Characterisation of copper oxide nanoparticles for antimicrobial ...

International Journal of Antimicrobial Agents 33 (2009) 587–590

Contents lists available at ScienceDirect

International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag

Short communication

Characterisation of copper oxide nanoparticles for antimicrobial applications Guogang Ren a , Dawei Hu b , Eileen W.C. Cheng b , Miguel A. Vargas-Reus c , Paul Reip d , Robert P. Allaker c,∗ a

School of Aerospace, Automotive Design and Engineering, University of Hertfordshire, Hatfield AL10 9AB, UK Queen Mary University of London, Department of Materials, London E1 2AT, UK c Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Blizard Building, 4 Newark Street, London E1 2AT, UK d Intrinsiq Materials Ltd., Farnborough, Hants GU14 0LX, UK b

a r t i c l e

i n f o

Article history: Received 28 November 2008 Accepted 1 December 2008 Keywords: Nanoparticle Copper oxide CuO Antimicrobial Cross-infection control

a b s t r a c t Copper oxide (CuO) nanoparticles were characterised and investigated with respect to potential antimicrobial applications. It was found that nanoscaled CuO, generated by thermal plasma technology, contains traces of pure Cu and Cu2 O nanoparticles. Transmission electron microscopy (TEM) demonstrated particle sizes in the range 20–95 nm. TEM energy dispersive spectroscopy gave the ratio of copper to oxygen elements as 54.18% to 45.26%. The mean surface area was determined as 15.69 m2 /g by Brunau–Emmet–Teller (BET) analysis. CuO nanoparticles in suspension showed activity against a range of bacterial pathogens, including meticillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli, with minimum bactericidal concentrations (MBCs) ranging from 100 ␮g/mL to 5000 ␮g/mL. The ability of CuO nanoparticles to reduce bacterial populations to zero was enhanced in the presence of sub-MBC concentrations of silver nanoparticles. Studies of CuO nanoparticles incorporated into polymers suggest release of ions may be required for optimum killing. © 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

1. Introduction As particles are reduced from a micrometre to a nanometre size, the resultant properties can change dramatically. For example, electrical conductivity, hardness, active surface area, chemical reactivity and biological activity are all known to be altered. The bactericidal effectiveness of metal nanoparticles has been suggested to be due to both their size and high surface-to-volume ratio. Such characteristics should allow them to interact closely with bacterial membranes, rather than the effect being solely due to the release of metal ions [1]. In theory, metal nanoparticles could be combined with polymers or coated onto surfaces, which may then have a variety of potential antimicrobial applications. The antimicrobial properties of both silver [2] and copper nanoparticles [3] have been previously reported, and both of these have been coated onto or incorporated into various materials [4]. Copper oxide (CuO)/copper (II) oxide/cupric oxide is a semiconducting compound with a monoclinic structure. CuO has attracted particular attention because it is the simplest member of the family of copper compounds and exhibits a range of potentially useful physical properties such as high temperature superconductivity, electron correlation effects and spin dynamics [5,6]. As an

∗ Corresponding author. Tel.: +44 20 7882 2388; fax: +44 20 7882 2191. E-mail address: [email protected] (R.P. Allaker).

important p-type semiconductor, CuO has found many diverse applications such as in gas sensors, catalysis, batteries, hightemperature superconductors, solar energy conversion and field emission emitters. In the energy-saving area, energy transferring fluids filled with nano CuO particles can improve fluid viscosity and enhance thermal conductivity [7]. CuO crystal structures possess a narrow band gap, giving useful photocatalytic or photovoltaic properties as well as photoconductive functionalities [8]. Limited information on the possible antimicrobial activity of nano CuO is available. CuO is cheaper than silver, easily mixed with polymers and relatively stable in terms of both chemical and physical properties. Highly ionic nanoparticulate metal oxides, such as CuO, may be particularly valuable antimicrobial agents as they can be prepared with extremely high surface areas and unusual crystal morphologies [9]. The aims of this study were to characterise physically and chemically nano CuO and to investigate this compound with respect to its potential antimicrobial applications. 2. Methods 2.1. Nanomaterial preparation Nanoparticles of CuO, Cu, Cu2 O, zinc oxide (ZnO) and silver (Ag) were prepared by Intrinsiq Materials Ltd. (Farnborough, UK) using thermal plasma (TesimaTM ) technology as shown in Fig. 1. This process allows the continuous gas phase production of bulk nanopowders.

