Green synthesis of multifunctional carbon coated copper oxide nanosheets and their photocatalytic and antibacterial activities

The studies of metal oxides in environmental remediation of chemical and biological pollutants are gaining colossal importance. Herein, we report the facile synthesis of multifunctional copper oxide nanosheets (CuO NS) using an aqueous extract of Rhazya stricta. The phytochemical investigation of R. stricta indicated the presence of saponins, tannins, and reducing sugars, responsible for the reduction and stabilization of CuO NS. A UV–Visible spectrophotometer initially confirmed the fabrication of CuO NS with specific Surface Plasmon Resonance at 294 nm. Field Emission Scanning Electron Microscopy (FE-SEM), Fourier-transform infrared spectroscopy FTIR, and XRD were further used to characterize the CuO NS. The obtained CuO NS were poly-dispersed with an average size of 20 nm. Interestingly these particles were aligned together in 3D cubical sheets layered above each other via self-assembly. The as-synthesized CuO NS showed enhanced antibacterial potential (17.63 mm, overall mean inhibition zone) in comparison to the known antibiotics (11.51 mm, overall mean inhibition zone) against both Solanaceous crop's wilt-causing bacteria (Ralstonia solanacearum and Clavibacter michiganensis). Furthermore, the appreciable photocatalytic potential of CuO NS has also been observed, causing 83% degradation of methylene blue (MB) upon solar irradiation. The synthesis methodology is devoid of any toxic waste or by-products. It could be used to produce eco-friendly CuO nanomaterial for industrial uses.

www.nature.com/scientificreports/ Recently copper oxide nanostructures have gained attention due to their low cost compared to the existing metal nanoparticles such as Ag and Au. They are also considered almost ten times cheaper than their other counterparts. Copper oxide nanostructures are believed to have high sensitivity against Gram-negative & Grampositive microorganisms. They have a high potential as an external microbial agent and a biocidal film over medical devices 13 . However, minimal studies are available for their use in agriculture to control pathogens.
Wilting of solanaceous crops caused by bacteria, including Ralstonia solanacearum and Clavibacter michiganensis, is of significant concern for the growers 14 . Both these bacteria spread rapidly on their outbreak and cause severe damage by clogging the vascular system through extracellular polysaccharides 15,16 . These pathogenic bacteria are generally controlled using commercially available antibiotics that are expensive and pose threats to the environment. Furthermore, rapid industrialization and excess use of dyes pose a severe threat to the water quality and biotic ecology. These organic pollutants are highly mutagenic and carcinogenic to human health. Dyes are poorly degradable, and their complex chemical structure makes them difficult to remove from water 17 . Therefore, it is of utmost importance to have an eco-friendly products for the control of these pathogens and for the removal of both biological and chemical pollutants.
To the best of our knowledge, there has been no report on the single-step synthesis of carbon-coated CuO-NS using an aqueous extract of R. stricta. Herein, we report a one-pot, environmentally friendly method for the fabrication of multifunctional CuO NS for photocatalytic degradation of methylene blue (MB) and their possible use as a bio-control agent against wilt causing bacterial pathogens of solanaceous crops. This research will ultimately open new avenues in the agriculture and environment sectors.

Materials and methods
Collection of plant material. The collection of plant material was carried out according to the relevant national and international guidelines 18 . Arial parts of Rhazya stricta (Wild) were collected with permission from local forest officers in the month of April 2020 from district Karak, Khyber Pakhtunkhwa, Pakistan, located at 33°7′12 N 71°5′41E (Fig. 1). The plant samples were stored in a paper bag and transferred to the laboratory within 48 h and identified by a botanist. A specimen of the sample was deposited in a special herbarium for weeds and medicinal plants (voucher No. MIK-5/20-397), Department of weed science and botany, The University of Agriculture Peshawar. The collected samples were rinsed with distilled water and air-dried under shade for 14 days. The dried samples were chopped into small pieces using a sterile scissor and heated in distilled water (10% weight to volume ratio) at 60 °C for 20 min on the heating mantle. The solution was cooled to room temperature, vacuum filtered to obtain the aqueous extract concentrate (brown color), and stored at 4 °C for further use.
