Cyclic voltammetry, photocatalytic and antimicrobial comparative studies of fabrication Fe3O4 and Fe3O4/PAN nanofibers

This study reports the properties of green mediated synthesized iron oxides nanoparticles (Fe3O4 NPs) from peel extracts of pomegranate plant and its polyacrylonitrile/iron oxide composite nanofibers (Fe3O4/PAN). The following were used to characterize the synthesized nanoparticles and its polymer nanofibers; FT-IR, UV-Visible spectroscopy, scanning electron microscope SEM, TEM and cyclic voltammetry. The antimicrobial activities of synthesized nanoparticles were investigated against selected bacterial pathogens. For the plant extract, FTIR revealed OH characteristics peaks at 3271 cm−1 and 1600 cm−1 while the absorption peaks at 577 and 430 cm1 showed successful reduction of the precursor to Fe3O4 nanoparticles. The SEM images showed a spherical morphology of Fe3O4 and that of the composite with entrapped Fe3O4 into the PAN nanofibers. Photocatalytic process showed that the synthesized Fe3O4 nanoparticles has degradation efficiency of 71.36% and the nanofibers exhibited efficiency of 22.68% towards methylene blue (MB) dye. However, further kinetic analysis of the degradation process put Fe3O4/PAN nanofibers (NF) at a better correlation coefficient of 0.9239 than the Fe3O4 nanoparticles. Electrochemical studies using cyclic voltammetry showed that PAN functionalized with Fe3O4 is more electroactive as compared to the other electrodes studied. The anodic peak potential at 599 mV also confirmed the presence of Fe3O4 in the nanocomposite Fe3O4/PAN. The antimicrobial studies revealed that as the concentration of the green mediated Fe3O4 nanoparticle increases in the composite Fe3O4/PAN an excellent antimicrobial activity against selected pathogens were observed, showing Fe3O4 nanoparticles potentials to control pathogens of public health significance.


Introduction
The development of inexpensive, environmentally and eco-friendly methods for the synthesis of nanoparticles and its composite has been made possible through nanotechnology [1][2][3]. Nanocomposites from metal oxides nanoparticles and polymer nanofiber are of great importance to researcher. Nanofibers from polymers have found various applications in the medical field such as in the detection of biological analytes [4], drug delivery [5], magnetic resonance imaging (MRI) [6], cancer therapy and diagnosis [7,8]. Other applications include; industrial, agricultural, environmental, and waste management.
The green method for the synthesis of metal oxides nanoparticles have been widely reported and also established [9]. Many metal oxides nanoparticles have been successfully synthesized using extracts from plants [10,11]. Metal oxide nanoparticles have chemical and physical properties such as particle size, density and surface area [12]. Metal oxide nanoparticles have found applications in areas like sensors, photochemical, and microelectronic circuit's fabrication. Nanoparticles of various metal oxide have been synthesized, these include; Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. nickel oxide (NiO) [13], zinc oxide (ZnO) [14], copper oxide (CuO) [15], and iron oxides (Fe 3 O 4 ) [16], to mention but few. Iron oxide nanoparticles (NPs) are of special interest among other nanomaterials because of its high surface area to volume ratio, fast kinetics, strong adsorption capacities, [17] and great magnetic properties [18].
Electrospinning method is simple and cost-effective in the production of nanofibers [31]. It gives a more convenient way to fabricate infinite, continuous fibers and has found applications in various field such as infiltration [32,33], biomedical films [34,35], and scaffolds for tissue engineering [36].
Pathogens are microorganisms which are capable of causing ill-health to humans and animals [43]. Pathogenic bacteria such as Salmonella Typhimurium, Salmonella Enteritidis, Escherichia coli, Vibrio cholera, Listeria monocytogenes has been blacklisted and reported to be of public health significance [43,44]. There have been several efforts to control bacteria pathogens through the use of antimicrobials such as antibiotics. However, in recent years, there have been cases of antibiotic resistance to this pathogens, thereby causing a search for method have shown excellent antimicrobial properties against pathogens [45]. The increasing threat posed to food safety and public health by evolving bacteria pathogens calls for concern, hence, it is necessary to investigate the potentials of Fe 3 O 4 and Fe 3 O 4 /PAN nanofibers as antimicrobial agents against pathogens of public health concerns.
This work, therefore, reports the investigation of the spectroscopic, morphological, and antimicrobial properties of green mediated based iron oxide nanoparticles and its functionalized PAN composite nanofibers.

