Synthesis, Electrochemical Studies, and Antimicrobial Properties of Fe3O4 Nanoparticles from Callistemon viminalis Plant Extracts

Less toxic, environmentally safe green-mediated iron (III) oxide nanoparticles (Fe3O4-NP) synthesized using Callistemon viminalis (C. viminalis) leaf (Fe3O4-NPL) and flower (Fe3O4-NPF) extracts is reported in this work for the first time. Total flavonoids and phenols present in the plant extracts were determined. Characterization of the nanoparticles was carried out using Fourier transform infrared (FTIR) spectroscopy, ultraviolet–visible spectroscopy (UV–VIS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and Malvern zeta sizer. Other properties of the nanoparticles were investigated using the thermogravimetric analyser and cyclic voltammetry. The average particle sizes obtained for Fe3O4-NPL and Fe3O4-NPF were 17.91 nm and 27.93 nm, respectively. Fe3O4-NPL exhibited an excellent electrochemical activity when compared with Fe3O4-NPF based on a stability study using cyclic voltammetry and regression value. Additionally, Fe3O4-NPF displayed excellent antimicrobial activity against Bacillus cereus, Salmonella enteritidis, and Vibrio cholerae with zones of inhibition of 13, 15, and 25 mm, respectively. Simple, cheap, and less toxic green-mediated iron (III) oxide nanoparticles synthesized from C. viminalis leaf (Fe3O4-NPL) and flower (Fe3O4-NPF) extracts hold the potential of being used to control the activity of pathogenic bacteria of health importance and as an electrochemical sensor for both biological and environmental analytes.


Introduction
Iron oxide nanoparticles have gained scientific interest due to their unique super-paramagnetic properties [1,2], large surface-area-to-volume ratio [1,2], ease of preparation [1], affordability, and less toxicity [2]. It has found application in water treatment [3], examination and treatment of cancerous

Preparation of C. viminalis Leaf and Flower Extracts
Harvested leaves and flowers of C. viminalis plant were washed thoroughly with tap water, rinsed with distilled water, and dried under sunlight for 48 h. About 5 g of the ground dried leaves was weighed and boiled with 100 mL distilled water for 15 min at 80 • C [13]. The leaf extract was allowed to cool under room temperature and filtered using a vacuum pump with a Büchner funnel and 125 mm Whatman filter paper. The colour of the leaf extract was an orange with 5.32 pH. The same procedure was followed to obtain a red-coloured flower extract, which gave a 3.89 pH value.

Phytochemical Test of Extracts
To determine the presence of flavonoids and phenolic compounds in the C. viminalis leaf and flower extracts, a preliminary test was conducted [23]. The presence of flavonoids in the extracts of the plant was determined using an alkaline reagent test with 2 mL of the extracts with four drops of 10% aqueous sodium hydroxide solution (10 g of NaOH in 90 g of distilled water) plus a lead acetate test of 0.1 M with 2 mL of the extracts. The presence of phenolic compounds in the extracts was determined by a ferric test using 0.1 M of alcoholic iron (III) chloride hexahydrate [23].

Total Determination of Phenol Content
An established TPC method with modification was employed in estimating the phenol content of the C. viminalis leaf and flower extracts using gallic acid as an internal standard as reported in the literature [24]. Five milligrams of dried ground C. viminalis leaves and flowers was dissolved in 5 mL methanol. Ten percent FCR and 6% Na 2 CO 3 were prepared by the addition of water in a 100 mL volumetric flask. An amount of 300 µL of each extract was taken into a glass test tube followed by the addition of 1.5 mL of 10% FCR solution. The mixture was sonicated for 5 min. Thereafter, 1.5 mL of 6% Na 2 CO 3 solution was added, and the test tubes were incubated in a dark place for 2 h at room temperature. Gallic acid standard at various concentrations (750, 650, 550, 450, 350, and 150 µg/mL) was prepared in the extracts simultaneously as described in the established protocol [24]. The C. viminalis extracts and gallic acid standard solutions were measured at 760 nm with a UV-visible spectrophotometer after a blank measurement (methanol). The phenol content of the extracts was estimated using the gallic acid standard calibration curve, and the total phenols were expressed as a microgram of gallic acid equivalent (GAEmg) per 5 g of dry extract.

