J Clust Sci (2017) 28:2097–2109 DOI 10.1007/s10876-017-1203-3 ORIGINAL PAPER

Nanosized Synthesis of Nickel Oxide by Electrochemical Reduction Method and their Antifungal Screening Ashwini A. Agale1 • Suresh T. Gaikwad1 Anjali S. Rajbhoj1

•

Received: 24 January 2017 / Published online: 3 April 2017  Springer Science+Business Media New York 2017

Abstract Nano-particle oxides of transition metals have attracted materials scientists. These materials have exceptional properties which stimulate many advanced applications. As like all other transition metal oxide, nickel oxide has been especially investigated in the degradation of several environmental pollutants due to its properties. The nickel oxide was synthesized by electrochemical reduction method using tetra-hexyl ammonium bromide as structure directing agent in an organic medium viz. tetra hydro furan and acetonitrile in 1:4 ratio by different current densities 10 and 14 mA/cm2. Such nanoparticles were prepared using simple electrolysis cell in which the sacrificial anode is a commercially available nickel metal sheet and platinum (inert) sheet act as a cathode. The synthesized nickel oxide nanoparticles were characterized by using UV–visible spectroscopy, X-ray diffraction (XRD), scanning electron microscopy, energy dispersive spectroscopy (EDS) and transmission electron microscope (TEM) analysis techniques. TEM analysis proved a cubic structure with size of 25–30 nm which was in agreement with the result calculated from the XRD analysis. EDS analysis revealed the presence of Ni and O element. The nanoparticles were tested for antifungal activity against human pathogens like A. alternaria, A. niger, F. oxysporum, etc., which showed excellent antifungal properties. Keywords Electrochemical cell  Tetra hexyl ammonium bromide  Nickel oxide nanoparticles  Antifungal study

& Anjali S. Rajbhoj [email protected] 1

Department of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, MS 431004, India

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Introduction The synthesis of nanoparticles is of current interesting endeavor for researcher due to their tremendous applications in different areas because of their physicochemical properties and small dimensions [1]. Nowadays, the synthesis of metal nanoparticles has received considerable attention because of their applications in the field of electrical, magnetic, thermal, sensor devices, biological pharmaceutical synthesis and chemical properties, especially catalytic properties which did not achieve from those of the bulk materials [2–5]. Properties of materials change as their size approaches the nanoscale and the percentage of atoms at the surface of a material becomes more significant. Stabilizing and protective agents in chemical synthesis of nanoparticles interact chemically with the surface of nickel oxide nanoparticles and modify their morphology, electronic and magnetic properties [6]. Nanostructures, whether synthetic or natural, exhibit fascinating properties e.g. quantum confinement semiconductor particles, surface plasmon resonance in noble metal particles. Therefore, various researchers are trying to prepare metal materials at the nano scales Particularly, for the synthesis of transition metal nickel oxide nanoparticles (NiO NPs) which have gained great importance in the last two decades as they possess good catalytic activity an efficient and reusability of catalyst for synthesis organic compounds also shows biological activities [7, 8]. Although many methods for treating fungal infections are currently available, there is an urgent requirement for new and improved approaches for microorganisms destruction. Owing to the extremely small size and large surface to volume ratio, metal nanoparticles have been getting much attention in recent times towards various applications; remarkably in the field of nanobiotechnology [9–11] Nanoparticles have an enlarged contact area with micro-organisms that facilitate their biological and chemical activity. Another important feature of metal nanoparticles is their ability to target different micro-organisms structures that can make them an efficient antimicrobial candidate for practical purposes [12]. Heavy metals are toxic and reactive with proteins; therefore, they bind protein molecules; as a result, cellular metabolism is inhibited causing death of microorganism [13]. It is believed that nickel oxide nanoparticles after penetration into micro-organism inactivate their enzymes, generate hydrogen peroxide and cause micro-organism cell death [14]. In addition, it is believed that nickel binds to functional groups of proteins, resulting in protein denaturation [15]. Fungi have become a major health threat because of the ever-increasing number of patients who have compromised immune systems, such as persons suffering from acquired immune deficiency syndrome and those receiving chemotherapy. Furthermore, fungal infections in non-immuno compromised patients are also on the rise, being the seventh leading cause of infection-related mortality [16]. The antimicrobial activities of nanoparticles have been attributed to their relatively smaller sizes and high amount of surface-area-to-volume ratio that facilitate interacting closely with membranes of viruses, fungi, and bacteria [17]. In recent years, resistance to commercially available fungicides by phytopathogenic fungi has been increasing and has become a serious problem [18]. Some metal nanoparticles have been

