Low‐temperature synthesis of hierarchical structures of copper oxide and their superior biological activity

Abstract

In this work, the authors report a facile low‐temperature wet‐chemical route to prepare morphology‐tailored hierarchical structures (HS) of copper oxide. The preparation of copper oxide collides was carried out using varying concentrations of copper acetate and a reducing agent at a constant temperature of 50°C. The prepared HS of CuO were characterised by powdered X‐rays diffraction that indicates phase pure having monoclinic structures. The morphology was further confirmed by field‐emission scanning electron microscope. It reveals a difference in shape and size of copper oxide HS by changing the concentration of reactants. In order to evaluate the effect of H₂ O₂ on CuO NPs, the prepared CuO are modified by treatment with H₂ O₂. In general trend, CuOH₂ O₂ collide showed enhanced protein kinase inhibition, antibacterial (maximum zone 16.34 mm against Staphylococcus aureus) and antifungal activities in comparison to unmodified CuO collides. These results reveal that CuO HS exhibit antimicrobial properties and can be used as a potential candidate in pharmaceutical industries.

1 Introduction

Microbes are a major source of infectious diseases throughout the world. Various efforts have been made to fight against microbial diseases. Majority of conventional drugs are unable to show their potential effects against microbial infections, this is due to the antibiotic resistance developed in microbes [1]. In recent years, inhibition of microbial infections has been the subject of universal concern in healthcare systems. This demands the development of effective material having antimicrobial properties. In this perspective, inorganic NPs exhibit greater potential due to their excellent catalytic, optical, and antibacterial characteristics [2]. Silver NPs are extensively studied as antibacterial agents against a variety of bacteria [3], but their cytotoxicity for normal cells restricts their uses as an antibacterial agent [4]. Besides silver, other NPs and their collides like Au‐NPs [5], ZnO‐NPs, carbon nanotubes, SiO₂ [6], TiO₂ ‐NPs [7], Co‐NPs, and Ni‐NPs [8] have shown toxicity towards normal cellular processes which limit their applications, especially in health sciences. CuO is an interesting multifunctional narrow bandgap p‐type semiconductor having tremendous physiochemical properties [9]. In general, copper oxide with diverse properties such as non‐toxicity, chemical stability, electrochemically active, copious availability of constituents, and little fabrication cost made them as a potential agent which have been extensively explored in plenty of fields such as gas sensors [10], catalysis [11], batteries [12], high‐temperature superconductors [13], solar energy conversion [14], antimicrobial agent etc. [15].

Owing to the fascinating properties, several methods have been in use for the synthesis of CuO‐NPs including hydrothermal [16], spin‐coating [17], electro‐deposition [18], sol–gel [19], solvothermal [20], thermal oxidation [21], and sonochemical [22]. The major limitation linked with these fabrication techniques is a synthesis of multi‐products such as CuO, Cu₂ O, Cu₃ O₄, and metallic copper that disturbs the properties of the product [23]. Furthermore, these methods are expensive and need special equipment for reactions. Wet chemical approaches are simpler and cost‐effective and use the aqueous medium for the synthesis of copper oxide hierarchical structures (HS) through calcination of copper hydroxide or hydroxyl carbonates [24]. These approaches are useful for the production of more homogeneous composition with better crystallite size [25].

The objective of our current research was to synthesise CuO NPs using low‐temperature‐mediated simplest chemical wet method and studied their influence on microbial growth. Furthermore, the colloidal formation of CuO with hydrogen peroxide and its applications as protein kinase inhibitor, antibacterial, and antifungal activity as compared to unmodified CuO NPs are also studied.

2 Materials and methods

2.1 Chemicals and glassware

Analytical grade solvents and chemicals were used without further purification. Hydrogen peroxide (35%), copper acetate monohydrate, DMSO, and NaOH was purchased from Merck. ISP4 medium (Difco Laboratories), TSB medium (Sigma‐Aldrich), and Luria broth and SDA (Oxide) were used as a medium in biological assays. X‐ray diffraction (XRD, X'pert PRO of PANalytical Company), centrifuge (Heraeus biofuge primo R. Thermo electron corporation), and scanning electron microscope (SEM) were the instruments used for the study.

