SYNTHESIS, CHARACTERIZATION OF POLYVINYL ALCOHOL-CHITOSAN-ZNO/CUO NANOPARTICLES FILM AND ITS BIOLOGICAL EVALUATION AS AN ANTIBACTERIAL AGENT OF Staphylococcus aureus

The polyvinyl alcohol-chitosan-ZnO/CuO nanoparticles film was researched. Synthesis, characterization, and its biological evaluation as an antibacterial of Staphylococcus aureus were the aims of this research . The biosynthesis of ZnO, CuO


INTRODUCTION
There are three methods in the synthesis process of metal nanoparticles. Physical, chemical, and biological methods are the major ones in this synthesis [1]. Previous literature [2] has stated that physical, chemical, and biological methods have their advantages and disadvantages, respectively. The advantages and disadvantages of the physical method are the absence of toxic chemicals and the requirement of expensive instrumentation in the synthesis process, respectively. The chemical method has advantages such as high yield, but the disadvantage of this synthesis method is the use of toxic chemicals. The last method is the biological method, where the synthesis process is easy but controlling the size of metal nanoparticles is challenging. The biological method has a challenge in the synthesis process such as the effect of pH, the concentration of reactant, reaction time, and temperature [3]. On the contrary, despite the challenges associated with the biological method, researchers have chosen this method because it is easy and cheap [4]. Plants (leaves etc), algae, bacteria, yeast, and fungus serve as the basic material in the biosynthesis of metal oxide nanoparticles through the biological method [5] and it was explored by previous literature [6] [7] [8] [9] [10]. However, the biosynthesis of these nanoparticles using extracts from plants is easier than with algae, bacteria, yeast, and fungus. The biosynthesis of metal oxide nanoparticles using algae, bacteria, yeast, and fungus requires specific conditions [11].
The common solvent for the biosynthesis process is a non-alcoholic or alcoholic solvent based on the polarity index. Non-alcoholic solvents (water) and alcoholic solvents (ethanol) have polarity indices of 9 and 5.2 [12], respectively. However, ethanol solvent is a universal solvent and demonstrates strength in the extraction process. A hydroxyl group and an alkyl group in the ethanol solvent affect the extraction process. The polar or non-polar properties of secondary metabolites in the plant can be extracted and dissolved by an ethanol solvent [ [20], and the product of ZnO/CuO nanoparticles can act as an antibacterial [21]. ZnO/CuO nanoparticles have a synergistic effect, on the other hand, the multifunctional bimetallic nanoparticles can be used in various fields [22].
Chitosan is derived from chitin. The advantages of chitosan are its non-toxicity and safety for use [23] [24]. The biological properties of chitosan include its antimicrobial activity [25]. To increase the film formation, chitosan can be mixed with polyvinyl alcohol (PVA). PVA is classified as a synthetic polymer and can be used as a film-forming agent [26] [27] [28], as well as easily forming hydrogels [29]. Furthermore, it enhances the chemical properties of chitosan [30]. In contrast, chitosan as an organic material, can be used as a supporting material to increase the effectiveness and properties of bimetallic nanoparticles [22].
We biosynthesized bimetallic nanoparticles (ZnO/CuO nanoparticles) following a procedure proposed by previous literature [18], with a slight modification. Here, we employed the casting method to synthesize the PVA-chitosan-ZnO/CuO nanoparticles film. The resulting film was used to inhibit the growth of the Staphylococcus aureus bacteria. The PVAchitosan-ZnO/CuO nanoparticles film was characterized by various techniques such as FTIR, XRD, and SEM. The biosynthesis (double metal oxide nanoparticles), as well as synthesis (PVAchitosan-ZnO/CuO nanoparticles film), are discussed.

Instrumentation
The Instruments used for characterization were as follows: FTIR Spectrophotometer (Shimadzu Prestige-21) for functional group analysis. X-Ray diffraction (XRD, Shimadzu 6000) for physical structure analysis. UV-Vis Spectrophotometer (Genesys 150 Thermo Scientific) for the analysis of metal nanoparticle wavelengths and Scanning Electron Microscopy (SEM, JEOL JSM 6510 LA) for surface morphology analysis

Maceration process of leaves guava seed
The ethanolic extract of leaves guava seed was prepared using the maceration method, as proposed by previous literature [16] with slight modification. Dried guava seed leaves (25 g) were soaked in ethanol 70 % (v/v, 250 mL) for 24 hours in a maceration bottle. After 24 hours, the filtrate was separated, and it was collected in a clean bottle, and stored at room temperature (filtrate I).
The residue was macerated again with 250 mL ethanol 70 % (v/v) (24 h). The filtrate was filtered, and the filtrate was collected and merged with filtrate I. All filtrate was stored a room temperature for further experiment.

