Document Type : Research Paper
1 Department of Chemistry, Faculty of Science, Ilam University, Ilam, Iran
2 Department of Chemistry, Faculty of Science, University of Zanjan, Zanjan, Iran
3 Department of Microbiology and Virology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
Physical, chemical, and environmentally friendly techniques have been utilized for the synthesis of nanoparticles. [1-5]. Recently, researchers focus on green synthesis techniques to prepare the various metal nanoparticles [6-10]. Metal oxide NPs and metal NPs have received great attention among other nanomaterials due to their promising properties [11, 12].
Industrial development and population expansion have led to increased environmental pollution which is a major global serious concern. Water pollution is a particularly pressing concern. Wastewater and water arriving from leather industries paper, textiles, and plastic are usually rich in organic dyes. Degradation using photocatalysis is one of the significant and effective methods for water and wastewater treatment [13-16].
Research on nanoparticles and their use as antimicrobial agents has increased due to their unique physical–chemical properties in limiting bacterial growth . Silver, gold , and Cu metal nanoparticles or oxides have been used in medicine, biology, and pharmacy in recent years . Nanoparticles’ antimicrobial action makes them useful in biology . Previous research have shown that nanoparticles’ antibacterial properties may change their structure, morphology, size, and functional group . Instead, inorganic nanoparticles may be employed more after being matched with organic nanoparticles owing to their stability and safety . Metal oxide nanoparticles possess antibacterial properties against both gram-negative and gram-positive bacteria. This is attributed to their ability to bind to biological macromolecules, which inactivates them and leads to the death of bacteria, viruses, and fungi [23, 24]. Among metal oxide nanoparticles, CuO is a p-type semiconductor having electrochemical, catalytic, photocatalytic, and antibacterial capabilities [25-26]. Even at low concentrations, it has a significant antimicrobial impact against a broad spectrum of pathogens.
This study addressed the green sol-gel synthesis of CuO nanoparticles with photocatalytic and antibacterial activates. The photocatalytic activity of CuO NPs in aqueous solution was examined by degrading RB21 dye under visible light irradiation. In addition, the antibacterial activity of CuO NPs was assessed by measuring their minimum inhibitory and bactericidal concentrations (MIC and MBC) using broth microdilution. To analyze the produced nanoparticles, XRD, FESEM, TEM, BET, and DRS were employed. The molecular structure of the RB21 dye is shown in Fig. 1 .
The tragacanth gum (TG) was acquired from a bio shop. The Cu(NO3)2.3H2O was acquired from Merck. CuO NPs were characterized by X-ray powder diffraction (XRD) utilizing Cu (K) radiation (wavelength: 1.5406 ) on an X’Pert-PRO advanced diffractometer. UV–Vis absorption spectra were obtained on a Metrohm (Analytical Jena-Specord 205) double-beam. The diffuse reflectance UV–vis spectroscopy (DRS) of the sample was determined by a UV–Vis spectrophotometer (Shimadzu, UV-2550, Japan). BET method was applied to calculate the specific surface area of CuO (Belsorp Mini II apparatus). The morphology and size of the synthesized copper oxide were considered by the TEM (Philips CM30) and FESEM (Zeiss EVO 18, Germany).
Synthesis of Copper Oxide NPs
Firstly, tragacanth gum (TG) gel was prepared based on our previous work . Then, 1.5 g of Cu(NO3)2.3H2O salt was added to the prepared gel. The container was moved to a sand bath with the temperature stabled at 75 °C for 12 h. Next, black color resin was calcined in air at 500 °C for 4 h to prepare copper oxide nanoparticles.
All photocatalytic experiments were considered under the fluorescent. The rate of dye degradation was monitored in various conditions to determine the impact of effective parameters. Thus, various amounts of copper oxide nanoparticles (0.02, 0.03, and 0.04) were applied in 50 ml of dye solution with different concentrations (10, 20, 30 mg/L). The degradation performance of reactive blue 21dye was followed by UV–Vis at a λmax = 664 nm.
Antibacterial Activity of CuO Nanoparticles
Staphylococcus aureus ATCC 29213 (Gram-positive), Enterococcus faecalis ATCC 29212 (Gram-positive), Escherichia coli ATCC 25922 (Gram-negative), Salmonella typhimurium ATCC 14028 (Gram-negative), Klebsiella pneumoniae ATCC 7881 (Gram-negative), Proteus mirabilis ATCC 7002 (Gram-negative), and Pseudomonas aeruginosa ATCC 27853 (Gram-negative) were selected as bacterial models for evaluating the antimicrobial activity of CuO nanoparticles.
Minimum Inhibitory Concentration (MICs)
In order to evaluate the antibacterial susceptibility test of CuO nanoparticles, the MIC method (microdilution method) was applied. Initially, bacterial strains were grown separately in 5ml of brain heart infusion (BHI) (Merck, Germany) at 37°C for 24 h. The bacterial suspension concentration was then adjusted to 0.5 McFarland standard (1.5×108 CFU/ml). Following, 200μl of Mueller-Hinton broth (Merck, Germany) was added to each of the 96 microplate wells. In order to obtain a concentration in the range of 1-5 μg/mL, the stock concentration of CuO nanoparticles was diluted serially in 1% dimethyl sulfoxide (DMSO). After that, 100μl of the prepared bacterial suspension along with 100μl of CuO were added to all microplate wells and incubated in a rotary shaker incubator (120rpm) at 37°C for 18 h. The MIC was defined as the minimum concentration of CuO, resulting in no visible microbial growth after incubation period.
