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Microwave-assisted synthesis of silver nanoparticles using sodium alginate and their antibacterial activity
Colloids and Surfaces A: Physicochem. Eng. Aspects 444 (2014) 180–188
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Microwave-assisted synthesis of silver nanoparticles using sodium alginate and their antibacterial activity Xihui Zhao a,c , Yanzhi Xia b,c,∗ , Qun Li a,c , Xiaomei Ma a,c , Fengyu Quan a,c , Cunzhen Geng a,c , Zhenyu Han a a
College of Chemistry and Chemical Engineering, Qingdao University, Qingdao 266071, China State Key Laboratory Cultivating Base for New Fiber Materials and Modern Textiles, Qingdao University, Qingdao 266071, China c Collaborative Innovation Center for Marine Biomass Fibers, Materials and Textiles of Shandong Province, Qingdao University, Qingdao 266071, China b
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• A green, microwave-assisted method was developed to synthesize silver nanoparticles using sodium alginate as stabilizer and reducer. • The reaction parameters significantly affected the formation of silver nanoparticles. • Sodium alginate played an important role in the synthesis and stabilization of silver nanoparticles.
a r t i c l e
i n f o
Article history: Received 28 June 2013 Received in revised form 26 November 2013 Accepted 7 December 2013 Available online 17 December 2013 Keywords: Sodium alginate Silver nanoparticles Microwave synthesis Antimicrobial activity
a b s t r a c t A simple, green, microwave-assisted method of synthesizing silver nanoparticles was developed using sodium alginate as stabilizer and reducer. A possible mechanism involved in the reduction and stabilization of nanoparticles was investigated. The effect of reaction conditions such as the concentration of sodium alginate and AgNO3 , irradiation time and pH on the synthesis of silver nanoparticle was studied. The silver nanoparticles were characterized by UV–vis spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD). The results indicated the formation of spherical, nanometer-sized particles. The reaction parameters significantly affected the formation rate, size and distribution of the silver nanoparticles. XRD analysis revealed that the particles were face-centered cubic. The silver nanoparticles prepared in this way were uniform and stable, which could be stored at room temperature for at least 6 months. The synthesized silver nanoparticles had significant antibacterial activity on two kinds of Gram bacteria. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Silver nanoparticles (AgNPs) have attracted extensive research interest due to their attractive optical, electronic properties and excellent antimicrobial activities [1–3]. AgNPs exhibit strong
∗ Corresponding author at: Collaborative Innovation Center for Marine Biomass Fibers, Materials and Textiles of Shandong Province, Qingdao University, Qingdao 266071, China. Tel.: +86 532 85953069; fax: +86 532 85952497. E-mail addresses: [email protected], [email protected] (Y. Xia). 0927-7757/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfa.2013.12.008
cytotoxicity toward a broad range of microorganisms and are widely used as an antibacterial agent [4]. The advantage of AgNPs compared to bulk metal or salts is the slow and regulated release of silver from nanoparticles, thereby causing long lasting protection against bacteria [5,6]. The antimicrobial activity of AgNPs is comparatively better than most prominent antibiotics used worldwide [7]. Recently, researchers have shown that AgNPs can interact with the human immunodeficiency virus type 1 and prevent the virus from binding to the host cells [8]. Numerous methods have been developed for the preparation of AgNPs. The most common method is chemical reduction of silver
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salt by a reducing agent in the presence of a stabilizing agent. The reducing agents such as sodium borohydride [9,10], hydrazine [11], N,N-dimethylformamide [12], or other organic compounds [13–15] have been used for the preparation of metal nanoparticles with high reactivity. However, many of them may be associated with potential environmental toxicity as well as biological risks [16]. On the other hand, the stabilizers including triphenylphosphine [17], citrate [18] and polyvinylpyrrolidone [19], are often toxic, difficult to dispose [20]. With the increasing awareness of environmental protection, people are inclined to develop the eco-friendly approach for the synthesis of nanoparticles. Three main steps in the preparation of nanoparticles that should be evaluated from a green chemistry perspective are the choice of the solvent medium, environmentally benign reducing agent, and nontoxic material for the stabilization of the nanoparticles. Currently, many natural polymers like chitosan [21], soluble starch [22,23], polypeptide [24], heparin [25], and hyaluronan [26] have been involved in the green preparation of nanoparticles as reducing and stabilizing agent. Wei et al. [21] reported the green synthesis of silver nanoparticles by thermal reduction methods in chitosan–acetic acid–water, and the obtained particles showed highly potent antibacterial activity toward both Gram-positive and Gram-negative bacteria. Yan et al. [24] developed a general strategy of fabricating gold nanoparticles using polypeptides as capping agent and reductant, the resulting positively charged polypeptide-conjugated gold nanoparticles can be applied for biomedical applications. In addition, the plant-based exudate, such as Gum acacia [27] and Gum kondagogu [1], and plant extracts [28–30] have been reported as reducing and stabilizing agents for the silver nanoparticles biosynthesis. Alginate is a naturally occurring carbohydrate polymer isolated from marine algae [31]. It can be characterized as an anionic copolymer comprising mannuronic acid (M block) and guluronic acid (G block) units arranged in an irregular block-wise pattern of varying proportions of GG, MG and MM blocks [32,33]. Sodium alginate is widely used for food and drink, pharmaceutical and bioengineering industries due to its biocompatibility, low toxicity, relatively low cost, and mild gelation by addition of divalent cations such as Ca2+ [34]. It has a number of free hydroxyl and carboxyl groups distributed along the back-bone. It can be well dissolved in water due to negatively charged carboxyl group. In the synthesis of the AgNPs, the carboxyl groups of sodium alginate can electrostatically interact with Ag+ to form a complex, and the hydroxyl groups reduce the Ag+ to Ag0 [35]. Pal et al. [36] and Yang and Pan [37] successfully fabricated metal nanoparticles using sodium alginate as a reducing and stabilizing agent. Compared to chitosan, alginate is stable at higher pH and compatible with other substances without coagulation in aqueous solution. The sodium alginate stabilized AgNPs offer numerous benefits of eco-friendliness and compatibility for pharmaceutical and biomedical applications. The aim of the present work is to develop a simple, rapid and totally green approach of synthesizing AgNPs using sodium alginate as reducing and stabilizing agent, water served as reaction medium. The synthesis was carried out in aqueous medium by microwave heating (without any additional chemical reducing and stabilizing agents). The emphasis was placed on the effects of the reaction conditions on the synthesis of AgNPs. The obtained particles were analyzed by UV–vis spectroscopy, transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). In addition, the antimicrobial activity of the synthesized AgNPs against Gram-positive and Gramnegative bacteria was also investigated. It is worth pointing that the microwave heating has been explored as a promising technique for nanoparticle synthesis. The advantage of microwave heating over the conventional heating are the rapid and uniform internal heating to the solution, microwave irradiation generates very fast nucleation sites in the solution, which significantly enhances the reaction
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rate [38]. To the best of our knowledge, the detailed research of using the microwave technique to prepare AgNPs with sodium alginate as reducing and stabilizing agents is limited in literature. 2. Experimental 2.1. Reagents Sodium alginate (SA) was supplied by Mingyue Seaweed Co. Ltd., Qingdao, Shandong Province, PR China. Silver nitrate (AgNO3 ) was analytical reagent grade. All solutions were prepared with deionized water. All glass wares were thoroughly cleaned with water and dried in an oven. 2.2. Synthesis of alginate-based silver nanoparticles In a typical preparation, sodium alginate was dissolved in distilled water in a 100 mL beaker using heating magnetic stirrer. After complete dissolution, the pH of the solution was adjusted within the range 5–11. Certain amount of AgNO3 precursor was then added dropwise (keeping in mind that the total volume of the reaction medium is 25 mL). Then the reaction mixture was placed in the microwave oven (Galanz WP750) for the reduction of silver ions. After the reaction, the solution was immediately cooled down to room temperature and adjusted to 25 mL by adding a small amount of distilled water to compensate for the loss of water during microwave irradiation. During the heating process, the color of the reaction mixture changed slowly from colorless to light brown due to reduction of Ag+ to Ag0 . The reaction mixture was analyzed by UV–vis spectroscopy to ascertain the formation of AgNPs. The reaction for the formation of AgNPs was systematically investigated by varying the irradiation time from 1 min to 10 min, initial sodium alginate concentration from 0.1% to 1.5%, pH values from 5 to 11, and AgNO3 concentration from 0.4 to 8.0 mmol/L. 2.3. Characterization UV–vis spectra of AgNPs in alginate solution were performed with a Shimadzu UV3150 UV–vis spectrophotometer operating in the transmission mode. A solution containing sodium alginate alone was used as a blank. Transmission electron microscopy (TEM) images were obtained by a JEM-1200EX microscope at an accelerating voltage of 100.0 kV. The samples for TEM studies were prepared by drop-casting a dispersion of AgNPs on carbon-coated copper grids, which were allowed to dry under ambient conditions. XRD measurements of sodium alginate and sodium alginate–AgNPs composites were carried out on a powder X-ray diffractometer (D/MAX-RB) using CuK␣ radiation ( = 0.15418 nm) over a 2 range of 5–80◦ with a step size of 0.05◦ . The aqueous solution of sodium alginate embedded with AgNPs was spread over a glass plate and coagulated in the air to form membrane and the yellowish-brown colored film obtained was used for XRD analysis. Fourier transform infrared spectroscopy (FTIR) was recorded on spectrometer (NICOLET5700) by KBr tablet method, and the spectra were scanned in the range of 400–4000 cm−1 at a resolution of 4 cm−1 . 2.4. Antibacterial assay The disk diffusion method was used to study the antibacterial activity of the synthesized silver nanoparticles. Staphylococcus. aureus (ATCC 25923) and Escherichia coli (ATCC 35218) were used as model test strains for Gram-positive and Gram-negative bacteria, respectively. Nutrient agar medium was prepared by using peptone (5.0 g), beef extract (3.0 g), and sodium chloride (5.0 g) in 1000 mL distilled water, the pH was adjusted to 7.0 and agar (15.0 g) was added to the solution. The agar medium was sterilized
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Fig. 1. Schematic illustration of a typical procedure for the synthesis of AgNPs in sodium alginate solution under microwave irradiation.