Qualitative analysis of phytochemicals. The phytochemical contents of Rhazya stricta, including alkaloid, phenols, flavonoids, terpenoid, saponins, steroids, tannins, and anthraquinones, were assessed using standard procedures 19,20 . Biosynthesis of CuO NS. For the synthesis of CuO NS, 60 mL of freshly prepared aqueous extract of Rhazya stricta was added to 80 mL of 0.5 M aqueous copper sulfate pentahydrate (CuSO 4 .5H 2 O) solution at room temperature. A noticeable change in color from blue to green was observed as soon as both solutions come  www.nature.com/scientificreports/ in contact, indicating the reduction of ionic copper. Upon heating at 80 °C for 12 h, the green solution turned brown. The reaction mixture was then allowed to cool at room temperature, and the brown suspension was collected through centrifugation (10,000 RPM for 10 min). The precipitates were thoroughly washed with distilled water and absolute ethanol to remove unreacted reagents and biomolecules. The obtained solid material was dried under N 2 flow and stored at 4 °C for further analysis.
Characterization copper oxide nanosheets. UV-Visible spectral analysis. The bio-reduction of Cu 2+ ion solution through an aqueous extract of Rhazya stricta was monitored using a split-beam UV-Vis spectrometer (Optima Sp3000 + Japan) for its maximum absorption v/s wavelength range against the aqueous extract of Rhazya stricta as a blank. Upon completion, the reaction mixture was centrifuged at 10,000 rpm for 10 min to eliminate any uncoordinated bio-molecules. The obtained solid material was re-suspended in double-distilled de-ionized water and scanned from 200-1100 nm wavelength.
Fourier transform infrared spectroscopy (FT-IR) analysis. The Fourier Transform Infrared spectra were recorded under identical conditions in the range 400-4000 cm −1 region using FTIR Spectrometer (SHIMADZHU; Japan).
X-ray diffraction analysis. The phase identity, crystalline structure, and crystallite size were determined from the XRD data using Cu-kα radiation source. The CuO NS powder was coated on a glass substrate and submitted for their crystal structure analysis. The results were recorded as a graph with 2θ vs. intensity at the x-axis and y-axis, respectively. Antibacterial assays. Synthesized CuO NS were tested for inhibition against Solanaceous crops' wilt-causing bacterial pathogens, including Ralstonia solanacearum and Clavibacter michiganensis. The antibacterial assay was carried out by the disc diffusion method 21 . Both bacterial strains were developed using nutrient broth at 38 °C for 24 h and then streaked over Potato Dextrose Agar's (PDA) surface using sterile cotton swabs. The sterile paper disk (5 mm) was adsorbed with 10 µL of the reaction mixture (6.23% in sterile water) and placed on the PDA surface. As a control 10 µL plant extract (10.00%), 10 µL 0.5 M aqueous CuSO 4 solution (6.23%), and a positive control 10 µL of 6.23% streptomycin (0.1 M in distilled water) were separately adsorbed on sterile paper discs (5 mm). All the prepared disks were placed on a prepared lawn of bacterial cultures on PDA to assess their effect on pathogens. The plates were incubated at 38 °C for 24 h, and bacterial growth inhibition was observed as clear halos (zones) around the discs.
Photocatalytic property. The photocatalytic potential of biogenic CuO NS was carried out at the end of May under sunlight irradiation. To 100 mL (10 µg mL −1 ) aqueous methylene blue (MB) solution in a transparent flask, 10 mg of synthesized CuO NS were added and allowed to stir for 2 h and 20 min under direct sunlight. A control experiment (in the absence of CuO NS) was also maintained simultaneously. After every 20 min of reaction duration, 5 mL sample aliquot was collected and centrifuged (10,000 rpm) for 6 min to remove suspended CuO NS. The supernatant was collected and examined by a UV-Vis spectrophotometer at λ max 664 nm to estimate available unreacted MB. The following formula was used to determine the percent photodegradation efficiency (PD%) of MB.
where PD% denote percent photodegradation, C 0 and C t represents the absorbance of MB at time 0 min and t min.
Statistical analysis. The data was obtained in triplicates. The antibacterial activity is presented as a mean zone of inhibition (mm) ± S.E, the different letters in columns are statistically significant at p < 0.05.

Results and discussion
The detection of phytochemical in aqueous extract of Rhazya stricta revealed the presence of various groups of bioactive natural products (Table 1). These natural products, especially polyphenols, are believed to reduce metal ions and the formation of metallic nanoparticles 22 . www.nature.com/scientificreports/ Biosynthesis of CuO NS. The synthetic procedure for CuO NS using an aqueous extract of Rhazya stricta is very simple, environmentally friendly, and cost-effective. Here the CuSO 4 dissolved in water dissociates into Cu 2+ and SO 4 2− ions. The Cu 2+ is then reduced to CuO by the action of biomolecules (predominantly polyphenols) present in the aqueous extract of Rhazya stricta. The generated metallic copper nuclei then start growing into nanosheets. Several biomolecules from Rhazya stricta distributed in a reaction then stabilized CuO NS. This copper NS then starts precipitating due to water insolubility. The unreacted biomolecules sticking the surface of nanoparticles were removed during washing with distilled water and ethanol, resulting in pure dark brown CuO nanosheets powder.
