International Journal of Minerals, Metallurgy and Materials V olume 20 , Number 4 , April 2013 , P age 403 DOI: 10.1007/s12613-013-0743-2
Preparation, characterization, and antibacterial activity of γ-irradiated silver nanoparticles in aqueous gelatin Majid Darroudi1) , Mansor B. Ahmad2) , Mohammad Hakimi3) , Reza Zamiri4) , Ali Khorsand Zak5) , Hasan Ali Hosseini3) , and Mohsen Zargar6) 1) Department of Modern Sciences and Technologies, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran 2) Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. 3) Chemistry Department, Payame Noor University, 19395-3697 Tehran, Iran 4) Department of Materials Engineering and Ceramic, CICECO, University of Aveiro, Campus Santiago, 3810-193 Aveiro, Portugal 5) Electroceramics and Materials Lab., Physics Department, Faculty of Science, Ferdowsi University of Mashhad, Iran 6) Faculty of Science, Qom Branch, Islamic Azad University, Qom, Iran (Received: 20 June 2012; revised: 13 August 2012; accepted: 23 August 2012)
Abstract: Colloidal silver nanoparticles (Ag-NPs) were obtained through γ-irradiation of aqueous solutions containing AgNO3 and gelatin as a silver source and stabilizer, respectively. The absorbed dose of γ-irradiation influences the particle diameter of the Ag-NPs, as evidenced from surface plasmon resonance (SPR) and transmission electron microscopy (TEM) images. When the γ-irradiation dose was increased (from 2 to 50 kGy), the mean particle size was decreased continuously as a result of γ-induced Ag-NPs fragmentation. The antibacterial properties of the Ag-NPs were tested against Methicillinresistant Staphylococcus aureus (MRSA) (Gram-positive) and Pseudomonas aeruginosa (P.a) (Gram-negative) bacteria. This approach reveals that the γ-irradiation-mediated method is a promising simple route for synthesizing highly stable Ag-NPs in aqueous solutions with good antibacterial properties for different applications. Keywords: silver; nanoparticles; silver nitrate; gelatin; gamma rays; irradiation; bacteria
1. Introduction Fabrication of nanoparticle clusters by the γirradiation induced technique has proved to be effective [1-3]. In this technique, the aqueous solution of metallic cations is exposed to γ-rays; the species hydrated electrons and hydrogen atoms prepared from water radiolysis are effective reducing agents and can reduce the metallic cations to metallic particles [4]. Recently, the use of irradiation techniques, such as γ-irradiation in the fabrication of metallic nanoparticles, has been demonstrated to have a number of extremely advantageous properties: (1) the controllable reduction of metal ions can be performed without using any reducing agent and/or producing any byproducts from the reductants (e.g., undesired oxidation products like metal oxides); (2) the rates of reactions initiated by radiation are well defined; (3) the reducing agent Corresponding author: Majid Darroudi
can be uniformly generated in the solution; (4) the process is easy and clean; (5) the γ-ray irradiation is harmless; and (6) during the γ-irradiation process, the metallic nanoparticles can become fully reduced, highly pure, and stable [5-6]. Previous work using the γ-irradiation method for preparing silver nanoparticles (Ag-NPs) has used different stabilizers [7-10]. In the current study, we introduce gelatin as an effective green stabilizer and clearly indicate how the γ-irradiation dose can influence the properties of the metallic nanoparticles (e.g., particle size). In previous our work, we used a stabilizer to prevent particle agglomeration in the fabrication of nanoparticles [11-18]. In this work, we reported the synthesis of Ag-NPs by γ-irradiation reduction method in aqueous gelatin solution as a capping agent. Gelatin has been used as a stabilizing agent in pre-
E-mail:
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c University of Science and Technology Beijing and Springer-Verlag Berlin Heidelberg 2013
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vious works for preparing Ag-NPs by pulsed laser ablation [11-12] and green synthesis method [15-18], but the resultant metal nanoparticles have not been prepared by γirradiation reduction method in gelatin and tested against antibacterial activity. The resulting Ag-NPs were characterized and tested on their efficacy in inhibiting the growth of Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P.a) bacteria.
2. Experimental 2.1. Chemicals All chemicals in this work were analytical grade and used as received, without further purification. AgNO3 (99.98%, Merck, Germany) and gelatin (Type B, SigmaAldrich, USA) were used as the silver precursor and stabilizer, respectively.
