Crystallography Facet-Dependent Antibacterial Activity: The Case of

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Crystallography Facet-Dependent Antibacterial Activity: The Case of Cu2O Jia Ren,† Wenzhong Wang,*,† Songmei Sun,† Ling Zhang,† Lu Wang,† and Jiang Chang‡ †

State Key Laboratory of High Performance Ceramics and Superfine Microstructure and ‡Biomaterials and Tissue Engineering Research Center, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, P. R. China

bS Supporting Information ABSTRACT: The dependence of antibacterial activities on different crystallography facets has been demonstrated by taking Cu2O crystals as an example. Owing to the different atomic arrangement of the exposed surfaces, Cu2O octahedral crystals bounded by {111} facets exhibited higher activity in killing E. coli than cubic ones bounded by {100} facets. Zeta potential measurement demonstrated that the electrostatic interaction between E. coli and octahedral crystals was more profitable in inactivating bacteria than that between E. coli and cubic ones. Moreover, the suspending medium, where the bactericidal process takes place, has been found to play a crucial role on the bactericidal efficiency. This work presents a way for the sterilization or killing of bacteria efficiently and selectively.

1. INTRODUCTION Over the past decades, a lot of work has been done in the study of chemical properties of definite crystal planes. It is generally considered that chemical reactivity of crystals can be significantly affected by their shapes, due to surface atom arrangement, bonding, and surface energy, etc.1 Inorganic single crystals with highly reactive surfaces are therefore desired. Unfortunately, surfaces with high reactivity usually possess high surface energy, and such high-surface energy surfaces tend to shrink rapidly during the crystal growth process to minimize total surface energy. Thus, crystals with a dominant high reactivity surface are of great interest yet challenging. Wang’s group successfully synthesized novel tetrahexahedral platinum bounded by highindex planes and achieved high electro-oxidation activity.2 Anatase TiO2 single crystals with exposed reactive {001} facets were obtained by Lu and co-workers.3,4 It is well-known that copper ions are toxic and are able to kill bacteria or mold effectively by denaturation or an oxidation mechanism.5,6 To our knowledge, there have been some studies on the production and application of the copper or copper oxide as antibacterial materials.7,8 Perelshtein et al. studied the antibacterial activities of the CuO fabric composite.9 The activity of CuO-doped phosphate glass fibers was reported by Abou Neel.10 Heinlaan et al. investigated the toxicity of nanosized and bulk CuO.11 Cu and CuO nanoparticles immobilized on silica thin films as antibacterial materials and photocatalysts were carefully researched by Akhavan et al.12 Cuprous oxide (Cu2O), a p-type semiconductor with a cubic crystal structure, has potential applications in catalysis, lithium ion batteries, solar energy conversion, gas sensors, and semiconductor technology.13 A lot of work has been done in the study of chemical properties of definite crystal planes. The special O Cu O 180-degree linear co-ordination made its crystalline surfaces of {111} and {100} possess distinctive chemical activities. The facet-dependent photocatalytic activities of Cu2O nanocrystals have been studied.14,15 However, rare studies are carried out on the investigation of the r 2011 American Chemical Society

morphology effect on the antibacterial activity of Cu2O bounded by different crystallography facets. Lu’s group studied the antibacterial activities of the Cu2O crystals for the first time and found that octahedral crystals had higher selectivity compared to cubic crystals. However, the mechanism that led to the difference in antibacterial activities remained unclear.16 In this work, cubicand octahedral-shaped Cu2O crystals exhibit distinctive antibacterial activity, and the mechanism is systematically studied. The interaction between the Cu2O crystals and the bacterium organisms was found to be affected by the exposed {111} and {100} surfaces of Cu2O. It is also revealed that the interaction environment has an effect on the antibacterial activity toward certain objects. This study presents a clue for the sterilization or killing of bacteria efficiently and selectively.

