SSRN Id4270332

A comparative study: Green Synthesis and Evaluation of ZnO-GO and ZnO-RGO
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Nanocomposites for Antibacterial Applications
Reza Ahmadia, Raziyeh Fattahi Nafchib, Parvaneh Sangpour 1*b, Mozhgan Bagherib, Elahe Badieib
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a Department of Materials Science and Engineering, Sharif University of Technology, Tehran, Iran
b Nanotechnology and Advanced Materials Department, Materials and Energy Research Center, Iran
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Abstract
ZnO nanoparticle, GO and RGO sheets, ZnO-GO, and ZnO-ROG nanocomposites synthesized to
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study their antibacterial activity. XRD and XPS confirmed that the nanocomposites successfully
synthesized. The UV-vis spectroscopy results showed the bonding between ZnO nanoparticles and GO
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sheets can engineer the semiconductor band gap and shift the absorption edge to longer wavelengths as
compared to pure ZnO. The antibacterial activity of ZnO nanoparticles, GO, and RGO sheets after 12 h of
contact with E. coli were 84%, 81%, and 73%, respectively. Antibacterial activity for Z-1GO, Z-5GO, Z-
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10GO, Z-1RGO, Z-5RGO, and Z-10RGO nanocomposites were 89%, 92%, 94%, 85%, 89%, and 91%,
respectively, which are improved compared to pure ZnO (under dark conditions). Enhanced antibacterial
activity in nanocomposites may be due to the simultaneous activation of mechanisms of dissolution of Zn2+
ions from ZnO and physical interaction between the sharp edges of GO or RGO sheets and the E. coli.
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Keywords: ZnO-GO and ZnO-RGO nanocomposite, Antibacterial activity, Antibacterial mechanisms, E.
coli
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1Corresponding Author: sangpour@merc.ac.ir

Tel/Fax:+98 26 36280040
This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4270332
1. Introduction
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Nowadays, many researchers are trying to develop new antibacterial agents that are not
harmful to humans. Pathogenic bacteria grow through the formation of colonies that are resistant
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to traditional antibacterial methods. Therefore, designing and synthesizing new materials as
antibacterial agents is critical to solving this challenge. It has been reported that metal oxide
nanoparticles (NPs) have shown significant activity against gram-positive and gram-negative
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bacteria [1,2]. The metal oxides such as TiO2, ZnO, and CuO are the most critical metal oxides
with antibacterial activity [3]. ZnO NPs have been considered for many applications, including
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solar cells, sensors, transistors, piezoelectric devices, anti-reflective coatings, and appropriate
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antibacterial activity. ZnO NPs nanoparticles have intense antibacterial activity against a wide
range of bacteria (such as E. coli and S. aureus [4]), and ZnO is cheaper and more biocompatible
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than silver [5]. The photocatalytic properties of ZnO NPs are one of the antibacterial mechanisms.
In this mechanism, radicals are created by electron-holes obtained from photoexcitation. Radicals
damage cell membranes and kill bacteria by attacking organic compounds in microorganisms [6].
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However, the photocatalytic efficiency of ZnO NPs remains low due to the rapid recombination
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of the electron-hole pairs produced. Therefore, to increase the antibacterial efficiency based on
photocatalytic performance, it is necessary to delay the recombination of charge carriers. The
design and synthesis of hybrid nanomaterials based on semiconductor oxide are the most important
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methods for solving the challenge [1]. Researchers used graphene oxide (GO) [7] and reduced
graphene oxide (RGO) [8–10] to increase the photocatalytic properties of metal oxides.
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Graphene-based materials such as GO and RGO have attracted the attention of many
researchers due to their unique structure and properties [11,12]. GO and rGO have unique
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applications in nanomedicine and antibacterial materials. Compared to other carbon materials such
as fullerene, carbon nanotubes, and graphene, these nanomaterials have less cytotoxicity [3].
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Semiconductor-based-GO nanocomposites have shown superior photocatalytic antibacterial
behavior under visible/UV light irradiation due to relatively lower recombination of electron-hole
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pairs and faster charge transfer in these photoactive [13]. Also, GO plates have sharp edges that,
through contact with bacteria, damage cell membranes and kill bacteria [14].
It has recently been reported that GO composite with CuO can increase antibacterial
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properties by 76% [3]. Alayande et al. [15] studied the antibacterial properties of RGO-CuO
nanocomposite films; they found that adding RGO to CuO could completely inactivate
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Pseudomonas aeruginosa PAO1. Liu et al. [16] investigated the antibacterial activity of graphite,
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graphite oxide, GO, and RGO. They reported that GO and RGO showed suitable antibacterial
activity against E. coli. In another study, ZnO NPs were shown to have significant antibacterial
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properties in contact with Staphylococcus aureus. Also, ZnO shows high biocompatibility against
human keratinocyte cells [17].

