Hebei, China). The other reagents and materials were commercially purchased and used without further purification or treatment. The morphologies of the samples were characterized with field emission scanning electron microscopy (FESEM, Inspect F, FEI) or transmission electron microscopy (TEM, Tecnai G2 20, FEI). The specific surface area was measured at 77 K by TriStar 3000 Analyzer (Micromeritics) according to the Brunauer−Emmett−Teller (BET) method. 2.2. Antimicrobial Assay. The antimicrobial effects of the three kinds of ZnO powders were first determined by disk diffusion assay using E. coli (ATCC 25922) as a model bacterium. ZnO powders with the mass of 0.1 g were pressured under 20 MPa at 25 °C to prepare a disc-shaped piece with diameter of 10 mm. Mueller−Hinton (MH) agar (20 mL) was poured into a sterilized Petri dish, and solidified within 10 min. The freshly prepared E. coli bacterial suspension (0.1 mL) with the concentration of 105 colony forming units per mL (CFU/mL) was uniformly inoculated on the solidified agar gel. Each disc-shaped ZnO piece was placed on the MH agar plate, and then incubated at 37 °C for 24 h. The antimicrobial activities were compared by the diameters of the zone of inhibition around each ZnO piece.9 The experiments were repeated three times for each sample. The antimicrobial activities of the three kinds of ZnO were also compared by the reduction in viability for 4 h. Each 0.05 g of ZnO sample was added into 95 mL of sterilized 0.85% saline in an Erlenmeyer flask and dispersed by ultrasonication. Each 5 mL of freshly prepared E. coli bacterial suspension with the concentration of 105 CFU/mL was poured into the ZnO suspension and mixed well. After 1 mL of each sample was immediately taken out to measure the bacteria concentration at the beginning of the treatment, the suspension in the Erlenmeyer flask was placed into a shaker and incubated at 150 shakes per min and 37 °C for 4 h. Next, 1 mL of each incubated sample was taken out to measure the bacteria concentration at the end of 4 h of treatment. Each of the 1 mL samples was diluted 10-fold in saline and then transferred onto nutrient agar plates to count the viable bacteria after being incubated at 37 °C for 24 h. The same amount of each level of the diluted sample was transferred onto three parallel plates. 2.3. Detection of Zn2+ Released from ZnO. Each 0.05 g of ZnO sample was added into 100 mL of sterilized 0.85% saline in an Erlenmeyer flask and dispersed by ultrasonication. The Erlenmeyer flasks with ZnO suspensions were placed on a shaker at 150 shakes per min and 37 °C for 24 h. The samples were then centrifugated at 8500 rpm for 5 min and then filtered with a 0.22 μm Millipore membranes. 5574

dx.doi.org/10.1021/la400378t | Langmuir 2013, 29, 5573−5580

Langmuir

Article

Figure 1. Micromorphologies of the samples: (a) SEM image of t-ZnO, (b) TEM image of n-ZnO, and (c) FESEM image of m-ZnO.

Figure 2. Optical images of inhibition zone for (a) t-ZnO, (b) n-ZnO, and (c) m-ZnO against E. coli without light irradiation. microwave power, 0.998 mW; modulation frequency, 100 kHz; modulation amplitude, 0.1 mT; microwave frequency, 9.18 GHz; sweep time, 1 min. 2.7. Adjustment of VO in ZnO. To adjust the concentration of VO in ZnO samples, reduction or oxidation treatments were performed at 600 °C in the atmospheres of oxygen, vacuum, or H2.30 The sample was put into a quartz tube in a pipe furnace and annealed isothermally for 0.5 h after heating to 600 °C with the heating rate of 3 °C/min. Next, the ZnO powders were cooled to room temperature in the quartz tube in a furnace.

