Laser-Assisted Synthesis of Mn 0 . 50 Zn 0 . 50 Fe 2 O 4 Nanomaterial : Characterization and In Vitro Inhibition Activity towards Bacillus subtilis Biofilm

1Department of Physics, University of Agriculture, Faisalabad 38040, Pakistan 2Department of Chemistry, University of Sargodha, Sargodha 40100, Pakistan 3Department of Pharmaceutical Chemistry, College of Pharmacy, Prince Sattam bin Abdulaziz University, P.O. Box 173, Al-Kharj 11942, Saudi Arabia 4Department of Biochemistry, University of Agriculture, Faisalabad 38040, Pakistan 5Department of Applied Chemistry & Biochemistry, Government College University, Faisalabad 38000, Pakistan 6Deanship of Scientific Research College of Engineering, King Saud University, P.O. Box 800, Riyadh, Saudi Arabia


Introduction
Preparation of nanosized fine spinal structure is an emerging research area in the field of nanotechnology.Presently, the applications of such nanomaterials are being explored to address the problems associated with water treatment, and so forth [1], catalysis [2], and microbial control [3,4].Nanosize metal particles can provide considerably large surface area as compared to bulk material.Theoretically, for spherical particles of identical size, a decrease in the particle size from 10 m to 10 nm can escalate the contact surface area by 10 9 [5,6].
Biofilms are aggregates of microorganisms formed by the attachment of microbial cells in polymeric substances [7].
Mostly, biofilms are highly resistant to antibiotics and are therefore being extensively studied in quite a lot of scientific disciplines, including evolutionary biology, biomedicine, and water treatment [8][9][10][11].The growth of a microbial biofilm initiates with the attachment of free-floating microorganisms to a surface.Subsequently, these adherent microbes are entrenched repeatedly within a self-produced medium of extracellular polymeric substances (EPS) usually composed of extracellular DNA, proteins, and polysaccharides.These bacterial biofilms can cause several infectious diseases and develop resistance manifold [11][12][13].
In the recent past, use of metal-based nanoparticles for curing bacterial infections has got great scientific interest [14].

2
Journal of Nanomaterials Some in vitro studies revealed that the metal nanoparticles exhibited potential antibiofilm activity as tested on biofilms formed by Pseudomonas aeruginosa and Staphylococcus epidermidis [14,15].The antibacterial efficiency of metal nanoparticles is not only due to metal-ion release but mostly based on their high surface-to-volume ratio [15].
Although coprecipitation is considered convenient method to prepare nanomaterials, it is rather difficult to prepare fine nanostructure with larger surface area using this approach.Therefore, we were motivated to prepare Mn 0.5 Zn 0.5 Fe 2 O 4 by novel coprecipitation cum laser ablation technique with the expectation of improved performance for inhibition of Bacillus subtilis biofilm.Furthermore, to the best of author's knowledge, Mn 0.5 Zn 0.5 Fe 2 O 4 has never been investigated by microtiter-plate assay for inhibition of Bacillus subtilis biofilm.To bridge the research gap in biofilm inhibition using novel Mn 0.5 Zn 0.5 Fe 2 O 4 nanomaterial is fascinating task; hence, we report synthesis of Mn 0.5 Zn 0.5 Fe 2 O 4 nanoparticles using coprecipitation approach followed by laser irradiation technique to assess their potential application in Bacillus subtilis biofilm inhibition by microtiterplate assay [16].O in 50 mL sterile deionized water.These stoichiometric quantities led to the formation of atomic ratios 1 : 1 : 4 for Mn : Zn : Fe in Mn 0.5 Zn 0.5 Fe 2 O 4 .These two solutions were homogenized by stirring at 65 ∘ C for 30 min using hot plate magnetic stirrer at moderate speed (55 rpm).For precipitation of chloride precursors, the solution pH was increased to 10 by adding dropwise 3.5 M NaOH with continuous stirring (it took about 120 min) until reddish brown precipitates formed.To get fine spinal structure of nanomaterial, thermal decomposition of Mn, Zn, and Fe chlorides was performed by irradiation of solution for 5 min with a 532 nm green laser beam (20 mW power) produced with Continuous Wave (CW) Diode Pumped Solid State Laser (DPSSL) Model CDPS532M-020 (JDS Uniphase Corp, USA).The synthesized material was then cooled to room temperature (30 ∘ C) and digested for 120 minutes to settle down the particles at the bottom of the beaker.The precipitate of Mn 0.5 Zn 0.5 Fe 2 O 4 was filtered using Grade 287 filter paper (GE Healthcare Life Sciences) and then washed using deionized water till chloride became free and the pH of the residual solution reached 7.0.It took 6 to 7 hours to make the residual neutral.The recovered precipitates were dried for 90 min at 100 ∘ C in an oven and then ground with pestle and mortar into a fine powder followed by calcination at 700 ∘ C for 4 hours in a muffle furnace (Vulcan, A550).