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This study aims to synthesize and develop novel ZnO-GO and ZnO-RGO nanocomposites
for antibacterial applications with excellent efficiency. ZnO nanoparticles interact with GO or
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RGO sheets through van forces in waltz, electrostatic, and hydrogen bonds. The antibacterial
properties of ZnO nanoparticles were compared with ZnO-GO and ZnO-RGO nanocomposites (1,
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5, and 10 %wt of GO or RGO sheets). The ZnO semiconductor nanocomposites with GO or RGO
showed improved antibacterial activity compared to pure ZnO, GO, and RGO. The highest
antibacterial activity under dark conditions after 12 h is related to Z-10GO nanocomposite, killing
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94% of E. coli bacteria. In nanocomposites, the bond between ZnO nanoparticles and GO or RGO
sheets leads to narrowing the energy gap and shifting the absorption edge to longer wavelengths
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than pure ZnO.
2. Materials and methods
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2.1. Preparation of ZnO NPs
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Synthesis of ZnO NPs using the green chemistry method consists of two steps. In the first
step, ionic liquid 1-butyl-3-methylimidazolium chloride was synthesized, and in the second step,
this liquid was used to synthesize ZnO NPs. For this purpose, 1-butyl chloride (Merck) and N-
methylimidazole (Merck) were poured into the balloon in a molar ratio of 1: 1.1, and the balloon
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was connected to the condenser. For one week, the balloon connected to the condenser was placed
under a magnetic stirrer (500 rpm) in an oil bath (60 °C). The obtained ionic liquid is in the form
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of a concentrated phase at the bottom of the balloon. To remove unreacted raw materials
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(immiscible with ionic liquid), the ionic liquid was cooled and washed with a diethyl ether (Merck)
solution. The diethyl ether residue was removed in an oven at 60 °C. In the second step, precursors
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of zinc acetate (Merck), sodium hydroxide (Merck), and synthesized ionic liquid with a molar ratio
of 1: 3: 1 were used. First, zinc acetate and sodium hydroxide were crushed separately in a mortar
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and then was mixed. The synthesized ionic liquid was added to the mixture of zinc acetate-sodium
hydroxide and was ultrasonicated for 30 min to obtain a homogeneous suspension. The resulting
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suspension was washed several times with water and ethanol and centrifuged. Finally, the
synthesized ZnO NPs were dried at 70 °C.

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2.2. Preparation of GO
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GO was synthesized by modifying the Hummer method. First, 0.5 gr of graphite powder
(Merck) and 0.3 gr of sodium nitrate (Merck) were added to 16.5 ml of cold sulfuric acid in a
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balloon and then stirred (1250 rpm) for 30 min. In the next step, an ice bath was used to bring the
temperature to 0 °C, and 1.5 gr of potassium permanganate (Merck) was gradually added to the
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suspension over 30 min. Next, the 35 °C oil bath replaced the ice bath, and the suspension was
stirred for 40 min. In the next step, 70 ml of deionized water was gradually added to the system,
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at which point the suspension temperature reached 85 °C, and brown vapors were released from
the suspension. After completion of the reaction, 1.75 ml of hydrogen peroxide (Merck) was added
drop wise to the suspension. The resulting suspension was then washed several times with
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hydrochloric acid, ethanol and centrifuged (4100 rpm) for 10 min. Finally, the resulting powder
was dried under vacuum for 24 h at 60 °C.
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2.3. Preparation of RGO er
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First, a suspension of GO with a 1 mg/mL concentration in deionized water was prepared
and ultrasonicated for 1 h to obtain the homogeneous suspension. Then, 1 ml of hydrazine hydrate
(Merck - 80% in water) was added to the suspension as a reducing agent. In order to complete the
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reduction reaction, an oil bath was used at 100 °C. At the end of the reduction reaction, the black
RGO is immersed on the water's surface and separated from the water by filtration. Finally, the
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synthesized RGO was washed several times with methanol and water and dried at ambient
temperature.
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2.4. Preparation of ZnO-GO and ZnO-RGO nanocomposites

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ZnO-GO and ZnO-RGO nanocomposites were synthesized by physical methods. The GO
and RGO in nanocomposites 1 %wt, 5 %wt, and 10 %wt were considered. For this purpose, three
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aqueous suspensions of GO or RGO and three aqueous suspensions of ZnO were prepared. Then,
the suspension containing GO or RGO was added to the ZnO suspension and subjected to vigorous
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stirring (1400 rpm) for 3 h. After that, the suspension containing GO or RGO and ZnO was
ultrasonicated for 30 min. Finally, the suspension was centrifuged, and the ZnO-GO or ZnO-RGO
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nanocomposite was dried at 80 °C for 4 h. The nanocomposites were coded as Z-xGO or Z-xRGO
with x =1, 5, and 10 %wt.
2.5. Characterization methods
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The study of chemical bonds and functional groups in nanocomposites synthesized by
Fourier transform infrared spectroscopy (FTIR, Perkin Elmer, Spectrum RXI device in the range
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of 400–4000 cm-1) analysis was performed. X-ray diffraction (XRD) analysis was used to evaluate
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the crystal structure and phases of the nanocomposites (Philips PW 3710 device, Cu Kα; λ = 1.5406
lamp, voltage = 50 kV, current = 250 mA, 2θ = 5–80 and scan rate = 0.5 °/min). Scanning electron
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microscopy (SEM, VEGA/TESCAN model) was used to study the nanostructure of ZnO and
Oxford Stereo Scan S360 to study the nanostructure of GO and RGO. The nanoparticles' surface
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area, size, and porosity distribution were evaluated by measuring the adsorption isotherm of
nitrogen gas molecules using the Brunauer–Emmett–Teller (BET, Belsorp-MiNI/Gemini 2375

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model). The nanomaterials’ optical properties were measured using ultraviolet-visible
spectroscopy (UV-vis, PerkinElmer Spectrometer made by LambdaIs USA). The X-ray