3. RESULTS AND DISCUSSION The micrographs of the ZnO samples used in this work are summarized in Figure 1. As it is shown in Figure 1a, each Table 1. Antimicrobial Activity of ZnO Samples against E. coli

Figure 3. Production of •OH in n-ZnO suspension measured by ESR method with DMPO at room temperature.

concentration of E. coli (CFU/L) samples

0h

4h

% reduction in viability

t-ZnO n-ZnO m-ZnO

2.25(±0.14) × 104 2.46(±0.11) × 104 2.37(±0.20) × 104

5.4(±1.1) × 103 8.5(±1.0) × 103 9.4(±1.2) × 103

76 65 60

crystalline body of the t-ZnO consists of a core part, from which four acicular parts spread out in spacial directions. The length of the acicular part is ca. 25 μm, and the basal diameter is ca. 3 μm. Morphology observation of the n-ZnO (Figure 1b) shows that most of them appear of hexagonal shape with the particle size of ca. 60 nm, and the m-ZnO (Figure 1c) has similar shape but larger particle size. The BET analysis results reveal that the specific surface areas of the t-ZnO, n-ZnO, and m-ZnO are 0.30, 12.43, and 3.74 m2/g, respectively. The antimicrobial effects of the samples against E. coli determined by disk diffusion method are shown in Figure 2. Each of the three kinds of ZnO shows a clear zone of inhibition around each disk, indicating obvious effect against E. coli. The inhibition zone diameters are measured as 2.31 ± 0.13, 1.91 ±

Figure 4. Production of •OH in n-ZnO suspension measured by fluorescence method with terephthalate; the inset shows the enlargement of the figure with the fluorescence intensity at 0−6.

5575

dx.doi.org/10.1021/la400378t | Langmuir 2013, 29, 5573−5580

Langmuir

Article

To verify the production of •OH in ZnO suspension in the dark or under illumination, fluorescence method, using 2hydroxy-terephthalate as the trap agent, was also employed to detect the concentration of •OH. Figure 4 shows the production of •OH determined by fluorescence method with 2-hydroxy-terephthalate. The intensity of the peak at 425 nm was related to the amount of •OH in ZnO suspension. Figure 4 indicated that the amount of •OH in the suspension with light irradiation increased with time, while few •OH was generated in the suspension in the dark, which coincided with the ESR results. It is almost certain that the antimicrobial activity of ZnO in the dark is not attributed to the production of •OH. Some research groups have reported that the antimicrobial activity of ZnO may be associated with the release of Zn2+ ions.23,31 Hereby, the concentration of Zn2+ ions dissolved from the three kinds of ZnO samples were determined by AAS. Table 2 shows the dissolved Zn2+ ions for 500 mg/L ZnO in aqueous media for 24 h. The amount of Zn2+ ions released from n-ZnO was the most among the three kinds of ZnO samples, while that from t-ZnO was the least. This is not consistent with the antimicrobial activity results. Moreover, the cytotoxicity testing results of various Zn2+ ion precursors, including Zn(CH3COO)2, ZnCl2, and ZnSO4, showed that the concentration of IC 50 value of these Zn2+ precursors approximately ranged from 10 to 20 mg/L.32 In the present work, the concentrations of Zn2+ ions released from ZnO samples were all less than 2.32 mg/L. Therefore, we believed that there was no significant direct relationship between released Zn2+ and the antimicrobial activity of ZnO. The H2O2 was proposed to be the main factor of manifesting antimicrobial activity for ZnO in aqueous suspension in the dark.23−26 Therefore, the production of H2O2 in suspension of the as-mentioned ZnO was detected without any light irradiation. Figure 5 showed the production of H2O2 in ZnO suspension determined by UV−vis spectrophotometry with KI and starch. Principally, the generated H2O2 can oxidize iodine ion to iodine, and when it is mixed with the colorless soluble starch, intensely colored starch−iodine complex forms, which has a maximum absorbance at 580 nm in the UV−vis absorption spectrum. So, in Figure 5a, the absorbance at 580 nm corresponds to the concentration of H2O2 in the filtrate of ZnO suspensions. On the basis of the standard curve, say relation between absorbance and H2O2 concentration, the

