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99FUNCTIONAL NANOSTRUCTURES

A B S T R AC T

X. Fukua,b*, K. Kasinathana,b, K. Kotsedia,b, N. Thovhogia,b, Malik Maazaa,b

Size and concentration influence of Cu/Cu2O/CuO/ZnO on selected environmental pathogenic bacteria: Escherichia coli, Staphylococcus aureus, and Proteus vulgaris

In view of simplicity, cost-effect nanoplatelets like structures of Cu/Cu2O/CuO/ZnO were prepared successfully via the green route. The structural and antimicrobial activities of Cu/Cu2O/CuO/ZnO nanocomposite were studied using analytical techniques. High resolution microscopy (HRTEM) and UV-VIS we able to confirm the morphological structure and optical band gap of the prepared nanocomposite. Meanwhile, TGA and DSC revealed the purity and crystalline nature of the material. RAMAN spectroscopy demonstrated coordination mechanism of metal oxides with the responsible polyphenols. Cu/Cu2O/CuO/ZnO nanoplatelets showed excellent antimicrobial activity against Escherichia coli (E. coli), Proteus vulgaris (P. vulgaris), and Staphylococcus aureus (S. aureus). Comparatively, P. vulgaris and S. aureus exhibited the highest sensitivity to Cu/Cu2O/CuO/ZnO nanocomposite whilst E. coli was the least sensitive. However, the hexagonal cube like structures decorated with spherical nanoparticles showed the minimum inhibitory concentration (MIC) to inhibit E.coli.

aUNESCO-UNISA Africa Chair in Nanosciences/Nanotechnology, College of Science, Engineering and Technology, University of South Africa, Muckleneuk ridge, PO Box 392, Pretoria-South Africa. bNanosciences African network (NANOAFNET), Materials Research Department, iThemba LABS-National Research Foundation of South Africa, Old Faure Road, PO Box 722, Somerset West 7129, Western Cape - South Africa. *Corresponding author

I. INTRODUCTION highly ordered nanoparticulates of any size and shape [8; 9; 10; 11], have led to the development of new biocidal agents [12; 13; 14; 15]. Due to their high surface area (surface-volume ratio), nanomaterials or the so-called ‘a wonder of modern medicine’ reveal good results compared to their counterparts which are being used in the detection and removal of chemicals and biological substances including metals in water treatment [7; 15]. These antibacterial materials can be classified into two types i.e., organic and inorganic [7; 16]. In this study inorganic metal oxides are been chosen over organic materials due to superior durability, less toxicity, greater selectivity, and heat resistance [16; 17; 18]. In addition, metal nanoparticles have been studied extensively because of their exclusive catalytic, optical, electronic, magnetic and antimicrobial wound healing and anti-inflammatory properties [16; 17; 19]. For those reasons, the purpose of the present paper is to demonstrate firstly the antimicrobial potential of Cu/Cu2O/CuO/ZnO on different pathogens (E. coli MTCC 443, S. aureus MTCC and

According to the World Health Organisation (WHO) about 1.8 million deaths and 61.9 million disability-adjusted life years (DALYs) are caused by unsafe water, sanitation and poor hygiene [1; 2]. Microbial contaminations especially with pathogenic microorganisms are known to be the main factors of water pollution and other medical application [1; 3]. Pathogenic bacteria such as E. coli O157:H7, P. vulgaris and S. aureus are known to be the route of groundwater and surface water contamination thus causing water borne diseases [2; 4; 5]. The latter substantiate that safe drinking water plays a pivotal role in the well-being and health of humans [4; 5; 6]. In this view, antibacterial agents are of great need in the number of sectors including environmental, water disposal, food, synthetic textiles, packaging, healthcare and medical care [2; 5; 7]. Nanoscience and nanotechnology/cleantech tenders the possibility of an efficient removal of pollutants and germs especially in the area of water purification. Recent advances in the field of nanotechnology, particularly the ability to prepare

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P. vulgaris MTCC 227), to determine the influence of nanocomposite particles size on its antimicrobial efficacy and lastly to evaluate the concentration effect on the minimum inhibitory concentration (MIC) of various pathogens. Our key objective was to find an alternative eco-friendly and cost-effective technology which could completely inhibit/remove pathogenic bacteria from test water and result in the production of safe drinking water for our communities.

