Introduction

Nano materials are widely synthesized for their properties like optical, mechanical and magnetic properties to counter the bulk materials [1–3]. Metal oxides are used in various applications like magnetic storage, catalysis and biological applications like bone tissue engineering [4–7]. The prolonged life expectation and aging of population has brought the escalating request of artificial material to regenerate diseased bones [8–11]. 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 (Fe3O4) 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) [22–26], 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 Fe3O4 nanoparticles with the surfactant; and it is also an attempt to study the antibacterial properties of Fe3O4 nanoparticles.

Materials and methods

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, gram-positive 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 (Fe3O4) 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 (Fe3O4). 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 μl 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 µg/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 Kα radiation (λ = 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.

Fig. 1
figure 1

XRD patterns of Fe3O4

Full size image
$$ D = \frac{0.9\lambda }{\beta \cos \theta } $$
(1)

Where λ is the X-ray wavelength (1.54 Å) for copper Kα. θ is the Bragg’s angle. β is full width half maximum value. Dislocation density (δ) is calculated with the crystalline size.

$$ \delta = \frac{1}{{D^{2} }} $$
(2)

From the calculated d spacing value, the lattice constants are calculated as follows: with the below formulae.

$$ d^{2} = \frac{{a^{2} }}{{h^{2} }} + \frac{{b^{2} }}{{k^{2} }} + \frac{{c^{2} }}{{l^{2} }} $$
(3)

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.

$$ \varepsilon = \frac{(\beta \cos \theta )}{4} $$
(4)

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.

Table 1 The values of standard ‘d’, observed ‘d’, absolute ‘a’ difference, percentage of lattice contraction, crystalline size, dislocation density, strain and h, k, l of Fe3O4
Full size table

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 was weight loss due to the loss of moisture in the sample. 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.

Fig. 2
figure 2

TG/DTA of Fe3O4

Full size image

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.

Fig. 3
figure 3

Particle analyzer of Fe3O4

Full size image

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.

Fig. 4
figure 4

SEM image of Fe3O4

Full size image

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.

Fig. 5
figure 5

TEM image of Fe3O4

Full size image

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).

Fig. 6
figure 6

EDAX of Fe3O4

Full size image
Fig. 7
figure 7

Antimicrobial activity of Fe3O4 at control level

Full size image

Antibacterial activity

Fe3O4 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 Fe3O4 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 Fe3O4 nanoparticles and the outer bilayer of bacteria could be the reason for the antibacterial activity of Fe3O4. The hydrogen peroxide produced enters the cell membrane of bacteria and kills them. 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 hydrogen peroxide to the medium [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.

Fig. 8
figure 8

Antimicrobial activity of different extracts with Fe3O4 at low concentration

Full size image
Fig. 9
figure 9

Antimicrobial activity of different extracts with Fe3O4 at high concentration

Full size image
Fig. 10
figure 10

Activation index against various microorganisms

Full size image

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 (\( {\text{O}}_{2}^{ - } \)), hydroxyl radicals (–OH) and hydrogen peroxide (H2O2); and singlet oxygen (1O2) 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 (H2O2) was generated when Fe2+ responded with oxygen. The ferrous irons reacted with the produced H2O2 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 Fe3O4 nanoparticles on 4 bacterial strains is summarized in Fig. 10. The Fe3O4 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 Fe3O4 for the first time with fuel urea. The XRD result and TEM results confirmed Fe3O4 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 Fe3O4 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 Fe3O4 is a very effective antibacterial agent.