Investigation of Morphological, Optical, and Antibacterial Properties of Hybrid ZnO-MWCNT Prepared by Sol-gel

*Corresponding Author: Selma M. H. Al-Jawad salma_aljawad@yahoo.com Abstract In this research, raw multiwalled carbon nanotubes (R-MWCNT) was successfully functionalized using sulfuric acid and nitric acid. Then a hybrid (ZnO-MWCNT) synthesized by the sol-gel method where diethylene glycol was used as a solvent and stabilizer that works to prevent the accumulation of nanoparticles and reduces the viscosity of the solution. A group of diagnostic techniques, including XRD, UV-Vis, EDX and microscopy has recognized the structural and optical properties of the prepared nanoparticles. High Resolution Electronic Scanner (FE-SEM) was also used in the investigation. FE-SEM images showed the formation of the hybrid (ZnO-MWCNT) by the growth of spherical clusters on the surface of the cross-linked tubes (MWCNT). In addition, FE-SEM images confirmed the success of a ZnO-MWCNT hybrid. The emergence of spherical shapes deposited on cylindrical tubes and associated with a wrinkled surface was recognized. In addition, the particle size ratio increased. The UV-Vis spectra revealed that all the composites had good absorbency with a shift towards short wavelengths. While it was observed from the analysis of X-ray diffraction (XRD) the formation of a hexagonal wurtzite crystal structure due to zinc oxide with a polycrystalline nature. The average crystal size calculated from the Debye-spark equation increased with the increase in the concentration of the streaked material. Antibacterial activity was studied for all prepared samples against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) at different μg/ml concentrations (500, 750, and 1000). It was observed that the highest inhibition Zone for functionalized multiwalled carbon nanotubes (FMWCNT) and ZnO-MWCNT hybrid was (17.3, 12.3mm), (22.5, 19mm) for Escherichia coli and Staphylococcus aureus, respectively.


Introduction
the R-MWCNTs, F-MWCNTs, and ZnO\MWCNT hybrid were studied by the antibacterial activity using diffusion well method against E. coli and P. aeruginosa bacteria with using different concentrations.

Experimental Part 2.1 Acid Treatment of R-MWCNTs
Multiwalled carbon nanotubes (MWCNTs, purity: 95%wt (USA), outside diameter: 5-15 nm) were covalently functionalized by chemical oxidation method to enhance the solubility of MWCNTs in aqueous media and next step for hybrid preparation. High dispersion results have reported with the method. In this part, Raw-MWCNTs were treated by using a mixture of sulphuric acid and nitric acid (3:1 v/v). Firstly, 2g of raw-MWCNTs was functionalized with 150 ml of sulphuric acid (95% H2SO4) and 50 ml of nitric acid (65% HNO3). The mixture was placed in ultra-sonically vibrated in water ultrasonic bath at temperature of 30 °C for 60 min. Then the mixture was diluted with 500 ml of distilled water, vacuum-filtered through a 0.22 μm polycarbonate membrane and then dried at 100 °C for 12 h. The resultant F-MWCNTs was treated with hydrogen peroxide (30% v/v H2O2) as reduction reagent, and the same procedure was repeated in order to achieve whole oxidative procedure started via H2SO4 and HNO3. As a second step, but in a gentler manner carboxyl groups were created on the surface of MWCNTs, which could subsequently be dispersed in composite preparation. Thereafter, the solid was washed by deionized water (DI), and then dried at 100 °C for 12 h to produce functional groups like -OH and/or -COOH functionalized F-MWCNTs. The flow chart representing the steps of functionalized F-MWCNTs by using mixture of sulphuric acid and nitric acid and filtrated method is shown in Figure 1.