Table 2. Concentration of Zn2+ in Suspension of the Three Kinds of ZnO samples

concentration of Zn2+ in the aqueous suspensions (mg/L)

t-ZnO n-ZnO m-ZnO

1.34 ± 0.11 2.32 ± 0.09 1.93 ± 0.07

0.15, and 1.48 ± 0.13 cm for t-ZnO, n-ZnO, and m-ZnO, respectively, exhibiting obvious differences in antimicrobial effect. The antimicrobial effects of the samples against E. coli determined by viability obtained through counting the number of CFU are given in Table 1. All of the ZnO samples inactivate E. coli by more than 60% after 4 h of treatment. The t-ZnO displays the highest antimicrobial activity among the three kinds of ZnO samples, which is consistent with the result determined by the disk diffusion method. It was suggested that ZnO with a smaller particle size had higher antimicrobial activity.9 In our experimental observations, the n-ZnO with smaller particle size exhibits better antimicrobial effect than mZnO, which is consistent with the literature.9 However, it seems curious that the t-ZnO with much larger size displays higher antimicrobial effect than the other two types of ZnO samples. The difference may be associated with the other mechanism in ZnO. As a typical reactive oxygen radical, •OH had been suggested by Applerot9 and Lipovsky21 et al. to be responsible for the antimicrobial activity of ZnO. Because the light conditions during the measurement were not specified in their works, the production of •OH determined by the ESR method may be attributed to the photocatalysis of ZnO. In the present work, we focus to specialize on the antimicrobial mechanism of ZnO in the dark. Figure 3 shows the production of •OH in ZnO suspension determined by ESR in the dark comparatively with that under light. ZnO suspension exposed to light for 20 min shows the representative DMPO/•OH adduct signal in the presence of DMPO. In contrast, ZnO suspensions kept in the dark for 20 min exhibited nearly no ESR signal. These data demonstrated that there was nearly no •OH generated in ZnO suspension in the dark, and the production of •OH in ZnO suspension was mainly attributed to the light irradiation.

Figure 5. Production of H2O2 in the suspensions of ZnO samples: (a) production of H2O2 in ZnO suspensions in dark for 2 h, and (b) variation of the amount of H2O2 due to time in the suspensions of ZnO samples in dark. 5576

dx.doi.org/10.1021/la400378t | Langmuir 2013, 29, 5573−5580

Langmuir

Article

Figure 6. High-resolution XPS spectra of Zn and O in ZnO: (a) Zn 2p of t-ZnO, (b) O 1s of t-ZnO, (c) Zn 2p of n-ZnO, (d) O 1s of n-ZnO, (e) Zn 2p of m-ZnO, and (f) O 1s of m-ZnO.

Table 3. VO in the Surface Layer of the Three Kinds of ZnO Samples Determined by the Deviation from the Stoichiometry Ratio with XPS samples

percent of Zn (%)

percent of OL (%)

percent of OH (%)

percent of Oad (%)

atomic ratio of OL/Zn

VO (%)

t-ZnO n-ZnO m-ZnO

39.78 40.30 37.50

30.11 32.84 31.25

25.81 22.39 25.00

4.30 4.48 6.25

0.76 0.81 0.83

24 19 17

m-ZnO, which coincides with the results of antimicrobial effect (Figure 2). These confirm that the generation of H2O2 is a main factor for the antimicrobial activity of ZnO. It has been reported that the amount of released H2O2 is related to the particle size of ZnO, and smaller particle size leads to more generation of H2O2.26 Herein, the same as the situation for antimicrobial activity, the n-ZnO with smaller particle size produces more H2O2 than m-ZnO, which is consistent with the literature. Yet, the situation is exceptional for t-ZnO. There may be some other factors affecting the generation of H2O2 in the ZnO suspensions. Early on, researchers found that catalytic activity of metal oxides in heterogeneous catalysts depends on the density of active sites.33 Although solid-state defects have been proposed to be the

changes of H2O2 concentrations with time are summarized in Figure 5b. It can be seen that the amount of H2O2 in each of the suspensions of the three kinds of ZnO increases with time. Moreover, the t-ZnO produces more H2O2 than do n-ZnO and 5577