2. EXPERIMENTAL SECTION Synthesis of Cu2O cubic crystals: In a typical procedure, 0.171 g CuCl2 3 2H2O was dissolved in 50 mL of deionized water, and NaOH solution (10.0 mL, 4 M) was added into the above solution under constant magnetic stirring for about 10 min. A 1.0 g portion of ascorbic acid was dissolved in 10 mL of deionized water, and this mixture was added to the solution. The resulting samples were collected, washed with deionized water, and dried at 60 °C in a vacuum oven. Synthesis of Cu2O octahedral crystals: The Cu2O octahedral crystals were prepared according to the procedure described in ref 13. The morphologies and microstructures of as-prepared samples were analyzed by field-emission scanning electron microscopy (JEOL JSM-6700F). Transmission electron microscopy (TEM) was obtained using a JEOL JEM-2100F field emission Received: March 18, 2011 Accepted: July 22, 2011 Revised: July 21, 2011 Published: July 22, 2011 10366

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Industrial & Engineering Chemistry Research

Figure 1. SEM images of the samples prepared with octahedral (a) and cubic morphology (b).

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Figure 3. Images of colonies on Petri dishes cultured with E. coli in 0.9% saline water dispersions: (a) control, (b d) samples containing octahedral Cu2O particles at concentrations of 5, 25, and 50 μg/mL, respectively, and (e g) samples containing cubic Cu2O particles at concentrations of 5, 25, and 50 μg/mL, respectively.

the above experiments were repeated three times and the average values were given.

Figure 2. Panels a and c are The TEM images and the corresponding selected area electron diffraction (SAED) patterns with the electron beams parallel to [100] and [110] of cubic-shaped Cu2O crystals, panels b and d are the counterpart with the electron beams parallel to [111] and [110] of octahedral-shaped Cu2O crystals.

electron microscope at acceleration voltage of 200 kV. Fourier transform infrared (FT-IR) spectra were collected on a NicoletNexus spectrometer. The KBr disk method was employed with a scan range of 2500 500 cm 1 and a resolution of 32 cm 1. E. coli, a Gram-negative bacterium, was used as model bacteria in this study. They were incubated in Lysogeny Broth (LB) medium at 37 °C for 18 h. Cells were harvested from an overnight culture by centrifugation at 4000 rpm for 5 min and then washed twice with 0.9% saline (wt %). The bactericidal activity of the samples was evaluated by the inactivation of E. coli. All materials used in the experiments were autoclaved at 121 °C for 40 min before use to ensure sterility. The treated cells were suspended and diluted to a cell suspension of ∼2  107 cfu/mL, with 0.9% saline and deionized water, respectively. The mass of photocatalyst were adjusted to 5, 25, and 50 μg/mL, respectively. The mixtures were stirred with a magnetic stirrer to prevent settling of the samples. The experiments were carried out at room temperature. Before and after the experiments, an aliquot of the reaction mixture was immediately diluted with 0.9% saline and plated on LB-agar plates. The colonies were counted after incubation at 37 °C for 24 h. All of

