INTRODUCTION

Nanotechnology is one of the most important achievements of the latest century which plays a key role in bond physics, chemistry, biology, and metallurgy together[1]. When components are brought to nanoscale, the way they operate changes and a lot of their physical properties changes[2]. The metal oxide structures that have an extensive bandgap are of importance as semi-conductors in chemistry and physics [3] and because of the important properties of metal oxides in nanoscale such as optical, electrical, magnetic, and structural properties, they have different applications in sensors, catalyzers, and photocatalysts, micro-electronics. nonlinear optics, photoelectrochemistry, imaging science, and electro-optical science[4-6]. Tin(IV)oxide (SnO₂) is one of the important nanostructures that has been given a lot of attention due to its transparency in the visible wavelength spectrum and other properties such as high carrier density, high-temperature tolerance, chemical stability, and low resistivity[4, 7]. SnO₂ is an n-type semiconductor with a bandgap of 3.6 eV[8]. 

In a study conducted by Boucherka Teldja et al., the effects of the combination of indium with tin oxide on the structure, morphology, optical and electrical properties of the combination were analyzed via the sol gel method. They have reported that the combination of indium with tin oxide improves the optical transmission and decreases the electrical conduction due to indium naturalness. In that research, thin films with high transmission and low resistance were obtained[9]. Ariya Nachiar and co-authors have reported that if the copper NPs are synthesized with tin oxide NPs using the co-precipitation method, the copper causes a change in the structural and optical properties of tin oxide NPs and reduces the bandgap of tin oxide NPs[2]. SnO₂ have many applications in gas and solid-state sensors, lithium batteries, electro-optical tools, as catalysts for the oxidation of organic components, ceramic and transparent conductors, biomedical and nanoelectronics[1, 3, 6, 10, 11], supercapacitors[12], plasma screens, touch screens[13], special coating for energy-conserving “low-emissivity” windows[14] and the electrodes of the solar cells [15]. 

There are different methods for accomplishing a chemical and physical synthesis of  SnO₂ such as co-precipitation, micro-emulation, sol-gel, and hydrothermal methods [8, 16], RF magnetron sputtering[17], spray pyrolysis[18], chemical vapor deposition [19] and deposition via laser[15]. Also, the strong light emission of tin oxide and other metal semi-conductors have important applications in remote communication and driving signs[20].

The manufacture of NPs in different shapes and sizes is more biologically and chemically active due to the fact that the number of surface atoms increases to volumetric atoms and causes antimicrobial agents. NPs can invade and disrupt bacteria in a variety of ways. NPs bind to the bacterial membrane by electrostatic reaction or by reacting with amines and carboxyl groups of the peptidoglycan layer of the bacterial wall, disrupting it and destroying the cell walls, completely destroying the bacterium [21-23]. Studies have been performed on the antibacterial properties of tin oxide NPs. According to the results of this work, tin oxide NPs synthesized by chemical methods show good antibacterial properties[24]. Kamaraj et al. synthesized tin (IV) oxide nanoparticles in a green method using the methanolic extract of Cleistanthus Collinus plant and investigated their biological activities. They investigated the antibacterial activity of the synthesized nanoparticles against Escherichia coli and Staphylococcus aureus bacteria. Antifungal activity of nanoparticles was also investigated. In this research, the average size of the synthesized nanoparticles was reported to be 49.26 nm, the synthesized nanoparticles showed significant antibacterial activity against Escherichia coli [25]. Khan et al were also able to synthesize tin (IV) oxide nanoparticles through the green method and using the extract of Clerodendrum Inerme. They also investigated the antibacterial and anticancer activities of synthetic nanoparticles. Morphological and crystalline changes of synthesized nanoparticles were also investigated by XRD and TEM analyses [26]. Studies on the biosynthesis of nanoparticles in the extracts of different plants were very different and it was found that in different plants, the synthesized nanoparticles show different effects. Plant extracts have antioxidant properties and a lot of secondary compounds [27]. The average size of nanoparticles synthesized using Turicum puliom plant in this research is approximately 20 nm, which has a greater effect on bacteria compared to the previous studies.

The chemical methods for synthesizing the NPs are expensive and dangerous and the NPs produced with these procedures are highly toxic and hazardous to the environment. The green synthesizes of the NPs, using plants, is cheaper and non-toxic, these methods are ecofriendly and the components used in the synthesis procedure are easily found in the nature[10, 28]. Medicinal plants also have a biological state, so they do not accumulate in the body and do not cause side effects and therefore have a significant advantage over chemical drugs. Teucrium polium plant is known for its medicinal uses and has properties such as liver protector, antioxidant, anti-inflammatory, anti-tumor, and antibacterial and contains important compounds including diterpenoids, flavonoids, iridoids, sterols, and terpenoids[29, 30]. The prevalence of drug-resistant microorganisms is a serious problem. Also, infections caused by these bacteria are leading causes of morbidity and mortality. Therefore, new research is needed for the development of antimicrobial compounds. One of the important antibacterial compounds is NPs. Herein, the Teucrium polium plant has been used for the first time to synthesize the tin oxide NPs using the co-precipitation method and the optical properties have been compared to the NPs produced with the chemical synthesis method. The procedure for producing the NPs with the green method was cheaper and was eco-friendlier than the one synthesized with the chemical method.