2.2 Synthesis of CuO HS

Fabrication of copper oxide HS was carried out in three reactions by varying concentration of precursor and reducing agent (NaOH) at a temperature of 50°C. Briefly, in reaction 1, an equimolar concentration of (0.5 M) precursor and the reducing agent was used, reaction 2 comprised 0.5 M precursor and 1 M NaOH, whereas reaction 3 contained 0.25 M precursor and 1 M NaOH (Table 1). Each reaction was carried out in 250 ml flask, the desired concentration of NaOH solution was initially warmed at required temperature following dropwise addition of copper acetate monohydrate under constant stirring at 500 rpm for 2 h. The appearance of brown colour indicates the completion of the reaction. The reaction was stopped by centrifugation of mixture at 3000 rpm for 5 min. The obtained pellets were washed thrice with deionised water and HS were dried at 80°C for 3 h.

Table 1.

Experimental conditions and size of CuO HS synthesised

Experiment no.	Copper acetate, M	NaOH, M	Temp., °C
1	0.5	0.5	50
2	0.5	1	50
3	0.25	1	50

2.3 Characterisation of CuO HS

Characterisation of CuO (HS) was made by powder X‐ray diffraction (PXRD) and field‐emission scanning electron microscopy (FE‐SEM). The XRD patterns with diffraction intensity versus 2 h were recorded using X'pert PRO of PANalytical company. The XRD pattern measurements of drop‐coated film of CuO NPs on glass substrate were documented in a wide range of Bragg angles θ at a scanning rate of 2θ angles/min. Operating voltage of the generator is of 40 kV with a 30 mA current provided at the room. The scanning range was selected between 10° and 100° using nickel monochromatic Cu Ka radiation (θ  = 1.5406 Å), NaI detector, variable slits, and a 0.050 step size. The result of PXRD was compared with standard JCPDS database values. The mean particle size and diameter of the copper oxide HS were calculated by the width of the XRD peaks, i.e. the data of full‐width at half‐maximum (FWHM) assuming that they are free from non‐uniform strains, using the Scherrer formula. D  = kλ /ß cos θ, where D is the average crystalline domain size perpendicular to the reflecting planes, K is the Scherrer coefficient (0.85), λ is the X‐ray wavelength (1.5406 Å), ß is the angular FWHM in radians, and θ is the diffraction angle [2θ (deg) is the measured angle of diffraction in degrees] or Bragg's angle. For morphological characterisation of CuO HS, SEM was carried out by using Jeol JSM 6610LV SEM.

2.4 Treatment of synthesised CuO HS with H₂ O₂

In order to analyse the effect of hydrogen peroxide on CuO HS, dried powder of CuO (25 mg) was mixed with 12 ml of distilled water and 8 ml of 36% H₂ O₂. Stirring of this mixture was done for 6 h at room temperature and centrifuged at 3000 rpm for 5 min.

2.5 Biological assays of CuO HS

For biological evaluation, the synthesised and modified CuO HS were suspended in DMSO at 4 mg/ml and sonicated for 10–15 min before each use.

2.6 Protein kinase inhibition assay

Protein kinase inhibition assay was performed by following a procedure developed earlier [26]. The ISP4 medium was used for the production of Streptomyces (largest genus of Actino‐bacteria) spores while liquid TSB medium was used for mycelium propagation. Streptomyces culture was refreshed on TSB medium in a shaker incubator at 28°C. An aliquot of 60 μl of refreshed culture was taken in an eppendrof tube and mixed with 540 μl of sterile TSB media. The ISP4 medium was autoclaved and 25 ml was poured into sterile Petri plates and allowed to solidify. Sterile cotton swabs were used to culture inoculum homogeneously over the entire surface of the Petri plates softly. The discs of 6 mm diameter were placed on the surface of Petri plate along with the 5 µl of CuO HS (4 mg/ml in DMSO) and negative control (DMSO) was poured carefully on the discs. After incubation of plates at 37°C for 24 h; the diameter of the zone of inhibition was measured.

2.7 Antibacterial assay

Antibacterial assay was performed against four bacterial strains, i.e. Gram‐positive bacterial strains like Staphylococcus aureus (ATCC 6633), Micrococcus luteus (ATCC 10240) and Gram‐negative bacterial strains include Salmonella typhimurium (ATCC) and Escherichia coli (ATCC). Disc diffusion assay was adopted for determination of antibacterial potential as described previously [26]. On each disc, 5 μl of 4 mg/ml of sample was poured. Discs were placed on the agar plate containing inoculated test bacterial strain. DMSO was used as negative control while roxrithromycin was used as positive control. Zone of inhibition was measured after 24 h.

