Synthesis of Fe3O4 nanoparticles and its antibacterial application

The Present work outlines the antibacterial activity of Fe3O4 nanoparticles synthesized through chemical combustion method where ferric nitrate is used as precursor material and urea as fuel with the assistant of Tween 80, a non-ionic surfactant. The obtained Fe3O4 nanoparticles were characterized by X-ray diffraction, differential thermal analysis/thermo gravimetric analysis (DTA/TGA), particle size analyzer, SEM with EDAX and TEM. Various parameters such as dislocation density, micro strain, analysis of weight loss and surface morphological studies were calculated. The particle size was calculated from XRD and it was found to be 33–40 nm. Using well diffusion method antibacterial activity of Fe3O4 nanoparticles was tested against gram-positive and gram-negative Staphylococus aureus, Xanthomonas, Escherichiacoli and Proteusvulgaris. Fe3O4 nanoparticles exhibited strong antibacterial activity against bacterial species.


Introduction
Nano materials are widely synthesized for their properties like optical, mechanical and magnetic properties to counter the bulk materials [1][2][3]. Metal oxides are used in various applications like magnetic storage, catalysis and biological applications like bone tissue engineering [4][5][6][7]. The prolonged life expectation and aging of population has brought the escalating request of artificial material to regenerate diseased bones [8][9][10][11]. Nanotechnology has responded to the situation with various ceramics with its bioactivity [12], mechanical properties [13,14] and ability to kindle bone growth. In particular iron oxide powder at nanometer is utilized at length because of the development in preparation technology. Monodispersed magnetite nanoparticles have given a new impetus in the application field where magnetic nanoparticles are extensively used in Ferro fluids, biological imaging and therapies [15,16]. Magnetic iron oxide (Fe 3 O 4 ) with oxygen forming face centred cubic has a cubic inverse spinal structure and in the interstitial tetrahedral sites and octahedral sites are occupied by iron (Fe) cations [17]. At the room temperature Fe ?2 and Fe ?3 ions flip between themselves in the octahedral sites giving rise to a class called half-metallic materials [18]. The desired physical and chemical properties of magnetite nanoparticles are synthesized by several chemical synthetic routes like co-precipitation of aqueous ferrous and ferric solutions [19], microemulsion technique [20] and hydrothermal synthesis [21]. Superparamagnetic nanoparticles are highly exciting materials because of their uses in magnetic resonance imaging (MRI) [22][23][24][25][26], drug delivery [27] and cell separation [28]. In the area of antibacterial agents metal nanoparticles are of a particular interest because they could be synthesized with high surface area with highly potential active sites [29]. A distinct class of metal oxide with distinctive magnetic properties and superior biocompatibility are is found in iron oxide nanoparticles.
In the past few years, a wide range of work has been done in producing new drugs due to the resistance of micro-organisms to the current drugs. This work is a novel way of synthesizing Fe 3 O 4 nanoparticles with the surfactant; and it is also an attempt to study the antibacterial properties of Fe 3 O 4 nanoparticles.

Materials
The chemical reagents used in this work were ferric nitrate, surfactant Tween 80, Urea and ammonia solution. Analytical grade chemical reagents were used throughout the experiment. We have taken four bacterial species, grampositive Staphylococus aureus and gram-negative Xanthomonas, Escherichia coli and Proteus vulgaris. The microbes were acquired from the biotechnology department of Jawaharlal Nehru Technological University Hyderabad.

Synthesis
The synthesis of magnetite (Fe 3 O 4 ) Nanoparticles was done by chemical combustion. The required amount of ferric nitrate (0.1 M) was dissolved in 20 ml of deionized water under the magnetic stirrer for 10 min. The fuel urea and ammonia (0.1 M) were dissolved separately in 30 ml of distilled water, respectively. The surfactant TWEEN80 (0.07 M) was dissolved in 20 ml of distilled water and was kept under stirring for 10 min separately. Fuel solution was mixed with oxidizer solution which was under stirring followed by mixing of surfactant solution. The whole solution was kept under stirring for 15 min for stirring. The solution was placed on a hot plate to initiate the reaction. When the temperature had started to increase, the solution boiled and fumes gushed forth from the solution; as the temperature increased above 100°C, the solution started to evaporated leading to an increase in the viscosity of the liquid and smouldering started eventually self-ignition took place forming the final product (Fe3O 4 ). The powder was collected from the beaker and calcinated for 1 h at 400°C. The powder It was collected for characterization and antibacterial application.

