Effect of silver (Ag) doping on structural, optical and antimicrobial properties of copper oxide (CuO) nanostructures

The present report investigates the effect of Ag doping on the structural, optical, and antimicrobial properties of CuO nanoparticles (NPs). CuO NPs were produced at optimized reaction conditions via hydrothermal synthesis. XRD study reveals a decrease in crystallite size with increased in Ag doping (2–6 wt %) in CuO. Ag-doped CuO NPs shows lower band gap values than undoped CuO NPs. FESEM analysis displays pure CuO NPs with spherical pellet-like structures, while Ag-doped CuO NPs have mixed morphologies, i.e., spherical, disc-like, and irregular shapes. EDX spectra confirm the purity of synthesized CuO NPs. Further, the antimicrobial properties of CuO nanostructures were studied against Escherichia coli and Enterococcus faecalis bacteria. CuO NPs functionalized with Ag dopant exhibit significant antibacterial potentials. The antibacterial activity of CuO NPs increased with increasing dopant concentration and in a dose-dependent manner. The gram positive (+ve) E. faecalis is more sensitive to the Ag-doped CuO NPs treatment than a gram negative (–ve) E. coli. Overall this study demonstrates a facile route of synthesizing Ag-doped CuO NPs that can materialize into effective broad-spectrum bactericidal agents.


Introduction
Nanotechnology is expanding at a remarkable pace and has the potential to provide promising opportunities for the development of human well-being [1].Nanotechnology has been employed to fabricate nanomaterials, having applications in different disciplines of science and technology [1].Research curiosity in nanomaterials has amplified significantly because of their exclusive structural, chemical, and physical properties [2].Scientists have focused a great deal of attention on metal oxide nanoparticles (MONPs) for their applications in semiconductors, catalysis, biomedical, antimicrobials, cosmetics, sensing devices, microelectronics, etc [3][4][5].MONPs owing to their unique structural, surface, and physiochemical properties, offer remarkable antibacterial activities [6].CuO NPs are known for their abundance, low-cost, and non-toxic nature [7].CuO NPs have applications in areas such as photocatalysis, solar cells, gas sensors, batteries, electronic devices, medicine (anticancer, antimicrobial potentials), etc [8].CuO NPs could be the source of cost-effective antimicrobial agents compared to Ag NPs and Au NPs [9,10].
Several scientists have used various metals for doping to alter the properties of metal oxide nanostructures [11,12].We can alter the structural, morphological, electrical, magnetic, optical, and chemical properties of CuO NPs with the doping of metal [11].Many researchers reported such changes in the properties of CuO NPs after doping with transition metal ions such as Mg [13], Mn [14], and Ag [15].Adding metal dopants promotes the antimicrobial properties of CuO NPs [6,9].Ag and CuO NPs offer a wide range of biomedical applications [16].Higher antibacterial activity of Ag-doped CuO NPs was observed compared to Zn and Ni-doped CuO NPs [6].However, it may depend upon several factors such as properties of synthesized CuO NPs, type of bacteria, experimental conditions, treatment dose of Cu NPs, etc.The antimicrobial activities of Ag-doped CuO NPs against different bacteria have been reported previously [6,9,[16][17][18][19].In the current study, the effect of Ag doping on the antibacterial activity of CuO NPs has been investigated against gram -ve E. coli and gram +ve E. faecalis.These bacteria in water bodies indicate faecal contamination.Both bacteria are reported to cause hospital-acquired infections like 'catheter-associated urinary tract' infections [20].Different strategies have been adopted so far for treating such infections.However, the ability to acquire antibiotic resistance via horizontal gene transfer [21] necessitated the search for novel antimicrobial platforms against such bacterial strains.
Several methods are reported to synthesize metal oxides nanostructures, like sol-gel, inert gas condensation, solvothermal and hydrothermal method [12].Among these, hydrothermal is an unique approach due to their efficiency and simplicity, high yield and scalability [12,17].The advantage of hydrothermal method are soft solution processing, single-step and low energy consumption.The hydrothermal approach has ability to control the morphology and size of the nanomaterials [18].
To the best of our knowledge and based on a literature survey, this is the first report on the antibacterial activity of hydrothermally synthesized Ag-doped CuO NPs against E. faecalis.The present study focuses on the antimicrobial potential of Ag-doped CuO NPs against two bacterial strains, i.e., E. coli and E. faecalis (associated with hospital-acquired/nosocomial infections).

Experimental section 2.1. Chemicals
The chemicals such as copper sulphate penta hydrate (CuSO 4 .5H 2 O), silver nitrate (AgNO 3 ), and sodium hydroxide (NaOH) were of analytical grade and utilized as such without any further purification for the experiment.All solutions used in the experimental work were prepared in double-distilled water (DDW).