The minimum bactericidal concentration (MBC)
The MBC is the minimum concentration of an antimicrobial agent required to kill 99.9% of the bacteria, measured by subculturing the 50μl of the broth dilutions applied for MIC determination on Mueller-Hinton agar (Merck, Germany) media and incubation for 18 h at 37°C. The absence of bacterial growth in the Mueller-Hinton agar medium is considered as MBC.
RESULTS AND DISCUSSION
Characterization of Copper Oxide NPs
Fig. 2 displays the X-ray diffraction patterns of the CuO NPs. Single-phase monoclinic structures were specified by the sharp and strong peaks. All diffraction peaks are indexed to the monoclinic structure of CuO (JCPDS card no. 80-1916).The crystallite size of the synthesized sample was calculated by the Scherrer formula  on the FWHM of the (002) diffraction peak and was obtained to be 21 nm.
The band gap of CuO NPs was calculated by UV-Vis DRS. The band gap energy was found by applying the Tauc model . The results confirm that the band gap of copper oxide nanoparticles is 2.04 eV (Fig. 3). Therefore, the copper oxide NPs are appropriate photocatalyst in a visible-light region.
The specific surface area, pore diameter, and total pore volume of CuO NPs were specified via the BET technique, which were obtained to be 29.294 m2g–1, 32.726 nm and 0.076 cm3 g−1, respectively. The nitrogen absorption/desorption curve of the sample was given in Fig 4. Isotherm is of type IV with hysteresis loops H1 based on the IUPAC classification of materials .
Fig. 5 shows the FESEM and TEM images of the CuO NPs synthesized by green sol-gel method. The images confirm the spherical shape of nanoparticles with a diameter of 25 nm.
Degradation of RB21 dye was done in three states to confirm the photocatalytic activity of CuO NPsL: visible light irradiation without CuO NPs (photolysis), nanoparticles under dark (adsorption), and CuO NPs under visible light irradiation (photocatalysis). For photolysis, adsorption, and photocatalysis states, the degradation efficiencies are 2%, 45%, and 86%, respectively. These results indicate that synthesized copper oxide nanoparticles have the photocatalytic activity in the visible light region for RB21 degradation (Fig. 6a). To found the best amount of photocatalyst, 0.02- 0.04 g of the CuO NPs was added in RB21 solution (50 ml, 20 mg/L), at a fixed time of 60 min (Fig. 6b). It is obvious that raising the catalyst dosage leads to an improvement in the degrading performance. The initial dye concentration is the significant parameter that affected the degradation efficiency . Thus, the photocatalytic degradation of RB21 dye was monitored by varying the RB21 concentrations (10-30 mg/L) with the optimum dosage of CuO (0.03 g). As given in Fig. 6c, the decolorization performance of RB21 dye is reduced by increasing the dye concentration from 20 to 30 mg/L. Then, UV-Vis absorbance spectra of selected dye were monitored in different time periods to examine the impact of irradiation time on decolorization efficiency. Significant decreases in absorbance intensity at 664 nm (λmax of RB21 dye) with increasing the irradiation time are shown in Fig. 6d. Based on the results, 86% of RB21 dye was removed in 60 min.
MIC Results: All of the bacteria studied, including S. aureus, E. faecalis, S. typhimurium, K. pneumoniae, P. mirabilis, and E. coli, were significantly inhibited by CuO nanoparticles. However, P. aeruginosa (1.5 μg/mL) were moderately inhibited by CuO (Table 1).
MBC Results: The highest MBC of CuO was found against gram-positive bacteria, E. faecalis and S. aureus (1 μg/mL). MBC of CuO against S. typhimurium, K. pneumoniae, P. mirabilis, and E. coli was 1.25 μg/mL. However, the lowest MBC was observed against P. aeruginosa (2 μg/mL) (Table 1).
This research examined the green synthesis of CuO nanoparticles with photocatalytic and antibacterial applications. The sample were studied by XRD, FESEM, BET, DRS, and TEM techniques. The XRD and FESEM results verified their spherical shape and nano-crystallite size. The bandgap energy of CuO-NPs was found to be 2.04 eV. The photocatalytic results confirmed the photocatalyst function of CuO NPs during the whole RB 21 degradation process under visible light irradiation, with an obtained 86% degradation rate. The CuO nanoparticles demonstrated a significant antibacterial effect against both gram-negative and positive bacteria. According to the results of this study and other previous studies, it can be concluded that these nanoparticles can be used in the treatment of multidrug-resistant bacteria in the future.
The authors would like to appreciate “Ilam University”.
CONFLICT OF INTEREST
The authors declare no conflicts of interest.