Total Determination of Flavonoid Content
The extracts were prepared following the same procedure in phenol content. Five percent NaNO 3 , 5% NaOH, and 7% AlCl 3 solutions were prepared by using water in a 100 mL volumetric flask. The leaf and flower (300 µL) extracts were taken into a glass test tube; 75 µL of 5% NaNO 3 was thereafter added and left for 5 min. Then 1.25 mL of AlCl 3 and 0.5 mL of NaOH were added. The mixture was sonicated for 5 min and incubated for 5 min at room temperature [24]. Concurrently, quercetin standard of different concentrations (500, 300, 100, 50, and 25 µg/mL) was also prepared. The absorbance of all the working solutions and standard solutions was measured at 510 nm against blank methanol [24].

Synthesis of Fe 3 O 4 -NP from Extracts
Fe 3 O 4 -NP was synthesized from the C. viminalis leaf extract (Fe 3 O 4 -NPL). Iron (III) chloride hexahydrate (10 mM) solution (99% purity) was added to the leaf extract in a 1:1 volume ratio. A change in colour from yellow to black was observed, indicating the formation of Fe 3 O 4 -NPL. The mixture was stirred for 3 h and allowed to stand at room temperature for 1 h to obtain colloidal suspension. Colloidal suspension was thereafter centrifuged at 25 • C, 6000 rpm, and an acceleration of 9 m s −2 for 30 min and washed several times with ethanol and dried in an oven at 40 • C for 2 h. Fe 3 O 4 -NP from the C. viminalis flower extract (Fe 3 O 4 -NPF) was synthesized following the same steps as in the Fe 3 O 4 -NPL synthesis [16].

Electrochemical Characterization
An electrochemical study was carried out by using cyclic voltammetry. The screen-printed carbon electrode (SPCE) was modified with

Antimicrobial Studies
Considering the properties and applications of Fe 3 O 4 -NP, it can be concluded that Fe 3 O 4 -NP hold the potential of inhibiting the replication of pathogens (antimicrobial agents) [25]. The antimicrobial properties of the green-mediated Fe 3 O 4 -NP were investigated by using agar disk diffusion methods as outlined by CLSI (2011) [26]. The antimicrobial activity of the nanoparticles was examined against eight categories of bacterial pathogens of public health importance belonging to both Gram-positive and Gram-negative types. Bacterial pathogens include Bacillus cereus (ATCC 10876) as Gram-positive, E. coli (ATCC 259622), Enterococcus faecalis (ATCC 29212), Enterococcus faecium (ATCC 700425), Enterococcus gallinarum (ATCC 701221), Salmonella typhimurium (ATCC 14028), Salmonella enteritidis, (ATCC 13076), and Vibrio cholerae. Preferred bacterial pathogens were reactivated in Luria-Bertani broth overnight before use, and their concentrations adjusted to 10ˆ6 CFU/mL. The antimicrobial disk used was sterilized in an autoclave at 121 • C for 15 min at 15 pascals. An antimicrobial disk was impregnated in dispersed nanoparticles of different concentrations (100 and 150 µg/mL). Mueller-Hinton agar (MHA) was prepared according to the manufacturer's design and was sterilized. MHA was poured into sterile petri dishes and made to solidify before its seeding with 10ˆ6 CFU/mL of chosen pathogens. To check mate overlapping, the nanoparticle-impregnated antimicrobial disk was placed in equidistance to each other while the antibiotic, ciprofloxacin (30 µg), was used as control. Plates were aerobically incubated for 24 h at 37 • C, after which zones of inhibition were observed and measured using a graduated meter rule in millimetres.

Phytochemical Test
On treating the leaf and flower extracts of Callistemon viminalis (C. viminalis) with the alkaline reagent of sodium hydroxide, brown-and an orange-coloured-precipitates were formed with yellowand blue-coloured precipitates, respectively, when the leaf and flower extracts were treated with 0.1 M lead acetate solution, indicating the presence of flavonoids. Grey-black and black solutions were formed on adding a few drops of 0.1 M alcoholic iron (III) chloride hexahydrate on the flower and leaf extracts, indicating the presence of phenols.

