Synthesis of ZnO nanoparticles by two different methods & comparison of their structural, antibacterial, photocatalytic and optical properties

All the starting raw materials including zinc acetate dihydrate [Zn(CH₃COO)₂.2H₂O, Merck Specialties, India], sodium hydroxide [NaOH, Merck Specialties, India] and absolute ethanol [CH₃CH₂OH, Merck Specialties, Germany) were maintained at a high purity level (>99%). However, in the biosynthesis method, another raw material was also used and that was the leaf of Azadirachta indica (Neem leaf).

At first, 20 gm Zn(CH₃COO)₂.2H₂O was mixed into 150 ml distilled water and stirred for 20 min at 35 °C to produce a zinc acetate solution. Again, 80 gm NaOH powder was weighed, mixed into 80 ml water and stirred for around 20 min at 35 °C for producing NaOH solution. After mixing both solutions, the titration reaction was performed by the addition of 100 ml ethanol into the drop-wise manner accompanied by vigorous stirring. The stirring was continued for around 90 min to complete the reaction for obtaining a gel-like product. Then the gel was dried at 80 °C overnight and calcined in an oven at 250 °C for 4 h. Finally, ZnO nanoparticles were prepared. However, the overall chemical reaction for the preparation of ZnO nanoparticles by using NaOH can be expressed as:

$$\begin{eqnarray}&&\begin{array}{l}{\rm{Zn}}{({{\rm{CH}}}_{3}{\rm{COO}})}_{2}.2{{\rm{H}}}_{2}{\rm{O}}+2{\rm{NaOH}}\to {\rm{ZnO}}+2{{\rm{NaCH}}}_{3}{\rm{COO}}+{{\rm{H}}}_{2}{\rm{O}}\end{array}\end{eqnarray} \tag{ 1 }$$

At first, the neem (A. Indica) leaves were collected from the Azadirachta Indica trees on the campus of Rajshahi University of Engineering and Technology, Bangladesh. After washing with distilled water, the leaves were dried into a dryer for 24 h. Then 20 gm dried leaves were smashed and mixed with 50 ml distilled water. After that, the mixture was stirred by a magnetic stirrer and heated at 60 °C for 1 h. As the mixture displayed a yellow color, it was filtered using the Whatman^TM filter paper. However, the extract solution was used for further preparation of ZnO nanoparticles. The overall process for the preparation of Neem leaf extract is stereotyped in figure 1.

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The next step included the preparation of the zinc acetate solution. For this, 21.94 gm Zn(CH₃COO)₂.2H₂O was mixed into 50 ml water and stirred for 20 min at 35 °C. Similarly, in order to prepare a NaOH solution, 4 gm NaOH powder was added into 50 ml distilled water and simultaneously stirred for 20 min at 35 °C. Both solutions were then mixed by vigorous stirring. During this stirring process, the neem leaf extract was drop-wise mixed with the solution. As the addition of neem leaf continued, white precipitation of nanoparticles appeared. Then the solution was filtered and the filtered product was dried at 80 °C for 4 h. After that, the dried powder was calcined at 250 °C for 4 h and grounded to obtain the desired ZnO nanoparticles.

X-ray diffraction was performed for structural analysis employing 40 kV-40 ma (scanning step of 0.02°) and Cu- K_α radiation having wavelengths of K_α1 = 1.54060 Å, K_α2 = 1.54439 Å (Bruker Advance D8, Germany). Morphological characterization was accomplished by scanning electron microscopy (ZEISS EVO 18, UK). The optical properties were determined through UV–vis spectroscopy (SHIMADZU UV/Vis-1650 PC, Japan) into a range of 200–800 nm.

Escherichia coli bacteria were mainly involved in the determination of the antibacterial performance of ZnO NPs. Initially, the bacteria was stock-cultured in brain heart infusion (BHI) growth medium at −20 °C. Around 3 ml of BHI broth was added to 300 ml of stock-culture and preserved the culture overnight at 36 °C ± 1 °C for 24 h. After 24 h of incubation, dilution of the bacterial suspension (inoculum) was accomplished by using sterile saline. To indicate the bacterial growth during the test, a solution of 2-(4-iodophenyl)−3-(4-nitrophenyl)−5-phenyltetrazolium chloride (INT) in ethanol was added to the bacterial inoculum. Then the inoculum was distributed on a Mueller Hinton Agar Petri Dish in a consistent manner. After that, ZnO A NPs and ZnO B NPs were placed into the wells (prepared by cutting the agar gel) and the systems were preserved at 36 °C ± 1 °C for 24 h to allow successive incubation. After 24 h, the growth of bacteria was monitored and finally, the zone of inhibition for bacterial growth was determined in mm scale.