2.3.
Characterization of Mn 0.5 Zn 0.5 Fe 2 O 4 Nanomaterial.The structural analysis of ultrafine homogeneous powder of Mn 0.5 Zn 0.5 Fe 2 O 4 nanomaterial was performed using X-ray diffractometer (D8 FOCUS 2220 Bruker AXS) with Cu K radiation ( = 1.5418Å).The X-ray diffraction (XRD) pattern was recorded at a scanning rate of 0.02 ∘ /s in 2 ranging from 10 ∘ to 80 ∘ .The gold coated specimen was utilized to appraise the morphologies of the nanoparticles by Scanning Electron Microscope (SEM, JEOL JSM 7401F JEOL Ltd., Akishima, Japan) operated at an accelerating voltage of 15 kV.The elemental analysis was performed by using energy dispersive X-ray microanalysis (EDX, Inca-FET-3, Oxford Instruments, UK Ltd.).The BET specific surface area was investigated by N 2 adsorption (77 K) using a Micromeritics ASAP 2000 system during the overnight treatment (130 ∘ C) under vacuum thereby degassing the sample.On average, BET specific surface area was found to be 78.3 m 2 g −1 .

Assessment of Biofilm Inhibition.
The inhibition of bacterial (Bacillus subtilis) biofilm formation was assessed by the microtiter-plate method as described by Stepanović et al. [16].The wells of a sterile 24-well flat bottomed plastic tissue culture plate were filled with 100 L of nutrient broth (Oxoid, UK).Two concentrations, that is, 2.5 and 5.0 g of testing samples (dissolved in 1 mL of DMSO), were added in different wells.Finally, 20 L of bacterial suspension containing 1 × 10 9 CFU/mL was inoculated.Positive control well contained Rifampicin and nutrient broth (Oxoid, UK) while negative control well contained nutrient broth and microbial strain.Afterwards, plates were covered and then incubated aerobically for 24 hours at 37 ∘ C. Thereafter, the contents of each well were behed thrice with 220 L of sterile phosphate buffer (pH: 7.2).To remove all nonadherent bacteria, plates were vigorously shaken.Then, attached leftover bacteria were fixed with 220 mL of 99% methanol per well.Next, after 15 min, plates were emptied and left to dry.Then, plates were stained for 5 min with 220 mL of 50% crystal violet per well.Surplus stain was rinsed off using distilled water.Then plates were air-dried and the bound dye was resolubilized with 220 L of 33% (v/v) glacial acetic acid per well.The optical density (OD) of each well was measured at 630 nm using microplate reader (Biotek, USA).All the tests were carried thrice against selected bacterial strain and the results were averaged.The bacterial growth inhibition (INH%) was calculated using the following formula:  3  632 Bulk density (gcm −3 ) ∼4.00 X-ray density (gcm −3 ) 5.07 Pore volume (cm 3 g −1 ) 0.27 ± 0.02 Crystallite size (nm) ∼12 diffraction peaks and single cubic phase with fcc structure of Mn 0.5 Zn 0.5 Fe 2 O 4 without any impurity phase (Figure 1), revealing the highly crystalline character of the sample indexed with JCPDS card number 74-2401.The crystallite size determined by Debye-Scherrer's formula was found to be ∼12 nm and the calculated lattice parameters  for Mn 0.5 Zn 0.5 Fe 2 O 4 sample were found to be 0.869 nm (Table 1).