Green synthesis of the Fe 3 O 4 nanoparticle
Pomegranate plant was washed thoroughly and the peel was cut into pieces and dried up in an oven for 48 h at 40°C. The dried peel was then blended to a very fine powder, and 20 g powdered was boiled in 250 ml distilled water. The extract was filtered using vacuum pump filter. About 50 ml of 0.05M FeSO4. 6H2O were add to 50 ml pomegranate extract drop wisely under continuous stirring. The solution was then heated at 80°C for some minutes until about 2 h at 40°C. The extract was then centrifuged for few minutes at 4°C at 6000 rpm. The suspension obtained were dried for 30 min at 70°C temperature obtain nanoparticles in powder [46].

Fabrication of PAN and its composites Fe 3 O 4 /PAN nanofibers
Polyacrylonitrile PAN was pre-dried at 60°C for 24 h and then dissolved DMF to make 16 wt%. Electrospinnable polymer solution was suctioned into 20 ml syringe and 22 kV voltage was applied, the flow rate and the tip-collector distance were fixed at 0.15 ml h −1 and 15 cm respectively. Nanofibers were placed on an aluminium foil plate and the electrospinning was performed at an ambient temperature. The nanofibers were dried overnight at room temperature to eliminate water and solvent from the produced nanofibers. The same procedure was followed for the composite by measuring 1:1 of PAN and Fe 3 O 4 nanoparticles to make 16 wt% required spinnable solution.

Characterization of Fe 3 O 4 , PAN, and its composites Fe 3 O 4 /PAN nanofibers
Spectroscopy characterization such as UV-vis was performed on Perkin Elmer Lambda 650 UV-visible spectrometer, and Fourier-transform infrared spectroscopy (FTIR.) The absorbance scans were run over the wavelength range of 200 to 800 nm. The morphological and size measurement were also performed on scanning electron microscope (SEM), transmission electron microscopy (TEM) characterization were performed. Image J software was used to measure the diameter of the nanofibers and the TEM image of the nanoparticle.

Cyclic voltammetry studies
Electrochemical characterization of the synthesized nanoparticles and its polymer nanofibers was carried out by using cyclic voltammetry (CV). Cyclic voltammetry measurements were carried out by using a dropsense software driven mini PGSTAT 302. The screen print electrode consists of three electrodes which are Ag/AgCl as reference electrode, counter electrode and a carbon working electrode. The carbon electrode (working electrode) was modified using the drop-dry method. Separate drop of Fe 3 O 4 NPs, PAN and Fe 3 O 4 /PAN were cast on the electrode respectively and dried in the oven for 2 min at temperature of 50°C. The voltammogram for the bare and modified SPC electrode were measured in 5 mM potassium hexacyano-ferrate (III) (K3Fe(CN)6) solution prepared in 0.1 M phosphate buffer solution (PBS) as a supporting electrolyte.

Photocatalytic studies
The photodegradation of methylene blue (MB) dye was studied using the synthesized Fe 3 O 4 nanoparticles and the PAN/Fe 3 O 4 nanofibers. The methylene blue solution (20 mg L −1 ) was prepared and 100 ml used for the photocatalytic study. The solution was first agitated for 30 min in a dark environment for proper equilibration after which 10 mg of the nanoparticles and 62.5 mg of the nanofibers (5×5 cm) were used separately. The dye and nanomaterial solution was stirred for 20 min and the UV light was switched on. The reaction was carried out in a photocatalytic reactor for 2h while aliquot samples were taken to study the rate of dye degradation using UVvis spectrophotometer. The degradation study was carried out within the wavelength of 350-740 nm and the MB absorption maximum recorded at 664 nm.