2.8 Antifungal assay

Antifungal assay was analysed against Aspergillus fumigatus (FCBP‐66), Mucor species (FCBP‐0300), Aspergillus niger (FCBP‐0198), and Aspergillus flavus (FCBP‐0064). Disc diffusion method was used for antifungal activity analysis [26]. SDA (Sabouraud dextrose agar, pH 5.7) was autoclaved and poured in Petri plates under sterile conditions. Fungal lawns were prepared by inoculating spores on the surface of the media after that discs loaded with 5 μl of 4 mg/ml sample and were placed on the surface of Petri plate. The plates were incubated at 25°C for 1 day and zone of inhibition was measured, respectively.

2.9 Statistical analysis

All these tests were done in triplicate and the results were examined statistically through SPS in order to define average difference between the mean values.

3 Results and discussion

CuO (HS) were synthesised by wet chemical method using different concentrations of copper acetate monohydrate and sodium hydroxide at 50°C. Three sets of reactions were carried out by varying concentrations of both precursor and reducing agent (NaOH). In reaction 1, equimolar ratio of both precursor and NaOH; in reaction 2, concentration of reducing agent was increased to twofolds while concentration of precursor remained constant, while in reaction 3, concentration of precursor was reduced to twofolds on the other site while reducing agent was kept constant as used in reaction 2. XRD analysis of these HS of CuO fabricated in reaction 1 showed six distinct peaks at 2θ degrees ranging from 35.52° to 68.80°, i.e. at 35.52°, 38.72°, 48.76°, 58.36° 61.20°, and 66.00° (Fig. 1). Phase formation was proved from characteristic peaks which can be recognised and indexed to the (002), (111), (202), (202), (113), and (311). The peak pattern of CuO HS synthesised in reaction 2 was ranged from 35.41° to 68.20° likewise the peak pattern ranged between 35.3° and 68.0° was observed for CuO HS synthesised in reaction 3. All the peaks of different samples of CuO NPs verify monoclinic geometry of CuO Hs (space group: C2/c, space group no: 15, reference code: 00‐001‐1117, JCPDS card no. 45‐0937) which were closely matched with the values reported by other scientists [27]. No characteristic peaks of any impurities were noticed, recommending that highly pure CuO NPs was synthesised. Particle size has been calculated from the XRD pattern using the Scherrer's equation, i.e. D  = Kλ /β cos θ, where K  = 0.9 is the constant factor, λ = 1.54°A is the wavelength of X‐ray which act as a radiation source of Cu Kα radiation, θ is the Bragg angle of diffraction, and β is the FWHM, i.e. angular FWHM of the respective diffraction peaks. Copper oxide HS formed by wet chemical method were of 18.06, 17.34, and 15.4 nm, respectively. The existence of sharp structural peaks in XRD patterns and crystalline size <100 nm recommended the nanocrystalline nature of CuO NPs.

Fig. 1.

XRD analysis of copper oxide (HS) synthesised by varying concentration of precursor and reducing agent

(a) XRD of CuO HS by using equimolar concentration (0.5 M) of precursor and reducing agent, (b) XRD of CuO HS by using 0.5 M precursor and 1 M reducing agent, (c) XRD of CuO HS obtained by using 0.25 M precursor and 1 M reducing agent

Synthesised CuO (HS) were subjected to SEM. By varying the concentrations of precursor and reducing agents, different morphological structures were observed are shown in Fig. 2. Fibre‐like structure was obtained in reaction 1 which arranges itself into a definite French fries‐like structure in reaction 2, when the concentration of precursor was further reduced in reaction 3 more regular sea urchin‐like structure was observed. It shows that when the concentration of copper acetate monohydrate was reduced more ordered structure was formed due to controlled reaction rate because of low concentration of reactants. While those of sodium hydroxide, in general basis, was also same as was observed for copper salt that low concentration was responsible for good morphology, but it was also observed that when the concentration of sodium hydroxide was increased, it favoured good structures. This might be due to the role of sodium hydroxide in HS synthesis. Sodium hydroxide combined with copper acetate monohydrate to form copper hydroxide, which is then decomposed to copper oxide. Since heating was used to speed up the reaction, no intermediate copper hydroxide precipitates were observed. On the other hand, as it is a strong electrolyte, and it may neutralise the surface charges of the CuO HS, it prevents them from possible crystalline aggregation. Finally, the use of high‐concentrated sodium hydroxide may create diffusion layers on certain surfaces of CuO, which may create an additional growth anisotropy, allowing only energetically favourable crystallographic planes to grow [28].