0924-8579/$ – see front matter © 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2008.12.004

588

G. Ren et al. / International Journal of Antimicrobial Agents 33 (2009) 587–590

suspension onto tryptone soya agar (Oxoid) plates. Plates were incubated at 37 ◦ C in air plus 10% CO2 for 24 h. 2.5. Time–kill determination To investigate possible synergistic antimicrobial activity and thus minimise potential toxicity and resistance problems, mixtures of nanoparticles were also tested. CuO alone and CuO combined with Ag at sub-MBC concentrations were used in killing assays against the following strains: S. aureus strains EMRSA-16, EMRSA-15 and S. aureus Oxford (NCTC 6571); S. epidermidis SE-51; E. coli NCTC 9001; P. aeruginosa PAOI; and Proteus spp. All nanoparticles were prepared in phosphate-buffered saline (PBS) with sonication as described above. At time zero, ca. 5 × 107 CFU/mL of each microorganism was added to the nanoparticle suspension at a dilution of 1 in 80. Incubation was then carried out in a shaking incubator (200 rpm at 37 ◦ C in air for up to 4 h). Inoculated nanoparticle-free suspensions in PBS were used as negative controls. Growth was assessed by plating serial dilutions of each nanoparticle/bacterial suspension at different time points onto tryptone soya agar plates. Plates were then incubated at 37 ◦ C in air with CO2 for 24 h. Fig. 1. TesimaTM plasma process to generate nanoparticles.

2.2. Transmission electron microscopy (TEM) and TEM energy dispersive X-ray spectroscopy (EDS) analysis To determine particle size, morphology and composition, CuO nanoparticles were visualised using a high-resolution transmission electron microscope (JEOL 2010; JEOL Ltd.) and subjected to TEMEDS for high-speed elemental analysis. 2.3. Surface area determination using Brunau–Emmet–Teller (BET) method analysis The BET method was employed to measure the surface area of nano CuO with a Micromeritics Gemini 2360 surface area analyser, whereby nitrogen gas molecules are adsorbed onto a solid surface, which allows measurement of the surface area of a material. The nano CuO samples were first prepared using a Gemini Vac Prep degasser. Dry and degassed samples were then analysed using the single/multipoint adsorption method for surface area assessment. 2.4. Minimum bactericidal concentration (MBC) determination CuO nanoparticles at 5000, 2500, 1000, 500, 250, 100, 50 and 20 ␮g/mL in suspension were used to determine the lowest bactericidal concentration required to prevent the growth of bacteria after transfer onto nanoparticle-free media. The following strains were used: Staphylococcus aureus EMRSA-16 (epidemic meticillinresistant S. aureus), EMRSA-15, meticillin-resistant S. aureus (MRSA) 252, S. aureus ‘Golden’ (laboratory isolate) and S. aureus Oxford (NCTC 6571); Staphylococcus epidermidis SE-51 and SE-4; Escherichia coli NCTC 9001; Pseudomonas aeruginosa PAOI (laboratory isolate); and Proteus spp. (laboratory isolate). For comparison, Cu, Cu2 O, ZnO and Ag nanoparticles were also tested. Nanoparticle suspensions were prepared in tryptone soya broth (Oxoid Ltd., Basingstoke, UK) and sonicated to ensure optimum nanoparticle dispersion. A Soniprep 150 MSE sonicator was used, with 3 s pulses and 6 s rests for a total of 90 s. Approximately 5 × 107 colony-forming units (CFU)/mL of each microorganism were added to the nanoparticle suspension at a dilution of 1 in 100. Incubation was then carried out in a shaking incubator (200 rpm at 37 ◦ C in air for 24 h). Inoculated nanoparticle-free broths were used as negative controls. Growth was then assessed by plating each nanoparticle/bacterial