dx.doi.org/10.1021/la400378t | Langmuir 2013, 29, 5573−5580

Langmuir

Article

2p peaks are symmetric, indicating that there is only one valence state of zinc in ZnO samples. Because the peak positions of Zn 2p3/2 in the three samples are all about 1021 eV, the zinc in all of the samples is in the Zn2+ valence state. The O 1s peaks (Figure 6b, d, and f) are asymmetric, suggesting that more than one kind of oxygen species exist in the near surface region of the ZnO samples. Some of the literature fitted the O 1s spectra into three peaks,39,43 while some of the reports fitted the spectra into two peaks.44,45 According to the shape of the spectra we obtained, the XPS spectra of O 1s of three ZnO samples were fitted into three peaks. The peak at ca. 530 eV is closely associated with the lattice oxygen (OL) of ZnO, the peak at ca. 531.7 eV is attributed to the oxygen of surface hydroxyl (OH), while the peak at ca. 533 eV is due to the adsorbed oxygen (Oad).39,43 Calculating with the peak areas and atomic sensitivity factors, the atomic percentages of Zn, OL, OH, Oad and relative ratio of OL/Zn are obtained and summarized in Table 3. The relative ratios of lattice oxygen to zinc are less than 1.0, indicating that VO exists in the surface layers of the samples. The concentration of VO in t-ZnO is the largest among the three kinds of ZnO, subsequently followed by n-ZnO and m-ZnO. ESR method is generally used to characterize the native defects in metal oxides.46,47 Single charged VO with unpaired electron is paramagnetic, and consequently is observable by ESR.48,49 Figure 7 shows the ESR spectra measured at 77 K. Because the signals of the three kinds of ZnO are obtained from equal mass of ZnO samples, it can be concluded that the sample with higher intensity of the signal associates with more VO in it.44 Therefore, according to Figure 7, it is known that the amount of VO in t-ZnO is the most among the three kinds of ZnO, and that in m-ZnO is the least, which coincides with the results determined by XPS. It is necessary to have a theoretical investigation on VO in ZnO crystals. According to the chemical equilibrium principle of crystal growth kinetics,50 the concentration of VO in ZnO lattice depending on the Gibbs free energy was calculated. The detailed deduction was given in the Supporting Information. Figure 8 shows the variation of Gibbs free energy with VO concentration at 873 K, the whiskers’ growth temperature.27 The result shows that the minimum value of Gibbs free energy appears at the VO concentration of 25−30%, meaning the stable value of VO in ZnO under this condition. Because the single crystalline t-ZnO is prepared with a slow growth rate, which is very close to the equilibrium state, it is reasonable that the VO concentration in this kind of single crystal is closest to the equilibrium concentration. The order of VO concentration of the three ZnO samples is in conformity with that of the production of H2O2, suggesting that the surface VO plays an important role in governing the H2O2 generation in ZnO. On the basis of the above results, we proposed a novel and facile approach to adjust the antimicrobial activity by controlling the concentration of VO in ZnO. Because the VO in t-ZnO was the most and the antimicrobial activity of t-ZnO was the highest of the three kinds of ZnO, t-ZnO was selected for further study. Experimentally, t-ZnO was thermally treated under different atmospheres such as H2, vacuum, and O2. Detection on the nonstoichiometric ratio of Zn and O by XPS (Table 4) shows that the heat-treatment in H2 could increase the VO in t-ZnO from 24% to 29%, which may have resulted from the reducing effect of H2 by seizing some oxygen from the surface layer of ZnO. The amount of VO in t-ZnO treated in a vacuum increases from 24% to 27%, which may be attributed to

Figure 7. ESR spectra of VO in the three kinds of ZnO samples at 77 K.

Figure 8. Dependence of Gibbs free energy as a function of oxygen concentration at 873 K. For the theoretical proposal, see the Supporting Information.