Int. J. Miner. Metall. Mater., V ol. 20 , No. 4 , Apr. 2013
were used for the antibacterial assay as Gram negative and Gram positive bacteria, respectively. Briefly, 6 mm sterile paper discs impregnated with 20 μL of colloidal Ag-NPs (5, 15, and 50 kGy), which prepared at different γ-doses, were suspended in the sterile distilled water and left to dry at 37◦ C for 24 h under sterile conditions. The bacterial suspension was prepared by making a saline suspension of isolated colonies selected over 18-24 h agar plates of tryptic soy. The suspension is adjusted to match the 0.5 McFarland turbidity standards, using the spectrophotometer at 600 nm [19], which is equivalent to 1.5×108 Colony Forming Units (CFU)/mL. The surface of the Mueller Hinton agar was completely inoculated using a sterile swab and was then steeped in the prepared suspension of bacteria. Finally, the impregnated discs were placed on the inoculated agar and incubated at 37◦ C for 24 h.
2.2. Synthesis of Ag-NPs
3. Results and discussion
All glass wares used in the experimental lab were cleaned with a fresh solution of HNO3 /HCl (3:1, v/v), washed thoroughly with doubly distilled water, and dried before use. For the synthesis of Ag-NPs, 1.0 g gelatin was added to 90 mL H2 O in a flask, and the solution was stirred to obtain a clear solution. Aqueous AgNO3 (10 mL, 0.1 mol/L) was added to a gelatin solution and continuously stirred to obtain Ag+ /gel-sol. The obtained solution was γ-irradiated with a 60 Co γ-ray source (Picker V9, Germany) at different absorbed doses (i.e., 2, 5, 10, 15, 30, and 50 kGy).
This section reports the results of Ag-NPs synthesized in gelatin solutions. In this study, we attempted the fabrication of nanometer silver using the γ-irradiation method and gelatin as a green stabilizer. The effect of γ-irradiation doses, as well as Ag+ , on the size of the as-synthesized AgNPs was investigated. Gelatin as a capping medium for silver ions and a stabilizing agent for γ-prepared Ag-NPs was employed. After initial preparation of the particles with γ-irradiation, the prepared particles could dissociate due to further γ-irradiation to form smaller particles stabilized by the amine pendant groups on the gelatin backbone, leading to the formation of gelatin-stabilized Ag-NPs [20]. Gelatin was used in the synthesis of AgNPs as both reducing and stabilizing agents due to its oxygen- and nitrogenrich structures in carboxyl and amine groups, respectively (Fig. 1), which led to a very tight bond with Ag-NPs by electrostatic interactions [21]. Under γ-irradiation, water molecules can be radiolyzed to hydrated electrons, which, in this case, reduced silver ions to form Ag-NPs. The produced hydroxyl radicals due to water radiolysis can also reduce Ag+ ions. The radiolytic-reduction method normally involves radiolysis of aquatic solutions, which provides an effective route for reducing the metallic ions of transition metals. Using this method, aquatic solutions are exposed to γ-irradiation, and the solvated electrons, e− aq , can be produced. In turn, the produced e− reduces the metallic cations to the metalaq lic atoms and finally coalesces to form agglomerates, as shortly explained by the following reactions [4, 10]:
2.3. Characterization of Ag-NPs The γ-irradiated Ag-NPs prepared at various γirradiation doses were characterized by using ultravioletvisible (UV-vis) spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD), and atomic force microscopy (AFM). The optical absorption properties of the prepared samples were characterized using a Lambda 35 (Perkin-Elmer) UV-vis spectrophotometer in the 300700 nm range. The TEM images were performed using a Hitachi H-7100 electron microscopy, and the particle size distributions of nanoparticles were determined using the UTHSCSA Image Tool software, Version 3.00 program. The XRD patterns were carried out on Philips (X’pert, Cu Kα ) and were recorded at a scan speed of 2◦ /min. The AFM image was also carried out on an Ambios, Q-scope (SPM) machine.
