Facile microwave‐assisted synthesis of NiO nanoparticles and its effect on soybean (Glycine max)

Abstract

NiO nanoparticles in high purity, 15 ± 0.5 nm in size, were prepared via solid‐state microwave irradiation. The [Ni(NH₃)₆](NO₃)₂ complex as a novel source was decomposed in the presence of microwave irradiation for a short time (10 min). The present method is facile, safe, and low‐cost. This method exhibits other advantages; there is no need of a solvent, fuel, surfactant, expensive material, or complex instrument. Synthesised NiO nanoparticles were determined by various analyses. Also, for the first time, NiO nanoparticle effects on biochemical factors in soybean were investigated. Seeds of soybean were grown in the Murashige and Skoog agar medium containing different concentrations of NiO nanoparticles (0, 200, and 400 mg/L) for 21 days under growth chamber conditions. Estimates of malondialdehyde, hydrogen peroxide contents, and antioxidant enzymes (catalase and ascorbate peroxidase) under treatment of NiO nanoparticles were assayed. The result showed that by significantly increasing the concentration of NiO nanoparticles, the activity of catalase and ascorbate peroxidase enzymes was enhanced. Malondialdehyde and hydrogen peroxide contents significantly increased in the presence of NiO nanoparticles. In this study, the increasing activity of catalase and ascorbate peroxidase was not enough for radical oxygen species detoxification.

1 Introduction

The nanoparticle effects on live species have received considerable attention in recent years [1, 2]. Many studies have shown that fungi [3, 4], bacteria [5, 6], and plants [7] are affected by various nanoparticles. Among living organisms, plants play a key role in food chains. The effects of many nanoparticles on anatomical, morphological, biochemical, and physiological characteristics of plants have been investigated. Their effects on plants were both negative and positive. Positive effects of nanoparticles such as enhanced water uptake capacity of seeds, seed germination, root elongation, and plant growth were reported. For example, silica nanoparticles increased the root and shoot length [8], dry weight, and photosynthesis of maize seedlings under field conditions [9]. Several studies reported the negative effects of nanoparticles such as reduced photosynthesis, lower content of photosynthetic pigments, and decreased uptake of nutrients and water. For example, Chlamydomonas reinhardtii exposed to Ag nanoparticles showed inhibition of electron transport activity and photochemical reactions of photosynthesis [10]. Overall, a few studies have been carried out to determine the effects of metal oxides on plant species. On the other hand, the effect of NiO nanoparticles on plants is not studied. In this study, we prepared NiO nanoparticles using a novel precursor under microwave irradiation and investigated their effects on biochemical parameters in soybean (Glycine max).

During the past several years, various physical and chemical synthetic techniques have been used and developed for synthesising crystalline powders in nanoscale dimensions, including physical vapour deposition [11], sonochemical method [12, 13], thermal decomposition [14], biosynthesis [15], microwave irradiation [16], and so forth. Among these techniques, the microwave‐assisted solid‐state method with controlling temperature and time of reaction is able to produce narrow size particle distribution and controlled morphology [17]. The main intention for choosing the synthetic method is to reduce the cost of physical and chemical syntheses and produce pure materials in nanometre scale in a short time. The microwave‐assisted solid‐state method quite provides these main purposes. From this viewpoint, we succeeded in preparing high‐purity NiO nanoparticles by the solid‐state microwave method as a novel method. The product was characterised and for the first time, NiO nanoparticle effects on biochemical factors in soybean (Glycine max) were investigated.

In this work, the [Ni(NH₃)₆](NO₃)₂ complex was prepared and used as a novel precursor for preparing NiO nanopowders. The precursor was placed under microwave irradiation without adding any additional template agents such as a surfactant in 10 min. The CuO powder was used as a secondary irradiation absorber.

The synthesised NiO was characterised by X‐ray diffraction (XRD), Fourier‐transform infrared (FT‐IR) spectroscopy, scanning electron microscopy (SEM), energy‐dispersive X‐ray (EDX) spectroscopy, and transmission electron microscopy (TEM). The electronic property of synthesised nanostructures was discussed by UV–Vis spectroscopy. In continuation, we investigate the effect of synthesised nanoparticles on biochemical parameters in soybean (Glycine max).

2 Experimental Section

2.1 Material

All the chemical reagents used in the experiments were of spectroscopic grade and used as received without further purification.