Screening of antibacterial activities
The well-diffusion technique [30] was used. 500 ll of microbes cultures of age 18-24 h were added to Petri plates and nutrient agar was poured. Once the medium was solidified, holes were made and each hole was packed with different concentrations of nanoparticles ranging from 20 to 150 lg/ml one after the other. The plates were wrapped in parafilm tape and transferred to incubator and maintained at 37°C for 24 h. Negative and positive controls were used. The inhibition zones of were then recorded in centimetres.

Results and discussion
XRD An X-ray diffraction (XRD) pattern of the sample was done at room temperature on D8 Advance Bruker diffractometer with a Cu Ka radiation (k = 0.154 nm). From Fig. 1, it can be observed that the diffraction peaks exhibit a phase face centred cubic structure and was in good agreement with the JCPDS [space group Fd3m (227), JCPDS #89-4319]. Using Scherrer formula [31,32], average crystallite sizes were estimated. It was found that the size of nanoparticles were 33-40 nm from the X-ray line broadening.
Where k is the X-ray wavelength (1.54 Å ) for copper Ka. h is the Bragg's angle. b is full width half maximum  value. Dislocation density (d) is calculated with the crystalline size.
From the calculated d spacing value, the lattice constants are calculated as follows: with the below formulae.
where a, b, c are lattice parameters and h, k, l are miller indices. Micro strain arises due to the lattice misfit which varies on the deposition conditions and thus it is calculated by the formula.
We have observed that dislocation density has decreased with the increase in the crystallite size. Similarly, the micro strain has increased with the decrease in the crystallite size. These results are shown in the Table 1.

TG/DTA
In the differential thermal analysis/thermogravimetric analysis (DTA/TG), we have observed both exothermic and endothermic graphs. There is a gradual weight loss in the TG graph as the temperature increases. At 100°C, there was a sudden fall in the graph indicating that there There was gradual weight loss due to the loss of carbon at 500°C. Correspondingly, in Fig. 2, there was DTA graph showing the endothermic peak at 350°C indicating that maximum heat was absorbed into the sample.

Particle size analyzer
The particle size was calculated at various cumulative factors using particle analyser. In ethyl alcohol at room temperature and at low concentration the sample was suspended and ultra sonicated for 10 min and subjected to laser of 245 nm wavelength in the particle analyser instrument. Thus in Fig. 3 average particle size for sample was shown with histogram.

SEM, TEM and EDAX
The morphological studies were done by using SEM. In Fig. 4, we have observed that the sample has many pores on the surface of the sample indicating that the obtained sample has porous nature.
The main benefit of a TEM is that it can simultaneously give evidence in real space (in the imaging mode) and reciprocal space (in the diffraction mode). Using the TEM image, both the size and shape of the obtained nanoparticles were observed. They were spherical in shape and porous surface as seen in the Fig. 5. The crystallite size obtained by the Scherrer's formula and the size from the TEM confirm each other.
The energy-dispersive X-ray spectroscopy results are shown in the Fig. 6. It shows the presence of oxygen and iron. This confirms the existence of oxygen and iron in the sample (Fig. 6).