2.4. Antibacterial assay of Ag-NPs The in vitro antibacterial assay of the synthesized AgNPs at different γ-doses was determined by the disc diffusion method using Mueller Hinton agar, which conformed to the recommended standards of the National Committee for Clinical Laboratory Standards (now known as the Clinical and Laboratory Standards Institute, 2000). Pseudomonas aeruginosa (P.a) (ATCC 10145) and Methicillinresistant staphylococcus aureus (MRSA) (ATCC 700689)
γ−ray
+ 0 0 H2 O −→ e− aq +H3 O +H +H2 +OH +H2 O2 + · · · +
+
Ag + [−Gel−]n → Ag [−Gel−]n +
Ag [−Gel−]n +
e− aq
0
→ Ag [−Gel−]n
Ag0 [−Gel−]n + Ag+ → [−Gel−]n Ag+ 2 0 + [−Gel−]n Ag+ 2 + Ag → [−Gel−]n Agm − 0 [−Gel−]n Ag+ m + eaq → [−Gel−]n Agm
(1) (2) (3) (4) (5) (6)
M. Darroudi et al., Preparation, characterization, and antibacterial activity of γ-irradiated silver ...
Fig. 1.
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Molecular structure of gelatin [21].
Therefore, the extensive number of hydroxyl and amine groups present in gelatin molecular structure, [−Gel−]n , can facilitate the complexation of silver cations (Ag+ ) to a molecular matrix, giving Ag+ [−Gel−]n . Under γ-irradiation, the produced complexes are reduced by solvated electrons, e− aq , produced in the aqueous solutions, giving Ag0 [−Gel−]n , and finally Ag0m [−Gel−]n species. Clearly, the main steps involve γ-irradiation of water molecules and formation of different ions and radicals (Eq. (1)). Reactions of these species with gelatin are anticipated to occur mostly in the secondary steps, resulting in crosslinkings, oxidations, radical and cation formations, etc. These phenomena enable gelatin to coat and stabilize Ag-NPs while inhibiting their excessive aggregations. Accordingly, we suggest that the size transformation of Ag-NPs from prolonged γ-irradiation was most likely the result of γ-irradiation-induced fragmentation of AgNPs, a process similar to the phenomenon observed when using UV and visible light as the irradiation source [22-23]. As shown by the insets in Fig. 2, the color of γ-irradiated gelatin solution containing silver ions due to the increased irradiation dose changed from brown to dark brown. UVvis absorption spectra are quite sensitive to the fabrication of Ag-NPs because the position of the SPR absorption peak, λmax , depends on particle size and shapes [24-26]. Thus, to indicate the influence of the γ-irradiation dose on the size of nanoparticles from AgNO3 solutions, we used UV-vis absorption spectroscopy to monitor Ag-NPs at different γ-irradiation doses. Six aqueous samples containing silver cations and 1wt% gelatin were exposed to various γ-ray doses: 2, 5, 10, 15, 30, and 50 kGy. Fig. 2 reveals the growth of the SPR of AgNO3 solutions as a function of the γ-irradiation dose. No characteristic SPR of Ag-NPs occurs before γirradiation as well as after a dose of 2 kGy. At a dose of 2 kGy, the appearance of a weak SPR peak at 450 nm (curve B in Fig. 2) displayed the fabrication of Ag-NPs at moderately low concentration. In contrast, after further γ-irradiation, the intensity of the SPR bands of Ag-NPs increased significantly as the
Fig. 2.
UV-vis spectra of Ag-NPs prepared in gelatin
solutions at different γ-irradiation doses.
γ-irradiation dose increased from 5 to 50 kGy, which indicated that correspondingly higher yieldsof Ag-NPs were obtained (curves C-G in Fig. 2) [27]. The gradated blue shift of the SPR bands of Ag-NPs (from 450 to 435 nm) occurred with increasing doses of γ-irradiation. These results suggest that smaller particles were obtained at higher γ-irradiation doses. It can be seen that larger Ag-NPs were obtained under a shorter γ-irradiation dose and were disintegrated under further γ-irradiation doses [28]. It has been suggested that an electron transformation from metallic silver to silver ions occurs on the surface of nanoparticles similar to photo-induced electron emission for Ag-NPs by illuminating light [23]. With increasing doses of visible irradiation, the SPR absorption peak tends to a blue shift. In this study, similar results were observed, i.e., increas-
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ing doses of γ-irradiation decreased NPs diameters as well as blue shifts of the corresponding SPR absorption bands. Therefore, the decrease in size that occurred in obtained Ag-NPs with further γ-irradiation is similar to the phenomenon observed in a visible-irradiation process [23]. In the last γ-irradiation at a dose of 50 kGy, no significant change in the λmax of the SPR absorption band occurred in these Ag-NPs relative to those of the particles formed at a dose of 30 kGy. This result indicates that the formation of Ag-NPs was complete after γ-irradiation at a dose of 30 kGy. Therefore, it is possible to control the size and quantity of Ag-NPs by varying the dose of γ-irradiation applied to the silver ion solution. In addition, the role
Fig. 3.