3. Results 3.1. TEM-EDS and TEM analysis TEM-EDS produced the trace spectrum of nano CuO as shown in Fig. 2A. The weight compositions for copper (Cu) and oxygen (O) were 80.65% and 16.96%, respectively. The atomic compositions were then calculated as 54.18% and 45.26%, respectively. A small proportion of impurities, such as pure Cu and Cu2 O nanoparticles, were detected owing to interactions with air after plasma processing. TEM analysis demonstrated that nano CuO particles exhibited an approximate equi-axes shape with no sharp edges observed (Fig. 2B). The particle size was determined to be in the range 22.4–94.8 nm. Almost all the particles had only one unique grain; very few particles contained two or more grains. 3.2. Surface area determination of nano CuO using BET method analysis The mean surface area was calculated as 15.6931 m2 /g. A mean particle size can then be estimated if the particles are assumed to have the same spherical shape and size dimensions. The average particle diameter (D) is given by D = 6/(S × ı), where S = surface area and ı = density (density of the bulk CuO was 6310 kg m−3 ). Thus, the CuO particle size was on average 60.6 nm [D = 6/(15.6931 × 1000 × 6310) = 60.6 nm]. This was in the same range as the result obtained from TEM analysis. The particles did not show any evidence of being porous. 3.3. Minimum bactericidal concentration and time–kill determinations CuO nanoparticles demonstrated antimicrobial activity against a range of Gram-positive and Gram-negative bacteria, including MRSA. In contrast to Ag, with MBC values of 100 ␮g/mL against all 10 strains tested, MBC values for CuO ranged from 100 ␮g/mL for S. aureus (Oxford) to 5000 ␮g/mL for P. aeruginosa and Proteus spp. With nano Cu, values of 250, 2500 and 2500 ␮g/mL, respectively, were observed with these bacteria (Table 1). Apart from nano Cu2 O against P. aeruginosa, all MBC values for nano Cu2 O and ZnO were equal to or above those determined for nano CuO. Using time–kill assays, populations of Gram-positive (×4 strains) and Gram-negative (×3 strains) organisms tested were reduced by 68% and 65%, respectively, in the presence of 1000 ␮g/mL nano CuO


589

4. Discussion

Fig. 2. (A) Transmission electron microscopy (TEM) energy dispersive spectroscopy analysis spectrum for nanoparticulate copper oxide (CuO) sample. Ta, traces of rare tantalum. (B) TEM image of nanoparticulate CuO.

within 2 h. This was increased to 88% (P < 0.05; Student’s independent t-tests) and 100% (P < 0.01), respectively, with the addition of a sub-MBC concentration (50 ␮g/mL) of nano Ag. Populations of P. aeruginosa, S. aureus (Oxford), EMRSA-16 and S. epidermidis SE-51 were reduced to zero by 4 h in the presence of 1000 ␮g/mL nano CuO. With the exception of EMRSA-15, addition of a sub-MBC concentration of nano Ag reduced all populations to zero by 4 h.

Table 1 Minimum bactericidal concentrations (MBCs) of nanoparticulate metals and metal oxides. Strain

EMRSA-16 EMRSA-15 MRSA 252 Staphylococcus aureus (Golden) S. aureus (Oxford) Staphylococcus epidermidis SE-51 S. epidermidis SE-4 Escherichia coli NCTC 9001 Proteus spp. Pseudomonas aeruginosa PAOI

MBC (␮g/mL) Ag

ZnO

Cu2 O

CuO

Cu

100 100 100 100 100 100 100 100 100 100

5000 5000 >5000 2500 5000 2500 2500 >5000 >5000 >5000

2500 2500 2500 2500 2500 2500 2500 500 5000 2500

1000 250 1000 2500 100 2500 2500 250 5000 5000

250 250 1000 1000 250 500 1000 250 2500 2500

EMRSA, epidemic meticillin-resistant S. aureus; MRSA, meticillin-resistant S. aureus.