Table 4. VO, Antimicrobial Activity, and H2O2 Production of t-ZnO Annealed in Different Atmospheres atmosphere

atomic ratio of OL/Zn

VO (%)

diameter of inhibition zone (cm)

concentration of H2O2 (μg/g)

H2 vacuum O2

0.71 0.73 0.87

29 27 13

2.0 ± 0.16 1.8 ± 0.18 1.3 ± 0.13

0.22 ± 0.028 0.20 ± 0.021 0.12 ± 0.024

active sites in heterogeneous catalysis,34 active centers have rarely been conclusively identified, and the rational design of catalysts is still out of reach. VO in ZnO has been indicated not only to play important roles in electrical and magnetic properties of ZnO,35−38 but also to be responsible for the catalytic activity of them.39−41 However, as far as we know, the effect of VO on the antimicrobial activity has not been studied to the present, although the particle size,18 orientation,19 and lattice constant20 have been found to affect the antimicrobial activity of ZnO. To investigate the effect of VO on antimicrobial activity, the VO in the surface layer of the three kinds of ZnO was determined by the deviation from stoichiometry ratio, which was obtained from the molar ratio of zinc and oxygen detected by XPS.42 The high-resolution XPS spectra of zinc and oxygen are shown in Figure 6. As shown in Figure 6a, c, and e, the Zn 5578

dx.doi.org/10.1021/la400378t | Langmuir 2013, 29, 5573−5580

Langmuir

Article

(2) Desselberger, U. Emerging and re-emerging infectious diseases. J. Infect. 2000, 40, 3−15. (3) Cohen, M. L. Epidemiology of drug resistance: Implications for a post-antimicrobial era. Science 1992, 257, 1050−1055. (4) Fang, M.; Chen, J. H.; Xu, X. L.; Yang, P. H.; Hildebrand, H. F. Antibacterial activities of inorganic agents on six bacteria associated with oral infections by two susceptibility tests. Int. J. Antimicrob. Agents 2006, 27, 513−517. (5) Li, Q. L.; Mahendra, S.; Lyon, D. Y.; Brunet, L.; Liga, M. V.; Li, D.; Alvarez, P. J. J. Antimicrobial nanomaterials for water disinfection and microbial control: Potential applications and implications. Water Res. 2008, 42, 4591−4602. (6) Cao, H. L.; Liu, X. Y.; Meng, F. H.; Chu, P. K. Biological actions of silver nanoparticles embedded in titanium controlled by microgalvanic effects. Biomaterials 2011, 32, 693−705. (7) Lv, M.; Su, S.; He, Y.; Huang, Q.; Hu, W. B.; Li, D.; Fan, C. H.; Lee, S. T. Long-term antimicrobial effect of silicon nanowires decorated with silver nanoparticles. Adv. Mater. 2010, 22, 5463−5467. (8) Ren, G. G.; Hu, D. W.; Cheng, E. W. C.; Vargas-Reus, M. A.; Reip, P.; Allaker, R. P. Characterisation of copper oxide nanoparticles for antimicrobial applications. Int. J. Antimicrob. Agents 2009, 33, 587− 590. (9) Applerot, G.; Lipovsky, A.; Dror, R.; Perkas, N.; Nitzan, Y.; Lubart, R.; Gedanken, A. Enhanced antibacterial activity of nanocrystalline ZnO due to increased ROS-mediated cell injury. Adv. Funct. Mater. 2009, 19, 842−852. (10) Sawai, J.; Kawada, E.; Kanou, F.; Igarashi, H.; Hashimoto, A.; Kokugan, T.; Shimizu, M. Detection of active oxygen generated from ceramic powders having antibacterial activity. J. Chem. Eng. Jpn. 1996, 29, 627−633. (11) Sawai, J. Quantitative evaluation of antibacterial activities of metallic oxide powders (ZnO, MgO and CaO) by conductimetric assay. J. Microbiol. Methods 2003, 54, 177−182. (12) Reddy, K. M.; Feris, K.; Bell, J.; Wingett, D. G.; Hanley, C.; Punnoose, A. Selective toxicity of zinc oxide nanoparticles to prokaryotic and eukaryotic systems. Appl. Phys. Lett. 2007, 90, 213902/1−3. (13) Sawai, J.; Shoji, S.; Igarashi, H.; Hashimoto, A.; Kokugan, T.; Shimizu, M.; Kojima, H. Hydrogen peroxide as an antibacterial factor in zinc oxide powder slurry. J. Ferment. Bioeng. 1998, 86, 521−522. (14) Xie, Y. P.; He, Y. P.; Irwin, P. L.; Jin, T.; Shi, X. M. Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni. Appl. Environ. Microb. 2011, 77, 2325−2331. (15) Akiyama, H.; Yamasaki, O.; Kanzaki, H.; Tada, J.; Arata, J. Effects of zinc oxide on the attachment of Staphylococcus aureus strains. J. Dermatol. Sci. 1998, 17, 67−74. (16) Jones, N.; Ray, B.; Ranjit, K. T.; Manna, A. C. Antibacterial activity of ZnO nanoparticle suspensions on a broad spectrum of microorganisms. FEMS Microbiol. Lett. 2008, 279, 71−76. (17) Sawai, J.; Igarashi, H.; Hashimoto, A.; Kokugan, T.; Shimizu, M. Effect of ceramic powder slurry on spores of Bacillus subtilis. J. Chem. Eng. Jpn. 1995, 28, 556−561. (18) Raghupathi, K. R.; Koodali, R. T.; Manna, A. C. Size-dependent bacterial growth Inhibition and mechanism of antibacterial activity of zinc oxide nanoparticles. Langmuir 2011, 27, 4020−4028. (19) Wang, X. L.; Yang, F.; Yang, W.; Yang, X. R. A study on the antibacterial activity of one-dimensional ZnO nanowire arrays: Effects of the orientation and plane surface. Chem. Commun. 2007, 43, 4419− 4421. (20) Yamamoto, O.; Komatsu, M.; Sawa, J.; Nakagawa, Z. E. Effect of lattice constant of zinc oxide on antibacterial characteristics. J. Mater. Sci.: Mater. Med. 2004, 15, 847−851. (21) Lipovsky, A.; Nitzan, Y.; Gedanken, A.; Lubart, R. Antifungal activity of ZnO nanoparticles-the role of ROS mediated cell injury. Nanotechnology 2011, 22, 105101/1−5. (22) Franklin, N. M.; Rogers, N. J.; Apte, S. C.; Batley, G. E.; Gadd, G. E.; Casey, P. S. Comparative toxicity of nanoparticulate ZnO, bulk ZnO, and ZnCl2 to a freshwater microalga (Pseudokirchneriella