II. EXPERIMENTAL PROCEDURES

Reagents and materials

From pomegranate (punica granatum L) fruit, peels were sourced and used by cleaning and drying them in the sun for few days (2 - 3 days). Metal precursors such as zinc acetate, copper acetate, 15 mL polyesterine sterile centrifuge tubes and 0.22 μm hydrophilic filters (Whatman) were used in the synthesis and characterisation of the metal oxides. Inocula of 105 and 106 cfu/ml and three different pathogens of S. aureus-MRSA-33591, E. coli-K12, and P. vulgaris- ATCC-49132 were purchased for antibacterial tests. Ultra pure water was used for the experiments.

Synthesis of Cu/Cu2O/CuO/ZnO nanaocomposite

Scheme 1. Schematic representation of the synthetic method and mechanism of interaction.

Reduced Cu/Cu2O/CuO/ZnO nanostructures were prepared through green process. Briefly, equimolar amounts of zinc acetate and copper acetate (8g - (0.4 mol L-1)) were added into a 250 mL round bottle containing yellow peel extract of punica granatum L or punicalagin-polyphenol (at 80 °C)- which acted as both reducing and capping/chelating agent helps form radicals which aid in formation of nanoscaled materials. A dark brown precipitate of copper oxide (CuO)/zinc oxide (ZnO) nanoparticles (NPs), pH 5)) was formed. The precipitate was washed (with distilled water) and collected via a combination of sonication (10 min) and centrifugation (10 min, 1000 rpm). 70% yield of the nano-powder was achieved and was dried in an oven at 65 °C, 1h. After crashing the prepared Cu/Cu2O/CuO/ZnO nanocomposite into a fine powder using mortar and pestle, the nanomaterials were annealed at different temperatures (100 °C, 200 °C, 300 °C, 400 °C, 500 °C and 600 °C) after which they were characterised by different microscopic and spectroscopic techniques. The metal salts/precursors interacted and formed a coordination with the polyphenols after which a possible mechanism of interaction was proposed (scheme 1). Briefly; (Zn(OAc) and Cu(OAc)) dissociate into matal ion (Zn2+ and Cu2+) when in solution. Thus,  polyphenolic compounds contained in the extract of punica granatum L have hydroxyl and ketonic groups which are able to bind to metals and reduce  the metal salt and provide stable-capped NPs while preventing agglomeration. After coordination and chelation of the ligands to the metal ions, a complex was formed (Zn2+/Cu2+-punicalagin). Furthermore, to substantiate this coordination, chelation and formation of the NPs, a comparison study of the Raman spectra between i.e., prepared NPs at RT, 100 °C, 200 °C, 300 °C, 400 °C, 500 °C and 600 °C, was carried out, Fig. 2.

Antibacterial activity

Antibacterial activity of Cu/Cu2O/CuO/ZnO nano-oxides was monitored against four bacterial strains i.e., gram positive (S. aureus-MRSA-33591, S. aureus-25923) and gram negative (E. coli-K12, P. vulgaris- ATCC-49132), bacteria species by modified Kirby-Bauer disk diffusion method [20; 21]. Briefly, the four bacterial strains were grown in nutrient broth at 37 °C until the bacterial suspension reached 1.5 × 108 CFU mL-1 whilst 20 mL of sterile autoclaved molten Mueller-Hinton agar was introduced into four petri-dishes after which was allowed to cool and solidify (30 min). The bacterial suspensions were swapped over the different specified petri-dishes containing the agar. Small sized disks were loaded in different size and shapes of the nanocomposite (Cu/Cu2O/CuO/ZnO) at different annealing temperatures ((RT, 100 °C, 200 °C, 300 °C, 400 °C, 500 °C and 600 °C). Furthermore, the disks were also loaded in different concentrations (5 – 30 mg mL-1) of the nanocomposite annealed at 600 °C. The compounds were dispersed in sterile water

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and were used as negative controls. Simultaneously the standard antibiotics were used as positive controls and were tested against the bacterial pathogens. Then the plates were incubated at 37 °C for 24 h, the inhibition zone was measured in millimeter (mm) for every dish. The obtained results were compared with the standard antibiotic-Gentamicin.