Antibacterial activity
Fe 3 O 4 showed antibacterial effect against gram-positive as well as gram-negative bacteria which clearly indicates that these nanoparticles are effective antibacterial agents. In Figs. 7, 8 and 9 the control, low and high concentrations of Fe 3 O 4 were shown, respectively. Many antibacterial studies were made using different nanoparticles. The reason for the bactericidal activity is due to the presence of reactive oxygen species (ROS) generated by different nanoparticles [33]. Chemical interaction between hydrogen peroxide and membrane proteins or between the chemical produced in the presence of Fe 3 O 4 nanoparticles and the outer bilayer It is also noted that nanoparticles continue to be in interaction with dead bacteria once the hydrogen peroxide is generated; thus foiling further bacterial action and continue to produce and release  [34]. In Figs. 8 and 9 we clearly see the antibacterial activity in brown and yellow colours indicating that the bacteria is completely destroyed and antibacterial activity is still active, respectively.
The possible mechanism of action is that the metal nanoparticles are carrying the positive charges and the microbes are having the negative charges which create the electromagnetic attraction between the nanoparticles and the microbes. When the attraction is made, the microbes get oxidized and die instantly [35]. Generally, the nanomaterials release ions, which react with the thiol groups (-SH) of the proteins present on the bacterial cell surface which leads to cell lysis [36].
The central mechanism that caused the antibacterial activity by the particles might be through oxidative stress caused by ROS [37,38]. ROS includes radicals like superoxide radicals (O À 2 ), hydroxyl radicals (-OH) and hydrogen peroxide (H 2 O 2 ); and singlet oxygen ( 1 O 2 ) could be the reason damaging the proteins and DNA in the bacteria. ROS could have been produced by the present metal oxide (iron oxide) leading to the inhibition of most pathogenic bacteria like S. aureus, Xanthomonas, E. coli and P. vulgaris. A related study was explained by Kim et al. [16] in which hydrogen peroxide (H 2 O 2 ) was generated when Fe 2? responded with oxygen. The ferrous irons reacted with the produced H 2 O 2 subsequently through Fenton reaction and thus leading to creating hydroxyl radicals which damage the biological macro-molecules [39].
The nanoparticles can also produce bactericidal effects as verified by a few authors have verified. A few authors like Lee et al. [40] stated that the iron nanoparticles caused the inactivation of E. coli by zero-valent and the diffusion of the small particles ranging from 10 to 80 nm into E. coli membranes. Nano scale zero valent iron could interact with intracellular oxygen thus generating oxidative stress and ultimately triggering the interference of the cell membrane. Nanoparticles of ZnO and MgO also have revealed that with a decrease in particle size, antibacterial activity increases [41,42]. Likewise, Taylor and Webster also made a study studies on iron oxide nanoparticles and its bactericidal effects of on S. epidermidis [43]. They also described that bacterial inhibition depends on concentration. We need to note that iron oxide nanoparticles do not negatively impact all cells but with an appropriate magnetic field of iron oxide, nanoparticles may be engaged to destroy bacteria.
The results revealed that the microorganisms are sensitive to the test samples in varying magnitudes. The Antibacterial activity of Fe 3 O 4 nanoparticles on 4 bacterial strains is summarized in Fig. 10. The Fe 3 O 4 Nanoparticle showed a good antibacterial activity on E. coli and P. vulgaris than the S. aureus bacterial strains. The gram-negative bacteria are more sensitive when compared to gram-positive bacteria. Earlier studies also indicate that gram-negative bacteria are less sensitive than gram-positive bacteria. A strong bactericidal activity was observed against E. coli and P. vulgaris.

Conclusion
The novel facile Surfactant TWEEN80 has been used to synthesis Fe 3 O 4 for the first time with fuel urea. The XRD result and TEM results confirmed Fe 3 O 4 has the crystallite size 35 nm. The differential thermal analysis/thermogravimetric analysis showed the weight due to vapour and carbon. The dislocation density has decreased with the increase in the crystallite size. Similarly the micro strain has increased with the decrease in the crystallite size. The Fe 3 O 4 nanoparticles showed their antibacterial properties on both gram positive and gram negative bacterial strains. As the diameter of the zone of inhibition is high, we can conclude that Fe 3 O 4 is a very effective antibacterial agent.
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