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studied and proved for their antifungal properties [19–21]. However, few studies are available on the effects of nickel oxide nanoparticles on fungal pathogens especially Fusarium causing pathogens. The nanoparticles penetrate easily into the fungal cell membrane due to their small particle size, bind to functional groups of proteins, and phosphorous- and sulfur-containing compounds such as DNA, and cause fungal cell death [22]. As observed, micro-organism growth log phase has a delayed trend at lower concentrations of NiO NPs loadings. Therefore, it was concluded that complete micro-organism inhibition depends upon the concentrations of NPs and on the number of fungal cells. Indeed, it suggest that NiO NPs could have significant biocide effect in reducing micro-organism growth for practical applications. In the present investigation, we have adapted the electrochemical reduction method to synthesis of nickel oxide nanoparticles in which a controlled current is used throughout the electrolysis process. The tetra hexyl ammonium bromide (THAB) salts were used which are highly soluble and dissociate in the solvent to play the role of electrolyte as well as capping agent. The bioevaluation assay of the inhibitory activity of synthesized nickel oxide on fungal strain A. niger, A. alternata, F. oxysporum were performed to establish the potential of these nanoparticles as antifungal agents at different concentration and was also compared with standard Flucanozol.

Experimental Materials All chemicals (up to 99.99% purity) were purchased from Sigma Aldrich. The HPLC grade tetra hexyl ammonium bromide salt THAB and tetrahydrofuran (THF), acetonitrile (ACN) were purchased from Sigma Aldrich and Rankem chemicals and used as such. The sacrificial anode in the form of nickel sheet and platinum sheet as inert cathode having thickness 0.25 mm and purity 99.99% were purchased from Alfa Aesar. Method The synthesis of nickel oxide nanoparticles by electrochemical reduction method is a previously reported method [23–25]. In the overall process the bulk metal is oxidized at the anode, the metal cations migrate to the cathode and reduction takes place with formation of metal or metal oxide in the zero-oxidation state. Agglomeration with formation of undesired metal powder is prevented by the presence of the ammonium stabilizer. The process makes uses an inexpensive two electrodes set up for 25–30 ml electrolyte solution which includes both oxidation of bulk metal and reduction of metal ions for size selective preparation of tetra alkyl ammonium salt stabilized metal nanoparticles. In the initial experiment, we have used a nickel metal sheet (1 9 1 cm) as anode and a platinum sheet (1 9 1 cm) as the cathode. These two electrodes were placed parallel to one another and were separated by 1 cm in 0.01 M solutions of tetra-hexyl ammonium salt (THAB) were prepared in ACN/THF (4:1) served as the supporting electrolyte. The electrolysis

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process was then carried out by applying current of 14 mA/cm2 for 2 h. Electrolysis was carried out in nitrogen atmosphere. These nanoparticles after electrolysis settled for one day. The agglomerated solid sample was separated from the solution by decantation and washed three to four times with THF. The washed samples were then dried under vacuum condition in desiccators and calcinated at 400 C before being used for characterizations (Fig. 1). Reaction mechanism for electrochemical synthesis of metal clusters: Anode  M bulk ! Mnþ þ ne Cathode  Mnþ þ ne þ stabilizer ! M coll/Stabilizer M bulk þ stabilizer ! Mcoll/stabilizer where, M bulk = metal bulk sheet, M coll/stabilizer = ammonium salt stabilized colloidal metal cluster.

Antifungal Screening Solidified PDA plates were labeled according to fungi, i.e. A. niger, A. alternata, F. oxysporum and 0.1 ml spore suspension of respective fungi were poured with a sterile 0.1 ml pipette on the respective labeled plates and spread with a sterile

Fig. 1 Representative diagram of the synthesis process of NiO nanoparticles

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spreader to form uniform layers of spores on the surface of agar. Plates were then labeled according to the metal nanoparticles. Then sterile Whatmann filter paper discs were dipped into respective nanoparticles solution prepared in ACN: THF solvent and placed on respective labels. The ACN: THF was used a control of the solvent incubated at 37 C for 24 h. Plates were kept for diffusion in refrigerator for 30 min and then incubated at room temperature for 48 h. After incubation, zones of inhibition were measured and observations were recorded in mm. The diameter of zone of inhibition around each disc was measured by scale and results were recorded in terms of mm. The antifungal activity was found to depend on zone of inhibition in mm, if inhibition zone \9 mm the sample showed poorer antibacterial activity while [9 mm then sample would have better antifungal activity. Keeping above mentioned points in view, the screening of the metal nanoparticles for antifungal activity has been performed. Nickel oxide nanoparticles have been checked against three fungi A. alternata, A. niger, F. oxysporum by Kirby Baur, s disc diffusion method, for two different concentrations of NiO nanoparticles i.e. 50, 100 ll and compared with well-known antibiotic Flucanozol. From the results in Table 1 it can be seen that all samples have better antifungal activity as all sample has zone of inhibition with size more than 9 mm. The ACN/THF control did not show any antifungal activity against the tested fungal strains.

