Silver nanoparticles (AgNPs) belong to a special group of materials with many applications in various fields such as dentistry, clothing, photography, catalysis, mirrors, optics, electronics, and food industry. Due to such potentials many methods of synthesis of AgNPs have been developed. Such methods should control the size of AgNPs. Efficient synthesis of small particles without bulking was favorable [1, 2]. The most important methods for the synthesis of AgNPs are follows: chemical reduction [3, 4], optical reduction , micelles , gamma irradiation , sol-gel  and biosynthetic methods [9–11]. When the size of silver particles decreases to nano-scale, their antibacterial efficacy increases because of their larger total surface area per unit volume [12, 13].
It is generally believed that heavy metals react with proteins by combining the thiol (SH) groups, leading to the inactivation of proteins . Recent, microbiological and chemical experiments implied that interaction of silver ions with thiol groups played an essential role in bacterial inactivation . However, the antimicrobial effects of silver nanoparticles (Ag-NPs) were not fully investigated.
The aim of this work is synthesis, characte-rization, and study the antibacterial activities of silver(I) complex [Ag(3-bpdh)(NO3)]n in nano-scale as well as bulk. The antibacterial activities of the Schiff base ligand and silver(І) oxide nanoparticles are also investigated against two Gram-positive and two Gram-negative bacteria.
MATERIALS AND METHOD
Materials and physical techniques
All reagents for the synthesis and analysis were commercially available and used as received. An ultrasonic bath (type; DT510H, 50–60 HZ 230 W) was used for the ultrasonic irradiation. Melting points were measured on an Elemental Engineering Ltd-IA9200 apparatus. IR spectra were recorded using Brucker FT-IR Tensor 27 spectrophotometer. X-ray powder diffraction (XRD) measurements were performed using a Philips X’pert diffractometer with monochromatized Cu kα radiation. The samples were characterized with a scanning electron microscope (SEM) (Company KYKY and model EM3200) with gold coating.
Synthesis of the Schiff base ligand
1.53 mL methyl 3-pyridyl ketone was dissolved in ethanol (25mL), followed by dropwise addition of 1.53 mL hydrazine monohydrate solution in ethanol (25 mL). After addition of two drops of formic acid, the mixture was stirred at ambient temperature for 24 h. The solvent was removed under vacuum, and upon removal of the solvent, bright yellow crystalline solid was obtained .
IR (υ, cm-1): 702(s), 814(s), 1018(s), 1075(w), 1120(w), 1366(s), 1412(s), 1603(s), 1699(w), 2969(w) and 3446(w). 1H-NMR (DMSO, δ): 9.1(s, 2H); 8.6 (d, 2H); 8.3 (d,2H); 7.5 (d.d, 2H) ppm
Synthesis of silver(I) complex [Ag(3-bpdh)(NO3)]n
2,5-Bis(3-pyridyl)-3,4-diaza-2,4-hexadiene (L=3-bpdh) (0.12 g, 0.5 mmol) was dissolved in methanol (10 mL) and poured dropwise into prepared alcoholic solution of silver(I) nitrate (0.085 g, 0.50 mmol) and sodium perchlorate (0.061 g, 0.50 mmol). After this addition, a light-yellow precipitate was obtained.
Synthesis of silver(І) [Ag(3-bpdh)(NO3)]n(1) nanorods by sonochemical method
10 ml of an alcoholic solution containing silver(І) nitrate (0.085g, 0.50 mmol) and sodium perchlorate (0.061 g, 0.50 mmol) in a round-bottom flask was placed into ultrasonic bath, then 5 ml solution of 3-bpdh (0.12g, 0.50 mmol) poured dropwise into this solution for 1 h. The nano-structure product was filtered, and then dried.
Synthesis of silver(І)oxide by direct calcination
Ag2O were prepared by heating compound 1 in an electrical furnace at 700 °C for 2 h.
Antibacterial activity test
Assessment of antibacterial effect of the components was carried out by using well diffusion method [17, 18]. It was determined against the two Gram-positive bacteria: Staphylococcus aureus PTCC1112, Enterococcus faecalis (isolated from clinical samples, Afzalipour Hospital in Kerman, Iran) and also two Gram-negative bacteria: Escherichia coli PTCC 1330 and Pseudomonas aeruginosa ATTC 27853.
In order to test the antimicrobial activity, the samples were dissolved in HNO3 (70%). Then, after adding 0.1 molar sodium bicarbonate to the solution, the pH value increased to a neutral range of 7-8. Media Agar (20 mL) was poured into each 15 cm Petri dish. Growth was adjusted to a turbidity equivalent to a 0.5 McFarland standard. 0.01 μL of suspension containing approximately 108 bacteria/mL was placed over Agar in Petri dishes and dispersed. Then, wells were cut and 50 μl of the compound was added. The plates were then incubated at 37°C for 24-48 h. The antimicrobial activity was assayed by measuring the diameter of the inhibition zone formed around the well. Two standard antibiotics [Penicillin (10 mg) and Trimethoprim/Sulfamethoxazole (SXT)] were used as the positive controls.