The photocatalytic analysis was performed by monitoring the degradation of Methylene Blue (MB) dye due to ZnO NPs under the influence of UV radiation (having intensity ∼120 μW cm⁻² and wavelength ∼300–400 nm). At first, 5 gm NPs were added into MB solution and mixed properly. The mixture was placed in the dark for 2 h and then irradiated with UV rays with subsequent stirring action and at a variation of time (0, 40, 80, 120, 160, 200 min). The absorbance of the mixture was measured by UV–vis spectroscopy (SHIMADZU UV/Vis-1650 PC, Japan). The efficiency of photodegradation was measured by the following equation:

$$\begin{eqnarray}&&{\rm{Degradation}}\,{\rm{efficiency}},\eta =\displaystyle \frac{{{\rm{C}}}_{0}-{{\rm{C}}}_{1}}{{{\rm{C}}}_{0}}\times 100\end{eqnarray} \tag{ 2 }$$

Where C₀ is the absorption of MB solution before the addition of ZnO NPs and C₁ is the absorption of the mixture solution with respect to time t.

ESR (electron spin resonance) analysis was performed using the EPR spectrometer (Bruker EMX MicroX, Germany) for the identification of the major factor that provides effective photocatalytic performance. During this characterization, DMPO (5,5-dimethyl-1-pyrroline-N-oxide) was used as a spin-trapped reagent in methanol and aqueous state. Moreover, the analysis was performed both in the presence and absence of light irradiation.

Neem leaf extract contains various phytochemicals such as flavones, quinines, organic acids, aldehyde and ketones which act as reducing agents and significantly reduces the particle sizes. After the successive reduction of particle sizes, the NPs are also affected by the terpenoids. Because of the interaction between the terpenoids and the ZnO NPs become stabilized as terpenoids are effective capping and stabilizing agents. The corresponding mechanism is graphically abstracted into figure 2. Moreover, the possible seven types of terpenoids that are present in Neem leaf extract are stereotyped in figure 3.

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Figure 4 represents the corresponding X-ray diffraction patterns of ZnO nanoparticles synthesized by sol-gel and bio-synthesis schemes respectively. The intense peaks at the crystal faces (100), (002), (101), (102), (110) assure the emergence of hexagonal wurtzite structure (as shown in figure 5) which belong to the space group of P6_3mc (JCPDS card no. 36-1451) [18]. The bio-synthesized ZnO nano-particles show more acute diffraction peak value introducing the appearance of the high percentage of crystalline phases. In addition, no impurity phases are present in the samples.

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However, considering the most severe diffraction peak (101), the crystallite size (D) can be calculated in accordance with the Debye Scherer formula [19]:

$$\begin{eqnarray}&&{\rm{D}}=\displaystyle \frac{{\rm{k}}\lambda }{\beta \,\cos \,\theta }\end{eqnarray} \tag{ 3 }$$

Hither, β is the Full Width at Half Maxima of the corresponding peak, k is a dimensionless shape factor (∼0.94), while λ is the wavelength of Cu K_α radiation (1.54 Å) and ϴ is the Bragg angle. D is mainly the mean size of the ordered domains which is considered to be equal to the particle size (applicable for only particles less than 100 nm). So, the average particle size of ZnO A NPs and ZnO B NPs is 33.20 nm and 25.95 nm respectively [19]. Again, there remains an inverse relationship between the β and the D which means that narrower peaks are resulted due to larger particles while broader particles are obtained because of smaller particles. The ZnO NPs showed a good agreement with this statement.

Since the crystallite size can be further employed for the determination of defect concentration within the specimen which is designated as the dislocation density (δ) and the leading formulae is adopted for this purpose [20]:

$$\begin{eqnarray}&&\delta =\displaystyle \frac{1}{{{\rm{D}}}^{2}}\end{eqnarray} \tag{ 4 }$$