in an autoclave at 121 ◦ C for 20 min. This nutrient agar medium was transferred into sterilized petri dishes. After solidification of the media, bacterial culture was inoculated on the solid surface of the media and swabbed with sterile cotton swab. The sterile paper discs (10 mm) were impregnated with 30 L sample solutions and then left to dry at 37 ◦ C for 12 h in sterile conditions. Then, the impregnated discs were placed on the inoculated agar and incubated for 24 h at 37 ◦ C. After incubation, the zone of inhibition (ZOI) was measured by subtracting the disk diameter from the total inhibition zone diameter. The silver nanoparticles used here were prepared with 0.5% and 1.0% sodium alginate solution containing 4 mmol/L AgNO3 , pH 9 and microwave irradiation for 8 min. 3. Results and discussion 3.1. Reaction mechanism for formation of silver nanoparticles Sodium alginate was used to perform dual role: as reducing agent for silver ions and as stabilizing agent during/after the formation of AgNPs. Previous reports [28,37,39] have disclosed that the solutions of polymers can be used for the synthesis and stabilization of nanoparticles. For the synthesis of AgNPs, the generally accepted mechanism suggests a two-step process, i.e. atom formation and then polymerization of the atoms. In the first step, a portion of metal ions in the solution is reduced by the available reducing groups. The atoms thus produced act as nucleation centers and catalyze the reduction of the remaining metal ions present in the bulk solution. Compared with other water-soluble polymers, alginate is an anionic polymer with high charge density, the negatively charged alginate facilitates the attraction of the positively charged silver ions to the polymeric chains, which were then reduced by the existing reducing groups. The preformed silver atoms adsorb Ag+ on the surface via dimerization or association with excess ions due to the binding energy between metal atoms. The surface ions are again reduced, subsequently the atoms coalesce leading to the formation of metal clusters. In this way, the aggregation process does not cease and finally results in larger particles. The process can be stabilized through sodium alginate so preventing further coalescence. These
metal cluster surfaces are likely to be anchored through strong association between AgNPs surface and “O” atom of the functional groups ( COO− and OH) of sodium alginate [40]. The resulting surface negative charge of alginate fragments containing carboxylic groups stabilizes nanoparticles against coalescing with the next one because of electrostatic repulsion and steric effects [41,42]. The overall synthetic procedure is presented in Fig. 1. Fig. 2 shows the FTIR spectra of sodium alginate and sodium alginate–AgNPs. In the case of sodium alginate, the peaks centered at around 3435 cm−1 can be attributed to the stretching vibration of hydroxyl group; the strong peak noted at 1609 cm−1 is due to the stretching vibration of CO2 − (carboxylate ion) group; the band at around 1414 cm−1 is ascribed to the hydroxyl group deformation vibration; the absorption band at 1083 cm−1 is corresponded to C O C stretching mode from the glucosidic units. In the case of sodium alginate–AgNPs, the band of CO2 − is shifted to 1613 cm−1 ,
Fig. 2. FTIR spectra of sodium alginate and sodium alginate–AgNPs.
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Fig. 3. Effect of reaction parameters on the synthesis of silver nanoparticles. (A) UV–vis spectra of AgNPs prepared at different alginate sodium concentration. Reaction conditions: 4 mmol/L AgNO3 , pH 9, irradiation 8 min. Inset: Digital photograph of sodium alginate solution (0.5%) mixed AgNO3 before (a) and after (b) microwave irradiation for 8 min. (B) UV–vis spectra of AgNPs prepared using different concentrations of AgNO3 . Reaction conditions: 0.5% sodium alginate, pH 9, irradiation 8 min. (C) UV–vis spectra of AgNPs prepared with different irradiation times. Reaction conditions: 4 mmol/L AgNO3 , 0.5% sodium alginate, pH 9. (D) UV–vis spectra of AgNPs prepared with different pH. Reaction conditions: 0.5% sodium alginate, 4 mmol/L AgNO3 , irradiation 8 min.
the peaks of 3435 and 1414 cm−1 shift to 3422 and 1384 cm−1 indicating that carboxyl groups and the hydroxyl group are involved in the synthesis and stabilization of AgNPs. The variations of the hydroxyl and carboxylate groups have been reported in the previous study on synthesis of AgNPs with another polysaccharide [1,27]. 3.2. Effect of reaction parameters on the synthesis of silver nanoparticles UV–vis spectroscopy is a simple and sensitive technique for the characterization of AgNPs due to the excitation of surface plasmon resonance (SPR) in the AgNPs. Theoretical studies on the dependence of the UV–vis absorption on the size of the metal spheres have been conducted [43]. The general trend is that the SPR absorption band shows a red shift with increasing particle size. In addition, the aggregation of colloidal silver causes a decrease in the intensity of the main peak, and also results in a long tail on the long-wavelength side of the peak. After microwave irradiation, the sodium alginate solutions containing silver nitrate changed from colorless to yellow (see Fig. 3A inset.), the appearance of yellow color in the reaction mixtures supports the formation of AgNPs [18,30]. In order to monitor the effect of different parameters to the formation of AgNPs, the UV–vis absorption spectra of synthesized AgNPs were recorded against respective sodium alginate blanks. Fig. 3A shows the UV–vis spectra of the AgNPs obtained using sodium alginate as reducing and stabilizing agent in different concentration (0.1–1.5%) at the fixed concentration of silver nitrate
(4 mmol/L AgNO3 ), pH 9 and microwave irradiation for 8 min. The data reveals a number of findings which can be presented as follows: (i) the spectra exhibits unique SPR absorption band at approximately 417 nm, which is associated with the formation of nanoparticles. This result implies that sodium alginate can reduce the Ag+ to Ag0 due to the OH groups present in the polymer. Hydroxyl group mediated reduction was reported for the synthesis of silver and gold nanoparticles in previous studies [1,36,39]; (ii) the peak acquires an ideal bell sharp which implies that negligible aggregation occurs in the reactive system and the nanoparticles are well dispersed; (iii) the absorption intensity increases rapidly as the concentration of sodium alginate increases from 0.1% to 0.5%. The increase in SPR band intensity could be attributed to the formation of more AgNPs. The possible reason is that the increased number of carboxyl groups and hydroxyl groups facilitates the complexation of Ag+ to the molecular matrix, meanwhile, more hydroxyl groups benefit the reduction of Ag+ [39]; (iv) further increase in sodium alginate concentration from 0.5% to 1.5% results in decrease in the peak intensity, the peak shifts toward longer wavelength at higher sodium alginate concentrations, which suggests aggregation of the AgNPs and formation of larger particles as the concentration of sodium alginate exceeds 0.5%. The increase in the polymer concentration could induce conflicting effects on the particle size. At higher polymer concentrations, more polymer molecules can cap the AgNPs to decrease the particle size. While, polymer acts as a reductant, it accelerates the nucleation and growth of the AgNPs to produce larger particles at relatively high polymer concentrations [43]. At higher sodium alginate concentrations
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Fig. 4. TEM image and histogram of silver nanoparticles prepared at different conditions. (A) 0.8 mmol/L AgNO3 , 0.5% sodium alginate, pH 9, irradiation 8 min. (B) 4 mmol/L AgNO3 , 0.5% sodium alginate, pH 9, irradiation 8 min. (C) 6 mmol/L AgNO3 , 0.5% sodium alginate, pH 9, irradiation 8 min. (D) 0.1% sodium alginate, 4 mmol/L AgNO3 , pH 9, irradiation 8 min. (E) 0.5% sodium alginate, 4 mmol/L AgNO3 , pH 9, irradiation 8 min. (F) 1.0% sodium alginate, 4 mmol/L AgNO3 , pH 9, irradiation 8 min.