of γ-irradiation at a selective dose was checked to follow the advance of the silver salt reduction in the presence of gelatin before γ-irradiation, as shown by curve A in Fig. 2. No change occurred in the UV-vis absorption spectrum of the sample prepared before γ-irradiation. The stability of fabricated Ag-NPs in gelatin solutions was studied and indicated that the produced Ag-NPs were very stable over a long time (i.e., 3 months) in aqueous solution without any sign of aggregation or precipitation. It was subsequently determined that in the gelatin solution, the γ-irradiation played a crucial role for the synthesis of Ag-NPs. The TEM images also confirmed that, with increasing irradiation dose, the size of Ag-NPs decreased (Fig. 3).
TEM images and their corresponding size distributions of prepared Ag-NPs in gelatin under different
γ-irradiation doses: (a) 5 kGy, (b) 30 kGy, and (c) 50 kGy.
M. Darroudi et al., Preparation, characterization, and antibacterial activity of γ-irradiated silver ...
Fig. 4(a) displays the XRD pattern of Ag-NPs prepared at 50 kGy. As shown in the XRD pattern, the wide peaks’ 2θ values were assigned to Ag-NPs. Four peaks can also been seen at 2θ of 38.2◦ , 44.4◦ , 64.6◦ , and 77.49◦ , which are characteristic diffraction peaks of Ag-NPs. These peaks correspond to the (111), (200), (220), and (311) four crystalline planes of face-centeredcubic (fcc) crystalline structures of metallic silver, respectively (JCPDS file No. 00-004-0783). The silver peaks are indexed and indicate that the considered sample was highly pure Ag-NPs without any impurities. The average crystalline size of the sample prepared at 50 kGy was estimated from the XRD pattern by Scherrer’s equation [29]. The average crystalline size of the considered sample was 14.17 nm, which is relatively close to the average size (16.38 nm) of Ag-NPs as displayed in the TEM image (Fig. 3(a)). The AFM result shows the surface morphology
Fig. 4.
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of the well-dispersed Ag-NPs formed in the gelatin matrix. As shown in Fig. 4(b), the value determined by AFM was close to that determined by TEM and the films of gelatin containing Ag-NPs displayed a dense and uniform packed structure. Thus, the (Ag-NPs)-gelatin films could provide a biocompatible and rough surface for special biological applications, such as cell immobilization. Inhibition zones were determined for the synthesized Ag-NPs/gelatin colloids. The images of the inhibition zones are presented in Fig. 5. The images show that the colloidal Ag-NPs/gelatin had high and similar antibacterial activity against Gram-negative and Gram-positive bacteria. The solution of gelatin (10 mg/mL) did not show any antibacterial activity. The solution of irradiated AgNPs/gelatin at 5 kGy shows high antibacterial activity and this effect in the further irradiation doses (15 and 50 kGy) were increased with increasing Ag-NPs concentrations.
XRD pattern of Ag-NPs prepared under γ-irradiation at 50 kGy (a) and AFM image of the surface
morphology of the prepared Ag-NPs in gelatin (b).
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Fig. 5.
Images of antibacterial activity of gelatin and prepared Ag-NPs at different γ-irradiation doses.
The simple, cost effective, and environmental friendly synthesis of Ag-NPs using gelatin in addition to their effective antibacterial activity underlines the potential of adopting this “green” method to fabricate Ag-NPs as antibacterial agents for various fields.
4. Conclusions Fabrication of Ag-NPs in aqueous gelatin solutions using γ-irradiation without any reducing agent or heat treatment is simply possible. The studies of UV-vis absorption spectra reveal that the SPR bands of silver clusters are certainly affected by the amount of γ-dose absorbed. The TEM images of Ag-NPs and their particle size distributions indicated that the larger Ag-NPs were obtained in a smaller γ-irradiation dose. Further experiments showed that an increase in the dose of γ-irradiation caused a decrease in the diameter of Ag-NPs. The XRD pattern also displays the fcc geometry for the obtained Ag-NPs. These fabricated Ag-NPs are very stable over a long period of time in an aqueous solution without any sign of agglomeration or precipitation. This (Ag-NPs)-gelatin film can be used in biological applications, such as cell immobilization, because it has a biocompatible and rough surface. The simple, cost effective, and environmental friendly synthesis of Ag-NPs using gelatin in addition to their effective antibacterial activity underlines the potential of adopting this “green” method to fabricate Ag-NPs as antibacterial agents for various fields.
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