Biosynthesis of Flower-Shaped CuO Nanostructures and Their Photocatalytic and Antibacterial Activities

Eugenol (4-allyl-2-methoxyphenol) extracted from O. sanctum leaves is used as a natural reducing agent for the synthesis of CuO nanoflowers (NFs). CuO-NFs can degrade methylene blue with an efficiency of 90%. CuO-NFs offer a new vision to deactivate multi-drug microorganisms. Eugenol (4-allyl-2-methoxyphenol) extracted from O. sanctum leaves is used as a natural reducing agent for the synthesis of CuO nanoflowers (NFs). CuO-NFs can degrade methylene blue with an efficiency of 90%. CuO-NFs offer a new vision to deactivate multi-drug microorganisms. Copper oxide nanoflowers (CuO-NFs) have been synthesized through a novel green route using Tulsi leaves-extracted eugenol (4-allyl-2-methoxyphenol) as reducing agent. Characterizations results reveal the growth of crystalline single-phase CuO-NFs with monoclinic structure. The prepared CuO-NFs can effectively degrade methylene blue with 90% efficiency. They also show strong barrier against E. coli (27 ± 2 mm) at the concentration of 100 µg mL−1, while at the concentration of 25 µg mL−1 weak barrier has been found against all examined bacterial organisms. The results provide important evidence that CuO-NFs have sustainable performance in methylene blue degradation as well as bacterial organisms.


HIGHLIGHTS
• Eugenol (4-allyl-2-methoxyphenol) extracted from O. sanctum leaves is used as a natural reducing agent for the synthesis of CuO nanoflowers (NFs).
• CuO-NFs can degrade methylene blue with an efficiency of 90%.
• CuO-NFs offer a new vision to deactivate multi-drug microorganisms.

Introduction
The micro-/nanostructure studies demand a better understanding of crystal facet engineering with tailored architecture that can be attained by the new design and facile synthesis methods [1][2][3]. In the past few decades, cupric oxide (CuO) is intensively studied binary transition metal oxide [4]. CuO nanostructures with large surface areas and potential size-effects possess superior physical and chemical properties that remarkably differ from those of their microor bulk counterparts [5]. It has excellent architectures with different shapes and dimensions, such as zero-dimensional (0D) nanoparticles, one-dimensional (1D) nanotubes, 1D nanowires/rods, two-dimensional (2D) nanoplates, 2D nanolayers as well as several complex three-dimensional (3D) nanoflowers, urchin-like and spherical-like nanostructures [6,7]. These nanostructures have been extensively used in various applications such as solar cells [8], photodetectors [9], field emissions [10], lithium-ion batteries (LIBs) [11], magnetic storage media [12], energetic materials [12], electrochemical sensors/bio-sensors [13], supercapacitors [14], nanofluid [15], removal of inorganic pollutants [16], photocatalysis [17], and so on. In addition, the complex geometry of ordered self-assembly of CuO nano/microscale building blocks is a hot topic in recent materials research [4]. Several important innovations have been directed toward the production of CuO, out of which many of them involve complexity of chemical reactions and problems associated with the reproducibility [1]. Thus, an alternative, environmentally approachable method is required. Green route-assisted CuO nanostructures have been recognized as a technologically imperative material with its several applications in the fields of cutting-edge science and technology [18]. The consumption of plants in the biosynthesis of CuO-NPs involves the content of secondary metabolites as reducing agents [19]. Apparently, biological agents act as reducers, stabilizers, or both in the process of making nanoparticles [20]. Several approaches for CuO synthesis and surface modification have been proposed through utilizing various parts of plants such as leaves, fruit, and flowers [21][22][23][24]. Several microorganisms, plants, and plant extracts have been extensively used to synthesize CuO nanoparticles (Table 1) to avoid the consumption of toxic chemicals [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38]. The O. sanctum (Tulsi) is supposed to contain oleanolic acid, rosmarinic acid, eugenol, carvacrol, Linalool, β-caryophyllene, and ursolic acid [39][40][41][42]. The oil extracted from O. sanctum leaves contains a higher amount of eugenol with the balance presence of numerous trace compounds, typically terpenes [43]. O. sanctum is a small herb that is seen all over India and extremely used in medicinal purpose. It is also known as phytomedicine plant and has been recognized as owning antioxidant, antimicrobial properties and non-toxicity [44], which has encouraged us to perform the current investigations.
CuO nanoparticles synthesized using leaf extracts had shown good photocatalytic efficiency against methylene blue (MB) dye [45][46][47]. Moreover, Sreeju [48] had reported that the CuO-NPs are effective against bacteria killing. Biosynthesized CuO nanoparticles exhibit good antibacterial property for both gram-positive and gram-negative microbes [35]. These reports reveal that the green chemistry-assisted CuO nanoparticles are highly promising candidates for photocatalytic as well as antimicrobial activity. However, to the best of author's knowledge, there have been no reports on a complete investigation of the photocatalytic and antibacterial properties of O. sanctum (Tulsi)-extracted Eugenol (4-allyl-2-methoxyphenol)-assisted CuO-NPs. Thus, the aim of the present work is to synthesize CuO nanostructures using eugenol extracted from O. sanctum leaves (the detailed eugenol isolation procedures are shown in Electronic Supporting Information (ESI)), and the obtained product was evaluated for the photocatalytic activity against the organic dye (methylene blue) for water rectification and bacteria killing.