2.2 Synthesis of [Ni(NH₃)6](NO₃)₂ precursor

The [Ni(NH₃)₆](NO₃)₂ precursor was prepared according to published paper [18]. Briefly, a stoichiometric amount of ammonia solution was added to an aqueous solution of Ni(NO₃)₂ ·6H₂ O in an ice bath. Then, 15 mL of cold ethanol was added to this mixture slowly. The mixture was resided in the cold for 2 h, and the crystals of [Ni(NH₃)₆](NO₃)₂ were completely formed. The precipitate was separated by filtration, washed with ethanol and ether, and dried under vacuum over anhydrous CaCl₂.

2.3 Synthesis of nickel oxide (NiO) nanoparticles

To prepare NiO nanopowder, 2 g of the [Ni(NH₃)₆](NO₃)₂ powder was added to a porcelain crucible and placed in another porcelain crucible that was filled with a microwave absorber (CuO powder). The assembly was placed in a microwave oven (LG, 2.45 GHz, 900 W) and was exposed to microwaves irradiation at 900 W (100%) in air. During irradiation, the temperature of the CuO powder was elevated from room temperature to 250°C. The temperature was determined by a resided chromel–alumel thermocouple in the mixture vessel. After 10 min, decomposition of the [Ni(NH₃)₆](NO₃)₂ powder was completed. The product was cooled to room temperature then washed with ethanol, and dried under vacuum over anhydrous CaCl₂.

2.4 Preparation of soybean (Glycine max) seeds

Soybean (Glycine max) seeds from Shahid Chamran University of Ahvaz (Iran) were surface sterilised using 10% sodium hypochlorite (NaOCl) solution for 15 min and rinsed with deionised water two times. Then, ten seeds per bottle were placed in the Murashige and Skoog medium (10 g/L) supplemented with different concentrations 0, 200, and 400 mg/L of NiO nanoparticles. After inoculation, the bottles were kept in an incubation chamber at 24 °C. After 21 days, plants were harvested and biochemical responses were evaluated.

2.5 Estimation of lipid peroxidation (MDA content)

The level of lipid peroxidation was measured by following Heath and Packer [19]. A 50 mg fresh plant sample was homogenised with 0.5% (w/v) 2‐thiobarbituric acid prepared in 20% (w/v) trichloroacetic acid, incubated for 30 min in a pre‐heated water bath (100°C) and transferred at 0°C for 30 min. Then, the homogenate was centrifuged at 10,000 rpm for 15 min and the volume of clear supernatant was made up to 3 ml with distilled water. The absorbance of the clear supernatant was read spectrophotometrically at 532 nm and correction for unspecific turbidity was done by subtracting the absorbance of the sample at 600 nm. The MDA content was calculated using an extinction coefficient of 155 mmol L⁻¹ cm⁻¹.

2.6 Estimation of hydrogen peroxide (H₂ O₂) content

Hydrogen peroxide content was determined according to the method of Velikova et al. [20]. Absorbance of reaction mixture was recorded at 390 nm. Hydrogen peroxide content was calculated using a standard curve based on the absorbance (A390 nm) of H₂ O₂ standards.

2.7 Enzyme activity assays

2.7.1 Ascorbate peroxidases (APX) activity

APX activity was measured by following the decrease in absorbance at 290 nm [21]. The APX activity was assayed in a reaction mixture containing 0.5 mM ascorbate and 0.1 mM EDTA dissolved in 100 mM K‐phosphate buffer (pH = 7.0) and enzyme extract.

2.7.2 Catalase activity (CAT)

CAT was assayed by monitoring the decrease in the absorbance of H₂ O₂ within 30 s at 240 nm [22]. The decrease in hydrogen peroxide was inferred from the decline in absorbance at 240 nm.

2.8 Instruments and statistical analysis

To determine the crystalline phase, powder X‐ray diffraction (PXRD) analysis was carried out on an X‐ray diffractometer with Ni‐filtered Cu Kα irradiation (λ  = 1.54 Å), when an angle range of 2θ  = 20°–80° was applied. Infrared spectrum was recorded on a FT‐IR 160 spectrophotometer (Shimadzu system) by KBr tablets. UV–Vis spectroscopy measurement over the wavelength range of 300–700 nm at room temperature was obtained using a double‐beam Shimadzu 1650 PC. The powder for UV–Vis analysis was dispersed in EtOH for 25 min to obtain a homogeneous suspension. Morphology of the obtained NiO was examined by an SEM image taken on a Hitachi s4160/Japan equipped with a link EDX analyser with gold coating. The particle size was specified by a transmission electron microscope (TEM, Philips CM10) at 80 kV (accelerating voltage). The sample was sonicated in EtOH and a drop of the suspension was dried on a carbon‐coated micro‐grid for the TEM analysis.