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photoelectron spectrometer (XPS - Al KR anode) was used to investigate the chemical bonds
present in the nanocomposites. XPS at energy of 1486.6 eV was employed to investigate the
surface atomic composition, chemical state, and stoichiometry of the Zinc Oxide containing the
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GO and RGO nanosheets. Atomic force microscopy (AFM, Veeco Autoprobe CP-Research model
with 10 nm silicone tip and non-contact mode) was engaged in measuring the size and thickness
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of graphene oxide sheets.
2.6. Antibacterial activity
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The antibacterial activity of the synthesized samples was evaluated by the unit colony
formation (CFU) method. The LB liquid culture medium was obtained via dissolving peptone: 10
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g/L, salt: 10 g/L, and yeast extract: 5 g/L. After setting pH = 7, agar: 2 g is added per 100 mL of
culture medium. The LB liquid culture medium was placed in 15 mL tubes in an autoclave for 15
min at 120 °C. Then 15 mg of sample, 4.5 ml of LB liquid, and 0.5 μl of E. coli were mixed and
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incubated with the control sample (without an antimicrobial agent) for 12 h at 37 °C. Then diluted
samples were prepared and incubated on culture plates at 37 °C for 12 h. Finally, the number of
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colonies formed was counted, and the percentage of antibacterial activity was calculated using
Eq.1 [3].
B‒A
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R= B
× 100 (1)
Where R: percentage of antibacterial activity, A: number of colonies counted in the sample, and
B: number of bacteria counted in the control sample.

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3. Results and discussion
3.1. FTIR spectroscopy

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The FTIR spectrum for the ZnO, GO, Zn-1GO, Zn-5GO, and Zn-10GO samples are shown
in Fig. 1a. According to the FTIR spectrum of ZnO, the broad absorption peak at position 3418
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cm-1 belongs to the hydroxyl group in water adsorbed in the ZnO structure. The absorption peak
of the Zn-O bond is observed at 420 cm-1 wavenumbers [18]. Peaks at positions 890 cm-1, 1382
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cm-1, and 1622 cm-1 are related to the bending vibrations of C-H bond, the unsymmetrical
stretching vibrations, and the symmetrical stretching vibrations of COO bond zinc acetate
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(corresponding to the remaining zinc acetate in ZnO). In general, unintense peaks are related to
zinc acetate, indicating that this substance's slight amount remains in ZnO [19]. However, the
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adsorption peak related to C-N binding at position 1170 cm-1 was not observed (corresponding to
ionic liquid).
According to the FTIR spectrum of GO, the unintense peak observed in position 919 cm-1
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is related to the C-H bending (Fig. 1a). The peaks appearing at wavenumbers 1053 cm-1, 1225
cm-1, and 1412 cm-1 correspond to the stretching vibrations of C-O bond, the stretching vibrations
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of C-O-C bond, and the bending vibrations of O-H bond in the carboxylic acid groups. Also, the
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peaks 1618 cm-1 and 1720 cm-1 are related to aromatic bonds C=C and carbonyl/carboxyl (C=O),
respectively. Also, the broad absorption peak related to the O-H stretching vibration is observed
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at position 3356 cm-1 [20]. No unknown peaks were observed in the ZnO and GO spectra,
indicating that these nanomaterials are highly pure. In addition, the FTIR spectra of Z-1GO, Z-
5GO, and Z-10GO nanocomposites are shown in Fig. 1a. In the Zn-1GO nanocomposite spectrum,
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the peak intensities of oxygen-containing functional groups relative to GO decreased, but with
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increasing GO value, the intensities of these peaks increased slightly and became similar to the
GO spectrum (for Zn-5GO and Zn-10GO nanocomposites). After bonding between ZnO and GO,
a slight shift is observed in some peaks relative to pure ZnO or pure GO, indicating that the
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electrostatic adsorption of ZnO on the GO plates affects the vibrational state of the GO functional
groups. For example, a displacement in the C=O peak at position 1720 cm-1 (in GO spectrum) to
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1650 cm-1 (in the nanocomposites spectrum) is due to the formation of a bond between zinc and
carbonyl or carboxyl groups in the GO structure [21]. Also, the nanocomposite formation causes
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shifts in the Zn-O absorption band at 420 cm-1 wavenumbers to about 500 cm-1. In general, the
intensity reduction and shift in the nanocomposite spectrum compared to the GO and ZnO spectra
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indicates the successful formation of GO-ZnO nanocomposites [22].
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a 1622 cm-1 1382 cm-1
3418 cm-1 890 cm-1
420 cm-1
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1618 cm-1
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1053 cm-1
3356 cm-1
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919 cm-1
1225 cm-1
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b
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1790 cm-1 1620 cm-1

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3380 cm-1
1053 cm-1
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Figure 1. a) FTIR spectra of ZnO, GO, Zn-1GO, Zn-5GO and Zn-10GO, and b) FTIR spectra of ZnO,
RGO, Zn-1RGO, Zn-5RGO and Zn-10RGO.
The FTIR spectra of the ZnO, RGO, Z-1RGO, Z-5RGO, and Z-10RGO samples are shown
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in Fig. 1b. In the RGO spectrum, the peak intensity related to oxygen-containing functional groups
in positions 1053 cm-1, 1225 cm-1, 1412 cm-1 and 1720 cm-1 has significantly decreased compared
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to the GO spectrum. The reduction reaction of GO eliminates all oxygen-containing functional
groups; however, the presence of these groups is still observed in the RGO spectrum. In this study,
the formation of RGO or GO nanocomposites with ZnO is done through functional groups; thus,
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the functional groups must not be eliminated entirely in RGO [23], [24]. In the nanocomposite
spectrum, the peak intensities of the functional groups of RGO decreased, indicating that the
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functional groups in RGO react with ZnO. Also, the shift at the Zn-O absorption peak in
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nanocomposites relative to pure ZnO (from 420 cm-1 to 490 cm-1) confirms the interaction between
ZnO and RGO [25].