MATERIALS AND METHODS

The morphology and the size of NPs were identified using a Philips Scanning electron microscopy (SEM) of TESCAN-SEM Mira3-XMU. Fourier transform infrared spectroscopy (FTIR) model MagnaIR550 was employed to study the functional groups. The optical properties of the materials were investigated using the PerkinElmer LS55 photoluminescence spectrometer.

Preparation method

Teucrium polium plant extract was used as a surfactant for the green synthesis of tin oxide NPs. First, 1ml of the extract was added to 30ml of twice distilled water. Then, two drops of tin chloride was dissolved in 50ml of twice distilled water. After 30 minutes, the solution containing the extract of Teucrium polium was added to the tin solution and brown-colored sediment was obtained. The precipitate was removed using centrifugation and washed with water and ethanol. The precipitate was dried in an oven for 24 hours. The dried precipitate was placed in a furnace for a period of time to remove the excess components. For the chemical synthesis of NPs, two-aqueous tin chloride, ammonia and CTAB were used.

Determination of the minimum inhibitory concentration (MIC)

In this study, the antibacterial activity of the SnO₂ was evaluated against Staphylococcus aureus ATCC43300 and Psoudomonas aeroginosa PAO1.

Minimum inhibitory concentration (MIC) determine by the broth microdilution method, according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. First, 100 μL of Mueller Hinton broths (MHB) loaded onto sterile 96-well plates.  Afterward, dilution series of SnO₂ was prepared and added to the wells. Finally, 10 μL of bacterial suspension (5×10⁵ CFU) was added to each well and then incubated at 37 °C for 24 h. The lowest concentration of NPs that inhibited the growth of bacteria was recorded as the minimum inhibitory concentration. 

Antibiofilm Activity of SnO₂ against MRSA and PAO1 Biofilm

Microtiter plate (MTP) assay is a quantitative method to determine the Antibiofilm Activity of SnO₂. Bacterial suspension is 10-fold (1/10) diluted to reach 5.106 CFU/ml and 100 μl of bacterial suspensions are inoculated into 96-well flat-bottomed sterile polystyrene microplate in the absence and presence of, 1/8 MIC 1/4MIC, 1/2 MIC, and MIC concentrations of SnO₂. Wells involving biofilms were washed with phosphate-buffered saline (PBS). Then wells were fixed with methanol for 20 minutes. Subsequently, Biofilm mass was stained with 200 μL of 0.1% crystal violet for 15 minutes. Following the air-drying process of wells of a microplate, the dye of biofilms that lined the walls of the microplate is resolubilized by 200 μL glacial acetic acid. Then microplate is measured spectrophotometrically at 570 nm by a microplate reader.

RESULT AND DISCUSSION

X-ray diffraction pattern (XRD)

XRD analysis was performed to determine the crystal structure and phase composition or phase purity of the sample. Fig. 1 shows the SnO₂ diffraction pattern at angles of 2θ=10-90 degrees which corresponds to the standard card number 1250-021-00 [31]. The observed peaks with the page numbers in 2θ=(002), (321), (202 (301), (310), (220), (211), (200), (101), (110) and confirms the diffraction pattern of nanoparticle synthesis which lattice structure is tetragonal [32]. The size of the crystals can be estimated from a maximum peak of the XRD spectrum.

The Scherer relation fairly shows the dependence of particle size on the amplitude and diffusion of diffraction lines:

In this respect, D is the approximate size of the crystallites, is the wavelength of the X-ray beam, and β is the full width at half maximum (FWHM) of the main peaks for the hkl plates and is the angle. Using the above relation, the size of tin oxide NPs for green and chemical synthesis was 10 nm and 17 nm, respectively. X-ray diffraction pattern analysis is the same for both green and chemical synthesis, except that the particle size in green synthesis is smaller, which can be detected by the enlargement of the peak.

X-ray energy diffraction spectroscopy (EDS) is a method that uses X-ray energy to analyze and determine the chemical composition of samples on a small scale [33]. Using the EDS spectrum, the phase purity of the NPs is obtained. The initial composition of the sample is shown in Fig. 2. According to this diagram, only SnO₂ existed and no additional impurities were observed.

Scanning electron microscope (SEM)

The SEM image of SnO₂ samples synthesized by co-precipitation is shown in Fig. 3. This image shows that the NPs are uniform and have spherical shape. Also, the size of NPs is in accordance with the size obtained from the Scherer formula. The size of the NPs obtained by the green synthesis (A) and chemical synthesis (B), obtained in the range of 15-20 nm and 20-30 nm, respectively according to the inset figures. 