Minimal inhibitory concentration (MIC) assay

The MIC were determined by a broth macro-dilution method, using LB broth and final inocula of 105 and 106 cfu/ml. Five different concentration of Cu/Cu2O/CuO/ZnO nanocomposite were prepared and tested against S. aureus-MRSA-33591, S. aureus-25923, E. coli-K12, and P. vulgaris- ATCC-49132. Sterile centrifuge tubes were prepared for each bacterial inoculum to cover the various concentrations (10, 5, 2.5, 1.25, 0.625 mg/ml) of Cu/Cu2O/CuO/ZnO nanocomposite in triplicate. One mL volumes of each concentration of Cu/Cu2O/CuO/ZnO were transferred to the tubes accordingly, containing final inoculums of 105 cfu/mL test bacteria and the tubes were mixed thoroughly and incubated at 37°C, overnight. Thus the MIC is the lowest concentration of antimicrobial agents (nanocomposite) that completely visually inhibits 99% growth of the microorganisms. The MIC measurement was done in triplicate to confirm the value of MIC for each tested bacteria.

III. INSTRUMENTATION AND APPARATUS

Morphological studies and particle distribution of Cu/Cu2O/CuO/ZnO nanocomposite was carried out using the high resolution transmission electron microscopy (HR-TEM -Philips Technai-FE 12 TEM instrument operated at an accelerating voltage of 120 kV). Structural properties and mechanism of the prepared nano-oxides was determined by using confocal Raman spectroscopy while TGA and DSC were used to determine the crystalline nature of the nanocomposite. UV-vis measurements were performed using the ocean view fibre optics LED.

IV. RESULTS AND DISCUSSIONS

Morphology and composition

To ascertain the shape and size of the prepared nanocomposite (Cu/Cu2O/CuO/ZnO) at different annealing temperatures (a): RT: room temperature, (b): 100 °C, (c): 200 °C, (d): 300 °C, (e): 400 °C, (f): 500 °C high resolution-TEM was used (Fig.1 (a-f)). The effect of temperature on the morphological structure of the prepared nanocomposite was also revealed. TEM image revealed nanoclusters (Fig.1 (a and b)) which changes to spherical nanoparticles (Fig.1 C) and then changes to a hexagonal cube like structures decorated with spherical nanoparticles (Fig 1 (a and b)), when subjected to different temperatures. From TEM images, the average diameter (n = 10) of the nanocomposite

was estimated to be 40 ± 5 nm meanwhile the uni-directional lattice fringes and poly-crystaline nature of the nanocomposite was explained in our previous work. Conclusively, the results provided evidence that a change in temperature played a role in the shape and structural-morphology of the prepared nanocomposite. In their paper, Mageshwari and others [22; 23; 24] found similar behaviour of the synthesised NPs and nanocomposites.

Figure 1 HRTEM images of Cu/Cu2O/CuO/ZnO nano-composite at (a) RT, (b) 100 ºC, (c) 200 ºC, (d) 300 ºC, (e) 400 ºC and (f) 600 ºC.