Experimental Details
All the details such as the extraction of eugenol from O. sanctum leaves, synthesis of copper oxide nanostructures, characterizations, and photocatalytic and antibacterial measurements are reported in ESI.

Synthesis Mechanism and Morphological Analysis
The plant extracts derived from various plants as shown in Table 1 have been reported for CuO-NSs synthesis by the green approach. It is well known that the most preferred 1 3 green approach method is bio-reduction that includes the reaction between the biologically active produces isolated from plants with CuO in the reduced state [49]. In view of those ideas, we have chosen O. sanctum (Tulsi) leaf for the extraction of eugenol as a capping agent well as a stabilizing agent. In the beginning stage experiment, we have used the steam distillation setup to isolate eugenol oil from O. sanctum leaf extract, the mass of product isolated from O. sanctum leaf extract was examined through gas chromatography-mass spectrometry and confirmed the isolated product is 4-allyl-2-methoxyphenol (eugenol) (Fig. S1). The eugenol has a phenylpropene and an allyl chain-substituted guaiacol [40] and six reaction sites (acts as a hexadentate ligand) to form Cu 2+ ion complex [50]. Based on the above assumptions and using the Job's method, we have explained the possible growth mechanism schematically as shown in Fig. 1a. The OH − ions coordinate with Cu 2+ ions and control the reaction process under alkaline conditions, leading to nucleation and hence the growth of CuO micellar structures [51]. These structures form a network with each other through van der Waals forces and hydrogen bonding resulting in the formation of observed geometry. From the examination of eugenol structure we have found, it had replaceable hydrogen and a neighboring donor in the oxygen of the o-methoxy group [52] and generally shares two eugenol molecules to one copper in the formation of Cu 2+(eugenol) − 2 complex [53]. This process is led by the active reduction of Cu 2+ ions through acid-base reactions and followed by nanoparticle formation, presented as Eqs. 1 and 2: As the time elapses, few free molecules in the reaction mixture start to redeposit on the faintly larger particles to attain a thermodynamically stable state [4]. This condition leads to the complete exhaustion of the smaller particulates, further resulting in a large flower-like shape. The evolution of the flower-shaped CuO-NSs is believed to be the result of eugenol capping, and the growth mechanism can also be understood through the microstructural investigation. The FESEM image for surface morphology of CuO flower-shaped structures is shown in Fig. 1a (magnification 10.0 k×, scale 2 μm). It was clearly seen that the flowershaped branches of the single product grow in different directions and are formed in large quantity with almost uniform sizes. The rich assessment of the single flower-shaped structure is illustrated in the inset of Fig. 1c, which exposes   shows the SAED pattern of the circled portion of single petal shown in Fig. 1e. The bright spots reveal that the made petals have crystalline features [54,55].