From the exploration of diffraction data, the lattice constant (a & c), inter-planar spacing (d) and unit cell volume (V) of the specimens (table 1) can also be enumerated by utilizing the following formulas respectively [21]:

$$\begin{eqnarray}&&{\rm{a}}=\sqrt{\displaystyle \frac{{\lambda }^{2}}{4{\mathrm{Sin}}^{2}\theta }\left({{\rm{h}}}^{2}+{{\rm{k}}}^{2}+{{\rm{l}}}^{2}\right)}\end{eqnarray} \tag{ 5 }$$

$$\begin{eqnarray}&&{\rm{c}}=\displaystyle \frac{\lambda }{\mathrm{Sin}\,\theta }\end{eqnarray} \tag{ 6 }$$

$$\begin{eqnarray}&&{\rm{d}}=\displaystyle \frac{{\rm{a}}}{\sqrt{\left.{{\rm{h}}}^{2}+{{\rm{k}}}^{2}+{{\rm{l}}}^{2}\right)}}\end{eqnarray} \tag{ 7 }$$

And,

$$\begin{eqnarray}&&{\rm{V}}=\displaystyle \frac{\sqrt{3}}{2}{{\rm{a}}}^{2}{\rm{c}}\end{eqnarray} \tag{ 8 }$$

Where, h, k, l belong to Miller indices.

Besides, the lengthening of the stricture (L) between Zn and O can be enumerated by the following equation [20]:

$$\begin{eqnarray}&&{\rm{L}}=\sqrt{\left(\displaystyle \frac{{{\rm{a}}}^{2}}{3}+{\left(\displaystyle \frac{1}{2}-{\rm{u}}\right)}^{2}{{\rm{c}}}^{2}\right)}\end{eqnarray} \tag{ 9 }$$

Where u corresponds to parameterized constant belonging to wurtzite structure and can be expressed as:

$$\begin{eqnarray}&&{\rm{u}}=\displaystyle \frac{{{\rm{a}}}^{2}}{3{{\rm{c}}}^{2}}+0.25\end{eqnarray} \tag{ 10 }$$

In accordance with the Williamson-Hall proposition, the lattice strain was calculated by adopting the undermentioned equation [20]:

$$\begin{eqnarray}&&\beta \,\cos \,{\theta }_{{\rm{hkl}}}=\displaystyle \frac{{\rm{K}}\lambda }{{\rm{D}}}+4\varepsilon \,\sin \,{\theta }_{{\rm{hkl}}}\end{eqnarray} \tag{ 11 }$$

Whither, λ belongs to the wavelength of dispersion, β stands for the FWHM (full width at half maximum) of the corresponding peak, θ and ε correspond to the diffraction angle and strain residing into the specimen respectively. Actually, the strain value is attained from the slope of the linearly fitted $\beta \,\cos \,{\theta }_{{\rm{hkl}}}$ versus $4\varepsilon \,\sin \,{\theta }_{{\rm{hkl}}}$ plot which is stereotyped in figure 6 and the acquired outcomes are shown in table 1. However, the positive slope value for ZnO A NPs denotes the presence of tensile strain into the crystal, while the negative slope value for ZnO B NPs ensures the presence of compressive strain [22]. The compressive strain in ZnO B NPs is caused due to the reduction of the lattice parameters as compared to ZnO A NPs.

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Figures 7(a) and (b) shows the scanning electron micrographs of ZnO A and ZnO B NPs respectively. From the previous section, we have learned that the average particle size of ZnO B NPs (25.97 nm) is smaller than that of ZnO A NPs (33.20 nm). This can be also caused due to the presence of terpenoids in the Neem leaf extract. The terpenoid act not only as a stabilizing agent but also as a powerful reducing agent that interacts with ZnO NPs and reduces its size significantly [8, 17]. Moreover, the maximum particles of ZnO A NPs remain between the range of 15 nm to 68 nm, whereas for ZnO B NPs the range lies from 10 nm to 70 nm.

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Antibacterial activity of ZnO A NPs and ZnO B NPs was analyzed by adopting the agar well diffusion method using Escherichia coli O157: H7 as the bacterial medium. Generally, there involve three mechanisms behind the interaction between the bacteria and the NPs. The first one involves the formation of extremely active hydroxyls and the second one involves the deposition of NPs on the bacteria surface. In addition, for the last one, the NPs accumulates in the cytoplasm or in the periplasmic region of bacteria cell which disrupts the cellular operations and simultaneously disorganizes the membrane. However, in consideration of E. coli, ZnO NPs firstly disorganize the membrane of E. coli and enters into the cytoplasmic region. Positioning themselves into the cytoplasm, the NPs neutralizes the respiratory enzymes and increases the emersion of cytoplasmic contents into the outward direction which impairs the membrane and finally kills the E. coli bacteria resulting in a zone of inhibition of bacterial growth around itself [3, 23].