To assess the crystal quality or the amount of defects and the nature of the electron phonon interaction Raman analysis has been carried out on the prepared samples. Raman spectrum showed characteristic phonon frequencies, spin–phonon interaction and size dependent electron phonon scattering of the annealed Cu/Cu2O/CuO/ZnO nanocomposite. The nanocomposite revealing a combination of ZnO has a wurtzite structure which belongs to the space group C6 with two formula units per primitive cell where all atoms occupy C3v while CuO NCs belong to the C62h space group with two molecules per primitive cell [25], Figure 3. Zone centre optical phonons predicted by group theory of each nanomaterial and nanocomposite are as follows [25; 26; 27; 28];

ΓCuO = A1+2E2+E1ΓZnO = A + 2B + 4A + 5B

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ΓCu2O/CuO/ZnO = A + 2B + 4A + 5B + A1+2E2+E1

Figure 2 Raman spectra of Cu/Cu2O/CuO/ZnO nanocomposite annealed at different temperature (RT- 600 °C).

Figure 2 shows weak Raman peak at 96 cm−1, which corresponds to vibration modes of Cu2O [27; 29]. Meanwhile, the relative weaker peaks of 180 and 307 cm−1 can be contributed to infrared vibration mode of Cu/Cu2O/CuO which have phonons of Γ = 2ELow + Bg whilst at 433 cm−1 there’s clear evidence of ZnO corresponding to Γ = 2Ehigh and 2ELow. The low frequency mode E2 is associated with the non-polar vibration of the heavier Zn sub-lattice while the high E2 mode involves the displacement of lighter oxygen atoms. Meanwhile, between 596-633 cm−1 two prominent peaks were observed and due to CuO (Γ = 2ELow + Bg) and mixture of Cu2O/CuO/ZnO (Γ = Quasi 1ELow + Bg) [19; 22]. The vibrational modes at 1200 - 1400 cm-1 were associated with the main ingredients within the extracts i.e., polyphenolic or vibrational ligands. In addition, the nanocomposite show visible characteristic peaks at high annealed temperatures such 300 - 600 °C. The presence of ZnO within the nanocomposite was noted at 200 °C. Meanwhile, the mixture of the bimetallic nano-oxides was eminent as we increase the calcinations temperatures (from 300 - 600 °C). From the obtained results there were no peak shifting observed due to size effects. Thus, it was scientifically sound to say the formation and crystallinity of the prepared nanocomposites is dependent to temperature. The results were found to be in union with others findings [27; 30]. Finally, the present study indicates the possibility of preparing high quality nanocomposite of metal oxides from a multiple precursor using the simple method as adopted in the present study.

Stability and crystallinity (DSC and TGA)

To determine the purity, mechanism of interaction and confirm the crystalline nature, differential scanning colorimetry (DSC) and thermal gravimetric analysis (TGA) of Cu/Cu2O/CuO/ZnO nanocomposite were carried out, Fig.3. Fig. 3 (a-e)

shows combined plots of DSC and TGA at different annealing temperatures (RT, 200 °C, 300 °C, 500 °C and 600 °C). From the TGA curve, it is evident that there’s weight loss (peak 1 - 4) from the nanocomposite (RT- 300 °C). Peak 1 - 4 may be due to the decomposition of the condensation dehydration of the polyphenols (especially kenotic (–C=O) and hydroxyl (OH-) group). Notably, from 500 °C and 600 °C the TGA plots reveal no weight loss of the nanocomposite. Thus, it is plausible to conclude that raising the calcination temperatures from 500 °C to 600 °C simultaneously eliminates the strong OH- and the already rather weak –C=O absorptions, suggesting thermally stable and pure Cu/Cu2O/CuO/ZnO nanocomposite. The results are in agreement with our previous FTIR results [31]. The DSC measurements of the prepared Cu/Cu2O/CuO/ZnO nanocomposite were also investigated at different calcination temperatures, Fig.3 (a-e). From the thermograms four exothermic peaks were evident at 90 °C, 250 °C (Fig. 3 (a-c) peaks 1-3), 300 °C and 330 °C (Fig. 3 (d-e) peaks 1-2), respectively. These peaks are attributed to the evaporation of water and organics. A large exothermic peak at 330 °C (Fig.3 (d) peak 2) exhibits the poly-crystallization of Cu/Cu2O/CuO/ZnO nanocomposite. Therefore, the DSC measurements proved the poly-crystallization of Cu/Cu2O/CuO/ZnO nanocomposite occurred at temperatures over 350 0C which is in agreement with the data acquired from both SEAD and XRD measurements [31].