Table 1 In vitro antifungal screening of synthesized nickel oxide nanoparticles Human pathogenic fungi

Mean zone of inhibition diameter (mm) ± SD Conc. (ll)

NiO 10 mA/cm2 NiO 14 mA/cm2 ACN ? THF (4:1)

A. alternaria

A. niger

F. oxysporum

50

16.95 ± 0.26

15.27 ± 0.89

20.56 ± 1.33

100

19.36 ± 1.00

17.18 ± 0.72

23.58 ± 0.60

50

21.29 ± 0.62

20.81 ± 0.72

24.14 ± 1.13

100

22.04 ± 1.44

22.80 ± 0.99

26.61 ± 1.43 0.0 ± 0.0

50

0.0 ± 0.0

0.0 ± 0.0

100

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

Flucanozol

50

17.63 ± 0.44

17.34 ± 0.58

22.16 ± 0.36

10 mA/cm2

100

22.92 ± 0.33

22.81 ± 0.78

26.07 ± 1.74

Flucanozol

50

24.03 ± 0.97

22.65 ± 0.52

26.82 ± 0.97

14 mA/cm2

100

25.96 ± 0.28

24.68 ± 1.05

27.72 ± 1.10

F value

903.291

841.660

918.713

P value

\0.01**

\0.01**

\0.01**

Test applied—one way ANOVA with post hoc Tukey HSD test, P \ 0.05 statistically significant Post hoc: values with same letter superscripted do not vary significantly SD Standard deviation ** P value \0.01—the test is highly significant

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1.0

1.2 1.0 364

Absorbance

Absorbance

0.8 0.6 0.4 0.2

0.8 360

0.6 0.4 0.2

0.0

0.0 300

400

500

Wavelength(nm)

600

300

350

400

450

500

550

600

Wavelength(nm)

Fig. 2 Optical absorption spectrum for Ni nanoparticles a 10 mA/cm2 with THAB and b 14 mA/cm2

Characterization Techniques The synthesized nickel oxide nanoparticles were characterized by UV–Visible spectrophotometry, X-Ray Diffraction, Transmission electron microscope, Scanning electron microscope–Energy dispersive spectroscopic techniques. In UV–visible spectroscopic analysis, supernatant of synthesized nanoparticles were used to record colour and study surface plasmon absorption in the UV–visible region using Jasco UV–Visible Spectroscopy. The powdered X-ray diffraction patterns were obtained on X-ray powder diffractometer PW-1840, using CuKa radiation (k = 1.54 A0). The samples were scanned from 20 to 820 at the scan rate of 5 9 104 CPS. The scanning electron microscopy study was carried out on JEOL make JSM 63608A microscope to study the morphology of the synthesized nanoparticles. Transmission Electron Microscopy of synthesized nanoclusters were ultrasonicated in ethanol and then a drop of the dispersed nanoparticles was placed onto a carbon coated 400 mesh copper grid with format coating over it, followed by natural evaporation. Transmission electron microscopic study and electron diffraction were carried out on Philips CM 200 kV. Results and Discussion UV Visible Spectroscopic Analysis The reduction of nickel ions was visibly evident from the color changes associated with it. As the phenomenon of Surface Plasmon occurs only in the case of nanoparticles and not in case of bulk metallic particles, hence unique optical properties of nanoparticles can be studied using UV–Visible spectroscopy [26]. The optical spectrum shows absorption bands in the range of 360–370 nm which confirms the metallic nature. The increasing in intensity of the UV band and shifting to higher wavelength may be resulting due to presence of capping agent. As the electrochemical process is a function of current density, the formation of nanoparticles is diffusion controlled and it also depends on the concentration of ions at the electrode surface and in the bulk (Fig. 2).

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XRD Estimation of Crystallite Size from X-ray Diffraction X-ray line broadening analysis provides a method of finding bulk average size of coherently diffracting domains. The average crystallite size (D) of solid material can be estimated from X-ray line broadening using the Debye-Scherer equation [27].