Fig. 2.

SEM images of CuO HS

(a) Fibre‐like structure of CuO HS obtained by using equimolar concentration (0.5 M) of precursor and reducing agent, (b) French fries‐like structure of CuO HS was observed with 0.5 M precursor and 1 M reducing agent, (c) Sea urchin‐like structure of CuO HS were obtained by using 0.25 M precursor and 1 M reducing agent

3.1 PK assay of modified and unmodified CuO (HS)

The results show that modified copper oxide HS are more effective as compared to unmodified copper oxide HS. Maximum zone of inhibition of 28.7 mm was shown by CuO 2 (H₂ O₂), while its unmodified form shows 16.4 mm that clearly showed that modification leads to increased lethality against Streptomyces. Least activity was observed for unmodified CuO 1 (11 mm) and its modified form showed high zone of inhibition of 21.8 mm and intermediate activity was observed for CuO 3, i.e. 19.8 and 22.6 mm for unmodified and modified nanoparticles (Fig. 3). Higher activity was observed for CuO 2 that might be due to the effective particle size, i.e. 17.34 nm which was calculated from XRD analysis by using Scherrer and Debey's equation. The Streptomyces exhibit mycelial growth on solid media. When nutrient supply will be limited, it undergoes differentiation which leads to the development of septic aerial hyphae to generate spores. All such instructions reside at transcription level and perhaps an alteration of proteins occurred after translation [29]. All cellular processes such as differentiation, chemical reactions or growth and development were dependent or regulated by protein phosphorylation in Streptomyces. On the basis of this knowledge, a wide range of protein kinase inhibitors had been isolated which play their role in monitoring systems for signal transduction and differentiation of cell [30]. Protein kinase involved in the transfer of the phosphoryl groups to their substrate protein from adenosine triphosphate. As protein phosphorylation is a major process which regulates its action, and hence is involved in cell signalling. If protein kinase got anything which distrusts its functional activity, it, in short, results in loss of regulation of processes that are taken place in the cells [31]. Due to this loss of regulation kinases being vital therapeutic targets which stimulate present‐day interest in the development of kinase inhibitors [32].

Fig. 3.

Protein kinase assay of modified and unmodified CuO (HS). Mean values (± standard errors) followed by the same letter are not significantly different after LSD test (p ≤ 0.05)

3.2 Antibacterial activity of CuO NPs

Following four strains, i.e. S. aureus, E. coli, M. luteus, and S. typhi, were tested against 4 mg/ml DMSO concentration of CuO HS. In Table 2 (zone of inhibition), designated CuO exhibited better antimicrobial activity against Gram‐positive bacteria. The inequality in the resistance and sensitivity towards both Gram‐negative and Gram‐positive bacterium might be owing to the diversity in the metabolic pathways, cell organisation, body processes, or the extent of interaction between HS and bacterium. In our case, greater activity was observed against S. aureus among Gram‐positive bacteria that might be due to the presence of a large number of carboxyl groups and amines on the surface of their cells that exhibit maximum affinity of Cu towards these functional groups. While on the other hand, mild to low activity was observed against E. coli, a Gram‐negative bacteria that might be due to unique cell membrane structure that help bacterium to resist against antimicrobial agents. Besides all this, other features such as the rate at which HS diffuses also play a crucial role against various bacterial strains [33]. HS of CuO were active against a wide range of bacterial strains that could be picked up from hospitals [34], but such bactericidal action needs a high concentration of nano CuO. Some studies had been carried out in order to explain the machinery of bactericidal action which HS play. Gram‐positive bacteria S. aureus are more resistant to silver HS in comparison to the Gram‐negative bacteria E. coli [35] and reveals that perhaps this might have been caused by the cell‐wall thickness due to the peptidoglycan layer that might avert, to some degree, the action of HS. On the other hand, Ruparelia et al. [36] established that some types of E. coli were unaffected as compared to S. aureus to silver HS. Hence, the bactericidal potential of HS and CuO HS not only rely on the membrane's structure; CuO could also bind with DNA molecules and cause distortions in the helical structure by interacting with DNA molecule. Cu ions may also disturb metabolic processes but the actual mechanism of the bactericidal effect of CuO HS is not known until now and needs further attentions in this regard (Table 2).

Table 2.