From figure 8, it is observed that the zone of inhibition of bacterial growth due to ZnO A NPs is different from the zone of inhibition that is caused by ZnO B NPs. However, ZnO B NPs introduce a higher zone of inhibition than ZnO A nanoparticles and the measurements of the inhibition zone of bacterial growth are tabulated in table 2. According to Krishna R Rangupathi, the antibacterial activity of nanoparticles is a size-dependent property and the property enhances with the reduction of particle size [23]. As the ZnO B NPs have smaller particle size as well as higher surface area, they show more antibacterial potential than that of ZnO A NPs [2].

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In the photocatalytic analysis, the ZnO nanoparticles were used as a photocatalyst in the aqueous solution of methylene blue (MB) dye and the subsequent degradation tendency of these particles was examined significantly. As ZnO is irradiated with UV light having energy equal or higher than the band gap energy (E_g) of ZnO nanoparticles, the electrons (e⁻) from the valence band (VB) move forward to the nearest empty conduction band (CB) to create pairs of electron-hole (e⁻/h⁺). The e⁻/h⁺ pairs then diffuse to the surface of the particles and perform redox reactions which are stereotyped in equations (12)–(14). During this reaction, extremely active hydroxyl radicals are formed by the reaction of H⁺ with H₂O and ${{\rm{OH}}}^{-}.$ In the meantime, the reaction between e⁻ and O₂ produces superoxide radical anions which finally converts to hydrogen peroxide as displayed in equation (15). Then the free OH· radicals interact with the MB dye and correspondingly decomposes it which results in the disappearance of blue color from the dye solution. A schematic representation of the photodegradation phenomenon of MB dye by ZnO NPs is displayed in figure 9. Again, figure 10 demonstrates the results obtained from the electron spin resonance (ESR) analysis. It is observed that no identical ESR signals are present in the dark environment while significant signals appear in the presence of visible light. However, the characteristic peaks at figures 10(a) and (b) correspond to the DMPO- OH·, and DMPO-·${{\rm{O}}}_{2}^{-}$ respectively. The trapping experiment is further continued to find out the most active species that causes the degradation of MB dye by using several scavengers. Where, sodium oxalate, isopropanol, and benzoquinone were used to scavenge ${{\rm{H}}}^{+},$ OH· and ·${{\rm{O}}}_{2}^{-}$ radicals respectively. However, the addition of these scavengers causes a significant reduction of the MB degradation efficiency of ZnO B NPs as shown in figure 10(c). In addition, it is observed that the lowest degradation efficiency of ZnO B NPs is observed due to the accession of isopropanol which proves that OH· radical provides a greater contribution to the degradation of MB dye.

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However, the corresponding reactions in the photodegradation scheme can be summarized as below [24, 25]:

$$\begin{eqnarray}&&{\rm{ZnO}}\mathop{\to }\limits^{h\upsilon }{\rm{ZnO}}\left({{\rm{e}}}_{\left({\rm{CB}}\right)}^{-}\right)+\left({{\rm{h}}}_{\left({\rm{VB}}\right)}^{+}\right)\end{eqnarray} \tag{ 12 }$$

$$\begin{eqnarray}&&{\rm{ZnO}}\left({{\rm{h}}}_{\left({\rm{VB}}\right)}^{+}\right)+{{\rm{H}}}_{2}{\rm{O}}\to {\rm{ZnO}}+{{\rm{H}}}^{+}+{\rm{OH}}\cdot \end{eqnarray} \tag{ 13 }$$

$$\begin{eqnarray}&&{\rm{ZnO}}\left({{\rm{e}}}_{\left({\rm{CB}}\right)}^{-}\right)+{{\rm{OH}}}^{-}\to {\rm{ZnO}}+\cdot {{\rm{O}}}_{2}^{-}\end{eqnarray} \tag{ 14 }$$

$$\begin{eqnarray}&&\cdot {{\rm{O}}}_{2}^{-}+{{\rm{H}}}^{+}\to {{\rm{HO}}}_{2}\cdot \end{eqnarray} \tag{ 15 }$$

$$\begin{eqnarray}&&{{\rm{HO}}}_{2}\cdot +{{\rm{HO}}}_{2}\cdot \to {{\rm{H}}}_{2}{{\rm{O}}}_{2}+{{\rm{O}}}_{2}\end{eqnarray} \tag{ 16 }$$

$$\begin{eqnarray}&&{\rm{ZnO}}\left({{\rm{e}}}_{\left({\rm{CB}}\right)}^{-}\right)+{{\rm{H}}}_{2}{{\rm{O}}}_{2}\to {{\rm{OH}}}^{-}+{\rm{OH}}\cdot \end{eqnarray} \tag{ 17 }$$

$$\begin{eqnarray}&&\cdot {{\rm{O}}}_{2}^{-}{{\rm{H}}}_{2}{{\rm{O}}}_{2}\to {\rm{OH}}\cdot +{{\rm{OH}}}^{-}+{{\rm{O}}}_{2}\end{eqnarray} \tag{ 18 }$$

$$\begin{eqnarray}&&{{\rm{H}}}_{2}{{\rm{O}}}_{2}+h\upsilon \to \,2{\rm{OH}}\cdot \end{eqnarray} \tag{ 19 }$$