(>0.5%), the stabilizing effect of the sodium alginate molecules is enhanced. However, the role of sodium alginate as a reductant is dominant at relatively high concentrations to produce large particles by further nucleation and growth. Therefore, it should be mentioned that a low concentration of sodium alginate in the reaction medium (0.5%) is enough for full reduction of the Ag+ to Ag0 nanoparticles. The production of AgNPs with 0.5% sodium alginate was evaluated with varying AgNO3 concentrations and the UV–vis spectra of the AgNPs obtained are shown in Fig. 3B. It can be observed that the strongest SPR band is centered at around 418 nm when the AgNO3 concentration ranged from 0.4 to 8.0 mmol/L, the peak is symmetric and there is no obvious absorption in the range of 450–700 nm, which indicates that negligible aggregation occurs in this reactive system and the nanoparticles are well dispersed [44]. The absorption peak intensity gradually increases with the increase of AgNO3 concentration reflecting the formation of more and more AgNPs. However, the particle size does not become larger due to the stabilizing effect of sodium alginate macromolecules. Therefore, it is possible that a large amount of AgNPs with small
particles size can be achieved using sodium alginate as the reducing and stabilizing agent. It was further confirmed by the TEM images shown in Fig. 4A–C. The synthesis was also monitored by different irradiation times (1–10 min) with 0.5% sodium alginate and 4 mmol/L AgNO3 at pH 9. Fig. 3C shows the UV–vis spectra of the AgNPs obtained after different irradiation times. The data in Fig. 3C reveals several important findings which can be presented as follows: (i) at the early stage reaction duration (<3 min), the plasmon band is broad and weak indicating low conversion of Ag+ to AgNPs at this irradiation duration. (ii) prolonging the reaction duration up to 5 min, the strongest SPR band occurs at 418 nm implying that large amounts of silver ions are reduced and used for cluster formation; (iii) further increase in the irradiation time to 8 min, there is a gradual increase in the absorption intensity and without any shift in the peak wavelength which indicates that the AgNPs content increases with increased irradiation time and the mean diameter of the AgNPs does not change much; (iv) raising irradiation time to 10 min is accompanied by the absorption band shift to longer wavelength (427 nm) which could be attributed to enlargement in the AgNPs size.
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Fig. 5. Average particle size of AgNPs synthesis at different reaction conditions. (A) Different concentration of AgNO3 . (B) Different concentration of sodium alginate. (C) Different pH.
Sodium alginate is a weak reductant, the reaction need long period with common heating method. Yang and Pan [37] reported the hydrothermal synthesis of AgNPs with sodium alginate as a reducing and stabilizing agent, it needed more than 6 h at 100–180 ◦ C. Sodium alginate would be degraded after a long period of heating, resulting in the decrease of the stabilizing ability to AgNPs, the prepared AgNPs should be centrifuged and redispersed in sodium alginate aqueous solution. Compared with the conventional heating method, the advantages of microwave irradiation are the rapid heating rate and uniformly heating ability, the reaction can be finished in a few minutes due to rapid internal heating, as well as the formation of a special reaction system that enables selective crystallization and homogeneous nucleation [42]. The stabilizing ability of sodium alginate to AgNPs is better because of the weak degradation in a short period of microwave heating.pH value is usually another key factor to the Ag+ reduction. Fig. 3D shows the UV–vis spectra of the AgNPs prepared at different pHs. At pH 5, only a band at about 270 nm was observed, which corresponded to the existence of Ag+ clusters [28,41]. With the increase of pH value, a new band appeared at about 418 nm on the UV–vis spectra, suggesting the formation of AgNPs. It also can be seen, the peak intensity increases gradually with the increase of pH value, that is because, higher pH value of sodium alginate solution would lead to more exposed carboxyl groups electrostatic interacting with Ag+ , and enhance the reduction rate of Ag+ by sodium alginate. When the solution pH value was increased to 11, the peak intensity increases, but the symmetry of plasmon band is not well implying a broad particle size distribution. 3.3. TEM and XRD analysis To understand better the effects of the synthesis conditions on the shape and size of AgNPs, TEM was used to evaluate the morphology and size of some representative silver particles obtained. Fig. 4 shows the typical TEM images of AgNPs synthesized at different conditions. As Fig. 4A demonstrates, fewer AgNPs were observed in the solution when the AgNO3 concentration was 0.8 mmol/L. Fig. 4B and C illustrates the TEM images and particle size distribution histograms of AgNPs prepared with the AgNO3 concentration of 4 and 6 mmol/L, respectively. As is evident from TEM micrographs, large numbers of nanoparticle formed at high AgNO3 concentration. The obtained AgNPs are in the nano range with uniform and spherical shapes. The histograms (the inset of Fig. 4A–C) clearly illustrate that the prepared particle size are around 10 nm. The AgNPs are well dispersed at higher AgNO3 concentration which indicates a good stabilization effect of the sodium alginate. Fig. 5 shows the average particle size distribution of AgNPs synthesized at different
reaction conditions. As Fig. 5A presents, the average particle size is about 12 nm at AgNO3 concentration of 8 mmol/L, the negligible change of the particle size at higher AgNO3 concentration further confirms the stabilizing effect of sodium alginate. Fig. 4D–F presents TEM micrograph and histogram of silver nanoparticles prepared at different sodium alginate concentrations. These silver nanoparticles observed are spherical in shape and well separated in aqueous medium. When the concentration of sodium alginate is increased from 0.1% to 0.5%, the particle size of the silver nanoparticles formed decreases and the uniformity of particle size distribution increases (Fig. 4D and E). The average particle size of AgNPs decreases from about 20 nm (0.1% sodium alginate) to 10 nm (0.5% sodium alginate) (Fig. 5B). These findings are similar to a previous study on biosynthesis of gold nanoparticles with sodium alginate, in which the concentration of the biopolymer increased from 0.01% to 0.1% [36]. The possible explanation is at higher alginate concentration, the interaction between ionic silver and functional groups on alginate as well as the rate of nanoparticles capping is excellent. In addition, the aggregation is lower due to less collision of silver nanoparticles. Compared with the preparation with 0.5% sodium alginate (Fig. 4E), the preparation with 1.0% sodium alginate contains much less silver nanoparticles and the particle size formed has a slight increase (Fig. 4F). This finding is consistent with the UV–vis analysis results, which shows that the absorption peak intensity of the obtained sample with 1.0% sodium alginate is weaker than that of the obtained sample with 0.5% sodium alginate (Fig. 3A). This study indicates that the particle size of the silver nanoparticles can be controlled by varying the concentration of alginate. The average particle sizes of AgNPs synthesized at pH 8, 9, and 11 are shown in Fig. 5C. The average particle size increases from about 10 nm (pH 9) to 15 nm (pH 11), which can be ascribed to the rapid reaction rate at higher pH value. The reductive properties of sodium alginate are substantially enhanced owing to the base hydrolysis with the formation of low molecular weight reducing fragments, and therefore, reflecting the dual role of sodium alginate as stabilizing and reducing agent in alkaline medium [41]. XRD analysis was performed to confirm the crystal phase of the prepared AgNPs. Fig. 6 shows typical XRD pattern of the alginate–AgNPs prepared with 0.5% sodium alginate and the AgNO3 concentration 4 mmol/L, at pH 9, microwave irritation for 10 min. The broad reflexion at 14◦ is due to the crystallinity of sodium alginate. Several distinct diffraction peaks at approximately 38.1◦ , 44.2◦ , 64.3◦ and 77.4◦ are assigned to reflections from the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) planes of the silver crystal, respectively, which confirms the existence of silver and further on the basis that they can be indexed as face-centered-cubic (FCC) structure of silver
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Fig. 6. XRD pattern of the as-prepared AgNPs: (a) sodium alginate–AgNPs and (b) sodium alginate.