The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined by macro dilution assay (NCCLS, 2008). The cultures were prepared in 24 h and 72 h broth cultures of microorganisms, respectively. The MIC was defined as the lowest concentration of the compound to inhibit the growth of microorganisms and the MBC was defined as the lowest concentration of compound to kill the microorganisms. Serial dilutions ranging from 0.195– 50 mg/ml were prepared in medium.
RESULTS AND DISCUSSION
Elemental analysis and spectroscopy data show complex (1), [Ag(3-bpdh)(NO3)]n, has been synthesized. The Schiff-base ligand was attached to silver (I) by the nitrogen atoms of pyridine groups (Fig. 1). The IR spectra of compound 1 in bulk form and nanorod are the same. The IR spectrum of compound 1 (Fig.2) in nanoparticle show a weak broad band at around 3400 cm–1 and the relatively weak absorption bands at around 3038–3072 cm–1, due to the O–H (water molecules) and C–H modes (aromatic rings), respectively. The variable intensity absorption bands in the frequency range 1300–1613 cm–1 correspond to ring vibrations of the ‘‘py” moiety of the ligands.
The morphology, structure and size of the compound 1 were investigated by Scanning Electron Microscopy (Fig.3). Fig. 3 clearly indicates the nanorod morphology for the particles, prepared by sonochemical process, with the average diameter of about 53 nm. Structural dispersion was estimated by using measurement software. The obtained data were used to sketch a histogram plot (Fig. 5a).
silver(І) oxide was obtained with thermolysis in air atmosphere at 700˚C for 2 h. The morphology and structure of the silver(І)oxide were investigated by Scanning Electron Microscopy (Fig. 4). From the histogram plot in Fig. 5b, the average size of 155 nm was estimated for the particles and structural dispersions.
The energy-dispersive X-ray (EDAX) spectroscopy of Ag2O nanoparticles shows the presence of silver and oxygen as the only elementary components (Fig.6).
Fig. 7a shows the XRD pattern of Ag2O nanoparticles that is the residue from the calcination of compound 1. The diffraction intensities were recorded 2θ angles from 10˚ to 80˚. Four additional broad bands are observed at 2θ = 37˚ (111), 48˚ (200), 68˚ (220) and 77˚ (311) planes of silver(I) oxide.
The average size (24.4 nm) of Ag2O nanoparticles can be estimated using the Scherrer equation from the line broadening of the (111) reflection. D=kλ/βcosθ, where D=thickness of the nanocrystal, k(Constant)= 0.94, λ= wavelength of X-rays, β=width at half maxima of (111) reflection at 2θ Bragg’s angle.
IR spectroscopy of silver(I) oxide
Fig. 7b shows the IR spectrum of silver(I) oxide. The absorption bands at 520 cm-1 shows Ag–O bond, and the absorption bands at 3430 and 1638 cm-1 are attributed to the ν(OH) stretching and bending vibrations, respectively, indicating the presence of physisorbed water molecules linked to Ag2O nanoparticles .
Table 1 shows the antibacterial activity results of Schiff bases ligand, bulk and nanorod complex and silver (I) oxide nanoparticles evaluated by well diffusion method against Staphylococcus aureus, Enterococcus faecalisand Escherichia coli and Pseudomonas aeruginosa. We found that the ligand shows lower antibacterial activity compared to the other compounds (bulk complex, nano-rod complex and silver (I) oxide nanoparticles). It seems that the nano-rod complex was more effective than the bulk complex against all tested bacteria; this is probably due to the diminished size of particles in these complexes. The MIC values for the compounds were in the range of 0.195-50 mg/ml. The results show that the compounds were effective on bacteria (Table 2). According to the results, the complex was more effective against bacteria. It is probably due to the positive charge of central atom shared with donor atoms of ligand and pi electron delocalization in over the whole chelate moiety, leading to the lipophilic nature and positive charge of complex. This property makes it stronger in penetrating through the lipid layers of microbial membranes and therefore, a better anti-bacterial agent.
The Schiff base ligand (L), 2,5-bis(3-pyridyl)-3,4-diaza-2,4-hexadiene (3-bpdh), the bulk and nanorod complex, and silver(І) oxide nanoparticles were synthesized and characterized by spectroscopic methods such as IR, SEM, XRD and EDAX. In addition, experimental investigations showed that these compounds have an antibacterial activity against E. coli, S. aureus, Enterococcus faecalis and P. aeruginosa. Results showed that compound 1 in nano-scale and silver(I) oxide nanorods have stronger antibacterial effects against E. coli, S. aureus, and P. aeruginosa bacteria in comparison with Schiff base ligand, and that nanorod silver(І) particles have a good antibacterial activity in comparison with bulk complex.
The authors acknowledge financial support by Shahid Bahonar University of kerman
CONFLICT OF INTEREST
The authors confirm that this article content has no conflict of interest.