Photoluminescence Spectroscopy (PL)

One of the well-known types of luminescence is PL, in which excitation is done by photons. The process of excitation of electrons to a higher energy level and then their transition to a lower energy level is accompanied by the absorption and emission of photons.  In the PL process, the sample is excited by a laser or a lamp and the spectrum is obtained by recording the emission as a function of wavelength. The study of PL is one of the important methods in studying the optical properties related to the adsorption and dispersion of nanomaterials, determining the optical quality, electronic structure, and photochemical properties of semiconductor materials and investigating the structure and properties of sites active on metal oxides and zeolites [34, 35]. In addition, PL can be used to study Crystal defects[36] and in the field of photocatalytic semiconductors, it is useful for understanding surface processes[37]. In nanostructured metal oxides, PL emission is divided into two main parts, including emission in the ultraviolet (UV) region and emission in the visible region. Emission in the UV region is associated with direct decay (band-band PL). Also, the emission in the visible region is the result of the radiative recombination of a hole with electrons in the oxygen and metal voids (excitonic PL) [38]. In semiconductor NPs, the sign of excitonic PL occurs, but the band-band PL signal is rarely observed [37] High emission intensity in the visible area is related to high concentrations of impurities and crystalline defects[39]. Defects such as oxygen voids are one of the most important and common defects in nanocrystalline oxides and act as luminous centers in the luminescence process[40]. The PL emission spectrum of tin oxide NPs in Fig. 4 was accomplished with the two methods mentioned, under excitation at the wavelength of 280nm, at room temperature, indicating that the maximum PL peak was about 398 nm for both syntheses.

Fourier Transform Infrared Spectroscopy

Infrared spectroscopy is a well-known technique in the qualitative identification of materials and is a method in which the absorption of radiation and vibrational mutations of molecules and ions of polyatoms are examined. This method is used as a powerful and developed technique for measuring chemical species and is mainly used to identify organic compounds because the spectra of these compounds are usually complex and have a large number of maximum and minimum peaks that can be used for comparison. In both spectra of Fig. 5, the broad absorption band is at 3430 cm^-1, which is related to the tensile vibrations of the O-H bond caused by the water adsorbed on the SnO₂ surface[41]. The adsorption band at 11630 cm^-1 corresponds to C = C groups or aromatic or tensile rings in the C = O carboxyl group [42]. The absorption bands shown in the 1200-400 cm-1 range are known as fingerprint bands, which are different for each material. The tensile bonds of Sn-O-Sn, O-Sn-O, and Sn-O are located in this fingerprint region. The presence of these functional groups can be considered as light centers at the nanoparticle levels.

Investigation of antibacterial properties of NPs

Determination of the Minimum Inhibitory Concentration (MIC) and MBC

According to the data shown in Table 2, among the tested bacteria maximum antibacterial activity were demonstrated against Pseudomonas aeruginosa PAO1.

Staphylococcus aureus is one of the most common and important gram-positive bacteria in the hospital, which can range from skin infections to life-threatening diseases and is increasing nowadays [43, 44]. Pseudomonas aeruginosa is a gram-negative bacterium that is scattered all over the world and is one of the most important bacteria in hospital[45].

In an experiment of interaction between NPs and two bacteria, Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC 43300, with an average MIC and MBC of milligrams per milliliter, the NPs turned out to be capable of being used as drugs to remove bacterial strains, as shown in Tables 1 and 2.

Antibiofilm Activity of SnO₂ against MRSA and PAO1 Biofilm

SnO₂ NPs showed obvious inhibition of attachment and biofilm formation (Fig. 6). According to the result, the SnO₂ nanoparticle in concentration MIC of SnO₂ nanoparticle inhibited the biofilm formation by MRSA and PAO1 with an inhibition rate of 60% and 61% respectively. The effect of different concentrations of NPs on the reduction of biofilm formation in the standard strain of Staphylococcus aureus ATCC 43300 and the standard strain of Pseudomonas aeruginosa PAO1, each microplate used for two bacteria, and the nanoparticle for each stock bacterium was prepared with MIC concentration. The fractional concentration of NPs in sumps is 1/8 MIC, 1/4 MIC, and 1/2 MIC and zero. They were studied in the same way as shown in Fig. 6, with an increase in the percentage of formation error, Biofilm is reduced for two bacteria.

However, different studies may suggest that MIC may vary due to the size of the NPs and the preparation methods, as well as differences in the strains of the studied bacteria and that the effect of NPs on bacteria depends not only on cell wall structure but also on fat peroxidation and producing the active species of oxygen[46, 47].

CONCLUSION

Tin oxide NPs were synthesized by the co-precipitation method, using plants and chemicals as surfactants. The optical and antibacterial properties of SnO₂ NPs were investigated using SEM, PL, EDX, XRD, and FTIR methods. The results of both methods are the same. Due to the fact that the use of chemical methods causes great harm to human health and the environment, plant-based materials and green synthesis are essential for the production of NPs that are non-toxic and less hazardous for the environment. The antimicrobial results of tin oxide NPs in inhibiting the formation of bacterial biofilms can be used as a suitable solution in the use of NPs for microbial purification and environments that are more exposed to this bacterium.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interests regarding the publication of this manuscript.