[45]. XRD pattern thus clearly illustrates that the silver nanoparticles formed in this present synthesis are crystalline in nature. The other unidentified crystalline peak at 2 = 27.19◦ , 32.24◦ are also apparent in many works in which the XRD pattern includes the relevant 2 range. These peaks are due to the crystalline and amorphous organic phases, accompanying crystallized AgNPs [46–48].
3.4. Stability analysis The aggregation degree of metal nanoparticles could be effectively reflected by changes of the absorption characteristics, especially the shift of SPR peak in the UV–vis spectrum [49]. If strong aggregation occurred for AgNPs after a long storage period, strong red shift of SPR band would be observed in the UV–vis spectra. In this work, the AgNPs in sodium alginate solution was stored for 6 months at room temperature (25 ± 5 ◦ C), UV–vis spectroscopy was used to measure the stability (Fig. 7), as shown in Fig. 6, the absorbance intensity of the AgNPs changes only slightly, without a significant change in the SPR band position or the spectral shape, The as-prepared AgNPs is still clear yellow with no obvious change in color (see Fig. 7 inset). The results indicate that the as-prepared AgNPs is stable at room temperature for months with negligible aggregation, which further confirms the sodium alginate play an important role in stabilizing the AgNPs.
Fig. 7. UV–vis absorption spectra of AgNPs stabilized with sodium alginate (a) after reaction termination and (b) after 6 months storage at room temperature. The arrow indicates the maximum absorbance at 418 nm. Inset: Digital photograph of sodium alginate-stabilized AgNPs (1) after reaction termination and (2) after 6 month storage at room temperature.
3.5. Antibacterial activity Silver in an aqueous solution releases silver ions, which are biologically active and have bactericidal effects [50]. Big changes in the membrane structure of bacteria as a result of the interaction with silver cations lead to the increased membrane permeability of the bacteria. In the case of AgNPs, they interact extensively with the bacteria cell walls and cause lysis [51]. Besides, recent study indicated that the bactericidal effect of AgNPs mostly depends on the size of particles and the smaller is the better, the nanoparticles smaller than 10 nm have a direct interaction with the bacteria and produce electronic effects, which enhance the reactivity of nanoparticles [52]. In this study, the synthesized AgNPs with 0.5% and 1.0% sodium alginate exhibit good antibacterial activity against both Gram-negative and Gram-positive bacteria (Fig. 8), but it shows higher antibacterial activity against E. coli (Gram-negative) then S. aureus (Gram-positive) (Table 1). The differential sensitivity of Gram-negative and Gram-positive bacteria toward AgNPs is possible due to the difference in their structure of the cell wall. The cell wall of the Gram-positive bacteria is composed of a thick layer of peptidoglycan, consisting of linear polysaccharide chains cross-linked by short peptides thus forming more rigid structure leading to difficult penetration of the silver nanoparticles, but the cell walls of Gram-positive bacteria possesses thinner layer
Fig. 8. Zone of inhibition of silver nanoparticles against (A) S. aureus, (B) E. coli. (0) is control, (1) is AgNPs prepared with 1.0% sodium alginate, (2) is AgNPs prepared with 0.5% sodium alginate.
X. Zhao et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 444 (2014) 180–188 Table 1 Zone of inhibition (mm) of synthesized silver nanoparticles against bacterial pathogens. Test organism
E. coli S. aureus
Silver nanoparticles in 30 L Synthesized with 0.5% sodium alginate
Synthesized with1.0% sodium alginate
9.4 5.8
7.0 4.3
of peptidoglycan [29]. On the other hand, silver nanoparticles synthesized with 0.5% sodium alginate have comparatively higher antibacterial activity than those synthesized with 1.0% sodium alginate because of smaller in size. Sodium alginate as control was not found any of antibacterial patterns. 4. Conclusions In this study, a facile and efficient method for the green synthesis of well-distributed spherical AgNPs was developed. The synthesis was carried out in an aqueous medium treated by microwaves using sodium alginate as stabilizing and reducing agent. The sodium alginate concentration, the initial AgNO3 concentration, as well as microwave irradiation time and pH had obvious effects on the amount of AgNPs produced and particle sizes and size distributions. The silver nanoparticles prepared in this way are uniform and stable in solution over a period of six months at room temperature (25 ◦ C) and show no signs of aggregation. The utilization of environmentally benign and renewable materials like sodium alginate for the synthesis of AgNPs in an aqueous medium offers numerous benefits, including eco-friendliness and compatibility with biomedical, pharmaceutical, and textile applications. Microwave irradiation can accelerate the formation rate of particles. Acknowledgements The authors appreciate the financial support to this research by National High Technology Research Development Plan (863 Plan) of China (No. 2010AA093701), the Natural Science Foundation of China (No. 51203083) and Special Fund for Self-directed Innovation of Shandong Province of China (No. 2013CXB80201). References [1] A.J. Kora, R.B. Sashidhar, J. Arunachalama, Gum kondagogu (Cochlospermum gossypium): a template for the green synthesis and stabilization of silver nanoparticles with antibacterial application, Carbohydr. Polym. 82 (2010) 670–679. [2] D. Li, Q. He, Y. Cui, J. Li, Fabrication of pH-responsive nanocomposites of gold nanoparticles/poly(4-vinylpyridine), Chem. Mater. 19 (2007) 412–417. [3] D. Wei, W. Qian, Facile synthesis of Ag and Au nanoparticles utilizing chitosan as a mediator agent, Colloid Surf. B: Biointerfaces 62 (2008) 136–142. [4] T. Textor, M. Fouda, B. Mahltig, Deposition of durable thin silver layers onto polyamides employing a heterogeneous Tollen’s reaction, Appl. Surf. Sci. 256 (2010) 2337–2342. [5] S.Y. Luo, J. Chen, M. Chen, W.C. Xu, X.L. Zhang, Antibacterial activity of silver nanoparticles colloidal sol and its application in package film, Adv. Mater. Res. 380 (2012) 254–259. [6] P. Totaro, M. Rambaldini, Efficacy of antimicrobial activity of slow release silver nanoparticles dressing in post-cardiac surgery mediastinitis, Interact. Cardiovasc. Thorac. Surg. 8 (2009) 153–154. [7] R. Roy, M.R. Hoover, A.S. Bhalla, T. Slaweekl, S. Dey, W. Cao, Ultradilute Ag-aquasols with extraordinary bactericidal properties: role of the system Ag–O–H2 O, Mater. Res. Innov. 11 (2008) 3–18. [8] J.L. Elechiguerra, J.L. Burt, J.R. Morons, A. Camachobragado, X.X. Gao, H.H. Lara, M.J. Yacaman, Interaction of nanoparticles with HIV-1, J. Nanobiotechnol. 3 (2005) 6–16. [9] K. Vimala, K.S. Sivudu, Y.M. Mohan, B. Sreedhar, K.M. Raju, Controlled silver nanoparticles synthesis in semi-hydrogel networks of poly(acrylamide) and carbohydrates: a rational methodology for antibacterial application, Carbohydr. Polym. 75 (2009) 463–471.