All statistical analysis was subjected to ANOVA test and means were compared by the Duncan test. Comparisons with P values < 0.05 were considered significantly different.

3 Results and discussion

After 30 min of microwave irradiation, the [Ni(NH₃)₆](NO₃)₂ precursor was unaltered, thus this compound does not absorb microwaves. Therefore, it requires a secondary irradiation absorber. In the present investigation, the NiO nanopowder was synthesised under microwave irradiation on the [Ni(NH₃)₆](NO₃)₂ compound in the presence of CuO powder. The heat of CuO absorber was transferred to the precursor and was completely decomposed until the nano‐sized NiO powder was formed. The redox reaction was occurred between the NH₃ ligands (reductants) and the NO₃ ^_ groups (oxidants), and the nano‐sized product was prepared within a short time. Decomposition of [Ni(NH₃)₆](NO₃)₂ precursor was accompanied with the emission of different gases such as N₂, NO, N₂ O, and H₂ O. The explosive reaction ensued in the nano‐sized NiO according to the following reaction:

$$\left[\mathrm{Ni}{\left(\mathrm{N}{\mathrm{H}}_3\right)}_6\right]{\left(\mathrm{N}{\mathrm{O}}_3\right)}_2\mathrm{complex}\to \mathrm{NiO}+\left({\mathrm{N}}_2+\mathrm{NO}+{\mathrm{N}}_2\mathrm{O}\right)+{\mathrm{H}}_2\mathrm{O}$$

3.1 XRD analysis

Fig. 1 displays the XRD pattern of the obtained product from decomposition of the [Ni(NH₃)₂](NO₃)₂ precursor at the power level of 900 W for 10 min in a microwave cavity. All the diffraction peaks of the XRD pattern could be indexed to the pure face‐centered cubic NiO phase (space group: Fm ˗3m; No. 225, JCPDS Card No. 73‐1523) at 2θ  = 37.3135°, 43.3609°, 62.9612°, 75.4474°, and 79.4686°, which can be perfectly related to (1 1 1), (2 0 0), (2 2 0), (3 1 1), and (2 2 2) crystal planes, respectively. Lattice constants of NiO phase are calculated as a  = b  = c  = 4.18 nm. Since no diffraction peaks were observed from impurities in the XRD pattern, it was revealed that pure face‐centered cubic NiO phase was synthesised through the fast and simple microwave irradiation method. By Debye–Scherrer formula (1) [23] the size (D _XRD) of the synthesised NiO particles was calculated to be 16 nm.

$$D=k\lambda /\beta \cos \theta$$ (1)

where D _XRD is the average crystalline size, K is a constant (ca. 0.9), λ is the wavelength of Cu Kα (λ  = 0.154 nm), β is the full width of the diffraction peak at the half‐maximum (FWHM) radians on 2θ scale, and θ is the Bragg angle.

Fig. 1.

XRD pattern of NiO nanopowder

3.2 FT‐IR spectrum analysis

The FT‐IR spectrum of the NiO nanoparticles is shown in Fig. 2. All bands of the precursor [18] were eliminated after the microwave irradiation to this precursor. A strong band is seen at around 450 cm⁻¹, which is related to the Ni–O stretching vibration in the face‐centred cubic (fcc) structure of the synthesised NiO [24]. Also, the very weak bands at ∼1380 and 3300 cm⁻¹ in the spectrum are corresponded to the H₂ O molecules. H₂ O molecules are absorbed by the KBr disk or sample [25].

Fig. 2.

FT‐IR spectrum of NiO nanopowder

3.3 Field emission scanning electron microscopy (SEM) analysis

The SEM image of the obtained NiO is presented in Fig. 3. The morphology of NiO is semi‐spherical in shape, which was loosely aggregated. The shape of nano‐sized NiO is compared to the precursor that clearly shows that the shape of the synthesized nanoparticles is different from the precursor [18]. In the SEM image of the obtained NiO, the morphology of the precursor is not seen, due to the complete decomposition of the precursor into the product particles. Therefore, solid‐state microwave irradiation is a good method for synthesising various nano‐sized compounds in high purity.