Biosynthesis of metal oxide nanoparticles [18]
The ethanolic extract of guava seed leaves (75 mL) in a beaker glass 250 mL was added to the solution of zinc acetate dihydrate (0.5500 g, 25 mL). The process of biosynthesis was done on a hot plate (80°C) with continuous stirring (60 minutes). After 60 minutes, sodium hydroxide solution (0.1 M, 10 mL) was added to this mixture and stirred continuously up to pH 10. The mixture was stored for one night until the precipitation appeared. The filtrate and residue (ZnO nanoparticles) were separated, and ZnO nanoparticles were washed with aquadest (15 ml) and absolute ethanol (15 mL). The product of ZnO nanoparticles was dried in the oven (60° C) until dry. The same procedure was used to prepare CuO and ZnO/CuO nanoparticles and summarized in Table 1.

Synthesis of the PVA-chitosan-bimetallic oxide nanoparticles film
PVA-chitosan-ZnO/CuO nanoparticles film (film C) was synthesized using an earlier procedure [31] with slight modification. Chitosan solution in 250 mL of beaker glass (0.1 g of chitosan powder and 10 mL of acetic acid solution 1% v/v). ZnO/CuO nanoparticles (0.1 g) were added to the chitosan solution by continuously stirring (room temperature, 1 h). Then the PVA solution (2.5 % (w/v), 10 mL) was added to this mixture. All mixture was stirred continuously for 30 minutes at room temperature. The PVAchitosan-ZnO/CuO nanoparticles were poured into a petri dish until dry at room temperature. The film D and E were prepared with the same procedure. Film A and B were prepared by adding PVA solution and metal oxide nanoparticles without chitosan as explained in Table 2.

The antibacterial procedure of the film PVAchitosan-metal oxide nanoparticles
Three Petri plates were used in this evaluation. The antibacterial agent of PVAchitosan-metal oxide nanoparticles film was evaluated by the agar disc diffusion method and the antibacterial procedure was adopted from our studies [32]. The procedure from Isnaeni et al. [33] was used to prepare the inoculum of the bacterial suspension. The film A, B, C, D, and E (± 6 mm in diameter respectively) was pasted in the second layer of nutrient agar. The second layer of nutrient agar contained the bacterial suspension. Incubation process at 37°C for 24 h. After 24 hours, the inhibition zone was observed.

Biosynthesis of Metal Oxide Nanoparticles, Synthesis of the PVA-Chitosan-Bimetallic Oxide Nanoparticles Film, and The Products
The process of biosynthesis of nanoparticles was described in Figure 1 as reported by Fouda et al [18]. The materials were used in the biosynthesis of metal oxide nanoparticles such as Zn 2+ , Cu 2+ ion, media of biosynthesis (ethanolic extract), and NaOH solution. Ethanolic extract of guava seed leaves are containing active biomolecules [34]. The biomolecules were activated at pH 10 and at this pH, the biomolecules will increase them. A previous paper [35] has explained that at alkaline pH, the electrical charges of biomolecules can be converted into capping and stabilizing agents.

Characterization of Metal Oxide Nanoparticles by UV-Vis Spectrophotometer
The UV−visible spectra of ZnO, CuO, and ZnO/CuO nanoparticles were analyzed in the range of 200-800 nm and shown in Figure 4. The specific absorbance for ZnO, CuO, and ZnO/CuO nanoparticles were synthesized by using the ethanolic extract of guava seed leaves at 318, 274, and 252 wavelengths (nm), respectively. For ZnO nanoparticles, the absorption bands at 318 nm confirmed the existence of ZnO nanoparticles [7]. At this peak, there is a transfer of electrons from the valence band (VB) to the conduction band (CB) [8].
The absorption band at 274 nm for CuO nanoparticles has similarities with previous literature [36] due to the core electrons of the CuO nanoparticles running into the interband transition [37]. The absorption peak for ZnO/CuO nanoparticles at 252 nm and its appearance in a single absorption band [38].