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3.2. X-Ray diffraction
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The XRD analysis was used for phase and structural analysis of the synthesized samples,
and the results are presented in Fig. 2. The XRD pattern obtained for pure ZnO has a Wurtzite
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crystal structure (hexagonal), and the impurity peak is not observed. The ZnO peaks with Wurtzite
structure are present in positions 2θ= 36.39, 40.19, 42.29, 55.72, 66.60, 74.40, and 78.84 [26],
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[27]. The network constants are related to the resulting structure a = b = 3.26 A° and c=5.21 A°.
The crystal size was calculated using Scherrer Eq. 2. The crystal size calculated for ZnO was about
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23 nm.
(0.9λ)
D = β cosθ (2)
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According to the XRD pattern for GO, the peak at positions 2θ= 11.34 and 49.34
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corresponds to plates (002) and (100), respectively (Fig. 2). Using the Bragg equation, plate
spacing (d = 0.9 nm) was calculated for the plate (002), which increased compared to graphite (d
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= 0.34 nm). This could be due to oxygen-containing functional groups such as carboxyl, hydroxyl,
epoxy, and water molecules introduced between the plates [28], [29]. In the RGO pattern (Fig. 1),
the peak at positions 2θ= 24.15 and 42.59 corresponds to plates (002) and (100), respectively, in
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which the peak of impurity is not observed. The plate spacing (002) in RGO (d = 0.36 nm) is
relatively close to the graphite plate spacing, which is less than the GO plate spacing. This may be
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due to removing oxygen-containing functional groups such as carboxyl, hydroxyl, and epoxy from
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the RGO plates during the reduction process. Also, the peak shift of the plate (002) in the RGO
pattern relative to the GO confirms the change in the spacing of these plates (Fig. 2) [29].
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According to the XRD pattern for Z-10GO and Z-10RGO nanocomposites (Fig. 2), GO,
and RGO peaks are absent, and the pattern obtained for these nanocomposites is similar to the pure
ZnO pattern. This may be due to the low amounts of GO and RGO in the structure of these
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nanocomposites. Also, the network constants and crystal size in Z-10GO and Z-10RGO
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nanocomposites were insignificantly different from pure ZnO [30], [31].

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(101)
(100)
(002)
(110)
(103)
(102)
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ZnO
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Z-10GO
(002)
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(100)
GO
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Z-10RGO
(002)
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(100)
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RGO
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Figure 2. XRD pattern for ZnO, GO, RGO, Z-10GO, and Z-10RGO synthesized samples.
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3.3. X-ray photoelectron spectrometer
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Because antibacterial activity is a surface activity and depends on the surface bandings,
hence, XPS analysis was used to investigate the elemental surface chemical composition of Z-
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10GO nanocomposite. The XPS spectra measured for the nanocomposite are shown in Fig. 3.
According to the XPS survey of Z-10GO (Fig. 3a), the peaks of Zn (2p), C (1s), and O (1s) indicate
the presence of zinc, carbon, and oxygen elements in the nanocomposite, respectively. Therefore,
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there is no peak corresponding to the impurity elements in the XPS spectrum. The Zn (2p) window
is shown in Fig. 3b, in which the peak in binding energies of 1022 eV and 1045 eV corresponds to
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Zn (2p3/2) and Zn (2p1/2), respectively. The shift of these peaks relative to the pure Zn peaks (1021
eV and 1044 eV) indicates an interaction between ZnO and GO [32], [33]. C (1s) spectra (Fig. 3c),
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decovoluted in four peaks corresponding to carbon bondings. The peak observed at positions
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282.30 eV and 284.20 eV are related to C-O-Zn and C-C bonding [34], [35]. In addition, the peaks
at binding energies 285.80 eV and 288.20 eV are assigned to the C-CO and O=C-O-Zn and π-π*
bands. The presence of C-O-Zn and O=C-O-Zn peaks in the C (1s) spectrum confirms the
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formation of ZnO-GO nanocomposite [36]. The O (1s) spectrum is shown in Fig. 3d, that the peak
at positions 529.48 eV corresponds to oxygen ions (O2-) in the ZnO network [37]. While the
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presence of peaks at binding energy 531.70 eV and 532.80 eV are assigned to surface oxygen
species in ZnO-GO nanocomposite, including C-O, C-OH, and adsorbed water molecules. In
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addition, the presence of C-O bond indicates the binding of ZnO to GO plates. Therefore, the
results of FTIR, XRD, and XPS characterizations showed that the nanocomposite was successfully
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formed and surface bonding is completed for the chemical or antibacterial activity on the surface
of nanocomposite [32].
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a b
Zn 2p
2p3/2
Zn loss
2p1/2
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C Auger
O 1s
Zn 3p
C 1s
Zn loss
O 2s
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c d
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Zn-O
C-O
C-C
O=C-O-Zn
C-CO
C-O-Zn er C-O
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Figure 3. (a) XPS survey spectrum of the Z-10GO nanocomposite; high-resolution core-level XPS
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windows spectra of : (b) Zn (2p), (c) C (1s) and (d) O (1s).