Identification of the precise elemental composition, particle size range and surface morphology of nanoparticulate CuO is a prerequisite to a full understanding of its potential application capabilities. TEM-EDS analysis demonstrated that the atomic composition of the Cu and O elements was 54.18% and 45.26%, respectively. The mean ratio of Cu and O was therefore 54.18:45.26 and an accurate compound formula based on the atomic ratio of Cu and O can thus be given as Cu1.2 O or CuO0.84 . Therefore, it is estimated that most of the nanoparticulate sample, generated by thermal plasma technology, was indeed CuO. CuO nanoparticles were effective in killing a range of bacterial pathogens involved in hospital-acquired infections. However, in comparison with nano Ag and nano Cu, higher concentrations of nano CuO were required to achieve a bactericidal effect. It has been suggested that the reduced amount (between 3- and 20-fold) of negatively charged peptidoglycans would make Gram-negative bacteria less susceptible to such positively charged antimicrobials [10]. This is supported by the MBC findings with nano CuO in this study for P. aeruginosa and Proteus spp. However, in the time–kill experiments the Gram-negative strains tested showed a greater susceptibility to nano CuO combined with nano Ag. Preliminary research on the antiviral (versus influenza A and SARS viruses) capabilities of nanomaterials has shown that nanoparticles such as Ag and Cu release Ag+ and Cu2+ ions that cause local pH and conductivity changes. This liberation of metal ions into solution will then have the capability to inactivate or kill viruses. Cu2+ and Ag+ ions are also small enough to disrupt bacterial cell membranes and gain entry in order to disrupt enzyme function. Indirect effects through changes in the surrounding charge environment may also impact on the effectiveness of nanoparticulate metals against microorganisms [2]. Studies to assess the potential of nano CuO embedded within a range of polymer materials have shown a lower contact-killing ability in comparison with releasekilling ability against MRSA strains (Allaker, Vargas-Reus and Ren, unpublished observations), which would suggest a release of ions into the local environment is required for optimal antimicrobial activity. Atomic force microscopy (AFM) and TEM are useful techniques to investigate the changes in bacteria upon exposure to antimicrobials. AFM enables precise three-dimensional mapping of the microbial cell surface and detection of very subtle changes, whilst TEM provides direct visualisation of any morphological changes in the microbial cell [11]. Studies using AFM and TEM with ‘aerogel’generated nano magnesium oxide and Escherichia coli have shown that the cell wall of this bacterium is extensively damaged, allowing the contents to leak out and nanoparticles to gain entry [9]. Likewise, Ag nanoparticles have been shown to attach to the microbial cell surface and penetrate inside, where intracellular targets, including respiratory enzymes, are disrupted [2]. The precise mechanism of action of nano CuO is the subject of ongoing investigations. Hospitals and transport are two particular areas that offer opportunities for the use of nanoparticulate metals and metal oxides to prevent the spread of infection. With the increase in air travel and greater mobility in general of people, airborne, vector-borne and zoonotic spread of infectious agents are important public health issues. Within the hospital environment, wall coverings, equipment, clothing and bedding are all potential risk areas for spread of infection [12]. Therefore, in terms of potential use, the incorporation of nanoparticulate metals and metal oxides, including nano CuO, into surfaces and other objects could be envisaged. Funding: South East England Development Agency. Competing interests: None declared. Ethical approval: Not required.

590


References [1] Morones JR, Elechiguerra JL, Camacho A, Holt K, Kouri JB, Ramirez JT. The bactericidal effect of silver nanoparticles. Nanotechnology 2005;16:2346–53. [2] Sondi I, Salopek-Sondi B. Silver nanoparticles as an antimicrobial agent: a case study on E. coli as a model for gram-negative bacteria. J Colloid Interface Sci 2004;275:177–82. [3] Cioffi N, Torsi L, Ditaranto N, Tantillo G, Ghibelli L, Sabbatini L, et al. Copper nanoparticle/polymer composites with antifungal and bacteriostatic properties. Chem Mater 2005;17:5255–62. [4] Li Z, Lee D, Sheng X, Cohen RE, Rubner MF. Two-level antibacterial coating with both release-killing and contact-killing capabilities. Langmuir 2006;33:9820–3. [5] Cava RJ. Structural chemistry and the local charge picture of copper oxide superconductors. Science 1990;247:656–62.

[6] Tranquada JM, Sternlieb BJ, Axe JD, Nakamura Y, Uchida S. Evidence for stripe correlations of spins and holes in copper oxide superconductors. Nature 1995;375:561. [7] Kwak K, Kim C. Viscosity and thermal conductivity of copper oxide nanofluid dispersed in ethylene glycol. Korea–Australia Rheol J 2005;17:35–40. [8] Xu JF, Ji W, Shen ZX, Tang SH, Ye XR, Jia DZ, et al. Preparation and characterization of CuO nanocrystals. J Solid State Chem 1999;147:516–19. [9] Stoimenov PK, Klinger RL, Marchin GL, Klabunde KJ. Metal oxide nanoparticles as bactericidal agents. Langmuir 2002;18:6679–86. [10] Kawahara K, Tsuruda K, Morishita M, Uhida M. Antibacterial effect of silverzeolite on oral bacteria under anaerobic conditions. Dent Mater 2000;16:452–5. [11] Allaker RP, Grosvenor PW, McAnerney DC, Sheehan BE, Srikanta BH, Pell K, et al. Mechanisms of adrenomedullin antimicrobial action. Peptides 2006;27:661–6. [12] Allaker RP, Ren G. Potential impact of nanotechnology on the control of infectious diseases. Trans R Soc Trop Med Hyg 2008;102:1–2.