Antibacterial activity (zone of inhibition) of copper oxide HS

Zone of inhibition
Sample	E. coli	Sa. typhimurium	M. lutues	S. aureus
CuO 1	8 ± 0.6	9.34 ± 0.3	7.34 ± 0.3	10.17 ± 0.6
CuO 2	8.34 ± 0.3	10.67 ± 0.6	7.17 ± 0.3	16.34 ± 0.6
CuO 3	8.67 ± 0.3	8.34 ± 0.3	7.34 ± 0.3	13.34 ± 0.6
CuO 1(H2 O2)	7.67 ± 0.3	10.34 ± 0.3	9.34 ± 0.3	16.34 ± 0.3
CuO 2 (H2 O2)	7.17 ± 0.3	10.67 ± 0.3	7 ± 0.5	16.17 ± 0.3
CuO 3‐(H2 O2)	7.34 ± 0.3	8.34 ± 0.3	10.34 ± 0.3	16 ± 0.5

3.3 Antifungal activity of modified and unmodified CuO (HS)

The fungal strains used, A. fumigatus, A. flavus, A. niger, and F. solani, were tested against 4 mg/ml solution of CuO (HS). DMSO was used as a solvent. Weak to mild activity was observed against all strains (Table 3). CuO (HS) synthesised by wet chemical method exhibit antifungal activity against various strains showing their use in future as antifungal agents. CuO HS, in fact, have no antifungal activity but as they slowly oxidise into cupric ions which are then available in the biological system and able to produce lethal hydroxyl free radicals in the immediate vicinity of the lipid membrane. These free radicals generate oxidative deterioration of lipids which form cell membrane [37] which, in fact, cause outflow of K⁺ ions and other substances from the cell [38], which lead to disturbance due to major chemical reactions inside cells [39] causing cell death. In our results, enhanced activity was observed with modified CuO HS, it was because H₂ O₂ oxidises CuO HS quickly or to a greater extent in comparison to unmodified HS. The efficiency of the CuO NPs as antifungal agents is also evidenced by the previous results [40]. While in case of A. niger, different behaviour was observed that unmodified HS are more effective against A. niger than modified one, this may be due to the tendency of A. niger to favour accumulation of H₂ O₂ which triggers the production of secondary metabolites like phytoalexin [41]. Various suspensions of plant cultures show the accumulation of H₂ O₂ during stimulation by the elicitor from the fungus [42].

Table 3.

Antifungal activity (zone of inhibition) of copper oxide HS

Antifungal activity of modified and unmodified CuO HS
	A. fumigatus	F. solani	A. niger	A. flavus
CuO 1	7.833 ± 0.3	6.67 ± 0.3	9 ± 0.3	7.17 ± 0.3
CuO 2	8.17 ± 0.3	7.84 ± 0.3	7.84 ± 0.3	7.17 ± 0.3
CuO 3	9.67 ± 0.3	7.84 ± 0.3	10.84 ± 0.3	10.5 ± 0.3
CuO 1 (H2 O2)	8.67 ± 0.3	7.67 ± 0.3	7.67 ± 0.3	7.34 ± 0.3
CuO 2 (H2 O2)	8.34 ± 0.3	8.34 ± 0.3	7.34 ± 0.3	7.67 ± 0.3
CuO 3 (H2 O2)	9.84 ± 0.3	9.84 ± 0.3	9.67 ± 0.3	8.84 ± 0.3

4 Conclusion

CuO HS were successfully synthesised by chemical wet method and then they were modified by hydrogen peroxide treatment. The effect of different concentrations of copper acetate monohydrate and sodium hydroxide under the constant temperature conditions on the shape and size of particles were investigated. Different sizes and shapes of CuO HS could be obtained by simple variations of concentration. The XRD results confirm the formation of pure‐phase CuO with monoclinic structure. Comparative study was done to study effect of unmodified and modified CuO HS. For this purpose, unmodified and modified CuO HS were tested against Streptomyces (protein kinase inhibition assay), antibacterial activity against both Gram‐positive and Gram‐negative bacteria and antifungal activity. The results showed that all the modified CuO HS exhibited good inhibitory effect against Streptomyces, bacteria, and fungi as compared to unmodified CuO HS. Except in case of antifungal activity where unmodified CuO HS are found to be more active against A. niger. Antibacterial activity of the CuO was found to be size dependent. Hence, it was shown that modification of CuO HS by hydrogen peroxide leads to enhanced activities and hence can be applicable in pharmaceutics.