Figure 3 Thermal analysis of Cu/Cu2O/CuO/ZnO nano-composite subjected to different temperatures (RT- 600 °C).

Optical properties

The optical properties of the nanocomposite with average particle sizes of 5 - 20 nm were

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revealed and confirmed by UV–visible spectra, Figure 4 (a). A comparison was made between different calcination temperatures with the aim of showing a decrease or increase in optical band gap energies of the nanocomposite. Two main absorption peaks were noted at λmax = 288.7 and 436.4 nm (RT - 200 °C), respectively. However, when subjected to high calcination temperatures the two absorption peaks disappear and a new absorption peak at (λmax = 367.5 nm) with new products materialized. The peaks at λmax = 288.7 and 436.4 nm are matched to the inter/band edge transition of Cu/Cu2O (RT, 100 °C and 200 °C) from deep level electrons of valance band and at λmax = 367.5 nm are attributed due to the complete formation of Cu/Cu2O/CuO/ZnO when subjected to 300 °C, 400 °C and 600 °C temperatures. The corresponding optical band gap energies were calculated using the planck’s equation [24] and were found to be 4.3 eV (288.7 nm), 3.3 eV (367.5 nm) and 2.8 eV (436.4 nm), Figure 4 (b). Worth to note there’s a remarkable decrease in absorption peak intensities with increasing temperature and as we move from low to higher wavelength the conductivity of the nanocomposite (NC) become obvious, evident by a decrease in band gap energies. Figure 4 (b), shows an energy diagram which distinguishes the effect of temperature and the energy required to move electrons. It is clear from the diagram that at low temperature p-type Cu/Cu2O requires more energy for an electron to emit/move from low (LUMO) to higher levels (HOMO) due to high band gap energies 4.3 eV. However, the photon energy required to emit electrons decreased due to the more conducing Cu/Cu2O (2.8 eV) and Cu/Cu2O/CuO/ZnO (3.4 eV) NCs as we go higher in temperatures. Conclusively, it is evident that preparing the NCs at high temperature is a need due lower photon energy required to shuttle an electron from ground state to the conduction state. Further, the nanostructured materials showed high conductance and absorptive properties thus confirming their use in different fields such as solar absorber, optoelectronics, catalysis and many others.

Figure 4 UV-VIS spectra of (a) Cu/Cu2O/CuO/ZnO nano-composite at different calcinations temperatures (RT, 100 ºC, 200 ºC, 300 ºC, 400 ºC and 500 ºC) and (b) Energy diagram of the as-synthesised nanocomposite.

Antimicrobial activity

The study was also aimed to determine the antimicrobial efficacy of green synthesized Cu/Cu2O/CuO/ZnO against various bacterial pathogens such as E coli-K12, P Vulgarias-ATCC-49132, S aureas-MRSA-33591 and also when subjected to streptomycin and gentamicin antibiotics. Antimicrobial tests (Zone of inhibition and minimum inhibitory concentration) were performed on three different bacterial pathogens. The applications were performed by varying calcination temperatures but same concentration of the prepared Cu/Cu2O/CuO/ZnO nanocomposite, Figure 5.

Figure 5 Represent zone of inhibition of Cu/Cu2O/CuO/ZnO nanocomposite tested on various bacterial species (E coli-K12, P Vulgarias-ATCC-49132, S aureas-MRSA-33591).

The antimicrobial activity of green prepared Cu/Cu2O/CuO/ZnO nanostructures at different calcinations temperatures (RT, 100 ºC, 200 ºC, 300 ºC, 400 ºC, 500 ºC and 600 ºC but same concentration 10 mg mL-1 , see table 1) towards various bacterial pathogens were tested by the well and disc diffusion agar methods (Fig. 5). The presence of inhibition zone clearly indicates that the mechanism of the biocidal action of Cu/Cu2O/CuO/ZnO nanostructures involves disruption of the membrane while generating surface oxygen species to kill pathogens [15].