Figure 11 displays the discoloration of MB dye due to the photocatalytic action of ZnO NPs at different times (0, 40 and 120 min). However, figures 12(a) and (b) illustrates the absorption spectra of MB dye as a function of wavelength under the influence of UV radiation at a variation of time i.e. 0, 40, 80, 120, 160, 200 min. From the graph, it is observed that the absorption rate of MB containing ZnO B NPs decreases more rapidly than that of ZnO A NPs. Moreover, the degradation efficiency (η) of ZnO NPs (biosynthesized and sol-gel synthesized) with respect to time is illustrated in figure 13. The degradation of MB for sol-gel synthesized ZnO are 35.3%, 45.7%, 56.1% 62.4%, 68.9% at 40, 80, 120, 160 and 200 min respectively. Again, the values for biosynthesized ZnO are 36.9%, 47.5%, 62.7%, 72.1%, and 80.2% at 40, 80, 120, 160 and 200 min respectively. So, MB dye degraded more rapidly in the presence of ZnO B NPs backing the reason for smaller particle sizes than that of ZnO A NPs. As the particles become smaller, the active surface area for the photocatalysis increased which results in enhanced degradation of MB [26]. Moreover, there remain terpenoids in the neem leaf extract which stabilizes the nanoparticles by capping themselves which also causes in the increment of photocatalytic action [27].

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Figures 14(a) and (b) displays the room temperature absorption spectrum of ZnO nanoparticles fabricated by sol-gel and biosynthesis methods correspondingly. Here, the absorption wavelengths are remaining within the maximum allowable limit of the absorption band of bulk ZnO (∼373 nm). Although the absorption slightly increases up to a wavelength of 363 nm for ZnO A NPs, the maximum incremental value for ZnO B NPs is 356 nm. The slight shift of the absorption peak may be caused due to the variation of particle size and their configuration [28]. However, this phenomenon results in the presence of a wide range of particle size distribution of ZnO [29]. Moreover, the redshift of ZnO A NPs compared to ZnO B NPs corresponds to the formation of agglomeration in the specimens significantly. Furthermore, in accordance with Gunanlan Sangeetha et al the shifting of absorption band to the higher wavelength as well as higher energy was associated with the increment of the size of nanoparticles [30]. Moreover, considering the direct interband transition between the valence band and the conduction band, the absorption band gap energy was measured by adopting the following Tauc's formula [31]:

$$\begin{eqnarray}&&{(\alpha {\rm{h}}\upsilon )}^{2}=A\,({\rm{h}}\upsilon -{{\rm{E}}}_{{\rm{g}}})\end{eqnarray} \tag{ 20 }$$

Where A is an energy-independent constant, α is the absorption coefficient, hυ is for the photon energy, and E_g is the optical band gap energy. The E_g of the ZnO NPs was obtained from the (αhυ)² versus hυ plot (as shown in the inset of figures 14(a) and (b). Where the extrapolation of the linear segment of the graph to (αhυ)² = 0 provides the value of E_g for ZnO NPs. It is observed that the optical band gap energy of ZnO B NPs (3.25 eV) is higher than that of ZnO A NPs (3.23 eV). This incremental phenomenon is mainly attributed to the quantum confinement effect. According to this theory, as the particle size decreases, the electrons in the valence band and the holes in the conduction band confine themselves within a space having a dimension of the de Broglie wavelength. However, this confinement influences the quantization of the energy and the momentum of the corresponding carriers and also enhances the optical transition energy between the valence band and the conduction band resulting in a broad band gap [32].

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Figure 15 displays the UV visible transmittance spectrum of ZnO A NPs and ZnO B NPs. Here, the transparency of ZnO B NPs is greater than that of ZnO due to the reduced particle size of ZnO B NPs. From the research of Takuya Tsuzuki, it is clear that smaller particles are capable to show higher transparency at the visible range of spectrum [33]. However, the UV blocking characteristics are almost similar for each of the variants of NPs.

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