Antimicrobial activity of PAN, Fe 3 O 4 and Fe 3 O 4 /PAN nanofibers
The antimicrobial activity of synthesized nanoparticles was evaluated using the agar disc diffusion method [47]. Selected pathogens of public health importance such as; Bacillus cereus, E. coli, Enterococcus faecalis, Enterococcus faecium, Enterococcus galinarium, Salmonella Typhimurium, Salmonella Enteritidis, Vibrio cholera were obtained from the Molecular Microbiology Laboratory, North West University, South Africa. Fresh overnight cultures of test pathogens were adjusted to 0.5 McFarland concentrations and was then diluted twenty-fold (1:20) to obtain 5×10 6 CFU ml −1 . Diluted cultures (10 6 CFU ml −1 ) of selected pathogens were seeded on Muller Hinton agar prepared according to manufacturer's specifications and was poured on petri dishes. Fe 3 O 4 nanoparticles and PAN/Fe 3 O 4 nanofibers sols were prepared by dispersing equal mass of nanoparticles/nanofibres in five times volume of dimethylformamide (DMF). The antibiotic (ciprofloxacin 30 μg) was used as positive control while dimethylformamide was used as negative control. Empty antimicrobial disc was singly impregnated with ciprofloxacin (30 μg), DMF, synthesized Fe 3 O 4 nanoparticles and PAN/Fe 3 O 4 nanofibers and were allow to stand for an hour. The impregnated discs were placed at equidistance to each other to prevent overlap on the seeded petri dishes. The experiment was determined in triplicates. Inhibitions zones were observed on plates incubated aerobically at 37°C for 24 h. Inhibition zones of nanoparticles against selected pathogens were measured using a graduated meter rule in millimetres (mm) and values were recorded.

Transmission Electron Microscopy (TEM)
In figure 4, the TEM image of the syntheised Fe 3 O 4 nanoparticles showed that the nanoparticles were well dispersed in colloidal solution with cubic shape ranging from 10 to 50 nm. The average well distributed particle size of 30 nm.