the negative pressure. However, in O2 atmosphere, the amount of VO decreases from 24% to 13%, due to the potential incorporation of oxygen. The antimicrobial activities and the generation of H2O2 of tZnO annealed in different atmospheres were detected, and the results are summarized in Table 4. The t-ZnO annealed in H2 exhibits the largest zone of inhibition attributed to the highest concentration of VO and the most production of H2O2, and that annealed in vacuum shows a little smaller zone of inhibition attributed to less H2O2, while the t-ZnO annealed in O2 displays little antimicrobial activity to E. coli due to the little production of H2O2. These results indicate that the VO in crystal has a critical effect on the antimicrobial activity of tZnO, and it acted through influencing the production of H2O2. It was indicated that VO in the ZnO crystal represented the active sites in the hydrogenative conversion of CO to methanol.40 So, it is not difficult to understand the effect of VO on the production of active antimicrobial species. However, more details should be explored to understand the formation process of active species.

4. CONCLUSION The antimicrobial mechanism of ZnO without light irradiation was studied with three kinds of ZnO samples. The generation of H 2 O 2 was suggested to be contributed from the antimicrobial activity of ZnO in the dark. The particle size and specific surface area, which are generally regarded as a main factor affecting the production of H2O2 from ZnO, do not have the obvious effect on the generation of H2O2. Yet the concentration of VO in ZnO crystal has a distinct effect on the generation of H2O2 and, consistently, the antimicrobial activity. The adjustment of VO in t-ZnO through heat-treating in different atmospheres convinces us that the VO may play an important role in the production of H2O2. These results provide the basis for developing a more detailed mechanism for H2O2 generation catalyzed by ZnO and for taking greater advantage of this type of antimicrobial agent.