Synthesis of ZnO-MWCNTs Hybrid
In this part, ZnO-MWCNTs hybrid was prepared by sol-gel method. Zinc acetate (Zn(CH3COO)2.2H2O), multiwalled carbon nanotube (MWCNTs), diethylene glycol (C4H10O3), and absolute ethanol were used as reactants. Initially, 0.33g of zinc acetate (Zn(CH3COO)2.2H2O) was dissolved in 75 ml diethylene glycol (DEG, C4H10O3), then 3 ml deionized water was added to the solution. Then, the solution of zinc acetate was constantly stirred at 160-180 °C for 12 min. According to the appropriate proportion, the zinc acetate is completely dissolved in a beaker and stirred constantly to get a homogenous solution, and then kept at room temperature for 2h to get ZnO sol. Specific amount of functional MWCNTs was added into the running synthetic solution of ZnO NPs after the gel formation and sonicated for 20 min. The mass of MWCNTs was 0.12 g to obtain the mass ratio of (MWCNTs:ZnO = 1:1). After that, the solution stirred at 160-180 °C for 2 h and then cooled down to the room temperature. Finally, and yet importantly, black precipitate centrifuged and washed with deionized water and absolute ethanol, then dried in an oven at temperature of 60 °C for 48 h. The mixture then calcined at 450 °C for 2h in atmospheric pressure to obtain powder of ZnO-MWCNTs hybrid.

Inhibition Zone Test (IZ)
The test of Antibacterial effect by measuring the inhibition Zone on agar plates was achieved by using well diffusion method to determine the (IZ) for preparation of different samples and also hybrid materials like ZnO-MWCNTs, F-MWCNTs and R-MWCNTs, respectively, against Gram negative (E. coli) bacteria and Gram positive (S. aureus) bacteria. Three different concentrations of each sample were used (500, 750, 1000 μg/ml) against the bacteria. The activated bacterial culture (S. aureus and E. coli) was subcultured on nutrient agar at 37 o C for overnight, and then bacteria suspended in normal saline (0.9%w\v) to prepare initial concentration of 107-108 CFU/ml of each bacteria strain using (standard McFarland tube 0.5). Each culture spread on nutritious agar plates. Then, different concentrations (500, 750, and 1000 μg/ml) of each sample were poured into well in the whole plates and incubated over night at 37 °C. The inhibition zone diameter of each sample was pendent in millimeter; also, the average of inhibition zones around three wells was determined.

Results and Discussion
X-ray diffraction analysis is a technique for identifying the crystal structure and grain size of the synthesized nanomaterials. Figure 2 shows the XRD patterns of diffraction for the R-MWCNTs, F-MWCNTs and ZnO/MWCNTs hybrid, respectively. The XRD patterns of the R-MWCNTs display the appearance of a broad peak of high intensity at 2θ = 25.8° corresponding to the interlayer distance of 3.44 Å. Additionally, the diffraction peak is low intensity at 2θ = 43 o and both are related to hkl planes of (002), (100), respectively. After acid treatment of F-MWCNTs, the XRD patterns display a broad diffraction peak at 2θ = 26 o corresponding to the interlayer distance of 3.45 Å and a low intensity diffraction peak at 2θ = 43.20 o corresponding to the interlayer distance of 2.07 Å, both are related to the hkl planes of (002), (100), respectively, and in good agreement with (JCPDS-0646). Where: D represents particle size; k is a dimensionless shape factor, with a value close to unity. The shape factor has a typical value of about 0.9, λ is the X-ray wavelength and its value = 0.15056 nm, FWHM (β) is the full width at half maximum of the peak, and θ is diffraction angle (deg.).   It was noticed that the R-MWCNTs appears as cylindrical tubes shape with different directions. In addition, the particles tend to agglomerate according to Van der Waals forces between the tubes. After the acid treatment of the F-MWCNTs, it was notable that there was a significant difference in the FE-SEM image as shown in Figure 4 a & 4b. The microscopic images indicate the appearance of nanoparticles in the shape of tangled tubes that look similar a rope. Furthermore, the F-MWCNTs tubes show shorter and have less agglomeration compared to the R-MWCNTs. Additionally, F-MWCNTs structure exhibits irregularity with defects on the surface of the tubes. The mean particle size of R-MWCNTs and F-MWCNTs is (21.06 nm, 33.99 nm), respectively [20]. Figure 5 a & 5 b represents the FE-SEM analysis of the ZnO\MWCNTs hybrid at different magnifications. It was observed that zinc oxide nanoparticles appear in the form of spherical clusters that grow on the surface of tangled tubes that represent the MWCNTs as densely and not uniform. Besides, the distribution of ZnO NPs does not occur in uniform way on MWCNTs. The strong attachment between ZnO and MWCNTs is also due to the interaction between the oxygen groups (functional groups) of both samples. As shown in the image, the hybrid possesses a high surface to volume ratio, which is an important factor for catalysis or surface mediated applications [21]. The results show the success of the ZnO-MWCNTs hybrid synthesis process using the Sol-Gel method [22].   The UV-Vis spectra of R-MWCNT displays a broad absorption band at a wavelength of 291 nm attributable to the π-π* electronic transitions of the C=C bonds forming the aromatic rings in the MWCNT structure. After acid treatment of the F-MWCNT, the UV-Vis spectra display a shifting to shorter wavelength (blue shift) for the absorption peak at 264 nm. Figure 6 a shows the blue-shift region. These shifts in the absorption peak are due to changes in the density of states of electronic structure of F-MWCNTs after covalent functionalization method could lead to disruption in the aromatic system [23,24]. Finally, the absorption spectrum of ZnO-MWCNT hybrid illustrates two absorption peaks at wavelength of 282nm, which is due to the π-π* electron transitions of the double bonds in the MWCNTs composition. Whereas, the absorption peak at 334 nm is due to the electron transitions in the Zn-O bond. This indicates the strong interaction between ZnO and the MWCNTs and thus the formation of the hybrid materials. In state of measurement, the optical energy band gap (Eg) for all samples can be calculated by Tauc's equation 2 [25]:

……… (2)
Where hν is the photon energy, Eg is the optical energy band gap, and A is a constant. Bedsides, the optical band gaps (Eg) of R-MWCNT, F-MWCNT and hybrid materials have been calculated by using plotting (αhν) 2 versus the incident photon energy (hν) as shown in Figure 6 b. It was found that the energy band gap values determined from the Tauc's equation eq. 2 for the R-MWCNT and F-MWCNT is up to 3.28 and 3.18 eV, respectively.

Conclusions
In summary, ZnO\MWCNTs hybrid was prepared using the sol-gel method. Then, X-ray diffraction showed that the presence of functional oxygen groups that increase the interlayer distance between the F-MWCNTs layers. This reveals the success of the functional process using sulfuric and nitric acid without altering the structure of the F-MWCNTs. It was observed that the average crystal size of the ZnO\MWCNTs hybrid, R-MWCNTs and F-MWCNTs are (32.73 nm, 3.27 nm, 3.19 nm), respectively. The FE-SEM images showed that the F-MWCNTs tubes are shorter and have less agglomeration compared to the R-MWCNTs. Additionally, F-MWCNTs structure exhibited irregularity with defects on the surface of the tubes. The hybrid possesses a high surface to volume ratio, which is an important factor for catalysis or surface mediated applications that the average particle size of ZnO MWCNTs hybrid, R-MWCNTs and F-MWCNTs are (32.51 nm, 21.06 nm, 33.99 nm), respectively. The UV-Vis spectra showed that the band gap for the R-MWCNT and F-MWCNT was up to 3.28 and 3.18 eV, respectively. In addition, the MWCNT with ZnO NPs show a significant decrease in the energy band gap with up to 2.98 eV. Antibacterial test at different concentrations (500, 750, and 1000 μg/ml) of R-MWCNTs, F-MWCNTs and hybrid samples against E. coli and S. aureus bacteria by determining the inhibition zone (IZ) in mm. Finally, results demonstrate that F-MWCNTs and ZnO\MWCNTs hybrid recorded highest IZ (17.3 mm and 22.5 mm) for E. coli and IZ (12.3 mm and 19 mm) for S. aureus, respectively at 1000 μg/ml of each sample after incubation overnight.