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Microwave-assisted synthesis of silver nanoparticles using sodium alginate and their antibacterial activity Xihui Zhao a,c , Yanzhi Xia b,c,∗ , Qun Li a,c , Xiaomei Ma a,c , Fengyu Quan a,c , Cunzhen Geng a,c , Zhenyu Han a a
College of Chemistry and Chemical Engineering, Qingdao University, Qingdao 266071, China State Key Laboratory Cultivating Base for New Fiber Materials and Modern Textiles, Qingdao University, Qingdao 266071, China c Collaborative Innovation Center for Marine Biomass Fibers, Materials and Textiles of Shandong Province, Qingdao University, Qingdao 266071, China b
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• A green, microwave-assisted method was developed to synthesize silver nanoparticles using sodium alginate as stabilizer and reducer. • The reaction parameters significantly affected the formation of silver nanoparticles. • Sodium alginate played an important role in the synthesis and stabilization of silver nanoparticles.
a r t i c l e
i n f o
Article history: Received 28 June 2013 Received in revised form 26 November 2013 Accepted 7 December 2013 Available online 17 December 2013 Keywords: Sodium alginate Silver nanoparticles Microwave synthesis Antimicrobial activity
a b s t r a c t A simple, green, microwave-assisted method of synthesizing silver nanoparticles was developed using sodium alginate as stabilizer and reducer. A possible mechanism involved in the reduction and stabilization of nanoparticles was investigated. The effect of reaction conditions such as the concentration of sodium alginate and AgNO3 , irradiation time and pH on the synthesis of silver nanoparticle was studied. The silver nanoparticles were characterized by UV–vis spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD). The results indicated the formation of spherical, nanometer-sized particles. The reaction parameters significantly affected the formation rate, size and distribution of the silver nanoparticles. XRD analysis revealed that the particles were face-centered cubic. The silver nanoparticles prepared in this way were uniform and stable, which could be stored at room temperature for at least 6 months. The synthesized silver nanoparticles had significant antibacterial activity on two kinds of Gram bacteria. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Silver nanoparticles (AgNPs) have attracted extensive research interest due to their attractive optical, electronic properties and excellent antimicrobial activities [1–3]. AgNPs exhibit strong
∗ Corresponding author at: Collaborative Innovation Center for Marine Biomass Fibers, Materials and Textiles of Shandong Province, Qingdao University, Qingdao 266071, China. Tel.: +86 532 85953069; fax: +86 532 85952497. E-mail addresses: [email protected], [email protected] (Y. Xia). 0927-7757/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfa.2013.12.008
cytotoxicity toward a broad range of microorganisms and are widely used as an antibacterial agent [4]. The advantage of AgNPs compared to bulk metal or salts is the slow and regulated release of silver from nanoparticles, thereby causing long lasting protection against bacteria [5,6]. The antimicrobial activity of AgNPs is comparatively better than most prominent antibiotics used worldwide [7]. Recently, researchers have shown that AgNPs can interact with the human immunodeficiency virus type 1 and prevent the virus from binding to the host cells [8]. Numerous methods have been developed for the preparation of AgNPs. The most common method is chemical reduction of silver
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salt by a reducing agent in the presence of a stabilizing agent. The reducing agents such as sodium borohydride [9,10], hydrazine [11], N,N-dimethylformamide [12], or other organic compounds [13–15] have been used for the preparation of metal nanoparticles with high reactivity. However, many of them may be associated with potential environmental toxicity as well as biological risks [16]. On the other hand, the stabilizers including triphenylphosphine [17], citrate [18] and polyvinylpyrrolidone [19], are often toxic, difficult to dispose [20]. With the increasing awareness of environmental protection, people are inclined to develop the eco-friendly approach for the synthesis of nanoparticles. Three main steps in the preparation of nanoparticles that should be evaluated from a green chemistry perspective are the choice of the solvent medium, environmentally benign reducing agent, and nontoxic material for the stabilization of the nanoparticles. Currently, many natural polymers like chitosan [21], soluble starch [22,23], polypeptide [24], heparin [25], and hyaluronan [26] have been involved in the green preparation of nanoparticles as reducing and stabilizing agent. Wei et al. [21] reported the green synthesis of silver nanoparticles by thermal reduction methods in chitosan–acetic acid–water, and the obtained particles showed highly potent antibacterial activity toward both Gram-positive and Gram-negative bacteria. Yan et al. [24] developed a general strategy of fabricating gold nanoparticles using polypeptides as capping agent and reductant, the resulting positively charged polypeptide-conjugated gold nanoparticles can be applied for biomedical applications. In addition, the plant-based exudate, such as Gum acacia [27] and Gum kondagogu [1], and plant extracts [28–30] have been reported as reducing and stabilizing agents for the silver nanoparticles biosynthesis. Alginate is a naturally occurring carbohydrate polymer isolated from marine algae [31]. It can be characterized as an anionic copolymer comprising mannuronic acid (M block) and guluronic acid (G block) units arranged in an irregular block-wise pattern of varying proportions of GG, MG and MM blocks [32,33]. Sodium alginate is widely used for food and drink, pharmaceutical and bioengineering industries due to its biocompatibility, low toxicity, relatively low cost, and mild gelation by addition of divalent cations such as Ca2+ [34]. It has a number of free hydroxyl and carboxyl groups distributed along the back-bone. It can be well dissolved in water due to negatively charged carboxyl group. In the synthesis of the AgNPs, the carboxyl groups of sodium alginate can electrostatically interact with Ag+ to form a complex, and the hydroxyl groups reduce the Ag+ to Ag0 [35]. Pal et al. [36] and Yang and Pan [37] successfully fabricated metal nanoparticles using sodium alginate as a reducing and stabilizing agent. Compared to chitosan, alginate is stable at higher pH and compatible with other substances without coagulation in aqueous solution. The sodium alginate stabilized AgNPs offer numerous benefits of eco-friendliness and compatibility for pharmaceutical and biomedical applications. The aim of the present work is to develop a simple, rapid and totally green approach of synthesizing AgNPs using sodium alginate as reducing and stabilizing agent, water served as reaction medium. The synthesis was carried out in aqueous medium by microwave heating (without any additional chemical reducing and stabilizing agents). The emphasis was placed on the effects of the reaction conditions on the synthesis of AgNPs. The obtained particles were analyzed by UV–vis spectroscopy, transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). In addition, the antimicrobial activity of the synthesized AgNPs against Gram-positive and Gramnegative bacteria was also investigated. It is worth pointing that the microwave heating has been explored as a promising technique for nanoparticle synthesis. The advantage of microwave heating over the conventional heating are the rapid and uniform internal heating to the solution, microwave irradiation generates very fast nucleation sites in the solution, which significantly enhances the reaction
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rate [38]. To the best of our knowledge, the detailed research of using the microwave technique to prepare AgNPs with sodium alginate as reducing and stabilizing agents is limited in literature. 2. Experimental 2.1. Reagents Sodium alginate (SA) was supplied by Mingyue Seaweed Co. Ltd., Qingdao, Shandong Province, PR China. Silver nitrate (AgNO3 ) was analytical reagent grade. All solutions were prepared with deionized water. All glass wares were thoroughly cleaned with water and dried in an oven. 2.2. Synthesis of alginate-based silver nanoparticles In a typical preparation, sodium alginate was dissolved in distilled water in a 100 mL beaker using heating magnetic stirrer. After complete dissolution, the pH of the solution was adjusted within the range 5–11. Certain amount of AgNO3 precursor was then added dropwise (keeping in mind that the total volume of the reaction medium is 25 mL). Then the reaction mixture was placed in the microwave oven (Galanz WP750) for the reduction of silver ions. After the reaction, the solution was immediately cooled down to room temperature and adjusted to 25 mL by adding a small amount of distilled water to compensate for the loss of water during microwave irradiation. During the heating process, the color of the reaction mixture changed slowly from colorless to light brown due to reduction of Ag+ to Ag0 . The reaction mixture was analyzed by UV–vis spectroscopy to ascertain the formation of AgNPs. The reaction for the formation of AgNPs was systematically investigated by varying the irradiation time from 1 min to 10 min, initial sodium alginate concentration from 0.1% to 1.5%, pH values from 5 to 11, and AgNO3 concentration from 0.4 to 8.0 mmol/L. 2.3. Characterization UV–vis spectra of AgNPs in alginate solution were performed with a Shimadzu UV3150 UV–vis spectrophotometer operating in the transmission mode. A solution containing sodium alginate alone was used as a blank. Transmission electron microscopy (TEM) images were obtained by a JEM-1200EX microscope at an accelerating voltage of 100.0 kV. The samples for TEM studies were prepared by drop-casting a dispersion of AgNPs on carbon-coated copper grids, which were allowed to dry under ambient conditions. XRD measurements of sodium alginate and sodium alginate–AgNPs composites were carried out on a powder X-ray diffractometer (D/MAX-RB) using CuK␣ radiation ( = 0.15418 nm) over a 2 range of 5–80◦ with a step size of 0.05◦ . The aqueous solution of sodium alginate embedded with AgNPs was spread over a glass plate and coagulated in the air to form membrane and the yellowish-brown colored film obtained was used for XRD analysis. Fourier transform infrared spectroscopy (FTIR) was recorded on spectrometer (NICOLET5700) by KBr tablet method, and the spectra were scanned in the range of 400–4000 cm−1 at a resolution of 4 cm−1 . 2.4. Antibacterial assay The disk diffusion method was used to study the antibacterial activity of the synthesized silver nanoparticles. Staphylococcus. aureus (ATCC 25923) and Escherichia coli (ATCC 35218) were used as model test strains for Gram-positive and Gram-negative bacteria, respectively. Nutrient agar medium was prepared by using peptone (5.0 g), beef extract (3.0 g), and sodium chloride (5.0 g) in 1000 mL distilled water, the pH was adjusted to 7.0 and agar (15.0 g) was added to the solution. The agar medium was sterilized
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Fig. 1. Schematic illustration of a typical procedure for the synthesis of AgNPs in sodium alginate solution under microwave irradiation.