Photocatalytic activity
In recent times, nanomaterials are greatly serving as photocatalysts in the degradation of dyes. Figure 9 shows the degradation of the dyes in absorbance terms with respect to irradiation time. From figure 9, there was a remarkable decrease in the first 30 min of the reaction but subsequently, the rate got slower. In figure 10, the degradation rate was gradual and the concentration of the dyes continued to decrease. During the degradation process, the blue colour of the dye tends to be lighter which was due to the destruction of the chromophores in     the structure of the compound dye. Further, the degradation efficiencies were calculated using the absorbance results obtained from the Uv-vis at the wavelength of absorption of the dye, 664 nm. The following equation was used; Where C o and C t are concentration value at the initial time and at a particular irradiation time t. The percentage degradation of the nanoparticles and the nanofibers were compared and it was expected that the nanofibers could have greater efficiency due to the impregnation of the Fe 3 O 4 into the PAN. On the contrary, the results showed 71.36% for the Fe 3 O 4 and 22.68% for the composite nanofibers at the 2 h of the reaction which reflects higher degradation efficiencies with the nanoparticles than the nanofibers. The reason may be due to the different shapes of the nanomaterials as their morphologies determines their activities and applications. In addition, photocatalytic process also depends on the available active species which determine the reduction reactions [51][52][53].
When UV light falls on the photocatalysts, energized electrons are excited leaving holes behind. The holes interact with water molecules to form active radicals ºOH which act as the redox agents while the excited electrons react with oxygen to form superoxide radicals ºO 2 [51]. The presence of more holes therefore enhances degradation process. It is therefore presumed that there were enough active sites or holes in the nanoparticles than in the nanofibers. Previous studies using PAN/TiO 2 and PAN/ZnO nanofibers have shown 93% and 91% degradation efficiencies of malachite green dye respectively [54]. In another report of adsorption of tetracycline using Fe 3 O 4 and Fe 3 O 4 /PAN enhanced activity was observed with the composite nanofibers than the nanoparticles as against the result recorded in this study as shown in figure 11(b). It is therefore, proposed that the green mediated nanoparticle does has improved photocatalytic activities due to the presence of phytochemicals in the extract used for the synthesis. The nanocomposite may prevent the aggregation of the incorporated nanoparticles thereby still enhancing the surface area, however, such may not have occurred in this study and this surface area is reduced leading to poor dye adsorption [55].
The degradation reaction rate constant for the nanomaterials were obtained from the pseudo first order equation or Langmuir-Hinshelwood model [54], . Bacillus cereus is a gram positive bacteria that colonizes food substrate and on ingestion can cause food borne infections. The zones of inhibition of composite nanoparticles against Escherichia coli ranged from 9-17 mm and were highest in PANF 3. As expected, ciprofloxacin (positive control) inhibited the growth of the test pathogens as a standard antibiotic. Ciprofloxacin is one of the main streams of antibiotics often prescribed in the treatment of infections caused by enteric pathogens.
Dimethylformamide (DMF) did not inhibit the growth of all tested pathogens justifying that the observed effect was a product of the activity of the synthesized nanoparticles. The zones of inhibition in Fe 3 O 4 ranged from 6-14 mm, PAN (7-18 mm), PANF1 (9-16 mm), PANF2 (11-19 mm), PANF3 (11-22 mm). The zones of inhibition observed against the test pathogens were lower than those obtained in PANF. The antimicrobial activity of the nanoparticles supports the previous observation reported in literature in AgNPs synthesized with R. chinensis galls extract and marine algae (Ecklonia cava) respectively [55,56]. Contrary to the above observations, antibacterial activities of the synthesized nanoparticles against Salmonella Typhimurium and Salmonella Enteritidis were not dependent on concentration of Fe 3 O 4 NPs. Antibacterial activity was optimum against Salmonella pathogens at 1.4 g of Fe 3 O 4 NP's substitution with PAN. The observed antibacterial activity of the synthesized nanoparticles from pomegranates peel could imply its ability to alter the formation of ROS, DNA replication, aggregation of proteins and interfere with 0leakages in the cell wall of bacterial pathogens [57][58][59].

Conclusion
The successful synthesis of Fe 3 O 4 nanoparticles from pomegranate peel extract and fabrication of Fe 3 O 4 /PAN nanofibers composite were confirmed by using spectroscopy and morphological techniques. The fabrication of Fe 3 O 4 /PAN nanofibers composite were performed by electrospinning technique. For characterization of nanoparticles and nanofibers SEM and TEM images were used and it was shown that Fe 3 O 4 nanoparticles were uniformly combined with the PAN nanofibers and FT-IR spectroscopy and UV-Visible spectra were used to confirm the functional groups and characteristics wavenumber of the nanoparticle. The electrochemical characterization of the of the electrodes from bare to the modified shows that the modified electrodes of Fe 3 O 4 /PAN gave a better enhancement and catalytic activities than the bare and other electrode studied. The application of the synthesized nanoparticles and nanofibers in the photodegradation of MB dye has shown some promising effects which can be further developed towards waste water treatment. However, Fe 3 O 4 /PAN nanofibers composite also exhibited higher antibacterial activity, showing their potential application in water purification, and capable of destroying pathogens or bacteria from waste water. Fe 3 O 4 /PAN nanofibers composite with contact between nanomaterial and pathogens results in a high antimicrobial efficiency, because the DNA binds when Fe 3 O 4 nanoparticles come in contact with pathogens and destroy their metabolism process by damaging the cells and eventually cause death.