Structural and Optical Analysis
The crystallographic phase of the as-prepared flower-shaped CuO-NSs was investigated via powder (D8 advance) X-ray diffraction pattern (XRD) technique. Rietveld analysis of XRD pattern is shown in Fig. 2a. Refinement was undertaken in space group C 6 2h , C2/C for monoclinic CuO with all atoms in general positions [57,58]. The marked (110), After numerous recursive refinements, the possible bestrefined lattice parameters obtained are as follows (weighted profile factor (R wp ) = 12.3, profile factor (R p ) = 11.9, expected R-factor (R exp ) = 7.8, Bragg R-factor (R Bragg ) = 7.02, goodness of fit (GOF) = 1.03 and χ 2 = 1.48) with unit cell parameters a = 4.6878 Ǻ, b = 3.4269 Ǻ, and c = 5.14567 Ǻ, and crystallite size ~ 15.7 nm, by using Scherrer's formula [56]. Additionally, refinement data (solid line) are in good agreement with experimental (• circle) data as the difference between these two is very less without any variations (solid line). Thus, the formation of CuO phase is predominant in the prepared sample.
To further support and clarify the crystallographic information, Raman spectroscopy was performed on the prepared sample (Fig. 2b). The spectrum was taken at 533 nm excitation wavelength with He-Ne laser at room temperature (RT). The peak located at 277.3 cm −1 is assigned to be A g mode at high frequency, which corresponds to the in-phase/ out rotation of the Cu and O atoms in the monoclinic phase [44]. The occurrence of the B g 1 and B g 2 modes discloses the  bending and the symmetric oxygen stretching of the Cu-O assigned to the monoclinic crystal structure of CuO that is consistent with the XRD result [59]. Further, in-depth analysis of chemical compositions and X-ray spectroscopy was performed. No impurity was observed for the prepared sample through the XPS survey spectrum (Fig. S1). The high-resolution core-level spectrum of Cu 2p and O 1s is schematically shown in Fig. 2c, d. Conferring to Fig. 2c, the Cu 2p peak of CuO was fitted into four peaks, consisting of two kinds of spin-orbit lines, named as SP-1 and SP-2 which were located at higher binding energies as compared to the main peaks which infer the occurrence of an empty Cu-3d9 shell and consequently approve the existence of Cu 2+ in the sample [60]. The characteristic peaks located at 934.27 and 954.26 eV were assigned to the Cu 2p3/2 and Cu 2p1/2 peaks with the binding energy difference between ~ 19.9 eV which further confirms the formation of CuO [61]. Figure 1d shows a high-resolution O 1s spectrum of flower-shaped CuO-NSs. Broad asymmetric curves were fitted to three sub-peaks named as Oa, Ob, and Oc for binding energies between 529-530, 530-531, and 532-533 eV, respectively [62]. There co-existed lattice oxygen (Oa ~ 529.98 eV), Cu(OH) 2 (Ob ~ 531.4 eV) and adsorbed oxygen from hydroxyl groups (Ob ~ 532.2 eV) of CuO-NFs formation via green route synthesis method. The UV-vis-NIR absorption spectrum of the flower-shaped CuO-NSs evaluated optical properties (Fig. S2). The absorption edge of the flower-shaped CuO-NSs is ≈ 560 nm. Inset of Fig. S2 shows that the E g of the as-prepared flowershaped CuO-NSs is ≈ 2.31 eV, as projected by applying Kubelka-Munk theory to the absorption spectrum [63].