Determination of Total Phenolic Content (TPC) in Callistemon viminalis
The TPCs of the C. viminalis leaf and flower extracts estimated from the calibration curve of gallic acid standard ( Figure 1) using the Folin-Ciocalteu assay at 760 nm were 12.636 and 29.320 GAEmg/g, respectively, based on Equation (1), where C mg/ml is the total phenolic content in gallic acid equivalent (GAE), c is the concentration of gallic acid established from the calibration curve, V is the volume of extract, and m is the mass of extracts used. The calibration curves complied with the Beer-Lambert law with 0.9998, 0.002 µg/mL, and 0.1066 regression, slope, and intercept values accordingly.

Determination of Total Flavonoid Content (TFC) in Callistemon viminalis
The total flavonoid compositions of the C. viminalis leaf and flower extracts from the quercetin standard calibration curve as presented in Figure 2 were 19.371 and 0.634 QEmg/g using Equation (1). QE is total flavonoid content in quercetin equivalent. The content of flavonoids was higher in the C. viminalis leaf extract than in the flower extract. The calibration curves obeyed the Beer-Lambert law. The regression, slope, and intercept values obtained from the curve were 0.9647, 0.0007 and 0.5701 µg/mL, respectively. Comparing the TFCs of the leaf and flower extracts of C. viminalis, the content of phenol in the flower extract was greater than in the leaf contract, which is in agreement with the literature [27].
The higher TFC in the leaves than in the flowers was supposedly due to the abundant shikimic acid in the leaves compared with those in other organs, while the lower TFC in the flowers was indicative of the depletion of flavonoids due to colour development in the flower petals. Shikimic acid is a precursor in the flavonoid biosynthetic pathway and has been reported to be higher in leaves than in other organs due to the photosynthetic activities in the leaves [27,28].
The higher TPC in the flowers in this study could have been influenced by the developmental phase. The associated colour change of the petals due to oxidation resulting in the easy rupture and release of the cell wall bonded phenolic compounds in the flowers. The higher TPC in this study corroborates the report on groundcover rose flowers [20]. An increase in total phenol content was reported to be influenced by the stages of development and colour change [29].