D¼

Kk b cos h

where, D = Average particle size, k = wavelength, h = diffraction angles, b = FWHM (Full width half maximum). Particle Size Studies

*(200)

1600 1400

600 400

#(220)

#(222)

800

*(311)

(b)

1000

#(220)

*(111)

,QWHQVLW\DX

1200

#(111)

X-ray diffraction (XRD) pattern of the prepared compound reveals the crystalline nature, phase purity and structure details. Figure 3a, b show the powder XRD pattern recorded for the prepared nickel oxide NPs. The powder X-ray diffraction ˚ ) radiation. From the figure three (XRD) with nickel filtered CuKa (l = 1.5405 A characteristic peaks for NiO having 2h = 37.29, 43.35, 62.97, 75.46, 79.49 with lattice parameter a = 4.176 corresponding to the (111), (200), (220), (311) and (222) plane for the FCC phase of NiO Nps [JCPDS No: 04-0835] [28, 29]. The presence of nickel oxide in the sample may have arisen due to the possible oxidation of nickel during drying, storing and analysis. In the electrochemical synthesis of

(a)

200 0 10

20

30

40

50

60

70

80

2Θ(Degree) Fig. 3 X-ray diffraction pattern of prepared nickel oxide nanoparticles. a 10 mA/cm2, b 14 mA/cm2

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these nanoparticles THAB are used as capping agent to control the particle size, stabilize nanoparticle dispersion and limit further oxidation on the particle surface. Maximum intensity peak (111) was used to estimate the crystallite size and it was found to be to be 28 and 22 nm respectively using Scherer equation. At the current density 14 mA/cm2 with THAB, sharp peaks were observed as compared to THAB 10 mA/cm2. SEM Figure 4 (a) THAB at 10 mA/cm2 and (b) THAB at 14 mA/cm2 show the low and high magnification images of NiO. The shapes of the NiO particles are spherical and are linked together. The formation of particle aggregates was observed due to strong interaction among nickel oxide nanoparticles. The microgram appears to be diffused and shows uneven morphology. EDX The energy dispersive X-ray analysis (EDX) was performed to know the elemental composition of Nickel oxide NPs. Nickel oxide nanoparticles synthesized with capping agent THAB were analyzed qualitatively and quantitatively by EDS and are shown in Fig. 5a, b. The Ni and O peaks can be obviously found in both EDS spectra, no peak of Br was found which indicates that the pure NiO particles are successfully prepared and there was complete removal of capping agent.

Fig. 4 SEM images of nickel oxide nanoparticles: 5000 and 10,000 lm magnification with a corresponding to current density 10 mA/cm2, b corresponding to current density 14 mA/cm2

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Mass%

Atom%

Ni

96.05

88.96

O

3.95

11.04

Total

100

100

Fig. 5 EDS spectrum of NiO nanoparticles and composition of elements

TEM Micrograph obtained from transmission electron microscope clearly indicate the decrease in particle size with increase in current density which resembles to the phenomenon observed in XRD pattern. The size, shape and phase composition of particles were studied by TEM. The sample for TEM analysis was obtained by evaporation of very dilute alcoholic suspensions onto carbon-coated copper grids. The appearance of some darker particles results from an enhanced diffraction contrast due to their orientation with respect to electron beam. The average particles size was measured to be 32 nm for THAB capping agent with different current density, which was in good agreement with calculated particles size by XRD analysis. TEM image (Fig. 6a, b) along with histogram of particle size distribution of the typical product showed that NiO nanoparticles were dispersed and no aggregation was observed. TEM images shows that the particles are crystalline and cubic in shape. The electron diffraction studies indicated that NiO nanoparticles were highly crystalline (right inset of Fig. 6a, b).

Antifungal Screening Table 1 indicates maximum zone of inhibition was observed for nickel oxide nanoparticles against F. oxysporum sp. (26.61 ± 1.43), A. niger sp. (22.80 ± 0.99) and A. alternaria (22.04 ± 1.44) at 100 ll concentration which were compared with standard Flucanozol. Similar results obtained for F. oxysporum sp. (24.14 ± 1.13) A. niger sp. (20.81 ± 0.72) and A. alternaria (21.29 ± 0.6) at 50 ll concentration at current density 14 mA/cm2. Similarly, maximum zone of inhibition was observed at current density 10 mA/cm2. F. oxysporum sp. (20.56 ± 1.33) A. niger sp. (15.27 ± 0.89) and A. alternaria (16.95 ± 0.26) at 100 ll concentration and results obtained for F. oxysporum sp. (23.58 ± 0.60) A. niger sp. (17.18 ± 0.72) and A. alternaria (19.36 ± 1.00) at 50 ll concentration. As compared with both current density results 14 mA/cm2 shows better antifungal activity.