Characterization of Metal Oxide Nanoparticles and All Films by FTIR Spectrophotometer
The FTIR spectra of ZnO, CuO, and ZnO/CuO nanoparticles as seen in Figure 5. In Figure 5 a, b, and c, the O-H and N-H groups (stretching vibration) can be observed in the wavenumber at 3423-3448 cm -1 [39]. This wavenumber appeared from a secondary metabolite as a bioactive compound from the ethanolic extract of guava seed leaves. The group of C=C, C=N, and C=O can be observed at 929-1620 cm -1 . The band below 1000 cm -1 is the characteristic of metal oxide absorption because of interatomic vibration [39]. The Cu-O group (stretching vibration) appeared at 619 cm -1 [40], [41], [42]. The band at 433-673 cm -1 is the stretching vibration Zn-O group [9], [43], [44] Figure 6 shows the FTIR spectral characteristic of PVA film, films A and B. The FTIR spectra of pure PVA film (Figure 6 a) showed -OH (stretching, 3410 cm -1 ), -CH2 (asymmetric, 2941 cm -1 ), -CH2 (symmetric stretching, 2908 cm -1 ), C=O (stretching in the acetate group, 1720 cm -1 ), -CH2 (stretching, 1330 cm -1 ), C-H (wagging,1240 cm -1 ), C-O (stretching of an acetyl group,1097 cm -1 ) and C-C group (850 cm -1 ) [45]. The FTIR spectra of the PVA-ZnO nanoparticles film (Figure 6b) and PVA-CuO nanoparticles (Figure 6c) showed that the stretching vibration of the OH group shifted to a higher wave number at 3450 cm -1 . The band at 468-675 cm -1 in Figure 6b showed that the Zn-O group appeared in this spectra. The Cu-O group was observed at 619 cm -1 (Fig. 6 c). Kumaraswamy et al [46] reported the shift in wave number, a new peak can be observed, and the intensities of the peak in the FTIR spectra between Figure 6a and 6b, c showed a specific interaction between ZnO or CuO nanoparticles and a functional group of PVA. The FTIR spectra of chitosan film, PVA film, film C, D and E as seen in Figure 7. Chitosan film (Figure 7a) showed bands at 3448 cm -1 (stretching vibration of O-H/N-H groups), 2920 cm -1 (stretching vibration of C-H groups), 1654 cm -1 (amide-I groups) and 1598 cm -1 (amide-II groups) [47]. The bands at FTIR spectra of PVA film (Figure 7b) were explained above. The wave number in the FTIR spectra of films C, D, and E (Figure 7c-e) has a wave number at 3429, 3441, and 3446 cm -1 respectively. The bands are the overlap of the stretching vibration of -NH and -OH groups from chitosan and PVA but all these bands are lower band than the band of stretching vibration of -NH and -OH groups chitosan film (3448 cm -1 ) and higher than stretching vibration of -OH groups PVA film (3410 cm -1 ). The functional groups of chitosan (-NH2 and -OH) react with the functional groups of PVA (-OH) to form a hydrogen bonding and it can change this wave number [48], [49]. On the other hand, the peaks at amide-I of PVAchitosan-ZnO/CuO nanoparticles film (C, D, E) shifted to lower wavenumber (1654 to 1587-1629 cm -1 ). The change in the wavenumber shows the interaction of reactive functional groups (-NH, -OH), and amide-I groups of chitosan with ZnO or CuO nanoparticles through a hydrogen bond [50] [51] and illustrated in Figure 8.
The FTIR spectra of C, D, and E films have differences with the FTIR spectra of chitosan and PVA, the bands at C, D, and E films, were observed at wavenumbers 617, 615, and 617 cm -1 respectively. Previous literature reported that the band between 433 and 673 cm -1 is the stretching vibration of the Zn-O or Cu-O group [9], [40], [41], [42], [43], [44] and the presence of Zn-O or Cu-O group was confirmed in this FTIR spectra of film C, D, and E.

Characterization of Metal Oxide Nanoparticles and All Films by XRD
The diffractogram of ZnO, CuO, and ZnO/CuO nanoparticles is seen in Figure 9. The diffraction peaks from ZnO nanoparticles (Figure 9 a) with 2θ = 23.7°, 31.46°, and 36.40°. These peaks can be connected to the crystal plans of (100) and (101) [52] and they have been used to estimate the average crystalline size. In the diffractogram of CuO nanoparticles (Figure  9 b), the diffraction peaks appeared at 2θ = 31.56° as a high-intensity peak [53]. The diffractogram ZnO/CuO nanoparticles (Figure 9c) at 2θ = 28.31°, 32.59° and 59.98° [53]. The high-intensity peaks at 2θ = 32.59° are the peaks of CuO nanoparticles. The crystallite size of nanoparticles is calculated by Debye Scherrer's formula [54] as in equation 1.
D, λ, β, and θ are the crystallite size (nm), the wavelength of the X-ray used, the full width at half maximum (FWHM), and Bragg's angle respectively. The crystallite size of the biosynthesized ZnO, CuO, and ZnO/CuO nanoparticles was estimated at 1.0572, 6.6315, and 2.3333 nm, respectively. All these sharp narrow peaks at the diffractogram of ZnO, CuO, and ZnO/CuO nanoparticles are indicated that the biosynthesis of ZnO, CuO, and ZnO/CuO nanoparticles is crystalline form. The description of the physical structure of films C, D, and, E can be seen in Figure 10. The XRD pattern of film C (Figure 10a) shows the peak at 2θ = 10.50°, 19.65°, 25.50° and 70.60°. The peak at 2θ = 10.50° and 19.65° can be indicated as an amorphous phase of chitosan-PVA [46], [49]. The peaks of ZnO, and CuO nanoparticles can be observed at 2θ = 25.50° and 70.60° [47], [55] and the presence of ZnO or CuO nanoparticles in the PVA-chitosan network although the peak intensity of the ZnO or CuO nanoparticles is low due to the less concentration of ZnO or CuO nanoparticles. The peaks appeared at 2θ = 10.40°, 18.83° and 25.73° was film D (Figure 10b), and in film E, the peaks appeared at 2θ = 14.01° and 16.91° (Figure 10c). Compared to the crystallite size of ZnO/CuO nanoparticles, the crystallite size of film PVAchitosan-ZnO/CuO nanoparticles (film E) was 1 nm and there is a reduction to 1.3333 nm. All diffractogram in Figure 10