Results and Discussion
It is worth mentioning here that laser irradiation brings confined thermal modifications in the light absorbing structures; nevertheless it evades thermal destruction to the nearby structure due to its directionality.Besides, diode laser output is narrowband; hence most of it is absorbed and exploited during irradiation of the material producing the fine crystal structure.
The average particle size ∼12 nm was estimated from ten particles at ×50,000 magnification from the SEM micrographs (Figure 2).Besides, it is evident from the SEM micrograph that MnZnFe 2 O 4 sample has uniform and spherical structural morphology with a narrow size distribution of the particles.
For quantitative analysis, the as synthesized ultrafine homogeneous nanomaterial sample (in bulk, flat, and polished form) was exposed to electron beam of accelerating voltage 15 kV in electron microscope under high vacuum.The EDXA spectrum (Figure 3) of the Mn 0.5 Zn 0.5 Fe 2 O 4 nanomaterial shows that it contains only Fe, Mn, Zn, and O with no traces of byproducts.Table 2 lists the relative abundance of each of the elements which is analogous to the theoretical stoichiometric values.No contamination is detected due to high purity of the starting materials used for the synthesis of the Mn 0.5 Zn 0.5 Fe 2 O 4 nanoparticles.4 depicted that 5 g of Mn 0.5 Zn 0.5 Fe 2 O 4 nanoparticles dissolved in 1 mL of DMSO showed excellent biofilm inhibitory activity 91.23% ± 1.87 against Bacillus subtilis (Table 3).This antibacterial potential of the tested nanomaterial can be attributed to the fact that the Mn 0.5 Zn 0.5 Fe 2 O 4 nanoparticles have greater surface area and unique crystal form having more sharp edges and site to enhance their activity by joining up with bacterial membrane to disrupt the membrane [14].
There may be different possible mechanisms of action for the observed antibacterial activity of Mn 0.5 Zn 0.5 Fe 2 O 4 nanoparticles.For example, the tested nanoparticles stick to the bacterial cell wall and breach the cell membrane [17] and cause degradation and lysis of the cytoplasm, resulting in cell death.As nanoparticles possess large surface area, their bactericidal efficacy is enhanced contrary to the bulky particles with lesser surface area.Furthermore, the Mn 0.5 Zn 0.5 Fe 2 O 4 nanoparticles keep a large surface-to-volume ratio, which leads to an increase in their bioactivity making them active and potent bactericidal agents [15,18].Previous studies on zinc nanoparticle structures revealed that zinc fixes to the membranes of microbes, analogous to mammalian cells by delaying their growth, thereby, prolonging the cell division time of the organism.Similarly, according to some other reports, the main chemical species linked to the antibacterial activity of such nanomaterials were supposed to be active oxides; for example, hydrogen peroxide (H 2 O 2 ) formed from the surface of the ceramic zinc [19,20].Hydroxyl radicals are very strong oxidants and when nanoparticles directly stick to the microbes, their surface undergoes crucial oxidative attack.The active oxides eagerly penetrate through the bacterial cell wall and cause cell destruction and thus inhibit the bacterial growth [20,21].).The holes and OH • both are extremely reactive towards organic complexes [22,23] and thus might have caused disruption of the cell membrane/cell wall of Bacillus subtilis and leakage of intracellular K + ions leading to cell death (Figures 4 and  5).Previously, outer cell membrane damage was noted by Sunada et al. [24] in case of E. coli wherein they found that the endotoxin, a vital part of the outer cell membrane, was smashed while photocatalysis was performed with TiO 2 .
Polyunsaturated phospholipids are vital constituents of the bacterial cell membrane and high vulnerability of these organic compounds to be attacked by reactive oxygen species (ROS) is an established fact [25,26].Many functions, such as semipermeability, oxidative phosphorylation reactions, and respiration, are influenced by the cell membrane composition and structure.Therefore, lipid peroxidation is detrimental to all forms of life.Besides, the usual functionalities linked with an intact membrane, for example, respiratory activity, are vanished by lipid peroxidation reaction.Hence, any damage to bacterial cell membrane structure or modification

Conclusions
The bacterial biofilms can cause several infectious diseases and develop resistance against antibiotics.In the present study, for the first time, the antibacterial properties of Mn 0.5 Zn 0.5 Fe 2 O 4 nanoparticles were established in an in vitro trial against biofilm formed by Bacillus subtilis using microtiter-plate assay.Mn 0.5 Zn 0.5 Fe 2 O 4 nanoparticles showed excellent inhibitory effect against the highly multidrug-resistant Gram-positive bacterial strain Bacillus subtilis.The results indicate that the Mn 0.5 Zn 0.5 Fe 2 O 4 nanocomposite is a promising disinfection material that can also be used in surface coatings to effectively inhibit bacterial growth and proliferation.

Figure 4 :
Figure 4: Phase contrast microscopic view of inhibition of Bacillus subtilis biofilm by Mn 0.5 Zn 0.5 Fe 2 O 4 nanoparticles at 100x ((a) controlled (native) Bacillus subtilis biofilm; (b) Rifampicin treated control; (c) biofilm treated by 5.0 g of Mn 0.5 Zn 0.5 Fe 2 O 4 nanoparticles dissolved in 1 mL of DMSO; (d) biofilm treated by 2.5 g of Mn 0.5 Zn 0.5 Fe 2 O 4 nanoparticles dissolved in 1 mL of DMSO).
Typically, in the case of Mn 0.5 Zn 0.5 Fe 2 O 4 nanoparticles, upon excitation by light, the photon energy produces a pair of electron and hole on the Mn 0.5 Zn 0.5 Fe 2 O 4 surface.The hole (in the valence band) might have reacted with H 2 O or hydroxide (OH) ions adsorbed on the surface producing hydroxyl radicals (OH • ) while the electron (in the conduction band) reduces O 2 to generate superoxide ions (O 2 −