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3.4. Specific surface area and porosity measurement
The high surface-to-volume ratio factor in nanomaterials makes nanomaterials more

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efficient than bulk materials. Therefore, measuring the specific surface area and porosity of ZnO
nanoparticles is essential for studying interaction with GO and RGO, antibacterial activity is also
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a surface reaction which dependent to surface area of the nanocomposite, So, determination of
pore size and its surface area is important. BET is a suitable method for determining the specific
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surface area, porosity volume, and porosity size distribution of nanomaterials with spherical,
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cylindrical, or layered shapes. The adsorption and desorption curves for ZnO nanoparticles as a
function of relative pressure (P/P0, P: applied pressure, and P0: saturation pressure N2) are shown
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in Fig. 4a. The adsorption and desorption curves in nanomaterials appear as hysteresis loops with
various shapes depending on porosity shape. The behavior of adsorption and desorption curves of
ZnO nanoparticles shows that ZnO is mesoporous nanomaterials with limited adsorption, and
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adsorption is stepwise (according to IUPAC standard) [38]. The data obtained from BET analysis
for ZnO nanoparticles include the average porosity diameter, porosity volume, and specific surface
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area, which were calculated as 26.74 (nm), 0.2554 (cm3g1-), and 38.199 (m2g1-), respectively. Also,
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the porosity size distribution of ZnO nanoparticles was obtained using the BJH theory (Fig. 4b).
According to the pore size distribution curve, ZnO nanoparticles have a wide pore size distribution
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(the maximum porosity size is about 46 nm). Therefore, the adsorption-desorption and the porosity
size distribution curves showed that ZnO nanoparticles are mesoporous.
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Figure 4. The obtained results from BET analysis for ZnO nanoparticles. a) absorption and desorption
curves, and b) porosity size distribution curves.

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3.5. Microstructure study
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The topography of synthesized ZnO, GO, and RGO samples were evaluated using SEM,
and the resulting images are presented in Figs 5a-c. Pure ZnO nanoparticles are mostly spherical
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with uniform shape and particle size. The average particle size of ZnO was calculated by Image J
software to be about 60 nm (Fig. 5a). The growth mechanism of ZnO nanoparticles is divided into
the following three steps: 1) In the first step, zinc hydroxide gel is formed by zinc acetate and
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sodium hydroxide reaction. This step usually occurs at high molar concentrations of sodium
hydroxide. The resulting gel is complexed with 𝑂𝐻 ‒ ions, and 𝑍𝑛(𝑂𝐻)24‒ species is formed
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according to the following reactions.
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𝑍𝑛(𝐶𝐻3𝐶𝑂𝑂)2.2𝐻2𝑂 + 2𝑁𝑎𝑂𝐻 → 𝑍𝑛(𝑂𝐻)2 + 2𝐶𝐻3𝐶𝑂𝑂𝑁𝑎 + 2𝐻2𝑂 (3)
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𝑍𝑛(𝑂𝐻)2 + 2𝐻2𝑂→ 𝑍𝑛(𝑂𝐻)24‒ + 2𝐻 + (4)
2) The second step involves the formation of 1-butyl-3-methylimidazole - 𝑍𝑛(𝑂𝐻)24‒ . The 1-

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butyl-3-methylimidazolium chloride ionic liquid contains 1-butyl-3-methylimidazolium cation
and 𝐶𝑙 ‒ anion. Liquid ionic cations and 𝑍𝑛(𝑂𝐻)24‒ can also be absorbed by electrostatic force. 3)
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In the third step, the pairs of ions formed between the ionic liquid cations and 𝑍𝑛(𝑂𝐻)24‒ anion is
dehydrated, and ZnO nuclei are formed. The electrostatic attraction between the ionic liquid cation
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and the oxide ions in ZnO nuclei deactivated the surface of ZnO nuclei. Thus, the growth of the
nuclei stops in different directions, and the spherical structure of ZnO is observed [39], [40].
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According to the topography of GO sheets (Fig 5b), it is observed that the synthesized GO
has a layered structure that is formed from the accumulation of graphene sheets. Also, graphene
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layers have sharp edges that can affect the antibacterial properties of GO (internal Fig. 5b). The
use of ultrasonic in the synthesis and dispersion of GO sheets reduces the number of graphene
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layers (exfoliation of GO). Also, AFM image in tapping mode was used to study the microstructure
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of GO sheets in more detail. Tapping mode is an alternating mode of contact and non-contact mode
used to prevent damage to GO sheets. Figures 5d and e show the results obtained from AFM.
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According to Fig. 5e, the GO sheets have a height profile of 1.5 to 3 nm, and the width of the
sheets is about 250 nm. Comparing the height profiles of synthesized GO sheets with the
theoretical thickness of GO, observed that the GO microstructure is mainly two or three layers
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[41]. Also, according to the SEM and AFM images in Figs 5b and e, respectively, sharp edges in
the GO sheets are confirmed.
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Figure 5c shows the SEM images of the RGO microstructure. There is no change in the
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RGO microstructure compared to the GO microstructure. In the graphene oxide reduction process,
functional groups are eliminated (Fig. 1b), which reduces the distance between the graphene
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sheets. The RGO microstructure is made from stacking graphene sheets, that the sheets have sharp
edges (similar to the GO microstructure). Comparing the SEM images of GO and RGO, it is clear
that the size of RGO sheets is larger than that of GO sheets, which may be due to the van der Waals
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force between the RGO sheets, which causes graphene sheets to agglomeration in RGO [16].
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a b c
ed
es
dg
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sha
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200 nm 500 nm 500 nm
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500500
nmnm 2 µm 2 µm
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d e
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Figure 5. SEM images for a) ZnO, b) GO, and c) RGO. AFM results for GO, d) 2D AFM image of GO,
and e) height profile.