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The study reveals that as we change the calcination temperatures and/or structure of the prepared nanocomposite, the growth of inhibition increased as a function of temperature and structural change. Worth to note, temperatures at 500 ºC and 600 ºC show high zone of inhibition compared to low temperatures (RT, 100 ºC, 200 ºC, 300 ºC, 400 ºC). Amongst bacterial pathogens, maximum rate of activity was noted for S aureas-MRSA-33591 followed by P Vulgarias-ATCC-49132 then E coli-K12, Fig. 5. Compared to the nanocomposites at low calcination temperatures, the antibiotics reveal slightly higher zone of inhibition as was expected (Fig.5). However, the results confirm that the nanocomposite at higher calcinations temperature can be better inhibitors relative to the antibiotics.

Table 2 The minimal inhibitory concentration (MIC) of Cu/Cu2O/CuO/ZnO nanocomposite on various bacterial pathogens.

Bacteria MIC (Conc. mg mL)E coli K12 1.25P Vulgarias-ATCC-49132 2.5S. aureus-25923 5

To determine the minimum inhibitory concentration (MIC) of the prepared Cu/Cu2O/CuO/ZnO nanocomposite at 600 ºC (chosen for high inhibitory activity), different concentrations (10, 5, 2.5, 1.25, 0.625 mg mL-1) of the nanocomposite were investigated. Table 2 reveals the MIC values (Defined as the lowest concentration of NPs that inhibits the growth of a microorganism) of Cu/Cu2O/CuO/ZnO nanocomposite. From the MIC results (table 2), the green prepared Cu/Cu2O/CuO/ZnO nanocomposite with haxegonal-like structures (at 600 ºC) showed enhanced inhibitory activity at lower concentration for E coli (1.25 mg mL-1) and P vulgaraias (2.5 mg mL-1) whilst slightly higher for S aureus (5 mg mL-1). The calculated MIC values are in agreement and slightly lower than those reported by other researcher [3; 32; 33]. The results show that a change in structure, size, concentration and calcination temperatures of the nanocomposite play a vital role in the inhibition studies of different bacterial pathogens.

V. CONCLUSIONS

Conclusively an enhanced antimicrobial agent of Cu/Cu2O/CuO/ZnO nanocomposite was achieved through bio-reduction process. TGA and DSC measurement were able to determine the purity and crystalline nature of the nanocomposites. Further, the conductivity and optical band gap of the ternary oxides were achieved by UV-vis spectroscopy. The results prove that the ternary-metal oxide can be used in catalysis and solar absorbers. Cu/Cu2O/CuO/ZnO nanocomposite showed excellent antimicrobial activity against three pathogenic bacteria. Further, the nanocomposites showed the least MIC compared to other studies. Thus, Cu/Cu2O/CuO/ZnO nanocomposite confirmed its potential for external uses as an environmental and biomedical antibacterial agent. Furthermore, nanocomposite can aid in surface coatings on various substrates to prevent microorganisms from attaching, colonizing, spreading, and forming biofilms in indwelling medical devices. The results show that a change in structure, size, concentration and calcination temperatures of the nanocomposite play a vital role in the inhibition studies of different bacterial pathogens.

VI. ACKNOWLEDGEMENTS

The authors are thankful to the UNISA/UNESCO African chair and NRF for funding. We acknowledge the support extended by iThemba Labs and the Department of Materials Division (MRD) for providing us with state-of-the-art facilities and instrumentation.

VII. COMPETING INTERESTS

The authors declare that they have no competing interests.

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Table 1 Antibacterial activity of different size and shapes of CuO NPs on various bacterial species (E. Coli-K12, P. Vulgarias-ATCC-49132, S. Aureas-MRSA-33591).

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