Fig. 3.

SEM image of the NiO nanoparticles

3.4 TEM image

TEM image of the synthesised NiO is given in Fig. 4. The sample was dispersed in ethanol by ultrasonic vibration to prepare TEM analysis. The particle size distribution of NiO powder is 13–16 nm (Fig. 5).

Fig. 4.

TEM images of the NiO nanoparticles

Fig. 5.

Particle size distribution of NiO nanoparticles

3.5 UV–Vis spectrum analysis

The electronic property of the prepared NiO nanoparticles was surveyed by UV–Vis analysis. Fig. 6 shows the electronic spectrum of the NiO nanoparticles. The sharp band appears at wavelength 360 nm. Fig. 7 shows electronic spectrum of the [Ni(NH₃)₆](NO₃)₂ complex precursor. Clearly, the UV–Vis spectrum of the NiO nanoparticles is quite different from that of the complex precursor, thus the sharp band appeared at 360 nm is due to NiO nanoparticle not precursor.

Fig. 6.

Electronic spectrum of the NiO nanoparticles (a) and (Ahʋ)² –hʋ curve (b)

Fig. 7.

Electronic spectrum of the [Ni(NH₃)₆](NO₃)₂ precursor

In the NiO semiconductor, the electronic transition from the valence shell to the conduction shell is accrued. By (2) the band gap (E_g ) of the NiO nanoparticles can be obtained.

$${\left(Ah\upsilon \right)}^2=B\left(h\upsilon -Eg\right)$$ (2)

where A is the absorption coefficient, hυ is the energy of photon, B is a constant relevant to the type of materials. Fig. 6 b shows the (Ahυ)² α hυ curve for the NiO nanoparticles. The band gap of the synthesised NiO nanoparticles was estimated to be 3.2 eV by extrapolation of this curve. The value of the band gap has a shift toward previous reported papers [26, 27]. That is depended on type precursor and synthetic method. In this work, NiO nanoparticles were synthesised by the microwave‐assisted solid‐state decomposition method. Difference of this method with the conventional heating methods is due to chemical or physical changes that happen during the microwave irradiation. This mechanism is interaction microwave wavelength with the reactants at the molecular level, so that the electromagnetic energy is converted to heat by generated kinetics from the rapid motion of the molecules [28]. By this method pure nanoscale materials can be produced in a short time.

3.6 EDX analysis

Fig. 8 shows the EDX analysis of the NiO nanoparticles. Signals of Ni and O elements were observed. The Si and Au signals appeared due to the coating material of the instrument. Hence, NiO nanoparticles were prepared in high purity by the solid‐state microwave method.

Fig. 8.

EDX spectrum of the NiO nanoparticles

Therefore, NiO nanostructures were prepared via different methods. For example, NiO nanoflower was synthesised from Ni(NO₃)₂ ·6H₂ O or NiCl₂ ·6H₂ O as a precursor compound by the hydrothermal method in the presence of surfactant [29]. Similarly, NiO nanostructures were synthesised by a hydrothermal method assisted with a non‐ionic surfactant (poly(ethylene glycol)) [30]. Furthermore, NiO nanoplates were synthesised from a Ni⁺² aqueous solution under microwave irradiation, meanwhile the final product was obtained at 450°C for 2 h [31]. NiO was prepared by solvothermal reaction assisted with a microwave hydrothermal system [32]. In this research, we synthesised NiO nanoparticles under mild conditions, at low particle size distribution (13–16 nm), using the [Ni(NH₃)₆](NO₃)₂ complex as a novel source under solid‐state microwave irradiation (the modified microwave method). Compared to the other methods, the solid‐state microwave method has more advantages. This method is facile and fast. In this work, there was no of use a solvent, fuel, surfactant, expensive material, or complex instrument; therefore, this method is safe and low‐cost.