ASSOCIATED CONTENT

S Supporting Information *

Theoretical calculation of VO in ZnO. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*Tel./fax: +86-028-8760-0454. E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (nos. 50272056, 51173148), the National High Technology Research and Development Program (“863” Program) of China (no. 2009AA03Z427), the Fundamental Research Funds for the Central Universities (SWJTU11ZT10), and Fund of Leshan Science & Technology Bureau (10801K10096006).



REFERENCES

(1) French, G. L. The continuing crisis in antibiotic resistance. Int. J. Antimicrob. Agents 2010, 36, S3−S7. 5579

dx.doi.org/10.1021/la400378t | Langmuir 2013, 29, 5573−5580

Langmuir

Article

subcapitata): The importance of particle solubility. Environ. Sci. Technol. 2007, 41, 8484−8490. (23) Li, M.; Zhu, L. Z.; Lin, D. H. Toxicity of ZnO nanoparticles to Escherichia coil: Mechanism and the influence of medium components. Environ. Sci. Technol. 2011, 45, 1977−1983. (24) Yin, H.; Casey, P. S.; McCall, M. J.; Fenech, M. Effects of surface chemistry on cytotoxicity, genotoxicity, and the generation of reactive oxygen species induced by ZnO nanoparticles. Langmuir 2010, 26, 15399−15408. (25) Perelshtein, I.; Applerot, G.; Perkas, N.; Wehrschetz-Sigl, E.; Hasmann, A.; Guebitz, G. M.; Gedanken, A. Antibacterial properties of an in situ generated and simultaneously deposited nanocrystalline ZnO on fabrics. ACS Appl. Mater. Interfaces 2009, 1, 363−366. (26) Tam, K. H.; Djurisic, A. B.; Chan, C. M. N.; Xi, Y. Y.; Tse, C. W.; Leung, Y. H.; Chan, W. K.; Leung, F. C. C.; Au, D. W. T. Antibacterial activity of ZnO nanorods prepared by a hydrothermal method. Thin Solid Films 2008, 516, 6167−6174. (27) Zhou, Z. W.; Deng, H.; Yi, J.; Liu, S. K. A new method for preparation of zinc oxide whiskers. Mater. Res. Bull. 1999, 34, 1563− 1567. (28) Schopfer, P. Histochemical-demonstration and localization of H2O2 in organs of higher-plants by tissue printing on nitrocellulose paper. Plant Physiol. 1994, 104, 1269−1275. (29) Boon, A. Q. M.; Huisman, H. M.; Geus, J. W. Influence of surface oxygen vacancies on the catalytic activity of copper-oxide: Part 2. Oxidation of methane. J. Mol. Catal. 1992, 75, 293−303. (30) Børseth, T. M.; Svensson, B. G.; Kuznetsov, A. Y.; Klason, P.; Zhao, Q. X.; Willander, M. Identification of oxygen and zinc vacancy optical signals in ZnO. Appl. Phys. Lett. 2006, 89, 262112/1−3. (31) Xia, T.; Kovochich, M.; Liong, M.; Madler, L.; Gilbert, B.; Shi, H. B.; Yeh, J. I.; Zink, J. I.; Nel, A. E. Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties. ACS Nano 2008, 2, 2121− 2134. (32) Park, S. J.; Park, Y. C.; Lee, S. W.; Jeong, M. S.; Yu, K. N.; Jung, H.; Lee, J. K.; Kim, J. S.; Cho, M. H. Comparing the toxic mechanism of synthesized zinc oxide nanomaterials by physicochemical characterization and reactive oxygen species properties. Toxicol. Lett. 2011, 207, 197−203. (33) Taylor, H. S. A theory of the catalytic surface. Proc. R. Soc. A 1925, 108, 105−111. (34) Thomas, J. M.; Evans, E. L.; Williams, J. O. Microscopic studies of enhanced reactivity at structural faults in solids. Proc. R. Soc. A 1972, 331, 417−427. (35) Kumar, E. S.; Venkatesh, S.; Rao, M. S. R. Oxygen vacancy controlled tunable magnetic and electrical transport properties of (Li, Ni)-codoped ZnO thin films. Appl. Phys. Lett. 2010, 96, 232504/1−3. (36) Hofmann, D. M.; Pfisterer, D.; Sann, J.; Meyer, B. K.; TenaZaera, R.; Munoz-Sanjose, V.; Frank, T.; Pensl, G. Properties of the oxygen vacancy in ZnO. Appl. Phys. A: Mater. Sci. Process. 2007, 88, 147−151. (37) Janotti, A.; Van de Walle, C. G. Oxygen vacancies in ZnO. Appl. Phys. Lett. 2005, 87, 122102/1−3. (38) Djurisic, A. B.; Leung, Y. H. Optical properties of ZnO nanostructures. Small 2006, 2, 944−961. (39) Chang, Y. G.; Xu, J.; Zhang, Y. Y.; Ma, S. Y.; Xin, L. H.; Zhu, L. N.; Xut, C. T. Optical properties and photocatalytic performances of Pd modified ZnO samples. J. Phys. Chem. C 2009, 113, 18761−18767. (40) Polarz, S.; Strunk, J.; Ischenko, V.; van den Berg, M. W. E.; Hinrichsen, O.; Muhler, M.; Driess, M. On the role of oxygen defects in the catalytic performance of zinc oxide. Angew. Chem., Int. Ed. 2006, 45, 2965−2969. (41) Dow, W. P.; Huang, T. J. Yttria-stabilized zirconia supported copper oxide catalyst: II. Effect of oxygen vacancy of support on catalytic activity for CO oxidation. J. Catal. 1996, 160, 171−182. (42) Wei, X. Q.; Man, B. Y.; Liu, M.; Xue, C. S.; Zhuang, H. Z.; Yang, C. Blue luminescent centers and microstructural evaluation by XPS and Raman in ZnO thin films annealed in vacuum, N2 and O2. Physica B 2007, 388, 145−152.