3. RESULTS AND DISCUSSION The composition and phase purity of the products were examined by XRD, and the results reveal the pure Cu2O is obtained in both of the samples (Supporting Information, Figure S1). The representative XRD pattern of Cu2O octahedra corresponds with the standard (JCPDS No. 05-0667). The XRD pattern of cubic Cu2O crystals obtained by ascorbic acid is also indexed to be cubic-phase Cu2O. No impurities, such as CuO or Cu(OH)2, were detected in the patterns. Meanwhile, the intensity ratio of the (111) and (200) diffraction peaks of the octahedral morphology is higher than that of the cubic. The typical scanning electron microscopy (SEM) images of the sample obtained by using hydrazine hydrate as the reducer show that the sample is composed of many octahedral crystals and the rhombic length is in the range of 600 700 nm (Figure 1). The sample prepared by using ascorbic acid as the reducer exhibited uniform cubic morphology with average edge length of about 400 nm. TEM and SAED analysis are also used to characterize the morphologies and structures of the samples furthermore. As shown in Figure 2, when the electron beam is aligned to be perpendicular to {100} of cubic-shaped Cu2O crystals, a two-dimensional square-shaped projection is observed, the selected area electron diffraction (SAED) patterns taken along the [100] direction indicate that the sample is single crystal (Figure 2a). Meanwhile, when the electron beam is aligned to be perpendicular to {111} of the octahedron, a hexagon-shaped projection is observed, and the SAED patterns indicate that the sample is single crystal (Figure 2b). Viewed along [110] zone axis, the projection drawing of the cube is rectangle-shaped with length versus width ratio being about 1.4 (Figure 2c). However, the projection drawing of the octahedron is parallelogramshaped (Figure 2d). This observation could further discriminate the cubic crystals from the octahedra. The antibacterial ability of Cu2O octahedra in the disinfection of E. coli was compared with cubic ones according to the decrease of the colony number formed on an agar plate. Both octahedral and cubic Cu2O crystals could inhibit the growth of E. coli efficiently, and their bactericidal activities become stronger with increased sample concentrations (Figure 3). It is worthy noting that up to 85% E. coli are killed in the presence of octahedral Cu2O particles (25 μg/mL), while the bactericidal percentage 10367

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Industrial & Engineering Chemistry Research

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Table 1. Zeta Potentials of Octahedral- And Cubic-Shaped Cu2O Crystals, and E. coli Suspended in Deionized Water Dispersions and 0.9% Saline Water Dispersions, Respectively zeta-potential (mV) suspending liquid

Figure 4. The antibacterial efficiency of E. coli with Cu2O octahedral crystals and cubic crystals in deionized water dispersions and 0.9% saline water dispersions (Cu2O concentration, 50 μg/mL; interaction time, 5 min).

Figure 5. The atomic arrangement in (100) and (111) planes of the Cu2O structure, respectively.

was only 21% after being treated with cubic Cu2O particles with the same concentration (Supporting Information, Figure S2). In addition, the antibacterial activity is affected by different suspending medium. As shown in Figure 4, the antibacterial efficiencies over the two kinds of crystals are lower in the deionized water. It is also demonstrated that Cu2O octahedral crystals exhibit higher antibacterial ability than the cubic ones. When the Cu2O crystals are suspended in 0.9% saline water (wt %), E. coli inactivation efficiency is up to 100% over the Cu2O octahedral crystals, while only about 50% of E. coli are killed by the Cu2O cubic crystals after 5 min interaction. To figure out why the difference of the inactivation activities existed between the Cu2O cubic crystals and the octahedral ones, the atom arrangements of exposed crystallographic facets is proposed as an important factor. Cu2O has a cuprite crystal structure, which is composed of a body-centered cubic packing of oxygen atoms with copper atoms occupying half of the tetrahedral sites. As shown in Figure 5, the structural arrangement perpendicular to the [100] direction alternates between copperand oxygen-containing planes. The two layers repeat alternatively to maintain stoichiometry and charge neutrality. An ideal {100} plane should be either copper or oxygen terminated. However, considering that the synthesis process was carried out in aqueous medium, copper terminated would be rather unstable due to the active interaction with the hydroxyl group.17 Herein, the oxygen terminated {100} plane is expected for the Cu2O cubic crystals. As regarded in the [111] direction, an ideal {111} plane possesses hexagonal symmetry. Every “Cu”-containing plane is sandwiched between two “O”-containing planes. The