in an autoclave at 121 ◦ C for 20 min. This nutrient agar medium was transferred into sterilized petri dishes. After solidification of the media, bacterial culture was inoculated on the solid surface of the media and swabbed with sterile cotton swab. The sterile paper discs (10 mm) were impregnated with 30 L sample solutions and then left to dry at 37 ◦ C for 12 h in sterile conditions. Then, the impregnated discs were placed on the inoculated agar and incubated for 24 h at 37 ◦ C. After incubation, the zone of inhibition (ZOI) was measured by subtracting the disk diameter from the total inhibition zone diameter. The silver nanoparticles used here were prepared with 0.5% and 1.0% sodium alginate solution containing 4 mmol/L AgNO3 , pH 9 and microwave irradiation for 8 min. 3. Results and discussion 3.1. Reaction mechanism for formation of silver nanoparticles Sodium alginate was used to perform dual role: as reducing agent for silver ions and as stabilizing agent during/after the formation of AgNPs. Previous reports [28,37,39] have disclosed that the solutions of polymers can be used for the synthesis and stabilization of nanoparticles. For the synthesis of AgNPs, the generally accepted mechanism suggests a two-step process, i.e. atom formation and then polymerization of the atoms. In the first step, a portion of metal ions in the solution is reduced by the available reducing groups. The atoms thus produced act as nucleation centers and catalyze the reduction of the remaining metal ions present in the bulk solution. Compared with other water-soluble polymers, alginate is an anionic polymer with high charge density, the negatively charged alginate facilitates the attraction of the positively charged silver ions to the polymeric chains, which were then reduced by the existing reducing groups. The preformed silver atoms adsorb Ag+ on the surface via dimerization or association with excess ions due to the binding energy between metal atoms. The surface ions are again reduced, subsequently the atoms coalesce leading to the formation of metal clusters. In this way, the aggregation process does not cease and finally results in larger particles. The process can be stabilized through sodium alginate so preventing further coalescence. These
metal cluster surfaces are likely to be anchored through strong association between AgNPs surface and “O” atom of the functional groups ( COO− and OH) of sodium alginate [40]. The resulting surface negative charge of alginate fragments containing carboxylic groups stabilizes nanoparticles against coalescing with the next one because of electrostatic repulsion and steric effects [41,42]. The overall synthetic procedure is presented in Fig. 1. Fig. 2 shows the FTIR spectra of sodium alginate and sodium alginate–AgNPs. In the case of sodium alginate, the peaks centered at around 3435 cm−1 can be attributed to the stretching vibration of hydroxyl group; the strong peak noted at 1609 cm−1 is due to the stretching vibration of CO2 − (carboxylate ion) group; the band at around 1414 cm−1 is ascribed to the hydroxyl group deformation vibration; the absorption band at 1083 cm−1 is corresponded to C O C stretching mode from the glucosidic units. In the case of sodium alginate–AgNPs, the band of CO2 − is shifted to 1613 cm−1 ,
Fig. 2. FTIR spectra of sodium alginate and sodium alginate–AgNPs.
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Fig. 3. Effect of reaction parameters on the synthesis of silver nanoparticles. (A) UV–vis spectra of AgNPs prepared at different alginate sodium concentration. Reaction conditions: 4 mmol/L AgNO3 , pH 9, irradiation 8 min. Inset: Digital photograph of sodium alginate solution (0.5%) mixed AgNO3 before (a) and after (b) microwave irradiation for 8 min. (B) UV–vis spectra of AgNPs prepared using different concentrations of AgNO3 . Reaction conditions: 0.5% sodium alginate, pH 9, irradiation 8 min. (C) UV–vis spectra of AgNPs prepared with different irradiation times. Reaction conditions: 4 mmol/L AgNO3 , 0.5% sodium alginate, pH 9. (D) UV–vis spectra of AgNPs prepared with different pH. Reaction conditions: 0.5% sodium alginate, 4 mmol/L AgNO3 , irradiation 8 min.
the peaks of 3435 and 1414 cm−1 shift to 3422 and 1384 cm−1 indicating that carboxyl groups and the hydroxyl group are involved in the synthesis and stabilization of AgNPs. The variations of the hydroxyl and carboxylate groups have been reported in the previous study on synthesis of AgNPs with another polysaccharide [1,27]. 3.2. Effect of reaction parameters on the synthesis of silver nanoparticles UV–vis spectroscopy is a simple and sensitive technique for the characterization of AgNPs due to the excitation of surface plasmon resonance (SPR) in the AgNPs. Theoretical studies on the dependence of the UV–vis absorption on the size of the metal spheres have been conducted [43]. The general trend is that the SPR absorption band shows a red shift with increasing particle size. In addition, the aggregation of colloidal silver causes a decrease in the intensity of the main peak, and also results in a long tail on the long-wavelength side of the peak. After microwave irradiation, the sodium alginate solutions containing silver nitrate changed from colorless to yellow (see Fig. 3A inset.), the appearance of yellow color in the reaction mixtures supports the formation of AgNPs [18,30]. In order to monitor the effect of different parameters to the formation of AgNPs, the UV–vis absorption spectra of synthesized AgNPs were recorded against respective sodium alginate blanks. Fig. 3A shows the UV–vis spectra of the AgNPs obtained using sodium alginate as reducing and stabilizing agent in different concentration (0.1–1.5%) at the fixed concentration of silver nitrate
(4 mmol/L AgNO3 ), pH 9 and microwave irradiation for 8 min. The data reveals a number of findings which can be presented as follows: (i) the spectra exhibits unique SPR absorption band at approximately 417 nm, which is associated with the formation of nanoparticles. This result implies that sodium alginate can reduce the Ag+ to Ag0 due to the OH groups present in the polymer. Hydroxyl group mediated reduction was reported for the synthesis of silver and gold nanoparticles in previous studies [1,36,39]; (ii) the peak acquires an ideal bell sharp which implies that negligible aggregation occurs in the reactive system and the nanoparticles are well dispersed; (iii) the absorption intensity increases rapidly as the concentration of sodium alginate increases from 0.1% to 0.5%. The increase in SPR band intensity could be attributed to the formation of more AgNPs. The possible reason is that the increased number of carboxyl groups and hydroxyl groups facilitates the complexation of Ag+ to the molecular matrix, meanwhile, more hydroxyl groups benefit the reduction of Ag+ [39]; (iv) further increase in sodium alginate concentration from 0.5% to 1.5% results in decrease in the peak intensity, the peak shifts toward longer wavelength at higher sodium alginate concentrations, which suggests aggregation of the AgNPs and formation of larger particles as the concentration of sodium alginate exceeds 0.5%. The increase in the polymer concentration could induce conflicting effects on the particle size. At higher polymer concentrations, more polymer molecules can cap the AgNPs to decrease the particle size. While, polymer acts as a reductant, it accelerates the nucleation and growth of the AgNPs to produce larger particles at relatively high polymer concentrations [43]. At higher sodium alginate concentrations
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Fig. 4. TEM image and histogram of silver nanoparticles prepared at different conditions. (A) 0.8 mmol/L AgNO3 , 0.5% sodium alginate, pH 9, irradiation 8 min. (B) 4 mmol/L AgNO3 , 0.5% sodium alginate, pH 9, irradiation 8 min. (C) 6 mmol/L AgNO3 , 0.5% sodium alginate, pH 9, irradiation 8 min. (D) 0.1% sodium alginate, 4 mmol/L AgNO3 , pH 9, irradiation 8 min. (E) 0.5% sodium alginate, 4 mmol/L AgNO3 , pH 9, irradiation 8 min. (F) 1.0% sodium alginate, 4 mmol/L AgNO3 , pH 9, irradiation 8 min.