Synthesis of undoped and Ag-doped CuO Nanoparticles
The undoped and Ag-doped CuO NPs were synthesized by low-temperature hydrothermal route using CuSO 4 .5H 2 O and NaOH as precursor salt and mineralizer, respectively.To synthesize CuO NPs, different concentration of copper sulphate was dissolved in 100 ml DDW, and added 1M NaOH drop by drop under mixing for 2 h.
The prepared solution was retained instainless steel autoclave (teflon lined) and kept in a hot air oven at 200 °C for 7 h as shown in figure 1.After that solution was allowed to cool at room temperature, and a precipitate was obtained.This precipitate was splashed multiple times with DDW and acetone to remove contaminants and dried in a hot air oven at 150 °C for 2 h.Finally, the fine powder of pure CuO NPs was obtained by grinding.For Ag-doped CuO NPs, AgNO 3 and CuSO 4 were used as precursors using the above procedure.

Characterization
The crystallite properties (size, structure, and lattice plane) of synthesized Ag-doped CuO NPs were examined by an x-ray diffractometer (Bruker AXS, D8 Advance).The XRD pattern was recorded by Cu-Kα radiation source of 1.54060 Å in the range of 20°to 80°.Field emission scanning electron microscopy (FESEM) was used to examine the surface morphology of CuO NPs (FEI, Quanta 200F), and for compositional analysis, an energy dispersive x-ray analysis (EDX) was performed.UV-vis spectroscopy was used to observe the optical properties of the synthesized undoped and Ag-doped CuO nanostructures.

Antibacterial studies
The antibacterial activity of undoped and Ag-doped CuO NPs was tested against gram +ve E. faecalis and gramve E. coli using a disc diffusion assay.The procedure was adopted, as mentioned in Ghorbi et al [18], with some modifications.The pure bacterial cultures were inoculated to freshly prepared nutrient broth media and maintained at 37 °C for ∼24 h.The respective bacterial inoculum was spread homogeneously on solidified nutrient agar media in Petri plates.The uniform-sized sterile filter paper discs (about 6 mm in diameter each) were dipped in different concentrations of NPs (0 to 1000 ppm) and placed on Petri plates.The discs dipped in DDW were taken as negative control.The Petri dishes were kept in an incubator for ∼24 h at 37 °C, and the zone of inhibition (ZOI) was recorded in mm [6].220) and (311), respectively [22].The observed reflections confirm the presence of a monoclinic phase of the CuO NPs (JCPDS card no.80-1917), which signifies that no other crystalline phase exists related to the Ag-assisted CuO NPs [23].Here, we can state that Ag metal ions are substituted at the appropriate lattice site within the crystal lattice of CuO NPs.The crystallite size (CS) of synthesized doped and undoped CuO NPs was determined by the Debye-Scherer equation given below equation (1):

Result and discussion
Where, D is the average crystallite size, K is the Debye-Scherrer's Constant (0.94), is λ the CuKα-radiation (0.154 nm), β Full width at half maximum (FWHM) of the peak, and θ is the Bragg's angle [23].
The average crystallite size of the undoped CuO NPs was deduced to be ∼33 nm.The average crystallite size is systematically reduced to ∼32 nm, ∼30 nm, and ∼27 nm when Ag doping is increased by 2, 4, and 6%, respectively.We have used 220 plane-oriented diffraction peaks to calculate the crystallite size by Debye-Scherer equation.In addition, we observed the reduction in peak intensity corresponding specifically to the (220) reflection and a subtle shift towards a higher angle in the (111) reflection with increasing dopant content.This suggests that the ionic radii of Ag (0.126 nm) with respect to Cu (0.073 nm) play an important role in tuning the host lattice strain, which could be a major cause for the variation in NPs crystallite size [22].

Optical analysis of undoped and Ag-doped CuO nanostructures
The bandgap (Eg) of the synthesized CuO NPs was calculated by using the Tauc's relation: Where, a= absorption coefficient, n h = incident energy, and the exponent n (n = 2 and n = 1/2 for direct and indirect transition) depends on the type of transition, respectively.
Extrapolation of the linear portion of the curve to the energy intercept yields band gap value of the materials.In figure 3, Using Tauc's relation [24], the interception makes an indirect band gap, E g 2.85 eV for CuO and 2.11 eV for 6 wt% Ag-doped CuO.The optical band gap energy (E g ) of CuO NPs achieved 2.85 eV, which is higher than Ag-doped CuO (∼2.11 eV) and in concordance with the literature [15].We have observed the shifting of UV-visible peak towards a higher wavelength (redshift), corresponding to a decrease in bandgap energy value (E g = 2.11 eV).It can be argued that the redshift results from lattice distortions, localization of charge carriers, and electron-phonon coupling due to defect-interface interactions [15,25].

FESEM studies on undoped and Ag-doped CuO nanoparticles
The FESEM morphologies of CuO NPs with and without Ag doping are shown in figure 4. As exhibited in figure 4(a), the undoped CuO NPs have a spherical pellet-like morphology, and figures 4(b)-(d) of Ag-doped CuO NPs showed mixed morphologies, i.e., such as spherical and disc-like [26].Parvathi raja et al [27] also reported spherical shaped Ag/CuO NPs with size ranges from 20-25 nm.The presence of elemental compositions, i.e., copper (Cu), oxygen (O), and silver (Ag), were determined by EDAX spectra.The major peaks for Cu, O, and Ag confirmed the Ag doped CuO [27].