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Also, microstructural changes of nanocomposites due to the combination of ZnO with GO
and RGO were investigated using SEM. The SEM images for Z-5GO, Z-10GO, Z-5RGO, and Z-
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10RGO nanocomposites are presented in Fig. 6a-d (scales: 2 µm and 500 nm), respectively.
According to the SEM image of Z-5GO and Z-10GO nanocomposites (Fig. 6a and b), ZnO
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nanoparticles cover the surface of GO sheets, which occurs by bonding ZnO with functional groups
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on GO. ZnO nanoparticles show high accumulation on GO sheets, which by reducing the
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percentage of ZnO in the Z-10GO nanocomposite, ZnO accumulation is reduced (Fig. 6a and b).
According to Fig. 6c and d, ZnO nanoparticles are accumulated on GO sheets, which is due to the
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bonding of ZnO with a limited amount of functional groups on GO sheets. In general, the
accumulation of ZnO nanoparticles on GO sheets is higher than RGO sheets (Fig. 6). This may be
due to the presence of a more significant amount of functional groups on GO than RGO.
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ZnO
a nanoparticles b
r
GO sheets
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2 µm 500 nm 2 µm 500 nm
c d
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ZnO
nanoparticles
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RGO sheets 2 µm 500 nm 2 µm 500 nm
Figure 6. SEM images for a) Z-5OG, b) Z-10OG, c) Z-5ROG, and d) Z-10ROG (scales: 2 µm and 500
nm).
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Pr
19
The mechanism of nanocomposite formation can be explained as follows. Through van der
ed
Waals, electrostatic, and hydrogen bond interactions, ZnO nanoparticles can be bonded to GO or
RGO sheets. The interaction between ZnO and GO or RGO can be based on hydrogen bonds
iew
between the oxygen in ZnO and the hydroxyl groups on GO. Also, ionization of functional groups
such as carboxylic acid at the edges of graphene sheets (negative charge) absorbs ZnO
nanoparticles (positive charge) through electrostatic force. In addition, the interaction between zinc
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atoms in the ZnO structure and oxygen atoms related to carbonyl groups in the GO or RGO
structure can affect the formation of nanocomposites [42]. Also, another mechanism involved in
r
the formation of nanocomposites is the use of ultrasonic waves in the synthesis process of
er
nanocomposites. Ultrasonic causes the formation, growth, and destruction of a large number of
microbubbles in the suspension. When these microbubbles bursts, high heat (about 5000 °K) is
pe
generated locally around the microbubbles for a few microseconds. These local heats are suitable
sites for the chemical interaction of ZnO with GO or RGO sheets [43].
ot
3.6. Ultraviolet-visible spectroscopy

tn
The study of the optical properties of the synthesized samples was performed using UV-
vis spectroscopy, and the results are presented in Fig.7. In the UV-vis spectrum of pure GO (Fig.
rin
7a), an absorption peak at wavelength 240 nm is related to the C=C aromatic bond (due to 𝜋→𝜋 ∗
transfer). Also, a shoulder at position 300 nm is observed, which is belong to the transfer of 𝑛→𝜋 ∗
ep
at C=O bonds in the carboxylic acid groups. The absorption peak and shoulder in the GO spectrum
confirmed the formation and high purity of GO [44]. In the RGO spectrum (Fig. 7b), an absorption
Pr
peak at the wavelength 267 nm corresponding to the transfer of 𝜋→𝜋 ∗ in aromatic bonds C=C.
The absorption peak in the RGO spectrum shifts to longer wavelengths than the GO spectrum,
20
which may be due to the removal of functional groups between the RGO sheets and the increase
ed
in conjugated bonds during the reduction process. According to the UV-vis spectrum of ZnO in
Fig. 7c, the absorption peak that appeared at position 358 nm confirmed the formation of ZnO
iew
nanoparticles [45]. In this study, the adsorption peak of ZnO nanoparticles was shifted to shorter
wavelengths than ZnO bulk (384 nm), which could be due to the smaller size of the nanoparticles.
The UV-vis spectrum of Z-1GO, Z-5GO, and Z-10GO nanocomposites are shown in Fig.
ev
7c. The bonding between ZnO nanoparticles and GO sheets leads to narrowing the semiconductor
energy gap and shifting the absorption edge to longer wavelengths than pure ZnO. This behavior
r
is due to the high electron acceptor of GO and is also observed in n-type semiconductors such as
er
TiO2. GO sheets act as electron acceptors in ZnO-GO nanocomposites, and ZnO semiconductors
act as electron donors [8]. Through the π electron rotation system, excited electrons to the
pe
conductivity level of ZnO nanoparticles are transferred to GO sheets (electron injection). The
binding of ZnO nanoparticles to GO sheets via carboxyl groups leads to shortenning the electron
transfer path from ZnO to GO [46]. Increasing GO from 1% to 10% in nanocomposites makes the
ot
energy gap narrower, and the absorption edge shifts to longer wavelengths (red-shift) (Fig. 7c).
tn
According to the UV-vis spectrum of Z-1RGO, Z-5RGO, and Z-10RGO nanocomposites in Fig.
7d, the absorption edge is shifted by increasing the percentage of RGO to higher wavelengths (red-
shift). The reason for this behavior in ZnO-RGO nanocomposites is similar to the behavior of ZnO-
rin
GO nanocomposites. Also, the red-shift in ZnO-RGO nanocomposites is higher than in ZnO-GO
nanocomposites, which may be due to the higher electron acceptor RGO than GO (Fig. 7e).
ep
Pr
21
ed
a b
iew
ev
c d
r
er
pe
e
ot
tn
rin
ep
Figure 7. UV–vis spectra for a) GO, b) RGO, c) ZnO, Z-1GO, Z-5GO, Z-10GO, d) ZnO, Z-1RGO, Z-
5RGO, Z-10RGO, and e) ZnO, Z-10GO, Z-10RGO.