(43) Lu, W. W.; Gao, S. Y.; Wang, J. J. One-pot synthesis of Ag/ZnO self-assembled 3D hollow microspheres with enhanced photocatalytic performance. J. Phys. Chem. C 2008, 112, 16792−16800. (44) Xu, X. Y.; Xu, C. X.; Dai, J.; Hu, J. G.; Li, F. J.; Zhang, S. Size dependence of defect-induced room temperature ferromagnetism in undoped ZnO nanoparticles. J. Phys. Chem. C 2012, 116, 8813−8818. (45) Aljawfi, R. N.; Mollah, S. Properties of Co/Ni codoped ZnO based nanocrystalline DMS. J. Magn. Magn. Mater. 2011, 323, 3126− 3132. (46) Panigrahy, B.; Aslam, M.; Misra, D. S.; Ghosh, M.; Bahadur, D. Defect-related emissions and magnetization properties of ZnO nanorods. Adv. Funct. Mater. 2010, 20, 1161−1165. (47) Gallino, F.; Di Valentin, C.; Pacchioni, G.; Chiesa, M.; Giamello, E. Nitrogen impurity states in polycrystalline ZnO. A combined EPR and theoretical study. J. Mater. Chem. 2010, 20, 689−697. (48) Vlasenko, L. S. Magnetic resonance studies of intrinsic defects in ZnO: Oxygen vacancy. Appl. Magn. Reson. 2010, 39, 103−111. (49) Kasai, H. P. Electron spin resonance studies of donors and acceptors in ZnO. Phys. Rev. 1963, 130, 989−995. (50) Dutta, S.; Chattopadhyay, S.; Sarkar, A.; Chakrabarti, M.; Sanyal, D.; Jana, D. Role of defects in tailoring structural, electrical and optical properties of ZnO. Prog. Mater. Sci. 2009, 54, 89−136.

5580

dx.doi.org/10.1021/la400378t | Langmuir 2013, 29, 5573−5580