Excellent Anti-bacterial Activity of Poly(o-toluidine)-DBSA/ ZnO Nanocomposite

This work presents a study of the biological application (antibacterial activity) of ZnO/poly(o-toluidine) (POT) doped with organic acids dodecylbenzene sulfonate acid (DBSA) nanocomposites synthesized by in-situ polymerization of (o-toluidine) monomer in presence of 5% ZnO. The FTIR spectroscopy confirms the existence of an interaction between POT-DBSA matrix and ZnO particles. Scanning electron microscopy reveals the nanostructure nature of the obtained composite. The antibacterial activity of POT-DBSA/ZnO nanocomposite and POT-DBSA studied by agar well diffusion method, was found to increase with increasing concentration meanwhile POT/DBSA/ZnO exhibits better antibacterial activity compared to POT/DBSA and POT separately.

2.2 Synthesis of POT-DBSA/ ZnO nanocomposite POT-DBSA/ZnO nanocomposite was prepared by in situ oxidative polymerization of o-toluidine in the presence of ZnO (Sigma Aldrich, USA) nanoparticles (NPs). 5wt% of ZnO NPs were poured into solution of o-toluidine-DBSA and polymerization was affected by the addition of solution containing oxidant ammonium persulphate prepared in 1 M HCl. The addition of oxidant solution led to the polymerization of adsorbed o-toluidine on ZnO NPs resulting in the appearance of light greenish black precipitate colored solution which was kept under continuous stirring for 24 h. The reaction mixture was then filtered, washed with double distilled water to remove excess acid. The nanocomposite thus prepared was dried at 70oC for 6 h in an oven, converted into powders and stored in desiccator for further investigations.
2.3 Antibacterial activity The Muller Hinton agar was used for the growth of Escherichia coli and Pseudomonas aerogmosa (Gram negative) and staphylococcus aurous (Gram positive) bacteria to study the antibacterial activity of POT-DBSA/ ZnO nanocomposite and POT-DBSA, by using the diffusion in the agar method. The bacterial strain was cultured on the nutrient agar broth to produce a new colony of age 24 h, then to obtain a bacterial growth one new colony from the growth was inoculated on a 5 ml of nutrient broth and was incubated in 37C for 6 h. A 0.1 ml from the growth was separated on the Muller-Hinton agar. The inoculated agar surface was poured in 4 mm diameter for every pore. The pores of cultured media were filled by 100, 200, 300, 400 and 500 µg/ml of POT-DBSA/ZnO nanocomposite and POT-DBSA, the controlled solution was 10 ml of Dimethyl-sulpha oxide. Then the dishes were kept in the incubator at 37°C for a period of 18-24 h. The inhibition zone diameters in mm were measured around the pores.

Characterizations
The morphology of nano composites films was investigated by field emission scanning electron microscopy (FESEM) using FEINova Nano SEM 450. Fourier transform infrared (FT-IR) spectra were recorded on FT-IR 4200 Jasco.

FTIR spectroscopy analysis
The FT-IR spectrum of the POT-DBSA/ (5 wt% ZnO) nanocomposite is shown in Fig 1. The characteristic bands of POT-DBSA are as follows: the peak at 1604 cm−1 is ascribed to the stretching vibration of quinoid rings; the peak at 1406 cm−1 depicts the presence of benzenoid ring units (Kulkarni, Viswanath et al. 2004, Ebrahim, Gad et al. 2010; the peak at 1170 cm−1 is assigned to a vibration band of the dopant ion, which is formed during protonation of POT with DBSA (Rao, Sathyanarayana et al. 2002, Kulkarni, Viswanath et al. 2004; the peak at 3121 cm−1 is attributed to the free N-H stretching vibration (Kulkarni, Viswanath et al. 2004), the peak at 1004 cm-1 corresponds to the C-H in plan bending vibration (Kulkarni, Viswanath et al. 2004); while the peaks at 682 and 582 cm-1 are associated to C-H out of plane bending vibration (Kulkarni, Viswanath et al. 2004) and C-N-C bonding mode of aromatic ring, respectively. The characteristic peak at 436 cm−1 belongs to the Zn-O stretching mode (Hamedani and Farzaneh 2006, Kakazey, Vlasova et al. 2007, Geetha and Thilagavathi 2010, Zhao, Cai et al. 2011, Nosrati, Olad et al. 2012. The frequency data obtained, and their assignments are presented in Table 1.

Determination the inhibition zone diameter
The inhibition zone diameters against Staphylococcus aurous, Escherichia coli and Pseudomonas aerogmosa bacteriaby POT-DBSA/ZnO nanocomposite and POT-DBSA which were compared with POT taken separately and with standard drug. There are four types of antibacterial activities (R. Kavitha 2011): (i) the inhibition zone diameter >>12 mm resulting in high sensitive bacteria; (ii) moderate sensitive bacteria for the inhibition zone diameter in the range 9-12 mm; (iii) if the inhibition zone diameter is in the range 6-9 mm, then the sensitivity of bacteria is less; and (iv) bacteria becomes resistant if then inhibition zone diameter is < 6 mm. From Table 3 and photographic pictures shown in Figure 4, it can be indicated that the inhibition zone diameter is highest for POT-DBSA/ZnO nanocomposite and POT-DBSA; and that the inhibition zone diameter increases as the concentration of the nanocomposite increases.
From Table 3 the following points can be highlighted: 1. POT-DBSA/ZnO nanocomposite exhibits a better antibacterial activity than POT-DBSA, POT and Oxytetracyclin drug. 2. The inhibition zone diameter against Pseudomonas aerogmosaand Escherichia coli (gram negative) is relatively higher than against Staphylococcus aureus (gram negative) due to the difference in the cell wall between gram positive and gram-negative bacteria. The increase in the inhibition zone diameter may be due to the presence of ZnO NPs in POT-DBSA/ZnO nanocomposite because the antibacterial activity has been reported to be size dependent (Boomi, Prabu et al. 2014), then the bacterial death due to electromagnetic Advances in Physics Theories and Applications www.iiste.org ISSN 2224-719X (Paper) ISSN 2225-0638 (Online) Vol.81, 2019 20 attraction between the bacteria (negative charge) and ZnO nanocomposite (positive charge) (A. Thomas1 2014). In addition, the effect of DBSA in POT-DBSA may destruct bacterial enzymes by coordinating to the electron donating groups, thus DBSA causes deformities in bacterial cell wall to render them leaky (Shakir, Khan et al. 2014).

Conclusion
POT-DBSA/5% ZnO were successfully prepared using in-situ polymerization of (o-toluidine) monomer in the presence of 5% weight ratio of ZnO NPs. Characteristic bands of ZnO and POT-DBSA were identified by FTIR and confirming the interaction between ZnO and polymer matrix. FESEM observations reveal rock salt structure embedded within the polymeric structure, signifying that ZnO NPs are fully covered by POT, with an average diameter of POT-DBSA/ZnO grains around 37 nm. The comparative study of biological application (antibacterial activities) of POT, POT-DBSA, POT-DBSA/ZnO nanocomposite and oxytetraacyclin drug have been investigated. The results confirm that POT-DBSA/ZnO nanocomposite exhibits enhanced antibacterial activity against E.coli and Pseud. aerogmosa Gram negative and Staph. aureus Gram positive bacteria, as well that the inhibition growth of bacteria increases when POT-DBSA are coordinated with ZnO NPs. The antibacterial properties could make POT-DBSA/ZnO nanocomposite potentially useful as novel drug, thereby future in-vivo measurements are underway.