Pr
22
3.7. Antibacterial activity under dark conditions
ed
The antibacterial activity of the synthesized samples was evaluated using the CFU counting
method (Eq. 1) for E. coli bacteria (for 12 h at 37 °C); the results are presented in Fig. 8. Figure
iew
8a shows a blank sample (control sample); there is no antibacterial agent in the E.coli culture
medium. Figure 8b shows the CFUs of E. coli in contact with pure ZnO nanoparticles, which kill
about 84% of the bacteria (Fig. 8k). Generally, various mechanisms have been proposed for the
ev
antibacterial activity of ZnO nanoparticles, such as the photocatalytic production of reactive
oxygen species (ROS) that destroy bacteria's cellular components or cell membranes. The energy
r
gap of the synthesized ZnO nanoparticles is in the ultraviolet range, which requires ultraviolet light
for antibacterial action (to excite electrons and create electron-hole pairs). In this study,
er
antibacterial activity was performed in a dark and ultraviolet light-free environment, suggesting
pe
that another mechanism is involved in the antibacterial properties of ZnO. The mechanism of
antibacterial activity of ZnO nanoparticles can be due to the dissolution and release of Zn2+ ions
from ZnO in the bacterial culture medium. Joe et al. [47] believed that ZnO nanoparticles attached
ot
to the bacterial cell wall dissolve, and Zn2+ ions are transfected into the cytoplasm membrane.
Lipoteichoic acid on the outer membrane of E.coli bacteria contains large amounts of negatively
tn
charged lipopolysaccharides that may provide attachment sites for ZnO nanoparticles. Therefore,
lipoteichoic can ZnO dissolution by the formation of ionic salts with Zn2+ [48]. Finally, Zn2+ ions
rin
enter the cytoplasm of E. coli through various membrane metalloproteins such as members of
ATP-binding cassette (ABC) and natural resistance-associated macrophage protein (NRAMP)

ep
[49]. The cytoplasm of E.coli disintegrates after the arrival of Zn ions; thus, the antibacterial
mechanism of ZnO nanoparticles under dark conditions is based on dissolved Zn2+ ions [5].
Pr
23
According to Fig. 8c and d, GO sample (81%) kills more E. coli colonies after 12 hours
ed
than RGO (71%). The antibacterial activity of GO sheets under dark conditions may be via
physical contact with the bacterium, which causes stress and destruction of the E.coli cell wall.
iew
The SEM and AFM images (Fig. 5) showed that the GO sheets had sharp edges, which destroyed
the bacterial cell membrane when bacteria came in physical contact with them. Also, Liu et al.
[16] reported that bacterial cells are severely damaged after direct contact of E. coli with graphene-
ev
based nanomaterial. Also, the oxidative stress caused by ROS has a minor role in the antibacterial
activity of GO, because graphene materials produce little superoxide. Another mechanism involves
r
the destruction of E.coli cell membrane by oxidizing the vital structure of the bacterial cell without
er
ROS intervention [50]. In this mechanism, the bacterial antioxidant (γ-L-glutamyl-L-cysteinyl-
glycine, GSH) compounds are decomposed, leading to bacterial death. GSH is a tripeptide with
pe
thiol groups and an antioxidant in bacterial cells. The thiol groups (-SH) in GSH can be oxidized
to disulfide bonds (-S-S-). The antibacterial mechanism of RGO sheets is similar to GO sheets.
The lower antibacterial activity of RGO (hydrophobic due to the removal of functional groups)
ot
than GO (hydrophilic due to functional groups) may be due to the agglomeration of RGO sheets,
which is due to the elimination of functional groups in the RGO reduction process. The van der
tn
Waals force is created between RGO sheets and prevents the graphene sheets from dispersing,
while GO sheets disperse more than RGO. Therefore, the sharp edges on RGO sheets overlap,
rin
which reduces the number of active RGO sites (reduce contact of sharp edges with bacterial
membranes) compared to GO (Fig. 8 c-d and k) [3], [51].

ep
Pr
24
a b c d
ed
iew
e f g
r ev
h i j
er
pe
100 k
ot
92 94
89 89 91
Antibacterial activity (%)
84 85
81
80
73
tn
60
40
rin
20
0
RGO
Z-10RGO
Z-5GO
Z-5RGO
ZnO
Z-1GO
Z-10GO
Z-1RGO
GO
ep
Figure 8. The antibacterial test images for a) blank, b) ZnO, c) GO, d) RGO e) Z-1GO, f) Z-5GO, g) Z-
Pr
10GO, h) Z-1RGO, i) Z-5RGO, j) Z-10RGO and k) antibacterial activity for all samples, after 12 h of
contact with E.coli at 37 °C.
25
Generally, ZnO-GO and ZnO-RGO nanocomposites show enhanced antibacterial activity
ed
compared to ZnO, GO, or RGO (Fig. 8k). According to Figs e-g, which are related to Z-1GO, Z-
5GO, and Z-10GO nanocomposites, increasing the weight percentage of GO has reduced the
iew
number of E.coli colonies. Increasing the weight percentage of GO from 1% to 10% leads to an
increase in the antibacterial activity of ZnO nanocomposites from 89 to 94% (Fig. 8k). Figures h-
j show the results of antibacterial activity of Z-1RGO, Z-5RGO, and Z-10RGO nanocomposites,
ev
which killed 85%, 89%, and 91% of E. coli bacteria after 12 h, respectively. The ZnO-GO
nanocomposites destroyed more CFUs than ZnO-RGO, which may be due to the agglomeration
r
of RGO sheets (due to the removal of functional groups). The antibacterial mechanism of ZnO-
er
GO or ZnO-RGO nanocomposites involves a combination of the following mechanisms: 1)
dissolution of Zn2+ ions from ZnO and penetration into the cytoplasm of E.coli; and 2) physical
pe
interaction between the sharp edges of GO or RGO sheets and the E. coli cell membrane.
Therefore, the increase in antibacterial activity of nanocomposites compared to pure samples is
due to the simultaneous activation of the two mechanisms mentioned in nanocomposite samples.
ot
tn
4. Conclusion
In this study, novel ZnO-GO and ZnO-RGO nanocomposites were synthesized in which
rin
ZnO nanoparticles interacted with GO or RGO sheets through van-waltz forces, electrostatic, and
hydrogen bonds. The results of XRD, FTIR, and XPS analysis confirmed that the nanocomposites
ep
were successfully synthesized. The SEM images showed that ZnO nanoparticles accumulated
more on GO than RGO due to removing oxygen-containing functional groups on RGO sheets. In
the UV-vis spectrum, increasing the GO or RGO from 1 to 10 %wt in the nanocomposites leads
Pr
to a narrowing of the energy gap, and the absorption edge shifts to longer wavelengths (red-shift)
26
relative to pure ZnO. The ZnO-GO and ZnO-RGO nanocomposites showed improved antibacterial
ed
activity under dark conditions compared to pure ZnO. The synthesized ZnO, GO, RGO, Zn-1GO,
Zn-5GO, Zn-10GO, Zn-1RGO, Zn-5RGO, and Zn-10RGO samples kill 84%, 81%, 73%, 89%,
iew
92%, 94%, 85%, 89%, and 91% of E. coli bacteria after 12 h at 37 °C, respectively. The enhanced
antibacterial activity of nanocomposites is due to the simultaneous activation of two antibacterial
mechanisms, including dissolution of Zn2+ ions from ZnO and physical interaction between the
ev
sharp edges of GO or RGO sheets and E. coli.
r
Declaration of interests
er
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
pe
☐ The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
ot
tn
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A comparative study: Green Synthesis and Evaluation of ZnO-GO and ZnO-RGO