Characterization of Metal Oxide Nanoparticles and All Films by SEM
The SEM images of ZnO, CuO, and ZnO/CuO nanoparticles are shown in Figure 11. The morphological study of ZnO nanoparticles shows that these nanoparticles have irregular morphology (Figure 11a). CuO nanoparticles have agglomerate based on the SEM images ( Figure 11b) and the SEM images of ZnO-CuO nanoparticles showed the agglomeration of these bimetallic nanoparticles (Figure 11c). The SEM images of films C, D, and E are displayed in Figure 12. Figure 12a shows the synthesis of film C from the mixture of 0.1 g (ZnO/CuO nanoparticles), 10 mL (PVA 2.5%), and 0.1 g (chitosan) caused the surface morphology of film C to become rough, have a cavity and fracture. The surface morphology of Film D (the amount of chitosan is twice that of ZnO/CuO nanoparticles) is different than film C and E. The surface morphology of film D was less homogeneous causing the high concentration of chitosan [56]. The amount of ZnO/CuO nanoparticles is twice that of chitosan at film E, the fracture surface is seen the film E (Figure 12c). The fracture surface was caused by the binding effect between metal nanoparticles and chitosan structure as reported by Susilowati et al [57].

Evaluation of All Films as Antibacterial
The antibacterial effect of all films was studied and seen in Figure 13. The calculation result of the inhibition zone is displayed in Table  3.  Table 3 showed the average inhibition zone of films C, D, and E is higher than films A and B. The addition of bimetallic nanoparticles (ZnO or CuO nanoparticles) and chitosan resulted in better antibacterial properties of films C, D, and E against Staphylococcus aureus bacteria. An antibacterial of chitosan through the penetration of chitosan structure into the microorganisms' nuclei and the electrostatic forces [58]. The electrostatic forces can be defined as the positive charge in the chitosan structure (NH3 + groups) that can bond with the negative charge of the bacterial cell membrane. ZnO or CuO nanoparticles can act as antibacterial reported by previous literature. The bonding between the film and the negatively charged bacteria results in electrostatic interactions and the activity of ZnO or CuO nanoparticles through photocatalytic activity can contribute to enhancing the antibacterial effect [47]. Akintelu and Folorunso [59] reported that the reactive oxygen species (ROS) and the particle size of ZnO and CuO nanoparticles also can contribute to the antibacterial effect. The al Kimiya: Jurnal Ilmu Kimia dan Terapan p-ISSN: 2407-1897, e-ISSN: 2407-1927 Vol. 10, No. 1 (1-12), Juni 2023/Dzulhijah 1444 8 release of Cu 2+ and Zn 2+ ions and their interaction with the negatively charged cell of bacteria may also enhance antibacterial [60]. The effect of the release of Zn 2+ ions, followed by reactive oxygen species such as OH -, H2O2, also O2 2- [61], and the difference of ionic radius between Cu 2+ and Zn 2+ ions can enhance antibacterial activity because the effect of crystallite size [62].

CONCLUSION
ZnO, CuO, and ZnO/CuO nanoparticles can be biosynthesized successfully using the ethanolic extract of guava seed leaves as a medium. The synthesis of films A-E requires ZnO, CuO, and ZnO/CuO nanoparticles. The Zn-O and Cu-O groups can be observed in the FTIR spectrum of ZnO/CuO nanoparticles and PVAchitosan-ZnO/CuO nanoparticles. The physical structure of ZnO, CuO, and ZnO/CuO nanoparticles is crystalline, whereas the physical structure of the PVA-chitosan-ZnO/CuO nanoparticles film is amorphous. ZnO/CuO nanoparticles can change the surface morphology of chitosan in the PVA-chitosan-ZnO/CuO nanoparticles film. The antibacterial activity of film D is higher than C and E.