Effect of Ag doping on CuO NPs synthesis
The copper sulphate solution produces Cu + ions to make a blue precipitate.With an increasing temperature, the colour of the precipitate gradually moved into dark brown, indicating dehydration of copper sulphateand formation of CuO NPs.After Ag doping, the change in the colour of the precipitate was not observed but appeared to have a consecutive impact on the CuO NPs growth rate [9].The Ag ions are adsorbed on the CuO basal planes and thus alter surface properties, for example, relative surface free energy on the facets; these results in preventing and blocking the incorporation of other molecules from solution into the CuO crystal lattice and thus slow down the growth in a particular direction [28].The higher Ag concentration also affects the dehydration of copper sulphate and retards its phase transformation to CuO.

Antibacterial activity of undoped and Ag-doped CuO NPs
The antibacterial potential of synthesized CuO NPs against E. faecalis (gram +ve) and E. coli (gram -ve) bacteria has been investigated.Ag-doped CuO NPs show higher ZOI than undoped CuO NPs in both bacteria.The results are in agreement with the findings of [6,17].The antibacterial potential of CuO NPs was increased with the dopant concentration [18] (figure 5), which could be due to the antimicrobial properties of Ag [17] and variation in structural and physicochemical characteristics of CuO NPs as a result of Ag-doping [16,29,30].We observed a decrease in the size of NPs with an increase in dopant concentration and higher antibacterial activity of 6 wt% Ag-doped CuO NPs could also be attributed to their reduced size and higher surface area to volume ratio [6,[29][30][31].The antibacterial activity of Ag-doped CuO NPs in the present report follows an increasing trend in a dose-dependent manner, with maximum ZOI at 1000 ppm in both bacteria.In the case of undoped CuO NPs treatment, no significant inhibition zones are seen in E. coli at all the tested doses, while E. faecalis showed minor inhibition at 1000 ppm.
Hence, the prepared Ag-doped CuO NPs showed a differential response, with higher sensitivity against gram +ve E. faecalis than gram -ve E. coli.The lipopolysaccharide cell membrane layer in E. coli is believed to offer some degree of restriction against the NPs [9].The cell wall of gram +ve and gram -ve bacteria has a negative charge due to teichoic acids (bonded to peptidoglycan/underlying plasma membrane) and phospholipids/ lipopolysaccharides (present at the outer cell surface), respectively.CuO NPs release Cu 2+ ions that strongly bind to the negatively charged bacterial cell surface and cause damage to the membranous structural framework [6].The schematic view depicting the action of NPs on the bacterial cell is illustrated in figure 6.The antibacterial activity of NPs is based on the generation of 'reactive oxygen species (ROS) (like hydroxyl radical, hydrogen peroxide, superoxide anions, etc) that can damage the cell wall, and consequently, the NPs gained entry inside the bacterial cell, and the ROS further prevents the DNA replication, deactivates protein, inhibits enzymatic activity, and can cause electrolyte imbalance [6,16].Death is inevitable due to the leakage of internal cellular components of bacteria [6].

Conclusion
A facile synthesis of Ag-doped and undoped CuO nanostructures was carried out via the alkaline hydrothermal method.Ag doping substantially affects the band gap, morphology, and size of NPs.The Ag-doped CuO NPs  were comparatively smaller in size than undoped CuO NPs.The present investigation is the first report on the antibacterial potential of Ag-CuO NPs against E. faecalis.The effect of Ag-CuO NPs treatment was more pronounced on gram +ve E. faecalis than a gram -ve E. coli.The novelty of this work is the significant bactericidal action of Ag-CuO NPs even at lower treatment doses, which could be due to the presence of antimicrobial Ag and modification in structural and optical properties of CuO NPs upon Ag-doping.The antibacterial activity of Ag-doped CuO NPs in the present work followed an increasing trend in a dose-dependent manner against both bacteria.The maximum ZOI values were recorded at a 1000-ppm dose of 6wt% Ag-doped CuO NPs against both bacteria.The studied Ag-doped CuO NPs can potentially prevent the various types of illness caused by these bacteria, such as 'hospital-acquired infections' and 'gastro-intestinal infections'.Such investigations can open up new opportunities in the area of functionalized nanomaterials for developing potentially effective antimicrobial agents with a wide-spectrum of activities.

Figure 1 .
Figure 1.Schematic diagram indicating important steps of Ag-doped CuO NPs synthesis.

Figure 3 .
Figure 3. Tauc's plot to calculate the band gap of pure and Ag-doped CuO NPs.

Figure 4 .
Figure 4. SEM micrographs and EDAX spectra of pure and Ag-doped CuO NPs.