(>0.5%), the stabilizing effect of the sodium alginate molecules is enhanced. However, the role of sodium alginate as a reductant is dominant at relatively high concentrations to produce large particles by further nucleation and growth. Therefore, it should be mentioned that a low concentration of sodium alginate in the reaction medium (0.5%) is enough for full reduction of the Ag+ to Ag0 nanoparticles. The production of AgNPs with 0.5% sodium alginate was evaluated with varying AgNO3 concentrations and the UV–vis spectra of the AgNPs obtained are shown in Fig. 3B. It can be observed that the strongest SPR band is centered at around 418 nm when the AgNO3 concentration ranged from 0.4 to 8.0 mmol/L, the peak is symmetric and there is no obvious absorption in the range of 450–700 nm, which indicates that negligible aggregation occurs in this reactive system and the nanoparticles are well dispersed [44]. The absorption peak intensity gradually increases with the increase of AgNO3 concentration reflecting the formation of more and more AgNPs. However, the particle size does not become larger due to the stabilizing effect of sodium alginate macromolecules. Therefore, it is possible that a large amount of AgNPs with small
particles size can be achieved using sodium alginate as the reducing and stabilizing agent. It was further confirmed by the TEM images shown in Fig. 4A–C. The synthesis was also monitored by different irradiation times (1–10 min) with 0.5% sodium alginate and 4 mmol/L AgNO3 at pH 9. Fig. 3C shows the UV–vis spectra of the AgNPs obtained after different irradiation times. The data in Fig. 3C reveals several important findings which can be presented as follows: (i) at the early stage reaction duration (<3 min), the plasmon band is broad and weak indicating low conversion of Ag+ to AgNPs at this irradiation duration. (ii) prolonging the reaction duration up to 5 min, the strongest SPR band occurs at 418 nm implying that large amounts of silver ions are reduced and used for cluster formation; (iii) further increase in the irradiation time to 8 min, there is a gradual increase in the absorption intensity and without any shift in the peak wavelength which indicates that the AgNPs content increases with increased irradiation time and the mean diameter of the AgNPs does not change much; (iv) raising irradiation time to 10 min is accompanied by the absorption band shift to longer wavelength (427 nm) which could be attributed to enlargement in the AgNPs size.
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Fig. 5. Average particle size of AgNPs synthesis at different reaction conditions. (A) Different concentration of AgNO3 . (B) Different concentration of sodium alginate. (C) Different pH.
Sodium alginate is a weak reductant, the reaction need long period with common heating method. Yang and Pan [37] reported the hydrothermal synthesis of AgNPs with sodium alginate as a reducing and stabilizing agent, it needed more than 6 h at 100–180 ◦ C. Sodium alginate would be degraded after a long period of heating, resulting in the decrease of the stabilizing ability to AgNPs, the prepared AgNPs should be centrifuged and redispersed in sodium alginate aqueous solution. Compared with the conventional heating method, the advantages of microwave irradiation are the rapid heating rate and uniformly heating ability, the reaction can be finished in a few minutes due to rapid internal heating, as well as the formation of a special reaction system that enables selective crystallization and homogeneous nucleation [42]. The stabilizing ability of sodium alginate to AgNPs is better because of the weak degradation in a short period of microwave heating.pH value is usually another key factor to the Ag+ reduction. Fig. 3D shows the UV–vis spectra of the AgNPs prepared at different pHs. At pH 5, only a band at about 270 nm was observed, which corresponded to the existence of Ag+ clusters [28,41]. With the increase of pH value, a new band appeared at about 418 nm on the UV–vis spectra, suggesting the formation of AgNPs. It also can be seen, the peak intensity increases gradually with the increase of pH value, that is because, higher pH value of sodium alginate solution would lead to more exposed carboxyl groups electrostatic interacting with Ag+ , and enhance the reduction rate of Ag+ by sodium alginate. When the solution pH value was increased to 11, the peak intensity increases, but the symmetry of plasmon band is not well implying a broad particle size distribution. 3.3. TEM and XRD analysis To understand better the effects of the synthesis conditions on the shape and size of AgNPs, TEM was used to evaluate the morphology and size of some representative silver particles obtained. Fig. 4 shows the typical TEM images of AgNPs synthesized at different conditions. As Fig. 4A demonstrates, fewer AgNPs were observed in the solution when the AgNO3 concentration was 0.8 mmol/L. Fig. 4B and C illustrates the TEM images and particle size distribution histograms of AgNPs prepared with the AgNO3 concentration of 4 and 6 mmol/L, respectively. As is evident from TEM micrographs, large numbers of nanoparticle formed at high AgNO3 concentration. The obtained AgNPs are in the nano range with uniform and spherical shapes. The histograms (the inset of Fig. 4A–C) clearly illustrate that the prepared particle size are around 10 nm. The AgNPs are well dispersed at higher AgNO3 concentration which indicates a good stabilization effect of the sodium alginate. Fig. 5 shows the average particle size distribution of AgNPs synthesized at different
reaction conditions. As Fig. 5A presents, the average particle size is about 12 nm at AgNO3 concentration of 8 mmol/L, the negligible change of the particle size at higher AgNO3 concentration further confirms the stabilizing effect of sodium alginate. Fig. 4D–F presents TEM micrograph and histogram of silver nanoparticles prepared at different sodium alginate concentrations. These silver nanoparticles observed are spherical in shape and well separated in aqueous medium. When the concentration of sodium alginate is increased from 0.1% to 0.5%, the particle size of the silver nanoparticles formed decreases and the uniformity of particle size distribution increases (Fig. 4D and E). The average particle size of AgNPs decreases from about 20 nm (0.1% sodium alginate) to 10 nm (0.5% sodium alginate) (Fig. 5B). These findings are similar to a previous study on biosynthesis of gold nanoparticles with sodium alginate, in which the concentration of the biopolymer increased from 0.01% to 0.1% [36]. The possible explanation is at higher alginate concentration, the interaction between ionic silver and functional groups on alginate as well as the rate of nanoparticles capping is excellent. In addition, the aggregation is lower due to less collision of silver nanoparticles. Compared with the preparation with 0.5% sodium alginate (Fig. 4E), the preparation with 1.0% sodium alginate contains much less silver nanoparticles and the particle size formed has a slight increase (Fig. 4F). This finding is consistent with the UV–vis analysis results, which shows that the absorption peak intensity of the obtained sample with 1.0% sodium alginate is weaker than that of the obtained sample with 0.5% sodium alginate (Fig. 3A). This study indicates that the particle size of the silver nanoparticles can be controlled by varying the concentration of alginate. The average particle sizes of AgNPs synthesized at pH 8, 9, and 11 are shown in Fig. 5C. The average particle size increases from about 10 nm (pH 9) to 15 nm (pH 11), which can be ascribed to the rapid reaction rate at higher pH value. The reductive properties of sodium alginate are substantially enhanced owing to the base hydrolysis with the formation of low molecular weight reducing fragments, and therefore, reflecting the dual role of sodium alginate as stabilizing and reducing agent in alkaline medium [41]. XRD analysis was performed to confirm the crystal phase of the prepared AgNPs. Fig. 6 shows typical XRD pattern of the alginate–AgNPs prepared with 0.5% sodium alginate and the AgNO3 concentration 4 mmol/L, at pH 9, microwave irritation for 10 min. The broad reflexion at 14◦ is due to the crystallinity of sodium alginate. Several distinct diffraction peaks at approximately 38.1◦ , 44.2◦ , 64.3◦ and 77.4◦ are assigned to reflections from the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) planes of the silver crystal, respectively, which confirms the existence of silver and further on the basis that they can be indexed as face-centered-cubic (FCC) structure of silver
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Fig. 6. XRD pattern of the as-prepared AgNPs: (a) sodium alginate–AgNPs and (b) sodium alginate.