Keywords: ZnO-GO and ZnO-RGO nanocomposite, Antibacterial activity, Antibacterial mechanisms, E.

1Corresponding Author: sangpour@merc.ac.ir

photocatalytic performance, it is necessary to delay the recombination of charge carriers. The

human keratinocyte cells [17].

than pure ZnO.

synthesized ZnO NPs were dried at 70 °C.

was dried under vacuum for 24 h at 60 °C.

2.4. Preparation of ZnO-GO and ZnO-RGO nanocomposites

ZnO-GO and ZnO-RGO nanocomposites were synthesized by physical methods. The GO

with x =1, 5, and 10 %wt.

2.5. Characterization methods

nitrogen gas molecules using the Brunauer–Emmett–Teller (BET, Belsorp-MiNI/Gemini 2375

model). The nanomaterials’ optical properties were measured using ultraviolet-visible

spectroscopy (UV-vis, PerkinElmer Spectrometer made by LambdaIs USA). The X-ray

of graphene oxide sheets.

B: number of bacteria counted in the control sample.

3. Results and discussion

3.1. FTIR spectroscopy

1790 cm-1 1620 cm-1

RGO, Zn-1RGO, Zn-5RGO and Zn-10RGO.

ZnO and RGO [25].

nanocomposites were insignificantly different from pure ZnO [30], [31].

windows spectra of : (b) Zn (2p), (c) C (1s) and (d) O (1s).

3.4. Specific surface area and porosity measurement

The high surface-to-volume ratio factor in nanomaterials makes nanomaterials more

size distribution curves showed that ZnO nanoparticles are mesoporous.

curves, and b) porosity size distribution curves.

2) The second step involves the formation of 1-butyl-3-methylimidazole - 𝑍𝑛(𝑂𝐻)24‒ . The 1-

butyl-3-methylimidazolium chloride ionic liquid contains 1-butyl-3-methylimidazolium cation

the GO sheets are confirmed.

and e) height profile.

Also, microstructural changes of nanocomposites due to the combination of ZnO with GO

RGO sheets 2 µm 500 nm 2 µm 500 nm

3.6. Ultraviolet-visible spectroscopy

GO nanocomposites. Also, the red-shift in ZnO-RGO nanocomposites is higher than in ZnO-GO

5RGO, Z-10RGO, and e) ZnO, Z-10GO, Z-10RGO.

ATP-binding cassette (ABC) and natural resistance-associated macrophage protein (NRAMP)

membranes) compared to GO (Fig. 8 c-d and k) [3], [51].

contact with E.coli at 37 °C.

Therefore, the increase in antibacterial activity of nanocomposites compared to pure samples is

antibacterial activity of nanocomposites is due to the simultaneous activation of two antibacterial

antibacterial behavior of in situ grown CuO-GO nanocomposites,” Mater. Today Commun.,

Photocatalytic Performance in Degradation of Methylene Blue,” ECS J. Solid State Sci.

[11] Y. Liu et al., “Synthesis of a poly(N-methylthionine)/reduced graphene oxide

methylthionine at electrochemically reduced graphene oxide electrodes,” Electrochim.

“Chemical bath synthesis of CuO-GO-Ag nanocomposites with enhanced antibacterial

[14] I. Sengupta, P. Bhattacharya, M. Talukdar, S. Neogi, S. K. Pal, and S. Chakraborty,

[21] M. Nasrollahzadeh, B. Jaleh, and A. Jabbari, “Synthesis, characterization and catalytic

[24] H. R. Azimi, M. Ghoranneviss, S. M. Elahi, and R. Yousefi, “Photovoltaic and UV detector

[43] E. K. Goharshadi, Y. Ding, M. N. Jorabchi, and P. Nancarrow, “Ultrasound-assisted green

May 2009, doi: 10.1016/J.JALLCOM.2008.07.123.

34, no. 4, pp. 621–640, Apr. 2013, doi: 10.1016/S1872-2067(12)60530-0.

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