Photocatalytic and Antibacterial Activities
Methylene blue (MB, C 16 H 18 N 3 SCl) [64] dye degradation was carried using the as-prepared flower-shaped CuO-NSs. The setup and testing are provided in ESI. MB is a thiazine cationic dye which has an absorption peak at λ max ≈ 663 nm (π → π*) (Fig. 3a). Additionally, the absorption spectra of an MB solution photocatalyzed through H 2 O 2 (alone) and flower-shaped CuO (alone) are shown in Fig. S3. was used to improve the degradation rate of the MB dye [65]. The absorbance depends on the number of molecules reacted with it. The photocatalytic activity (absorption spectra) of the flower-shaped CuO + peroxide (H 2 O 2 ) was observed when it is used as a photo-catalyzer of the methylene blue dye (MB) solution under UV light. It is seen from Fig. 3b that the intensity of absorption peak at λ max decreases from 0.66 to 0.04 a.u. as reaction time increases from 0 min to 120 min and had no new absorption peak during the entire reaction process. This exhibits the comprehensive photodegradation of MB. Also, the histogram (Fig. 3c) shows that around 90% degradation was reached after 120 min of exposure of light which have a strong proof that the flower-shaped CuO effectively degraded the MB dye molecules. The graph of radiation time against ln(C 0 /C) (Fig. 3d) shows kinetics [64][65][66]  When the light (photon) strikes the surface of flower-shaped CuO-NSs, it gets absorbed. The photon (hv) with energy greater than or equal to the band-gap energy (Eg) of flower-shaped CuO creates an electron-hole (e − ↔ h + ) pair, and both the valence band (VB) and conduction band (CB) receive equal amounts of photon generating h + and e − , respectively, as shown in Eq. 3 [66]. These photoexcited carriers move to the surface of the flower-shaped CuO and react with oxidants such as O 2 and reductants such as OH − , respectively [67]. Generally, the dissolved pollutants and O 2 will be more prone to being adsorbed on the surface of the flower-shaped CuO in the mixed solution due to its larger specific surface area calculated through N 2 adsorption-desorption analysis (Fig.  S4). In the presence of photocatalyst, H 2 O 2 oxidizes the CB and condenses itself to be extremely reactive • OH oxidizing potential. However, when it reacts with water molecules, which further oxidizes the stable MB into reactive intermediates, it stops the recombination process of electron-hole pairs [64]. Thus, the intermediate species (OH radicals, O 2− , H 2 O 2 , and O 2 ) interacted by surface charges of photocatalyst and caused a speed-up in the mineralization of dye molecules (OM) into the end-product carbon dioxide (CO 2 ) and water (H 2 O) with less toxic inorganic acids [65]. Also to achieve our basic objective, we have utilized the as-prepared flower-shaped CuO-NSs as an antibacterial agent and tested their antibacterial efficiency using agar well diffusion process report by Naika et al. [26] and Sharma [68] against E. coli, S. aureus, and P. fluorescens bacterial strains. Figure 4 illustrates the inhibition tendency of varied concentrations CuO-NFs. The O. sanctum leaves-extracted eugenol mediated synthesized flower-shaped CuO-NSs that played like a potential inhibitor at 100 µL concentration for all examined bacterial organisms, which is exposed from Moreover, plant extract also shows noteworthy results (zone of inhibition) in contrast to the tested pathogenic organisms due to the existence of antibacterial efficiency in O. sanctum leaves extract [70,71]. Also, due to the size of the as-synthesized flower-shaped CuO-NSs, strong electrostatic interaction between bacterial organisms could have been developed which oxidized the bacterial cell wall to destruct leading to immediate death [72][73][74]. The as-synthesized CuO-NFs show strong barrier against E. coli (29 ± 2 mm) at the concentration of 100 µg mL −1 , while at concentration of 25 µg mL −1 weak barrier was found in all examined bacterial organisms. The obtained results confirm that the prepared flower-shaped CuO-NSs showed good antibacterial activity.

Conclusion
On the basis of the results and discussion of the present study, we can summarize that the flower-shaped CuO-NSs can successfully be synthesized via green route using Ocimum sanctum (Tulsi) leaves-extracted Eugenol (4-allyl-2-methoxyphenol) as a capping agent as well as the stabilizing agent. The results obtained from XPS analysis corroborated with the crystallographic (XRD, Raman) results, revealing the formation of pure monoclinic CuO nanostructure. The detailed morphological characterizations revealed that the Eugenol created OH − ions which lead to a high percentage exposure of active planes that encourage the formation of flower-shaped CuO nanostructures with high precision. The synthesized flower-shaped CuO-NSs possess photocatalytic activity with H 2 O 2 oxidant against degradation of methylene blue. Moreover, the antibacterial activity of flower-shaped CuO-NSs has proven the biological importance in ecological and antimicrobial applications. The present work highlights the attractive benefits of O. sanctum-extracted Eugenol (4-allyl-2-methoxyphenol), e.g., high yield, less time, and an inexpensive and nontoxic route to synthesize flower-shaped nanostructures with excellent ecological properties. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.

Electronic supplementary material
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