Characterization of CuO NS. UV-Vis spectroscopy analysis. The addition of aqueous extract of Rhazya stricta to the CuSO 4 solution led to a change in the color from blue to green which turned brown after heating at 80 °C, indicating the formation of CuO NS's ( Fig. 2A). This change in the color is due to the excitation of surface plasmon vibration of CuO NS, which generated a high-intensity peak appearing at 294 nm. A minor peak at 314 could be from the different sizes/shapes of CuO NS (Fig. 2B Inset) 23 . We opined that this is also attributed to the n → π* transition of C=O bonds 24 . The peak appearing at 226 nm, 244 nm and 258 nm (Fig. 2-B inset) in the lowfrequency region are the characteristic peaks of graphene nanosheets corresponding to the π conjugation network or π → π* transition 25 . The presence of low-frequency peaks (Fig. 2B-inset) confirms that the biomolecules present in the aqueous extract of Rhazya stricta might have played a role in reducing and stabilizing CuO NS. Amorphous carbon has a less sharp peak and an extended band tail than graphitic carbon 26 . The peak positions of onion-like nano carbon depended on the shell structure. For example, the spherical, polyhedral, and ribbonlike onion structures had peaks at 233, 241, and 249 nm, respectively 27 . Tan and co-workers 28 have attributed the peak at 220 nm to a disordered and defective carbon. The high-frequency peaks might be attributed to the existence of nanoribbon like carbon sheets surrounding the CuO NS.
The bandgap of CuO NS was determined using the Tauc plot method 29 , by plotting (αhν) 2 versus hν, as shown in Fig. 2C, where α is the absorption coefficient, hv is the energy of incident light, h is Planck's constant (6.626 × 10 −34 Js), and v is the frequency of light. Based on tauc's plot, the bandgap was calculated as 3.84 eV. A similar energy band gap (3.85 eV) was reported by Dhineshbabu and co-workers while studying the structural and optical properties of cupric oxide nanoparticles synthesized via sonochemical method 30 . More recently, a slightly higher bandgap of 4.01 eV of CuO nanoparticles has been reported using melanin as a stabilizer agent 31 .
X-ray diffraction analysis. Figure 3 shows the X-ray diffraction (XRD) pattern of the carbon-coated CuO NS. Overall the spectrum shows an amorphous nature of the CuO NS. However, the appearance of some small diffraction peak at 2θ = 23.8°, 2θ = 31.90°, 2θ = 38.52° and a weak shoulder at 2θ = 41.22°, confirms the growth of crystals and formation of some crystalline planes. The peaks appearing at 2θ = 23.8° and 2θ = 41.22° are believed to be due to the emergence of (002) and (102) respectively for graphene oxide, and the peaks appearing at 2θ = 31.90°, 2θ = 38.52° are attributed to the (110) and (002) planes for CuO 32,33 . The lattice spacing determined from the (002)* plane is of the order of 0.37 nm, while for crystalline graphite, the lattice spacing remains about 0.34 nm. The slightly higher d spacing value suggests the presence of some oxygen functional groups between the carbon layers. Besides this, the broadened peak corresponding to the (002)* plane is believed to be due to the disordered carbon structure indicating the formation of graphene sheets as observed by Ashish and Sundara 34 , which might be due to the self-assembly of carbon atoms.
Fourier transform infrared spectroscopy (FT-IR) analysis. Various bio-molecules having different functional groups were responsible for reducing CuSO 4 and then stabilizing CuO NS derived from bioactive molecules. Various functional groups were found to be attached to nanosheets' surface during synthesis. FTIR is one of the most important techniques used to identify functional groups attached to nanomaterials' surface. Figure 4 shows a sharp band appearing at 630 cm −1 , the characteristics band of pure monoclinic CuO, as reported earlier 35 . The peak appearing at 1040 cm −1 is attributed to the C-H bending vibrations. A sharp absorption band at 1600 cm −1 is believed to be due to the self-assembled disordered carbon sheets. The broad peak at 3297 cm −1 is believed to be due to OH groups' existence on the surface of CuO NS. A sharp absorption band at 2930 cm −1 is because of the CH and CH 2 groups. The appearance of bands at 1600 cm −1 and that of the 2930 cm −1 is believed to predict graphene sheets interacting with CuO NS 36 . Chemical linkages on the surface of CuO NS suggest that the hydroxyl and carbonyl groups might have reacted as reducing and stabilizing agents for the fabrication of CuO material and, consequently, accumulating CuO NS.