Spectroscopic and Morphological Characterization
Fourier transform infrared (FTIR) analysis was carried out on the extracts and synthesized Fe 3 O 4 -NP in order to determine the compounds and the bonding properties present. In the FTIR spectra (Figure 3a), the leaf and flower extract (Figure 3b) spectrum displayed important vibration peaks at about 3300, 1729, and 1724, which are assigned to the O-H stretch of the alcohol/phenol group and C = O of the carbonyl group (carboxylic acid, aldehyde, ketone) accordingly. These peaks are possible reducing agents that are responsible for the formation of iron (III) oxide nanoparticles. The observed peak at 1735 in FeCl 3 .6H 2 O is ascribed to the deformation of water molecules, suggesting the existence of physio sorbed water on the surface of ferric chloride [30]. The FTIR spectrum of    [12], and nearly spherical in shape Fe 3 O 4 -NP with rough surfaces, which suggests absorption of the carboxylate group existing in protein, acting as surfactant to adhere to the nanoparticle surface, resulting in the Fe 3 O 4 -NP stabilization as can be seen in Figure 5a,b. The mechanism of absorption is presented in Figure 6.
The energy-dispersive X-ray (EDX) analysis results revealed the presence of iron and oxygen in the   Figure 7ai indicate the shape of iron in Fe 3 O 4 -NPL to be nearly spherical, spatially distributed with some particles agglomerated, and the size distribution is shown in Figure 7aii using the ImageJ software. The mean diameter and average particle size for N = 237 of the synthesized particles (Fe 3 O 4 -NPL) were 5.313 nm and 5.3 ± 1.9. Figure 7bi shows the TEM image for noticed. Figure 7bii indicates the size distribution of Fe 3 O 4 -NPF with a mean diameter and average particle size of 1.127 nm and 1.1 ± 0.8 for N = 2163 (N = number of counts).  Zeta potential analysis of Fe 3 O 4 -NP was conducted to obtain information on the surface charge of the dispersed particles (Fe 3 O 4 -NP). Particles with zeta potential values within the range of ±30 mV are widely considered stable [12,23]. The zeta potential values obtained for Fe 3 O 4 -NP synthesized from the leaf (Fe 3 O 4 -NPL) and flower (Fe 3 O 4 -NPF) extracts were −20.0 and −28.4 mV, respectively. The values are well comparable to the values reported in the literature [34]. Figure 8a,b gives a summary of the zeta potential analysis of Fe 3 O 4 -NPL and Fe 3 O 4 -NPF, respectively. The result of the zeta potential analysis showed good stability for Fe 3 O 4 -NP. A higher stability was observed for Fe 3 O 4 -NPF than Fe 3 O 4 -NPL due to greater surface charge (−28.4 mV), which could be attributed to the existence of denser electrostatic forces within the synthesized particles and compositions of the reducing agent (phenolic compounds) present in the flower extract [23,34].
TGA analysis of Fe 3 O 4 -NP was conducted with SDT Q600 V20.9 Build 20 thermogravimetric analyser of DSC-TGA standard in an unreactive nitrogen gas environment at a high furnace temperature using the RAMP method and a heating rate of 10 • C min −1 . The thermogravimetric curves of Fe 3 O 4 -NP synthesized from the C. viminalis leaf (Fe 3 O 4 -NPL) and flower (Fe 3 O 4 -NPF) extracts are shown in Figure 9 with three stages of weight loss. In Fe 3 O 4 -NPL and Fe 3 O 4 -NPF, the first stage lies within 25-65 • C and 29-58 • C with an insignificant weight loss (1%), which could be attributed to loss of water and volatile solvents. The second stage started at 65 and 58 • C for Fe 3 O 4 -NPL and Fe 3 O 4 -NPF accordingly and ended at 197 • C with a weight loss of 13% and 10%, respectively, and could be assigned to loss of water due to crystallization and gas desorption. Finally, the third stage at 636 and 654 • C for Fe 3 O 4 -NPL and Fe 3 O 4 -NPF had a significant weight loss of 44% and 50%, which could be assigned to the transition phase of Fe 3 O 4 to FeO since FeO is stable (thermodynamically) at temperatures above 570 • C [35] with a reaction formula at a possible equilibrium phase: The XRD spectra (Figure 10a) of synthesized Fe 3 O 4 -NP from the C. viminalis leaf (Fe 3 O 4 -NPL) and flower (Fe 3 O 4 -NPF) extracts conducted from 20 • to 80 • at 2 theta were closely similar to those reported in the literature [16,36].
The presence of a hump instead of a peak observed at 28.56 • and 26.62 • for Fe 3 O 4 -NPL and Fe 3 O 4 -NPF, respectively, could possibly be due to the covering of Fe 3 O 4 -NP with organic substances [35]. Therefore, the XRD pattern for the Fe 3 O 4 -NP showed deficiency in distinctive peaks, which suggests the amorphous nature of Fe 3 O 4 -NPL and Fe 3 O 4 -NPF and proves that the surface of the iron oxide is coated by organic materials from the extracts, and a similar XRD pattern was reported in the literature [36,37]. The XRD spectrum depicts Fe 3 O 4 -NP as confirmed by the colour of the precipitates formed during synthesis (Figure 10b) as opposed to the brick red/brown colour expected for Fe 2 O 3 (haematites).     (12.820 µA). The SPCE-modified electrodes displayed higher anodic current (Ipa) response than the bare SPCE, which proved a successful modification of the bare screen-printed carbon electrode. The Ipa for the SPCE-Fe 3 O 4 -NPL was found to be six times higher than that for the bare SPCE and three times higher than that for SPCE-Fe 3 O 4 -NPF, suggesting SPCE-Fe 3 O 4 -NPL to be more electrically conductive than SPCE-Fe 3 O 4 -NPF, suggesting excellent ionic interaction among the nanoparticles' large surface area, which in turn allowed free flow of ions/electrolytes in and out of the electrode surface. The high diffusivity of ions at the same electrode is ascribed to higher surface area for effective diffusion. SPCE-Fe 3 O 4 -NPF was two times higher than the bare SPCE. The large surface-to-volume ratio and high electrical conductivity of iron (III) oxide nanoparticles (Fe 3 O 4 -NP) were responsible for the high electron transport at the modified