cube

octahedron

E. coli

deionized water

14.7 ( 1.1

4.0 ( 1.1

37.6 ( 0.5

0.9% NaCl

16.4 ( 1.6

11.1 ( 1.9

23.7 ( 3.9

three-plane unit repeats to satisfy stoichiometry and charge neutrality. Thus the ideal {111} plane should be terminated by an outer layer of oxygen anions, with a second atomic layer of Cu+ cations, and a third atomic layer of oxygen anions. Every two Cu atoms has a dangling bond perpendicular to the {111} planes. Thus, the {111} plane was expected to possess a higher energy status compared with the {100} plane with 100% oxygen terminated. On the basis of the analysis about the relationship between the morphology with different exposed surfaces and chemical activities, it is predicted that the octahedral crystals bounded by the {111} facet are more active than the cubic crystals exposed with the {100} plane. And the results of experiments are in good accordance with the above analysis. To investigate the effect of the environment on the interaction between E. coli and Cu2O crystals, the antibacterial experiments were carried out in deionized water and 0.9% saline water, respectively. The zeta potential measurement was performed (Table 1). The overall charges of E. coli are negative, and the zeta potential is more negative in the presence of deionized water than in 0.9% saline water. On the other hand, the zeta potential of Cu2O cubic crystals in the presence of deionized water approximates that in the presence of 0.9% saline water, and the inactivation efficiencies are correspondingly close. When the environment varied, however, the zeta potential of Cu2O octahedral crystals changed from negative to positive. It would affect the surface charging of metal oxides when electrolyte is added in the aqueous system. Octahedral-shaped Cu2O crystals may be more favorable for the adsorption of Na+. Consequently, the inactivation efficiency was enhanced from 60% to as high as 100% for Cu2O octahedral crystals. The notable improvement of the antibacterial activity is ascribed to the electrostatic attraction occurring between E. coli and Cu2O octahedral crystals. The zeta potential of Cu2O cubic crystals was negative, while it was positive for the Cu2O octahedral crystals. The different adsorption behavior of cubic- and octahedral-shaped Cu2O crystals may be related with their different atomic arrangement of the exposed surfaces.18 Thus the electrostatic attraction between E. coli and Cu2O octahedral crystals could make bacteria contact with Cu2O excellently, leading to the higher antibacterial activity. The electrostatic interaction between charged particles and the charged groups on the cell membrane surface of bacteria is crucial to the inactivation efficiency. Herein, the FT-IR measurements were carried out on the bacterial cells before and after treatment with octahedral Cu2O particles (Figure 6). The peaks at ∼1242 and ∼1080 cm 1 are assigned to PdO asymmetric and symmetric stretches of the phosphodiester backbone of nucleic acids DNA and RNA.19 And the peaks at ∼1242 and ∼1080 cm 1 are almost undetectable after treatment with octahedral Cu2O particles, which indicated that the bacteria were destroyed. Nanosized solid oxidized copper states such as Cu2O could be a proper substitute for 10368

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Industrial & Engineering Chemistry Research

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’ REFERENCES

Figure 6. FT-IR measurements were carried out on the bacteria cells: (a) E. coli without Cu2O particles, and (b) E. coli after treatment with octahedral Cu2O particles for 5 min.

the copper ion solution, since they can provide a more sustained release of copper ions. The morphology-dependent activities of the Cu2O crystals have the potential application in the selective inactivation of bacteria under special condition.

4. CONCLUSIONS In summary, the antibacterial activities of cubic- and octahedral-shaped Cu2O crystals have been studied. Cu2O octahedral crystals exhibited higher activity in inactivating E. coli than the Cu2O cubic ones. The difference in the antibacterial activity of Cu2O crystals can be ascribed to the atomic arrangements of different exposed surfaces. The results of zeta potential measurement also indicated that the electrostatic interaction between E. coli and Cu2O octahedral crystals is stronger, which is favorable for inactivating bacteria. In addition, the suspending aqueous dispersions affect the bactericidal efficiency obviously. The surface-dependent property of the Cu2O crystals is expected to potentially be applied in catalysis, sensors, and optoelectronics. ’ ASSOCIATED CONTENT

bS

Supporting Information. Details of synthesis of Cu2O octahedral crystals; additional figures as described in the text. This material is available free of charge via the Internet at http:// pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Fax: +86-21-5241-3122. E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (50972155, 50902144), National Basic Research Program of China (2007CB613305, 2010CB933503) and Science Foundation for Youth Scholar of State Key Laboratory of High Performance Ceramics and Superfine Microstructures (SKL200904).

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