Field emission electron microscopy of CuO NS. The field emission scanning electron microscopy (FE-SEM) was used to analyze the morphology of as-prepared CuO nanosheets, as shown in Fig. 5. It can be observed that CuO crystallizes in the form of smaller nanoparticles, which are polydispersed and aligned together in the proper sequence. These nanoparticles, upon diffusion, lead to the formation of nanosheets. The sheet's width appears to be less than 20 nm, which leads to highly reactive edges and corners.
Antibacterial potential of CuO NS against wilt causing pathogens. The antibacterial properties of CuO NS were evaluated against the wilt-causing bacterial pathogens of Solanaceous crops. Two bacterial strains, including Clavibacter michiganensis (Gram-positive) and Ralstonia solanacearum (Gram-negative), were tested using the disk diffusion method. The synthesized CuO NS showed significant inhibition zones against bacterial strains. The Cu ions are known to disrupt various biochemical processes 37 . The bacterial cell wall comprises both amine and carboxyl groups 38 , which shows a high affinity towards the copper ions 39   www.nature.com/scientificreports/ to volume ratio may also bind with the nucleic acid of bacteria destroying the helical structure of DNA. The cationic Cu released from nanoparticles can attach to the negatively charged portion of the bacterial cell wall resulting in denaturation of protein and subsequently causing cell death 40 .
The zone of inhibition of CuO NS (6.23%) against solanaceous wilt causing bacterial strains, namely, Ralstonia solanacearum and Clavibacter michiganensis, is shown in Fig. 6. It is visible that Clavibacter michiganensis was more affected than Ralstonia solanacearum. The synthesized CuO NS were tested compared to the aqueous extract of Rhazya stricta, the CuSO 4 aqueous solution (6.23%), and known broad-spectrum antibiotic streptomycin sulfate (6.23%). The result is presented in Table 2 shows the biosynthesized CuO NS possessed higher antibacterial activity against both R. solanacearum and C. michiganensis with an inhibition zone measured as 17.30 mm and 17.97 mm, respectively (p < 0.05). Significantly improved antibacterial activity of the CuO NS against tested bacterial strains was recorded compared to standard antibiotics (p < 0.05).
Interestingly both selected bacteria started to develop resistance after 18 h against streptomycin, and clear hallow around disk became turbid after 24 h. In contrast, selected bacteria did not show any growth around CuO NS, and clear hallow remained persistent after 24 h (Fig. 6). The aqueous extract of Rhazya stricta (blank) showed no activity against both R. solanacearum and C. michiganensis. The dense growth of both bacterial isolates was seen (Fig. 6, blank) around the disk containing an aqueous extract of R. stricta. A reason could be that the extract contains organic compounds suitable for the growth of bacterial isolates. The aqueous CuSO 4 solution did not affect R. solanacearum, while against C. michiganensis, it showed a 6.43 mm inhibition zone. Overall, Clavibacter michiganensis was more susceptible to all experimental conditions than Ralstonia solanacearum. Similarly, the mean observed effect (17.63 mm) of CuO NS on bacterial isolates was recorded higher than all other treatments, including positive control experiments.    www.nature.com/scientificreports/ These free radicals are highly reactive species causing oxidation of aromatic rings of MB dye to degrade into CO 2 , H 2 O and other degradation products.
A detailed mechanism for the photocatalytic degradation of MB in the presence of CuO NS is presented in Fig. 8.

Conclusions
The successful one-pot eco-friendly synthesis of Copper NS was achieved using an aqueous extract of Rhazya stricta in 24 h under mild conditions. Regular self-assembly of synthesized CuO NS was recorded in the form of three-dimensional cubical sheets with significant photocatalytic degradation efficiency against methylene blue. The nanosheets show a prominent bactericidal potential against wilt causing bacterial pathogens of solanaceous plants. The Cu liberated in the field as a by-product could also be used as a micronutrient by the crops.

Data availability
Data supporting this study's findings will be available from the corresponding author upon request.  Figure 8. Mechanism of photocatalytic degradation of methylene blue using CuO NS. Image generated using ChemDraw professional v.17.