electrodes (SPCE-Fe 3 O 4 -NPL and SPCE-Fe 3 O 4 -NPF). The ratio of the anodic-to-cathodic peak currents was greater than 1 and the unity for SPCE-Fe 3 O 4 -NPL and SPCE-Fe 3 O 4 -NPF, suggesting an irreversible and reversible redox process accordingly. The influence of varying scan rates on the modified electrodes was determined for further understanding of the kinetics of the electron transport process at the electrodes (SPCE-Fe 3 O 4 -NPL and SPCE-Fe 3 O 4 -NPF) using cyclic voltammetry in PBS containing 5 mM K 3 Fe(CN) 6 solution. Increase in scan rates resulted in an increased anodic current response as seen in Figure 12ai,bi. Regression values of 0.9968 and 0.9969 for the respective modified electrodes were obtained from the plot of current (Ipa) against the square root of the scan rate (Figure 12aii,bii), which confirmed a diffusion-controlled process at the modified SPCE electrodes.
Materials 2020, 13, x; doi: FOR PEER REVIEW www.mdpi.com/journal/materials understanding of the kinetics of the electron transport process at the electrodes (SPCE-Fe3O4-NPL and SPCE-Fe3O4-NPF) using cyclic voltammetry in PBS containing 5 mM K3Fe(CN)6 solution. Increase in scan rates resulted in an increased anodic current response as seen in Figure 12ai,bi. Regression values of 0.9968 and 0.9969 for the respective modified electrodes were obtained from the plot of current (Ipa) against the square root of the scan rate (Figure 12aii,bii), which confirmed a diffusioncontrolled process at the modified SPCE electrodes. The parameters used to compare the electrochemical properties of the modified electrodes were electron transport coefficients (α), which were calculated to be 0.53 and 0.56 for the SPCE-Fe 3 O 4 -NPL and SPCE-Fe 3 O 4 -NPF electrodes from the slope values of the linear plots of Ep (peak potentials) versus the logarithm of scan rates (log mVs −1 ) (graph not shown) equal to −2.3RT ∝nF and 2.3RT (1−∝)nF assigned to cathodic and anodic peaks, respectively, by Lavarion [38], where Ep is the slope, R represents the gas constant (8.3142 JK −1 mol −1 ), and T is room temperature in K (298). The number of electrons transferred (n) at the respective electrodes was one for both electrodes. The values of the slope from the plots of the respective modified electrodes were found to be 0.11235 and 0.13916 Vdec −1 . The Tafel value "b" was obtained using Equation (2).
The values of b for the modified electrodes SPCE-Fe 3 O 4 -NPL and SPCE-Fe 3 O 4 -NPF were 0.224 and 0.278 Vdec −1 , respectively. The Tafel value obtained for both electrodes were found higher than the theoretical value (0.118 Vdec −1 ) for one-electron transport. The differences in the value of "b" in theory and practice suggest surface assimilation of reactants or intermediates on the electrode surfaces [39].
In the Ip maximum current (amperes), A is the electrode area of SPCE (0.1257 cm 2 ), C is the concentration of the bulk solution (mol/cm 3 ), and v is the scan rate in Vs −1 . The surface concentrations of the respective modified electrodes (SPCE-Fe 3 O 4 -NPL and SPCE-Fe 3 O 4 -NPF) were calculated to be 3.47 and 1.91 µmol/cm 2 by applying Equation (4).  Table 1 presents the zones of inhibition of green-mediated Fe 3 O 4 -NP activity against selected bacterial pathogens (microbes) exposed to varying concentrations (100 and 150 µg/mL) of nanoparticles. The antimicrobial activity of Fe 3 O 4 -NPL against Bacillus cereus ranged from 8 to 13 mm and was highest in Fe 3 O 4 -NPF at 150 µg/mL. However, there was no significant difference between the antimicrobial activities of Fe 3 O 4 -NPL and Fe 3 O 4 -NPF. A similar trend was observed in the antimicrobial activities of nanoparticles against E. coli, Enterococcus faecalis, Enterococcus faecium, Salmonella enteritidis, and Vibrio cholerae. At 150 µg/mL, the antimicrobial activity of Fe 3 O 4 -NPF (14 mm) was highest against the tested pathogens except in Salmonella typhimurium (8 mm). In Vibrio cholerae, it could be suggested that the antimicrobial activity of Fe 3 O 4 -NP was influenced not only by the concentration of nanoparticles but also by the part of the plant used and the phytochemical present because higher zones of inhibition were obtained in Fe 3 O 4 -NP synthesized by the flower extract compared with those obtained in Fe 3 O 4 -NP synthesized by the leaf extract in most cases, which could be attributed to high phenolic content in the flower extract, leading to an improved reducing ability of nanoparticles. Differences in bacterial cell structures also play a role in the inhibition of microorganisms [41], which could be the case of high antimicrobial activity of Fe 3 O 4 -NPL against Salmonella typhimurium compared with that of Fe 3 O 4 -NPF. The structure of the bacterial cell wall and the nanoparticle concentration influence the ability of nanoparticles to alter the replication process in bacteria and the toxic activities of nanoparticles against pathogens [41]. Nanoparticles could generate a pit within the cell wall, resulting to an improved permeability, which could lead to the death of bacterial cells [39]. Nanoparticles have the ability to switch off the activity of cellular enzymes and DNA through the electron-donating group (thiols, carboxylates, amides, imidazoles, indoles, and hydroxyls) [42]. This electron-donating group could be from the phytochemical components of the plant used in the green synthesis of Fe 3 O 4 -NP. Previous studies have reported the activity of nanoparticles against pathogenic and drug-resistant bacteria [43]. The result shown in Table 1 reveal that Vibrio cholerae was more susceptible to Fe 3 O 4 -NPF, followed by Salmonella enteritidis, Enterococcus faecalis, and E. coli, and from leaves, Vibrio cholerae, Salmonella typhimurium, Enterococcus faecium, E. coli, and Enterococcus faecalis. The inhibition zones of selected bacterial pathogens (microbes) increased with increase in the concentration of Fe 3 O 4 -NP in µg/mL.