3.7 Assay NiO nanoparticle effect on biochemical parameters of soybean (Glycine max)

3.7.1 Effect on hydrogen peroxide (H₂ O₂) and malondialdehyde (MDA) contents

Compared to the control, concentrations of 200 and 400 mg/L NiO nanoparticles significantly increased hydrogen peroxide (Fig. 9) and malondialdehyde (MDA) contents (Fig. 10) during the 21 day treatment. The H₂ O₂ and MDA contents increased significantly (P  < 0.05) with increasing NiO nanoparticles concentration. At a concentration of 400 mg/L, the contents of H₂ O₂ and MDA were higher than the concentration of 200 mg/L.

Fig. 9.

Effect of NiO nanoparticles on H₂ O₂ content in soybean. Different letters are significantly different (P < 0.05) between treatments

Fig. 10.

Effect of NiO nanoparticles on MDA content in soybean. Different letters are significantly different (P < 0.05) between treatments

3.7.2 Effect on catalase and APX activity

Catalase (CAT) (Fig. 11) and APX (Fig. 12) enzymes showed a similar response to NiO nanoparticles at 21 day. CAT and APX activities increased significantly at 200 and 400 mg/L concentrations of NiO nanoparticles compared to the control. An analysis of biochemical parameters was done to evaluate toxicity of NiO nanoparticles in soybean plant. Results of the present research demonstrated that significant additive effects were observed in malondialdehyde (MDA) and hydrogen peroxide contents. Similarly, CeO₂ nanoparticle treatments increased accumulation of H₂ O₂ and MDA in maize leaves [33].

Fig. 11.

Effect of NiO nanoparticles on CAT activity in soybean. Different letters are significantly different (P < 0.05) between treatments

Fig. 12.

Effect of NiO nanoparticles on APX activity in soybean. Different letters are significantly different (P < 0.05) between treatments

The MDA formation in plants exposed to adverse environmental conditions is an indicator of lipid peroxidation in biological systems [19]. Ma et al. [34] and Arruda et al. [35] have reported nanoparticle effects on phytotoxicity caused by the production of reactive oxygen species (ROS), which subsequently result into oxidative stress, lipid peroxidation, proteins, and DNA damage in plants. ROS is a term encompassing molecules such as hydrogen peroxide, hydroxyl radical, and superoxide as well as singlet oxygen.

By producing ROS, metal or metal oxide nanoparticles caused oxidative stress in plants. When nanoparticles are dissolved, they can release ions, react with proteins and fatty acids, and produce ROS. Plants can employ several defense mechanisms against oxidative stress and the harmful effects of ROS. By modulating the plant antioxidant defense systems, plants can also alleviate oxidative damage under stress. The activity of catalase and ascorbate peroxidase is one of the strategies of the plants to alleviate and repair the damage caused by ROS [36]. Catalase (2H₂ O₂ → 2H₂ O + O₂) and ascorbate peroxidase (ascorbate + H₂ O₂ → dehydroascorbate + H₂ O) are the major players in H₂ O₂ metabolism. These enzymes slow down or inhibit the oxidative processes by interrupting the free‐radical chain reaction [37]. In the present research, CAT and APX activities increased significantly in the presence of NiO nanoparticles at 200 and 400 mg/L. A similar increase in CAT and APX activities was observed under nanoparticles in reported investigations. By producing ROS, metal or metal oxide nanoparticles caused oxidative stress in different plants [38]. ZnO [39], Al₂ O₃ [40], and CeO [41] nanoparticles in Pisum sativum, Tobacco and Oryza sativa plants, respectively, produced ROS such as superperoxides and H₂ O₂, which resulted in oxidative stress in plants. Also, Lei et al. demonstrated that TiO₂ nanoparticles had improved the activity of APX and CAT in spinach [42]. In this study, we show that although the activities of catalase and ascorbate peroxidase were increased in the soybean plant, but they were not enough for ROS detoxification.

4 Conclusions

In conclusion, NiO nanoparticles, 13–16 nm, were successfully prepared via solid‐state decomposition of the [Ni(NH₃)₆](NO₃)₂ precursor under microwave irradiation in the presence of secondary absorber microwave (CuO). The main advantage of our approach is that there is no need for synthesis of complex metal organic precursors; meanwhile, the NiO nanoparticles were prepared in a short time with low energy consumption. Another advantage of the microwave‐assisted solid‐state method is that there is no need for a solvent, fuel, surfactant, or any expensive. The results show that the NiO nanoparticles in soybean plant may be toxic by ROS production. Increasing activity of catalase and ascorbate peroxidase enzymes in soybean plant could not scavenge the active oxygen species.