[45]. XRD pattern thus clearly illustrates that the silver nanoparticles formed in this present synthesis are crystalline in nature. The other unidentified crystalline peak at 2 = 27.19◦ , 32.24◦ are also apparent in many works in which the XRD pattern includes the relevant 2 range. These peaks are due to the crystalline and amorphous organic phases, accompanying crystallized AgNPs [46–48].
3.4. Stability analysis The aggregation degree of metal nanoparticles could be effectively reflected by changes of the absorption characteristics, especially the shift of SPR peak in the UV–vis spectrum [49]. If strong aggregation occurred for AgNPs after a long storage period, strong red shift of SPR band would be observed in the UV–vis spectra. In this work, the AgNPs in sodium alginate solution was stored for 6 months at room temperature (25 ± 5 ◦ C), UV–vis spectroscopy was used to measure the stability (Fig. 7), as shown in Fig. 6, the absorbance intensity of the AgNPs changes only slightly, without a significant change in the SPR band position or the spectral shape, The as-prepared AgNPs is still clear yellow with no obvious change in color (see Fig. 7 inset). The results indicate that the as-prepared AgNPs is stable at room temperature for months with negligible aggregation, which further confirms the sodium alginate play an important role in stabilizing the AgNPs.
Fig. 7. UV–vis absorption spectra of AgNPs stabilized with sodium alginate (a) after reaction termination and (b) after 6 months storage at room temperature. The arrow indicates the maximum absorbance at 418 nm. Inset: Digital photograph of sodium alginate-stabilized AgNPs (1) after reaction termination and (2) after 6 month storage at room temperature.
3.5. Antibacterial activity Silver in an aqueous solution releases silver ions, which are biologically active and have bactericidal effects [50]. Big changes in the membrane structure of bacteria as a result of the interaction with silver cations lead to the increased membrane permeability of the bacteria. In the case of AgNPs, they interact extensively with the bacteria cell walls and cause lysis [51]. Besides, recent study indicated that the bactericidal effect of AgNPs mostly depends on the size of particles and the smaller is the better, the nanoparticles smaller than 10 nm have a direct interaction with the bacteria and produce electronic effects, which enhance the reactivity of nanoparticles [52]. In this study, the synthesized AgNPs with 0.5% and 1.0% sodium alginate exhibit good antibacterial activity against both Gram-negative and Gram-positive bacteria (Fig. 8), but it shows higher antibacterial activity against E. coli (Gram-negative) then S. aureus (Gram-positive) (Table 1). The differential sensitivity of Gram-negative and Gram-positive bacteria toward AgNPs is possible due to the difference in their structure of the cell wall. The cell wall of the Gram-positive bacteria is composed of a thick layer of peptidoglycan, consisting of linear polysaccharide chains cross-linked by short peptides thus forming more rigid structure leading to difficult penetration of the silver nanoparticles, but the cell walls of Gram-positive bacteria possesses thinner layer
Fig. 8. Zone of inhibition of silver nanoparticles against (A) S. aureus, (B) E. coli. (0) is control, (1) is AgNPs prepared with 1.0% sodium alginate, (2) is AgNPs prepared with 0.5% sodium alginate.
X. Zhao et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 444 (2014) 180–188 Table 1 Zone of inhibition (mm) of synthesized silver nanoparticles against bacterial pathogens. Test organism
E. coli S. aureus
Silver nanoparticles in 30 L Synthesized with 0.5% sodium alginate
Synthesized with1.0% sodium alginate
9.4 5.8
7.0 4.3
of peptidoglycan [29]. On the other hand, silver nanoparticles synthesized with 0.5% sodium alginate have comparatively higher antibacterial activity than those synthesized with 1.0% sodium alginate because of smaller in size. Sodium alginate as control was not found any of antibacterial patterns. 4. Conclusions In this study, a facile and efficient method for the green synthesis of well-distributed spherical AgNPs was developed. The synthesis was carried out in an aqueous medium treated by microwaves using sodium alginate as stabilizing and reducing agent. The sodium alginate concentration, the initial AgNO3 concentration, as well as microwave irradiation time and pH had obvious effects on the amount of AgNPs produced and particle sizes and size distributions. The silver nanoparticles prepared in this way are uniform and stable in solution over a period of six months at room temperature (25 ◦ C) and show no signs of aggregation. The utilization of environmentally benign and renewable materials like sodium alginate for the synthesis of AgNPs in an aqueous medium offers numerous benefits, including eco-friendliness and compatibility with biomedical, pharmaceutical, and textile applications. Microwave irradiation can accelerate the formation rate of particles. Acknowledgements The authors appreciate the financial support to this research by National High Technology Research Development Plan (863 Plan) of China (No. 2010AA093701), the Natural Science Foundation of China (No. 51203083) and Special Fund for Self-directed Innovation of Shandong Province of China (No. 2013CXB80201). References [1] A.J. Kora, R.B. Sashidhar, J. Arunachalama, Gum kondagogu (Cochlospermum gossypium): a template for the green synthesis and stabilization of silver nanoparticles with antibacterial application, Carbohydr. Polym. 82 (2010) 670–679. [2] D. Li, Q. He, Y. Cui, J. Li, Fabrication of pH-responsive nanocomposites of gold nanoparticles/poly(4-vinylpyridine), Chem. Mater. 19 (2007) 412–417. [3] D. Wei, W. Qian, Facile synthesis of Ag and Au nanoparticles utilizing chitosan as a mediator agent, Colloid Surf. B: Biointerfaces 62 (2008) 136–142. [4] T. Textor, M. Fouda, B. Mahltig, Deposition of durable thin silver layers onto polyamides employing a heterogeneous Tollen’s reaction, Appl. Surf. Sci. 256 (2010) 2337–2342. [5] S.Y. Luo, J. Chen, M. Chen, W.C. Xu, X.L. Zhang, Antibacterial activity of silver nanoparticles colloidal sol and its application in package film, Adv. Mater. Res. 380 (2012) 254–259. [6] P. Totaro, M. Rambaldini, Efficacy of antimicrobial activity of slow release silver nanoparticles dressing in post-cardiac surgery mediastinitis, Interact. Cardiovasc. Thorac. Surg. 8 (2009) 153–154. [7] R. Roy, M.R. Hoover, A.S. Bhalla, T. Slaweekl, S. Dey, W. Cao, Ultradilute Ag-aquasols with extraordinary bactericidal properties: role of the system Ag–O–H2 O, Mater. Res. Innov. 11 (2008) 3–18. [8] J.L. Elechiguerra, J.L. Burt, J.R. Morons, A. Camachobragado, X.X. Gao, H.H. Lara, M.J. Yacaman, Interaction of nanoparticles with HIV-1, J. Nanobiotechnol. 3 (2005) 6–16. [9] K. Vimala, K.S. Sivudu, Y.M. Mohan, B. Sreedhar, K.M. Raju, Controlled silver nanoparticles synthesis in semi-hydrogel networks of poly(acrylamide) and carbohydrates: a rational methodology for antibacterial application, Carbohydr. Polym. 75 (2009) 463–471.
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