Conclusions
This work reports a comparative study of Fe 3 O 4 -NP (Fe 3 O 4 -NPL and Fe 3 O 4 -NPF) synthesized by using C. viminalis leaf and flower extracts typical in the North West province of South Africa. The extracts acted as a reducing agent, reducing iron (III) ions (Fe 3+ ) to zero valence metal. Energy band gap calculation through UV-visible spectra revealed synthesized nanoparticles to be semiconductors. FTIR spectra showed functional groups such as phenols and ketones to be present in plant extracts, which are believed to be responsible in the formation of synthesized iron (III) oxide nanoparticles. SEM micrographs revealed rough surfaces of synthesized nanoparticles, confirming the absorption of extracts on the surface of the nanoparticles. TEM analysis revealed the shape of the synthesized particles to be spherical, especially Fe 3 O 4 -NPF. Zeta potential analysis revealed negative surface charges of the synthesized nanoparticles and their electrical stability with Fe 3 O 4 -NPF being more stable due to its high surface charge, which could be attributed to the existence of denser electrostatic forces within the synthesized particles and compositions of the reducing agent (flower extracts). Additionally, antimicrobial studies conducted on synthesized nanoparticles against selected pathogens (microbes) revealed good antimicrobial activity for both Fe 3 O 4 -NPL and Fe 3 O 4 -NPF. Electrochemical studies at a modified screen-printed electrode confirmed better electrochemical activity at a modified SPCE of Fe 3 O 4 -NPL than Fe 3 O 4 -NPF considering the current (Ipa) response, although better stability was noticed at SPCE/Fe 3 O 4 -NPF. However, both modified Fe 3 O 4 -NPL and Fe 3 O 4 -NPF can be good catalysts for fabricating electrochemical sensors for biological and environmental analytes.