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(a)

160

Particles Counts

140

Counts:1496 Mean:16.912 Std.Dev:4.478

120 100 80 60 40 20 0 10

20

30

Dimeter in nm

(b)

Counts:3744

250

Mean:21.356 Std.Dev:8.182

Particles Counts

200

150

100

50

0 10

20

30

40

Dimeter in nm

Fig. 6 a TEM image of NiONPs shows the related SAED pattern, and histogram at 10 mA/cm2, b TEM image shows the related SAED pattern at 14 mA/cm2 along with histogram

As compared with both current density results 14 mA/cm2 shows better antifungal activity. The particles size decreases reactivity of nanoparticles increases. The antifungal data have been analyzed by data analysis tool [30–32]. One way ANOVA followed by the Tukey’ HSD test. The antifungal studies of nickel oxide nanoparticles against fungi, the statistical analysis data was obtained in Table 1. The result obtained from Turkey’ HSD test showed that the P value corresponding to the F-statistic of ANOVA is \0.01 which strongly suggest that statistically significant different occur in antifungal screening. Mechanism of Antimicrobial Activity of Nickel Oxide Nanoparticles Figure 7 summarizes the interaction of NiO NPs with microbial cells. The antimicrobial activity of nickel oxide nanoparticles relies on generation of ROS and release of nickel ions Ni (II). Diffusion and endocytosis of nickel nanoparticles accumulation in cell membrane alter membrane permeability and destroy membrane

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Fig. 7 Mechanism of antimicrobial activity of nickel oxide nanoparticle

proteins. NiONps react with water forming ROS that penetrate the cell membrane causing protein disruption and cell membrane damage with subsequent leakage of cellular contents. Dissolution of Ni (II) ions and free radicals interrupt electron transport in the microbial cell resulting in cell death. These observations are in good agreement with earlier reports [33] for antimicrobial activity of silver nanoparticles. Earlier studies provide substantial evidence that nanoparticles produce reactive oxygen species (ROS) in bacterial cells and ROS accumulation intracellularly regulates apoptosis [34]. Oxidative stress-induced respiratory cells damage can be determined by measuring respiratory chain lactate dehydrogenase activity in microbial cells and nanoparticles enhance protein leakage by increasing membrane permeability [35]. The electron and hole interacts with water (H2O) to produce OH and H?. In addition, O2 molecules (suspended within the mixture of fungi and NiO) yield superoxide anion (O_2), which reacts with H? to produce HO2. Afterward, HO2 interferes with electrons generating hydrogen peroxide (HO2); which combines with H? giving hydrogen peroxide (H2O2) molecules. The latter are capable to enter the membrane where they either damage or kill the bacteria. H2O2 generation mainly relies on the surface of NiO-NPs to yield additional active molecules. There is a linear proportionality between the concentrations of H2O2 produced in NiO slurry and the NiO particle size [36]. The superoxides and hydroxyl radicals cannot penetrate the membrane due to their negative charges [37]. Thus, these species are found on the outer surface of the bacteria, in contrast, H2O2 molecules are able to pass through the bacterial cell wall, subsequently leading to injuries and destroy, and finally triggering cell death [38].

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Conclusion We have synthesized nickel oxide nanoparticles by electrochemical reduction method with different current densities. The optical spectra show absorption bands in the range 360–370 nm which confirm the metallic nature in both stabilizers. From XRD analysis, particle size decreased with increase in current density as the current density is proportional to the rate of formation of Ni ion reduction to Ni adatoms. TEM and SEM analysis proved a nearly cubic morphology of particles. EDX analyses revealed the presence of Ni, O elements demonstrating that pure NiO particles were successfully prepared, with there was complete removal of capping agent. In this work, we have demonstrated the antifungal effects of NiO nanoparticles in vitro conditions. The results of this study demonstrated the potentiality of nanoparticles as inhibitor agent of fungal growth. Nickel oxide nanoparticles showed good antifungal activity with THAB as compared with different current densities, concentrations and various microorganisms. Obtained results recommend the use of NiONPs as effective antifungal agent in practical application. Thus, it can be concluded that nickel oxide nanoparticles can be used as fungicide after further confirmatory and biosafety studies. Acknowledgements The authors are grateful do Department of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad and UGC-SAP-DRS-1 scheme New Delhi for providing laboratory facility. One of the authors (ASR) thankful for financial assistance from Major Research project University Grants Commission, New Delhi. The author (AAA) is also thankful to the University Grants Commission, New Delhi for Rajiv Gandhi National Fellowship.

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